JP2002191364A - Method for detecting or determining protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an epitope peptide contained in human II type DNA topoisomerase α, suitable for use in an epitope tag method, an antibody to be specifically combined with the peptide and to provide a method for using the antibody. SOLUTION: A target protein can be efficiently detected and determined by specifying the epitope of an anti-human II type DNA topoisomerase α antibody and by using reactivity as an index between a fusion protein of a polypeptide containing the amino acid sequence of the epitope and the target protein with the antibody. The target protein can be efficiently purified by using a solid carrier supporting the antibody and the fusion protein.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a fusion protein of a target protein and a polypeptide having an amino acid sequence containing an epitope of an anti-human type II DNA topoisomerase α antibody, and the target protein is identified by using the reactivity with the antibody as an index. The present invention relates to a method for detecting and quantifying a target protein, which is characterized by performing detection or quantification. For more information,
Target protein and anti-human II for use in the above method
Fusion protein with a polypeptide having an amino acid sequence containing an epitope of type-DNA topoisomerase α antibody,
A vector for use in expressing the fusion protein, the vector including a DNA encoding a polypeptide having an amino acid sequence including an epitope,
A kit for performing the method, and contacting a fusion protein of an anti-human type II DNA topoisomerase α antibody supported on a solid carrier with a target protein and a polypeptide having an amino acid sequence containing an epitope of the antibody. The present invention relates to a method for purifying a characteristic target protein.

[0002]

2. Description of the Related Art Elucidation of the functions of proteins and their interactions is based on the accumulation of the primary structure of genomic DNA elucidated by a genomic project centered on human beings, which is currently proceeding very rapidly. It is indispensable to apply to advanced technology such as medicine. Even in bacteria and yeasts whose complete genomic DNA sequence was determined early, it is clear that the rate-limiting step in research and application is to speed up functional analysis.
There is a strong need for a comprehensive and comprehensive means for protein function analysis.

[0003] In the functional analysis of proteins, detection, quantification, or analysis of intracellular dynamics of a specific protein is a major constituent technique. These are generally performed by labeling an antibody that specifically recognizes a protein and using the amount of the label as an index. However, if the antibody of a specific protein is not available, the reactivity with the antibody is low, or the amount of the protein to be measured is small, it may be difficult to analyze by the above method. In such a case, as a means for achieving the above object, for example, when an antibody cannot be obtained, and when a protein preparation as an antigen is not obtained, a primary DNA encoding a protein to be an antigen is used. A method has been adopted in which an antibody is prepared by using a polypeptide synthesized as a hapten having a sequence predicted from its structure as a hapten bound to a carrier such as keyhole limpet hemocyanin (KLH) as an antigen. However, antibodies obtained by such a method do not always have high reactivity with an antigen, and each antibody shows specific reactivity. Consideration was needed.

Accordingly, as a second means, a polypeptide having a specific amino acid sequence and a target protein are prepared as a fusion protein, and a known antibody specifically recognizing the specific polypeptide contained in the fusion protein is used. A method for detecting a protein based on the reactivity of the protein is used. This method is called the epitope tag method (Jarvik, JW e
tal., Annu.rev.Genet., 32, 601-618 (1998)).

[0005] The epitope tag method can be applied to detection of the above-mentioned target protein inside and outside the cell, analysis of intracellular dynamics, protein purification using an immobilized antibody, or analysis of protein interaction.

In the epitope tag method, specifically, for example, a His6 tag and an anti-His monoclonal antibody, c-M
yc tag and anti-Myc monoclonal antibody (9E10)
(Evan, G. et al., Mol. Cell. Biol., 5, 3610-3616 (198
5)), and a FLAG tag and an anti-FLAG antibody (M1, M
2, M5) (Hopp, TP et al., Biotechnology, 6, 1205-1).
210 (1988)).

[0007] These epitopes are usually 6 to 30
And a portion of a protein that does not bind to a protein present in a cell that induces the expression of the fusion protein. For example, for specific detection of a target protein that has been induced to be expressed in cells such as humans and mice, a part of the virus protein that is not normally expressed in the cells, specifically, for example, hematoagglutin of influenza virus: HA-1, bovine papillomavirus: BPV-1, polyomavirus: midd
leT and SV40 viruses: largeT and the like and artificial peptides (His6, RYIRS, and HTTPHH)
Etc.) are used.

[0008] Antibodies and epitope peptides used in the epitope tag method are selected based on the sensitivity of detection of the fusion protein by the antibody and the specificity of its binding, and the effect on the target protein itself to be fused with the epitope peptide is small. Not taken into account. Therefore, whether or not the epitope can be bound so that it can be used for functional analysis experiments without impairing the original function of the target protein even if it is effective when used simply for detection by immunoblotting. Was extremely difficult to predict. For example, the amino acid sequence of an epitope conventionally used in the epitope tag method contains many hydrophobic amino acid residues and charged amino acid residues,
It was inevitable that the binding of these epitopes would cause a large change in the three-dimensional structure of the target protein. In addition, the epitope peptide bound as a fusion protein often loses its binding property to an antibody because of the three-dimensional structure of the fusion protein, which takes a structure embedded inside.

Therefore, the number of hydrophobic amino acid residues is as small as possible.
Moreover, a combination of an epitope peptide having an amino acid sequence having no extreme charge and an antibody having high reactivity with such an epitope peptide and specifically binding thereto has been desired. Whether or not the binding of a specific epitope peptide does not inhibit the original function of the protein and takes a structure in which the epitope is exposed on the surface of the protein mainly depends on the combination of the structure of the target protein and the epitope peptide. Therefore, in the epitope tag method, the wider the choice of the epitope peptide and the antibody that specifically recognizes the epitope peptide, the easier it is to use. Therefore, the more types of the epitope peptide, the better.

The present inventors have determined that among antibodies prepared using human type II topoisomerase α as an antigen, there are antibodies having high binding ability to the protein and high selectivity.
Kimura, et al., J. Biol. Chem., 271, 21439-21455 (1996)
Are disclosed. DNA topoisomerase is DN
It is an enzyme that catalyzes the conversion of the higher order structure of A, and is an essential enzyme for cell growth. Further, there are a type I enzyme which induces temporary cleavage and reconnection of only one strand of a DNA chain, and a type II enzyme which induces the above-mentioned cleavage and reconnection simultaneously on both chains and requires ATP. In higher eukaryotes, including humans, I
Type I topoisomerase has α and β isoenzymes. In particular, type II DNA topoisomerase α is frequently found in cells in the growth phase, and is absent in cells in the stationary phase. No expression is observed in nerve cells.
The details of the epitope recognized by the anti-human type II DNA topoisomerase α antibody up to the peptide level have not been analyzed so far.

[0011]

SUMMARY OF THE INVENTION The present invention provides an epitope peptide contained in human type II DNA topoisomerase α, an antibody that specifically binds to the peptide, and a method of using the same, which is suitable for use in the epitope tag method. The task is to provide.

[0012]

Means for Solving the Problems The present inventors have conducted intensive studies to achieve the above object, and as a result, have found that the hybridoma (8D
2, 7B9, 7D5, 6H8), the epitope of the anti-human type II DNA topoisomerase α monoclonal antibody produced is analyzed, and the epitope peptide and target protein are produced as a fusion protein using Escherichia coli. When immunoblotting analysis was performed using a human type II DNA topoisomerase α monoclonal antibody, it was found that the target protein could be efficiently detected by the antibody. The present invention has been completed based on these findings.

That is, according to the present invention, (1) a fusion protein of a target protein and a polypeptide having an amino acid sequence containing an epitope of an anti-human type II DNA topoisomerase α antibody, and (2) an amino acid sequence of the epitope A fusion protein according to the above (1), which is an amino acid sequence represented by No. 3, 4, 5, or 6, (3) a DNA encoding the fusion protein according to the above (1) or (2), (4) A fusion protein expression vector comprising the DNA of (3) above, (5) D encoding at least any one of the amino acid sequences of SEQ ID NOs: 3 to 6
A vector for use in expressing the fusion protein according to the above (1) or (2), which comprises NA; (6) a polypeptide having an amino acid sequence containing an epitope of a target protein and an anti-human type II DNA topoisomerase α antibody; Detecting or quantifying the target protein using the reactivity of the fusion protein with the antibody as an index, and a method for detecting and quantifying the target protein, wherein (7) the amino acid sequence of the epitope is SEQ ID NO: 3, 4, The method according to the above (6), wherein the antibody is any one of the amino acid sequences shown in 5 and 6, and (8) the antibody is a hybridoma 8D2, 7B9, 7
The method according to the above (6), which is a monoclonal antibody produced by either D5 or 6H8, (9) the amino acid sequence containing an epitope is a sequence containing the amino acid sequence shown in SEQ ID NO: 3, and the amino acid sequence is The method according to (6) above, wherein the antibody that specifically recognizes is a monoclonal antibody produced by hybridoma 8D2;
(10) The above (6), wherein the amino acid sequence containing the epitope is a sequence containing the amino acid sequence shown in SEQ ID NO: 4, and the antibody that specifically recognizes the amino acid sequence is a monoclonal antibody produced by hybridoma 7B9. (11) the amino acid sequence containing the epitope is a sequence containing the amino acid sequence shown in SEQ ID NO: 4 or 5, and the antibody that specifically recognizes the amino acid sequence is a monoclonal antibody produced by hybridoma 7D5. (6) the method according to (6), wherein the amino acid sequence containing the epitope is a sequence containing the amino acid sequence shown in SEQ ID NO: 6, and an antibody that specifically recognizes the amino acid sequence is a monoclonal antibody produced by hybridoma 6H8 (13) the method of (6), wherein the human I supported on a solid carrier is -Type DNA topoisomerase α antibody, method for purifying a target protein which comprises contacting a fusion protein with a polypeptide having an amino acid sequence comprising an epitope of the target protein and antibody,
(14) The antibody is a hybridoma 8D2, 7B9, 7D
(15) the method according to (13) above, which is a monoclonal antibody produced by any of 5 or 6H8;
The method according to (13), wherein the fusion protein is the protein according to (1) or (2), (16) an antibody that specifically recognizes any of the amino acid sequences shown in SEQ ID NOs: 3 to 6 is carried. (17) an epitope peptide having any of the amino acid sequences shown in SEQ ID NOs: 3 to 6, (18) an antibody that specifically recognizes at least any of the amino acid sequences shown in SEQ ID NOs: 3 to 6 And a kit for use in the detection or quantification of a protein, characterized by comprising:

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION (1) Anti-human type II DNA topoisomerase α antibody The anti-human type II DNA topoisomerase α antibody used in the present invention specifically binds to human type II DNA topoisomerase α and its epitope Means an antibody for which is specified. The antibody may be either monoclonal or polyclonal as long as it specifically recognizes a polypeptide having an amino acid sequence containing the epitope.
As a method for producing an antibody that specifically recognizes a specific amino acid sequence contained in human type II DNA topoisomerase α, for example, Harlow, E. et al., Antibodies, A
Laboratory Manual, Cold Spring Harbor Laboratory (19
Although the method described in 88) can be used, when a peptide is used as an antigen, it is preferable to select a part facing the surface in the three-dimensional structure of human type II DNA topoisomerase α protein.

As a specific preparation method, for example, when a monoclonal antibody is prepared using human type II DNA topoisomerase α protein as an antigen, (i) antibody-producing cells are prepared from an animal immunized with human type II DNA topoisomerase α protein. The step of collecting (ii) the step of fusing the collected antibody-producing cells and myeloma cells to produce a hybridoma (iii) the step of selecting monoclonal antibody-producing cells that recognize the target antigen from the hybridoma.

