JP2000050885A - Antibody against cathepsin and its utilization - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new antibody for elucidation of onset mechanism, treatment or the like for Alzheimer disease or the like which is specific to the cathepsin Z protein arising from human being or the protein which is homologous to this protein and shows cysteine protease activity or antigenicity. SOLUTION: This antibody is a new antibody against cathepsin Z protein arising from human being, cathepsin protein (salt) arising from human being which has at least 40% homology to the amino acid sequence in this protein and shows cysteine protease activity or equal antigenicity, proteins containing the amino acid sequence of 62-303 positions, the amino acid sequence of 21-303 positions, the amino acid sequence of 1-303 positions among the amino acid sequence represented by the formula, protein (salt) which contains the amino acid sequence substantially identical with one of them and shows biological activity or these partial peptides (salts) and is useful in the elucidation of onset mechanism or the development of the treatment and the therapeutic drug for diseases, e.g. Alzheimer disease, pulmonary emphysema, rheumatic arthritis, cancer and the like.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a polynucleotide (or nucleic acid) and a polypeptide (or protein) (or a part thereof) or a salt thereof which have been newly identified; Mutants and derivatives of the polynucleotide (or nucleic acid) and polypeptide (or protein), and methods for producing the mutants and derivatives thereof; agonists and antagonists of the polypeptide (or protein); (Or proteins), in particular monoclonal antibodies; and the use of said polynucleotides (or nucleic acids), polypeptides (or proteins), variants, derivatives, agonists and antagonists. The present invention relates to many normal cellular processes such as intracellular protein metabolism, activation of hormone precursors, and bone modification (remodeling), including Alzheimer's disease, emphysema, rheumatoid arthritis, muscular dystrophy,
Novel proteins, cysteine protease enzymes belonging to the papain family, which are thought to play an important role in many diseases such as osteoporosis, neurodegenerative diseases and cancer invasion and metastasis, and which are useful in such studies, especially The present invention relates to a human-derived cathepsin protein (or a part thereof) or a salt thereof and a gene encoding the same. More specifically, the present invention relates to a novel human cysteine protease having a papain-like structure in an active site cloned from a human cDNA library (the novel human cysteine protease disclosed in the present invention is referred to as cathepsin Z). No.), a DNA containing the nucleotide sequence encoding the same, a host cell transformed with the DNA, a method for producing the human cysteine protease using the host cell, a monoclonal antibody binding to the cysteine protease protein, The present invention relates to uses of these proteins and antibodies and nucleic acid fragments.

[0002]

BACKGROUND OF THE INVENTION Cysteine proteases form a family of enzymes involved in many normal cellular processes, such as intracellular protein metabolism, activation of hormone precursors, and bone modification (Berti, PJ, et a
l., J. Mol. Biol., 246: 273-283, 1995). These proteolytic enzymes have been suggested to play important roles in many diseases such as Alzheimer's disease, emphysema, rheumatoid arthritis, muscular dystrophy, osteoporosis and cancer invasion and metastasis (Berti, PJ, et al. ., J. Mol.
Biol., 246: 273-283, 1995; Berquin, IM, et al.,
Perspect. DrugDiscov. Des.,. 2: 371-388, 1994).
At present, nine kinds of cathepsins, namely cathepsin B (Chan, SJ, et
l., Proc. Natl. Acad. Sci. USA, 83: 7721-7725,
1986), cathepsin L (Gal, S., et al., Biochem.
J., 253: 303-306, 1988), cathepsin H (Ritonja,
A., et al., FEBS Lett., 228: 341-345, 1988), cathepsin S (Shi, GP, et al., J. Biol. Chem., 267:
7258-7262, 1992), cathepsin C (Paris, A., et al.,
FEBS Lett., 369: 326-330, 1995), cathepsin O (Ve
lasco, G., et al., J. Biol. Chem., 269: 27136-2714
2, 1994), cathepsin K (Inaoka, T., et al., Bioche
m. Biophys. Res. Commun., 206: 89-96, 1995), Cathepsin W (Linnevers, C., et al., FEBS Lett., 405:
253-259, 1997) and cathepsin L2 (Santamaria, I,
et al., Cancer Res., 58; 1624-1630, 1998) have been isolated and analyzed to the amino acid sequence level. All of these contain a cysteine residue in the active site, but differences have been observed in the enzymatic characteristics such as the expressed tissue, substrate specificity, and pH stability.

Further, the presence of cathepsins M, N, P and T as cysteine proteases has been reported.
These were originally separated by differences in specificity for substrates such as aldolase, collagen, insulin precursor, or tyrosine aminotransferase, but analysis at the level of these molecules has not yet been reported. No (Pontremoli, S., et al., Arch. Bio
chem. Biophys., 214: 376-385, 1982; Maciewicz, R.,
et al., Biochem. J., 25: 433-440, 1988; Docherty,
K., et al., Proc. Natl. Acad. Sci. USA, 79: 461.
3-4617, 1982; Gohda, E., et al., J. Biol. Chem., 25
6: 2567-2572, 1981). From the structural comparison between cysteine proteases, it is known that cysteine protease is synthesized as a preproenzyme, cleaved into proenzymes, and further incorporated into lysosomes by addition of mannose 6-phosphate. Furthermore, these enzymes are composed of a single polypeptide or SS-linked heavy and light chains.
It becomes a main chain and matures (Berti, PJ, et al., J. Mol.
Biol. 246; 273-283, 1995). However, in some cases, these precursor enzymes move out of this cleavage pathway and into the secretory system, where they are ultimately released into extracellular space via storage granules (Sloane, BF, et al., Science,
212: 1151-1153, 1981). Several lysosomal cysteine proteases are released from cancer cells of different origin and are thought to play an important role in the progression of cancer malignancy (Sloane, BF, et al., Science 212: 1151-1153, 1
981; Recklies, AD, et al., Biochem. J. 207: 633-
636, 1982; Gal, S., et al., J. Biol. Chem. 261: 17
60-1765, 1986).

[0004] On the other hand, lysosomal cysteine protease is activated macrophages in patients with I-cell disease,
It is also secreted by osteoblast or fibroblast (Chapman, HA, et al., Annu Rev. Ph.
ysiol. 59: 63-88 1997; Kornfeld, S., et al., Annu.
Rev. Cell Biol. 5: 483-525, 1989) The fact predicted a wide range of physiological and pathological distributions of these enzymes. Because cysteine proteases play an important role in diseases including normal and cancerous, the search for unknown proteolytic enzymes belonging to this family and produced by human tissues has been advanced. In the process, a new human cysteine protease, cathepsin O, derived from breast cancer and widely distributed in human tissues (Velasco,
G., et al., J. Biol. Chem., 269: 27136-27142, 1994)
Was isolated. Human bleomycin hydrolase, a cysteine protease present in the cytoplasm, has also been cloned (Ferrando, AA, et al., Cance.
r Res., 56: 1746-1750, 1996). Furthermore, cathepsin L2 which is structurally similar to cathepsin L but has a unique expression tissue distribution (Santamaria, I, et al., Cancer Res.,
58: 1624-1630, 1998).

[0005]

Cysteine proteases are involved in many normal cellular processes, including intracellular protein metabolism, activation of hormone precursors, and bone modification, while Alzheimer's disease, emphysema, rheumatism, It has been suggested that it plays an important role in many diseases such as osteoarthritis, muscular dystrophy, osteoporosis, neurodegenerative diseases and cancer invasion / metastasis. Identifying and isolating novel cysteine proteases and elucidating the function of cysteine proteases associated with various diseases, particularly cancer, is extremely important for elucidation of the pathogenesis of these diseases, treatment and development of therapeutic drugs. is there.

[0006]

Means for Solving the Problems The present inventors consider that there is a novel member of the cysteine protease family that has not yet been reported, and have conducted various studies using genetic engineering techniques. Succeeded in cloning the human gene, clarifying all of the gene base sequence and amino acid sequence,
The present invention has been completed. When the amino acid sequence of the predicted novel cysteine protease was compared with that of the previously reported cathepsin, the amino acid sequence was less than 40% homologous to the previously reported cathepsin. It had all the structural features. Based on this amino acid sequence feature, the new member was named "cathepsin Z". Accordingly, the present invention provides a novel protein (or polypeptide) that is a human-derived factor having cathepsin Z activity, a method for producing the same and its use, a gene encoding the protein, a use thereof, and the like. The present invention further provides an antibody that specifically reacts with cathepsin Z, a method for immunologically measuring cathepsin Z using the antibody, and a reagent for measuring the same. Further, the present invention provides a hybridization probe specific to the cathepsin Z gene.

The present invention relates to a novel human cathepsin Z gene, a human-derived protein cathepsin Z, and analogs thereof. The invention further relates to DNA sequences encoding all or part of the novel human cathepsin Z, vectors having such DNA sequences, and host cells transformed or transfected with such vectors. Further, the present invention also encompasses a method for producing recombinant human cathepsin Z and its use. The present invention also relates to an antibody that binds to human cathepsin Z, particularly a monoclonal antibody and a hybridoma cell that produces the antibody. A nucleic acid that hybridizes to the human cathepsin Z gene;
For example, DNA and RNA probes. Further, the present invention provides a reagent for detecting and measuring human cathepsin Z using the above product, and a method for detecting and measuring using the reagent.

Further, the present invention provides a human cathepsin Z agonist. Particularly preferred agonists are
Cathepsin Z that binds to a cathepsin Z binding molecule or a cathepsin Z receptor molecule and / or causes or enhances a cathepsin Z-induced reaction
It is a pseudo molecule. A preferred agonist is a molecule that interacts with cathepsin Z or a cathepsin Z polypeptide, or another cathepsin Z activity regulator, and enables or enhances the action or effect of cathepsin Z or more. is there. The present invention provides a human cathepsin Z antagonist. Particularly preferred antagonists are cathepsin Z mimetic molecules that bind to a cathepsin Z binding molecule or a cathepsin Z receptor molecule but do not cause or inhibit one or more cathepsin Z-induced reactions. Also preferred antagonists are cathepsin Z or cathepsin Z polypeptide, or other cathepsin Z
It is a molecule that interacts with an activity controlling substance and inhibits or suppresses one or more actions and effects of cathepsin Z. Furthermore, the present invention provides a method for detecting a substance that inhibits cysteine protease activity and a substance that inhibits proteolytic activity of human cathepsin Z, and provides a drug containing the substance or derivative as an active ingredient, for example, a cysteine protease activity or proteolytic activity. It also relates to neutralizing antibodies that inhibit, especially monoclonal antibodies that bind to human cathepsin Z. The present invention also relates to a partial peptide of human cathepsin Z that binds to an antibody against human cathepsin Z and has no human cathepsin Z activity, and a derivative thereof or a salt thereof. The present invention also relates to an antisense nucleotide that blocks expression of human cathepsin Z and a derivative thereof, and a drug containing the same as an active ingredient.

[0009] The present invention provides: [1] an antibody against: (i) a human-derived cathepsin Z protein or at least 40% of the amino acid sequence of the cathepsin Z protein;
A human-derived cathepsin protein or a salt thereof, which has the above homology and has cysteine protease activity or equivalent antigenicity, (ii)
Among the amino acid sequences represented by SEQ ID NO: 1 in the sequence listing, those having the amino acid sequence of positions 62 to 303, those having the amino acid sequences of positions 21 to 303, and those of the first
The amino acid sequence having the amino acid sequence of positions from position 303 to position 303, the amino acid sequence having an amino acid sequence substantially equivalent to any one of them, and the amino acid sequence having a biological activity substantially equivalent to any one of them. A protein or a salt thereof, which is a protein selected from the group consisting of: or (iii) a partial peptide of the protein according to the above (i) or (ii) or a salt thereof; The antibody of the above-mentioned [1], which is a monoclonal antibody; [3] (1) the propeptide region of human cathepsin Z (for example, the region of positions 21 to 61 in SEQ ID NO: 1 in the sequence listing) Area, or part of, or near)
(2) A region containing a propeptide cleavage site of human cathepsin Z (for example, positions 58 to 66 of SEQ ID NO: 1 in the sequence listing)
Region or a part thereof or its vicinity), (3) a region of activated human cathepsin Z of human cathepsin Z (for example, a region of positions 70 to 89 of SEQ ID NO: 1 in the sequence listing, 95th to 126th, 136th to 154th, 165th to 178th, 179th to 246th, or 273rd to No.
(1) The antibody according to (1) or (2) above, which is an antibody against a portion selected from the group consisting of the region at position 296 or a portion thereof or its vicinity; The antibody according to any one of the above [1] to [3], which is:

[5] the antibody of any one of the above [1] to [3], which is a labeled antibody; [6] the antibody of any one of the above [1] to [5] (I) using a human-derived cathepsin Z protein or an amino acid sequence of the cathepsin Z protein of at least 40%
A cathepsin protein of human origin or a salt thereof having the above homology and cysteine protease activity or equivalent antigenicity, or (ii) represented by SEQ ID NO: 1 in the sequence listing Among the amino acid sequences, those having the amino acid sequences of positions 62 to 303, those having the amino acid sequences of positions 21 to 303,
Having the amino acid sequence of positions 1 to 303, having substantially the same amino acid sequence as any one of them, and having substantially the same biological activity as any one of them A protein or a salt thereof, which is a protein selected from the group consisting of those having an amino acid sequence; [7] at least two of (a) a solid-phased antibody and (b) a labeled antibody; The method of the above-mentioned [6], wherein a seed is used;

[8] An immunoassay reagent for the following protein or a salt thereof, which comprises the antibody according to any one of [1] to [5]: (i) cathepsin Z derived from human At least 40% of the amino acid sequence of the protein or the cathepsin Z protein
A cathepsin protein of human origin or a salt thereof having the above homology and cysteine protease activity or equivalent antigenicity, or (ii) represented by SEQ ID NO: 1 in the sequence listing Among the amino acid sequences, those having the amino acid sequences of positions 62 to 303, those having the amino acid sequences of positions 21 to 303,
Having the amino acid sequence of positions 1 to 303, having substantially the same amino acid sequence as any one of them, and having substantially the same biological activity as any one of them A protein or a salt thereof, which is a protein selected from the group consisting of those having an amino acid sequence;

[9] a medicament comprising the antibody of any one of the above [1] to [3]; and [10] the antibody of any one of the above [1] to [5]. An antibody that promotes or inhibits the biological activity of: (i) a human-derived cathepsin Z protein or at least 40% amino acid sequence of said cathepsin Z protein
A human-derived cathepsin protein or a salt thereof, which has the above homology and has cysteine protease activity or equivalent antigenicity, (ii)
Among the amino acid sequences represented by SEQ ID NO: 1 in the sequence listing, those having the amino acid sequence of positions 62 to 303, those having the amino acid sequences of positions 21 to 303, and those of the first
The amino acid sequence having the amino acid sequence of positions from position 303 to position 303, the amino acid sequence having an amino acid sequence substantially equivalent to any one of them, and the amino acid sequence having a biological activity substantially equivalent to any one of them. Or a salt thereof, or (iii) a partial peptide of the protein according to the above (i) or (ii) or a salt thereof; and [11] the above [ [1] A method and a screening kit for screening a compound that promotes or inhibits the biological activity of the following, characterized by containing the antibody according to any one of [1] to [5]: (i) Human-derived At least 40% of the cathepsin Z protein or the amino acid sequence of the cathepsin Z protein
A human-derived cathepsin protein or a salt thereof, which has the above homology and has cysteine protease activity or equivalent antigenicity, (ii)
Among the amino acid sequences represented by SEQ ID NO: 1 in the sequence listing, those having the amino acid sequence of positions 62 to 303, those having the amino acid sequences of positions 21 to 303, and those of the first
The amino acid sequence having the amino acid sequence of positions from position 303 to position 303, the amino acid sequence having an amino acid sequence substantially equivalent to any one of them, and the amino acid sequence having a biological activity substantially equivalent to any one of them. (Iii) a partial peptide of the protein described in (i) or (ii) above, or a salt thereof.

[0012] The present invention further relates to: [12] a human-derived cathepsin Z protein or an amino acid sequence of the cathepsin Z protein of at least 40
% Of a novel cathepsin protein or a salt thereof derived from human, which has a homology of not less than 30% and a cysteine protease activity or equivalent antigenicity; [13] SEQ ID NO: 1 Among the amino acid sequences represented, those having the amino acid sequence of positions 62 to 303, those having the amino acid sequence of positions 21 to 303,
Having the amino acid sequence of positions 1 to 303, having substantially the same amino acid sequence as any one of them, and having substantially the same biological activity as any one of them A protein or a salt thereof, which is a protein selected from the group consisting of those having an amino acid sequence; [14] a partial peptide of the protein according to the above [12] or [13] or a salt thereof; [15] the above [12] or a nucleic acid having a nucleotide sequence encoding the protein of [13]; [16] an open reading frame portion or a substantial portion thereof in the nucleotide sequence represented by SEQ ID NO: 2 in the sequence listing A nucleic acid according to the above [15], characterized by having a substantially equivalent nucleotide sequence; [17] a vector comprising the nucleic acid according to the above [15] or [16] -[18] a transformant comprising the nucleic acid of [15] or [16] or the vector of [17]; [19] a host cell comprising Escherichia coli, yeast, CHO cells and COS
A transformant according to the above [18], which is selected from the group consisting of cells; [20] a transformant according to the above [19]. A protein according to the above [12] or [13]; [21] a human-derived cathepsin Z gene comprising all or a part of the base sequence represented by SEQ ID NO: 2 in the sequence listing; [22] The hybridization probe according to the above [21], which comprises an untranslated region of the base sequence represented by SEQ ID NO: 2 in the sequence listing; [23] The above [12] to [12] 14] The protein according to any one of the above, a partial peptide thereof, or a salt thereof; or a medicament characterized by containing the nucleic acid according to the above [15] or [16]; 〕~〔14〕 [25] The above [12], wherein the drug comprises a compound or a salt thereof that promotes or inhibits the biological activity of the protein, a partial peptide thereof, or a salt thereof according to any one of the above. A method and a kit for screening a compound that promotes or inhibits the biological activity of the protein according to any one of [14] to [14], a partial peptide thereof, or a salt thereof.

Further, according to another embodiment, the present invention relates to [26] a natural human cathepsin Z or a salt thereof, which has substantially the same cysteine protease activity or antigen activity, or [27] the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, wherein the protein or its salt or a part thereof according to the above [12], which has substantially the same primary structure conformation; Among them, those having the amino acid sequence of positions 62 to 303, those having the amino acid sequence of positions 21 to 303,
And a homology higher than 50%, preferably 80% or higher, more preferably 90% or higher, to an amino acid sequence selected from the group consisting of A protein or a salt thereof or a part thereof according to the above [12], which has homology; [28] in the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, positions 62 to 62; Those having the amino acid sequence of position 303, those having the amino acid sequence of positions 21 to 303,
And a human cathepsin Z protein having an amino acid sequence selected from the group consisting of those having amino acid positions 1 to 303, or one or more amino acid residues unique to the protein (for example, 1 to 100 , Preferably 1 to 60, more preferably 1 to 40, more preferably 1 to 20, especially 1 to 10, etc.) deleted deletion analogs, amino acid residues unique to the human cathepsin Z One or more of the groups (e.g., 1 to 100, preferably 1 to 60, more preferably 1 to 40, more preferably 1 to 20, especially 1 to 10) are other residues Substituted analogs, and one or more (eg, 1 to 100, preferably 1
To 60, more preferably 1 to 40, still more preferably 1 to 20, and especially 1 to 10 amino acid residues. (29) a protein having the nucleotide sequence encoding the protein according to any one of (26) to (28); [30] a vector comprising the nucleic acid of the above-mentioned [29];

[31] a transformant comprising the nucleic acid of [29] or the vector of [30]; [32] a host cell comprising Escherichia coli, yeast, CHO cells and COS
A transformant according to the above [31], which is selected from the group consisting of cells; [33] a transformant according to the above [31] or [32], which is obtained by expression. [34] The protein according to any one of [26] to [28], wherein the protein is labeled with an enzyme, [1] to [3], and [5]. [35] the antibody of any one of the above [1] to [3], [5] and [34], wherein Fab 'of a monoclonal antibody is labeled; [36] a base comprising at least a part of the base sequence represented by SEQ ID NO: 2 in the sequence listing and capable of hybridizing to the human cathepsin Z gene under stringent conditions; Labeled, characterized in that it contains a sequence A hybridization probe; [37] the base sequence represented by SEQ ID NO: 2 in the sequence listing, which contains a fragment specific to at least the 3′-side untranslated region. [38] The probe described in [38] In the nucleotide sequence represented by SEQ ID NO: 2 in the sequence listing, 270 bp FokI / Eco specific to the 3′-side untranslated region portion
The above-mentioned (36), which contains an RI digested fragment.
Or a probe according to [37]; [39] a method of inhibiting a biological activity such as cysteine protease activity or antigenicity of the protein according to [12] or [13]; and [40] an inhibitor comprising a peptide, A protein, a non-peptidic compound, a synthetic compound, a fermentation product, a cell extract, a plant extract, an animal tissue extract and an antibody, for example, the one selected from the group consisting of monoclonal antibodies; 39] The method described above.

Further, according to another embodiment, the present invention is characterized in that [41] a monoclonal antibody which specifically immunoreacts with the protein of the above [12] or [13] or a salt thereof is used as a measuring reagent. [42] a method for immunologically measuring the protein of the above [12] or [13] or a salt thereof; [42] comprising a monoclonal antibody specifically immunoreactive with the protein of the above [12] or [13] or a salt thereof. The reagent according to any one of the above-mentioned [12] to [14] and [26] to [28], The method according to the above [6] or [7], wherein the salt is quantitatively measured; [44] the measurement sample is blood, serum, plasma, synovial fluid, urine,
The above-mentioned [6], [7] or [43], which is selected from the group consisting of saliva, cerebrospinal fluid, amniotic fluid, tissue extract, cultured cell extract and cell culture supernatant [45] A method used in the method according to any one of the above [6], [7], [43] and [44], wherein (i) the antibody according to the above [1], (Ii) a reagent set for measuring human cathepsin Z having at least two reagents: a reagent containing a phased reagent and (ii) a reagent containing the antibody of the above-mentioned [1] and a labeled reagent; 46] (a) producing a monoclonal antibody against a protein or a salt thereof according to the above (12) or (13) and (b) a partial peptide or a salt thereof selected from the group consisting of: (12) or the protein according to (13) or a salt thereof and (b) a partial peptide thereof Or a hybridoma obtained by cell fusion of spleen cells of an animal such as a mouse immunized with a member selected from the group consisting of salts thereof and cells capable of continuous culture such as myeloma cells of an animal such as a mouse; And (47) (a) spleen cells of an animal such as a mouse immunized with a protein or a salt thereof according to the above (12) or (13) and (b) a peptide selected from the group consisting of a partial peptide or a salt thereof. Cell fusion was performed with cells that can be cultured continuously such as myeloma cells of animals such as mice, and (a)
2) or the protein of (13) or a salt thereof and (b)
The present invention provides the method for producing a hybridoma according to the above [46], which comprises selecting cells capable of continuous culture that produce a monoclonal antibody against a member selected from the group consisting of the partial peptide or a salt thereof.

