JP2017201918A - Fusion protein, polynucleotide encoding the same, and uses thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a fusion protein of a functional protein capable of measurement (detection, quantification, etc.) based on a change in luminescent property and an antibody Fc region-binding domain, which can be produced in a high yield by a simple production process.SOLUTION: According to the present invention, there is provided a fusion protein that comprises: a first region consisting of an Fc region-binding domain having the ability to bind to the Fc region of the antibody; and a second region consisting of a GST active domain having glutathione-S-transferase (GST) activity. According to the present invention, the fusion protein is used for measurement of antigen, antibody, or GSH in a sample.SELECTED DRAWING: Figure 7

Description

  The present invention relates to a fusion protein of an antibody binding domain and glutathione-S-transferase (also referred to herein as “GST”), a polynucleotide encoding the same, and uses thereof. More specifically, the present invention relates to a fusion protein of a ZZ domain capable of binding to an Fc region of an antibody and GST, a polynucleotide encoding the same, and uses thereof.

  Glutathione-S-transferase (GST) is an enzyme that catalyzes the formation of a conjugate between an electrophilic compound and reduced glutathione (also referred to as “GSH” in this specification). It is known to be involved in glutathione conjugation of endogenous active metabolites. In vivo, GST plays a role of detoxifying or discharging electrophilic compounds, for example, by producing these conjugates. In addition to this, GST has various functions such as steroid hormone biosynthesis, amino acid degradation, and prostaglandin biosynthesis. In addition, several subtypes of GST are overexpressed in cancer cells, and inhibitor treatment and knockdown induce cell death, and thus are considered important targets in drug discovery.

  For this reason, there is also a need for measuring the activity of GST to help diagnose specific diseases. Furthermore, in the biochemical research field, many kits using GST have been developed, and the demand for accurately measuring the activity of GST is very high.

  Here, conventionally proposed methods for measuring GST activity include absorption method (colorimetric method), bioluminescence method, fluorescence method and the like. As a method for measuring GST activity by an absorption method (colorimetric method), for example, a method is known in which a change in absorbance at 340 nm of a glutathione product is detected using 2,4-dinitrochlorobenzene (CDNB). . On the other hand, as a method for measuring GST activity by bioluminescence method, for example, Non-Patent Document 1 discloses luciferin produced by nitrobenzenesulfonic acid ester of cagedluciferin undergoing aromatic nucleophilic substitution reaction (deprotection reaction) by GST. , And a method for measuring GST activity from the luminescence intensity by emitting light in the presence of ATP and luciferase. Furthermore, several fluorescent probes have been developed as methods for measuring GST activity by the fluorescence method. For example, Non-Patent Document 2 discloses a series of fluorescent probes such as DNAFs and DNAT-Me as probes having a maximum absorption wavelength at 490 nm and a maximum fluorescence wavelength near 510 nm. These fluorescent probes show a significant increase in fluorescence intensity due to glutathioneization due to GST activity and the accompanying denitration reaction. Thus, it can be said that the fluorescent probe disclosed in Non-Patent Document 2 has a very high reactivity in the presence of GST and is a relatively useful fluorescent probe.

  However, the fluorescent probes (DNAFs and DNAT-Me) disclosed in Non-Patent Document 2 have a high reactivity with GST-independent glutathione, so that a sufficient S / N ratio cannot be obtained. There was a problem that the subsequent fluorescence quantum yield was as low as about 0.1 (when the quantum yield of fluorescein in 0.1N NaOH aqueous solution was 0.85). Furthermore, according to the conventional studies by the present inventors, it has been found that when DNAFs are used as fluorescent probes, there is a problem that the fluorescence intensity of the product is greatly reduced under pH conditions in a weakly acidic region. As a technique for solving this problem, the present inventors have used a fluorescein derivative in which a fluorine atom or a chlorine atom is introduced at a specific site as a fluorescent probe for GST measurement. -It has been reported that the problems of Me can be solved (Non-patent Document 3).

  By the way, protein A is a protein derived from the cell wall of Staphylococcus aureus and is known to specifically bind to an Fc region of an immunoglobulin (particularly, immunoglobulin G (IgG)). Using this property, it has been proposed to use a fusion protein of protein A and aequorin, which is a calcium-binding photoprotein, in an immunoassay (for example, Non-Patent Document 4).

  The ZZ domain is a synthetic IgG binding region developed based on the IgG binding region of protein A (for example, Non-Patent Document 5). It has also been proposed to use a fusion protein of ZZ domain and calcium-binding photoprotein oberin (Non-patent Document 6) or a fusion protein of ZZ domain and aequorin (Patent Document 1) for immunoassay. However, there has been no report on the technique for fusing the ZZ domain with GST.

JP 2008-48607 A

Chem Commun (Camb). 2006 (44): 4620-4622 J Am Chem Soc. 2008 130 (44): 14533-14543 Chem Commun (Camb). 2015 (57): 11459-11462 Biochem. Biophys. Res. Commun. 1990, (171): 169-174 Protein Eng. 1987 (1): 107-113 Biochem. Biophys. Res. Commun. 1996, (219): 475-479

  Here, in the technique for constructing aequorin or oberin, which is a calcium-binding photoprotein, disclosed in Non-Patent Document 4, Non-Patent Document 6 and Patent Document 1 as a fusion protein with protein A or ZZ domain, a recombinant technique, etc. The fusion protein obtained by this method cannot be used for antibody quantification as it is, but it can be converted into a holoprotein by bringing the apoprotein part (apoaequorin or apoovelin) contained in the fusion protein into contact with coelenterazine, a luminescent substrate. Processing is necessary. For this reason, the production process of the fusion protein is complicated, and when the apoprotein is converted into a holoprotein, some of the apoproteins are not converted into a holoprotein, and as a result, all the fusion proteins obtained cannot be used. There was also.

  Therefore, the present invention is a fusion protein of a functional protein that can be measured (detection, quantification, etc.) based on a change in luminescence properties and an antibody Fc region binding domain, and can be produced in a high yield by a simple production process. The purpose is to provide.

  The present inventors have intensively studied to solve the above problems. As a result, the above problem is solved by a fusion protein in which a region comprising a domain having GST activity (GST active domain) is fused to a region comprising a domain having an ability to bind to an Fc region of an antibody (Fc region binding domain). As a result, the present invention has been completed.

  That is, according to one aspect of the present invention, a first region comprising an Fc region-binding domain capable of binding to an Fc region of an antibody and a GST active domain comprising glutathione-S-transferase (GST) activity. A fusion protein comprising two regions is provided.

  Further, according to another aspect of the present invention, a polynucleotide comprising a polynucleotide encoding the fusion protein according to the above aspect, a recombinant vector containing the polynucleotide, and a transformant introduced with the recombinant vector Also provided is a method for producing a fusion protein comprising the steps of culturing the transformant to produce a fusion protein according to the above-described form.

  Furthermore, according to still another aspect of the present invention, in addition to a method of measuring “an antibody containing an Fc region”, “antigen”, and “GSH” in a sample using the fusion protein according to the above aspect, A kit for use in the method is provided. In particular, the above-described method for measuring “an antibody comprising an Fc region” can be applied to a screening method for a preventive and / or therapeutic agent for cancer.

  According to the present invention, a fusion protein of a functional protein and a ZZ domain that can be measured (detection, quantification, etc.) based on a change in luminescent properties, and can be produced in a high yield by a simple production process. Can be provided.

FIG. 1 is a vector map of various pCOLD-I-ZZ-GST expression vectors, which are expression vectors of various ZZ-GSTs constructed in Examples. FIG. 2 shows (A) ZZ-hGSTA1 fusion protein, (B) ZZ-hGSTM1 fusion protein, (C) ZZ-hGSTP1 fusion protein, and (D) ZZ-DmGSTe13 fusion protein, which are ZZ fusion type GSTs prepared in Examples. It is an electrophoresis photograph which shows the result of having carried out Coomassie brilliant blue (CBB) dyeing | staining by SDS-PAGE about each of these. In addition, each lane in FIGS. 2 (A) to 2 (D) is, respectively, 1: before protein expression induction, 2: after protein expression induction, 3: His-tag column non-adsorbed fraction, 4: after purification It is. FIG. 3 shows (A) ZZ-hGSTA1 fusion protein, (B) ZZ-hGSTM1 fusion protein, (C) ZZ-hGSTP1 fusion protein, (D) ZZ-DmGSTe13 fusion protein, which are ZZ fusion type GSTs prepared in the examples. It is a graph of Michaelis Menten plot obtained as a result of examining enzyme reaction kinetics about each of these. FIG. 4 is a diagram illustrating a complex in which various ZZ-fused GSTs according to the present invention are adsorbed via a ZZ domain to an anti-transferrin receptor antibody immobilized on agarose beads via protein L in Examples. It is a graph which shows the result of having measured GST activity using 3, 4-DNADCF which is a fluorescent probe for GST measurement. FIG. 4A is a graph in which the change in fluorescence intensity in this experiment is plotted against the elapsed time after the addition of 3,4-DNADCF. FIG. 4B is a graph showing the initial reaction rate in this experiment. FIG. 4 (B) shows an experiment using an anti-transferrin receptor antibody (αTR) necessary for adsorbing ZZ-fused GST to agarose beads (via a ZZ domain). The results for the case where the test is performed (αTR is “+”) and the case where the experiment is performed without using this (the αTR is “−”) are also shown. FIG. 5 is a graph showing the results of an experiment similar to that shown in FIG. 4 in a system using ZZ-DmGSTe13 when different antibody concentrations were used. FIG. 5 (A) is a graph in which the change in fluorescence intensity in this experiment is plotted against the elapsed time after the addition of 3,4-DNADCF. FIG. 5 (B) is a graph showing the correlation of GST activity with the antibody concentration used in this experiment. FIG. 6 is a diagram showing a complex obtained by adsorbing various ZZ fusion-type GSTs according to the present invention via a ZZ domain to an anti-transferrin receptor antibody immobilized on a plate via protein L in Examples. It is a graph which shows the result of having measured GST activity using 3, 4-DNADCF which is a fluorescent probe for a measurement. FIG. 6 (A) is a graph showing changes over time in the increase in fluorescence intensity when different concentrations of anti-transferrin receptor antibodies are incubated. Here, in the series in which ZZ-DmGSTe13 was not added, 0 μM and 10 μM anti-transferrin receptor antibody treatment was used as a control. FIG. 6 (B) is a graph plotting the initial reaction rate against the concentration of the anti-transferrin receptor antibody. FIG. 7 shows a fluorescent probe for GST measurement in which the fusion protein of the present invention is applied to the detection and quantification of an antibody in an on-cell ELISA against a transferrin receptor expressed on the cell surface of MCF7 cells in Examples. -It is a graph which shows the result of having measured GST activity using DNADCF. FIG. 7A is a graph showing temporal changes in fluorescence intensity associated with different treatments. Here, the marker marked with □ is untreated, the marker marked with ◆ is only treated with anti-transferrin receptor antibody, the marker marked with △ is treated only with ZZ-DmGSTe13, the marker marked with ● is anti-transferrin receptor antibody and ZZ- Both processes of DmGSTE13 are performed. FIG. 7B is a graph showing an increase in fluorescence intensity per unit time in each process (the slope of the graph shown in FIG. 7A).

  Embodiments of the present invention will be described below.

1. One form of the present invention comprises a first region comprising an Fc region-binding domain capable of binding to an Fc region of an antibody, and a GST active domain having glutathione-S-transferase (GST) activity. And a second region. In the present specification, the fusion protein is also referred to as “the fusion protein of the present invention”.

(First area)
The first region in the fusion protein of the present invention is a region consisting of an Fc region-binding domain capable of binding to the Fc region of an antibody. Here, there is no restriction | limiting in particular about the specific form of a 1st area | region, What is necessary is just to consist of a domain which has an amino acid sequence which has the binding ability with respect to the Fc region of an antibody. Here, the “antibody” in the present invention means one having an Fc region, and the class (isotype) may be any of IgG, IgM, IgA, IgD, and IgE. The Fc region-binding domain constituting the first region is the first region, particularly when the Fc region binding domain specifically binds to the Fc region of IgG, such as the protein A and ZZ domains described in detail below. An embodiment in which the Fc region-binding domain constituting the domain is a domain capable of binding to the Fc region of an IgG antibody (that is, an embodiment in which the antibody is an IgG antibody) is a preferred embodiment of the present invention.