The production and purification of the human type II DNA topoisomerase α protein in step (i) can be performed by a commonly used method. For example, fractionation of immunized animal plasma with a low concentration of ammonium sulfate, denaturation of fibrinogen by heat treatment, ion exchange chromatography, and gel filtration can be performed. Animals immunized with the purified protein include mice, rats, rabbits, guinea pigs, sheep, and the like.
/ C is preferably used. The immunization method can use Freund's adjuvant tuberculosis-killed bacterium, nucleic acid, alum, or the like as an adjuvant, and can be performed by intraperitoneal injection or subcutaneous injection. The administration may be performed once, or may be performed 2 to 4 times at appropriate intervals as needed. BA
When administered intraperitoneally to LB / c mice, it is preferably performed four times at two week intervals. When the antibody titer of the immunized animal increases, 3 to 10 days after the final immunization, the antibody-producing cells of the animal are collected. Antibody-producing cells can be isolated from spleen, lymph nodes, peripheral blood, etc. Among them, cells obtained from spleen are most preferred.

The myeloma cells used in step (ii)
Although not particularly limited, it is preferable to use a cell line of an animal of the same species as the animal used for preparing the antibody-producing cells. Antibody-producing cells immunized with mice are mouse myeloma cells, specifically, P3X63-Ag8.653 (ATCC: CRL-1580),
P3-NS1 / 1Ag4.1 (RIKEN Cell Bank: RCB009
5) is preferred. Cell fusion is performed by mixing antibody-producing cells and myeloma cells at an appropriate ratio and using a cell fusion medium such as RPMI1640 or MEM in which 50% polyethylene glycol (PEG) is dissolved. be able to. The electrofusion method (U. Zimmerma
nn. et al., Naturwissenschaften, 68, 577 (1981)).

In the step (iii), screening of a hybridoma producing an antibody can be carried out by culturing the above-mentioned P3X63-Ag8.653 as a myeloma cell in a HAT medium to grow only the hybridoma. . The screening can be carried out by cloning the hybridoma using a limiting dilution method or the like, and then using a microtiter plate or the like by enzyme-linked immunosorbent assay (ELISA) using the reactivity with the antigen as an index.

Anti-human type II D thus prepared and selected
As a hybridoma producing an NA topoisomerase α antibody, specifically, for example, 8D2, 7B9, 7D5,
6H8 and the like.

The method of preparing the antibody of the present invention can be carried out by a commonly used method known per se. For example, as a method of preparing a monoclonal antibody produced by the above-mentioned hybridoma, first, the hybridoma is intraperitoneally injected into a mouse. The antibody production is induced in the hybridoma by injection and culturing, or culturing in an appropriate medium. When culture is performed by intraperitoneal administration to a mouse, pristane (2,6,10,14-tetramethylpentadecane) is injected intraperitoneally, and then the hybridoma cloned as described above is intraperitoneally administered for an appropriate period. By breeding, a tumor is formed by the hybridoma in the body of the mouse, and an antibody is produced at a high concentration in the ascites. Accordingly, the antibody can be prepared by collecting the ascites. The collected ascites fluid can be used as it is as an antibody-containing solution, but it can also be purified by, for example, ammonium sulfate fractionation, ion chromatography, or a protein A-bound carrier.

Suitable culture media for culturing hybridomas include Ham 'medium, MCDB153 medium, low calcium MEM medium, MCD104 medium, MEM medium,
-MEM medium, RPMI1640 medium, RD medium and the like, and for example, serum, hormones, cytokines, inorganic / organic substances, etc. can be added according to the purpose.

The antibody thus prepared can be confirmed for its binding to the antigen by Western blotting or the like. In the anti-human type II DNA topoisomerase α antibody of the present invention, human type II D
An NA topoisomerase α protein or a partial peptide fragment thereof can be used. The hybridoma 8 selected as described above as an antibody that specifically binds to human type II DNA topoisomerase α protein
Antibodies produced and prepared by D2, 7B9, 7D5, 6H8 and the like.

(2) Epitope analysis of anti-human type II DNA topoisomerase α antibody As a method for identifying the epitope of the anti-human type II DNA topoisomerase α antibody thus obtained, the antibody specifically binds to the obtained antibody. There are a method of determining by specifying an amino acid sequence and a method of using an antibody prepared by immunization with a specific peptide antigen.

As a method for determining an amino acid sequence which specifically binds to an antibody, Western blotting using the antibody (Proc. Natl. Acad. Sci. USA 76, 3116 (1979))
There is a method of specifying by a method. In particular,
For example, human type II DNA topoisomerase α as an antigen
When using the full length protein (SEQ ID NO: 2 etc.)
First, DNA encoding a protein is cut into fragments encoding about 50 to 200 amino acids with a restriction enzyme or the like, and each DNA fragment is inserted into an appropriate expression vector, which is transcribed and translated by an appropriate host. The protein is expressed, and the binding between the protein and the antibody is examined. The appropriate expression vector may be any suitable as long as it is suitable for the host into which it is introduced. For example, when the host uses Escherichia coli or a cell-free transcription / translation system, a pET-based vector such as pET3a (TAKARA) Company)
Etc. can be used. As a system for performing transcription and translation, living cells such as Escherichia coli, yeast, animal cells, and insect cells were also prepared based on rabbit reticulocytes, extracted wheat germ, Escherichia coli extract (Escherichia coli S30 extract), and the like. A cell-free transcription / translation system can also be used. Thus, the epitope is narrowed down to a certain peptide fragment, and subsequently, a DNA fragment encoding about 5 to 50 amino acid sequences in the peptide fragment is prepared by polymerase chain reaction (PCR), synthetic oligonucleotides, etc. Is expressed as a fusion protein, and the binding to the antibody is examined. The appropriate protein used for this fusion protein may be any protein that does not bind to the antibody to be analyzed. Thus, the epitope that is the smallest unit of polypeptide necessary and sufficient to bind the antibody can be identified.

On the other hand, the method using an antibody prepared by immunization with a specific peptide antigen is a method in which a peptide having a specific amino acid sequence is used as an antigen in preparing an antibody, and the antibody is also screened using the peptide. It is. Since the epitope of the antibody thus prepared has already been determined, the peptide can be specified as the epitope of the antibody.

The anti-human type II D thus determined
Specific examples of the amino acid sequence of the epitope of the NA topoisomerase α antibody include, for example, hybridoma 8D2
Monoclonal antibody (hereinafter referred to as "8D2
Antibody)) and a polypeptide having the amino acid sequence of SEQ ID NO: 3, hybridoma 7B9
Monoclonal antibody (hereinafter referred to as “7B9
A polypeptide having the amino acid sequence of SEQ ID NO: 4, hybridoma 7D5
Monoclonal antibody (hereinafter referred to as “7D5
SEQ ID NO: 4 or 5
And the monoclonal antibody produced by hybridoma 6H8 (hereinafter sometimes referred to as “6H8 antibody”), the polypeptide having the amino acid sequence of SEQ ID NO: 6, and the like.

(3) Polypeptide Having an Amino Acid Sequence Containing an Epitope of Human Type II DNA Topoisomerase α A polypeptide having an amino acid sequence containing an epitope of human type II DNA topoisomerase α is defined as human type II DNA topoisomerase α Means a polypeptide having the amino acid sequence of the epitope of the above-described anti-human type II DNA topoisomerase α antibody (hereinafter sometimes referred to as “epitope peptide”). The length of the polypeptide is not particularly limited, but is desirably large enough not to affect the structure of the target protein when prepared as a fusion protein with the target protein, and is usually composed of 6 to 30 amino acids. Is preferred.

It is not necessary to use the epitope peptide only with the amino acid sequence of the epitope specified in the above (2).
As long as it contains this amino acid sequence, any amino acid added before and after it can be used as the epitope peptide of the present invention. As the amino acid to be added, an amino acid located before and after the amino acid sequence of the above epitope in the amino acid sequence of human type II DNA topoisomerase α is preferably used. In addition, a polypeptide in which the amino acid sequence of the above-described epitope is repeated several times, or a polypeptide in which the amino acid sequence before and after the amino acid sequence of the epitope is added several times, can also be used. In any case, it can be used as the epitope peptide of the present invention by confirming that it has the property of binding to an antibody that specifically recognizes the amino acid sequence by the method described in (2) above.

(4) Target protein and anti-human type II DN
A fusion protein with a polypeptide having an amino acid sequence containing an epitope of an A topoisomerase α antibody The target protein used in the present invention is a DNA capable of obtaining a DNA encoding the protein, and expressing the DNA. Any protein may be used as long as it can be produced as a protein. In addition, any of naturally occurring proteins, variants thereof, artificial proteins, variants thereof, and the like can be used. Naturally occurring proteins may be those obtained by transcription and translation from cDNA libraries derived from organs, tissues or cells of various organisms. Examples of the artificial protein include a sequence obtained by combining all or a part of the amino acid sequences of naturally occurring proteins, or a protein containing a random amino acid sequence. As a method for producing a fusion protein of such a target protein and a polypeptide having an amino acid sequence containing an epitope of an anti-human type II DNA topoisomerase α antibody, a protein and a DNA fragment encoding an epitope peptide are placed in frame. A method in which the protein is expressed by binding and introducing the protein into an appropriate expression vector, followed by transcription and translation by an appropriate host, and expression of the protein. The order of binding of the epitope peptide and the target protein is not particularly limited, and the epitope peptide may be bound to either the 5 ′ side or the 3 ′ side.

[0030] DN encoding the above epitope peptide
A may be a DNA encoding the same amino acid sequence in human type II DNA topoisomerase α gene,
The amino acid constituting the epitope peptide may be replaced with an appropriate DNA sequence according to codons. Specifically, for example, the DNA encoding the epitope peptide of SEQ ID NO: 3 is the DNA of SEQ ID NO: 7, the DNA encoding the epitope peptide of SEQ ID NO: 4 is the DNA of SEQ ID NO: 8, and the epitope peptide of SEQ ID NO: 5 is encoded. The DNA of SEQ ID NO: 9 is preferably used, and the DNA of SEQ ID NO: 6 is preferably used as the DNA encoding the epitope peptide of SEQ ID NO: 6.

The suitable expression vector may be any suitable as long as it is suitable for the host into which the vector is to be introduced. For example, when the host is Escherichia coli or a cell-free transcription / translation system, a pET system such as pET3 is used. A vector (manufactured by TAKARA) or the like can be used. As a system for performing transcription and translation, living cells such as Escherichia coli, yeast, animal cells, and insect cells were also prepared based on rabbit reticulocytes, extracted wheat germ, Escherichia coli extract (Escherichia coli S30 extract), and the like. A cell-free transcription / translation system can also be used.

The culture of the host and the separation and extraction of the fusion protein from the culture can be carried out by conventional methods known per se. In a cell-free transcription / translation system, it can be carried out by a method generally used.

According to another aspect of the present invention, there is provided a vector for expressing the fusion protein of the present invention. The DNA contained in this vector includes the epitope peptide described in (2) above. DN to code
A containing A. As the epitope peptide, those shown in SEQ ID NOs: 3 to 6 are preferably used.

[0034] DN encoding the above epitope peptide
As A, the same one as in the case of preparing the above fusion protein can be used. The DNA thus obtained is inserted into a suitable vector so that it is under the control of a suitable promoter. At this time, a plasmid for expressing the fusion protein of the present invention can be more easily constructed by creating a cloning site for inserting the target protein into the 5 ′ and 3 ′ sides of the DNA. As the promoter, the same promoter as in the case of preparing the fusion protein can be used.

As a cloning site for the target protein, a DNA encoding the target protein and the above-mentioned epitope peptide must be bound so as to match the frame. Is preferred. Such a restriction enzyme site may be formed by synthesizing and annealing an oligo DNA having a target restriction enzyme site and its complementary strand, and then, between the 5 ′ end of the DNA strand encoding the peptide and the promoter, and / or It can be provided by inserting into the 3 ′ side of the DNA chain encoding the above peptide. The thus-produced fusion protein expression vector of the present invention includes, for example, a vector having the configuration shown in FIG.