According to another embodiment, the present invention relates to the following: [48] (i) the protein according to any one of the above [12] to [14] and [26] to [28]; (12) to (14) and (14) characterized in that a peptide and salts thereof and (ii) those selected from the group consisting of the antibodies according to any one of the above (1) to (5) are used. 26] A method of screening for a compound that promotes or inhibits the biological activity of the protein according to any one of [28], a part of the peptide or a salt thereof; [49] The above [12] to [14] and A method for screening a compound that promotes or inhibits the biological activity of the protein according to any one of [26] to [28], a partial peptide thereof, or a salt thereof, comprising: 〔14〕
And (26) to (28) when a substrate is brought into contact with a protein, a partial peptide thereof or a salt thereof, and (ii) the above (12) to (14) and (26) to [28] a screening method comprising comparing a protein, a partial peptide thereof, or a salt thereof with a substrate and a test compound; and [50] the above [49]. (I) and / or (ii) are carried out in the presence of the antibody according to any one of the above [1] to [5], wherein the screening method of the above [49]; (I) the protein according to any one of the above [12] to [14] and [26] to [28], a partial peptide thereof and a salt thereof, and (ii) the protein according to the above [1] to [5]. The above (12) to (14), characterized in that it contains one selected from the group consisting of the antibodies according to any one of the above. And a screening kit for a compound that promotes or inhibits the biological activity of the protein according to any one of [26] to [28], a partial peptide thereof or a salt thereof; [52] The above [1] to [5] [53] (i) the above [11], [25], [48], [49] or [50], which comprises the antibody of any one of [1] to [3]. The screening method described above or (ii)
(1), (25), (51), or (52), which is obtained by using the screening kit according to any one of (12) to (14) and (26) to (28). A compound or a salt thereof which promotes or inhibits the biological activity of the described protein, a part of the peptide or a salt thereof;
And (54) (i) the screening method described in (11), (25), (48), (49) or (50), or (ii) the (1)
1), (25), (51) or the protein according to any one of (12) to (14) and (26) to (28), obtained using the screening kit according to (52). A medicament characterized by containing a compound or a salt thereof that promotes or inhibits the biological activity of some peptides or salts thereof is provided.

Further, according to another aspect, the present invention provides a method for screening according to the above [11], [25], [48], [49] or [50] or (ii) The above [1
1], (25), (51), or characterized by being obtained using the screening kit according to (52), the protein according to any one of (12) to (14), (56) (i) the screening method according to (11), (25), (48), (49) or (50) or a compound or a salt thereof that promotes the biological activity of the peptide or a salt thereof; ii) The above [1
1], (25), (51), or characterized by being obtained using the screening kit according to (52), the protein according to any one of (12) to (14), A compound or a salt thereof that inhibits the biological activity of the peptide or a salt thereof; [57] the biological activity according to the above [11], [25], [48], [49] or [50], wherein the protease activity is The screening method according to the above [11], [25], [48], [49] or [50], which is characterized by being; [58] The above [11], [25], [51], or [ 52] The screening kit according to the above [11], [25], [51], or [52], wherein the biological activity according to the above is a protease activity; [59] The organism according to the above [53]. The compound of the above-mentioned (53), wherein the biological activity is a protease activity, or a salt thereof. [60] above [12] a protein of any one according to [14], above, characterized in that those that inhibit a part of the peptide or the biological activity of their salts

[9]
[61] The pharmaceutical according to [24] or [60], wherein the biological activity according to [24] or [60] is a protease activity; -The antibody according to any one of [5] to the test sample and the labeled protein according to any one of [12] to [14], a partial peptide thereof or a salt thereof. A test, characterized in that the ratio of the labeled protein according to any one of the above (12) to (14) bound to the antibody, a partial peptide thereof or a salt thereof is measured. The above [12]-in the sample
[14] a method for quantifying the protein according to any one of [14], a partial peptide thereof or a salt thereof; and [63] a test sample and the above-mentioned [1]-
The antibody according to any one of [3] and the labeled antibody according to any one of the above [1] to [3] are reacted, and the label on the insolubilized carrier or the label not bound to the insolubilized carrier is measured. A method for quantifying the protein according to any one of the above [12] to [14], a partial peptide thereof, or a salt thereof in a test sample is provided.

Other objects, features, advantages and aspects of the present invention will be apparent to those skilled in the art from the following description. However, it is understood that the description of the present specification, including the following description and the description of the specific examples and the like, shows preferred embodiments of the present invention and is given only for explanation. I want to. Various changes and / or alterations (or modifications) within the spirit and scope of the present invention disclosed herein will be apparent to those skilled in the art, based on the following description and knowledge from other portions of the present specification. Will be readily apparent. All patents and references cited in this specification are cited for explanatory purposes, and they are to be construed as being incorporated herein by reference. is there. In this specification,
The term "and / or" means that both (1) merged connection and (2) selective connection exist, for example, in the case of "treatment and / or prevention" (1) It is used to mean both treatment and prevention and (2) both treatment and prevention. Elsewhere, the term “and / or” is similarly used to encompass both (1) merged connectivity and (2) selective connectivity.

[0019]

DETAILED DESCRIPTION OF THE INVENTION According to the present invention, a human-derived cathepsin Z protein or a cathepsin Z, which is a member of the papain family having cysteine protease activity.
A protein or a salt thereof having at least 40% homology with the amino acid sequence of the protein and having cysteine protease activity or equivalent antigenicity, a characteristic partial peptide of the protein or a salt thereof, and a gene encoding them A vector or a plasmid containing the gene so that it can be manipulated by genetic recombination technology, such as DNA or RNA, a host cell transformed with such a vector, and a transgene expressing the gene. A transgenic animal such as a transgenic mouse, a knockout animal such as a knockout mouse in which its gene is specifically inactivated, a method for producing the protein or a salt thereof by culturing a transformed cell thereof, and a method for producing the protein or a salt thereof. Said protein or its salt or its protein Antibodies obtained using characteristic partial peptides of quality or salts thereof, particularly monoclonal antibodies, hybridoma cells producing the antibodies, the isolated genes, such as DNA, RNA, etc., are used as probes, or Measurement and diagnostic means and reagents using the antibody are provided. Furthermore, the present invention provides use of the active ingredient disclosed and described herein, for example, a drug or a reagent containing the active ingredient is provided, and a disease, disease or abnormal condition using such an active ingredient is provided. And / or a method for preventing and / or screening the same.

More specifically, the human cathepsin Z of the present invention includes those having the amino acid sequence at positions 62 to 303 of the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, and those having the same sequence. Those having the amino acid sequence of positions 21 to 303, those having the amino acid sequence of positions 1 to 303,
And at least 40% homology, preferably at least 50% homology, preferably at least 80% homology, particularly preferably at least 90% homology with any one of them, and cysteine protease activity or All those having an amino acid sequence having substantially the same biological activity such as the same antigenicity and the like can be mentioned. The human cathepsin Z of the present invention may be any one of the papain family having cysteine protease activity and having a novel amino acid sequence. More preferably, the present invention provides an amino acid sequence represented by SEQ ID NO: 1 having an amino acid sequence of positions 62 to 303,
A group having the amino acid sequence of positions 21 to 303; a group having the amino acid sequence of positions 1 to 303; and a group having an amino acid sequence substantially equivalent to any one of them A cathepsin Z protein of human origin characterized by being a protein selected from the group consisting of: Further, the human cathepsin Z of the present invention may have a part or the whole of the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing. Any having such an arrangement may be included.

The gene encoding human cathepsin Z of the present invention typically has a base sequence represented by SEQ ID NO: 2 in the sequence listing, and a base sequence represented by SEQ ID NO: 2 in the sequence listing. A sequence containing a base sequence consisting of ATG at positions 241 to 243 and TAA at positions 1150 to 1152 (the stop codon TAA at positions 1150 to 1152 is TGA
Or TAG), or 11 to 424 of the base sequence represented by SEQ ID NO: 2 in the sequence listing.
A sequence containing the nucleotide sequence at position 52, SEQ ID NO: 2 in the sequence listing
Those containing the nucleotide sequence from position 301 to position 1152 of the nucleotide sequence represented by, initiation codon in those nucleotide sequences, for example,
Met-encoded codons, or those having an amino acid sequence having at least 40% homology with the protein encoded by the base sequence and having substantially the same cysteine protease activity or equivalent antigenicity as the protein. Any protein may be used as long as it contains a nucleotide sequence equivalent to that of a protein having a biological activity. The gene encoding the human cathepsin Z includes single-stranded DNA, double-stranded DNA, RNA, and DNA:
It is a nucleic acid such as an RNA hybrid or a synthetic DNA, and may be any of a human genomic DNA, a human genomic DNA library, a cDNA derived from human tissues / cells, and a synthetic DNA. The nucleotide sequence of the gene encoding human cathepsin Z can be modified (eg, added, removed, substituted, etc.), and such modified ones can be included.
Further, as described below, the nucleic acid of the present invention may encode the protein of the present invention or a part thereof, and DNA is preferable. The “equivalent nucleotide sequence” is, for example, one which hybridizes under stringent conditions to the nucleotide sequence represented by SEQ ID NO: 2 in the sequence listing, or an amino acid sequence substantially equivalent to human cathepsin Z And the like.

The DNA of the present invention containing the base sequence represented by SEQ ID NO: 2 in the sequence listing or a base sequence equivalent thereto
Can be obtained, for example, by the following method. In addition,
Genetic recombination techniques are described, for example, in T. Maniatis et al., "Mole
cular Cloning ", 2nd ed., Cold Spring Harbor Laborat
ory, Cold Spring Harbor, NY (1989); DM Glove
r et al. ed., "DNA Cloning", 2nd ed., Vol. 1 to 4,
(ThePractical Approach Series), IRL Press, Oxford
University Press (1995); edited by The Biochemical Society of Japan, “Seismic Chemistry Experiment Course 1, Genetic Research Method II”, Tokyo Chemical Dojin (1
986); edited by The Biochemical Society of Japan
II (Recombinant DNA technology) ", Tokyo Kagaku Doujin (1992); R. Wu
ed., "Methods in Enzymology", Vol. 68, Academic Pr
ess, New York (1980); R. Wu et al. ed., "Methods
inEnzymology ", Vol. 100 & 101, Academic Press, New
York (1983); R. Wu et al. Ed., "Methods in Enzymol
ogy ", Vol. 153, 154 & 155, Academic Press, New York
(1987); JH Miller ed., "Methods in Enzymolog
y ", Vol. 204, Academic Press, New York (1991); R.
Wu et al. Ed., "Methods in Enzymology", Vol. 218, A
cademic Press, New York (1993), or the method described in the literature cited therein, or a method substantially similar to or a modification thereof.
(The statements therein are included in the disclosure herein by reference thereto).

First, a sequence showing significant similarity with the amino acid sequence of a known human cysteine protease family (more preferably papain family, particularly preferably human cathepsin) from which the entire sequence or a partial sequence has been cloned is obtained from a gene database. Identify by search. Examples of sequences showing significant similarity include, for example, cDNA sequences derived from animals, and more preferably cDNA sequences derived from ovarian cancer (AA283747; registered by Hillier et al., Wa
shU-Merck EST Project). Appropriate primers are designed and synthesized based on the sequence thus identified, and the target sequence is PCR-amplified, preferably using a cDNA library derived from an animal that is the origin of the identified sequence. The primer can be prepared by a method known in the art. For example, the primer can be synthesized using an automatic DNA synthesizer by a phosphodiester method, a phosphophotoester method, a phosphoramidite method, or the like. Obtained DNA
Using the fragment as a probe, a human genomic DNA library or a human-derived cDNA library constructed from various human tissues or cultured cells is screened, a clone that hybridizes to the probe is selected, and the inserted sequence of the DNA in the clone is selected. Is determined, and a DNA fragment having a novel human cysteine protease gene sequence is determined and obtained. If necessary, the inserted sequence of the DNA in the clone can be subcloned. The nucleotide sequence can be determined using the dideoxy method, such as the M13 dideoxy method, the Maxam-Gilbert method, or the like.
ev 2.0 kit, etc., automatic base sequencer,
For example, it can be performed using a fluorescent DNA sequencer. Examples of the polymerase used in the dideoxy method include Klenow fragment of DNA polymerase I, AMV reverse transcriptase, Taq DNA polymerase, T7D
NA polymerase, modified T7 DNA polymerase and the like.

Based on the DNA sequence which is considered to have the novel human cysteine protease gene thus analyzed, a gene encoding the human cysteine protease is obtained. The gene can be obtained by preparing a specific hybridization probe from the clone identified as described above, screening a human-derived DNA library, and selecting a clone that hybridizes to the probe. it can. In addition, based on the DNA sequence of the analyzed novel human cysteine protease gene, sense primers and antisense primers are synthesized, and these primers and various human-derived tissues or cultured cells (particularly, human White blood cells, colon, small intestine, ovary, testis, prostate, thymus, spleen, pancreas, kidney, skeletal muscle, liver, lung, placenta, brain, heart, etc. Leukemia such as MOLT-4, Burkitt's lymphoma such as Raji, colorectal cancer such as SW480, lung cancer such as A549, melanoma cell lines such as G361, etc.) Polymerase chain reaction (PCR ) To amplify the cDNA. A cDNA library used as a template can be prepared as described below, or a commercially available cDNA library derived from various tissues can be directly used. Can be used.

The PCR reaction can be carried out by a method known in the art or a method substantially similar to or a modification thereof. For example, R. Saiki, et al., Science, 230: 13
50, 1985; R. Saiki, et al., Science, 239: 487, 198
8; HA Erliched., PCR Technology, Stockton Pres
s, 1989; DM Glover et al. ed., "DNA Cloning", 2
nd ed., Vol. 1, (The Practical Approach Series), I
RLPress, Oxford University Press (1995); MA In
nis et al. ed., "PCRProtocols", Academic Press, Ne
w York (1990) and the like. The obtained PCR product is cloned, the nucleotide sequence of the obtained PCR product is determined, and a DNA fragment having a novel human cysteine protease gene sequence can be obtained. Also, various cDNA libraries can be screened in the same manner using this DNA fragment as a probe to isolate a target DNA. For cloning PCR products, for example, p-Direct (Clontech), pCR-Script
^TM SK (+) (Stratagene), pGEM-T (Promega), pAmp ^TM
A commercially available plasmid vector such as (Gibco-BRL) can be used.

In order to obtain a full-length novel human cysteine protease gene sequence containing a complete open reading frame, the novel human cysteine protease gene obtained as described above may be constructed, if necessary. Using the DNA fragment as a probe, a cDNA library constructed from various human tissues or cultured cells as described above is screened, a clone that hybridizes to the probe is selected, and the cDNA insertion sequence in the clone is The nucleotide sequence is determined, and a novel DNA fragment constituting the novel human cysteine protease gene is identified and obtained. Of course, if necessary, the cDNA in the clone
Can be subcloned. The target DNA can be isolated based on the determined base sequence. Preferably, a cDNA library derived from human brain or prostate is screened, and the nucleotide sequence is determined to isolate the target DNA. To label a probe or the like with a radioactive isotope, a commercially available labeling kit,
For example, random primed DNA labeling kit (Boeh
ringer Mannheim).

In order to construct a cDNA library, the cD
It is necessary to obtain NA, which can be obtained, for example, as follows. First, mRNA is isolated from various human tissues or cultured cells as described above. The isolation of mRNA can be performed by a method known in the art or a method substantially similar to or a modification thereof.
l., "Molecular Cloning", 2nd ed., Chapter 7, Cold S
pringHarbor Laboratory, Cold Spring Harbor, NY
(1989); DM Glover etal. Ed., "DNA Cloning", 2n
d ed., Vol. 1, (The Practical ApproachSeries), IRL
Press, Oxford University Press (1995); L. Grossm
an et al.ed., "Methods in Enzymology", Vol. 12, Pa.
rt A & B, Academic Press, NewYork (1968); SL Be
rger et al. ed., "Methods in Enzymology", Vol. 152,
p.33 & p.215, Academic Press, New York (1987); Bio
Chem., 18: 5294-5299, 1979, etc., for example, guanidine-cesium chloride method, guanidine thiocyanate method, phenol method and the like.
Kits used for mRNA isolation include, for example, Phar
Examples include those commercially available from macia, Stratagene, Gibco-BRL, and the like. If necessary, the total RNA obtained is an oligo (dT) -cellulose column, spin column, oligo (dT)
Purified using bound magnetic beads, etc. to poly (A) ⁺ mRNA
Can be obtained.

Using the mRNA and reverse transcriptase (RNA-dependent DNA polymerase), a cDNA is prepared. In the reverse transcription reaction, an oligo (dT) primer can be used. An oligo (dT) primer having preferably 12 to 18 T residues can be used. When performing directional cloning, it is also preferable to use a synthetic oligonucleotide primer in which a restriction enzyme site is linked to the 5 'side of 12 to 18 T residues. Examples of such primers include the Xba I oligo
(dT) primer adapter and the like. When a random hexamer primer is used, the possibility of obtaining the 5 'end of mRNA is increased. This random hexamer primer can be used alone or in combination with an oligo (dT) primer. In the reverse transcription reaction, RNas
e Inhibitors such as RNasin (Boehringer Mannheim) can be added. cDNA using mRNA and reverse transcriptase
The synthesis can be performed by a method known in the art or a method substantially similar thereto or a modified method.
et al., Nucleic Acids Res., 9: 2251, 1981; U. Gub
ler et al., Gene, 25: 263-269, 1983; SL Berger
et al. ed., "Methods in Enzymology", Vol. 152, p.3
07, Academic Press, New York (1987).

Based on the cDNA thus prepared, a cDNA is prepared by using a phage vector or a plasmid vector.
Can build libraries. In addition to using a phage vector, transformation of host cells such as E. coli can be performed by, for example, the calcium method, the rubidium / calcium method, the calcium / manganese method, the TFB high efficiency method, the FSB freezing competent cell method, the rapid colony method. Method, electroporation, or any other method known in the art or a method substantially similar thereto (D. Hanahan, J.
Mol. Biol., 166: 557, 1983). Further, using the thus prepared cDNA as a template, a novel DNA fragment constituting the novel human cysteine protease gene, primers are designed based on the determined base sequence, and PCR is further performed. This also makes it possible to obtain a gene encoding human cathepsin Z of the present invention.
Typically, the amino acid sequence obtained from the papain family is a known cysteine protease enzyme, the entire sequence or a partial sequence of which is cloned.
More preferably, a highly conserved amino acid sequence selected from the sequenced or deduced amino acid sequence of human cathepsin Z (for example, a sequence near the cleavage site of the precursor (pro form) and the active form) or An amino acid sequence of a region specifically present in cathepsin (eg, an amino acid sequence of a C-terminal region, an amino acid sequence of a region existing between a propeptide domain and a catalytic domain,
Based on the amino acid sequence of the active site region of the catalytic domain, a degenerated primer is prepared. Using this primer and the cDNA prepared above, PCR
I do. PCR can be performed as described above. The obtained PCR product is cloned in the same manner as described above, the nucleotide sequence of the obtained PCR product is determined, and a DNA fragment having a novel human cathepsin Z gene sequence can be obtained. Also, various cDNA libraries can be screened in the same manner using this DNA fragment as a probe to isolate a target DNA.

In order to isolate the desired DNA, reverse transcription PCR (polymerase chain reaction coupled reverse tr
anscription; RT-PCR), RACE (rapid amplification o
f cDNA ends) can be applied. RACE is described, for example, in MA Innis et al. Ed., "PCR Protocols" (MA
Frohman, "a guide to methods and applicarions"),
pp. 28-38, Academic Press, New York (1990). The RT-PCR product can be cloned into a plasmid vector, which can be introduced into highly efficient competent cells. Furthermore, a method capable of isolating and purifying mRNA from a small amount of cells or tissues,
For example, REXkit, United States Biochemical; Glass M
Using a commercially available kit, such as the ^{AX TM RNA spin cartridge system, Gibco} -BRL, resulting mRNA was reverse transcribed using oligo (dT) primers were synthesized 1st strand DNA, and then 3 of 1st strand DNA ' After attaching a homopolymer tail (for example, G residue) to the end or attaching an adapter to the DNA, the cDNA is converted to a P-DNA using an oligo (dT) primer and an oligo (dC) primer or an adapter primer.
CR amplification can also be performed. Commercially available kits suitable for this include the SuperScript ^TM pre-amplification system (G
ibco-BRL); cDNA Cycle ^™ kit (Invitrogen) and the like.

This will be described in more detail below. Probe DN
Using A, a novel human cathepsin Z gene is screened and identified from a gene library, and the nucleotide sequence of a DNA insert encoding the novel human cathepsin Z gene obtained by cloning is determined. Probe DNA
As, for example, ovarian cancer-derived cDNA sequence (AA283747; Hi
Using primers designed based on the WashU-Merck EST Project, registered by llier et al., and PCR-amplified using λgt11-phage DNA of a commercially available human brain cDNA library (Clontech) as a template, the DNA thus amplified is If necessary, it can be cloned, labeled and used as a probe. For labeling, a DNA labeling kit can be used. For example, a random-priming kit (Phar
Pharmacia LKB) the DNA probe using such [α- ³² P] d
By labeling using CTP (Amersham) or the like, a probe having radioactivity can be obtained. A gene library prepared from the labeled DNA fragment and human tissues / cells, for example, human
P1 artificial chromosome genomic library (H
Hybridization is performed using a Human Genome Mapping Resource Center) or a human brain or prostate cDNA library (available from, for example, Clontech). Human brain
The cDNA library can be constructed, for example, in a phage such as λgt10, and can be obtained by infecting host E. coli such as E. coli C600hfl strain to form plaques.