  The Fc region-binding domain constituting the first region can be, for example, a protein A amino acid sequence or a domain having substantially the same amino acid sequence. Here, “Protein A” is a protein derived from the cell wall of Staphylococcus aureus, and is known to specifically bind to the Fc region of immunoglobulin (particularly, immunoglobulin G (IgG antibody)). ing. The amino acid sequence of protein A is described in, for example, JP-A-07-51086.

  Another example (preferred embodiment) of the first region is a ZZ domain of protein A. The “ZZ domain of protein A” is a synthetic IgG binding region developed based on the IgG binding region of protein A, and details thereof are described in, for example, Non-Patent Document 5 (Protein Eng. 1987 (1): 107-). 113).

  More specifically, the “ZZ domain of protein A” as the first region includes, for example, (a) a region consisting of the amino acid sequence of SEQ ID NO: 1. Here, SEQ ID NO: 1 shows the amino acid sequence of the ZZ domain of protein A, and SEQ ID NO: 2 is the DNA encoding the amino acid sequence represented by SEQ ID NO: 1 (amino acid sequence of the ZZ domain of protein A) The base sequence is shown.

  The “ZZ domain of protein A” as the first region is, for example, (b) from an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 1. And a region having substantially the same activity or function as the region consisting of the amino acid sequence set forth in SEQ ID NO: 1. In addition, “substantially the same activity or function as the region consisting of the amino acid sequence of SEQ ID NO: 1” means, for example, the binding ability of the IgG antibody to the Fc region. Whether or not an IgG antibody has an ability to bind to an Fc region can be measured, for example, according to a method such as Western blotting or surface plasmon resonance.

  In the present specification, the range of “1 to plural” in “an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added” is, for example, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6 (1 to several), 1 to 5, 1 to 4, 1 to 3, 1 to 2 One. Generally, the smaller the number of amino acids deleted, substituted, inserted or added, the better. Two or more of the amino acid residue deletions, substitutions, insertions and additions may occur simultaneously. Such areas are described in Sambrook J. et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press (2001), Ausbel FM et al., Current Protocols in Molecular Biology, John Wiley and Sons (1987). -1997) “Nuc. Acids. Res., 10, 6487 (1982)”, “Proc. Natl. Acad. Sci. USA, 79, 6409 (1982)”, “Gene, 34, 315 (1985)”, “ Nuc. Acids. Res., 13, 4431 (1985) ”,“ Proc. Natl. Acad. Sci. USA, 82, 488 (1985) ”, etc. Can do.

  In addition, examples of the first region include (c) about 70% or more, 75% or more, 80% or more, 85% or more, 88% or more, 90% or more, 91% with respect to the amino acid sequence of SEQ ID NO: 1. % Or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more of the amino acid sequence, and SEQ ID NO: 1 And a region having substantially the same activity or function (for example, the binding ability of an IgG antibody to an Fc region). In general, the larger the numerical value of identity, the better. The identity of amino acid sequences and base sequences is determined by BLAST (see, for example, Altzshul SF et al., J. Mol. Biol. 215, 403 (1990)) or FASTA (Pearson WR, Methods in Enzymology 183, 63 (See (1990), etc.). When using BLAST or FASTA, the default parameters of each program are used.

  Furthermore, the first region comprises (d) an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a base sequence complementary to the base sequence of SEQ ID NO: 2; In addition, a region having substantially the same activity or function as the region consisting of the amino acid sequence of SEQ ID NO: 1 (for example, the binding ability of the IgG antibody to the Fc region) is also included. Polynucleotides that hybridize under stringent conditions will be described later.

That is, in a preferred embodiment of the fusion protein of the present invention, the first region is selected from the group consisting of the following (a) to (d):
(A) a region consisting of the amino acid sequence of SEQ ID NO: 1;
(B) a region consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 1 and having binding ability to the Fc region of an IgG antibody;
(C) a region comprising an amino acid sequence having 70% or more identity to the amino acid sequence of SEQ ID NO: 1 and having binding ability to the Fc region of IgG antibody; and
(D) Binding to the Fc region of an IgG antibody consisting of an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a base sequence complementary to the base sequence of SEQ ID NO: 2. An area with ability.

In a more preferred embodiment of the fusion protein of the present invention, the first region is selected from the group consisting of the following (a), (b ′), (c ′) and (d ′):
(A) a region consisting of the amino acid sequence of SEQ ID NO: 1;
(B ′) a region consisting of an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 1, and having a binding ability to the Fc region of an IgG antibody;
(C ′) a region consisting of an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 1 and having an ability to bind to the Fc region of an IgG antibody; and
(D ′) an Fc region of an IgG antibody comprising an amino acid sequence encoded by a polynucleotide that hybridizes with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 2 under highly stringent conditions; A region that has the ability to bind to.

(Second area)
The second region in the fusion protein of the present invention is a region consisting of a GST active domain having glutathione-S-transferase (GST) activity. Here, there is no restriction | limiting in particular about the specific form of a 2nd area | region, What is necessary is just to consist of a domain which has an amino acid sequence which has glutathione-S-transferase (GST) activity. Here, the “GST activity” in the present invention means an activity that catalyzes the formation reaction of a conjugate between an electrophilic compound and reduced glutathione (GSH).

  The domain having GST activity (GST active domain) can be, for example, a conventionally known amino acid sequence of GST or a domain having substantially the same amino acid sequence. Here, conventionally, as GST derived from a mammal, alpha (α), kappa (κ), mu (μ), omega (ω), pi (π), sigma (σ), theta (θ), Eight isoforms of zeta (ζ) are known. Examples of GSTs derived from organisms other than mammals include DmGSTe13 and DmGSte14 derived from Drosophila melanogaster, and SjGST derived from Schistosoma japonicum, which is widely used as a so-called GST tag. Are known. In the fusion protein according to this embodiment, the GST active domain constituting the second region may be an amino acid sequence of any of these conventionally known GSTs or a domain having substantially the same amino acid sequence.

  Here, a preferred embodiment of the second region is composed of human-derived hGSTA (alpha) 1, hGSTM (mu) 1, hGSTP (pi) 1 and DmGSTe13 derived from Drosophila among the above-mentioned conventionally known GSTs. It consists of a domain having the amino acid sequence of any GST selected from the group or a substantially identical amino acid sequence thereto. Among these, from the viewpoint of excellent GST activity, the second region is an amino acid sequence of any GST selected from the group consisting of hGSTM1, hGSTP1, and DmGSTe13 or an amino acid sequence substantially identical thereto. It is preferably a domain having a domain having the amino acid sequence of DmGSTe13 or a domain having substantially the same amino acid sequence as this.

  More specifically, “the domain having the amino acid sequence of any GST selected from the group consisting of hGSTA1, hGSTM1, hGSTP1, and DmGSTE13 or a substantially identical amino acid sequence thereof” as the second region is more specifically, For example, (e) a region consisting of the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9. Here, SEQ ID NO: 3 shows the amino acid sequence (GenBank Accession Number: NP_665683.1) of human GSTA1 (hGSTA1), and SEQ ID NO: 4 shows the amino acid sequence represented by SEQ ID NO: 3 (human GSTA1 (hGSTA1) ) Amino acid sequence)), the nucleotide sequence of the DNA (including the stop codon; GenBank Accession Number: NM — 145740.4). SEQ ID NO: 5 shows the amino acid sequence of human GSTM1 (hGSTM1) (GenBank Accession Number: NP_000552.2), SEQ ID NO: 6 shows the amino acid sequence represented by SEQ ID NO: 5 (amino acid of human GSTM1 (hGSTM1)) The base sequence (including the stop codon; GenBank Accession Number: NM_000561.3) of the DNA encoding the (sequence). SEQ ID NO: 7 shows the amino acid sequence (GenBank Accession Number: NP_000843.1) of human GSTP1 (hGSTP1), SEQ ID NO: 8 shows the amino acid sequence represented by SEQ ID NO: 7 (amino acid of human GSTP1 (hGSTP1)) DNA sequence encoding (sequence) (including stop codon; GenBank Accession Number: NM_000852.3). SEQ ID NO: 9 represents the amino acid sequence (GenBank Accession Number: NP — 610457.1) of Drosophila melanogaster GSTe13 (DmGSTe13), and SEQ ID NO: 10 represents the amino acid sequence represented by SEQ ID NO: 9 (Drosophila melanogaster GSTe13 (DmGSTe13) DNA sequence (including the stop codon; GenBank Accession Number: NM — 136613.3).

  In addition, as the second region, “the amino acid sequence of any GST selected from the group consisting of hGSTA1, hGSTM1, hGSTP1, and DmGSTE13 or a domain having an amino acid sequence substantially the same” (f) sequence It consists of an amino acid sequence in which one to a plurality of amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, and SEQ ID NO: : 3, SEQ ID NO: 5, SEQ ID NO: 7, or a region having substantially the same quality of activity or function as the region consisting of the amino acid sequence set forth in SEQ ID NO: 9. In addition, “substantially the same activity or function as the region consisting of the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9” means, for example, GST activity. Whether or not it has GST activity can be measured, for example, according to the method described in Examples described later.

  Examples of the second region include (g) about 70% or more, 75% or more, 80% or more of the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9. % Or more, 85% or more, 88% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more And having substantially the same activity or function as a region consisting of the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9 (for example, A region having (GST activity) is also included. In general, the larger the numerical value of identity, the better.

  Furthermore, the second region includes (h) a polynucleotide comprising a base sequence complementary to the base sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10 and stringent conditions Consisting of an amino acid sequence encoded by a polynucleotide that hybridizes underneath and substantially homogenous with a region consisting of the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9. Also included are regions having activity or function (eg, GST activity).

That is, in a preferred embodiment of the fusion protein of the present invention, the second region is selected from the group consisting of the following (e) to (h):
(E) a region consisting of the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9,
(F) consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, And a region having GST activity;
(G) a region consisting of an amino acid sequence having 70% or more identity to the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9 and having GST activity; and,
(H) encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10. A region having a GST activity.

In a more preferred embodiment of the fusion protein of the present invention, the second region is selected from the group consisting of the following (e), (f ′), (g ′) and (h ′):
(E) a region consisting of the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9,
(F ′) consisting of an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9. And a region having GST activity;
(G ′) a region consisting of an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, and having GST activity ;and,
(H) by a polynucleotide that hybridizes under high stringency conditions with a polynucleotide comprising a base sequence complementary to the base sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10. A region consisting of an encoded amino acid sequence and having GST activity. The fusion protein of the present invention may further contain an amino acid sequence for promoting translation and / or a peptide sequence for purification. As an amino acid sequence for promoting translation, a peptide sequence used in this technical field (for example, a TEE sequence) can be used. As a peptide sequence for purification, a peptide sequence used in this technical field (for example, a histidine tag (His-tag having an amino acid sequence of 4 or more, preferably 6 or more histidine residues) is used. tag) sequences, etc.) can be used. Such a sequence can be included at any position of the fusion protein. The fusion protein (ZZ fusion type GST) prepared in the examples described later has a histidine tag (His-tag) sequence at the N-terminus, but may be located at the C-terminus.

  In the fusion protein of the present invention, the first region and the second region may be directly connected or indirectly connected. In the latter case, for example, a linker sequence may be provided between the first region and the second region. In this case, the amino acid length of the linker sequence is not particularly limited, and those skilled in the art appropriately determine the amino acid length of the linker sequence and its specific sequence in consideration of the convenience during production of the fusion protein and the balance with the restriction site. Can be determined. There is no restriction | limiting in particular about the acquisition method of the fusion protein of this invention. The fusion protein of the present invention may be a fusion protein synthesized by chemical synthesis or a recombinant fusion protein produced by a gene recombination technique. When the fusion protein of the present invention is chemically synthesized, for example, it can be synthesized by Fmoc method (fluorenylmethyloxycarbonyl method), tBoc method (t-butyloxycarbonyl method) or the like. Chemical synthesis can also be performed using a commercially available peptide synthesizer. When the fusion protein of the present invention is produced by a gene recombination technique, it can be produced by an ordinary gene recombination technique. More specifically, the fusion protein of the present invention can be prepared by introducing a polynucleotide (eg, DNA) encoding the fusion protein of the present invention into an appropriate expression system. The polynucleotide encoding the fusion protein of the present invention and the expression of the fusion protein of the present invention in the expression system will be described later.

2. Polynucleotide of the Present Invention The present invention also provides a polynucleotide (hereinafter also referred to as “polynucleotide of the present invention”) comprising the polynucleotide encoding the fusion protein of the present invention described above. The polynucleotide of the present invention may be any polynucleotide as long as it contains a base sequence encoding the fusion protein of the present invention, but is preferably DNA. Examples of DNA include genomic DNA, genomic DNA library, cell / tissue-derived cDNA, cell / tissue-derived cDNA library, and synthetic DNA. The vector used for the library is not particularly limited and may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Further, the polynucleotide of the present invention may be obtained by directly amplifying the total RNA or mRNA fraction from the above cells / tissues by RT-PCR.