(4) Method for detecting and quantifying target protein The obtained fusion protein was subjected to a conventional Western blotting method (Proc. Natl. Acad. Sci. USA 76, 3116 (19)
The presence of the target protein can be confirmed by examining the reactivity with an appropriate anti-human type II DNA topoisomerase α antibody according to 79)) and the like. Specifically, the fusion protein prepared in the above (3) is separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and the protein in this gel is transferred to a nitrocellulose membrane using a semi-dry blotting device or the like. This nitrocellulose membrane can be used for skim milk, bovine serum,
Horse serum / TBS (20 mM Tris-HCl, pH 7.5, 150 mM NaC
After blocking using l) and the like, the epitope peptide used for the fusion protein is brought into contact with an anti-human type II DNA topoisomerase α antibody having antigenicity for an appropriate time. The concentration of the antibody is usually about 0.1 to 10 μg / ml, but is appropriately selected depending on the antibody titer, the amount of the fusion protein, and the like. It is common to use the same buffer as used for blocking.

After contacting with the primary antibody in this manner,
A suitable buffer, such as 0.05% Tween 20 / TB
Washing is performed several times using S (hereinafter sometimes referred to as “TTBS”) or the like. Subsequently, after washing the primary antibody, the secondary antibody is brought into contact for an appropriate time. As the secondary antibody, an antibody or the like that specifically binds to the IgG of the animal in which the used anti-human type II DNA topoisomerase α antibody was prepared, and an antibody for staining, such as horseradish peroxidase, are used. it can. Such a secondary antibody may be prepared by a method known per se, or a commercially available one may be used. The secondary antibody can usually be used at a concentration of about 0.01 to 1 μg / ml, but can be appropriately selected depending on the antibody titer, the amount of the fusion protein, and the like. After the reaction with the secondary antibody, the nitrocellulose membrane is washed several times using an appropriate buffer, for example, TTBS, and the like, and then contacted with an appropriate substrate to detect the fusion protein bound to the antibody. Since the fusion protein contains the target protein, the target protein can be detected by this detection.

At this time, if the anti-human type II DNA topoisomerase α antibody used in the present invention reacts with another protein or a peptide fragment thereof in the host that produced the fusion protein, it becomes difficult to detect the target protein. Therefore, it is preferable that the anti-human type II DNA topoisomerase α antibody used in the present invention has a binding property only to the epitope peptide and does not have a binding property to a protein or a peptide fragment thereof existing in the host.

In the above detection method, a fusion protein of an epitope peptide to be used and an appropriate protein, the amount of which has already been determined (hereinafter sometimes referred to as “standard fusion protein”) may be used as a standard. By comparing the amount of the antibody that binds to the standard fusion protein with the amount of the antibody that binds to the fusion protein of the present invention as the amount of the staining concentration of the nitrocellulose membrane, the target protein can be quantified. . The assay of the staining concentration of the protein bound to the nitrocellulose membrane can be performed by a commonly used method known per se, and specifically, for example, by a commercially available imaging analyzer or the like.

As a kit for detecting or quantifying such a target protein, at least an anti-human I
An antibody that specifically binds to a polypeptide having an amino acid sequence containing an epitope of type I DNA topoisomerase α antibody, preferably an antibody that specifically recognizes any one of the amino acid sequences shown in SEQ ID NOs: 3 to 6 is used. Can be

An antibody that specifically binds to a polypeptide having an amino acid sequence containing an epitope of human type II DNA topoisomerase α refers to an antibody produced by hybridomas 8D2, 7B9, 7D5, and 6H8 described in (1) above. . The kit of the present invention can be constituted by adding not only these antibodies but also appropriate ones as needed. Specifically, for example, a polypeptide having an amino acid sequence containing an epitope of the above antibody can be added as a positive control to a kit for detecting a target protein. The kit for quantifying a target protein is a fusion protein of a polypeptide having an amino acid sequence containing the epitope of the antibody as described in (3) above and an appropriate protein, and has already been quantified. One can be added as a standard protein.

(5) Method for Purifying Target Protein According to still another aspect of the present invention, the anti-human II of the present invention
The present invention provides a solid carrier having an antibody that specifically binds to a polypeptide containing the amino acid sequence of the epitope of type-DNA topoisomerase α antibody on the surface thereof.

The antibody that specifically binds to the epitope peptide of the present invention refers to an antibody produced by the hybridomas 8D2, 7B9, 7D5, and 6H8 described in (1) above. These antibodies are used for purifying the fusion protein of the present invention by immunoaffinity chromatography. As the solid carrier, a water-insoluble carrier usually used for immunoaffinity chromatography can be used. Specifically, for example, Protein G
-Sepharose (manufactured by Amersham Pharmacia Biotech) and the like.

The method for binding the antibody to such a solid carrier is described in Harlow, E. et al., Antibodies, A Laboratory.
As described in Manual, Cold Spring Harbor Laboratory (1988) and the like, a commonly used method known per se can be used.

The solid carrier having the antibody of the present invention carried on its surface is filled in a commonly used column tube or the like to prepare an affinity chromatography column.
After a solution containing the fusion protein described above, for example, a suspension of cells expressing the fusion protein of the present invention, is added to the column, the column is washed an appropriate number of times with an appropriate buffer such as PBS (-). . Thereafter, purification can be performed by adding an eluate having a pH of 2 to 3 to the column or eluting the fusion protein with a synthetic epitope peptide chain.

[0046]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the following examples should be regarded as helping to obtain a specific understanding of the present invention, and the scope of the present invention is not limited to the following examples. Is not limited in any way.

[0047]Example 1 Anti-human type II DNA topoisomer
Preparation of Rahse α monoclonal antibody  (1) Acquisition of antigen protein The DNA fragment of SEQ ID NO: 1 was converted into pET3a (TAKA
RA Co., Ltd.) and E. coli BL
Heat shock method for 21 / DE3 (manufactured by TAKARA)
To obtain a transformant. This transformation
The body is cultured and the beginning of the logarithmic growth phase (OD590 = 0.2)
Isopropyl-β-D-thiogalactopyranoside (hereinafter referred to as
Below, sometimes referred to as “IPTG”)
The cells are collected by centrifugation, and human type II DN
Obtain the inclusion body of A topoisomerase α
Was.

(2) Immunization of mice and preparation of hybridoma BALB / c mice were immunized using this protein as an antigen. Mice were immunized with the antigen by injecting a mixture of antigen protein and Freund's complete adjuvant four times at two-week intervals into the peritoneal cavity of the mouse. Three days after the final immunization, spleen cells were removed, and the cells were transferred to a myeloma cell line, P3-X63Ag8 (ATCC: C
RL-1580) by the polyethylene glycol method to prepare a hybridoma.

The obtained hybridoma was subjected to the limiting dilution method 2
Human B cloned and purified
HL60 which is a cell lymphoma (RCB: 0041)
Antibody-producing cells were screened using the human type II DNA topoisomerase protein purified from E. coli as an antigen. The specificity of the obtained monoclonal antibody was determined using the above-described nuclear extract of human type II DNA topoisomerase α or HeLa cells (ATCC: CCL-22) expressed in Escherichia coli. By this screening, hybridomas 8D2, 7B9, 7D5, and 6H8 were selected.

[0050]Example 2 Recognized by monoclonal antibodies
Epitope identification  (1) Production of peptide fragments (50-100 amino acid units)
Synthetic and anti-human type II DNA topoisomerase α-monochrome
Investigation of reactivity with internal antibodies The DNA of SEQ ID NO: 1 was digested with several
From the 5 'side, T1, T2, T3, T4, T5,
T6, T7, P8, P9, P10, P11, P12, Q
DNA fragments containing the 13, Q14, Q15, and C fragments (FIG. 2)
I got a piece. The total length of the obtained DNA fragment was determined as pET3a (T
AKARA) and the recombinant vector
Bacteria BL21 / DE3 (manufactured by TAKARA)
The transformant was obtained by the recombinant method to obtain a transformant. This form
Transformants are cultured and in the early phase of the logarithmic growth phase (OD590 =
0.2), add IPTG, and further culture for 2 hours at 37 ° C.
After culturing, the cells are recovered by centrifugation, and
I got protein.

Each of the obtained recombinant proteins was separated by SDS-polyacrylamide gel electrophoresis, and blotted on a nitrocellulose membrane by a semi-dry method.

The nitrocellulose membrane subjected to the blotting was 5% skim milk / TBS (20 mM Tris).
-HCl, pH 7.5, 150 mM NaCl), and the culture supernatant of each hybridoma obtained in Example 1 (2) was diluted with 5% skim milk / TBS for 10 minutes.
The antibody-containing solution dissolved in 1 to 30 times was reacted as a primary antibody, and HRP-labeled anti-mouse I was used as a secondary antibody.
The gG antibody (manufactured by Bio-Rad) was diluted 2500-fold in the same buffer as the primary antibody and allowed to react, and then stained by Konica Immunostain-HRP-1000 (manufactured by Konica).

This Western blotting method (Proc. N
USA, 76, 3116 (1979)). The antibody produced from 8D2 is Q14 (StuI-EcoR) in FIG.
I) Amino acid sequence encoded by DNA fragment, 7B9, 7
Antibodies produced from D5 and 6H8 were T2 (B
(spEI-ApoI) DNA fragment was found to specifically bind to the amino acid sequence encoded by the fragment.

(2) Preparation of peptide fragment (5 to 50 amino acid units) and anti-human type II DNA topoisomerase α
Examination of Reactivity with Monoclonal Antibody (2) -1 Examination of Reactivity with 8D2 Producing Monoclonal Antibody The Q14 fragment was cleaved in more detail with restriction enzymes, and the binding to 8D2 antibody was examined by the same method as described above. As a result, the RsaI-EcoRI fragment described in SEQ ID NO: 12 was identified. The DNA encoding the secretory signal was deleted from the DNA encoding the branched-chain amino acid binding protein (BraC protein) of Pseudomonas aeruginosa, which was confirmed to have no antigenicity between the RsaI-EcoRI fragment and each antibody prepared above. DNA (SEQ ID NO: 11, GENBANK
ACCESSION NO.M31071,172-1212b, hereafter referred to as "Bra
C-DNA ") was inserted into pET15b (manufactured by TAKARA) to prepare a recombinant vector. This recombinant vector was named pET3C4, and its structure is shown in FIG. The RsaI-EcoRI fragment was BraC-DNA
Using the DNA encoding human type II DNA topoisomerase α as a template using SEQ ID NOs: 13 and 14 so that the
Two types of constructs, which were bound to the terminal side and the C-terminal side, were prepared. N-terminal is BglII-XhoI in FIG.
The C-terminal side was inserted into the BamHI-SalI site in FIG. Using this recombinant vector, the 8D2 antibody and Br were used in the same manner as described in Example 2 (1).
When the binding to the fusion protein encoded by aC-DNA-RsaI-EcoRI was examined, the N-terminal,
It was confirmed that both fusion proteins bound to the C-terminal bound to the 8D2 antibody.

In order to identify peptides serving as epitopes in more detail, DNA fragments encoding the various peptides shown in FIG. 3 (FIG. 3: peptide numbers 1-7) contained in the peptide encoded by the RsaI-EcoRI fragment Was prepared by polymerase chain reaction (PCR) using a full-length cDNA of human type II DNA topoisomerase α as a template and a synthetic oligonucleotide (requested by Espec Oligo) as a primer. The sequence of the oligonucleotide primer used to prepare the DNA fragment is as shown in SEQ ID NO: shown in FIG. This PCR product and the BraC-DNA of SEQ ID NO: were ligated so as to be in frame, and the fusion protein and 8D were ligated in the same manner as described above.
FIG. 3 shows the results of examining the binding to the two antibodies.