In the hybridization, the plaque formed as described above was transferred to a membrane such as a nylon filter, denaturation treatment, immobilization treatment, washing treatment and the like were performed as necessary, and then transferred to the membrane. The reaction is carried out by reacting with a denatured labeled probe DNA fragment in a hybridization buffer if necessary. The hybridization process is usually performed at about 35 ° C. to about 80 ° C., more preferably about 50 ° C. to about 65 ° C., for about 15 minutes to about 36 hours, more preferably about 1 hour to about 24 hours. It can be carried out by selecting optimal conditions as appropriate. For example, the hybridization treatment is performed at about 55 ° C. for about 18 hours. The hybridization buffer can be selected from those commonly used in the art, and for example, a Rapid hybridization buffer (Amersham) or the like can be used. The denaturation of the transferred membrane
A method using an alkali denaturing solution is mentioned, and after the treatment, it is preferable to treat with a neutralizing solution or a buffer solution. The immobilization treatment of the membrane is usually performed at about 40 ° C. to about 100 ° C., more preferably about 70 ° C. to about 90 ° C., for about 15 minutes to about 24 hours, more preferably about
Baking is carried out for 1 hour to about 4 hours, but it can be carried out by appropriately selecting preferable conditions. For example, the immobilization is performed by baking the filter at about 80 ° C. for about 2 hours. The washing treatment of the transferred membrane includes a washing liquid commonly used in the art, for example, 1M N
aCl, 1mM EDTA and 0.1% SodiumDodecyl sulfate
It can be performed by washing with (SDS) -containing 50 mM Tris-HC1 buffer, pH 8.0, etc. The membrane such as a nylon filter can be selected from those commonly used in the art, and examples thereof include a nylon filter [Hybond-N, Amersham].

As the above-mentioned alkali denaturing solution, neutralizing solution and buffer solution, those commonly used in the art can be selected and used.
A solution containing 0.5M NaOH and 1.5M NaCl can be mentioned. As the neutralizing solution, for example, a solution containing 1.5M NaCl
0.5M Tris-HCl buffer, pH 8.0 and the like. As the buffer, for example, 2 × SSPE (0.36M NaCl,
20 mM NaH ₂ PO ₄ and 2 mM EDTA). Prior to the hybridization treatment, the transferred membrane is preferably subjected to a pre-hybridization treatment, if necessary, in order to prevent a non-specific hybridization reaction. This pre-hybridization treatment is performed, for example, using a pre-hybridization solution [50%
formamide, 5 × Denhardt's solution (0.2% bovine serum albumin, 0.2% polyvinyl pyrrolidone), 5 × SSPE,
0.1% SDS, 100 µg / ml heat-denatured salmon sperm DNA] and the like, and allowed to react at about 35 ° C to 50 ° C, preferably about 42 ° C, for about 4 to 24 hours, preferably for about 6 to 8 hours. However, those skilled in the art can determine more preferable conditions by repeating experiments as appropriate. Labeled probe DNA used for hybridization
The denaturation of the fragments is carried out, for example, at about 70 ° C to 100 ° C, preferably at about
It can be carried out by heating at 100 ° C. for about 1 minute to about 60 minutes, preferably for about 5 minutes.

After the completion of the hybridization, the filter is sufficiently washed to remove the labeled probe other than the labeled probe DNA fragment that has undergone the specific hybridization reaction. The filter can be washed and selected from those commonly used in the art. For example, 0.5 × SSC containing 0.1% SDS (O.15M NaCl,
(15 mM citric acid) solution. The hybridized plaque can be typically detected by autoradiography, but can also be appropriately selected from methods used in the art and used for plaque detection. The plaque corresponding to the detected signal is added to an appropriate buffer such as SM solution (100 mM N 2
aCl and 10 mM MgSO ₄ containing 50 mM Tris-HCl buffer, pH 7.
The phage suspension is appropriately diluted, infected with Escherichia coli, and the obtained Escherichia coli is cultured to obtain a desired recombinant phage from the cultured Escherichia coli. The process of screening for a target recombinant phage from a gene library or a cDNA library by hybridization using the above probe DNA as necessary can be repeatedly performed. The target recombinant phage can be obtained by subjecting the cultured Escherichia coli to extraction treatment, centrifugation treatment and the like.

The obtained phage particles can be purified and separated by a method commonly used in the art.
Glycerol gradient ultracentrifugation (Molecular cl
oning, a laboratory manual, ed.T. Maniatis, Cold
Spring Harbor Laboratory, 2nd ed. 78, 1989). From the phage particles, DNA can be purified and separated by a method commonly used in the art.
Suspend in 50 mM Tris-HCl buffer containing O ₄ (pH 7.8)
After treatment with ase I and RNase A, 20mM EDTA, 50μ
g / ml Proteinase K and 0.5% SDS mixture, etc.
After incubating at about 65 ° C for about 1 hour, this was extracted with phenol and extracted with diethyl ether, and DNA was precipitated by ethanol precipitation. The obtained DNA was washed with 70% ethanol, dried, and TE solution (containing 10 mM EDTA 10 mM Tris-HC1 buffer,
pH 8.0). The target DNA can be obtained in large quantities by subcloning or the like. For example, subcloning can be performed using Escherichia coli as a host and a plasmid vector. Obtained by such subcloning
For DNA, centrifugation, phenol extraction,
It can be purified and separated by a method such as ethanol precipitation.

The base sequence of the DNA thus obtained, for example, the base sequence of single-stranded DNA, can be sequenced in the same manner as described above. For example, the fluorescent DNA sequencer Mo
del373A (Applied Biosystems), Taq Dye Primer Cycle Sequencing Kit (Applied Biosystems)
s) can be used to determine the sequence. By analyzing the determined sequence, it is possible to identify and analyze a gene that has a structural characteristic typical of the known human cathepsin sequence but is presumed to be a novel human cathepsin. Based on the nucleotide sequence of the analyzed gene, primers are designed to obtain the cDNA of the novel putative human cathepsin. The sense primer is
Preferably, it can be synthesized by selecting from the exon site at the 5 ′ end of the putative novel human cathepsin analyzed, and the antisense primer is preferably putatively analyzed as the novel human cathepsin. Gene
It can be synthesized by selecting from the 3 'end exon site. The cDNA of a gene may aim to obtain the full length at once, but using the analyzed exon site (multiple exon sites), design and synthesize multiple primers, and perform multiple PCRs. The DNA fragment whose nucleotide sequence was determined in this way was analyzed,
The entire nucleotide sequence of the cDNA is determined, and the cDNA of the gene can be obtained from the cloned DNA fragment based on the determined nucleotide sequence. The primer is preferably an oligonucleotide consisting of 5 or more bases, more preferably an oligonucleotide consisting of 18 to 25 bases. As described above, the synthesis of the primer is performed by an automatic DNA synthesizer, for example,
It is synthesized using model 381A DNA synthesizer (Applied Biosystems).

Using mRNA isolated from human tissues, particularly human brain, human prostate, etc., the first transcriptase
Make strand cDNA. Brain or prostate tissue
Treat using a commercially available mRNA extraction / separation kit such as izolReagent (Life Technologies) or a cDNA synthesis kit (mRNA extraction / separation reagents and systems are attached), etc.
Prepare NA. For the 1st strand cDNA, oligo Script dT _12-18 primer was used and the Super Script kit (Life Technolog
ies), using a commercially available cDNA synthesis kit and the mRNA as a template. 1st strand cDNA synthesis, for example,
Poly (A) ⁺ RNA was added to 5 × RT buffer (250 mM Tris-HCl (pH 8.
_{3), 50mM MgCl 2, 375mM} KCl, 50mM DTT -containing), dNTPs
(Deoxynucleoside triphosphate dATP, dGTP, dCTP, dT
Mixtures TP), oligo dT ₁₆ primer, RNase inhibitor
(Boehringer Mannheim), MMLV RT (Gibco-BRL) or
This can be achieved by mixing with Superscript RT plus (Life Technologies) and deionized distilled water and incubating at about 37 ° C. for about 1 hour.

The obtained 1st strand DNA and a primer designed based on the exon of the analyzed gene were combined with 10 × reaction buffer (attached to Taq DNA polymerase), dNTPs (deoxynucleoside triphosphate dAT
P, dGTP, dCTP, dTTP), Taq DNA polymerase and deionized distilled water. The mixture is, for example, Ge
The cycle is repeated 25 to 60 times under general PCR cycle conditions using an automatic thermal cycler such as the neAmp 2400 PCR system, Perkin-Elmer / Cetus, but the number of cycles for amplification is appropriate according to the purpose. It can be the number of times. PCR cycle conditions include, for example, denaturing 90
~ 95 ° C 5 ~ 100 seconds, annealing 40 ~ 60 ° C 5 ~ 150 seconds,
Elongation 65-75 ° C 30-300 seconds cycle, preferably denaturation
94 ° C for 15 seconds, annealing 58 ° C for 15 seconds, extension 72 ° C
A 45-second cycle can be mentioned, but the annealing reaction temperature and time can be appropriately selected by experiments, and the denaturation reaction and extension reaction time can also be set to appropriate values according to the expected PCR product chain length. Can be selected. The annealing reaction temperature is usually preferably changed according to the Tm value of the hybrid of the primer and the template DNA. The time for the extension reaction is generally about 1 minute per 1000 bp chain length, but a shorter time can be selected in some cases.

The obtained PCR product is usually subjected to 1 to 2% agarose gel electrophoresis, cut out from the gel as a specific band, and the DNA is extracted using a commercially available extraction kit such as a gene clean kit (Bio 101). I do. Extracted
The DNA is cleaved with an appropriate restriction enzyme, and purified if necessary.
Ligation is performed with an appropriate plasmid vector such as a system vector, and an appropriate competent cell is transformed.
The nucleotide sequence of the cloned PCR product is analyzed. As a result of the nucleotide sequence analysis, the 5 'end and / or 3'
When it is necessary to obtain the sequence on the end side, it can be achieved by applying a rapid amplification of cDNA ends (RACE).

Further, the primers of the cDNA of the gene are utilized by using primers designed based on the nucleotide sequence of the exon site at the 5 'end analyzed among the exon sites of the gene.
Obtain the cDNA at the 5 'end, while using primers designed based on the nucleotide sequence of the exon site at the 3' end analyzed among the exon sites of the gene, the 3 'end of the cDNA of the gene Side cDNA, and then, if necessary, these primers and the cDNA of the 5 'end of the cDNA of the gene obtained.
Using a primer designed by using information on the base sequence of the NA and the 3′-end cDNA, 1st strand cDNA prepared by reverse transcriptase from mRNA isolated from human tissues, particularly human brain tissues, etc. (Or human cDNA library) as a template and amplify by PCR,
NA can be obtained.

The primer at the 5 'end contains at least a start codon or is selected so that it can be amplified including the start codon, and the primer at the 3' end contains at least a stop codon. Alternatively, it is preferable to select so as to enable amplification including the stop codon. In obtaining the full-length cDNA of the gene, PCR can be performed as described above. Preferred PCR cycle conditions include, for example,
~ 95 ° C for 10-20 seconds, annealing 55-60 ° C for 10-30 seconds,
Elongation 65-75 ° C 150-300 seconds cycle, more preferably denaturation 94 ° C 15 seconds, annealing 58 ° C 15 seconds, elongation 68
Cycle for 4 minutes. The obtained PCR product is cloned in the same manner as described above, and its nucleotide sequence is analyzed and determined. In addition, primers are designed based on the determined nucleotide sequence of DNA, and screening is performed by using these primers and cDNA libraries derived from various animal cells (for example, cDNA libraries derived from various human cells). Similarly, a desired clone can be obtained. PCR using these primers
By performing amplification, a target gene, a novel gene, a fragment thereof, and the like can be obtained. Thus, a PCR product having homology to human cathepsin Z but having a novel sequence can also be searched.

Thus, according to the present invention, the desired DNA
(For example, as a recombinant phage). For example, we screened a human brain cDNA library and a human prostate cDNA library, cloned a novel cysteine protease, and clarified the entire nucleotide sequence of the cDNA encoding it. The total length of the determined nucleotide sequence of the DNA of the isolated gene is 1541 bp
It can be seen that the sequence was obtained as shown in SEQ ID NO: 2 in the sequence listing. GenBank ^TM / EMBL
When the base sequence described in SEQ ID NO: 2 in the sequence listing was searched using DNA Data Base, no identical sequence was found. In the isolated and identified 1541 bp DNA sequence, the presence of an open reading frame encoding an estimated 303 amino acids was observed. The deduced amino acid sequence is shown in SEQ ID NO: 1 in the sequence listing. Is recognized as such. This putative protein has less than 40% homology to other known human cathepsins, such as about 34% homology to human cathepsin C,
It shows the structural features typical of such cathepsins and is a novel human cathepsin, which was named "human cathepsin Z". It is clear that the human cathepsin Z gene encodes a protein belonging to the papain family of a novel cysteine protease, and all recombinant plasmids prepared using the human cathepsin Z gene are novel recombinants. In addition, a transformant or transfectant obtained by transformation or transfection with the plasmid is also novel. The nucleic acid having all or a part of the base sequence represented by SEQ ID NO: 2 in the sequence listing can be obtained by chemical synthesis. In that case, the fragments may be chemically synthesized and ligated with an enzyme. In addition, as described above, a chemically synthesized fragment can be used as a primer or a probe to obtain a target sequence.

The gene for this novel cysteine protease is located on chromosome 20q13, where the presence of existing cathepsins is not known. A recombinant protein produced by genetic engineering based on this cDNA is a synthetic substrate Z- characteristic of the cysteine protease family.
Decomposes Phe-Arg-AMC. Cathepsin Z is a human tissue of leukocytes, colon, small intestine, ovary, testis, prostate, thymus, spleen, pancreas, kidney, skeletal muscle, liver, lung, placenta, brain and heart, as well as leukemia, Burkitt's lymphoma, colorectum / rectum cancer,
It was also found to be widely expressed in cancer cell lines and cancer tissues such as lung cancer and melanoma. And Cathepsin Z
Is also a lysosomal enzyme involved in the digestion of intracellular proteins and a proteolytic enzyme involved in the transformation of human cells into malignant transformation. Obtained DN
The A fragment is incorporated into a suitable vector as described in detail below, for example, a plasmid pEX, pMAMneo, pKG5, etc., and a suitable host cell as described in detail below, for example, E. coli, yeast, CHO cell, It can be expressed in COS cells and the like. The DNA fragment may be used as it is or as a DNA fragment to which an appropriate control sequence is added, or may be inserted into an appropriate vector and introduced into an animal to introduce a cathepsin Z gene, for example, human cathepsin Z.
Transgenic animals can be produced. Animals include mammals, for example, mice, rats, rabbits, guinea pigs, cows, and the like. Preferably, the DNA is contained in a fertilized egg of an animal such as a mouse.
Fragments can be introduced to create transgenic animals.

The confirmation of the human cathepsin Z gene product was confirmed by transfection of the human cathepsin Z gene with COS-
This can be performed using animal cells suitable for it, such as one cell. This foreign gene can be introduced into animal cells such as mammals by a method known in the art or a method substantially similar thereto. For example, the calcium phosphate method (for example, FL Graham et al., V.
irology, 52: 456, 1973), DEAE-dextran method (for example, D. Warden et al., J. Gen. Virol., 3:37).
1, 1968), the electroporation method (for example,
E. Neumann et al., EMBO J, 1: 841, 1982), microinjection method, ribosome method, virus infection method, phage particle method and the like. Gene products produced by animal cells transfected with the human cathepsin Z gene in this manner can also be analyzed by immunoprecipitation experiments using an anti-human cathepsin Z monoclonal antibody or by Western blotting.

As the plasmid incorporating the human cathepsin Z gene, host cells commonly used in genetic engineering (eg, prokaryotic host cells such as Escherichia coli and Bacillus subtilis, eukaryotic host cells such as yeast, CHO cells, COS cells, and Sf21) Insect cell host)
Any plasmid may be used as long as it can express the DNA. Such a sequence may contain, for example, codons suitably modified for expression in the selected host cell, may have a restriction enzyme site, and may have a target gene. Control sequences for facilitating expression, promoters, etc.
Furthermore, it may contain sequences useful for controlling antibiotic resistance and the like, controlling metabolism, and selecting. Preferably, a suitable promoter, for example in plasmid E. coli as a host, a tryptophan promoter (trp), lactose promoter (lac), the tryptophan lactose promoter (tac), lipoprotein promoter (lpp), the λ phage P _L promoter For a plasmid using animal cells as a host, the SV40 rate promoter, MMTV LTR promoter, RSV LTR promoter, CMV promoter, SRα promoter and the like can be used. For a plasmid using yeast as a host, GAL1 and GAL10 promoters can be used.

As a plasmid using Escherichia coli as a host,
For example, pBR322, pUC18, pUC19, pUC118, pUC119, pSP6
4, pSP65, pTZ-18R / -18U, pTZ-19R / -19U, pGEM-3, pG
EM-4, pGEM-3Z, pGEM-4Z, pGEM-5Zf (-), pBluescrip
t KS ^TM (Stratagene), pGEX-3X (Pharmacia LKB) and the like. Plasmid vectors suitable for expression in E. coli include pAS, pKK223 (Pharmacia), pMC1403,
pMC931, pKC30, pRSET-B (Invitrogen) and the like are also included. Plasmids using animal cells as hosts include SV40 vectors, polyoma virus vectors, vaccinia
Viral vectors, retroviral vectors and the like, for example, pcD, pcD-SRα, CDM8, pCEV4, pME18S,
pBC12BI, pSG5 (Stratagene) and the like. As plasmids using yeast as a host, YIp-type vectors, YE
Examples include a p-type vector, a YRp-type vector, and a YCp-type vector, such as pGPD-2. When the host cell is E. coli, examples of the host cell include those derived from E. coli K12 strain, such as NM533 XL1-Blue, C6
00, DH1, DH5, DH11S, DH12S, DH5α, DH10B, HB
101, MC1061, JM109, STBL2, BL21 (DE3) pLysS and the like. When the host cell is an animal cell, for example, African green monkey fibroblast-derived COS-7 cells, COS-1 cells, CV-1 cells, mouse fibroblast-derived COP cells, MOP
Cells, WOP cells, Chinese hamster cells
CHO cells, CHO DHFR ^- cells, human HeLa cells, mouse cell-derived C127 cells, mouse cell-derived NIH 3T3 cells, and the like. Insect cells include silkworm nuclear polyhedrosis virus
(Bombyx mori nuclear polyhedrosis virus) or a vector derived therefrom, using silkworm larvae or silkworm culture cells, such as BM-N cells. It is also possible to use plant cells as host cells, together with suitable vectors, which are widely known in the art.

In the genetic engineering method of the present invention, a restriction enzyme, reverse transcriptase, or a DNA fragment known or widely used in the art is modified or converted into a structure suitable for cloning. DNA modifying / degrading enzymes, DNA polymerases, terminal nucleotidyl transferases, DNA ligases, etc. can be used. Restriction enzymes include, for example, RJ Roberts, Nuc
leic Acids Res., 13: r165, 1985; S. Linn et al. ed.
Nucleases, p. 109, Cold Spring Harbor Lab., Cold
Spring Harbor, New York, 1982; RJ Roberts, D. M
acelis, Nucleic Acids Res., 19: Suppl. 2077, 1991
And the like. As a reverse transcriptase,
For example, mouse Moloney leukemia virus (mouse Moloney
leukemiavirus (MMLV) derived reverse transcriptase (reverse tra
nscriptase), chicken myeloblastosis virus (avian mye
loblastosis virus (AMV) -derived reverse transcriptase. As the reverse transcriptase, an RNase H-deficient mutant or the like can be preferably used, and particularly, a modified MM lacking RNase H activity.
LV RT can be preferably used, and more preferably those having high thermal stability. Suitable reverse transcriptases include MMLV RT (G
ibco-BRL), Superscript RT plus (Life Technologie
s).

As a DNA polymerase, for example, Escherichia coli
Klenow Flag, a DNA polymerase and its derivatives
Phage T4 DNA polymerase, E. coli phage
T7 DNA polymerase, thermostable DNA polymerase
And so on. Terminal nucleotidyl transferer
For example, R. Wu et al. Ed., "Methods in Enz
ymology ", Vol. 100, p. 96, Academic Press, New Yor
k (1983) 3'-OH terminal deoxynucleotide
TdTase to which (dNMP) is added. DNA modification
Degradative enzymes include exonuclease, endonuclease
Such as snake venom phosphodiestera
, Spleen phosphodiesterase, E. coli DNA exonuclease
Nuclease I, E. coli DNA exonuclease III, large
Enterobacteriaceae DNA exonuclease VII, λ exonuclease
, DNase I, nuclease S1, micrococcus
 (Micrococcus) nuclease and the like. DNA
Examples of ligases include E. coli DNA ligase, T4 DNA
Ligase and the like. Clone DNA gene
Vector suitable for constructing a DNA library
Plasmid, lambda phage, cosmid, P1 fur
Di, F factor, YAC and the like.
Di-derived vectors such as Charon4A and Charo4A.
n 21A, λgt10, λgt11, λDASHII, λFIXII, λEMBL3
 , ΛZAPII ^TM (Stratagene).

Further, the human cathepsin Z according to the present invention
By using a method commonly used in genetic engineering based on the nucleotide sequence of the gene, one or more amino acid substitutions, deletions, insertions, transfers, or additions were appropriately made in the amino acid sequence of human cathepsin Z. Corresponding proteins having such mutations introduced can be produced. Examples of such mutation, conversion, and modification methods include the edition of the Biochemical Society of Japan, “Seismological Chemistry Laboratory Course 1, Gene Research Methods II”, p105 (Susumu Hirose), Tokyo Kagaku Dojin (1986);
"Lecturer on Experimental Chemistry 2, Nucleic Acid III (Recombinant DNA Technology)
", P233 (Susumu Hirose), Tokyo Doujinshi (1992)
R. Wu, L. Grossman, ed., "Methods in Enzymolog
y ", Vol. 154, p. 350 & p. 367, Academic Press, New
York (1987); R. Wu, L. Grossman, ed., "Methods in
Enzymology ", Vol. 100, p. 457 & p. 468, Academic P
ress, New York (1983); JA Wells et al., Gene,
34: 315, 1985; T. Grundstroem et al., Nucleic Acids
Res., 13: 3305, 1985; J. Taylor et al., Nucleic
Acids Res., 13: 8765, 1985; R. Wu ed., "Methods in
Enzymology ", Vol. 155, p. 568, Academic Press, Ne
w York (1987); AR Oliphant et al., Gene, 44: 1
77, 1986 and the like. For example, methods such as position-directed mutagenesis using synthetic oligonucleotides (site-directed mutagenesis), Kunkel method, dNTP [αS] method (Eckstein), and region-directed mutagenesis using sulfite or nitrite are used. No.