The polynucleotide of the present invention includes (1) a first coding sequence selected from the group consisting of the following (A) to (D):
(A) a polynucleotide comprising a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2;
(B) a polynucleotide that hybridizes with a polynucleotide consisting of a base sequence complementary to the base sequence of SEQ ID NO: 2 under stringent conditions and encodes a region capable of binding to an Fc region of an antibody A polynucleotide;
(C) a polynucleotide containing a polynucleotide encoding a region consisting of the amino acid sequence of SEQ ID NO: 1; and
(D) a polymorphism encoding a region consisting of an amino acid sequence in which one to a plurality of amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 1 and having an ability to bind to an Fc region of an antibody A polynucleotide comprising nucleotides; and
(2) A second coding sequence selected from the group consisting of the following (E) to (H):
(E) a polynucleotide comprising a polynucleotide consisting of the base sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10;
(F) It hybridizes under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, and GST A polynucleotide comprising a polynucleotide encoding a region having activity;
(G) a polynucleotide comprising a polynucleotide encoding a region consisting of the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9; and
(H) consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, And a polynucleotide comprising a polynucleotide encoding a region having GST activity;
And polynucleotides and the like. In the present specification, the “first coding sequence” means a base sequence including a base sequence encoding the first region, and the “second coding sequence” encodes the second region. Means a base sequence containing the base sequence

In a preferred embodiment of the polynucleotide of the present invention, the first coding sequence is a group consisting of the following (A), (B ′), (C) and (D ′):
(A) a polynucleotide comprising a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2;
(B ′) a polynucleotide that hybridizes with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 2 under a highly stringent condition and encodes a region capable of binding to an Fc region of an antibody A polynucleotide comprising:
(C) a polynucleotide comprising a polynucleotide encoding a region consisting of the amino acid sequence of SEQ ID NO: 1; and
(D ′) an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 1, and encodes a region having an ability to bind to an Fc region of an antibody A polynucleotide comprising a polynucleotide;
Selected from
(2) The second coding sequence is composed of the following (E), (F ′), (G) and (H ′):
(E) a polynucleotide comprising a polynucleotide consisting of the base sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10;
(F ′) hybridizes under high stringency conditions with a polynucleotide comprising a base sequence complementary to the base sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, and A polynucleotide comprising a polynucleotide encoding a region having GST activity;
(G) a polynucleotide comprising a polynucleotide encoding a region consisting of the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9; and
(H ′) consisting of an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9. And a polynucleotide comprising a polynucleotide encoding a region having GST activity,
Selected from.

  Here, the “polynucleotide (eg, DNA) that hybridizes under stringent conditions” refers to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10. A polynucleotide consisting of a base sequence complementary to the base sequence or a polynucleotide encoding the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9 A polynucleotide (for example, DNA) obtained by using a colony hybridization method, a plaque hybridization method, a Southern hybridization method, or the like using a portion as a probe.

  In the present specification, “stringent conditions” may be any of low stringency conditions, moderate stringency conditions, and high stringency conditions. “Low stringent conditions” are, for example, conditions of 5 × SSC, 5 × Denhardt's solution, 0.5% (w / v) SDS, 50% (v / v) formamide, 32 ° C. The “medium stringent conditions” are, for example, conditions of 5 × SSC, 5 × Denhardt's solution, 0.5% (w / v) SDS, 50% (v / v) formamide, 42 ° C. “High stringent conditions” are, for example, conditions of 5 × SSC, 5 × Denhardt's solution, 0.5 (w / v)% SDS, 50% (v / v) formamide, 50 ° C. The more severe the conditions, the higher the degree of complementation required for duplex formation. Specifically, for example, it can be expected that a polynucleotide (eg, DNA) having higher homology (or identity) can be efficiently obtained as the temperature is increased under these conditions. However, multiple factors such as temperature, probe concentration, probe length, ionic strength, time, and salt concentration can be considered as factors that affect hybridization stringency, and those skilled in the art can appropriately select these factors. By doing so, it is possible to achieve the same stringency.

Hybridization is described in Sambrook J. et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press (2001), Ausbel FM et al., Current Protocols in Molecular Biology, Supplement 1-38, John Wiley and Sons (1987-1997), Glover DM and Hames BD, DNA Cloning 1: Core Techniques, A practical Approach, Second Edition, Oxford University Press (1995), etc. .

  As other polynucleotides that can be hybridized, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: when calculated using default parameters by an analysis program such as FASTA and BLAST. 7 or a polynucleotide encoding the amino acid sequence of SEQ ID NO: 9 and about 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 88% or more, 90% or more, 92 % Or higher, 95% or higher, 97% or higher, 98% or higher, 99% or higher, 99.3% or higher, 99.5% or higher, 99.7% or higher, 99.8% or higher, 99.9% or higher And DNA having sex. The identity of amino acid sequences and base sequences can be determined using the method described above.

  A polynucleotide encoding a region having an amino acid sequence in which one or a plurality of amino acids are deleted, substituted, inserted and / or added to a certain amino acid sequence can be obtained by site-directed mutagenesis (see, for example, Gotoh, T. et al. et al., Gene 152, 271-275 (1995), Zoller, MJ, and Smith, M., Methods Enzymol. 100, 468-500 (1983), Kramer, W. et al., Nucleic Acids Res. 12, 9441-9456 (1984), Kramer W, and Fritz HJ, Methods.Enzymol. 154, 350-367 (1987), Kunkel, TA, Proc. Natl. Acad. Sci. USA. 82, 488-492 (1985), Kunkel, Methods Enzymol. 85, 2763-2766 (1988), etc.), a method using amber mutation (for example, see Gapped duplex method, Nucleic Acids Res. 12, 9441-9456 (1984), etc.) Can be obtained.

  PCR (for example, Ho SN et al., Gene 77, 51 (for example, Ho SN et al., Gene 77, 51 ( 1989), etc.) can also introduce mutations into polynucleotides.

  In addition, a polynucleotide encoding a partial fragment of a protein that is a kind of deletion mutant has a sequence that matches the base sequence at the 5 ′ end of the region encoding the partial fragment to be prepared in the polynucleotide encoding the protein. It can be obtained by performing PCR using an oligonucleotide and an oligonucleotide having a sequence complementary to the base sequence at the 3 ′ end as a primer and using a polynucleotide encoding the protein as a template.

  Specific examples of the polynucleotide of the present invention include, for example, proteins consisting of the amino acid sequence of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 17 (these proteins are derived proteins of the present invention). A polynucleotide comprising a polynucleotide that encodes, for example, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, or a sequence. A polynucleotide comprising a polynucleotide consisting of the nucleotide sequence of No. 18 is exemplified. On the other hand, the polynucleotide of the present invention is preferably a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 17, and more Preferably, it consists of a polynucleotide consisting of the base sequence of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 18 (see Examples described later).

  The polynucleotide of the present invention may include a polynucleotide containing a base sequence encoding an amino acid sequence for promoting translation and / or a polynucleotide containing a base sequence encoding a peptide sequence for purification. The polynucleotide containing a base sequence encoding an amino acid sequence for promoting translation includes a base sequence encoding an amino acid sequence (for example, a TEE sequence) for promoting translation used in the art. Polynucleotides can be used. Examples of the amino acid sequence for promoting translation include those described above. As a polynucleotide encoding a peptide sequence for purification, a polynucleotide containing a base sequence encoding a peptide sequence for purification (for example, a histidine tag sequence) used in this technical field may be used. it can.

3. Recombinant vector and transformant of the present invention Furthermore, the present invention provides a recombinant vector and a transformant containing the above-described polynucleotide of the present invention.

(1) Preparation of recombinant vector The recombinant vector of the present invention can be obtained by ligating (inserting) the polynucleotide (DNA) of the present invention into an appropriate vector. More specifically, it can be obtained by cleaving a purified polynucleotide (DNA) with a suitable restriction enzyme, inserting it into a restriction enzyme site or a multiple cloning site of a suitable vector, and ligating the vector. The vector for inserting the polynucleotide of the present invention is not particularly limited as long as it can replicate in the host, and examples thereof include plasmids, bacteriophages, animal viruses and the like. As the plasmid, for example, various expression vectors or cloning vectors can be used. Plasmids derived from E. coli (eg, pBR322, pBR325, pUC118, pUC119, etc.), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, etc.), yeast Derived plasmids (eg, YEp13, YEp24, YCp50, etc.). Examples of the bacteriophage include λ phage. Examples of animal viruses include retroviruses, vaccinia viruses, insect viruses (eg, baculoviruses) and the like.

  In the present invention, a pCOLD-I vector, a pCOLD-II vector, a pCOLD-III vector, a pCOLD-IV vector (manufactured by Takara Bio Inc.) and the like can also be suitably used. As specifically shown in the examples described below, when these vectors are used to express prokaryotic cells as a host, the fusion protein of the present invention is produced as a soluble protein in the cytoplasm of the host cell. Can be made.

  The polynucleotide of the present invention is usually operably linked downstream of a promoter in an appropriate vector. The promoter used is preferably an SV40-derived promoter, a retrovirus promoter, a metallothionein promoter, a heat shock promoter, a cytomegalovirus promoter, an SRα promoter or the like when the host to be transformed is an animal cell. When the host is Escherichia, Trp promoter, T7 promoter, lac promoter, recA promoter, λPL promoter, lpp promoter, cspA promoter and the like are preferable. When the host is Bacillus, SPO1 promoter, SPO2 promoter, penP promoter and the like are preferable. When the host is yeast, PHO5 promoter, PGK promoter, GAP promoter, ADH1 promoter, GAL promoter, etc. are preferable. When the host is an insect cell, polyhedrin promoter, P10 promoter and the like are preferable.

  In addition to the above, the recombinant vector of the present invention may contain those containing an enhancer, a splicing signal, a poly A addition signal, a ribosome binding sequence (SD sequence), a selection marker, and the like as desired. Examples of the selection marker include a dihydrofolate reductase gene, an ampicillin resistance gene, a neomycin resistance gene, and the like.

  In addition, the recombinant vector of the present invention includes a polynucleotide containing a base sequence encoding an amino acid sequence for promoting translation and / or a polynucleotide containing a base sequence encoding a peptide sequence for purification. Also good. The polynucleotide containing a base sequence encoding an amino acid sequence for promoting translation includes a base sequence encoding an amino acid sequence (for example, a TEE sequence) for promoting translation used in this technical field. Polynucleotides can be used. In addition, as a polynucleotide encoding a peptide sequence for purification, a polynucleotide containing a nucleotide sequence encoding a peptide sequence for purification (such as a histidine tag sequence) used in this technical field is used. can do.

(2) Preparation of transformant A recombinant vector containing the polynucleotide of the present invention (that is, the polynucleotide encoding the fusion protein of the present invention) thus obtained is introduced into a suitable host. Thus, a transformant can be prepared. The host is not particularly limited as long as it can express the polynucleotide (DNA) of the present invention. For example, Escherichia, Bacillus, Pseudomonas, Rhizobium, yeast, animal cells or Insect cells. Examples of the genus Escherichia include Escherichia coli. Examples of the genus Bacillus include Bacillus subtilis. Examples of the genus Pseudomonas include Pseudomonas putida. Examples of the genus Rhizobium include Rhizobium meliloti. Examples of the yeast include Saccharomyces cerevisiae and Schizosaccharomyces pombe. Examples of animal cells include COS cells and CHO cells. Examples of insect cells include Sf9 and Sf21.

  The introduction method of the recombinant vector into the host and the transformation method thereby can be performed by various general methods. As a method for introducing a recombinant vector into a host cell, for example, calcium phosphate method (Virology, 52, 456-457 (1973)), lipofection method (Proc. Natl. Acad. Sci. USA, 84, 7413 (1987) ), Electroporation method (EMBO J., 1, 841-845 (1982)). Examples of the method for transforming Escherichia include the methods described in Proc. Natl. Acad. Sci. USA, 69, 2110 (1972), Gene, 17, 107 (1982), and the like. Examples of the method for transforming Bacillus include the method described in Molecular & General Genetics, 168, 111 (1979). Examples of yeast transformation methods include the method described in Proc. Natl. Acad. Sci. USA, 75, 1929 (1978). Examples of animal cell transformation methods include the method described in Virology, 52, 456 (1973). Examples of the method for transforming insect cells include the method described in Bio / Technology, 6, 47-55 (1988). Thus, a transformant transformed with a recombinant vector containing a polynucleotide encoding the fusion protein of the present invention (polynucleotide of the present invention) can be obtained.