From the above results, peptide No. 6 in FIG.
Since it was found to be the shortest peptide chain that binds to the D2 antibody, it contains the amino acid sequence near this peptide,
Double-stranded DNA obtained by synthesizing two complementary oligonucleotides (requested by Espec Oligo) having a DNA sequence encoding a shorter-chain peptide (FIG. 3: peptide Nos. 8 to 15) and annealing them. To BraC-DN
The fragment A was bound so as to be in frame, and the binding between the fusion protein and the 8D2 antibody was examined in the same manner as described above. FIG. 3 shows the results of the binding, SEQ ID NOs indicating the peptides, and the oligonucleotides used to prepare the peptides. This result revealed that the polypeptide of SEQ ID NO: 3 (FIG. 3: peptide No. 12) was the shortest and was necessary and sufficient as an epitope of the 8D2 antibody.

From the results, at least SEQ ID NO: 3
It was shown that when the DNA encoding the target protein was bound to and expressed in such a manner that the DNA encoding the polypeptide shown in (1) and the DNA encoding the polypeptide matched, the expressed fusion protein was detectable with the 8D2 antibody.

(2) -2 Investigation of Reactivity with 7B9, 7D5, 6H8 Producing Monoclonal Antibody From the results of the above (1), the 7B9, 7D5, 6H8 antibody is encoded by the T2 (BspEI-ApoI) fragment of FIG. Since the amino acid (113Asn-177Phe) was found to have an epitope, this T2 fragment was divided into three T2-
1 (SEQ ID NO: 38), T2-2 (SEQ ID NO: 39), T2
-3 (SEQ ID NO: 40) was prepared by chemical synthesis (requested by Sawada Technology). The reactivity between these peptides and each antibody was examined as follows by an experiment for inhibiting the binding between human II type DNA topoisomerase α protein and each antibody.

The human type II DNA topoisomerase α was prepared in the same manner as in Example 1 (1). This protein is SD
Separation was performed by S-polyacrylamide gel electrophoresis, and blotting was performed on a nitrocellulose membrane by a semi-dry method. The nitrocellulose membrane subjected to blotting was blocked with 5% skim milk / TBS, and then reacted with each antibody solution (dilute each hybridoma supernatant 20 to 30 times with 5% skim milk / TBS). At a concentration of 10 μg / ml. Thereafter, the nitrocellulose membrane was reacted with the secondary antibody in a solution in which an HRP-labeled anti-mouse IgG antibody (manufactured by Bio-Rad) was diluted 2500-fold in the same buffer as the primary antibody. And Konica Immunostain-HRP-1000 (manufactured by Konica). As a result, the 7B9, 7D5, 6H8 antibody and human II
The binding of all types of DNA topoisomerase α was inhibited by the T2-2 peptide.

In order to analyze the epitope in more detail, two complementary oligonucleotides (requested by Espec Oligo Inc.) containing the amino acid sequence near this peptide and having a DNA sequence encoding a shorter peptide were obtained. The double-stranded DNA synthesized and annealed was
T3C4 was combined with BraC-DNA so that the frame matched, and the binding between the fusion protein and each antibody was examined in the same manner as described above. The result of the binding and each peptide,
Table 1 shows the sequence numbers of the oligonucleotides used to prepare the peptides.

[0061]

[Table 1] This result revealed that the polypeptide of SEQ ID NO: 4 is the shortest peptide that binds to the 7B9 antibody, and is necessary and sufficient as an epitope of the 7B9 antibody. As the peptide that binds to the 7D5 antibody, the polypeptide of SEQ ID NO: 4 or 5 is the shortest
It was found that the epitope was necessary and sufficient for 5 antibodies. Further, as a peptide that binds to the 6H8 antibody, the polypeptide of SEQ ID NO: 6 is the shortest, and 7B
It turned out that it was necessary and sufficient as an epitope for 9 antibodies.

From the results, it can be seen that at least SEQ ID NO: 8
When the DNA encoding the target protein is bound and expressed in such a manner that the DNA and the DNA shown in the frame match, the expressed fusion protein can be detected by the 7B9 antibody. DNA encoding the target protein to fit
Is expressed by detecting the fusion protein with the 7D5 antibody, and expressing the fusion protein by binding the DNA encoding the target protein at least in frame with the DNA shown in SEQ ID NO: 10. It was shown that the expressed fusion protein was detectable by the 6H8 antibody.

Example 3 8D2 epitope peptide and B
Purification of raC fusion protein by antibody column (1) Preparation of 8D2 antibody column After washing 250 μl of Protein G-Sepharose (manufactured by Amersham Pharmacia Biotech) three times with a physiological saline solution containing a phosphate buffer solution (PBS (−)) Then, 0.5 mg of the purified 8D2 antibody was added, and the mixture was stirred for 1 hour at room temperature with a rotary shaker to bind the 8D2 antibody to the carrier. Thereafter, the plate is washed three times with PBS (-), and further, sodium borate buffer (pH 9.0).
Wash twice with 2 ml, suspend with 1.2 ml of the same solution, 8D
Dim for chemically cross-linking antibody and Protein G
Ethylpilimidate, 7.7 mg was added. After shaking for 30 minutes on a rotary shaker, 0.2
After washing with 1.2 ml of mM ethanolamine (pH 8.0) to stop the reaction, 1.2 ml of the same solution was added, and the mixture was further shaken with a rotary shaker for 2 hours. The carrier prepared in this manner is converted to a commercially available mini column (manufactured by Bio-Rad).
Alternatively, a chip with a filter (10 μl) was filled using a syringe or the like, and further washed with PBS (−) to obtain an antibody column.

(2) Purification of fusion protein BL21 / D into which the plasmid for fusion protein expression of the epitope peptide for 8D2 (SEQ ID NO: 3) and BraC protein prepared in Example 1 (2) -1 was introduced.
The E3 cells were used as a sample. IPTG in 10 ml of culture
To induce protein expression, and then 4 ° C., 700
Centrifuge at 0 rpm for 7 minutes, remove the medium, and add 50 mM
NaCl 10 mM Tris-HCl buffer (pH
7.5) and sonicated. This is 4 ° C, 7
What was centrifuged at 000 rpm for 15 minutes was replaced with an ultracentrifuge tube, and further centrifuged at 4 ° C and 10 ⁵ × G for 1.5 hours, and the supernatant was used as a sample.

The antibody column prepared in Example 2 (1) was washed with 1 ml of PBS (-), and buffer A (20
mM Tris-HCl (pH 7.5), 0.5 mM
EDTA, 1 mM MgCl ₂ , 0.01 mM DD
T, 50 mM NaCl) and washed twice with 500 μl of protease inhibitor (1 μM PMSF, 1 μg / ml each)
After washing twice with 500 μl of buffer A (hereinafter sometimes referred to as “buffer B”) to which antipain, pepstatin A, and leupepsin were added, the sample was adsorbed. After this, further bufferB,
Wash once with 500 μl, bufferA, 500 μl
After washing twice, the synthetic peptide having the amino acid sequence of peptide No. 9 in FIG. 3 (prepared by Sawada Technology Co., Ltd.) was started to flow, and after the peptide solution reached the lower end of the column, elution was stopped and 20 to 20 was stopped. Leave for 30 minutes. Thereafter, the elution was restarted, and the column was washed three times with 500 μl of buffer A. Next, elution was performed with a 20 mM glycine-HCl buffer (pH 2.2), and each fraction was immediately neutralized.

These fractions were separated by SDS-PAGE and silver-stained. The results are shown in FIG. 4A. In the column of FIG. 4, M represents a molecular weight marker, XPD represents a crude extract of Escherichia coli expressing the fusion protein prepared above, which was applied to the antibody column, and the unadsorbed fraction of the antibody column was passed through without washing. The washing fraction with Buffer A, the peptide elution represents the fraction eluted with the above peptide, and the acid elution represents the fraction eluted with the 20 mM glycine-HCl buffer. The fusion protein in which the peptide of SEQ ID NO: 3 was bound to the target protein BraC protein was about 40 KDa, and as shown by the arrow in the figure, it was found that the fusion protein was adsorbed to the antibody column and purified by elution with the peptide and acid. .

(3) Purification of fusion protein In order to find conditions for purifying and obtaining a fusion protein of the 8D2 epitope peptide and the target protein from all extracts of cells and cells, RIPA buffer (50 mM) was used.
Tris-HCl (pH 7.5), 150 mM Na
Cl, 0.1% SDS, 1% Tritonx-10
0, 0.01 mg aprotinin) with 8D
An experiment of purification using a two-antibody column was performed. The 8D2 epitope peptide and the BraC fusion protein, the expression of which was induced in the same manner as in Example 3 (2) above, were transformed into a RIPA buff.
in er in the same manner as in Example 3 (2) was sonicated, 10 ⁵ × G, subjected to 1.5 hours ultracentrifugation, and the supernatant was used as a sample. The 8D2 antibody column prepared in Example 3 (1) was also equilibrated with RIPA buffer in the same manner as described above. Subsequently, the sample is placed on the column, and the RIPA buff
After washing with er, elution with the same peptide as in Example 3 (2) was performed. Each fragment obtained by this chromatography was separated by SDS-PAGE, and the result of silver staining is shown in FIG. 4B. In the column of FIG. 4, M represents a molecular weight marker, and XPD represents Bra.
A crude extract of Escherichia coli expressing a fusion protein in which an epitope peptide is bound to the N-terminus of the C protein,
SPD is a crude extract of Escherichia coli expressing a fusion protein in which an epitope peptide is bound to the C-terminus of the BraC protein, the unadsorbed fraction of the antibody column is passed directly, and the peptide elution is the fraction eluted by the above peptide. Respectively. The fusion protein in which the peptide of SEQ ID NO: 3 was bound to the target protein BraC protein was about 42 KDa, and as indicated by the arrow in the figure, even when the whole extract by RIPA buffer was applied to this antibody column, Purification was achieved by elution, indicating that the target protein can be purified by one-step purification.

In this case, a fusion protein in which an epitope peptide is bound to the N-terminal of the BraC protein,
It was confirmed that a fusion protein in which an epitope peptide was bound to the C-terminal of BraC protein was similarly purified.

[0069]

The human II containing the epitope of the present invention
Type-DNA topoisomerase α is suitable for use as an epitope peptide because it does not exist in cells other than cells in the growing phase and cancer cells. In addition, the anti-human type II DNA topoisomerase α antibody used in the present invention has high selectivity for human antigens and has no cross-linking property with the same enzyme derived from other cells. Therefore, according to the present invention, there is provided a human type II DNA topoisomerase α antibody used in an epitope tag method which has high reactivity and extremely low background due to a non-specific reaction, and an amino acid sequence which specifically binds thereto. You.