The obtained protein of the present invention can be modified by modifying the amino acid residues contained in the protein by a chemical method, or a peptidase such as pepsin, chymotrypsin, papain, bromelain, endopeptidase, exopeptidase and the like. Can be modified or partially degraded using the above enzyme to obtain a derivative thereof. Alternatively, it may be expressed as a fusion protein when produced by a genetic recombination method, and converted and processed into a substance having a biological activity substantially equivalent to that of natural human cathepsin Z in vivo or in vitro. . A fusion production method commonly used in genetic engineering can be used, and such a fusion protein can be purified by affinity chromatography or the like using the fusion portion. Modification and alteration of the structure of the protein are described in, for example, "Chemistry of Experimental Chemistry 1, Protein VII, Protein Engineering", edited by The Biochemical Society of Japan, Tokyo Kagaku Dojin (1993), and the method described therein or cited therein. The method can be carried out in the manner described in the literature, or in a manner substantially similar thereto. As described below, the biological activity may include having an immunological activity, for example, having antigenicity.

Thus, the human-derived protein of the present invention is one in which one or more amino acid residues differ from the natural one in terms of identity, and one in which the position of one or more amino acid residues differs from the natural one. There may be. The human-derived protein of the present invention has one or more amino acid residues unique to human cathepsin Z (for example, 1 to 100, preferably 1 to 60).
, More preferably 1 to 40, more preferably 1 to
20 deletions, especially 1 to 10 deletion analogs, one or more amino acid residues specific to the human cathepsin Z (eg, 1 to 100, preferably 1 to 60, more preferably Is 1 to 40, more preferably 1 to 20, especially 1 to
Substituted analogs in which 10 residues (e.g., 10) have been replaced with other residues, 1
Or more (e.g., 1 to 100, preferably 1 to 60, more preferably 1 to 40, more preferably 1 to 20,
(In particular, 1 to 10 amino acids). As long as the domain structure or enzyme active center structure characteristic of natural human cathepsin Z is maintained, all of the above mutants are included in the present invention. It is considered that the human cathepsin Z of the present invention may include those having a primary structural conformation substantially equivalent to natural human cathepsin Z or a part thereof. It is considered that those having substantially the same biological activity (eg, cysteine protease activity or antigenicity) may be included. It can also be one of the naturally occurring variants. The human-derived protein of the present invention is, for example, the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing.
Selected from the group consisting of those having the amino acid sequences of positions 62 to 303, those having the amino acid sequences of positions 21 to 303, and those having the amino acid sequences of positions 1 to 303 Those having a homology higher than 50%, preferably 80% or more with respect to the amino acid sequence are mentioned, and those having 90% or more homology with the amino acid sequence are more preferable.

As used herein, “substantially equivalent” means that the activities of proteins, for example, catalytic activity, physiological activity, and biological activity are substantially the same. Furthermore, the meaning of the term may include the case where the substance has substantially the same activity. Examples of the substantially same activity include, for example, protease activity, degradation to a synthetic substrate for cysteine protease. Activity and the like. The substantially equivalent activity indicates that those activities are the same in nature, for example, physiologically,
Indicates that it is pharmacologically or biologically equivalent. For example, activities such as protease activity are equivalent (for example, about 0.001 to 1000 times, preferably about 0.01 to 100 times,
More preferably about 0.1 to 20 times, even more preferably about 0.5
However, the quantitative factors such as the degree of these activities and the molecular weight of the protein may be different. Second, amino acid substitutions, deletions, or insertions often do not result in significant changes in the physiological or chemical properties of the polypeptide, in which case the substitutions, deletions, or insertions are made. The polypeptide is
Would be substantially identical to those without such substitutions, deletions, or insertions. Substantially identical substitutions of amino acids in the amino acid sequence may be selected from other amino acids of the class to which the amino acid belongs. For example, non-polar (hydrophobic)
Examples of the amino acid include alanine, phenylalanine, leucine, isoleucine, valine, proline, tryptophan, and methionine. Amino acids having a positive charge (basic amino acids) include arginine, lysine, histidine and the like, and amino acids having a negative charge (acidic amino acids) include aspartic acid and glutamic acid.

The protein of the present invention and a partial peptide thereof can be synthesized by a method known in the field of peptide synthesis,
For example, a chemical synthesis method such as a liquid phase synthesis method and a solid phase synthesis method can be used. In such a method, for example, a resin for protein or peptide synthesis is used, and appropriately protected amino acids are sequentially bonded to a desired amino acid sequence on the resin by various condensation methods known per se. For the condensation reaction, various known activating reagents are preferably used. As such a reagent, for example, carbodiimides such as dicyclohexylcarbodiimide can be preferably used. When the product has a protecting group, the desired product can be obtained by appropriately removing the protecting group.
When the protein of the present invention and some of its peptides are obtained in a free form, they can be converted into salts by a method known per se or a method analogous thereto, and they can be converted into salts. When obtained, it can be converted to a free form or another salt by a method known per se or a method analogous thereto. The salts of the protein of the present invention and some of its peptides are preferably physiologically acceptable or pharmaceutically acceptable, but are not limited thereto. Such salts include:
For example, salts with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc., for example, acetic acid, formic acid, maleic acid, fumaric acid, succinic acid, citric acid, tartaric acid, malic acid, benzoic acid, methanesulfonic acid And salts with organic acids such as p-toluenesulfonic acid and benzenesulfonic acid. Examples of the salt further include ammonium salts, for example, salts with organic bases such as ethylamine, dimethylamine, trimethylamine, and hydroxyethylamine. Such human cathepsin Z of the present invention, its mutants, modifications, derivatives and the like can be separated and purified as described below. In the present invention, the terms "fragment", "derivative" and "analog" are transcribed from the polypeptide of SEQ ID NO: 1, the sequence of SEQ ID NO: 2 and are unspliced or specifically spliced. When referring to a polypeptide encoded by hnRNA or mRNA, or a polypeptide encoded by deposited genomic DNA, as a “fragment”, “derivative” or “analog”, such a polypeptide is essentially Polypeptides having the same biological function or activity. Thus, analogs include activatable proproteins and the like, where the proprotein portion is cleaved to produce an active mature polypeptide. The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide. The thus-obtained natural human cathepsin Z of the present invention or a protein or a salt thereof having substantially the same activity as the protein or a salt thereof, or a partial peptide thereof can be used to develop or search for an enzyme inhibitor using the same. Research, drug development research, and biological phenomena and reactions that are thought to involve human cathepsin Z, and can be used to generate antibodies against it, Alternatively, it can be used for researching a measurement object. Human cathepsin Z is a type of cysteine protease that is thought to be involved in many normal cellular processes, including intracellular protein metabolism, activation of hormone precursors, and bone modification. And is useful for studying reactions. Also,
Human cathepsin Z is useful for elucidating its functions in relation to many diseases such as Alzheimer's disease, emphysema, rheumatoid arthritis, muscular dystrophy, osteoporosis, neurodegenerative diseases and cancer invasion / metastasis, particularly cancer. It is expected to be extremely useful for elucidating the pathogenesis of these diseases, treating and developing therapeutic drugs. On the one hand, the invention thus also encompasses DNA sequences encoding the above-mentioned polypeptides, and also human cathepsin Z polypeptides having all or part of the natural characteristics, as well as DNA sequences encoding analogs or derivatives thereof.

Since the DNA sequence of the present invention provides information on the amino acid sequence of a previously unknown mammalian protein, the use of such information is also encompassed by the present invention. Such uses include, for example, the design of probes for the isolation and detection of genomic DNA and cDNA from mammals, particularly humans, encoding human cathepsin Z and related proteins.
The DNA sequences of the present invention are useful, for example, as probes for the isolation and detection of genomic DNA and cDNA from mammals, particularly preferably mice and humans, encoding human cathepsin Z and related proteins. For the isolation of genes, PCR and PCR using reverse transcriptase (RT)
(RT-PCR) can be used. Human cathepsin Z
The cDNA and its related DNA were selected from the amino acid sequence deduced from the cloned and sequenced human cathepsin Z cDNA sequence, a characteristic sequence region was selected, a DNA primer was designed and chemically synthesized, and the obtained DNA primer was Can be used for the isolation and detection of a human cathepsin Z-related gene using PCR, RT-PCR, and other methods. For example, the expression of human cathepsin Z mRNA in human tissues can be examined by Northern blot analysis on poly (A) ⁺ RNA derived from various tissues. When the cDNA of the present invention is used as a probe, for example, Northern blotting, Southern blotting, in situ
u Detection and measurement of human cathepsin Z mRNA expression and human cathepsin Z gene itself in human tissues by hybridization, including intracellular protein metabolism, activation of hormone precursors, and bone modification in human tissues The role of cysteine proteases involved in many normal cellular processes, Alzheimer's disease, emphysema, rheumatoid arthritis, muscular dystrophy, osteoporosis,
It can contribute to the development of research on many diseases such as neurodegenerative diseases and cancer invasion and metastasis. It can also be used for genetic diagnosis of diseases related to human cathepsin Z. Such a diagnosis is
It can be used to diagnose abnormalities in nucleic acids encoding the human cathepsin Z and related proteins, for example, damage, mutation, decreased expression, overexpression, and the like.

When transferring the DNA obtained in the present invention (for example, DNA encoding human cathepsin Z) to a target animal, the DNA is ligated as a DNA fragment or downstream of a promoter capable of expressing the DNA in animal cells. It is generally advantageous to use them. For example, when human cathepsin Z DNA is introduced into a mouse, a gene construct in which human cathepsin Z DNA derived from an animal having a high homology to the gene is bound downstream of various promoters capable of expressing the same in animal cells is used as a fertilized egg of a target animal. For example, gene injection (transgenic) mice that highly produce human cathepsin Z can be produced by microinjection into fertilized mouse eggs. Although the mouse is not particularly limited to a pure mouse, for example, C57BL / 6, Balb / C, C3H, (C
57BL / 6 × DBA / 2) F ₁ (BDF ₁ ). As this promoter, for example, a virus-derived promoter, a ubiquitous expression promoter such as metallothionein and the like can be preferably used. In addition, when the human cathepsin Z DNA is introduced, it can be recombined with a recombinant retrovirus and used. Preferably, a mouse fertilized egg into which the target DNA has been introduced can be grown using a foster mother mouse such as ICR. Transfer of the DNA obtained in the present invention (for example, DNA encoding human cathepsin Z) at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target animal. DN
A The presence of the DNA encoding human cathepsin Z in the germinal cells of the produced animal after transposition indicates that all the offspring of the produced animal have the DNA encoding the human cathepsin Z in all of its germ cells and somatic cells. means. The progeny of such animals that have inherited the gene have the potential to express the human cathepsin Z in all of their germinal and somatic cells.

The animal into which the human cathepsin Z DNA was introduced was confirmed to stably maintain the gene by mating.
The breeding subculture can be performed in a normal breeding environment as a holding animal. Furthermore, by crossing male and female animals having the target DNA, homozygous animals having the transgene on both homologous chromosomes are obtained, and by crossing the male and female animals, all offspring have the DNA. Breeding can be subcultured. Since the animal into which the human cathepsin Z DNA is introduced has high expression of the human cathepsin Z protein, it is useful as an animal for screening for an inhibitor (inhibitor) for the human cathepsin Z protein. It is also useful as an animal for screening an antisense oligonucleotide capable of inhibiting the expression of the human cathepsin Z gene, for example, antisense DNA. This transgenic animal can also be used as a cell source for tissue culture. For example, a protein related to the papain family of cysteine protease can be analyzed by directly analyzing DNA or RNA in the tissue of a transgenic mouse or by analyzing a protein tissue expressed by the gene. The cells of the tissue having cathepsin Z can be cultured by standard tissue culture techniques and used to study the function of cells derived from, for example, thymus, testis, brain, intestine, kidney and other tissues. Further, by using the cells, it is possible to contribute to the development of a drug that enhances the functions of various tissues, for example. Further, if there is a high expression cell line, cathepsin Z can be isolated and purified therefrom. Technologies related to transgenic mice and the like include, for example, Br
inster, RL, et al.,; Proc. Natl. Acad. Sci. US
A, 82: 4438, 1985; Costantini, F. & Jaenisch, R.
(eds): Genetic manipulation of the early mammal
The method can be performed by a method described in a document such as embryo, Cold Spring Harbor Laboratory, 1985, a method described in a document cited therein, or a modified method thereof.

The gene obtained in the present invention (for example, coding for mouse cathepsin Z corresponding to human cathepsin Z)
A mutant mouse (knockout mouse) having a mutation in DNA) and not expressing mouse cathepsin Z at all can be produced. For example, 4k before and after the translation initiation codon of the gene
It is possible to construct a targeting vector having a mutant gene in which a gene cassette comprising a neo resistance gene-polyA addition signal is inserted into an exon located near the center of a genomic DNA of about 8 kb including b and located near the translation initiation codon. The gene cassette to be inserted includes a DT-A cassette, a tk cassette, a lacZ cassette and the like in addition to the neo resistance gene cassette. Open the targeting vector in a linear fashion,
Established mouse embryonic stem cells (ES)
Cells) by electroporation and further cultivation to select ES cells that have acquired neo resistance. ES cells are 129
, C57BL / 6, F1 (C57BL / 6 × CBA) mice and the like. It is assumed that the ES cells that have acquired neo resistance have undergone homologous recombination with a targeting vector into which a gene cassette has been inserted in the mouse cathepsin Z gene region, and at least one of the mouse cathepsin Z gene alleles has been disrupted and the mouse cathepsin Z has been disrupted. Cannot be expressed normally. For the selection, an appropriate method is selected depending on the inserted gene cassette, and the introduction of the mutation can be confirmed by a method such as PCR, Southern hybridization or Northern hybridization.

The ES cells into which the mutation was introduced were C57BL / 6, BALB
/ c, injected into 8-cell stage embryos taken from ICR mice, etc.
Individuals can be grown by culturing for one day and then developing the blastocyst and transplanting it to a foster parent such as ICR. Born offspring mice are chimeric mice derived from ES cells having mutations and normal host embryos, and the amount of cells derived from ES cells is determined by the coat color of the individual. Therefore, it is desirable that the ES cell and the host embryo are a combination of lineages having different coat colors. Mutations in the obtained chimeric mice are heterozygous, and homozygous mutant mice can be obtained by crossing them appropriately.
The homozygous mutant mouse obtained in this manner has only the mouse cathepsin Z gene disrupted in all germ cells and somatic cells and does not express mouse cathepsin Z at all. Have. This knockout mouse is useful for analyzing the role of cathepsin Z in the life cycle of an individual such as development, growth, reproduction, aging and death, and the function of cathepsin Z in various organs and tissues, as compared with normal mice. In addition, it can be applied to drug development related to cysteine protease. Knockout mice can be used not only as model animals but also as a cell source for tissue culture, and can be used for functional analysis of cathepsin Z at the cell level. Techniques relating to knockout mice and the like include, for example, Mansour, SL, et al.,; Nature, 3
36: 348-352, 1988; Joyner, AL, ed .; Gene target
ing, IRL Press, 1993; Shinichi Aizawa, Generation of Mutant Mice Using Gene-Targeting ES Cells, Methods Described in Literatures such as Yodosha, 1995, or Methods Described in Literatures Cited There, It can be performed by a modified method.

According to the present invention, an antisense oligonucleotide (nucleic acid) capable of inhibiting the expression of the cathepsin Z gene is designed based on the nucleotide sequence information of the cloned or determined DNA encoding cathepsin Z. And can be synthesized. Such an oligonucleotide (nucleic acid) can hybridize with the mRNA of the cathepsin Z gene and can inhibit the function of the mRNA, or can inhibit the function of the cathepsin Z gene through an interaction with the cathepsin Z-related mRNA. Expression can be regulated and controlled. Oligonucleotides complementary to the selected sequence of the cathepsin Z-related gene and oligonucleotides capable of specifically hybridizing with the cathepsin Z-related gene regulate and control the expression of the cathepsin Z gene in vivo and in vitro It is also useful for treating or diagnosing diseases related thereto. The term "corresponding" means having homology or being complementary to a specific sequence of nucleotides, base sequences or nucleic acids including genes. "Corresponding" between nucleotides, base sequences or nucleic acids and peptides (proteins)
The term generally refers to the amino acids of a peptide (protein) as directed by the nucleotide (nucleic acid) sequence or its complement. 5 ′ end hairpin loop, 5 ′ end 6-base pair repeat, 5 ′ end untranslated region, polypeptide translation initiation codon, protein coding region, ORF translation initiation codon, 3 ′ end untranslated region, 3 ′ of the gene The terminal palindrome region and the 3′-end hairpin loop can be selected as preferred target regions, but any region within the gene can be selected as a target.

The relationship between the target nucleic acid and the oligonucleotide complementary to at least a part of the target region means the relationship between the target nucleic acid and the oligonucleotide capable of hybridizing with the target, which is “antisense”. It can be said. The antisense oligonucleotide has 2
Polydeoxynucleotides containing deoxy-D-ribose, polydeoxynucleotides containing D-ribose, other types of polynucleotides that are N-glycosides of purine or pyrimidine bases, or having a non-nucleotide backbone Other polymers (for example, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special bonds (provided that the polymer does not contain base pairing or base as found in DNA or RNA) (Including a nucleotide having a configuration that allows the attachment of nucleosides). They are,
It may be a double-stranded DNA, a single-stranded DNA, a double-stranded RNA, a single-stranded RNA, or a DNA: RNA hybrid, and may be further modified with an unmodified polynucleotide or an unmodified oligonucleotide, or a known modification. E.g., labeled, capped, methylated, substituted for one or more naturally occurring nucleotides with analogs, modified with intranucleotides, as known in the art. Those having an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.), those having a charged or sulfur-containing bond (eg, phosphorothioate, phosphorodithioate, etc.), For example, proteins (nucleases, nuclease inhibitors, toxins, antibodies, signal peptides, poly-L Those having side groups, such as lysine) or sugars (e.g., monosaccharides), those having an interactive compound (e.g., acridine, psoralen, etc.), chelating compounds (e.g., metals, radioactive Metals, boron, oxidizing metals, etc.), those containing an alkylating agent, and those having a modified bond (for example, α-anomeric nucleic acid). Here, "nucleoside", "nucleotide", and "nucleic acid" may include not only those containing known purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. The modified nucleosides and modified nucleotides may also be modified at the sugar moiety, e.g., one or more hydroxyl groups are replaced with halogens, aliphatic groups, etc., or converted to functional groups such as ethers, amines, etc. May have been.

The antisense nucleic acid of the present invention can be used for RNA, DNA
Or a modified nucleic acid. Specific examples of modified nucleic acids include, but are not limited to, sulfur derivatives and thiophosphate derivatives of nucleic acids, and those that are resistant to degradation of polynucleoside amides and oligonucleoside amides. The antisense nucleic acid of the present invention can be preferably designed according to the following policy. That is,
Making antisense nucleic acids more stable in cells,
Increase the cell permeability of the antisense nucleic acid, increase the affinity for the target sense strand, and, if toxic, reduce the toxicity of the antisense nucleic acid. Many such modifications are known in the art, for example, J. Kawakami et al., Pharm Tech Ja
pan, 8: 247, 1992; 8: 395, 1992; ST Crooke et a
l. ed., Antisense Research and Applications, CRC Pr
ess, 1993. The antisense nucleic acid of the present invention may contain altered or modified sugars, bases or bonds, may be provided in a special form such as liposomes or microspheres, may be applied by gene therapy, It can be provided in an added form. Such additional forms include polycations such as polylysine, which acts to neutralize the charge of the phosphate backbone, and lipids, which enhance the interaction with cell membranes and increase the uptake of nucleic acids ( For example, hydrophobic substances such as phospholipids and cholesterol are exemplified. Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chloroformate, cholic acid, etc.). These can be attached to the 3 'end or 5' end of the nucleic acid, and can be attached via a base, sugar, or intramolecular nucleoside bond. Examples of other groups include cap groups specifically arranged at the 3 ′ end or 5 ′ end of a nucleic acid for preventing degradation by nucleases such as exonuclease and RNase. Such capping groups include, but are not limited to, hydroxyl-protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.

The inhibitory activity of the antisense nucleic acid is determined by using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention,
Alternatively, it can be examined using an in vivo or in vitro translation system of cathepsin Z. The nucleic acid can be applied to cells by various methods known per se. As described above, the research results of the present inventors provide a method for transferring a human cathepsin Z gene and a recombinant DNA molecule into a host, expressing human cathepsin Z, and obtaining a desired human cathepsin Z. Thus, according to the present invention, a recombinant or transfectant that substantially expresses the human cathepsin Z gene, a method for producing the same, and uses thereof are also provided. In another aspect, the present invention provides a protein or a salt thereof, which has substantially the same activity as native cathepsin Z.
More preferably, a polypeptide having at least a part or all of the protein having substantially the same activity as human cathepsin Z or a salt thereof, or having substantially the same primary structure conformation, is obtained by using E. coli or the like. The present invention relates to nucleic acids such as DNA and RNA that can be expressed in prokaryotes or eukaryotes such as mammalian cells. Also such nucleic acids, especially DNs
A is (a) a sequence capable of encoding the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing or a sequence complementary thereto;
(B) a sequence capable of hybridizing with the DNA sequence of (a) or a fragment thereof, and (c) a sequence having a degenerate code capable of hybridizing with the sequence of (a) or (b). Can be Here, the conditions for hybridization can be stringent conditions. Prokaryotes such as Escherichia coli or eukaryotes such as mammalian cells which can be transformed with such a nucleic acid and express the polypeptide of the present invention also feature the present invention.