4). Production of fusion protein of the present invention The present invention also provides a method for producing the fusion protein of the present invention, comprising the steps of culturing the transformant to produce the fusion protein of the present invention. In the fusion protein of the present invention, the transformant is cultured under conditions that allow expression of the polynucleotide (DNA) encoding the fusion protein of the present invention, and the fusion protein of the present invention is produced, accumulated, separated and purified. Can be manufactured.

(Culture of transformants)
The transformant of the present invention can be cultured according to a usual method used for host culture. By this culture, the transformant produces the fusion protein of the present invention, and the fusion protein of the present invention is accumulated in the transformant or in the culture solution.

  As a medium for cultivating transformants whose hosts are Escherichia or Bacillus genus, it contains carbon sources, nitrogen sources, inorganic salts, etc. necessary for the growth of the transformants, so that transformants can be cultured efficiently. Any natural or synthetic medium may be used as long as it can be performed automatically. As the carbon source, carbohydrates such as glucose, fructose, sucrose and starch, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol are used. As the nitrogen source, ammonia, ammonium salts of inorganic or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, or other nitrogen-containing compounds, peptone, meat extract, corn steep liquor, and the like are used. Examples of inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate. During culture, an antibiotic such as ampicillin or tetracycline may be added to the medium as necessary. When cultivating a transformant transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, when cultivating a transformant transformed with an expression vector using Lac promoter or cspA promoter, isopropyl-β-D-thiogalactopyranoside (IPTG) or the like is transformed with an expression vector using TRP promoter. When cultivating the transformant, indoleacrylic acid (IAA) or the like may be added to the medium.

  When the host is an Escherichia bacterium, the culture is usually carried out at about 15 to 43 ° C. for about 3 to 24 hours, and if necessary, aeration or agitation is added. When the host is a genus Bacillus, the culture is usually performed at about 30 to 40 ° C. for about 6 to 24 hours, and if necessary, aeration or agitation is added.

  As a medium for cultivating a transformant whose host is yeast, for example, Burkholder minimum medium (Proc. Natl. Acad. Sci. USA, 77, 4505 (1980)) or 0.5% (w / v ) SD medium containing casamino acid (Proc. Natl. Acad. Sci. USA, 81, 5330 (1984)). The pH of the medium is preferably adjusted to about 5-8. Culture is usually performed at about 20 ° C. to 35 ° C. for about 24 to 72 hours, and aeration and agitation are added as necessary.

  As a medium for culturing a transformant whose host is an animal cell, for example, a MEM medium (Science, 122, 501 (1952)) containing about 5 to 20% (v / v) fetal calf serum, a DMEM medium (Virology) , 8, 396 (1959)). The pH is preferably about 6-8. The culture is usually performed at about 30 ° C. to 40 ° C. for about 15 to 60 hours, and aeration and agitation are added as necessary.

  As a medium for cultivating transformants whose host is an insect cell, an appropriate addition of an additive such as 10% (v / v) bovine serum inactivated in Grace's Insect Medium (Nature, 195, 788 (1962)) Is used. The pH of the medium is preferably adjusted to about 6.2 to 6.4. The culture is usually performed at about 27 ° C. for about 3 to 5 days, and aeration and agitation are added as necessary.

(Separation and purification of the fusion protein of the present invention)
The fusion protein of the present invention can be obtained by separating and purifying the fusion protein of the present invention from the culture. Here, the culture means any of a culture solution, cultured cells or cultured cells, or disrupted cultured cells or cultured cells. Separation and purification of the fusion protein of the present invention can be carried out according to ordinary methods.

  Specifically, when the fusion protein of the present invention is accumulated in cultured cells or cultured cells, after culturing, the cells or cells are cultured by a usual method (for example, ultrasound, lysozyme, freeze-thawing, etc.). After crushing, a crude extract of the fusion protein of the present invention can be obtained by ordinary methods (eg, centrifugation, filtration, etc.). When the fusion protein of the present invention is accumulated in the periplasmic space, an extract containing the target protein can be obtained by a usual method (for example, osmotic shock method) after completion of the culture. When the fusion protein of the present invention is accumulated in the culture solution, the cells or cells and the culture supernatant are separated from each other by a conventional method (for example, centrifugation, filtration, etc.) after completion of the culture. A culture supernatant containing the fusion protein of the invention can be obtained.

  Purification of the fusion protein of the present invention contained in the extract or culture supernatant thus obtained can be performed according to a conventional separation / purification method. Separation / purification methods include, for example, ammonium sulfate precipitation, gel filtration chromatography, ion exchange chromatography, affinity chromatography, reverse phase high performance liquid chromatography, dialysis, ultrafiltration, etc., alone or in appropriate combination. be able to. When the fusion protein of the present invention contains the above-described peptide sequence for purification, it is preferable to use this for purification. Specifically, when the fusion protein of the present invention contains a histidine tag sequence, nickel chelate affinity chromatography can be used.

5. Uses of Fusion Protein of the Present Invention As described above, the fusion protein of the present invention has GST activity and the ability to bind to the Fc region of an antibody. Therefore, the fusion protein of the present invention can be used, for example, for measurement of an antibody (particularly IgG antibody) or an antigen in a sample in an immunoassay. In the present specification, the term “measurement” should be interpreted in the broadest sense including measurement for various purposes such as detection, quantification, and qualitative, but preferably means detection or quantification, more preferably Mean quantitative. Here, when the fusion protein of the present invention is used for antibody measurement, the antibody may be a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody. In this case, as a preferred embodiment of the antibody measurement method using the fusion protein of the present invention, for example, a monoclonal antibody prepared using a hybridoma can be quantified. The production of a monoclonal antibody using a hybridoma can be performed according to a conventional method. For example, an appropriate mammal (eg, mouse, rat, etc.) is immunized with an antigen mixed with an appropriate adjuvant as necessary. A hybridoma can be obtained by fusing antibody-producing cells such as spleen cells and B lymphocytes of the animal with myeloma cells derived from an appropriate animal (eg, mouse, rat, etc.). Cell fusion can be performed by, for example, a polyethylene glycol (PEG) method in which antibody-producing cells and myeloma cells are fused in the presence of polyethylene glycol in an appropriate medium. After cell fusion, the hybridoma is selected with a selective medium such as HAT medium (medium containing hypoxanthine, aminoprelin, and thymidine), and screened for the ability of the hybridoma to produce an antibody against the antigen according to a conventional method (eg, EIA method). Do. Subsequently, a hybridoma producing a desired antibody is cloned according to a conventional method (for example, limiting dilution method), and a hybridoma producing a monoclonal antibody is selected. In this way, a monoclonal antibody against a desired antigen can be prepared. The prepared monoclonal antibody-containing solution (buffer solution) is used as a sample, and the monoclonal antibody in the sample can be quantified by carrying out the antibody measuring method according to the present invention.

  Such antibody or antigen measurement can be performed by a usual method. Specifically, when the fusion protein of the present invention is used for antibody measurement, the fusion protein of the present invention (through the first region) is bound to the antibody (the Fc region thereof) and the antibody bound to the antibody By measuring GST activity derived from the second region of the fusion protein, the antibody can be measured. When the fusion protein of the present invention is used for antigen measurement, an antibody known to specifically bind to the antigen is bound to the antigen, and then the fusion protein of the present invention ( Indirectly measuring the antigen by binding to the antibody (through the Fc region) (via the first region) and measuring the GST activity derived from the second region of the fusion protein bound to the antibody. Can do.

  Furthermore, the GST activity derived from the second region of the fusion protein of the present invention is an activity of catalyzing the formation reaction of a conjugate between an electrophilic compound and reduced glutathione (GSH). Therefore, the fusion protein of the present invention can also be used for measurement of reduced glutathione (GSH).

  In any use of the fusion protein of the present invention, it is necessary to measure GST activity derived from the second region, but GST activity can be measured by a usual method. For example, a GST measurement probe that shows a change in luminescence characteristics by reacting with reduced glutathione (GSH) in the presence of GST is used, and the change in the luminescence characteristics of the GST measurement probe is used as an index in the second region. The derived GST activity can be measured (ie, the antigen in the sample, the antibody comprising the Fc region, or GSH). In addition, as a method for measuring GST activity using such a probe for GST measurement, there are an absorption method (colorimetric method), a bioluminescence method, a fluorescence method, etc., but any of them can be used here. As a method for measuring GST activity by an absorption method (colorimetric method), for example, a method is known in which a change in absorbance at 340 nm of a glutathione product is detected using 2,4-dinitrochlorobenzene (CDNB). . In addition, as a method for measuring GST activity by the bioluminescence method, for example, luciferin produced by nitrobenzenesulfonate ester of caged luciferin is subjected to aromatic nucleophilic substitution reaction (deprotection reaction) by GST, presence of ATP and luciferase A method is known in which light is emitted below and GST activity is measured from the luminescence intensity (Non-Patent Document 1). In addition, it is preferable to use the fluorescence method from the viewpoint of excellent S / N ratio and sensitivity as compared with the above-described absorption method (colorimetric method) and bioluminescence method. In particular, the “change in luminescence properties” of the GST measurement probe is “the fluorescence intensity increases before and after the GST measurement probe is brought into contact with GST in the presence of reduced glutathione (GSH)”. More preferably. As a method for measuring GST activity by such a fluorescence method, a series of fluorescent probes such as DNAFs and DNAT-Me have been disclosed as probes having a maximum absorption wavelength at 490 nm and a maximum fluorescence wavelength at around 510 nm (non-native). Patent Document 2). Moreover, as a fluorescent probe which solved the problem which the fluorescent probe disclosed by the nonpatent literature 2 has, it is represented by the following general formula (1) represented by the 3, 4-DNADCF disclosed by the nonpatent literature 3. It is preferable to use a fluorescein derivative or a salt thereof. When a fluorescein derivative represented by the following general formula (1) or a salt thereof is used as a GST measurement probe (fluorescent probe), it exhibits high fluorescence intensity even in a weakly acidic region such as pH 6.5, and is enzyme-independent GSH. There is an advantage that it shows a very excellent S / N ratio with a low reactivity.

In the general formula (1), R ¹ and R ² each independently represent 1 to 3 identical or different substituents bonded to a hydrogen atom or a benzene ring. Examples of the substituent include an alkyl group, an alkoxy group, a halogen atom (any of a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom), an amino group, a mono- or di-substituted amino group, and a substituted silyl group. Or an acyl group and the like, but is not limited thereto. Moreover, when two or more substituents are present on each benzene ring, they may be the same or different from each other. In addition, as R < ¹ > and R < ² >, a hydrogen atom is preferable.

In the general formula (1), R ³ , R ⁴ , R ⁵ and R ⁶ each independently represent a hydrogen atom, a hydroxy group, an alkyl group or a halogen atom. In a preferred embodiment, R ³ , R ⁴ , R ⁵ and R ⁶ are a hydrogen atom, a fluorine atom or a chlorine atom, more preferably all are hydrogen atoms.

In the general formula (1), X ¹ and X ² each independently represent a fluorine atom or a chlorine atom. In a preferred embodiment, at least one of X ¹ and X ² is a chlorine atom, and particularly preferably both X ¹ and X ² are a chlorine atom.

In a preferred embodiment of the present invention, the compound represented by the general formula (1) is such that R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are hydrogen atoms, and X ¹ and X ² are chlorine. It is a compound which is an atom or a salt thereof (3,4-DNADCF synthesized in Examples described later).

  In the present specification, the “alkyl group” may be any alkyl group composed of a straight chain, a branched chain, a ring, or a combination thereof. Although carbon number of an alkyl group is not specifically limited, For example, it is about C1-C6, Preferably it is C1-C4. In this specification, the alkyl group may have one or more arbitrary substituents. Examples of the substituent include an alkoxy group, a halogen atom (which may be a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom), an amino group, a mono- or di-substituted amino group, a substituted silyl group, or Examples of the acyl group include, but are not limited to, an acyl group. When the alkyl group has two or more substituents, they may be the same as or different from each other. The same applies to the alkyl moiety of other substituents containing an alkyl moiety (for example, an alkyloxy group or an aralkyl group).

  In the present specification, “alkoxy group” means —O-alkyl group, where “alkyl group” is a group having the above-mentioned definition.