[0070]

[Sequence list] <110> Mitsubishi Chemical Corporation <120> Methods for detection of a protein <130> human TopoisomeraseIIa <140> <141> <160> 64 <170> PatentIn Ver. 2.0 <210> 1 <211> 4593 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) .. (4593) <400> 1 atg gaa gtg tca cca ttg cag cct gta aat gaa aat atg caa gtc aac 48 Met Glu Val Ser Pro Leu Gln Pro Val Asn Glu Asn Met Gln Val Asn 1 5 10 15 aaa ata aag aaa aat gaa gat gct aag aaa aga ctg tct gtt gaa aga 96 Lys Ile Lys Lys Asn Glu Asp Ala Lys Lys Arg Leu Ser Val Glu Arg 20 25 30 atc tat caa aag aaa aca caa ttg gaa cat att ttg ctc cgc cca gac 144 Ile Tyr Gls Lys Thrs Gln Leu Glu His Ile Leu Leu Arg Pro Asp 35 40 45 acc tac att ggt tct gtg gaa tta gtg acc cag caa atg tgg gtt tac 192 Thr Tyr Ile Gly Ser Val Glu Leu Val Thr Gln Gln Met Trp Val Tyr 50 55 60 gat gaa gat gtt ggc att aac tat agg gaa gtc act ttt gtt cct ggt 240 Asp Glu Asp Val Gly Ile Asn Tyr Arg Glu Val Thr Phe Val Pro Gly 65 70 75 80 ttg tac aaa atc ttt gat gag att cta gtt aat gct gcg gac aac aaa 288 Leu Tyr Lys Ile Phe Asp Glu Ile Leu Val Asn Ala Ala Asp Asn Lys 85 90 95 caa agg gac cca aaa atg tct tgt att aga gtc aca att gat ccg gaa 336 Gln Arg Asp Pro Lys Met Ser Cys Ile Arg Val Thr Ile Asp Pro Glu 100 105 110 aac aat tta att agt ata tgg aat aat gga aaa ggt att cct gtt gtt 384 Asn Asn Leu Ile Ser Ile Trp Asn Asn Gly Lys Gly Ile Pro Val Val 115 120 125 gaa cac aaa gtt gaa aag atg tat gtc cca gct ctc atatt gga cag 432 Glu His Lys Val Glu Lys Met Tyr Val Pro Ala Leu Ile Phe Gly Gln 130 135 140 ctc cta act tct agt aac tat gat gat gat gaa aag aaa gtg aca ggt 480 Leu Leu Thr Ser Ser Asn Tyr Asp Asp Asp Glu Lys Lys Val Thr Gly 145 150 155 160 ggt cga aat ggc tat gga gcc aaa ttg tgt aac ata ttc agt acc aaa 528 Gly Arg Asn Gly Tyr Gly Ala Lys Leu Cys Asn Ile Phe Ser Thr Lys 165 170 175 ttt act gtg gaa aca gcc agt aga gaa tac aag aaa atg ttc aaa cag 576 Phe Thr Val Glu Thr Ala Ser Arg Glu Tyr Lys Lys Met Phe Lys Gln 180 185 190 aca tgg atg gat aat atg gga aga gct ggt gag atg gaa ctc aag ccc 624 Thrp Met Asp Asn Met Gly Arg Ala Gly Glu Met Glu Leu Lys Pro 195 200 205 ttc aat gga gaa gat tat aca tgt atc acc ttt cag cct gat ttg tct 672 Phe Asn Gly Glu Asp Tyr Thr Cys Ile Thr Phe Gln Pro Asp Leu Ser 210 215 220 aag ttt aaa atg caa agc ctg gac aaa gat att gtt gca cta atg gtc 720 Lys Phe Lys Met Gln Ser Leu Asp Lys Asp Ile Val Ala Leu Met Val 225 230 235 240 aga aga gca tat gat att gct gga tcc acc aaa gat gtc aaa gtc ttt 768 Arg Arg Ala Tyr Asp Ile Ala Gly Ser Thr Lys Asp Val Lys Val Phe 245 250 255 ctt aat gga aat aaa ctg cca gta aaa gga ttt cgt agt tat gtg gac 816 Leu Asn Gly Asn Lys Leu Pro Val Lys Gly Phe Arg Ser Tyr Val Asp 260 265 270 atg tat ttg aag gac aag ttg gat gaa act ggt aac tcc ttg aaa gta 864 Met Tyr Leu Lys Asp Lys Leu Asp Glu Thr Gly Asn Ser Leu Lys Val 275 280 285 ata cat gaa caa gta aac cac agg tgg gaa gtg tgt tta act atg agt 912 Ile His Glu Gln Val Asn His Arg Trp Glu Val Cys Leu Thr Met Ser 290 295 300 gaa aaa ggc ttt cag caa att agc ttt gtc aac agc att gct aca 960 Glu Lys Gly Phe Gln Gln Ile Ser Phe Val Asn Ser Ile Ala Thr Ser 305 310 315 320 aag ggt ggc aga cat gtt gat tat gta gct gat cag att gtg act aaa 1008 Lys Gly Gly Arg His Val Asp Tyr Val Ala Asp G ln Ile Val Thr Lys 325 330 335 ctt gtt gat gtt gtg aag aag aag aac aag ggt ggt gtt gca gta aaa 1056 Leu Val Asp Val Val Lys Lys Lys Asn Lys Gly Gly Val Ala Val Lys 340 345 350 cca cat cag gtg aaa aat cac atg tgg att ttt gta aat gcc tta att 1104 Pro His Gln Val Lys Asn His Met Trp Ile Phe Val Asn Ala Leu Ile 355 360 365 gaa aac cca acc ttt gac tct cag aca aaa gaa aac atg act tta caa 1152 Glu Asn Pro Thr Phe Asp Ser Gln Thr Lys Glu Asn Met Thr Leu Gln 370 375 380 ccc aag agc ttt gga tca aca tgc caa ttg agt gaa aaa ttt atc aaa 1200 Pro Lys Ser Phe Gly Ser Thr Cys Gln Leu Ser Glu Lys Phe Ile Lys 385 390 395 400 gct gcc att ggc tgt ggt att gta gaa agc ata cta aac tgg gtg aag 1248 Ala Ala Ile Gly Cys Gly Ile Val Glu Ser Ile Leu Asn Trp Val Lys 405 410 415 ttt aag gcc caa gtc cag tta agt aag aag tca gct gta aaa cat 1296 Phe Lys Ala Gln Val Gln Leu Asn Lys Lys Cys Ser Ala Val Lys His 420 425 430 aat aga atc aag gga att ccc aaa ctc gat gat gcc aat gat gca ggg 1344 Asn Arg Ile Lys Gly Ile Pr o Lys Leu Asp Asp Ala Asn Asp Ala Gly 435 440 445 ggc cga aac tcc act gag tgt acg ctt atc ctg act gag gga gat tca 1392 Gly Arg Asn Ser Thr Glu Cys Thr Leu Ile Leu Thr Glu Gly Asp Ser 450 455 460 gcc aaa act ttg gct gtt tca ggc ctt ggt gtg gtt ggg aga gac aaa 1440 Ala Lys Thr Leu Ala Val Ser Gly Leu Gly Val Val Gly Arg Asp Lys 465 470 475 480 tat ggg gtt ttc cct ctt aga gga aaa ata ctc aat gtt c gaa gct 1488 Tyr Gly Val Phe Pro Leu Arg Gly Lys Ile Leu Asn Val Arg Glu Ala 485 490 495 tct cat aag cag atc atg gaa aat gct gag att aac aat atc atc aag 1536 Ser His Lys Gln Ile Met Glu Asn Ala Glu Ile Asn Asn Ile Ile Lys 500 505 510 att gtg ggt ctt cag tac aag aaa aac tat gaa gat gaa gat tca ttg 1584 Ile Val Gly Leu Gln Tyr Lys Lys Asn Tyr Glu Asp Glu Asp Ser Leu 515 520 525 aag acg gtt cgt aag ata atg att atg aca gat cag gac caa 1632 Lys Thr Leu Arg Tyr Gly Lys Ile Met Ile Met Thr Asp Gln Asp Gln 530 535 540 gat ggt tcc cac atc aaa ggc ttg ctg att aat ttt atc cat cac aac Glp y Ser His Ile Lys Gly Leu Leu Ile Asn Phe Ile His His Asn 545 550 555 560 tgg ccc tct ctt ctg cga cat cgt ttt ctg gac caa ttt atc act ccc 1728 Trp Pro Ser Leu Leu Arg His Arg Phe Leu Asp Gln Phe Ile Thr Pro 565 570 575 att gta aag gta tct aaa aac aag caa gaa atg gca ttt tac agc ctt 1776 Ile Val Lys Val Ser Lys Asn Lys Gln Glu Met Ala Phe Tyr Ser Leu 580 585 590 cct gaa ttt gaa gag tgg aag agt tgt act cca aat cat aaa aaa tgg 1824 Pro Glu Phe Glu Glu Trp Lys Ser Ser Thr Pro Asn His Lys Lys Trp 595 600 605 aaa gtc aaa tat tac aaa ggt ttg ggc acc agc aca tca aag gaa gct 1872 Lys Val Lys Tyr Tyr Lys Gly Leu Gly Thr Ser Thr Ser Lys Glu Ala 610 615 620 aaa gaa tac ttt gca gat atg aaa aga cat cgt atc cag ttc aaa tat 1920 Lys Glu Tyr Phe Ala Asp Met Lys Arg His Arg Ile Gln Phe Lys Tyr 625 630 635 640 tct ggt cct gaa gat gat gct gct atc agc ctg gcc ttt agc aaa aaa 1968 Ser Gly Pro Glu Asp Asp Ala Ala Ile Ser Leu Ala Phe Ser Lys Lys 645 650 655 cag ata gat gat cga aag gaa tgg tta act aat ttc at gag gat aga 2016 Gln Ile Asp Asp Arg Lys Glu Trp Leu Thr Asn Phe Met Glu Asp Arg 660 665 670 aga caa cga aag tta ctt ggg ctt cct gag gat tac ttg tat gga caa 2064 Arg Gln Arg Lys Leu Leu Gly Leu Pro Glu Asp Tyr Leu Tyr Gly Gln 675 680 685 act acc aca tat ctg aca tat aat gac ttc atc aac aag gaa ctt atc 2112 Thr Thr Thr Tyr Leu Thr Tyr Asn Asp Phe Ile Asn Lys Glu Leu Ile 690 695 700 ttg ttc tca at t gat aac gag aga tct atc cct tct atg gtg gat 2160 Leu Phe Ser Asn Ser Asp Asn Glu Arg Ser Ile Pro Ser Met Val Asp 705 710 715 720 ggt ttg aaa cca ggt cag aga aag gtt ttg ttt act tgc ttc aaa cgg 2208 Gly Leu Lys Pro Gly Gln Arg Lys Val Leu Phe Thr Cys Phe Lys Arg 725 730 735 aat gac aag cga gaa gta aag gtt gcc caa tta gct gga tca gtg gct 2256 Asn Asp Lys Arg Glu Val Lys Val Ala Gln Leu Ala Gly Ser Val Ala 740 745 750 gaa atg tct tct tat cat cat ggt gag atg tca cta atg atg acc att 2304 Glu Met Ser Ser Tyr His His Gly Glu Met Ser Leu Met Met Thr Ile 755 760 765 atc aat ttg gct cag aat ttt gtg ggt agc aat aat cta aac ctc ttg 2352 Ile Asn Leu Ala Gln Asn Phe Val Gly Ser Asn Asn Leu Asn Leu Leu 770 775 780 cag ccc att ggt cag ttt ggt acc agg cta cat ggt ggc aag gat tct 2400 Gln Pro Ile Gly Phe Gly Thr Arg Leu His Gly Gly Lys Asp Ser 785 790 795 800 gct agt cca cga tac atc ttt aca atg ctc agc tct ttg gct cga ttg 2448 Ala Ser Pro Arg Tyr Ile Phe Thr Met Leu Ser Ser Leu Ala Arg Leu 805 810 815 tta ttt cca cca aaa gat gat cac acg ttg aag ttt tta tat gat gac 2496 Leu Phe Pro Pro Lys Asp Asp His Thr Leu Lys Phe Leu Tyr Asp Asp 820 825 830 aac cag cgt gtt gag cct gaa tgg tac att cct att att ccc atg gtg 2544 Asn Gln Arg Val Glu Pro Glu Trp Tyr Ile Pro Ile Ile Pro Met Val 835 840 845 ctg ata aat ggt gct gaa gga atc ggt act ggg tgg tcc tgc aaa atc 2592 Leu Ile Asn Gly Ala Glu Gly Ile Gly Thr Gly Trp Ser Cys Lys Ile 850 855 860 ccc aac ttt gat gtg cgt gaa att gta aat aac atc agg cgt ttg atg 2640 Pro Asn Phe Asp Val Arg Glu Ile Val Asn Asn Ile Arg Arg Leu Met 865 870 875 875 880 gat gga g aa gaa cct ttg cca atg ctt cca agt tac aag aac ttc aag 2688 Asp Gly Glu Glu Pro Leu Pro Met Leu Pro Ser Tyr Lys Asn Phe Lys 885 890 895 ggt act att gaa gaa ctg gct cca aat caa tat gtg att agt 2736 Gly Thr Ile Glu Glu Leu Ala Pro Asn Gln Tyr Val Ile Ser Gly Glu 900 905 910 gta gct att ctt aat tct aca acc att gaa atc tca gag ctt ccc gtc 2784 Val Ala Ile Leu Asn Ser Thr Thr Thr Ile Glu Ile Ser Glu Leu Pro Val 915 920 925 aga aca tgg acc cag aca tac aaa gaa caa gtt cta gaa ccc atg ttg 2832 Arg Thr Trp Thr Gln Thr Tyr Lys Glu Gln Val Leu Glu Pro Met Leu 930 935 940 aat ggc acc gag aag aca cct cct ctc ata aca gac tat agg gaa tac 2880 Asn Gly Thr Glu Lys Thr Pro Pro Leu Ile Thr Asp Tyr Arg Glu Tyr 945 950 955 960 cat aca gat acc act gtg aaa ttt gtt gtg aag atg act gaa gaa aaa 2928 His Thr Asp Thr Thr Val Lys Phe Val Val Lys Met Thr Glu Glu Lys 965 970 975 ctg gca gag gca gag aga gtt gga cta cac aaa gtc ttc aaa ctc caa 2976 Leu Ala Glu Ala Glu Arg Val Gly Leu His Lys Val Phe Lys Leu Gln 9 80 985 990 act agt ctc aca tgc aac tct atg gtg ctt ttt gac cac gta ggc tgt 3024 Thr Ser Leu Thr Cys Asn Ser Met Val Leu Phe Asp His Val Gly Cys 995 1000 1005 tta aag aaa tat gac acg gtg ttg gat att cta aga gac ttt ttt gaa 3072 Leu Lys Lys Tyr Asp Thr Val Leu Asp Ile Leu Arg Asp Phe Phe Glu 1010 1015 1020 ctc aga ctt aaa tat tat gga tta aga aaa aaa gaa tgg ctc cta gga atg 3120 Leu Arg Leu Lys Tyr Arg Lys Glu Trp Leu Leu Gly Met 1025 1030 1035 1040 ctt ggt gct gaa tct gct aaa ctg aat aat cag gct cgc ttt atc tta 3168 Leu Gly Ala Glu Ser Ala Lys Leu Asn Asn Gln Ala Arg Phe