The antibodies (for example, antisera and monoclonal antibodies) according to the present invention can be used to immunize animals by a known method using human cathepsin Z or a fragment thereof obtained according to the present invention as an immunogen, and are known in the art. Alternatively, it can be produced by a commonly used method, for example, the method of Keller and Milstein (Nature, 256: 495-97, 1975). In this method, the immunogen may be natural human cathepsin Z, recombinant human cathepsin Z
And a fragment obtained from a synthetic peptide or protein having an amino acid sequence selected from a region having low homology to a cysteine protease belonging to another papain family in human cathepsin Z, for example, consisting of at least three consecutive amino acids. A synthetic peptide having a partial amino acid sequence of human cathepsin Z, for example, 4-250
, Preferably a synthetic peptide having an amino acid sequence having 6 to 45, more preferably 10 to 35 amino acid residues, preferably a peptide having a sequence portion characteristic of human cathepsin Z. Can be. For example, a propeptide region of human cathepsin Z (for example, a region from position 21 to position 61 in SEQ ID NO: 1 of the sequence listing, or a part thereof, or a vicinity thereof), a region containing a propeptide cleavage site ( For example, the region of position 58 to position 66 in SEQ ID NO: 1 of the sequence listing, or a part or vicinity thereof, the region of activated human cathepsin Z (for example, the region of SEQ ID NO: 1 in sequence listing 1) Regions 70th to 89th, 95th to 126th, 136th to 154th, 165th to 178th, 179th to 246th, 273th to 296th Or a part thereof or its vicinity).

Human cathepsin Z can be obtained from production cells inside and outside the body, for example, cultured cells, excised tissues, cultured tissues, and the like. For example, human leukocytes, colon, small intestine, ovary, testis, prostate, thymus, Tissues and cells such as spleen, pancreas, kidney, skeletal muscle, liver, lung, placenta, brain, heart, etc.
Leukemia such as leukemia HL-60, leukemia K-562, leukemia MOLT-4,
It can be obtained from Burkitt's lymphoma such as Raji, colon / rectal cancer such as SW480, lung cancer such as A549, and melanoma cell line such as G361. Furthermore, human cathepsin Z
Can be obtained as recombinant human cathepsin Z, and can be obtained from human cathepsin Z-producing cells and tissues as described above by using genetic recombination technology.
Human cathepsin Z prepared according to the present invention or one derived therefrom can be suitably used as an immunizing antigen. These human cathepsins Z can be obtained from various raw materials, for example, cultured cells, cultured tissues, etc., from antigen-producing materials such as transformant cells by a conventionally known method, for example, salting out such as ammonium sulfate precipitation, gel filtration by Sephadex, etc. Ion exchange chromatography using a carrier having a diethylaminoethyl group or a carboxymethyl group, etc., for example, a hydrophobic chromatography method using a carrier having a hydrophobic group such as a butyl group, an octyl group, a phenyl group, a dye gel chromatography It can be obtained by purification by a chromatography method, electrophoresis method, dialysis, ultrafiltration method, affinity chromatography method, high performance liquid chromatography method and the like. Preferably, it can be purified and separated by treatment with polyacrylamide electrophoresis, affinity chromatography in which an antibody specifically reacting with an antigen such as a monoclonal antibody is immobilized, or the like. Examples include gelatin-agarose affinity chromatography, heparin-agarose chromatography, and the like. The purified recombinant human cathepsin Z can be suitably used as an immunizing antigen for producing a monoclonal antibody.

Further, a peptide is synthesized based on the information of the human cathepsin Z gene obtained in the present invention, and the synthesized peptide can be suitably used as an immunizing antigen for producing an antibody (for example, a monoclonal antibody). Further, the antibodies (including monoclonal antibodies) can be appropriately labeled by a commonly used method. As the label, luminescent substances such as enzymes, radioisotopes (radioactive substances), and chemiluminescent compounds, fluorescent substances, metal colloids, prosthetic molecules, coloring substances, biotin, and the like can be used. Hereinafter, production of the antibody will be described in detail.

Needless to say, the monoclonal antibody of the present invention may be a monoclonal antibody obtained by using a cell fusion technique using myeloma cells. The monoclonal antibody of the present invention can be produced, for example, by the following steps. 1. 1. Preparation of immunogenic antigen 2. Immunization of animals with immunogenic antigens. 3. Preparation of myeloma cells (myeloma cells) 4. Cell fusion between antibody-producing cells and myeloma cells 5. Selection of hybridomas (fused cells) and monocloning Production of monoclonal antibodies

1. Preparation of immunogenic antigen As the antigen, recombinant human procathepsin Z prepared according to the method described above can be used.
Based on the determined information on cathepsin Z, an appropriate oligopeptide can be chemically synthesized and used as an antigen. Human cathepsin Z is a human procathepsin Z
And active human cathepsin Z can be used. Further, it may be an immunogenic conjugate or the like, but it can be used as it is by mixing it with an appropriate adjuvant to immunize an animal. For example, an antigen used as an immunogen is obtained by fragmenting human cathepsin Z, or selecting a characteristic sequence region based on an amino acid sequence deduced from a cloned and sequenced cDNA sequence, and designing a polypeptide. It may be a synthetic polypeptide fragment obtained by chemical synthesis. In addition, the fragment is bound to various carrier proteins via an appropriate condensing agent to form an immunogenic conjugate such as a hapten-protein, which can be used to react with only a specific sequence (or only a specific sequence). Can be used to design monoclonal antibodies. A cysteine residue or the like can be added to the designed polypeptide in advance so that the immunogenic conjugate can be easily prepared. Upon binding to the carrier proteins, the carrier proteins can first be activated. For such activation, introduction of an activation bonding group may be mentioned. Examples of the activated linking group include (1) activated ester or activated carboxyl group such as nitrophenyl ester group, pentafluorophenyl ester group, 1-benzotriazole ester group, N-succinimide ester group, and the like. A dithio group, for example, a 2-pyridyldithio group and the like can be mentioned. As carrier proteins, keyhole limpet hemocyanin (KLH)
Bovine serum albumin (BSA), ovalbumin, globulin, polypeptides such as polylysine, and bacterial cell components such as BCG.

2. Immunization of Animals with Immunogenic Antigen To immunize animals, for example, Shigeru Muramatsu, et al., Experimental Biology Course 14, Immunobiology, Maruzen Co., Ltd., 1985, Japan Biochemical Society, Seikagaku Experimental Course 5. Immunobiochemical research method, Tokyo Chemical Dojin, 1986, edited by The Japanese Biochemical Society, New Chemistry Experiment Course 12, Molecular Immunology III, antigen / antibody / complement, Tokyo Chemical Dojin, 1992, etc. It can be performed according to it. Adjuvants used with antigens include, for example, Freund's complete adjuvant, Ribi adjuvant, pertussis vaccine, BCG,
Lipid A, liposomes, aluminum hydroxide, silica and the like. Immunization is performed using an animal such as a mouse such as BALB / c. The dose of the antigen is, for example, about 1 to 400 μg / animal for a mouse, which is generally injected intraperitoneally or subcutaneously into a host animal, and thereafter intraperitoneally every 1 to 4 weeks, preferably every 1 to 2 weeks. 2-10 boosters, subcutaneous, intravenous or intramuscular
Repeat about once. BALB as a mouse for immunization
In addition to / c mice, F1 mice of BALB / c mice and other mice can also be used. If necessary, an antibody titer can be prepared, and the antibody titer can be measured to confirm the degree of animal immunity. The antibodies of the present invention may be those obtained from the immunized animals thus obtained, and include, for example, antisera, polyclonal antibodies and the like.

3. Preparation of myeloma cells (myeloma cells) As an infinitely proliferative cell line (tumor cell line) used for cell fusion, a cell line that does not produce immunoglobulin can be selected. For example, P3-NS-1-Ag4-1 ( NS-1, Eur.J. Immuno
l., 6: 511-519, 1976), SP2 / 0-Ag14 (SP2, Nature, 27
6: 269-270, 1978), P3-X63-Ag8-U1 derived from mouse myeloma MOPC-21 cell line (P3U1, Curr.
biol. Immunol., 81: 1-7, 1978), P3-X63-Ag8 (X63, Na
ture, 256: 495-497, 1975), P3-X63-Ag8-653 (653,
J. Immunol., 123: 1548-1550, 1979). 8-Azaguanine-resistant mouse myeloma cell line is prepared by adding antibiotics such as penicillin and amikacin, fetal calf serum (FCS), etc. to a cell culture medium such as Dulbecco's MEM medium (DMEM medium) and RPMI-1640 medium.
The cells are passaged in a medium supplemented with azaguanine (for example, 5 to 45 μg / ml), and can be passaged in a normal medium 2 to 5 days before cell fusion to prepare a required number of cell lines. In addition, the cell strain to be used should be completely thawed at about 37 ° C. and then washed three times or more with a normal medium such as RPMI-1640 medium.
A required number of cell lines may be prepared by culturing in a normal medium.

4. Cell fusion between antibody-producing cells and myeloma cells. An animal, for example, a mouse immunized according to the above step is spleen removed 2 to 5 days after the final immunization to obtain a spleen cell suspension. In addition to spleen cells, lymph node cells from various parts of the body can be obtained and used for cell fusion. The thus obtained spleen cell suspension and 3. The myeloma cell line obtained according to the steps of
(MEM medium), DMEM medium, RPMI-1640 medium, etc., and add a cell fusion agent such as polyethylene glycol. Other various cell fusion agents known in the art can be used, such as inactivated Sendai virus (HVJ: Hemagglutina
ting Virus of Japan). Preferably,
For example, 0.5% of 30-60% polyethylene glycol
~ 2 ml can be added, and the molecular weight is 1,000-8,
000 polyethylene glycol can be used,
Further, polyethylene glycol having a molecular weight of 1,000 to 4,000 can be more preferably used. The concentration of polyethylene glycol in the fusion medium is, for example, 30-60%.
It is preferable that If necessary, fusion can be promoted by adding a small amount of, for example, dimethyl sulfoxide. The ratio of spleen cells (lymphocytes) to myeloma cell line used for fusion may be, for example, 1: 1 to 20: 1, and more preferably 4: 1 to 7: 1.
It can be. The fusion reaction is performed for 1-10 minutes, and then a cell culture medium such as RPMI-1640 medium is added. The fusion reaction treatment can be performed plural times. After the fusion reaction treatment, the cells are separated by centrifugation or the like, and then transferred to a selection medium.

5. Selection and Monocloning of Hybridomas (Fused Cells) Examples of the selection medium include FCS-containing MEM medium containing hypoxanthine, aminopterin and thymidine, RPMI-1
A medium such as 640 medium, a so-called HAT medium can be used. The method of replacing the selective medium can be generally performed by adding an equal volume to the volume dispensed to the culture plate on the next day, and thereafter replacing half the amount with the HAT medium every 1 to 3 days. Changes can be made to this. On the 8th to 16th days after the fusion, the medium can be replaced with a so-called HT medium excluding aminopterin every 1 to 4 days. As a feeder, for example, mouse thymocytes can also be used, which may be preferred. The culture supernatant of the culture well in which the growth of the hybridoma is extensive can be analyzed by, for example, a radioimmunoassay (RIA), an enzyme immunoassay (ELISA), or a fluorescence immunoassay (FIA), or a fluorescence-induced cell separation device (FACS). Then, screening is performed by using human cathepsin Z or a fragment peptide thereof as an antigen, or by measuring a target antibody using a labeled anti-mouse antibody. The hybridoma producing the desired antibody is cloned. Cloning can be done by picking up colonies in an agar medium or by limiting dilution. It can be more preferably performed by a limiting dilution method. Cloning is preferably performed multiple times.

6. Production of Monoclonal Antibody The obtained hybridoma strain was prepared using a MEM medium containing FCS,
After culturing in an appropriate growth medium such as RPMI-1640 medium, a desired monoclonal antibody can be obtained from the culture supernatant. In order to obtain a large amount of antibodies, ascites of the hybridoma can be mentioned. In this case, each hybridoma is transplanted into the peritoneal cavity of a histocompatible animal of the same type as the myeloma cell-derived animal, and is grown or transplanted, for example, each hybridoma is transplanted into a nude mouse or the like,
The monoclonal antibody produced in the ascites of the animal can be recovered and obtained. Animals can be intraperitoneally administered mineral oil, such as pristane (2,6,10,14-tetramethylpentadecane), prior to hybridoma transplantation, after which the hybridomas are allowed to grow and ascites are collected You can also. Ascites fluid as it is or conventionally known methods such as salting out such as ammonium sulfate precipitation, gel filtration using Sephadex, etc., ion exchange chromatography, electrophoresis, dialysis, ultrafiltration, affinity chromatography And purified by high performance liquid chromatography or the like and used as a monoclonal antibody. Preferably, the ascites containing the monoclonal antibody is subjected to DEAE after ammonium sulfate fractionation.
-An anion exchange gel such as Sepharose and an affinity column such as a protein A column can be used for purification and separation. Particularly preferably, affinity chromatography, in which an antigen or an antigen fragment (for example, a synthetic peptide, a recombinant antigen protein or peptide, a site specifically recognized by an antibody, etc.) is immobilized,
And immobilization of affinity chromatography.

It is also possible to determine the sequence of the antibody obtained in such a large amount, and to prepare an antibody by a gene recombination technique using a nucleic acid sequence encoding an antibody obtained from a hybridoma strain. Furthermore, these antibodies are treated with enzymes such as trypsin, papain, and pepsin, and optionally reduced to obtain Fab, Fab ', F (ab') _2.
May be used. As the antibody to be labeled, an IgG fraction, and a specific binding portion Fab ′ obtained by digestion with pepsin and then reduction can be used. Examples of labeled substances in these cases include enzymes (peroxidase, alkaline phosphatase, β-D-galactosidase, etc.), chemical substances, fluorescent substances, radioisotopes, and the like, as described below. Detection and measurement in the present invention can be performed by immunostaining, for example, tissue or cell staining, immunoassay, for example, a competitive immunoassay or a non-competitive immunoassay, and a radioimmunoassay, ELISA, or the like can be used.
The measurement may be performed with or without BF separation. Preferred are a radioimmunoassay and an enzyme immunoassay, and further include a sandwich type assay. For example, in a sandwich-type assay, one of the antibodies to human cathepsin Z is detectably labeled. Another antibody that can recognize the same antigen is immobilized on a solid phase.

Incubation is performed to react the sample with the labeled antibody and the solid-phased antibody as necessary, and after separating the non-bound antibody, the labeled substance is measured. The amount of label measured is proportional to the amount of antigen, namely human cathepsin Z. This assay is referred to as a simultaneous sandwich-type assay, a forward sandwich-type assay, or a reverse sandwich-type assay, depending on the order of addition of the insolubilized antibody or the labeled antibody. For example, washing,
Stirring, shaking, filtration, or pre-extraction of the antigen, etc., are appropriately employed in these measurement steps under specific circumstances. Other measurement conditions such as the concentration of specific reagents and buffers, temperature and incubation time can be changed according to factors such as the concentration of antigen in the sample and the properties of the sample. A person skilled in the art can perform the measurement by appropriately selecting the optimal conditions effective for each measurement while using a usual experimental method.

Many carriers on which an antigen or an antibody can be immobilized are known, and in the present invention, they can be appropriately selected and used. As the carrier, various carriers used for antigen-antibody reactions and the like are known, and in the present invention, of course, any of these known carriers can be used. Particularly preferably used are, for example, glass, such as activated glass, porous glass, silica gel, silica-
Inorganic materials such as alumina, alumina, magnetized iron, magnetized alloys, polyethylene, polypropylene, polyvinyl chloride,
Crosslinking such as polyvinylidene fluoride, polyvinyl acetate, polymethacrylate, polystyrene, styrene-butadiene copolymer, polyacrylamide, cross-linked polyacrylamide, styrene-methacrylate copolymer, polyglycidyl methacrylate, acrolein-ethylene glycol dimethacrylate copolymer Natural or denatured cellulose such as albumin, collagen, gelatin, dextran, agarose, cross-linked agarose, cellulose, microcrystalline cellulose, carboxymethyl cellulose, cellulose acetate, polyamide such as cross-linked dextran, nylon, and organic polymers such as polyurethane and polyepoxy resin Substances and those obtained by emulsion polymerization of these substances, cells, erythrocytes, etc. Which are introduced are mentioned. Further, filter paper, beads, inner walls of test containers, for example, test tubes, titer plates, titer wells, cells made of synthetic materials such as glass cells, synthetic resin cells, glass rods, rods made of synthetic materials, and thicker ends. Alternatively, the surface of a solid material (object) such as a thinned rod, a rod having a round or flat projection at its end, or a thin plate-shaped rod may be used.

An antibody can be bound to these carriers, and preferably, a monoclonal antibody that specifically reacts with human cathepsin Z obtained in the present invention can be bound thereto. The binding between the carrier and those involved in the antigen-antibody reaction is performed by a physical method such as adsorption, a condensing agent, or a chemical method using an activated material, or a mutual method. It can be performed by a method utilizing a chemical binding reaction or the like. Labels include enzymes, enzyme substrates, enzyme inhibitors, prosthetic molecules,
Coenzyme, zymogen, apoenzyme, fluorescent substance, dye substance,
Examples include a chemiluminescent compound, a luminescent substance, a coloring substance, a magnetic substance, a metal particle such as a gold colloid, and a radioactive substance. As enzymes, dehydrogenase,
Oxidoreductases such as reductases and oxidases, for example, transferases that catalyze the transfer of amino groups, carboxyl groups, methyl groups, acyl groups, phosphate groups, etc., for example, ester bonds, glycoside bonds, ether bonds, peptide bonds Hydrolases, lyases, isomerases, ligases, and the like, which hydrolyze and the like. Enzymes can be used for detection by using a plurality of enzymes in combination. For example, enzymatic cycling can be used.

Representative radioisotope labeling isotopes include [ ³² P], [ ¹²⁵ I], [ ¹³¹ I], and [ ¹³¹ I].
³ H], [ ¹⁴ C], [ ³⁵ S] and the like. Representative enzyme labels include peroxidase such as horseradish peroxidase, galactosidase such as Escherichia coli β-D-galactosidase, maleate dehydrogenase, glucose-6-phosphate dehydrogenase, glucose oxidase, glucoamylase, acetylcholinesterase, catalase, and bovine. Alkaline phosphatase such as small intestine alkaline phosphatase and Escherichia coli alkaline phosphatase. When alkaline phosphatase is used, umbelliferone derivatives such as 4-methylumbelliferyl phosphate,
Using a phosphorylated phenol derivative such as nitrophenyl phosphate, an enzymatic cycling system using NADP, or a substrate such as a luciferin derivative or a dioxetane derivative, it can be measured by the resulting fluorescence or luminescence. Luciferin and luciferase systems can also be used. When catalase is used, it reacts with hydrogen peroxide to generate oxygen, and the oxygen can be detected by an electrode or the like. The electrode may be a glass electrode, an ion electrode using a poorly soluble salt film, a liquid film electrode, a polymer film electrode, or the like. The enzyme label can be replaced with a biotin label and an enzyme-labeled avidin (streptavidin). The sign may use a plurality of different kinds of signs. In such a case, it may be possible to make multiple measurements continuously or discontinuously, and simultaneously or separately.

In the present invention, 4-hydroxyphenylacetic acid, 1,2-phenylenediamine, tetramethylbenzidine and the like and horseradish peroxidase, umbelliferyl galactoside, nitrophenyl galactoside and the like and β-D-galactosidase are used for signal formation. Combinations of enzyme reagents such as glucose-6-phosphate dehydrogenase can also be used, and quinol compounds such as hydroquinone, hydroxybenzoquinone and hydroxyanthraquinone, thiol compounds such as lipoic acid and glutathione, phenol derivatives, ferrocene derivatives and the like can be used as enzymes. What can be formed by the action of can be used. Examples of the fluorescent substance or the chemiluminescent compound include fluorescein isothiocyanate, for example, rhodamine derivatives such as rhodamine B isothiocyanate and tetramethylrhodamine isothiocyanate, dansyl chloride, dansyl fluoride, fluorescamine, phycobiliprotein, acridinium salt, lumiferin, and luciferase. And luminols such as equorin, imidazole, oxalate, rare earth chelate compounds, coumarin derivatives and the like. Labeling can be performed using a reaction between a thiol group and a maleimide group, a reaction between a pyridyl disulfide group and a thiol group, a reaction between an amino group and an aldehyde group, and the like. The method can be applied by appropriately selecting from the methods that can be used, and the methods that modify them. In addition, a condensing agent that can be used for preparing the immunogenic complex, a condensing agent that can be used for binding to a carrier, and the like can be used.

Examples of the condensing agent include glutaraldehyde, hexamethylene diisocyanate, hexamethylene diisothiocyanate, N, N'-polymethylenebisiodoacetamide, N, N'-ethylenebismaleimide, ethyleneglycolbissuccinimidyl succinate , Bisdiazobenzidine, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, succinimidyl 3- (2-pyridyldithio) propionate (SPDP), N-succinimidyl
4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), N-sulfosuccinimidyl 4- (N-
(Maleimidomethyl) cyclohexane-1-carboxylate, N-succinimidyl (4-iodoacetyl) aminobenzoate, N-succinimidyl 4- (1-maleimidophenyl) butyrate, N- (ε-maleimidocaproyloxy) succinimide (EMCS ), Iminothiolane, S-acetylmercaptosuccinic anhydride, methyl-3- (4'-dithiopyridyl) propionimidate, methyl-4-mercaptobutyrylimidate, methyl-3-mercaptopropionimidate, N-succinimidyl -S-acetylmercaptoacetate and the like.

According to the measurement method of the present invention, a labeled antibody reagent such as a monoclonal antibody in which a substance to be measured is labeled with an enzyme or the like and an antibody bound to a carrier can be sequentially reacted, or simultaneously reacted. You can also. The order in which the reagents are added depends on the type of carrier system chosen. In the case of using sensitized beads such as plastic, a labeled antibody reagent such as a monoclonal antibody labeled with an enzyme or the like is first put together with a sample containing the substance to be measured in an appropriate test tube, and then the sample is put into an appropriate test tube. The measurement can be performed by adding beads such as sensitized plastic. In the quantification method of the present invention, an immunological measurement method is used.In this case, as a solid support, polystyrene, polycarbonate, polypropylene or polyvinyl balls that well adsorb proteins such as antibodies,
Various materials and forms such as a microplate, a stick, a microparticle, or a test tube can be arbitrarily selected and used. When measuring, select the optimal pH, for example, pH
It can be performed in a suitable buffer system to keep it at about 4-9. Particularly suitable buffers include, for example, acetate buffers, citrate buffers, phosphate buffers, Tris buffers, triethanolamine buffers, borate buffers, glycine buffers, carbonate buffers, Tris-HCl. Buffers and the like can be mentioned. Buffering agents can be used by mixing them at an arbitrary ratio. The antigen-antibody reaction is approximately 0 ° C to 60 ° C.
It is preferred to work at a temperature between 0 ° C.