In the present specification, “amino group” means a —NH ₂ group, and “mono- or di-substituted amino group” means a group in which one or both of the hydrogen atoms of an amino group are substituted with a substituent. Examples of the substituent include, but are not limited to, an alkyl group.

In the present specification, the “substituted silyl group” means a group in which at least one hydrogen atom of a silyl group (—SiH ₃ group) is substituted with a substituent. Examples of the substituent include, but are not limited to, an alkyl group.

  In the present specification, the “acyl group” may be either an aliphatic acyl group or an aromatic acyl group, or may be an aliphatic acyl group having an aromatic group as a substituent. The acyl group may contain one or more heteroatoms. For example, as an acyl group, an alkylcarbonyl group (such as an acetyl group), an alkyloxycarbonyl group (such as an acetoxycarbonyl group), an arylcarbonyl group (such as a benzoyl group), an aryloxycarbonyl group (such as a phenyloxycarbonyl group), an aralkylcarbonyl group ( Benzylcarbonyl group), alkylthiocarbonyl group (such as methylthiocarbonyl group), alkylaminocarbonyl group (such as methylaminocarbonyl group), arylthiocarbonyl group (such as phenylthiocarbonyl group), or arylaminocarbonyl group (phenylaminocarbonyl group) And the like, but is not limited thereto. These acyl groups may have one or more arbitrary substituents. Examples of the substituent include, but are not limited to, an alkoxy group, a halogen atom, an amino group, a mono- or di-substituted amino group, a substituted silyl group, and an acyl group. When the acyl group has two or more substituents, they may be the same as or different from each other.

  The compound represented by the general formula (1) may exist as a salt. Examples of the salt include base addition salts, acid addition salts, amino acid salts and the like. Examples of the base addition salt include metal salts such as sodium salt, potassium salt, calcium salt, and magnesium salt, ammonium salts, and organic amine salts such as triethylamine salt, piperidine salt, and morpholine salt. Examples of the acid addition salt include mineral acid salts such as hydrochloride, sulfate, and nitrate, and organic acid salts such as methanesulfonate, paratoluenesulfonate, citrate, and oxalate. Examples of amino acid salts include glycine salts. However, the salt of the compound according to the present invention is not limited to these.

  The compound represented by the general formula (1) or a salt thereof may have one or more asymmetric carbons depending on the type of substituent, and there are stereoisomers such as enantiomers or diastereomers. There is a case. Pure forms of stereoisomers, any mixture of stereoisomers, racemates, and the like are all within the scope of the present invention.

  The compound represented by the general formula (1) or a salt thereof may exist as a hydrate or a solvate, and any of these substances is included in the technical scope of the present invention. Although the kind of solvent which forms a solvate is not specifically limited, For example, solvents, such as ethanol, acetone, isopropanol, are mentioned.

  The compound of the present invention represented by the general formula (1) or a salt thereof is preferably a hydrophilic compound (salt). In the present specification, “log P value” can be used as a hydrophilicity index of a compound (salt). Here, the “log P value” is also referred to as “octanol-water partition coefficient” or “log Pow”, and is a value of the ratio of the distribution concentration of a substance to each phase of a two-phase solvent system composed of n-octanol and water. The larger the value, the higher the lipophilicity (the lower the hydrophilicity). In the present specification, the value of the logP value can be measured by a flask shaking method described in JIS Z-7260-107: 2000. Further, the logP value can be estimated by a computational chemical method or an empirical method instead of the actual measurement.

  As a calculation method, Crippen's fragmentation method ("J. Chem. Inf. Comput. Sci.", 27, p21 (1987)), Viswanadhan's fragmentation method ("J. Chem. Inf. Comput. Sci."). , 29, p163 (1989)), Broto's fragmentation method (“Eur. J. Med. Chem.-Chim. Theor.”, 19, p71 (1984)), CLogP method (references) Leo, A., Jow, PYC, Silipo, C., Hansch, C., J. Med. Chem., 18, 865 1975) and the like are preferably used, but the Crippen's fragmentation method ( “J.Chem Inf.Comput.Sci. ", 27, p21 (1987)). However, when the measurement value by the flask shaking method mentioned above and the value estimated by the calculation chemical method or the empirical method are significantly different, the measurement value by the flask shaking method has priority.

  In the present invention, the logP value of the compound represented by the general formula (1) is preferably 5.2 or less, more preferably 5.0 or less, still more preferably 4.8 or less, and still more preferably 4.6 or less, particularly preferably 4.4 or less, and most preferably 4.2 or less. On the other hand, the lower limit value of the logP value of the compound of the general formula (1) is not particularly limited, but usually, the above-described effects can be sufficiently obtained if the logP value is about 1.5 or more. The logP value of 3,4-DNADCF, which is a compound synthesized in the examples described later, is 4.16.

  The compound of the present invention represented by the general formula (1) is, for example, a conventionally known document (Synthesis 2009, 7; 1224-1226 and J Am Chem Soc. 2008, 130 (44): 14533-14543 (non-patent document). It is possible to synthesize with reference to the method described in 2)). In the Examples section of the present specification, production methods for typical compounds included in the compound of the present invention represented by the general formula (1) are specifically shown. In addition, by arbitrarily selecting starting materials, reagents, reaction conditions, and the like by referring to the disclosure of the above, and taking into account the common general knowledge at the time of filing the application as necessary, any of those included in the general formula (1) This compound can be easily produced.

The compound represented by the general formula (1) is substantially non-fluorescent in a neutral region (for example, in the range of pH 5 to 9). On the other hand, in the presence of reduced glutathione (GSH) and glutathione-S-transferase (GST), the nitro group (—NO ₂ group) bonded to the para position (position 4) of the benzene ring in the compound is eliminated. At the same time, the para-position (position 4) is converted to glutathione and becomes a highly fluorescent compound. For example, the compound represented by the general formula (1) or a salt thereof hardly emits fluorescence when irradiated with excitation light of, for example, about 505 nm in the neutral region, but the compound that is glutathione as described above is It has the property of emitting extremely strong fluorescence (for example, a fluorescence wavelength of 525 nm) under the same conditions. Therefore, by using the compound as a GST measurement probe (fluorescent probe), the presence of GST can be measured by a change in fluorescence intensity.

  Here, in the technology of constructing aequorin or oberin, which is a calcium-binding photoprotein, disclosed in Non-Patent Document 4, Non-patent Document 6 and Patent Document 1 as a fusion protein with protein A or ZZ domain, aequorin or oberin is The apoprotein and coelenterazine peroxide are present in the form of a complex. When calcium ions are bound to aequorin or oberin in the form of this complex, coelenteramide and carbon dioxide, which are oxides of coelenterazine, are generated and light is emitted. By measuring the intensity of this luminescence, calcium ions and antibodies bound to the ZZ domain can be quantified. However, luminescence when calcium-binding photoprotein binds to calcium ions is instantaneous, and cannot be applied to assay systems that want to maintain luminescence continuously. There was also the problem of being limited. In contrast, the GST measurement probe as described above is used, and the GST activity derived from the second region is measured using the change in the luminescence characteristics of the GST measurement probe as an index (ie, the antigen in the sample, Fc According to a technique for measuring a region-containing antibody or GSH) (in particular, a fluorescence method), it is possible to maintain luminescence continuously over a long period of time. Therefore, there is an advantage that it can be applied to an assay system to which the technology based on the above-described calcium-binding photoprotein cannot be applied.

  In addition, the measurement of GST activity by the method using the compound represented by the general formula (1) or a salt thereof as a fluorescent probe can be performed under neutral conditions, for example, in the range of pH 5.0 to 9.0. It can be carried out. Here, when the activity of GST is measured using DNAF1, which is a fluorescent probe disclosed in Non-Patent Document 2, under weakly acidic conditions such as pH 6.5, the fluorescence intensity of the reaction product is reduced. In contrast, when the compound represented by the general formula (1) or a salt thereof is used as a fluorescent probe, such a decrease in fluorescence intensity is hardly observed even in measurement under acidic conditions such as pH 6.5. I can't. Therefore, with respect to the measurement of GST activity by the method using the compound represented by the general formula (1) or a salt thereof as a fluorescent probe, in a preferred embodiment, the measurement is performed in the range of pH 6.3 to 8.5, and more preferably. Is measured in the range of pH 6.4 to 6.6.

  As the fluorescent probe for GST measurement, the compound represented by the above general formula (1) or a salt thereof may be used as it is, but if necessary, a composition containing additives usually used for the preparation of reagents. It may be used as For example, additives such as solubilizers, pH adjusters, buffers, and isotonic agents can be used as additives for using the reagent in a physiological environment, and the amount of these additives is appropriately selected by those skilled in the art. Is possible. These compositions are provided as a composition in an appropriate form such as a mixture in a powder form, a lyophilized product, a granule, a tablet, or a liquid.

Of the uses of the fusion protein of the present invention, another preferred embodiment of the antibody measurement method relates to a method of measuring “an antibody against a predetermined antigen” in a sample. That is, a preferred embodiment of the method is
Contacting a predetermined antigen with a sample that may contain an antibody against said antigen;
Contacting the antigen after contacting the fusion protein with the sample in the absence of an antibody that is not bound to the antigen;
A probe for measuring GST showing a change in luminescence properties by reacting with reduced glutathione (GSH) in the presence of GST in the absence of the fusion protein not bound to the antibody and in the presence of GSH. Contacting with the antigen after contacting with the fusion protein;
Detecting or quantifying the antibody in the sample, using as an index the change in the luminescence properties before and after contacting the GST measurement probe with the antigen;
Is included.

  Here, in order to measure an antibody against a predetermined antigen in the above-described embodiment, an antibody immobilized on a solid phase is usually used as the predetermined antigen. At this time, a bead carrier or a substrate is preferably used as the solid phase. Examples of the bead carrier include magnetic beads, gold nanoparticles, agarose beads, plastic beads, and the like, and magnetic beads are preferable because of easy handling using magnetism. Examples of the substrate include a glass substrate, a silicon substrate, a plastic substrate, and a metal substrate.

  In the above embodiment, the predetermined antigen may be a cell surface antigen. At this time, it is preferable to use cancer cells as the cells. When the cell is a cancer cell, the surface antigen is preferably expressed specifically in the cancer cell. According to such a form, “an antibody against a surface antigen specifically expressed in cancer cells” in a sample can be measured (quantified). At this time, a sample that can contain the above-mentioned “antibody against a surface antigen specifically expressed in cancer cells” is used. In addition, “an antibody against a surface antigen specifically expressed in cancer cells” can be used as an agent for preventing and / or treating cancer. Therefore, cancer preventive and / or therapeutic agents or candidate antibodies thereof can be screened based on the result of measuring “antibodies against a predetermined antigen” in the sample. That is, still another embodiment of the use of the fusion protein of the present invention is to measure an antibody against the surface antigen in the sample using the above-described method for measuring “an antibody against a predetermined antigen” in the sample. And a method for screening cancer preventive and / or therapeutic agents or candidate antibodies thereof, comprising obtaining an antibody against the surface antigen based on the measurement result. In addition, the determination based on the measurement result may be performed using the presence or absence of “detection” of the antibody as an index, or the result of “quantification” of the antibody in light of a predetermined standard (for example, a cutoff value). A determination may be made.

6). Kit of the present invention According to the present invention, an antigen or antibody in a sample comprising any one selected from the fusion protein of the present invention, the polynucleotide of the present invention, the recombinant vector of the present invention and the transformant of the present invention. Alternatively, a kit for measuring GSH is also provided. The kit of the present invention can be produced by commonly used materials and methods. The kits of the invention may include, for example, sample tubes, plates, instructions for kit users, solutions, buffers, reagents, samples suitable for standardization or control samples.

  EXAMPLES Hereinafter, although preferable embodiment of this invention is described in detail using an Example, the technical scope of this invention is not necessarily limited by the following Example.

[Synthesis example]
Previously known literature (Synthesis 2009, 7; 1224-1226, J Am Chem Soc. 2008, 130 (44): 14533-14543 (Non-patent literature 2) and Chem Commun (Camb). 2015 (57): 11459-11462 (Non-patent Document 3)) Referring to the method described in the following, the fluorescein derivative according to the present invention (compound (5) described in the following synthesis scheme (also referred to as “3,4-DNADCF”)) according to the following synthesis scheme Was synthesized.

  The instrument data of the compounds (3) to (5) described in the above synthesis scheme are as follows.