Ile Leu 1045 1055 1055 ata gat ggc aaa ata atc att gaa aat aag cct aag aaa gaa 3216 Glu Lys Ile Asp Gly Lys Ile Ile Ile Glu Asn Lys Pro Lys Lys Glu 1060 1065 1070 tta att aaa gtt ctg att cag agg gga tat gat tcgat 3264 Leu Ile Lys Val Leu Ile Gln Arg Gly Tyr Asp Ser Asp Pro Val Lys 1075 1080 1085 gcc tgg aaa gaa gcc cag caa aag gtt cca gat gaa gaa gaa aat gaa 3312 Ala Trp Lys Glu Ala Gln Gln Lys Val Pro Asp Glu Glu Glu Asn Glu 1090 1095 1100 gag agt gac aac gaa aag gaa act gaa aag agt gac tcc gta aca gat 3360 Glu Ser Asp Asn Glu Lys Glu Thr Glu Lys Ser Asp Ser Val Thr Asp 1105 1110 1115 1120 tct gga cca acc ttc aac tat ctt ctt gat atg ccc ctt tgg tat tta 3408 Ser Gly Pro Thr Phe Asn Tyr Leu Leu Asp Met Pro Leu Trp Tyr Leu 1125 1130 1135 acc aag gaa aag aaa gat gaa ctc tgc agg cta aga aata gaa 3456 Thr Lys Glu Lys Lys Asp Glu Leu Cys Arg Leu Arg Asn Glu Lys Glu 1140 1145 1150 caa gag ctg gac aca tta aaa aga aga aag agt cca tca gat ttg tgg aaa 3504 Gln Glu Leu Asp Thr Leu Lys Arg Lys Ser Asp Leu Trp Lys 1155 1160 1165 gaa gac ttg gct aca ttt att gaa gaa ttg gag gct gtt gaa gcc aag 3552 Glu Asp Leu Ala Thr Phe Ile Glu Glu Leu Glu Ala Val Glu Ala Lys 1170 1175 1180 gaa ga caa gga ctt cct ggg aaa ggg ggg aag gcc 3600 Glu Lys Gln Asp Glu Gln Val Gly Leu Pro Gly Lys Gly Gly Lys Ala 1185 1190 1195 1200 aag ggg aaa aaa aca caa atg gct gaa gtt ttg cct tc t ccg cgt ggt 3648 Lys Gly Lys Lys Thr Gln Met Ala Glu Val Leu Pro Ser Pro Arg Gly 1205 1210 1215 caa aga gtc att cca cga ata acc ata gaa atg aaa gca gag gca gaa 3696 Gln Arg Val Ile Pro Arg Ile Thr Ile Glu Met Lys Ala Glu Ala Glu 1220 1225 1230 aag aaa aat aaa aag aaa att aag aat gaa aat act gaa gga agc cct 3744 Lys Lys Asn Lys Lys Lys Ile Lys Asn Glu Asn Thr Glu Gly Ser Pro 1235 1240 1245 caa gaa gat gtg gaa cta gaa ggc cta aaa caa aga tta gaa aag 3792 Gln Glu Asp Gly Val Glu Leu Glu Gly Leu Lys Gln Arg Leu Glu Lys 1250 1255 1260 aaa cag aaa aga gaa cca ggt aca aag aca aag aaa caa acta Gln Lys Arg Glu Pro Gly Thr Lys Thr Lys Lys Gln Thr Thr Leu 1265 1270 1275 1280 gca ttt aag cca atc aaa aaa gga aag aag aga aat ccc tgg cct gat 3888 Ala Phe Lys Pro Ile Lys Lys Gly Lys Lys Arg Asn Pro Trp Pro Asp 1285 1290 1295 tca gaa tca gat agg agc agt gac gaa agt aat ttt gat gtc cct cca 3936 Ser Glu Ser Asp Arg Ser Ser Asp Glu Ser Asn Phe Asp Val Pro Pro 1300 1305 1310 cga gaa a ca gag cca cgg aga gca gca aca aaa aca aaa ttc aca atg 3984 Arg Glu Thr Glu Pro Arg Arg Ala Ala Thr Lys Thr Lys Phe Thr Met 1315 1320 1325 gat ttg gat tca gat gaa gat ttc tca gat ttt gat gaa aaa act gat 4032 Asp Leu Asp Ser Asp Glu Asp Phe Ser Asp Phe Asp Glu Lys Thr Asp 1330 1335 1340 gat gaa gat ttt gtc cca tca gat gct agt cca cct aag acc aaa act 4080 Asp Glu Asp Phe Val Pro Ser Asp Ala Ser Pro Pro Lys Thr Lys Thr 1345 1350 1355 1360 tcc cca aaa ctt agt aac aaa gaa ctg aaa cca cag aaa agt gtc gtg 4128 Ser Pro Lys Leu Ser Asn Lys Glu Leu Lys Pro Gln Lys Ser Val Val 1365 1370 1375 tca gac ctt gaa gct gat gat gtt aag ggc agt gta cca ctg tct tca 4176 Ser Asp Leu Glu Ala Asp Asp Val Lys Gly Ser Val Pro Leu Ser Ser 1380 1385 1390 agc cct cct gct aca cat ttc cca gat gaa act gaa att aca aac cca 4224 Ser Pro Pro Ala Thr His Phe Pro Asp Glu Thr Glu Ile Thr Asn Pro 1395 1400 1405 gtt cct aaa aag aat gtg aca gtg aag aag aca gca gca aaa agt cag 4272 Val Pro Lys Lys Asn Val Thr Val Lys Lys Thr Al a Ala Lys Ser Gln 1410 1415 1420 tct tcc acc tcc act acc ggt gcc aaa aaa agg gct gcc cca aaa gga 4320 Ser Ser Thr Thr Ser Thr Thr Gly Ala Lys Lys Arg Ala Ala Pro Lys Gly 1425 1430 1435 1440 act aaa agg gat cca gct ttg aat tct ggt gtc tct caa aag cct gat 4368 Thr Lys Arg Asp Pro Ala Leu Asn Ser Gly Val Ser Gln Lys Pro Asp 1445 1450 1455 cct gcc aaa acc aag aat cgc cgc aaa agg aag cca tcc act tct gat 4416 Pro Ala Lys Thr Lys Asn Arg Arg Lys Arg Lys Pro Ser Thr Ser Asp 1460 1465 1470 gat tct gac tct aat ttt gag aaa att gtt tcg aaa gca gtc aca agc 4464 Asp Ser Asp Ser Asn Phe Glu Lys Ile Val Ser Lys Ala Val Thr Ser 1475 1480 1485 aag aaa tcc aag ggg gag agt gat gac ttc cat atg gac ttt gac tca 4512 Lys Lys Ser Lys Gly Glu Ser Asp Asp Phe His Met Asp Phe Asp Ser 1490 1495 1500 gct gtg gct cct cgg gca aaa tat gta cgg gca aag aaa cct ata aag 4560 Ala Val Ala Pro Arg Ala Lys Tyr Val Arg Ala Lys Lys Pro Ile Lys 1505 1510 1515 1520 tac ctg gaa gag tca gat gaa gat gat ctg ttt 4593 Tyr Leu Glu Glu Ser Asp Glu Asp Asp Leu Phe 1525 1530 <210> 2 <211> 1531 <212> PRT <213> Homo sapiens <400> 2 Met Glu Val Ser Pro Leu Gln Pro Val Asn Glu Asn Met Gln Val Asn 1 5 10 15 Lys Ile Lys Lys Asn Glu Asp Ala Lys Lys Arg Leu Ser Val Glu Arg 20 25 30 Ile Tyr Gln Lys Lys Thr Gln Leu Glu His Ile Leu Leu Arg Pro Asp 35 40 45 Thr Tyr Ile Gly Ser Val Glu Leu Val Thr Gln Gln Met Trp Val Tyr 50 55 60 Asp Glu Asp Val Gly Ile Asn Tyr Arg Glu Val Thr Thr Phe Val Pro Gly 65 70 75 80 Leu Tyr Lys Ile Phe Asp Glu Ile Leu Val Asn Ala Ala Asp Asn Lys 85 90 95 Gln Arg Asp Pro Lys Met Ser Cys Ile Arg Val Thr Ile Asp Pro Glu 100 105 110 Asn Asn Leu Ile Ser Ile Trp Asn Asn Gly Lys Gly Ile Pro Val Val 115 120 125 Glu His Lys Val Glu Lys Met Tyr Val Pro Ala Leu Ile Phe Gly Gln 130 135 140 Leu Leu Thr Ser Ser Asn Tyr Asp Asp Asp Glu Lys Lys Val Thr Gly 145 150 155 160 Gly Arg Asn Gly Tyr Gly Ala Lys Leu Cys Asn Ile Phe Ser Thr Lys 165 170 175 Phe Thr Val Glu Thr Ala Ser Arg Glu Tyr Lys Lys Met Phe Lys Gln 180 185 190 Thr Trp Met Asp Asn Met Gly Arg Ala Gly Glu Met Glu Leu Lys Pro 195 200 205 Phe Asn Gly Gl u Asp Tyr Thr Cys Ile Thr Phe Gln Pro Asp Leu Ser 210 215 220 Lys Phe Lys Met Gln Ser Leu Asp Lys Asp Ile Val Ala Leu Met Val 225 230 235 240 Arg Arg Ala Tyr Asp Ile Ala Gly Ser Thr Lys Asp Val Lys Val Phe 245 250 255 Leu Asn Gly Asn Lys Leu Pro Val Lys Gly Phe Arg Ser Tyr Val Asp 260 265 270 Met Tyr Leu Lys Asp Lys Leu Asp Glu Thr Gly Asn Ser Leu Lys Val 275 280 285 Ile His Glu Gln Val Asn His Arg Trp Glu Val Cys Leu Thr Met Ser 290 295 300 300 Glu Lys Gly Phe Gln Gln Ile Ser Phe Val Asn Ser Ile Ala Thr Ser 305 310 315 320 Lys Gly Gly Arly His Val Asp Tyr Val Ala Asp Gln Ile Val Thr Lys 325 330 335 Leu Val Asp Val Val Lys Lys Lys Asn Lys Gly Gly Val Ala Val Lys 340 345 350 Pro His Gln Val Lys Asn His Met Trp Ile Phe Val Asn Ala Leu Ile 355 360 365 Glu Asn Pro Thr Phe Asp Ser Gln Thr Lys Glu Asn Met Thr Leu Gln 370 375 380 Pro Lys Ser Phe Gly Ser Thr Cys Gln Leu Ser Glu Lys Phe Ile Lys 385 390 395 400 Ala Ala Ile Gly Cys Gly Ile Val Glu Ser Ile Leu Asn Trp Val Lys 405 410 415 Phe Lys Ala G ln Val Gln Leu Asn Lys Lys Cys Ser Ala Val Lys His 420 425 430 Asn Arg Ile Lys Gly Ile Pro Lys Leu Asp Asp Ala Asn Asp Ala Gly 435 440 445 445 Gly Arg Asn Ser Thr Glu Cys Thr Leu Ile Leu Thr Glu Gly Asp Ser 450 455 460 Ala Lys Thr Leu Ala Val Ser Gly Leu Gly Val Val Gly Arg Asp Lys 465 470 475 480 Tyr Gly Val Phe Pro Leu Arg Gly Lys Ile Leu Asn Val Arg Glu Ala 485 490 495 495 Ser His Lys Gln Ile Met Glu Asn Ala Glu Ile Asn Asn Ile Ile Lys 500 505 510 Ile Val Gly Leu Gln Tyr Lys Lys Asn Tyr Glu Asp Glu Asp Ser Leu 515 520 525 Lys Thr Leu Arg Tyr Gly Lys Ile Met Ile Met Thr Asp Gln Asp Gln 530 535 540 Asp Gly Ser His Ile Lys Gly Leu Leu Ile Asn Phe Ile His His Asn 545 550 555 560 Trp Pro Ser Leu Leu Arg His Arg Phe Leu Asp Gln Phe Ile Thr Pro 565 570 575 Ile Val Lys Val Ser Lys Asn Lys Gln Glu Met Ala Phe Tyr Ser Leu 580 585 590 590 Pro Glu Phe Glu Glu Trp Lys Ser Ser Thr Pro Asn His Lys Lys Trp 595 600 605 Lys Val Lys Tyr Tyr Lys Gly Leu Gly Thr Ser Thr Ser Lys Glu Ala 610 615 620 620 Lys Glu Tyr Phe A la Asp Met Lys Arg His Arg Ile Gln Phe Lys Tyr 625 630 635 640 Ser Gly Pro Glu Asp Asp Ala Ala Ile Ser Leu Ala Phe Ser Lys Lys 645 650 655 Gln Ile Asp Asp Arg Lys Glu Trp Leu Thr Asn Phe Met Glu Asp Arg 660 665 670 Arg Gln Arg Lys Leu Leu Gly Leu Pro Glu Asp Tyr Leu Tyr Gly Gln 675 680 685 Thr Thr Thr Tyr Leu Thr Tyr Asn Asp Phe Ile Asn Lys Glu Leu Ile 690 695 700 Leu Phe Ser Asn Ser Asp Asn Glu Arg Ser Ile Pro Ser Met Val Asp 705 710 715 715 720 Gly Leu Lys Pro Gly Gln Arg Lys Val Leu Phe Thr Cys Phe Lys Arg 725 730 735 Asn Asp Lys Arg Glu Val Lys Val Ala Gln Leu Ala Gly Ser Val Ala 740 745 750 Glu Met Ser Ser Tyr His His Gly Glu Met Ser Leu Met Met Thr Ile 755 760 765 Ile Asn Leu Ala Gln Asn Phe Val Gly Ser Asn Asn Leu Asn Leu Leu 770 775 780 Gln Pro Ile Gly Gln Phe Gly Thr Arg Leu His Gly Gly Lys Asp Ser 785 790 795 800 Ala Ser Pro Arg Tyr Ile Phe Thr Met Leu Ser Ser Leu Ala Arg Leu 805 810 815 Leu Phe Pro Pro Lys Asp Asp His Thr Leu Lys Phe Leu Tyr Asp Asp 820 825 830 Asn Gln Arg ValGlu Pro Glu Trp Tyr Ile Pro Ile Ile Pro Met Val 835 840 845 Leu Ile Asn Gly Ala Glu Gly Ile Gly Thr Gly Trp Ser Cys Lys Ile 850 855 860 Pro Asn Phe Asp Val Arg Glu Ile Val Asn Asn Ile Arg Arg Leu Met 865 870 875 880 Asp Gly Glu Glu Glu Pro Leu Pro Met Leu Pro Ser Tyr Lys Asn Phe Lys 885 890 895 Gly Thr Ile Glu Glu Leu Ala Pro Asn Gln Tyr Val Ile Ser Gly Glu 900 905 910 Val Ala Ile Leu Asn Ser Thr Thr Ile Glu Ile Ser Glu Leu Pro Val 915 920 925 925 Arg Thr Trp Thr Gln Thr Tyr Lys Glu Gln Val Leu Glu Pro Met Leu 930 935 940 Asn Gly Thr Glu Lys Thr Pro Pro Leu Ile Thr Asp Tyr Arg Glu Tyr 945 950 955 960 His Thr Asp Thr Thr Val Lys Phe Val Val Lys Met Thr Glu Glu Lys 965 970 975 Leu Ala Glu Ala Glu Arg Val Gly Leu His Lys Val Phe Lys Leu Gln 980 985 990 Thr Ser Leu Thr Cys Asn Ser Met Val Leu Phe Asp His Val Gly Cys 995 1000 1005 Leu Lys Lys Tyr Asp Thr Val Leu Asp Ile Leu Arg Asp Phe Phe Glu 1010 1015 1020 Leu Arg Leu Lys Tyr Tyr Gly Leu Arg Lys Glu Trp Leu Leu Gly Met 1025 1030 1035 1040 Leu Gly Ala Glu Ser Ala Lys Leu Asn Asn Gln Ala Arg Phe Ile Leu 1045 1050 1055 Glu Lys Ile Asp Gly Lys Ile Ile Ile Glu Asn Lys Pro Lys Lys Glu 1060 1065 1070 Leu Ile Lys Val Leu Ile Gln Arg Gly Tyr Asp Ser Asp Pro Val Lys 1075 1080 1085 Ala Trp Lys Glu Ala Gln Gln Lys Val Pro Asp Glu Glu Glu Asn Glu 1090 1095 1100 Glu Ser Asp Asn