An antibody reagent such as a monoclonal antibody labeled with an enzyme or the like, and an antibody reagent bound to a carrier;
In addition, the incubation of the substance to be measured
The reaction can be performed until equilibrium is reached, but at a much earlier time than the equilibrium of the antigen-antibody reaction is achieved, the solid phase and the liquid phase can be separated and the reaction can be stopped after a limited incubation treatment. The extent of the presence of a label, such as an enzyme, in either the phase or the solid phase can be measured. The measurement operation can be performed using an automated measurement device, and the measurement is performed by detecting a display signal generated by the substrate being converted by the action of an enzyme using a luminescence detector, a photo detector, or the like. You can also. In the antigen-antibody reaction, the reagent used, the substance to be measured, and further stabilize the label such as an enzyme,
Appropriate measures can be taken to stabilize the antigen-antibody reaction itself. In addition, proteins, stabilizers, surfactants, chelating agents, etc. should be added to the incubation solution to remove non-specific reactions and reduce inhibitory effects, or to activate measurement reactions. Can be added. As the chelating agent, ethylenediaminetetraacetic acid salt (EDTA) is more preferable. It may be subjected to a blocking treatment to prevent a non-specific binding reaction commonly used in the art or known to those skilled in the art, for example, normal serum proteins such as mammals,
Albumin, skim milk, fermented milk, collagen,
It can be treated with gelatin or the like. These methods can be used without any particular limitation as long as the purpose is to prevent a non-specific binding reaction.

As a sample to be measured by the measuring method of the present invention, any form of solution, colloid solution, non-fluid sample, etc. can be used, but a sample derived from a living organism such as thymus, testis, intestine, kidney, Brain, breast cancer, ovarian cancer, colorectal cancer, blood, serum, plasma, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, urine, other body fluid, cell culture, tissue culture, tissue homogenate, biopsy sample, Examples include a tissue and a cell. In applying these individual immunological assay methods to the assay method of the present invention, no special conditions, operations, and the like need to be set. The normal human cathepsin Z, which is a factor having cysteine protease activity of the present invention, or a human having substantially the same activity as the factor having cysteine protease activity of the present invention, by adding ordinary technical considerations to those skilled in the art to the ordinary conditions and operation methods in each method. What is necessary is just to construct the measurement system related to the protein of origin. For more information on these common technical measures,
Reviews and books can be referred to [for example, edited by Hiroshi Irie, "Radio Immunoassay", Kodansha, published in 1974; Hiroshi Irie, edited by "Radio Immunoassay", published in Kodansha, 1979; Eiji Ishikawa et al. Ed., “Enzyme Immunoassay”, Medical Shoin, published in 1978; Eiji Ishikawa et al., Ed., Enzyme Immunoassay (Second Edition), Medical Shoin, published in 1982; Eiji Ishikawa, et. Measurement Method "(3rd edition), Medical Shoin, published in 1987; HV Vunakis et al. (Ed.)," Meth
ods in Enzymology ", Vol. 70 (Immunochemical Techni
ques, Part A), Academic Press, New York (1980);
J. Langone et al. (Ed.), "Methodsin Enzymology",
Vol. 73 (Immunochemical Techniques, Part B), Acade
micPress, New York (1981); JJ Langone et al.
(ed.), "Methods in Enzymology", Vol. 74 (Immunochem
ical Techniques, Part C), AcademicPress, New York
(1981); JJ Langone et al. (Ed.), "Methods in En
zymology ", Vol. 84 (Immunochemical Techniques, Par
t D: SelectedImmunoassays), Academic Press, New Yo
rk (1982); JJ Langone et al. (ed.), "Methods in
Enzymology ", Vol. 92 (Immunochemical Techniques, P
art E: Monoclonal Antibodies and General Immunoass
ay Methods), Academic Press, New York (1983); JJ
Langone et al. (Ed.), "Methodsin Enzymology", Vo
l. 121 (Immunochemical Techniques, Part I: Hybrido
maTechnology and Monoclonal Antibodies), Academic
Press, New York (1986)). Epitope mapping can also be performed using the anti-cathepsin Z antibody of the present invention, in particular, a monoclonal antibody. If an antibody that recognizes each epitope is used, detection of proenzyme, active enzyme, etc.
Measurements can be made.

Thus, typically, it is an object of the present invention to use a human cathepsin Z gene, a probe derived therefrom, or a monoclonal antibody against human cathepsin Z, and a monoclonal antibody against human cathepsin Z for a solid support. Use, or, if necessary, an inhibitory substance against human cathepsin Z, an excellent method for detecting / fractionating and quantifying free pro-form or active human cathepsin Z or its gene in a test sample, and further producing cells; It is to provide a reagent kit for that purpose. The present invention detects such a free pro-form, active human cathepsin Z or its gene, and further detects a producing cell.
It is understood that each of the reagents in the reagent kit that can be differentially quantified is included in the embodiment. Furthermore, an object of the present invention is to detect and sort and quantify free human procathepsin Z or active human cathepsin Z or its gene using the above-mentioned method, and thereby to determine intracellular protein metabolism and hormone precursors. activation,
Role of cysteine proteases involved in many normal cellular processes such as bone and bone modification, and many diseases such as Alzheimer's disease, emphysema, rheumatoid arthritis, muscular dystrophy, osteoporosis, neurodegenerative diseases and cancer invasion and metastasis And to provide a reagent or a diagnostic agent that can be monitored for Therefore, various uses of the above reagents in the medical and physiological fields, and the use of the above reagents for the purpose of research, analysis and measurement of cells and tissues of animals such as humans such as tumors and cancers are all practicing the present invention. It is understood to be included in the embodiments.

The human cathepsin Z or a salt thereof of the present invention has protease activity and is considered to be an indispensable factor in, for example, protein metabolism, antigen presentation, bone modification, activation of hormone precursors, etc. in vivo. . Then, the protein is considered to be useful for treating human cathepsin Z-related dysfunction diseases that exhibit pathologies such as human cathepsin Z aplasia, cathepsin Z expression deficiency, and cathepsin Z gene deficiency. That is, human cathepsin Z,
Use of a medicament containing a mutant, a modified product, or a derivative makes it possible to bring a disease patient due to insufficient activity by cathepsin Z into a healthy state. The protein of the present invention is one of proteases, and is useful as a medicament such as an agent for treating and / or preventing various diseases caused when the expression level of the protein or the protease activity is reduced. Further, the protein of the present invention is useful as a medicament such as an agent for treating and / or preventing various diseases caused when the degrading activity for the protein is reduced. For example, in the case of a patient in which the biological activity in cells is not sufficiently obtained due to a decrease or deficiency of human cathepsin Z in a living body, or in a patient with an abnormal symptom, (A) the present invention (B) by administering a nucleic acid such as the DNA of the present invention to the patient to express the protein or the like of the present invention in a living body, or (C) By transplanting cells into which the nucleic acid such as the DNA of the present invention can be expressed so that the protein of the present invention can be replenished in a living body, the symptoms in the patient can be improved.

The compounds (agonists or promoters) of the present invention which promote the function of human cathepsin Z such as biological activity (eg, protease activity) or salts thereof may be used for human cathepsin Z dysfunction, emphysema, muscular dystrophy and the like. , Osteoporosis, Alzheimer's disease, neurodegenerative disease, rheumatoid arthritis, and various diseases such as cancer infiltration / metastasis. On the other hand, a compound (antagonist or inhibitor) or a salt thereof that inhibits a function such as a biological activity of human cathepsin Z (for example, protease activity) of the present invention or a salt thereof may be used for hyperhuman cathepsin Z function, pulmonary emphysema, muscular dystrophy, It can be used as a medicament such as an agent for treating and / or preventing various diseases such as osteoporosis, Alzheimer's disease, neurodegenerative disease, rheumatoid arthritis and cancer invasion / metastasis. For example, human cathepsin Z has a degrading activity on a cysteine protease substrate.
Inhibited by inhibitors specific for cysteine proteases. Thus, the inhibitor is cathepsin Z
Is useful as a medicament for the treatment of diseases caused by excessive degradation due to.

Thus, the protein such as human cathepsin Z of the present invention is a compound (agonist) that promotes a function such as biological activity (eg, protease activity) of the protein such as human cathepsin Z of the present invention. It is useful as a reagent for screening a compound (antagonist) or a salt thereof that inhibits or inhibits. Thus, using a protein such as human cathepsin Z of the present invention, a partial peptide thereof or a salt thereof, a protein such as the cathepsin Z of the present invention, a partial peptide thereof or a biological activity such as a salt thereof, etc. Compound promoting function (eg, protease activity) (agonist)
And a method of screening for a compound that inhibits or inhibits (antagonist) or a salt thereof. In the screening, for example, (i) a case where a substrate is brought into contact with the protein of the present invention, a partial peptide thereof, or a salt thereof (which may include a transformant expressing the protein; the same applies hereinafter); (ii) A comparison is made with the case where a substrate and a test sample are brought into contact with the protein of the present invention, some of its peptides, or salts thereof. Specifically, in the above-mentioned screening, the biological activity (for example, protease activity or the like) is measured and compared. In a preferred case, the reaction can be carried out in the presence of the antibody of the present invention. In the above cases (i) and / or (ii), the antibody of the present invention is used. As the substrate, any substrate can be used as long as it can serve as a substrate for the protein of the present invention. For example, casein, collagen, and synthetic oligopeptides can be used by selecting from those used for the purpose of measuring protease activity, but preferably a fluorescently-labeled synthetic peptide substrate for cysteine protease can be used, Examples of such peptide substrates include, for example, benzyl
oxycarbonyl-L-phenylalanyl-L-arginine-7-amido-4-me
thylcoumarin (Z-Phe-Arg-AMC) and the like. The substrate can be used as it is, but preferably a substrate labeled with a fluorescent, enzymatic or radioactive substance such as fluorescein can be used.

The test sample includes, for example, proteins, peptides, non-peptidic compounds, synthetic compounds, fermentation products, plant extracts, tissue extracts of animals and the like, cell extracts and the like. Examples of test compounds used in test samples may include cysteine protease inhibitors, compounds having inhibitory activity against the cysteine protease family, preferably synthetic compounds. These compounds may be novel compounds,
Known compounds may be used. The screening can be performed according to a usual method for measuring protease activity, for example, Biochemistry, 32, pp. 4330-4337 (1993)
The method can be carried out with reference to the method described in, for example. In addition, various labels, buffer systems and other appropriate reagents described in the above-mentioned measurement method using the antibody of the present invention can be used, or the measurement can be performed according to the operation described there. In the screening, the protein or the like of the present invention can be treated with an activator such as aminophenylmercuric acetate, or its pro form (precursor) or latent form can be converted into the active form in advance. . The measurement is usually performed in a buffer such as Tris-HCl buffer or phosphate buffer which does not adversely affect the reaction.
10 (preferably at a pH of about 6 to 8). In these individual screenings, human cathepsin Z of the present invention or a protein or peptide having substantially the same activity as the human cathepsin Z of the present invention is added to the ordinary conditions and operation methods of each method with ordinary technical considerations of those skilled in the art. What is necessary is just to build a related measurement system. For details of these general technical means, reference can be made to reviews, books, etc. (for example, Methods in Enzymol
ogy, Vol. 1,2, 5 & 6 (Preparation and Assay of Enz
ymes); Ibid, Vol. 3 (Preparationand Assay of Subst
rates); Ibid, Vol. 4 (Special Techniques for the E
nzymologist); Ibid, Vol. 19 (Proteolytic Enzymes);
Ibid, Vol. 45 (ProteolyticEnzymes, Part B);
Vol. 80 (Proteolytic Enzymes, Part C)
mic Press (USA)).

The compound or its salt obtained by using the screening method or the screening kit of the present invention can be a test compound such as a peptide, protein, non-peptide compound, synthetic compound, fermentation product, cell extract, plant It is a compound selected from extracts, animal tissue extracts, etc., and is a compound that promotes or inhibits the function of the protein or the like of the present invention. Examples of the salt of the compound include pharmaceutically acceptable salts. For example,
Examples thereof include salts with inorganic bases, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like. Preferred examples of the salt with an inorganic base include, for example,
Alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt,
And aluminum salts and ammonium salts. Preferred examples of salts with an organic base include, for example, trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine,
Salts with dicyclohexylamine, N, N'-dibenzylethylenediamine and the like are mentioned. Preferable examples of salts with inorganic acids include salts with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid and the like. Preferred examples of salts with organic acids include, for example, formic acid, acetic acid, propionic acid, fumaric acid,
Oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid,
Salts with malic acid, methanesulfonic acid, benzenesulfonic acid, benzoic acid and the like can be mentioned. Preferred examples of the salt with a basic amino acid include, for example, salts with arginine, lysine, ornithine, and preferred examples of the salt with an acidic amino acid include, for example, salts with aspartic acid, glutamic acid, and the like. Is mentioned.

Compounds or salts thereof that promote or inhibit the function of the protein or the like of the present invention can be used as safe and low-toxic treatments for various diseases, similarly to the above-mentioned protein of the present invention, partial peptides thereof or their salts. Or it is useful as a prophylactic agent. When a compound or a salt thereof obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned therapeutic and / or prophylactic agent, the protein of the present invention, its partial peptide or its salt may be used in a conventional manner. Can be implemented in accordance with Active ingredient of the present invention (e.g., (a) the protein of the present invention, a partial peptide thereof or a salt thereof,
(b) a nucleic acid such as a DNA of the present invention, (c) an antibody (including a monoclonal antibody) against a protein of the present invention or a partial peptide thereof, or a derivative thereof;
An antisense oligonucleotide against a nucleic acid such as NA, (d) a protein of the present invention, a compound or a salt thereof that promotes or inhibits the biological activity of a partial peptide or a salt thereof) as a medicament, For example, the protein such as human cathepsin Z of the present invention, a partial peptide thereof or a salt thereof is usually used alone or mixed with various pharmacologically acceptable adjuvants as a pharmaceutical composition or a pharmaceutical preparation. Can be administered. It is preferably administered in the form of a pharmaceutical preparation suitable for use such as oral administration, topical administration, or parenteral administration, and may be administered in any form (including inhalation or rectal administration) depending on the purpose. Good.

The parenteral administration form may include topical, transdermal, intravenous, intramuscular, subcutaneous, intradermal or intraperitoneal administration, but may also be administered directly to the affected area.
It is also suitable in some cases. Preferably orally to mammals including humans, or parenterally (eg, intracellular,
Intra-tissue, intravenous, intramuscular, subcutaneous, intradermal, intraperitoneal, intrathoracic, intraspinal, infusion, enema, rectal, ear, eye and nose, teeth, skin and mucous membranes, etc. ) Can be administered. Specific forms of pharmaceutical preparations include solution formulations,
Dispersed preparations, semisolid preparations, granular preparations, molded preparations, exudable preparations, etc., include, for example, tablets, coated tablets, sugar-coated preparations, pills, troches, hard capsules, soft capsules, microcapsules Capsules, implants, powders, powders, granules, fine granules, injections, solutions, elixirs, emulsions, irrigants, syrups, solutions, emulsions, suspensions, liniments, lotions , Aerosols, sprays,
Inhalants, sprays, ointments, plasters, patches, pastes, cataplasms, creams, oils, suppositories (eg, rectal suppositories), tinctures, skin solutions, eye drops, nasal drops, Powders for ear drops, liniments, infusions, injections, etc.
Lyophilized preparations, gel preparations and the like can be mentioned.

The pharmaceutical composition can be formulated according to a usual method. For example, if necessary, a physiologically acceptable carrier, a pharmaceutically acceptable carrier, an adjuvant, an excipient, a excipient, a diluent, a flavor, a flavor, a sweetener, a vehicle, a preservative, and a stable Agent, binder, p
H regulator, buffer, surfactant, base, solvent, filler,
Bulking agents, solubilizers, solubilizers, isotonic agents, emulsifiers, suspending agents, dispersants, thickeners, gelling agents, curing agents, absorbents,
Use adhesives, elasticizers, plasticizers, disintegrants, propellants, preservatives, antioxidants, sunscreens, humectants, emollients, antistatic agents, soothing agents, etc., alone or in combination, and By mixing the protein or the like of the present invention, it can be produced in a unit dosage form required for generally accepted practice of preparations. Formulations suitable for parenteral use include sterile solutions or suspensions of the active ingredients with water or other pharmaceutically acceptable vehicles, such as injections. In general, water, saline, aqueous dextrose, other related sugar solutions, and glycols such as ethanol, propylene glycol and polyethylene glycol are preferred liquid carriers for injections. When preparing an injection, distilled water, Ringer's solution, a carrier such as physiological saline, a suitable dispersant or wetting agent and a suspending agent, etc., using a method known in the art, , Suspensions and emulsions.

The aqueous solution for injection includes, for example, physiological saline, isotonic solution containing glucose and other auxiliary agents (eg, D-sorbitol, D-mannitol, sodium chloride, etc.). A suitable acceptable solubilizer, such as an alcohol (eg, ethanol),
It may be used in combination with a polyalcohol (eg, propylene glycol, polyethylene glycol, etc.), a nonionic surfactant (eg, Polysorbate 80 (TM), HCO-50, etc.). Examples of the oily liquid include sesame oil and soybean oil, which may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. Also, buffers (eg, phosphate buffer, sodium acetate buffer, etc.) or reagents for adjusting osmotic pressure, soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human Serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants such as ascorbic acid, absorption promoters and the like may be blended. The prepared injection is usually filled in a suitable ampoule.

For parenteral administration, in a sterile pharmaceutically acceptable liquid such as water, ethanol or oil, with or without the addition of surfactants and other pharmaceutically acceptable auxiliaries. In the form of a solution or suspension. Examples of the oily vehicle or solvent used in the preparation include natural or synthetic or semi-synthetic mono- or di- or triglycerides, natural, semi-synthetic or synthetic oils or fatty acids, such as peanut oil, corn oil, Vegetable oils such as bean oil and sesame oil are included. For example, this injection can be usually prepared so as to contain about 0.1 to 10% by weight of the compound of the present invention. Formulations suitable for topical, e.g., oral, or rectal use include, for example, mouthwashes, dentifrices, mouth sprays,
Examples include inhalants, ointments, dental fillers, dental coatings, dental pastes, suppositories and the like. Mouthwashes and other dental agents are prepared by a conventional method using a pharmacologically acceptable carrier. The oral spray or inhalant can be dissolved in a solution for aerosol or nebulizer together with the compound of the present invention itself or a pharmacologically acceptable inert carrier, or can be administered as fine powder for inhalation to teeth. The ointment is prepared by a conventional method by adding a commonly used base, for example, an ointment base (white petrolatum, paraffin, olive oil, macrogol 400, macrogol ointment, etc.).

Drugs for topical application to teeth and skin can be formulated as solutions or suspensions in suitably sterilized aqueous or non-aqueous vehicles. Additives include, for example, buffers such as sodium bisulfite or disodium edetate; preservatives including bactericidal and antifungal agents such as phenylmercuric acetate or benzalkonium chloride or chlorohexidine; And thickeners. Suppositories are carriers well known in the art, preferably non-irritating suitable excipients, such as polyethylene glycols, lanolin, cocoa butter, fatty acid triglycerides, etc., which are preferably solid at room temperature but at intestinal temperature. It is prepared by a conventional method using a liquid which melts in the rectum and releases the drug, and is usually prepared so as to contain about 0.1 to 95% by weight of the compound of the present invention. The drug, depending on the excipient and concentration used, can either be suspended or dissolved in the excipient. Adjuvants such as local anaesthetics, preservatives and buffering agents can be dissolved in the vehicle. Formulations suitable for oral use include, for example, solid compositions such as tablets, pills, capsules, powders, granules and troches, and liquid compositions such as solutions, syrups and suspensions. No. When preparing a formulation,
A formulation auxiliary known in the art is used. Tablets and pills can also be prepared with enteric coating. When the preparation unit form is a capsule, a liquid carrier such as oil and fat can be further contained in the above-mentioned type of material.

Further, when the nucleic acid such as the DNA of the present invention is used as a therapeutic and / or prophylactic agent as described above, the nucleic acid can be used alone, or it can be obtained by a genetic recombination technique as described above. It can be used by binding to an appropriate vector to be used, for example, a virus-derived vector such as a retrovirus-derived vector, or the like. The nucleic acid such as the DNA of the present invention can be administered by a commonly known method, and may be used as it is or together with a suitable auxiliary agent or a physiologically acceptable carrier so as to promote uptake into cells. And can be used as a pharmaceutical composition or a pharmaceutical preparation as described above. Also, a method known as gene therapy can be applied. The active ingredient of the present invention can be administered by selecting its dosage over a wide range.
Age, weight, general health, diet, administration time, administration method, excretion rate, combination of drugs, the degree of medical condition of the patient being treated at that time, and these or other factors are determined. . In the manufacture of pharmaceuticals, their additives and preparation methods are described in, for example, the Japanese Pharmacopoeia Commentary Editing Committee, 13th Revised Japanese Pharmacopoeia Commentary, published on July 10, 1996, Hirokawa Shoten Co., Ltd .; Ichigase Hisashi Other Development of Pharmaceuticals, Volume 12 (Formulation [I]), Heisei 2
Published on October 15, 2010, Hirokawa Shoten Co., Ltd .; Development of pharmaceuticals
Volume 12 (Pharmaceutical material [II]) published on October 28, 1990, can be applied by appropriately selecting from them according to need and referring to the description of Hirokawa Shoten Co., Ltd.

By utilizing the above-mentioned various aspects of the present invention, it is involved in the diagnosis of many diseases such as Alzheimer's disease, emphysema, rheumatoid arthritis, muscular dystrophy, osteoporosis, neurodegeneration and cancer invasion / metastasis. Various technical means can be provided as a test / measurement means useful for research or applied to other medical and physiological uses. In the description and drawings, the terms are based on IUPAC-IUB Commission on Biochemical Nomenclatu
re, or based on the meaning of a term commonly used in the art. The main abbreviations used below are shown. bp: base pair (s); E-64: trans-epoxysuccinyl-L-leucylamido- (4-guanidi
no) butane; EST: expressed sequence tag; GST: glutathione S-transferase; IPTG: isopropyl-1-thio-β-D-galactopyranoside; PAGE: polyacrylamide gel electrophoresis; PCR: polymerase chain reaction; SDS: sodium dodecyl sulfate; Z -Phe-Arg-AMC: benzyloxycarbonyl-L-phenylalanyl-L-
arginine-7-amido-4-methylcoumarin.