  (Equipment data of compound (3))

  (Equipment data of compound (4))

  (Equipment data of compound (5))

[Construction of ZZ-fused GST expression vector]
Five ZZ-GST expression vectors pCOLD-I-ZZ-GST were constructed by the following procedure. The breakdown of the five types uses GST (hGSTA1, hGSTM1, or hGSTP1) derived from humans, and two types use GST (Nobo-Dm / GSTe14 or DmGSTe13) derived from Drosophila melanogaster. It was what was there.

(Construction of pCOLD-I-ZZ-Nobo-Dm / GSte14)
A cDNA sequence encoding Nobo-Dm / GSTe14, which is a kind of Drosophila melanogaster-derived GST, was amplified by a PCR method using the following PCR primers.

  The obtained PCR product was cleaved with restriction enzymes (KpnI and XbaI), inserted into pEZZ18 Protein A Gene Fusion Vector (Amersham Biosciences), which is a vector encoding ZZ domain that is an IgG antibody binding domain, and Escherichia coli DH5α After transformation into the strain, it was cloned to obtain pEZZ18-Nobo-Dm / GSTe14.

  Subsequently, the ZZ-GST region (not including the leader sequence present at the N-terminus of the ZZ sequence) in the pEZZ18-Nobo-Dm / GSTe14 vector prepared above was amplified by the PCR method using the following PCR primers. .

  The obtained PCR product was cleaved with restriction enzymes (NdeI and XbaI) and inserted into a pCOLD-I vector (Takara Bio) to obtain a pCOLD-I-ZZ-Nobo-Dm / GSTe14 vector. The pCOLD-I-ZZ-Nobo-Dm / GSTe14 vector was constructed in the construction of the pCOLD-I-ZZ-hGSTA1 vector, the pCOLD-I-ZZ-hGSTP1 vector, and the pCOLD-I-ZZ-DmGSte13 vector described later. It is not used for experiments other than those used.

(Construction of pCOLD-I-ZZ-hGSTA1)
A cDNA sequence (SEQ ID NO: 4) encoding hGSTA1, which is a kind of human-derived GST, was amplified by a PCR method using the following PCR primers.

  The obtained PCR product was cleaved with restriction enzymes (SacI and XbaI), and ligated to the pCOLD-I-ZZ-Nobo-Dm / GSTe14 constructed above was similarly treated with a restriction enzyme. Thus, the pCOLD-I-ZZ-hGSTA1 vector was obtained by substituting the target sequence of pCOLD-I-ZZ-Nobo-Dm / GSTe14 from ZZ-Nobo-Dm / GSTe14 to ZZ-hGSTA1.

  FIG. 1 is an expression vector map of various pCOLD-I-ZZ-GSTs, which are expression vectors of various ZZ-GSTs including the pCOLD-I-ZZ-hGSTA1 vector. Here, SEQ ID NO: 12 is the base sequence (CDS; including stop codon) of the DNA encoding the ZZ-hGSTA1 fusion protein expressed by the pCOLD-I-ZZ-hGSTA1 vector. SEQ ID NO: 11 is the amino acid sequence of the ZZ-hGSTA1 fusion protein encoded by the coding DNA expressed by the vector. As is clear from SEQ ID NO: 11, since the fusion protein contains a hexahistidine tag (His-tag) on the N-terminal side of the ZZ domain, affinity purification by His-tag is possible (hereinafter, referred to as “SEQ ID NO: 11”). The same is true for the fusion protein.

(Construction of pCOLD-I-ZZ-hGSTP1)
A cDNA sequence (SEQ ID NO: 8) encoding hGSTP1, which is a kind of human-derived GST, was amplified by a PCR method using the following PCR primers.

  The obtained PCR product was cleaved with restriction enzymes (SacI and XbaI), and ligated to the pCOLD-I-ZZ-Nobo-Dm / GSTe14 constructed above was similarly treated with a restriction enzyme. Thus, the pCOLD-I-ZZ-hGSTP1 vector was obtained by substituting the target sequence of pCOLD-I-ZZ-Nobo-Dm / GSTe14 from ZZ-Nobo-Dm / GSTe14 to ZZ-hGSTP1. Here, SEQ ID NO: 16 is the base sequence (CDS; including stop codon) of the DNA encoding the ZZ-hGSTP1 fusion protein expressed by the pCOLD-I-ZZ-hGSTP1 vector. SEQ ID NO: 15 is the amino acid sequence of the ZZ-hGSTP1 fusion protein encoded by the coding DNA expressed by the vector.

(Construction of pCOLD-I-ZZ-DmGSte13)
A cDNA sequence (SEQ ID NO: 10) encoding DmGSTe13, which is a kind of Drosophila melanogaster-derived GST, was amplified by a PCR method using a PCR primer set consisting of a forward primer and a reverse primer. At this time, the forward primer contained a KpnI sequence, and an XbaI sequence was present inside the reverse primer of the target sequence.

  The obtained PCR product was cleaved with restriction enzymes (KpnI and XbaI), and ligated to the restriction enzyme-treated pCOLD-I-ZZ-Nobo-Dm / GSte14 constructed above. The pCOLD-I-ZZ-DmGSte13 vector was obtained by substituting the target sequence of pCOLD-I-ZZ-Nobo-Dm / GSTe14 from ZZ-Nobo-Dm / GSTe14 to ZZ-DmGSTe13. Here, SEQ ID NO: 18 is the base sequence (CDS; including stop codon) of the DNA encoding the ZZ-DmGSTe13 fusion protein expressed by the pCOLD-I-ZZ-DmGSTe13 vector. SEQ ID NO: 17 is the amino acid sequence of the ZZ-DmGSTe13 fusion protein encoded by the coding DNA expressed by the vector.

(Construction of pCOLD-I-ZZ-hGSTM1)
A cDNA sequence (SEQ ID NO: 6) encoding hGSTM1, which is a kind of human-derived GST, was amplified by a PCR method using the following PCR primers.

  The obtained PCR product was cleaved with restriction enzymes (SacI and XbaI), inserted into pEZZ18 Protein A Gene Fusion Vector (Amersham Biosciences), transformed into E. coli DH5α strain, cloned, and pEZZ18-hGSTM1 Got.

  Subsequently, the ZZ-GST region (not including the leader sequence present at the N-terminus of the ZZ sequence) in the pEZZ18-hGSTM1 vector prepared above was amplified by the PCR method using the following PCR primers.

  The obtained PCR product was cleaved with restriction enzymes (KpnI and XbaI) and inserted into a pCOLD-I vector (Takara Bio) to obtain a pCOLD-I-ZZ-hGSTM1 vector. Here, SEQ ID NO: 14 is the base sequence (CDS; including stop codon) of the DNA encoding the ZZ-hGSTM1 fusion protein expressed by the pCOLD-I-ZZ-hGSTM1 vector. SEQ ID NO: 13 is the amino acid sequence of the ZZ-hGSTM1 fusion protein encoded by the coding DNA expressed by the vector.

  In addition, about various pEZZ18-GST and various pCOLD-I-ZZ-GST obtained by said operation, the inserted DNA sequence was confirmed by determining a base sequence with a DNA sequencer (Eurofins).

[Expression of ZZ-GST by E. coli]
Escherichia coli BL21 strain was transformed with the various recombinant vectors pCOLD-I-ZZ-GST prepared above. The obtained transformant was cultured with shaking in 5 mL LB medium (containing ampicillin 100 μg / mL) at 37 ° C. for 16 hours. The culture was then added to 250 mL LB medium and shake cultured at 37 ° C. until the OD ₆₀₀ value of the culture reached 0.4-0.5. Thereafter, after cooling at 4 ° C. for 30 minutes, both IPTG (manufactured by Wako Pure Chemical Industries, Ltd.) and ampicillin were added to the culture solution to a final concentration of 100 μM and cultured at 15 ° C. for 24 hours. After culturing, the E. coli solution was suspended in B-PER Bacterial Protein Extraction Reagent (manufactured by Thermo Scientific) and stored at −80 ° C. until use.

[Purification of recombinant ZZ-GST]
A 50 ml E. coli solution pellet was suspended in 2.5 mL B-PER and dissolved by shaking at room temperature for 20 minutes. The E. coli lysate was centrifuged (12,000 × g, 4 ° C., 20 minutes) to remove the insoluble fraction, and the supernatant was collected. 2 mL of cComplete His-Tag Purification Resin (manufactured by Roche Diagnostics) was mixed therewith, and the protein was adsorbed at 4 ° C. for 1.5 hours. The column to which the protein was adsorbed was washed three times with 20 mM Tris-HCl (pH 8.0) containing 500 mM sodium chloride and 25 mM imidazole. The target protein was dialyzed overnight at 4 ° C. in a 20 mM sodium phosphate solution (pH 7.0) containing 3 mM EDTA and 3.75 mM β-mercaptoethanol. The dialyzed protein solution was then dialyzed against a 20 mM sodium phosphate solution (pH 7.0) containing 3.75 mM EDTA and 3.75 mM β-mercaptoethanol and 20% (w / v) glycerol for 6 hours. . The obtained protein solution was stored at −80 ° C. until use. The protein concentration was measured using the Bradford method, which is one of protein measuring methods, using bovine serum albumin (manufactured by Sigma) as a standard solution. The protein purity was measured using SDS-PAGE, and the GST specific activity was measured using CDNB and 3,4-DNADCF.

  FIG. 2 shows (A) ZZ-hGSTA1 fusion protein, (B) ZZ-hGSTM1 fusion protein, (C) ZZ-hGSTP1 fusion protein, and (D) ZZ-DmGSTe13 fusion protein by Coomassie Brilliant Blue. (CBB) It is a migration photograph which shows the result of having dye | stained. In addition, each lane in FIGS. 2 (A) to 2 (D) is, respectively, 1: before protein expression induction, 2: after protein expression induction, 3: His-tag column non-adsorbed fraction, 4: after purification It is. As is apparent from the results of each lane in FIGS. 2A to 2D, according to this method, the target fusion protein can be produced with high purity. In addition, the yield was as high as 100 mg / L or more depending on the molecular species.

[Comparison of specific activities of various ZZ-GSTs]
The specific activity (GST activity per 1 mg of protein) was compared for the four types of ZZ-fused GSTs (ZZ-hGSTA1, ZZ-hGSTM1, ZZ-hGSTP1, and ZZ-DmGSTE13) prepared above. Specifically, 1 mM CDNB or 1 μM 3,4-DNADCF is added to 100 mM phosphate buffer (pH 6.5, ethanol 1% (v / v) in the case of CDNB, DMSO0 in the case of 3,4-DNADCF. 1% (v / v) was used as a co-solvent) and reacted in the presence of 1 mM reduced glutathione (GSH) at 30 ° C. with stirring. At this time, the enzyme concentration in a region where the reaction rate is linear with respect to the change in the enzyme concentration was used. Then, using a multiwell plate reader (SH-9000, manufactured by Corona Electric Co., Ltd.), in the case of CDNB, the change in absorbance (340 nm), and in the case of 3,4-DNADCF, the fluorescence intensity is changed every 30 seconds for 1000 seconds. Measurement was performed (excitation / fluorescence wavelength = 505/525 nm). From the reaction rate obtained from each enzyme concentration, the specific activity (micromolar number of CDNB or 3,4-DNADCF catalyzed by 1 mg of GST per minute) was calculated. The results are shown in Table 1 below.

  As is clear from the results shown in Table 1, all the fusion proteins showed GST activity above the level required for practicing the present invention. Here, when the specific activities of various fusion proteins were compared, there was a difference in specific activity in the order of ZZ-DmGSTe13> ZZ-hGSTP1≈ZZ-hGSTM1> ZZ-hGSTA1. Among them, the ZZ-DmGSTe13 fusion protein showed a very high level of specific activity. From the above, it can be said that various fusion proteins (in particular, ZZ-DmGSTE13) can be expressed in large amounts and easily purified, and their activities can be measured with a corresponding high-sensitivity fluorescent probe.

[Comparison of enzyme kinetics of various ZZ-GSTs]
The enzyme kinetics of 3,4-DNADCF were compared for the four types of ZZ-fused GSTs (ZZ-hGSTA1, ZZ-hGSTM1, ZZ-hGSTP1, and ZZ-DmGSTe13) prepared above. Specifically, various concentrations of 3,4-DNADCF were dissolved in 100 mM phosphate buffer (pH 6.5; DMSO 0.1% (v / v) was used as a co-solvent), and 1 mM reduced glutathione (GSH) was dissolved. ) In the presence of ZZ-hGSTA1: 0.96 μg / mL, ZZ-hGSTM1: 30 ng / mL, ZZ-hGSTP1: 25 ng / mL, ZZ-DmGSTE13: 11 ng / mL). In addition, the fluorescence intensity was measured at room temperature (25 ° C.) for 5 minutes. The initial rate at each concentration (linear increase in fluorescence intensity) was calculated, and Michaelis-Menten equation V = V _max / ([S] + K _M ) (where V _max is the maximum reaction rate and [S] is the substrate the concentration, K _M has created a regression curve using a substrate concentration) to give the speed of V _{max / 2.} The obtained Michaelis Menten plot is shown in FIG. In addition, the graph of each Michaelis Menten plot of FIG. 3 is respectively shown in four types of ZZ-GST of (A) ZZ-hGSTA1, (B) ZZ-hGSTM1, (C) ZZ-hGSTP1, and (D) ZZ-DmGSTE13. It corresponds.