Glu Lys Glu Thr Glu Lys Ser Asp Ser Val Thr Asp 1105 1110 1115 1120 Ser Gly Pro Thr Phe Asn Tyr Leu Leu Asp Met Pro Leu Trp Tyr Leu 1125 1130 1135 Thr Lys Glu Lys Lys Asp Glu Leu Cys Arg Leu Arg Asn Glu Lys Glu 1140 1145 1150 Gln Glu Leu Asp Thr Leu Lys Arg Lys Ser Pro Ser Asp Leu Trp Lys 1155 1160 1165 Glu Asp Leu Ala Thr Phe Ile Glu Glu Leu Glu Ala Val Glu Ala Lys 1170 1175 1180 Glu Lys Gln Asp Glu Gln Val Gly Leu Pro Gly Lys Gly Gly Lys Ala 1185 1190 1195 1200 Lys Gly Lys Lys Thr Gln Met Ala Glu Val Leu Pro Ser Pro Arg Gly 1205 1210 1215 Gln Arg Val Ile Pro Arg Ile Thr Ile Glu Met Lys Ala Glu Ala Glu 1220 1225 1230 Lys Lys Asn Lys Lys Lys Ile Lys Asn Glu Asn Thr Glu Gly Ser Pro 1235 1240 1245 Gln Glu Asp Gly Val Glu Leu Glu Gly Leu Lys Gln Arg Leu Glu Lys 1250 1255 1260 Lys Gln Lys Arg Glu Pro Gly Thr Lys Thr Lys Lys Gln Thr Thr Leu 1265 1270 1275 1280 Ala Phe Lys Pro Ile Lys Lys Gly Lys Lys Arg Asn Pro Trp Pro Asp 1285 1290 1295 Ser Glu Ser Asp Arg Ser Ser Asp Glu Ser Asn Phe Asp Val Pro Pro 1300 1305 1310 Arg Glu Thr Glu Pro Arg Arg Ala Ala Thr Lys Thr Lys Phe Thr Met 1315 1320 1325 Asp Leu Asp Ser Asp Glu Asp Phe Ser Asp Phe Asp Glu Lys Thr Asp 1330 1335 1340 Asp Glu Asp Phe Val Pro Ser Asp Ala Ser Pro Pro Lys Thr Lys Thr 1345 1350 1355 1360 Ser Pro Lys Leu Ser Asn Lys Glu Leu Lys Pro Gln Lys Ser Val Val 1365 1370 1375 Ser Asp Leu Glu Ala Asp Asp Val Lys Gly Ser Val Pro Leu Ser Ser 1380 1385 1390 Ser Pro Pro Ala Thr His Phe Pro Asp Glu Thr Glu Ile Thr Asn Pro 1395 1400 1405 Val Pro Lys Lys Asn Val Thr Val Lys Lys Thr Ala Ala Lys Ser Gln 1410 1415 1420 Ser Ser Thr Ser Thr Thr Gly Ala Lys Lys Arg Ala Ala Pro Lys Gly 1425 1430 1435 1440 Thr Ly s Arg Asp Pro Ala Leu Asn Ser Gly Val Ser Gln Lys Pro Asp 1445 1450 1455 Pro Ala Lys Thr Lys Asn Arg Arg Lys Arg Lys Pro Ser Thr Ser Asp 1460 1465 1470 Asp Ser Asp Ser Asn Phe Glu Lys Ile Val Ser Lys Ala Val Thr Ser 1475 1480 1485 Lys Lys Ser Lys Gly Glu Ser Asp Asp Phe His Met Asp Phe Asp Ser 1490 1495 1500 Ala Val Ala Pro Arg Ala Lys Tyr Val Arg Ala Lys Lys Pro Ile Lys 1505 1510 1515 1520 Tyr Leu Glu Glu Glu Ser Asp Glu Asp Asp Leu Phe 1525 1530 <210> 3 <211> 6 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1) .. (6) <400> 3 Pro Ala Thr His Phe Pro 1 5 <210> 4 <211> 9 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1) .. (9) <400> 4 Tyr Val Pro Ala Leu Ile Phe Gly Gln 1 5 <210> 5 <211> 9 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1) .. (9) <400> 5 Pro Ala Leu Ile Phe Gly Gln Leu Leu 1 5 <210> 6 <211> 11 <212> PRT <213> Homo sapiens <400> 6 Tyr Val Pro Ala Leu Ile Phe Gly Gln Leu Leu 1 5 10 <210> 7 <211> 18 <212> DNA <213> Homo sapiens <400> 7 cctgctacac atttccca 18 <210> 8 <211> 27 <212> DNA <213> Homo sapiens <400> 8 tatgtcccag ctctcatatt tggacag 27 <210> 9 <211> 27 <212> DNA <213> Homo sapiens <400> 9 ccagctctca tatttggaca gctccta 27 <210> 10 <211> 33 <212> DNA <213> Homo sapiens <400> 10 tatgtcccag ctctcatatt tggacagctc cta 33 <210> 11 <211> 1047 <212> DNA <213> Pseudomonas aeruginosa <400> 11 gccgccgaca ccatcaagat cgccctggct ggcccggtca ccggtccggt agcccagtac 60 ggcgacatgc agcgcgccgg tgcgctgatg gcaatcgaac agatcaacaa ggcaggcggc 120 gtgaacggcg cgcaactcga aggcgtgatc tacgacgacg cctgcgatcc caagcaggcc 180 gtggcggtcg ccaacaaggt ggtcaacgac ggcgtcaagt tcgtggtcgg tcatgtctgc 240 tccagctcca cccaacccgc caccgacatc tacgaagacg aaggcgtgct gatgatcacc 300 ccgtcggcca ccgccccgga aatcacctcg cgcggctaca agctgatctt ccgcaccatc 360 ggcctggaca acatgcaggg cccggtggcc ggcaagttca tcgccgaacg ctacaaggtc 420 aagaccatcg cggtactgca cgacaagcag cagtacggcg aaggcatcgc caccgaggtg 480 aagaagaccg tggaagacgc cggcatcaag gttgccgtct tcgaaggcct gaacgccggc 540 gacaaggact tcaacgcgct gatcagcaag ctgaagaaag ccggcgtgca gttcgtctac 600 ttcggcggct accacccaga aatgggcctg ctgctgcgcc aggccaagca ggccgggctg 660 gacgcgcgct tcatgggccc ggaaggggtc ggcaacagcg aaatcaccgc gatcgccggc 720 gacgcttcgg aaggcatgct ggcgaccctg ccgcgcgcct tcgagcagga tccgaagaac 780 aaggccctga tcgacgcctt caaggcgaag aaccaggatc cgagcggcat cttcgtcctg 840 cccgcctact c cgcggtcac agtgatcgcc aagggcatcg agaaagccgg cgaggccgat 900 ccggagaagg tcgccgaggc cctgcgcgcc aacaccttcg agactcccac cgggaacctc 960 gggttcgacg agaagggcga cctgcag ggccac gccag ccccac gccag ccagca gccac gacca gccac gacca gccac gacca gccac gacca gccac gaccac <210> 12 <211> 185 <212> DNA <213> Homo sapiens <400> 12 gtaccactgt cttcaagccc tcctgctaca catttcccag atgaaactga aattacaaac 60 ccagttccta aaaagaatgt gacagtgaag aagacagcag caaaaagtca gtcttccacc 120 tccactaccg gtgccaaaaa aagggctgcc ccaaaaggacagtt 180g <210> 13 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 13 ggaagatctt accactgtct tcaagcc 27 <210> 14 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 14 gcctcgagat tcaaagctgg atcc 24 <210> 15 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 15 gcctcgagac cggtagtgga ggtg 24 <210> 16 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 16 ggaagatctt atcttccacc tccacta 27 <210> 17 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 17 gcctcgagtg tcttcttcac agtc 24 <210> 18 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 18 ggaagatctt aaaaaagaat gtgacagtga 30 <210> 19 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 19 gcctcgagtt cagtttcatc tggg 24 <210> 20 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 20 gatcacacat ttcccagatg aaactgaaat tacaaaccca gt 42 <210> 21 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 21 tcgagaggaa ctgggtttgt aatttcagtt tcatctggga aa 42 <210> 22 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 22 gatcccactg tcttcaagcc ctcctgctc 29 <210> 23 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 23 tcgagagcag gagggcttga agacagtgg 29 <210> 24 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 24 gatccctcct gctacacatt tcccagatc 29 <210> 25 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 25 tcgagatctg ggaaatgtgt agcaggagg 29 <210> 26 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 26 gatcacacat ttcccagatg aaactgaac 29 <210> 27 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 27 tcgagttcag tttcatctgg gaaatgtgt 29 <210> 28 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 28 gatccctcct gctacacatt tcc 23 <210> 29 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesuzed <400> 29 tcgaggaaat gtgtagcagg agg 23 <210> 30 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 30 gatccctgct acacatttcc cac 23 <210> 31 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 31 tcgagtggga aatgtgtagc agg 23 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 32 gatccctcct gctacacatc 20 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 33 tcgagatgtg tagcaggagg 20 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 34 gatccctgct acacatttcc 20 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: sunthesized <400> 35 tcgaggaaat gtgtagcagg 20 <210> 36 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 36 gatcgctaca catttcccac 20 <210> 37 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 37 tcgagtggga aatgtgtagc 20 <210> 38 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 38 Asn Asn Leu Ile Ser Ile Trp Asn Asn Gly Lys Gly Ile Pro Val 1 5 10 15 <210> 39 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 39 Val Glu His Lys Val Glu Lys Met Tyr Val Pro Ala Leu Ile Phe Gly 1 5 10 15 Gln Leu Leu <210> 40 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 40 Thr Ser Ser Asn Tyr Asp Asp Asp Glu Lys Lys Val Thr Gly Gly Arg 1 5 10 15 Asn Gly Tyr Gly Ala Lys Leu Cys Asn Ile Phe Ser Thr Lys Phe 20 25 30 <210> 41 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 41 gatctatgtc ccagctctca tatttggaca gctcc 35 <210> 42 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 42 tcgaggagct gtccaaatat gagagctggg acata 35 <210> 43 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 43 gatcccagct ctcatatttg gacagctcc 29 <210> 44 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 44 tcgaggagct gtccaaatat gagagctgg 29 <210> 45 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 45 gatctatgtc ccagctctca tatttggaca gg 32 <210> 46 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 46 tcgacctgtc caaatatgag agctgggaca ta 32 <210> 47 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 47 gatctatgtc ccagctctca tatttg 26 <210> 48 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 48 tcgacaaata tgagagctgg gacata 26 <210> 49 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 49 gatctatgtc ccagctctca tag 23 <210> 50 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 50 tcgactatga gagctgggac ata 23 <210> 51 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 51 gatctatgtc ccagctctcg 20 <210> 52 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 52 tcgacgagag ctgggacata 20 <210> 53 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 53 gatcgtccca gctctcatac 20 <210> 54 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 54 tcgagtatga gagctgggac 20 <210> 55 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 55 gatcccagct ctcatatttc 20 <210> 56 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 56 tcgagaaata tgagagctgg 20 <210> 57 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 57 gatcgctctc atatttggac 20 <210> 58 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 58 tcgagtccaa atatgagagc 20 <210> 59 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 59 gatcgtccca gctctcatat ttggacagg 29 <210> 60 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 60 tcgacctgtc caaatatgag agctgggac 29 <210> 61 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 61 gatcccagct ctcatatttg gacagt 26 <210> 62 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 62 tcgaactgtc caaatatgag agctgg 26 <210> 63 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 63 gatcgctctc atatttggac agg 23 <210> 64 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthesized <400> 64 tcgacctgtc caaatatgag agc 23