[0097]

The present invention will be described in detail with reference to the following examples. However, it is understood that the present invention is not limited to these examples, and that various embodiments based on the concept of the present specification are possible. It should be. In the following examples, unless otherwise specified, specific operations and processing conditions are not limited to those described in T. Maniatis et al., "Molec
ular Cloning ", 2nd ed., Cold Spring Harbor Laborat
ory, Cold Spring Harbor, NY (1989) and DM G
lover et al. ed., "DNA Cloning", 2nd ed., Vol. 1 t
o 4, (The Practical Approach Series), IRL Press, Ox
ford University Press (1995)
Erlich ed., PCR Technology, Stockton Press, 1989;
DM Glover et al. Ed., "DNA Cloning", 2nd ed.,
Vol. 1, (The Practical Approach Series), IRL Pres
s, Oxford University Press (1995) and MA Innis
et al. ed., "PCR Protocols", Academic Press, New Y
This method is performed according to the method described in ork (1990), and when commercially available reagents or kits are used, attached instructions (protocols) or attached chemicals are used.

Example 1 Screening of Human Brain and Prostate cDNA Libraries and Analysis of Base Sequence and Amino Acid Sequence (a) A sequence having homology to human cysteine protease
GenBank ^{TM for} human expressed sequence tags (ESTs)
Searched from the database. As a result, an ovarian cancer cDNA clone showing significant similarity to the existing cathepsin amino acid sequence (AA283747; registered by Hillier et al., WashU-Merck
EST Project) was identified. We searched for similar ESTs and found 20 overlapping ESTs. When these were reconstructed, a 1350 bp sequence having homology to the C-terminal region of the cysteine protease known hitherto was obtained. Based on this, a probe for searching for a novel cysteine protease was designed. A DNA fragment having the reconstructed ESTs sequence was prepared by performing cDNA PCR amplification from a commercially available brain cDNA library (Clontech, Palo Alto, CA). Λgt11-phage of brain cDNA library
Two specific primers designed based on overlapping ESTs using DNA as a template, 5'-CACCCGGAACCAGCACAT (primer Z1; SEQ ID NO: 3)
PCR was performed using 5′-CGATGGGGTCCCCAATG (primer Z2; SEQ ID NO: 4) to search for the presence of a sequence corresponding to the above-mentioned expected novel cysteine protease.
Primers were synthesized by the phosphoramidite method using a DNA synthesizer from Applied Biosystems (model 392A). The PCR reaction was performed using the Perkin-Elmer / Cetus Gene
Amp 2400 PCR system allows for denaturation (94 ° C, 15 seconds), annealing (56 ° C, 15 seconds) and extension (72 ° C, 60 seconds).
Performed in 35 cycles.

The resulting 708 bp PCR product was phosphorylated with T4 polynucleotide kinase (Boehringer Mannheim), inserted into a pBS vector cut with EcoRV (Boehringer Mannheim), and cloned. The nucleotide sequence of the cloned cDNA was analyzed, and it was confirmed that the sequence was identical to the expected sequence. This cDNA was excised from the vector and random-pri
Using ming kit ^{(Pharmacia), [α- 32 P} ] dCTP (3000
(Ci / mmol) (Amersham, UK). The radioactively labeled DNA fragment was used as a probe for screening a human brain cDNA library. Hybridization of the radioactive probe was performed with 6 x SSC (1 x = 1
50 mM NaCl, 15 mM sodium citrate, pH 7.0), 5 × De
nhardt's (1 × = 0.02% bovineserum albumin, 0.02%
polyvinyl pyrrolidone, 0.02% Ficoll), 0.1% SDS, 1
The test was performed at 55 ° C. for 18 hours in denatured herring sperm DNA at 00 μg / mL. 2x SSC, 0.1% SDS, 55 ° C, wash twice for 1 hour,
It was exposed to XAR-5 film (Kodak) at -70 ° C with an intensifying screen. About 1 × 10 ⁶ plaques were screened and four independent clones hybridizing with the probe were isolated. From these plaques, phage was purified, and the insert was ligated with EcoRI (Boehringer Mannheid).
m) Excised by digestion and subcloned into the EcoRI site of pUC18.

A DNA insert fragment cloned from the human brain cDNA library was cut out, inserted into the polylinker region of the phage vector M13mp19, and M13 universal primer or cDNA-specific primer.
Sequenase Version 2.0 kit (US Biochemicals, Cl
eveland, OH) and its nucleotide sequence was determined by the Sanger method. All sequence analysis was performed on both chains and confirmed. Computer analysis of DNA base sequences and protein amino acid sequences was performed at the University of Wisconsin Genetics Comp
This was done with the uter group's GCG software package. As a result of the sequencing, all four clones were determined to encode the same novel human cathepsin.
However, none of the isolated cDNA clones encoded the N-terminal side of this novel enzyme.

(B) In order to obtain the N-terminal base sequence of the novel enzyme, 85
The bp fragment was amplified by PCR to prepare a probe, and a human prostate cDNA library (Clontech, Palo Alto, CA) was screened. Hybridization of the radioactive probe was performed in the same manner as described above. About 1 × 10 ⁶ plaques were screened, one clone hybridizing with the 85 bp probe was isolated, and its DN was isolated.
The nucleotide sequence of the A insert was analyzed. The nucleotide sequence was analyzed in the same manner as described above. This positive clone contained a sequence about 300 bp further upstream from the 5'-end of the brain cDNA clone, and the N-terminal nucleotide sequence of the novel enzyme could be determined. Computer analysis of the 5'-terminal extension nucleotide sequence revealed a 909 bp open reading frame starting at ATG at position 241 and ending at TAA at position 1150 (FIG. 1 and SEQ ID NO: 1). The protein encoded by this open reading frame was composed of 303 amino acid residues, and was predicted to have a molecular weight of 33,881 (excluding the molecular weight of the added sugar chain). The homology between the obtained amino acid sequence and the known human cysteine protease varied from 34% for cathepsin C to 26% for cathepsin B. Figure 2
FIG. 4 shows a diagram in which the amino acid sequences of the novel protein and the known cysteine protease are aligned based on homology. Although the homology between the amino acid sequence encoded by the obtained cDNA clone and the known cysteine protease was not high, the novel protein had all the structural features typical of other cysteine proteases. That is, the sequence rich in hydrophobic amino acids present at the N-terminus corresponds to the signal peptide of the secretory enzyme. At the end of the 20-residue signal sequence is the Ala-Gly-Ala sequence that perfectly matches the Ala-X-Ala cleavage site motif for eukaryotic preproproteins. The protein cleaved at this predicted cleavage site consists of 283 amino acid residues.

The active form of the novel protein can be identified from the pro-region of the known cysteine protease and the cleavage site of the active form enzyme. As shown in FIGS. 2 to 4, the N-terminus of the active cysteine protease has, in all cases, the second amino acid is proline. According to this, it is presumed that the active form of the novel protein starts at the leucine residue at position 62 and has a molecular weight of 27,280 (excluding the molecular weight of the sugar chain to be added). Furthermore, the novel protein has a cysteine residue present in the active site of cysteine protease, a histidine residue and an asparagine residue which are important for the catalytic mechanism at positions 92 and 24, respectively.
It had 1st and 261st places. These three amino acid residues and their neighboring sequences are very well conserved compared to other cathepsins (Kamphuis, IG, et al.,
J. Mol. Biol. 182: 317-329, 1985; Musil, D., et a.
l., EMBO J. 10: 2321-2330, 1991; Coulombe, R., et.
al., EMBO J. 15: 5492-5503, 1996). In addition, in the N-terminal region, a glutamine residue (position 87) forming an oxyanion hole, a tryptophan residue and a hydrophobic segment are located immediately adjacent to the cysteine active site.
In addition, the C-terminal region contains a series of conserved aromatic residues and the Asn-Ser-Trp tripeptide within the active site. The novel protein has a potential N-linked glycosylation sequence (Asn-Tyr-Thr) at positions 184 and 224 of the mature region. Because all mammalian cysteine proteases reported to date have been glycosylated, there is at least one glycosylation required for lysosomal induction by the mannose-6-phosphate receptor system. It is estimated that.

Summarizing all these structural features, it can be concluded that the novel protein is a novel human cysteine protease. This new human cysteine protease was named "Cathepsin Z". This nucleic acid sequence has an EMBL accession number: A
Registered as F032906. Cathepsin Z has three characteristic amino acid sequence insertions. The first is the glutamine residue of the oxyanion hole (87th
His-Ile-Pro that exists in the immediate vicinity of The other two are 8-residues located almost in the middle of the molecule and 14-
Residue. The most characteristic structure of cathepsin Z is a short propeptide not found in other cysteine proteases. All cysteine proteases are synthesized as inactive precursors with an N-terminal propeptide that functions as an endogenous inhibitor of proteolytic activity. This propeptide is required for correct refolding in certain cysteine proteases and contributes to stability to pH environment (Tao, K., et al., Ar
ch. Biochem. Biophys. 311: 19-27, 1994; Carmona,
E., et al., Biochemistry 35: 8149-8157, 1996).

Although the homology between the cysteine protease propeptides is low, they can be divided into two groups according to their structural characteristics (Karrer, KM, et al., Proc. N
atl.Acad. Sci. USA 90: 3063-3067, 1993; Vernet,
T., et al., J. Biol. Chem. 270: 10838-10846, 199.
Five). The first is a group consisting of cathepsin L-like enzymes,
It has a pro-region consisting of more than 90 residues including two motifs called ERFNIN and GNFD. The second is a group consisting of cathepsin B, which has a pro-region of approximately 60 residues lacking the ERFNIN motif. However, cathepsin Z
The pro region consists of only 41 residues and lacks both motifs. In the pro-region of other cysteine proteases, the structure consisting of lysine is required for the addition of mannose-6-phosphate (Cuozzo, JW, et al., J. Biol. Chem.
270: 15611-15619, 1995) Nevertheless, the proregion of cathepsin Z does not contain any lysine residues. From these structural features, the pro-region of cathepsin Z is:
It deviates from the existing family and is considered to belong to a different subfamily.

Example 2: Chromosome mapping of human cathepsin Z Human P1 artificial chromosome (PAC) library (Human Genome Ma
The hybridization was performed using the full-length cDNA of cathepsin Z as a probe. Three independent clones were isolated as containing Cathepsin Z by PCR and Southern blot. DNA is prepared from these PAC clones by the alkaline lysis method,
Used for fluorescence in situ hybridization (FISH) mapping. 2 μg of PAC DNA was nick-translated in the presence of biotin-16-dUTP and hybridized with metaphase chromosomes prepared from normal male fetohemagglutinin-stimulated lymphocytes. The labeled probe was detected with two avidin-dyes. The chromosomes are banded with diamidine-2-phenylindole dihydrochloride (DAPI) and analyzed by a CCD camera.
(Photometrics) and a fluorescence microscope (Zeiss) connected. On the chromosome banded with DAPI, the yellow fluorescent signal derived from the biotinylated PCA clone is
3 (Fig. 5). It is already known that this site encodes adenosine deaminase, prostacyclin synthase or phosphoenol pilebate carboxidase (H
onig, J., et al., Ann.Hum.Genet. 48: 49-56, 198.
4; Yokoyama, C., et al., Genomics 36: 296-304, 199
6; Yu, H., et al., Genomics 15: 219-221, 1993). However, no previously reported subfamily of cysteine proteases was present at this chromosomal site.

Example 3 Construction of Expression Vector and Expression in Escherichia coli In order to express recombinant cathepsin Z in Escherichia coli, a 727 bp DNA fragment containing a sequence encoding active human cathepsin Z was prepared. Using the clone obtained in Example 1 as a template, using the following primers: PCR amplified. The PCR reaction uses Expand ^TM Long Template PCR to reduce errors.
System (Boehringer Mannheim), denaturation (95 ° C,
30 seconds), annealing (56 ° C, 30 seconds) and elongation (72
C., 1 minute) for 30 cycles. The PCR product is phosphorylated with T4 polynucleotide kinase (Boehringer Mannheim) and Klenow fragment (Boehringer Mannheim)
Repair with SmaI and alkaline phosphatase
(Boehringer Mannheim) -treated expression vector pGEX-3
X (Pharmacia LKB). Obtained plasmid
(pGEX-3X CathZ) was used to transform E. coli BL21 (DE3) strain. The transformant was cultured in LB medium containing 100 μg / mL ampicillin at 37 ° C. for about 16 hours, then diluted 1/100 in the same medium, and further grown until A ₆₀₀ reached 1.0. Isopropyl-1-thio-β-D-galactopyranoside (IPTG)
Was added at a final concentration of 1 mM and the culture was continued for a further 3 hours. The cells were collected by centrifugation, washed and resuspended in 0.05 volume of PBS, lysed with a French press, and centrifuged at 20,000 g (4 ° C., 20 minutes). The soluble extract of the supernatant was subjected to glutathione Sepharose 4B, and glutathione elution buffer (50 mM Tris-H containing 10 mM reduced glutathione) was used.
Cl pH 8.0). E. coli extracts and purified recombinant proteins were analyzed by SDS-PAGE. As seen in FIG. 6, E. coli transformed with the recombinant plasmid pGEX-3X CathZ contained a fusion protein of about 52 kDa that was not found in E. coli transformed with the control plasmid pGEX-3X. A 29 kDa band derived from glutathione-S-transferase (GST) was observed in the control E. coli extract. The purified fusion recombinant cathepsin Z is purified by SDS-PA
It showed a single band of the size expected for GE (about 52 kDa). E. coli DH5α with plasmid (pGEX-3X CathZ)
The strain was transformed. A transformant "pGEX-3X CathZ" was obtained.

Example 4 Analysis of Enzyme Activity of Recombinant Cathepsin Z Enzyme activity of cathepsin Z produced and purified in Escherichia coli was determined by using 20 μM Z-Phe-Arg-AMC and Z-Arg-Arg-A as substrates.
Using MC or Z-Arg-AMC (Bubendorf, Switzerland), Barrett and Kirschke (Barrett, AJ, et al., M
ethods Enzymol., 80: 535-561, 1981). The analysis was performed at 30 ° C. with 8 mM dithiothreitol, 2 mM EDTA, and 100 mM containing 0.05% Brij35.
Performed with sodium acetate buffer pH 5.5. Substrate hydrolysis was performed using the Cytofluor 2350 fluorometer (Millipore, Bedfor
d, MA) at an excitation wavelength of 360 nm and a fluorescence wavelength of 460 nm. Inhibition analysis was performed using recombinant cathepsin Z and 20 μM E-64 (S
igma, St. Louis, MO) was previously incubated at 30 ° C. for 15 minutes, and the residual activity of recombinant cathepsin Z was measured using the fluorescent substrate Z-Phe-Arg-AMC as described above. As a positive control for enzyme activity measurement, recombinant cathepsin O (Velasco, G., et al., J. Biol.
m., 269: 27136-27142, 1994). Recombinant cathepsin Z is 3 μmol / min / m to synthetic peptide Z-Phe-Arg-AMC, which is the optimal substrate for other cysteine proteases.
ol enzyme. The substrate degradation activity was slightly higher than the activity of recombinant human cathepsin O measured under the same conditions (FIG. 7).

[0108]

Embedded image

Further, a fluorescent substrate Z-Arg-A of cathepsin Z
The activity against rg-AMC and Z-Arg-AMC was practically undetectable. E-64 is an inhibitor that specifically inhibits cysteine protease. Cathepsin Z substrate degradation activity against Z-Phe-Arg-AMC was inhibited by E-64,
EDTA, a metalloprotease, serine protease and aspartic protease inhibitor, respectively
Phenylmethylsulfonyl fluoride and pepstatin A were not inhibited. These enzymatic analyzes revealed that the cloned cDNA encodes a true cysteine protease with substrate specificity and inhibitors specific to the cysteine protease family. Similarly, an enzyme inhibitor can be searched using cathepsin Z of the present invention.

Example 5: Cathepsin Z expression analysis in tissues and cancer cell lines 2 μg of poly (A) ⁺ RN from different human tissues and cancer cell lines
A or 20μg of total RNA isolated from cancer tissue
Was subjected to Northern blot analysis. Prehybridization was performed using 50% formamide, 5 × SSPE (1 × =
150 mM NaCl, 10 mM NaH ₂ PO ₄ , 1 mM EDTA, pH 7.4), 1
The test was performed at 42 ° C. for 3 hours in denatured herring sperm DNA at 0 × Denhardt's, 2% SDS and 100 μg / mL. After pre-hybridization, the filter is 3 'of human cathepsin Z.
Hybridization was carried out using a radioactively labeled probe of a FokI / EcoRI digested fragment of 270 bp specific to the -side untranslated region portion. This probe showed the lowest homology of less than 20% to other cathepsin families and was able to reduce cross-reactivity. After hybridization, filter the cells in 0.1X SSC, 0.1% S at 50 ° C.
Washed with DS for 2 hours and subjected to autoradiography.
Northern blot analysis of poly (A) ⁺ RNA from various human tissues revealed that all tissues examined (white blood cells, colon, small intestine, ovary, testis, prostate, thymus, spleen, pancreas, kidney, skeletal muscle, liver, One transcript of about 1.7 kb was strongly expressed in the lung, placenta, brain and heart) (FIG. 8).

The broad distribution of cathepsin Z is consistent with cathepsin Z being a lysosomal enzyme involved in the digestion of intracellular proteins that occurs in all cells.
Cathepsin Z is similar in that it is widely expressed in human tissues like cathepsins B, L, H and O, which have a housekeeping role like lysosomal enzymes,
Cathepsins K, S, W and L2 are distinguished from those that are over- or exclusively expressed in specific tissues. By the way, cathepsins K, S, W and L2 are each isolated from osteoclasts (Inaoka, T., et al., Biochem. Biophys. Res. Co.
mmun., 206: 89-96, 1995; Gelb, BD, et al., Scien
ce, 273: 1236-1238, 1996), lymphoid tissue (Shi, GP,
et al., J. Biol. Chem., 267: 7258-7262, 1992), T-
Lymphocytes (Linnevers, C., et al., FEBS Lett., 405: 2
53-259, 1997) and thymus and testis (Santamaria, I, et a
l., Cancer Res., 58: 1624-1630, 1998), and bone remodeling (cathepsin K)
It is involved in antigen presentation (cathepsin S), regulation of cytolytic activity of T-lymphocytes (cathepsin W), and immune response and fertilization process (cathepsin L2).

On the other hand, as a result of Northern blot analysis of poly (A) ⁺ RNA derived from a cancer cell line, most cell lines examined (leukemia HL-60, HeLa cell S3, leukemia K-562, leukemia MOLT-4, Burkitt lymphoma Raji, colorectal cancer
One transcript of about 1.7 kb was strongly expressed in SW480, lung cancer A549 and melanoma G361) (FIG. 9), indicating that cathepsin Z may be overexpressed by human tumors. In fact, examination of cathepsin Z expression in various cancer tissues revealed that this gene was widely expressed in various tumors (FIG. 9). From these facts, it is considered that cathepsin Z is one of the proteolytic enzymes involved in the transformation of human cells into malignant transformation. Similarly, cancers or tumors can be detected and measured using the nucleic acids disclosed in the present invention.

Example 6 Preparation of Monoclonal Antibody (a) Immunogen The purified recombinant recombinant cathepsin Z obtained in Example 3 can be used for immunization. Alternatively, a recombinant protein obtained by treating the fusion recombinant cathepsin Z with factor Xa and removing the glutathione-S-transferase (GST) portion can be used. Further, as the antigen used for immunization, it is possible to use recombinant cathepsin Z obtained by ligating the cDNA obtained in Example 1 to an animal cell expression vector and expressing this in CHO cells or COS cells. . These antigenic proteins can be purified by ion exchange, gel filtration or various other types of chromatography. The purified antigen for immunization is immunized by a general method, antibody-producing cells are induced, and the antibody-producing cells can be obtained as hybridomas by cell fusion. Furthermore, based on the reactivity with the purified antigen for immunization, it can be cloned and established as a monoclonal antibody-producing hybridoma. Since the hybridoma obtained using the fused recombinant cathepsin Z as an immunogen contains an antibody-producing strain against GST, the antibody-producing hybridoma that reacts with GST must be removed. As an immunogen for obtaining a cathepsin Z-specific monoclonal antibody, a haptenylated synthetic peptide having an amino acid sequence characteristic of cathepsin Z can be used. Human cathepsin
Positions 21 to 61 of the Z propeptide region (SEQ ID NO: 7),
Activated human cathepsin Z region at positions 70-89 (SEQ ID NO: 8), 95-126 (SEQ ID NO: 9), 136-
Positions 154 (SEQ ID NO: 10), Positions 165 to 178 (SEQ ID NO: 11), Positions 179 to 246 (SEQ ID NO: 12), Positions 273 to 296 (SEQ ID NO: 13) ) Has low homology with other cathepsins. These polypeptides, and synthetic peptides containing sequences near and in the vicinity of the polypeptide, are suitable as immunogens for obtaining cathepsin Z-specific monoclonal antibodies.

(B) Preparation of Antigen Polypeptide The amino acid sequence of human cathepsin Z shown in SEQ ID NO: 1 has lower homology to other human cathepsin families,
The following sequences were selected and synthesized as sequences characteristic of human cathepsin Z. [SEQ ID NO: 14] AsnTyrAlaSerIleThrArgAsnGlnHisIleProGlnTyrCys (sequence of Asn75 to Cys89 of SEQ ID NO: 1; “CathZ # 1”
[SEQ ID NO: 15] TrpArgValGlyAspTyrGlySerLeuSerGlyArgGluLysMet (Sequence of Trp188 to Met202 in SEQ ID NO: 1 in Sequence Listing; “Cath
Z # 2) [SEQ ID NO: 16] ThrTyrLysAspGlyLysGlyAlaArgTyrAsnLeuAlaIleGluGluHi
sCys (sequence of Thr279 to Cys296 in SEQ ID NO: 1; “Cath
Z # 3). These polypeptides were synthesized using a peptide synthesizer (Peptide Synthesizer 9600, MilliGen / Biosearch).
It was synthesized by the oc-bop method. Cysteine was introduced into the N-terminus of the polypeptide CathZ # 2. The synthesized peptide is μBondas
Purified by high performance liquid chromatography using a C18 column (Waters).