  Table 2 below shows the enzyme reaction kinetic parameters in each ZZ fusion type GST calculated from the results shown in FIG.

From the results shown in FIG. 3 and Tables 1 and 2, it has been clarified that ZZ-DmGSTe13 exhibits the highest specific activity and also exhibits extremely excellent specific activity exceeding 10 ⁷ M ⁻¹ s ⁻¹ .

[Immobilization of ZZ-GST to protein L beads]
In order to evaluate the applicability of the various ZZ-fused GSTs produced in the above to the quantification of antibodies, protein L having high affinity for antibody e-chains was immobilized on agarose beads by the following method. An anti-transferrin receptor antibody with a known concentration was immobilized, the various ZZ-fused GSTs prepared above were adsorbed to the antibody, and the activity was measured using 3,4-DNADCF.

  Specifically, first, 20 μL of protein L-agarose (manufactured by Santacruz biotech) was added in PBS containing 1% (w / v) bovine serum albumin and 0.2% (w / v) sodium azide for 30 minutes. Incubated at room temperature. Any concentration of anti-transferrin receptor antibody was then added and incubated for 1 hour at 4 ° C. Thereafter, the protein L-agarose was centrifuged (1000 × g, 3 minutes, 4 ° C.), washed 3 times with 1 mL of PBS containing 0.1% (w / v) Triton X-100, and then 1% It was suspended in 1 mL of PBS containing (w / v) BSA and 0.2% (w / v) sodium azide. 1.6 microgram / mL ZZ-GST was added there, and it incubated at 4 degreeC for 30 minutes. Thereafter, the protein L-agarose was centrifuged (1000 × g, 3 minutes, 4 ° C.), washed three times with PBS containing 0.1% (w / v) Triton X-100, and then the reaction buffer ( It was suspended in a 180 mM aqueous sodium phosphate solution, 600 mM sodium chloride, 0.2 mM EDTA, pH 6.5). Thus, the ZZ fusion type GST according to the present invention was adsorbed to the anti-transferrin receptor antibody immobilized on the agarose beads via protein L via the ZZ domain.

[Evaluation of GST activity using agarose beads-protein L-antibody-ZZ-GST complex]
For the agarose beads-protein L-antibody-ZZ-GST complex prepared using various ZZ-fused GSTs as described above, by measuring GST activity using 3,4-DNADCF, which is a fluorescent probe for GST measurement, ZZ fusion type GST present in each system was quantified. Then, the obtained measured value was converted into the amount of anti-transferrin receptor antibody present in each system.

  Specifically, the assay was performed as follows. First, 10 μL of beads immobilized with ZZ fusion type GST as described above were added to a well of a 96-well plate, 50 μL of water (including 0.2 mM GSH) was added thereto, and incubated at room temperature for 5 minutes. Thereto, 100 μL of a reaction buffer (containing 1 μM 3,4-DNADCF. 90 mM aqueous sodium phosphate, 300 mM sodium chloride, 0.1 mM EDTA, pH 6.5) was added, and room temperature (25 ° C.) for 3600 seconds (60 minutes) ) And the change in fluorescence intensity was measured. Then, the initial velocity (fluorescence intensity increase was linear) at each concentration was calculated and converted to the reaction product concentration per unit time.

  In this experiment, first, four types of ZZ-fused GST were performed under the condition that the antibody concentration was fixed at 40 nM. FIG. 4A is a graph in which the change in fluorescence intensity in this experiment is plotted against the elapsed time after the addition of 3,4-DNADCF. FIG. 4B is a graph showing the initial reaction rate in this experiment. FIG. 4 (B) shows an experiment using an anti-transferrin receptor antibody (αTR) necessary for adsorbing ZZ-fused GST to agarose beads (via a ZZ domain). The results for the case where the test is performed (αTR is “+”) and the case where the experiment is performed without using this (the αTR is “−”) are also shown. From the results shown in FIG. 4, the GST activity of the ZZ fusion type GST is not impaired under the condition that the protein L is adsorbed to the immobilized agarose beads. Among the four types of ZZ fusion type GST, ZZ-DmGSTe13 is the most. It turns out that it shows high activity.

  Moreover, this experiment was also conducted in a system using ZZ-DmGSTe13 when different antibody concentrations were used. At this time, the time for measuring the change in the fluorescence intensity was 2400 seconds (40 minutes). FIG. 5 (A) is a graph in which the change in fluorescence intensity in this experiment is plotted against the elapsed time after the addition of 3,4-DNADCF. FIG. 5 (B) is a graph showing the correlation of GST activity with the antibody concentration used in this experiment. As shown in FIG. 5 (A) and FIG. 5 (B), in this experiment, the antibody concentration-fluorescence intensity increase rate showed a proportional relationship, and excellent linearity was observed between the two up to the antibody concentration of 1.5 μg / mL. It became clear to show. Further, from this measurement, the detection limit of the antibody amount in 200 μL of the 96-well plate was calculated to be 4.1 ng, and it was revealed that the antibody has sufficient sensitivity for antibody detection.

[Immobilization of ZZ-GST on protein L plate]
The anti-transferrin receptor antibody with a known concentration was immobilized on a 96-well microplate on which protein L having high affinity for the e chain of the antibody was immobilized by the following procedure, and the antibody was prepared as described above. Various ZZ-fused GSTs were adsorbed and their activity was measured using 3,4-DNADCF.

  Specifically, first, a black 96-well microplate on which protein L is immobilized (manufactured by G Bioscience)) is 1% (w / v) bovine serum albumin (IgG-free Wako Pure Chemical Industries, Ltd.), Incubation was performed at 4 ° C. for 30 minutes in PBS containing 0.1% (w / v) Triton X-100 and 0.2% (w / v) sodium azide. Any concentration of anti-transferrin receptor antibody was then added and incubated at 4 ° C. for 1 hour. Then, after washing 3 times with 300 μL of PBS containing 0.1% (w / v) Triton X-100, 1% (w / v) bovine serum albumin (IgG free), 0.1% (w / v) Triton X 100 μL PBS containing −100, 0.2% (w / v) sodium azide was added. 43 μg / mL ZZ-DmGSTe13 was added thereto and incubated at 4 ° C. for 30 minutes. Then, after washing 3 times with PBS containing 0.1% (w / v) Triton X-100, reaction buffer (containing 0.4 mM GSH. 90 mM sodium phosphate aqueous solution, 300 mM sodium chloride, 0.1 mM EDTA, pH 6 .5). In this way, ZZ-DmGSTe13 was adsorbed to the anti-transferrin receptor antibody immobilized on the 96-well plate via protein L.

[Evaluation of GST activity using plate-protein L-antibody-ZZ-GST complex]
The plate-protein L-antibody-ZZ-GST complex prepared using various ZZ-fused GSTs as described above was measured by measuring GST activity using 3,4-DNADCF, which is a fluorescent probe for GST measurement. ZZ fusion type GST present in the system was quantified. Then, the obtained measured value was converted into the amount of anti-transferrin receptor antibody present in each system.

  Specifically, the assay was performed as follows. First, 100 μL of a reaction buffer (containing 1 μM 3,4-DNADCF. 90 mM sodium phosphate aqueous solution, 300 mM sodium chloride, 0.1 mM) is added to the well in which ZZ-DmGSTe13 is adsorbed as described above (including 100 μL of reaction buffer). EDTA, pH 6.5) was added, and the change in fluorescence intensity was measured at room temperature (25 ° C.) for 1200 seconds (20 minutes). Then, the initial velocity (fluorescence intensity increase was linear) at each concentration was calculated and converted to the reaction product concentration per unit time.

  FIG. 6 (A) is a graph showing changes over time in the increase in fluorescence intensity when different concentrations of anti-transferrin receptor antibodies are incubated. Here, in the series in which ZZ-DmGSTe13 was not added, 0 μM and 10 μM anti-transferrin receptor antibody treatment was used as a control. FIG. 6 (B) is a graph plotting the initial reaction rate against the concentration of the anti-transferrin receptor antibody. From the results shown in FIGS. 6 (A) and 6 (B), according to the present system, it was shown that detection and quantification are possible if the antibody concentration is 0.1 nM or more.

[Detection and quantification of antigen in cultured cells]
In this experiment, the fusion protein of the present invention is applied to the detection and quantification of an antibody in an on-cell ELISA (Transferin receptor) expressed on the cell surface of MCF7 cells, which is a fluorescent probe for GST measurement. GST activity was measured using 4-DNADCF.

  First, MCF7 cells were seeded on the day before the experiment in a transparent bottom black 96-well plate (manufactured by Greiner) at 24,000 cells / well. Cells were washed 3 times with PBS and incubated for 15 minutes at room temperature in PBS containing 4% (w / v) paraformaldehyde. Then, after washing 3 times with PBS, it was incubated at room temperature for 1 hour in PBS containing 5% (w / v) skim milk (Morinaga Milk Industry Co., Ltd.) and 0.1% (w / v) Triton X-100. . Thereafter, the mixture was shaken in PBS containing 0.1% (w / v) Triton X-100 for 5 minutes at room temperature, and this was washed three times. Next, PBS containing 5% (w / v) skim milk and 0.1% (w / v) Triton X-100 to which an anti-transferrin receptor antibody was added so as to be 0.24 μg / well at room temperature for 1 hour. Incubated. Then, it was washed by shaking with PBS containing 0.1% (w / v) Triton X-100 at room temperature for 5 minutes, and this was repeated three times. Next, incubation was performed at 4 ° C. for 1 hour in PBS containing 5% (w / v) skim milk and 2% μg / mL ZZ-DmGSTe13 and 0.1% (w / v) Triton X-100. Thereafter, the mixture was shaken with PBS containing 0.1% (w / v) Triton X-100 at room temperature for 5 minutes, and washed 5 times. Then, 100 μL reaction buffer (containing 200 μM GSH. 90 mM aqueous sodium phosphate solution, 300 mM) Sodium chloride, 0.1 mM EDTA, pH 6.5) was added.

  The assay was performed as follows. First, with respect to 100 μL of the cell solution on which ZZ-fused GST is immobilized as described above, 100 μL of a reaction buffer (containing 1 μM 3,4-DNADCF. 90 mM sodium phosphate aqueous solution, 300 mM sodium chloride, 0.1 mM EDTA, pH 6.5) ) And the change in fluorescence intensity was measured at room temperature (25 ° C.) for 5000 seconds (about 83 minutes). Then, the slope was calculated at the time when each increase in fluorescence intensity was linear, and the reactivity was compared as the increase in fluorescence intensity per unit time.

  FIG. 7A is a graph showing temporal changes in fluorescence intensity associated with different treatments. Markers marked with □ are untreated, markers marked with ♦ are only treated with anti-transferrin receptor antibody, markers marked with △ are treated only with ZZ-DmGSTe13, markers marked with ● are marked with anti-transferrin receptor antibody and ZZ-DmGSTe13 Both processes are performed. FIG. 7B is a graph showing an increase in fluorescence intensity per unit time in each process (the slope of the graph shown in FIG. 7A). As shown in FIG. 7 (A) and FIG. 7 (B), a significant increase in fluorescence intensity is observed only when both the anti-transferrin receptor antibody and ZZ-DmGSTe13 are treated. It is suggested that the increase in the fluorescence intensity in is dependent on the amount of transferrin receptor expressed on the cell surface of MCF7 cells.