[Brief description of the drawings]

FIG. 1 is a diagram showing a configuration of a vector for expressing a fusion protein of various peptides and a BraC protein for analyzing an epitope peptide of the present invention.

FIG. 2 is a diagram showing a restriction map of a human type II DNA topoisomerase α gene and a DNA fragment cut by the restriction enzyme.

FIG. 3 is a diagram showing an amino acid sequence near an epitope peptide of an 8D2 antibody and the antigenicity of each peptide to the 8D2 antibody. N-Terminal, C-Ter
“minal” indicates whether the epitope peptide is bound to the N-terminal or C-terminal of the BraC protein, respectively.

FIG. 4 is an electrophoretic photograph showing the degree of purification of BraC protein using an 8D2 antibody column.

FIG. 5 is a view showing a configuration of a fusion protein expression vector of the present invention.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12M 1/40 C12P 21/02 C C12N 15/02 G01N 33/53 D C12P 21/02 33/577 B G01N 33/53 C07K 16/12 33/577 C12N 15/00 ZNAA // C07K 16/12 CF term (reference) 4B024 AA13 BA07 BA31 BA44 BA50 BA80 CA04 DA06 EA04 GA03 GA11 GA18 GA19 HA15 4B029 AA21 BB17 CC03 4B064 AG27 AG31 CA02 CA10 CA19 CA20 CC24 CE02 CE12 DA15 4H045 AA11 AA20 AA30 BA41 BA42 CA11 CA40 DA76 DA86 EA50 EA52 FA72 FA74 GA01 GA26

Claims (18)

[Claims] 1. A fusion protein of a target protein and a polypeptide having an amino acid sequence containing an epitope of an anti-human type II DNA topoisomerase α antibody. 2. The fusion protein according to claim 1, wherein the amino acid sequence of the epitope is the amino acid sequence shown in SEQ ID NO: 3, 4, 5, or 6. 3. A DNA encoding the fusion protein according to claim 1 or 2. A vector for expressing a fusion protein, comprising the DNA according to claim 3. 5. A vector for use in expressing a fusion protein according to claim 1 or 2, comprising a DNA encoding at least one of the amino acid sequences of SEQ ID NOs: 3 to 6. 6. Detection or quantification of a target protein using a fusion protein of a target protein and a polypeptide having an amino acid sequence containing an epitope of an anti-human type II DNA topoisomerase α antibody, and the reactivity with the antibody as an index. A method for detecting and quantifying a target protein, characterized in that: 7. The method according to claim 6, wherein the amino acid sequence of the epitope is any of the amino acid sequences shown in SEQ ID NOs: 3, 4, 5, and 6. 8. The method according to claim 8, wherein the antibody is hybridoma 8D2, 7B.
7. The method of claim 6, which is a monoclonal antibody produced by any of 9, 7D5, or 6H8. 9. The amino acid sequence containing an epitope is a sequence containing the amino acid sequence shown in SEQ ID NO: 3, and an antibody that specifically recognizes the amino acid sequence is a hybridoma 8D2
The method according to claim 6, which is a monoclonal antibody produced by: 10. The amino acid sequence containing an epitope is a sequence containing the amino acid sequence shown in SEQ ID NO: 4, and an antibody that specifically recognizes the amino acid sequence is hybridoma 7B
9. A monoclonal antibody produced by the method of claim 9.
The method described in. 11. The amino acid sequence containing an epitope is a sequence containing the amino acid sequence shown in SEQ ID NO: 4 or 5, and the antibody that specifically recognizes the amino acid sequence is a monoclonal antibody produced by hybridoma 7D5. 7. The method according to 6. 12. The amino acid sequence containing an epitope is a sequence containing the amino acid sequence shown in SEQ ID NO: 6, and an antibody that specifically recognizes the amino acid sequence is hybridoma 6H.
8. A monoclonal antibody produced by the method of claim 6.
The method described in. 13. A target comprising contacting a fusion protein of an anti-human type II DNA topoisomerase α antibody carried on a solid carrier with a target protein and a polypeptide having an amino acid sequence containing an epitope of the antibody. A method for purifying a protein. 14. The method according to claim 14, wherein the antibody is hybridoma 8D2, 7B.
14. The method of claim 13, which is a monoclonal antibody produced by any of 9, 7D5, or 6H8. 15. The method according to claim 13, wherein the fusion protein is the protein according to claim 1 or 2. 16. A solid carrier carrying an antibody that specifically recognizes any one of the amino acid sequences shown in SEQ ID NOs: 3 to 6. 17. An epitope peptide having any one of the amino acid sequences shown in SEQ ID NOs: 3 to 6. 18. A kit for use in detecting or quantifying a protein, comprising an antibody that specifically recognizes at least any one of the amino acid sequences shown in SEQ ID NOs: 3 to 6.