[SEQ ID NO: 1]
The amino acid sequence of the ZZ domain of protein A is shown.
[SEQ ID NO: 2]
This shows the base sequence (CDS) of DNA encoding the amino acid sequence represented by SEQ ID NO: 1 (amino acid sequence of ZZ domain of protein A).
[SEQ ID NO: 3]
The amino acid sequence of human GSTA1 (hGSTA1) is shown.
[SEQ ID NO: 4]
2 shows the base sequence (CDS; including stop codon) of DNA encoding the amino acid sequence represented by SEQ ID NO: 3 (amino acid sequence of human GSTA1 (hGSTA1)).
[SEQ ID NO: 5]
The amino acid sequence of human GSTM1 (hGSTM1) is shown.
[SEQ ID NO: 6]
2 shows the base sequence (CDS; including stop codon) of DNA encoding the amino acid sequence represented by SEQ ID NO: 5 (amino acid sequence of human GSTM1 (hGSTM1)).
[SEQ ID NO: 7]
The amino acid sequence of human GSTP1 (hGSTP1) is shown.
[SEQ ID NO: 8]
2 shows the base sequence (CDS; including a stop codon) of DNA encoding the amino acid sequence represented by SEQ ID NO: 7 (the amino acid sequence of human GSTP1 (hGSTP1)).
[SEQ ID NO: 9]
The amino acid sequence of Drosophila melanogaster GSTe13 (DmGSTe13) is shown.
[SEQ ID NO: 10]
The base sequence (CDS; including a stop codon) of DNA which codes the amino acid sequence represented by sequence number: 9 (amino acid sequence of Drosophila melanogaster GSTe13 (DmGSTe13)) is shown.
[SEQ ID NO: 11]
The amino acid sequence of the ZZ-hGSTA1 fusion protein encoded by the DNA encoding the ZZ-hGSTA1 fusion protein inserted into the expression vector pCold-I-ZZ-hGSTA1 prepared in the example is shown.
[SEQ ID NO: 12]
The base sequence (CDS; including a stop codon) of DNA which codes ZZ-hGSTA1 fusion protein inserted in the expression vector pCold-I-ZZ-hGSTA1 produced in the Example is shown.
[SEQ ID NO: 13]
The amino acid sequence of the ZZ-hGSTM1 fusion protein encoded by the DNA encoding the ZZ-hGSTM1 fusion protein inserted into the expression vector pCold-I-ZZ-hGSTM1 prepared in the example is shown.
[SEQ ID NO: 14]
The base sequence (CDS; including a stop codon) of DNA which codes ZZ-hGSTM1 fusion protein inserted in the expression vector pCold-I-ZZ-hGSTM1 produced in the Example is shown.
[SEQ ID NO: 15]
The amino acid sequence of the ZZ-hGSTP1 fusion protein encoded by the DNA encoding the ZZ-hGSTP1 fusion protein inserted in the expression vector pCold-I-ZZ-hGSTP1 prepared in the example is shown.
[SEQ ID NO: 16]
The base sequence (CDS; including a stop codon) of DNA which codes ZZ-hGSTP1 fusion protein inserted in the expression vector pCold-I-ZZ-hGSTP1 produced in the Example is shown.
[SEQ ID NO: 17]
The amino acid sequence of DmGSTe13 fusion protein encoded by the DNA which codes ZZ-DmGSTe13 fusion protein inserted in the expression vector pCold-I-ZZ-DmGSTe13 produced in the Example is shown.
[SEQ ID NO: 18]
The base sequence (CDS; including a stop codon) of DNA which codes ZZ-DmGSTe13 fusion protein inserted in the expression vector pCold-I-ZZ-DmGSTe13 produced in the Example is shown.
[SEQ ID NO: 19]
It is a forward primer containing a KpnI sequence used to amplify the cDNA sequence of Nobo-Dm / GSTe14 in the examples.
[SEQ ID NO: 20]
It is a reverse primer containing the XbaI sequence used to amplify the cDNA sequence of Nobo-Dm / GSTe14 in the examples.
[SEQ ID NO: 21]
It is a forward primer containing an NdeI sequence used for amplifying the sequence of ZZ-Nobo-Dm / GSTe14 from pEZZ18-Nobo-Dm / GSTe14 in the Examples.
[SEQ ID NO: 22]
It is a forward primer containing a SacI sequence used for amplifying the cDNA sequence of hGSTA1 in Examples.
[SEQ ID NO: 23]
It is a reverse primer containing the XbaI sequence used for amplifying the cDNA sequence of hGSTA1 in the Examples.
[SEQ ID NO: 24]
It is a forward primer containing a SacI sequence used for amplifying the cDNA sequence of hGSTP1 in Examples.
[SEQ ID NO: 25]
It is a reverse primer containing the XbaI sequence used to amplify the cDNA sequence of hGSTP1 in the examples.
[SEQ ID NO: 26]
It is a forward primer containing a SacI sequence used for amplifying the hGSTM1 cDNA sequence in Examples.
[SEQ ID NO: 27]
It is a reverse primer containing the XbaI sequence used for amplifying the cDNA sequence of hGSTM1 in the examples.
[SEQ ID NO: 28]
It is a forward primer containing the KpnI sequence used to amplify the sequence from pEZZ18-hGSTM1 to ZZ-hGSTM1 in the Examples.

Claims (29)

  A fusion protein comprising a first region comprising an Fc region-binding domain capable of binding to an Fc region of an antibody, and a second region comprising a GST active domain having glutathione-S-transferase (GST) activity.   The fusion protein of claim 1, wherein the antibody is an IgG antibody.   The fusion protein according to claim 1 or 2, wherein the Fc region binding domain is a ZZ domain of protein A. The fusion protein according to any one of claims 1 to 3, wherein the first region is selected from the group consisting of the following (a) to (d):
(A) a region consisting of the amino acid sequence of SEQ ID NO: 1;
(B) a region consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 1 and having binding ability to the Fc region of an IgG antibody;
(C) a region comprising an amino acid sequence having 70% or more identity to the amino acid sequence of SEQ ID NO: 1 and having binding ability to the Fc region of IgG antibody; and
(D) Binding to the Fc region of an IgG antibody consisting of an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a base sequence complementary to the base sequence of SEQ ID NO: 2. An area with ability. The fusion protein according to claim 4, wherein the first region is selected from the group consisting of the following (a), (b '), (c') and (d '):
(A) a region consisting of the amino acid sequence of SEQ ID NO: 1;
(B ′) a region consisting of an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 1, and having a binding ability to the Fc region of an IgG antibody;
(C ′) a region consisting of an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 1 and having an ability to bind to the Fc region of an IgG antibody; and
(D ′) an Fc region of an IgG antibody comprising an amino acid sequence encoded by a polynucleotide that hybridizes with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 2 under highly stringent conditions; A region that has the ability to bind to. The fusion protein according to any one of claims 1 to 5, wherein the second region is selected from the group consisting of the following (e) to (h):
(E) a region consisting of the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9,
(F) consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, And a region having GST activity;
(G) a region consisting of an amino acid sequence having 70% or more identity to the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9 and having GST activity; and,
(H) encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10. A region having a GST activity. The fusion protein according to claim 6, wherein the second region is selected from the group consisting of the following (e), (f '), (g') and (h '):
(E) a region consisting of the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9,
(F ′) consisting of an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9. And a region having GST activity;
(G ′) a region consisting of an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, and having GST activity ;and,
(H ′) a polynucleotide that hybridizes under high stringency conditions with a polynucleotide comprising a base sequence complementary to the base sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10 A region consisting of the amino acid sequence encoded by and having GST activity.   The fusion protein according to any one of claims 1 to 7, further comprising an amino acid sequence for promoting translation and / or an amino acid sequence for purification.   A protein consisting of the amino acid sequence of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 17.   A polynucleotide comprising a polynucleotide encoding the fusion protein according to any one of claims 1 to 9. (1) A first coding sequence selected from the group consisting of the following (A) to (D):
(A) a polynucleotide comprising a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2;
(B) a polynucleotide that hybridizes with a polynucleotide consisting of a base sequence complementary to the base sequence of SEQ ID NO: 2 under stringent conditions and encodes a region capable of binding to an Fc region of an antibody A polynucleotide;
(C) a polynucleotide comprising a polynucleotide encoding a region consisting of the amino acid sequence of SEQ ID NO: 1; and
(D) a polymorphism encoding a region consisting of an amino acid sequence in which one to a plurality of amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 1 and having the binding ability to the Fc region of an antibody A polynucleotide comprising nucleotides; and
(2) A second coding sequence selected from the group consisting of the following (E) to (H):
(E) a polynucleotide comprising a polynucleotide consisting of the base sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10;
(F) It hybridizes under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, and GST A polynucleotide comprising a polynucleotide encoding a region having activity;
(G) a polynucleotide comprising a polynucleotide encoding a region consisting of the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9; and
(H) consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, And a polynucleotide comprising a polynucleotide encoding a region having GST activity;
And a polynucleotide comprising. (1) The group consisting of the following (A), (B ′), (C) and (D ′):
(A) a polynucleotide comprising a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2;
(B ′) a polynucleotide that hybridizes with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 2 under a highly stringent condition and encodes a region capable of binding to an Fc region of an antibody A polynucleotide comprising:
(C) a polynucleotide comprising a polynucleotide encoding a region consisting of the amino acid sequence of SEQ ID NO: 1; and
(D ′) an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 1, and encodes a region having an ability to bind to an Fc region of an antibody A polynucleotide comprising a polynucleotide;
Selected from
(2) the group consisting of the following (E), (F ′), (G) and (H ′):
(E) a polynucleotide comprising a polynucleotide consisting of the base sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10;
(F ′) hybridizes under high stringency conditions with a polynucleotide comprising a base sequence complementary to the base sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, and A polynucleotide comprising a polynucleotide encoding a region having GST activity;
(G) a polynucleotide comprising a polynucleotide encoding a region consisting of the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9; and
(H ′) consisting of an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9. And a polynucleotide comprising a polynucleotide encoding a region having GST activity,
The polynucleotide of claim 11 selected from.   A polynucleotide comprising a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 18.   A recombinant vector containing the polynucleotide according to any one of claims 10 to 13.   A transformant into which the recombinant vector according to claim 14 has been introduced.   A method for producing a fusion protein, comprising culturing the transformant according to claim 15 and generating the fusion protein according to any one of claims 1 to 9.   A method for measuring an antigen, an antibody containing an Fc region, or reduced glutathione (GSH) in a sample using the fusion protein according to any one of claims 1 to 9.   The method according to claim 17, wherein the antibody is a monoclonal antibody.   Using a GST measurement probe that exhibits a change in luminescence characteristics by reacting with reduced glutathione (GSH) in the presence of GST, and using the change in the luminescence characteristics of the GST measurement probe as an index, the antigen in the sample, Fc 19. A method according to claim 17 or 18, comprising measuring an antibody comprising the region, or GSH.   The method according to claim 19, wherein the change in the luminescence property is that the fluorescence intensity increases before and after contacting the GST measurement probe with GST in the presence of reduced glutathione (GSH). The GST measurement probe has the following general formula (1):
Wherein R ¹ and R ² each independently represent 1 to 3 identical or different substituents bonded to a hydrogen atom or a benzene ring; R ³ , R ⁴ , R ⁵ and R ⁶ are each Independently represents a hydrogen atom, a hydroxy group, an alkyl group or a halogen atom; X ¹ and X ² each independently represent a fluorine atom or a chlorine atom;
The method of Claim 20 containing the fluorescein derivative represented by these, or its salt. The method according to claim 21, wherein, in the general formula (1), R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are hydrogen atoms, and X ¹ and X ² are chlorine atoms. Contacting a predetermined antigen with a sample that may contain an antibody against said antigen;
Contacting the antigen after contacting the fusion protein with the sample in the absence of an antibody that is not bound to the antigen;
The GST measurement probe that exhibits a change in luminescence properties by reacting with reduced glutathione (GSH) in the presence of GST and the antigen after contacting with the fusion protein are not bound to the antibody. Contacting in the absence of the fusion protein and in the presence of GSH;
Detecting or quantifying the antibody in the sample, using as an index the change in the luminescence properties before and after contacting the GST measurement probe with the antigen;
23. The method of any one of claims 17-22, comprising:   The method according to any one of claims 17 to 23, wherein the predetermined antigen is immobilized on a solid phase.   The method according to any one of claims 17 to 24, wherein the predetermined antigen is a cell surface antigen.   26. The method of claim 25, wherein the cell is a cancer cell.   27. The method according to claim 26, wherein the surface antigen is specifically expressed in the cancer cell. Measuring the antibody to the surface antigen in the sample using the method of claim 27;
Obtaining an antibody against the surface antigen based on the result of the measurement;
A method for screening cancer preventive and / or therapeutic agents or candidate antibodies thereof.   The fusion protein according to any one of claims 1 to 9, the polynucleotide according to any one of claims 10 to 13, the recombinant vector according to claim 14, or the trait according to claim 15. A kit for measuring an antigen, antibody or GSH in a sample, comprising a transformant.