(C) Preparation of complex of polypeptide and BSA (1) Bovine serum albumin (BSA) via cysteine residue
To form an antigen conjugate. 10.1mg BSA
Was dissolved in 1 mL of 0.1 M phosphate buffer, pH 7.0 and 1.
14mg EMCS (N- (ε-maleimidecaproyloxy) -succinimide)
Was mixed with 24.9 μL of dimethylformamide, and reacted at 30 ° C. for 30 minutes.
Sephadex G-25 equilibrated with 0.1 M phosphate buffer, pH 7.0
(Pharmacia) Gel filtration was performed on a gel column (diameter 13 mm, length 120 mm). The resulting maleimide-bound BSA concentration was 3.02 mg.
/ mL. The polypeptide CathZ # synthesized in the above (b)
1 was dissolved in 0.1 M phosphate buffer, pH 7.0, and mixed with maleimide-bound BSA in a 50-fold molar amount. That is, 1440 nm
ol of the polypeptide CathZ # 1 was mixed with 28.8 nmol of maleimide-bound BSA, and the total amount was 1 mL, followed by incubation at 4 ° C. for 20 hours to prepare a BSA-polypeptide complex. The protein concentration of the obtained BSA-polypeptide complex is 4.6 mg / mL.
Met. This is 200 μg / 0.1M in 0.1 M phosphate buffer, pH 7.0.
After diluting to 150 μL, the mixture was dispensed in 150 μL aliquots and stored frozen at -30 ° C.

(2) The antigen was conjugated with bovine serum albumin (BSA) via a cysteine residue.
11.6 mg of BSA dissolved in 1 mL of 0.1 M phosphate buffer, pH 7.0, and 1.31 mg of EMCS (N- (ε-maleimidecaproyloxy)-
succinimide) in 31.3 μL of dimethylformamide, and reacted at 30 ° C. for 30 minutes.Then, the above mixture was equilibrated with 0.1 M phosphate buffer, pH 7.0
phadex G-25 (Pharmacia) gel column (diameter 13 mm, length
(90 mm). The resulting maleimide-bound BSA concentration was 2.70 mg / mL. The polypeptide CathZ # 2 synthesized in (b) was dissolved in 0.1 M phosphate buffer, pH 7.0,
A 50-fold molar amount was mixed with the maleimide-bound BSA. That is, 27.2 nmol for 1358 nmol of the polypeptide CathZ # 2
Maleimide-bonded BSA mixed at 4 ° C, 20 mL
After incubation for a time, a BSA-polypeptide complex was prepared. The protein concentration of the obtained BSA-polypeptide complex was 4.3 mg / mL. This was added to 0.1 M phosphate buffer, pH 7.
Dilute to 200 μg / 150 μL with 0, then 150 μl
Each L was dispensed and stored frozen at -30 ° C.

(3) The antigen was conjugated to bovine serum albumin (BSA) via a cysteine residue.
10.1 mg of BSA dissolved in 1 mL of 0.1 M phosphate buffer, pH 7.0, and 1.14 mg of EMCS (N- (ε-maleimidecaproyloxy)-
succinimide) in 24.9 μL of dimethylformamide, and reacted at 30 ° C. for 30 minutes.Then, the above mixture was equilibrated with 0.1 M phosphate buffer, pH 7.0
phadex G-25 (Pharmacia) gel column (diameter 13 mm, length
(120 mm). The concentration of the obtained maleimide-bound BSA was 3.02 mg / mL. The polypeptide CathZ # 3 synthesized in the above (b) was dissolved in 0.1 M phosphate buffer, pH 7.0,
A 50-fold molar amount was mixed with the maleimide-bound BSA. That is, 24.2 nmol for the polypeptide CathZ # 3 of 1210 nmol.
Maleimide-bonded BSA mixed at 4 ° C, 20 mL
After incubation for a time, a BSA-polypeptide complex was prepared. The protein concentration of the obtained BSA-polypeptide complex was 4.1 mg / mL. This was added to 0.1 M phosphate buffer, pH 7.
Dilute to 200 μg / 150 μL with 0, then 150 μl
Each L was dispensed and stored frozen at -30 ° C.

(D) Preparation of antibody-producing cells-1 (1) 187 μg of the BSA-polypeptide CathZ # 1 complex prepared in (c) above was added together with complete Freund's adjuvant.
Weekly Balb / c female mice were intraperitoneally administered and immunized for the first time. 18
On the day, mice that had been initially immunized were intraperitoneally administered with 200 μg of BSA-polypeptide complex dissolved in 0.1 M phosphate buffer, pH 7.5, and boosted. Further, on day 52, 200 μg of BSA-polypeptide complex dissolved in 0.1 M phosphate buffer, pH 7.5
Was administered intravenously to give a final immunization. Four days later, the spleen was excised and a spleen cell suspension was prepared. Immunization was performed on two mice.

(2) A 6-week-old Balb / c female mouse was intraperitoneally administered with 200 μg of the BSA-polypeptide CathZ # 2 conjugate prepared in the above (c) together with complete Freund's adjuvant, for the first immunization. On day 21, mice that had been initially immunized were intraperitoneally administered with 200 μg of BSA-polypeptide complex dissolved in 0.1 M phosphate buffer, pH 7.5, and boosted. 0.1M on day 56
200 μg of the BSA-polypeptide complex dissolved in phosphate buffer, pH 7.5, was intravenously administered for final immunization. Three days later, the spleen was removed to prepare a spleen cell suspension. Immunity is
Performed on two mice.

(3) 200 μg of the BSA-polypeptide CathZ # 3 complex prepared in the above (c) was intraperitoneally administered to a 6-week-old Balb / c female mouse together with complete Freund's adjuvant for the first immunization. On day 18, mice that had been initially immunized were intraperitoneally administered with 200 μg of BSA-polypeptide complex dissolved in 0.1 M phosphate buffer, pH 7.5, and boosted. 0.1M on day 52
200 μg of the BSA-polypeptide complex dissolved in phosphate buffer, pH 7.5, was intravenously administered for final immunization. Three days later, the spleen was removed to prepare a spleen cell suspension. Immunity is
Performed on two mice.

(E) Cell fusion-1 The following materials and methods were used for cell fusion. RPMI-1
640 medium: RPMI-1640 (Flow Lab.) With sodium bicarbonate (24 mM), sodium pyruvate (1 mM), penicillin G
Potassium (50 U / ml) and amikacin sulfate (100 μg / ml) were added, the pH was adjusted to 7.2 with dry ice, and sterile filtration was performed with a 0.2 μm Toyo membrane filter. NS-1 medium: RPMI above
Fetal bovine serum (FCS, MA Bi
oproducts) to a concentration of 15% (v / v). PE
G-4000 solution: RPMI-1640 medium with polyethylene glycol
A serum-free medium was prepared by adding -4000 (PEG-4000, Merk & Co.) to 50% (w / w). Fusion with 8-azaguanine-resistant myeloma cell SP2 (SP2 / 0-Ag14)
Method in culture immunology p351-372 (ed.BB
Mishell and SN Shiigi), WH Freeman and Compa
The method of Oi et al. described in ny (1980) was slightly modified.

The nucleated splenocytes (viable cell rate 100%) and myeloma cells (viable cell rate 100%) prepared in (1) of (d) were fused in the following procedure at a ratio of 5: 1. The polypeptide-immunized spleen cell suspension and myeloma cells were converted to RPMI1640
Washed with medium. Next, the cells were suspended in the same medium, and nucleated splenocytes and myeloma cells were mixed for fusion. That is, 8.0 × 10 ⁷ myeloma cells were mixed with 4.0 × 10 ⁸ nucleated spleen cells. Further, the nucleated splenocytes (viable cell rate: 100%) and myeloma cells (viable cell rate: 100%) prepared in (2) of (d) were fused by the following procedure at a ratio of 5: 1. The polypeptide-immunized spleen cell suspension and myeloma cells were each washed with RPMI1640 medium. Next, the cells were suspended in the same medium, and nucleated splenocytes and myeloma cells were mixed for fusion. That is, 5.0 × 10 ⁸ nucleated splenocytes were mixed with 1.0 × 10 ⁸ myeloma cells. The nucleated splenocytes prepared in (3) of (d) (viable cell rate 100%)
And myeloma cells (100% viable cell ratio) were fused in the following procedure at a ratio of 5: 1. The polypeptide-immunized spleen cell suspension and myeloma cells were each washed with RPMI1640 medium. Next, the cells were suspended in the same medium, and nucleated splenocytes and myeloma cells were mixed for fusion. That is, 6.0 × 10 ⁸
1.2 × 10 ⁸ myeloma cells were mixed with each nucleated spleen cell.

Next, cells were precipitated by centrifuging each cell mixture, and the supernatant was completely removed by suction. RPMI-1640 containing 50% PEG-4000 heated to 37 ° C on the precipitated cells
A medium (volume was determined to be 3 × 10 ⁷ myeloma cells / mL) was added dropwise over 1 minute, stirred for 1 minute, and the cells were resuspended and dispersed. Next added RP containing 50% PEG-4000
An RPMI-1640 medium heated to 37 ° C. twice the volume of the MI-1640 medium was added dropwise over 2 minutes. Then add 2-3 volumes of RPMI-1640 medium 7 times the volume of RPMI-1640 medium containing 50% PEG-4000.
The mixture was added dropwise with stirring over a period of minutes to disperse the cells. This was centrifuged, and the supernatant was completely removed by suction. Next, NS-1 medium heated to 37 ° C. was rapidly added to the precipitated cells so that the myeloma cells became 3 × 10 ⁶ cells / mL, and the large cell mass was carefully dispersed by pipetting. The same medium was further added and diluted, and the cells were inoculated into a 96-well polystyrene microwell so that the number of myeloma cells per well was 6.0 × 10 ⁵ . The microwells containing each cell were placed in 7% carbon dioxide / 93% air at a temperature of 37 ° C and a humidity of 100%.
And cultured.

(F) Selective growth of hybridoma on selective medium-1 (1) The medium used was as follows. HAT medium: To the NS-1 medium described in the above section (e), hypoxanthine (100 μM), aminopterin (0.4 μM) and thymidine (16 μM) were further added. HT medium: The same composition as the above HAT medium except that aminopterin was removed. (2) On the next day (day 1) after the start of the culture in the above (e), 2 drops (about 0.1 ml) of HAT medium were added to the cells using a Pasteur pipette. On days 2, 3, 5, and 8, replace half of the medium (approximately 0.1 ml) with fresh HAT medium, and on day 10, replace half of the medium with fresh HAT medium.
Replaced with HT medium. On day 14, positive wells were examined by solid phase-antibody binding test (ELISA) in all wells where hybridoma growth was visually observed. First, 20mM
Coat a polystyrene 96-well plate with antigen polypeptide diluted with carbonate buffer (pH 9.6) (100 ng / well),
Next, the peptide was washed with PBS containing 0.05% Tween 20 to remove unadsorbed peptides. To each well, add 0.1 ml of the culture supernatant of the well in which the growth of the hybridoma has been confirmed.
Let stand for 1 hour. After washing, horseradish peroxidase (HRP) -labeled goat anti-mouse immunoglobulin (Cappel) was added as a secondary antibody, and the mixture was allowed to stand at room temperature for about 1 hour. After washing, hydrogen peroxide as a substrate and o-phenylenediamine were added to develop color. 2N sulfuric acid was added to each well to stop the color development reaction, and the degree of color development was measured by absorbance at 492 nm using a microplate absorbance meter (MRP-A4, Tosoh).

(G) Cloning of hybridomas The hybridomas in the wells positive for the antigenic peptide obtained in the above section (f) were monocloned by the limiting dilution method. That is, the cloning medium containing 10 ⁷ mouse thymocytes were prepared as NS-1 medium 1 ml per feeder, five wells per hybridomas in a 96-well microtiter well, diluted one, to 0.5 units, 36 each
Hole, 36 holes, added to 24 holes. On day 5 and day 12, about 0.1 ml of NS-1 medium was added to all wells. After cloning starts
In 2 weeks, ELISA described in (f) was performed on the group in which the growth of hybridomas was visually observed and the number of colony formation negative wells was 50% or more. When all the wells examined were not positive, 4 to 6 wells having one colony in the antibody-positive well were selected and recloned.
Finally, hybridomas producing monoclonal antibodies against each polypeptide were obtained.

(H) Determination of Class and Subclass of Monoclonal Antibody According to the above-described ELISA, the hybridoma supernatant obtained in the above (g) was added to a polystyrene 96-well plate coated with each polypeptide. Next, after washing with PBS, isotype-specific rabbit anti-mouse IgG antibody (Zyme
d Lab.). After washing with PBS, horseradish peroxidase-labeled goat anti-rabbit IgG (H + L) was added, and the class and subclass were determined using hydrogen peroxide and 2,2'-azino-di (3-ethylbenzothiazolic acid) as substrates. . The monoclonal antibody-producing hybridoma clone numbers and subclasses against the human cathepsin Z polypeptide obtained in the above section (g) are shown in (Tables 1 to 3). A series of hybridoma clones obtained by immunizing the BSA-polypeptide CathZ # 1 complex
No. 4 is used as the identification number for a series of monoclonal antibodies obtained by immunizing the BSA-polypeptide CathZ # 2 complex.
No. 8 was identified as the identification number for a series of monoclonal antibodies obtained by immunizing the BSA-polypeptide CathZ # 3 complex.
No. 5 was added to the beginning of each clone name.

[0127]

[Table 1]

[0128]

[Table 2]

[0129]

[Table 3]

(I) Purification of GST-fused human cathepsin Z Escherichia coli strain JM109 transformed with the Escherichia coli expression vector pGEX-3X CathZ described in Example 3 was cultured on an LB agar medium containing 100 μg / mL ampicillin to form a single colony. Take 40 mL
The cells were inoculated into an LB medium containing 100 μg / mL ampicillin, and cultured at 37 ° C. for 16 hours. 400 mL of the obtained primary culture in 100
The LB medium containing μg / mL ampicillin was re-inoculated and cultured at 37 ° C. until the absorbance at 600 nm became 0.6. IP to this
TG was added to a final concentration of 0.5 mM, and the cells were cultured at 37 ° C. for 4 hours to induce the expression of recombinant GST-fused human cathepsin Z. Immediately cool the culture solution on ice and collect the cells by centrifugation (5,000 rpm, 20 min) 20 mL of 20 mM Tris-HCl containing 1 mg / mL lysozyme
And suspended on ice for 15 minutes to break the cell wall. Ultrasonic disruption of the cell suspension under ice cooling (30 seconds, 10
Times) and centrifugation (18,000 rpm, 10 min) to collect insoluble inclusion bodies. Resuspend and wash the insoluble inclusion bodies with 1 mL of PBS,
Collect the precipitate by centrifugation (15,000 rpm, 10 min), and add 2 mL of 8
It was dissolved in PBS containing M urea. 1/5 volume of 6 × S in eluted fraction
A DS-PAGE sample buffer (containing 2-mercaptoethanol) was added, the mixture was left at 37 ° C. for 1 hour, and then subjected to SDS-PAGE. A 0.6 mL sample was spread on a 90 × 70 × 1 mm gel. After the electrophoresis, the gel was immersed in 3M KCl to visualize the protein band, and the GST-cathepsin Z fusion protein band was cut out. The cut gel was chopped, placed in an electroelution column, energized at 300 V (constant voltage) for 5 hours, reversed the current and energized again for 3 minutes, and then the eluted fraction was collected. In the eluted fraction, deoxycholic acid (D
OC) and left at 4 ° C for 16 hours, followed by PBS containing 5mM DOC.
And then dialyzed against PBS (3 times for 24 hours). Insoluble matter generated during dialysis is removed by centrifugation (15,000 rpm, 10 min),
The supernatant was concentrated by ultrafiltration. Soluble recombinant GST-cathepsin Z fusion protein obtained from 400 mL of E. coli culture was approximately 4 mg (4 mg / mL x 1 mL). 0.1M NaCl
0.1M phosphate buffer, diluted with pH 7.0, 150μg / 75μL
And stored frozen at -20 ° C.

(J) Preparation of antibody-producing cells-2 GST-cathepsin Z fusion protein prepared in the above (i)
56.3 μg with complete Freund's adjuvant for 6 weeks
lb / c female mice were intraperitoneally administered and immunized for the first time. Day 21 and
On day 39, 63.8 μg and 56.3 μg of GST-cathepsin Z fusion protein dissolved in 0.1 M phosphate buffer, pH 7.5, respectively
Each mouse was intraperitoneally administered and boosted. Further 71
On the day, 63.8 μg of GST-cathepsin Z fusion protein dissolved in 0.1 M phosphate buffer, pH 7.5 was intravenously administered for final immunization. Three days later, the spleen was removed to prepare a spleen cell suspension. Immunization was performed on two mice.

(K) Cell fusion-2 The nucleated splenocytes (viable cell rate 100%) and myeloma cells (viable cell rate 100%) prepared in the above (j) were prepared in the following procedure at a ratio of 5: 1. Fused. GST-cathepsin Z fusion protein immunized spleen cell suspension and myeloma cells were converted to RPMI16
Washed with 40 media. Next, the cells were suspended in the same medium, and nucleated splenocytes and myeloma cells were mixed for fusion. That is, 8.0 × 10 ⁷ myeloma cells were mixed with 4.0 × 10 ⁸ nucleated spleen cells. Next, the cell mixture was centrifuged to precipitate the cells, and the supernatant was completely removed by suction. RPMI-164 containing 50% PEG-4000 heated to 37 ° C on the precipitated cells
0 medium (volume was determined to be 3 × 10 ⁷ myeloma cells / mL) was added dropwise over 1 minute, stirred for 1 minute, and the cells were resuspended and dispersed. Next added R containing 50% PEG-4000
RPMI-1640 heated to 37 ° C twice the volume of PMI-1640 medium
The medium was added dropwise for 2 minutes. 50% PEG-4000 further added
2-3 times the volume of RPMI-1640 medium containing 7 times the volume of RPMI-1640 medium
The mixture was added dropwise with stirring over a period of minutes to disperse the cells.
This was centrifuged, and the supernatant was completely removed by suction. next,
NS-1 medium heated at 37 ° C. was rapidly added to the precipitated cells so that the myeloma cells became 3 × 10 ⁶ cells / mL, and the large cell mass was carefully dispersed by pipetting. The same medium was further added and diluted, and the cells were inoculated into a 96-well polystyrene microwell so that the number of myeloma cells per well was 6.0 × 10 ⁵ . 7% of the microwells containing cells
The cells were cultured in carbon dioxide / 93% air at a temperature of 37 ° C and a humidity of 100%.

(L) Selective Propagation of Hybridoma in Selection Medium-2 The next day (day 1) after the start of the culture described in the above (k), 2 drops (about 0.1 ml) of HAT medium was added to the cells using a Pasteur pipette. . Two
On days 3, 5 and 8, half of the medium (approximately 0.1 ml) was
The medium was replaced with AT medium, and on day 10, half of the medium was replaced with fresh HT medium. On the eleventh day, positive wells were examined by solid phase-antibody binding test (ELISA) in all wells where hybridoma growth was visually observed. First, a 96-well plate made of polystyrene was coated with a GST-cathepsin Z fusion protein diluted with 20 mM carbonate buffer (pH 9.6) (50 ng / well), and then washed with PBS containing 0.05% Tween20 and unadsorbed. Was removed. 0.1 ml of the culture supernatant of the well in which the growth of the hybridoma was confirmed was added to each well, and the mixture was allowed to stand at room temperature for about 1 hour. After washing, horseradish peroxidase (HRP) -labeled goat anti-mouse immunoglobulin (Cappel) was added as a secondary antibody, and the mixture was allowed to stand at room temperature for about 1 hour. After washing, hydrogen peroxide as a substrate and o-phenylenediamine were added to develop color. 2N sulfuric acid was added to each well to stop the color development reaction, and the degree of color development was measured by absorbance at 492 nm using a microplate absorbance meter (MRP-A4, Tosoh). The above-mentioned screening was performed on 848 wells, and 32 wells having an absorbance at 492 nm of 0.5 or more were selected, scaled up to a 24-well plate, and the culture was continued.

(M) Selection of anti-cathepsin Z antibody-producing hybridoma Escherichia coli BL21 (DE3) transformed with pGEX-3X CathZ was cultured according to the method described in Example 3, and IPTG was added to a final concentration of 1 mM.
To induce GST expression. The cells were collected by centrifugation, washed and resuspended in PBS, lysed with a French press, and centrifuged at 20,000 g (4 ° C, 20 minutes). The soluble extract of the supernatant was subjected to Glutathione Sepharose 4B for GST
After adsorption and washing, the mixture was eluted with a glutathione elution buffer (50 mM Tris-HCl containing 10 mM reduced glutathione, pH 8.0).
The purified GST was coated (50 ng / well) on a polystyrene 96-well plate as described in (1) above, and then 0.05%
Unwashed proteins were removed by washing with Tween 20-containing PBS. 0.1 ml of the culture supernatant of the hybridoma (32 species) on day 13 selected and scaled up in the above (1) was added,
It was left at room temperature for about 1 hour. After washing, horseradish peroxidase (HRP) -labeled goat anti-mouse immunoglobulin (Cappel) was added as a secondary antibody, and the mixture was allowed to stand at room temperature for about 1 hour. After washing, hydrogen peroxide as a substrate and o-phenylenediamine were added to develop color. 2N sulfuric acid was added to each well to stop the color development reaction, and the degree of color development was measured by absorbance at 492 nm using a microplate absorbance meter (MRP-A4, Tosoh). As a result, it was found that 8 out of 32 hybridomas reacted with GST. In addition, the results of ELISA performed simultaneously on the GST-cathepsin Z fusion protein revealed that nine types of hybridomas lost antibody-producing ability. Excluding these, 15 hybridomas were selected as candidates for anti-cathepsin antibody-producing hybridomas.

(N) Selection of hybridoma by Western blotting The GST-cathepsin Z fusion protein purified in Example 3 was
It was subjected to SDS-PAGE and transferred to a nitrocellulose membrane. Membrane
3% skim milk, 50 mM phosphate buffer containing 0.1 M NaCl, pH
Blocking was performed at 7.5 for 1 hour at room temperature, and the resultant was cut into 2.5 mm wide strips. The hybridoma culture supernatant selected in the above section (m) was added to each strip, and reacted at room temperature for 16 hours. HRP-labeled goat anti-mouse immunoglobulin (Cappel) diluted with 3000% 3% skim milk, 50 mM phosphate buffer containing 0.1 M NaCl, pH 7.5 after washing with 50 mM phosphate buffer containing 0.1 M NaCl, pH 7.5 Was reacted at room temperature for 1 hour. After washing, color was developed using hydrogen peroxide and 3,3'-diaminobenzidine as substrates. From the 15 hybridomas selected in section (m) above, it was confirmed that 11 of them reacted with the GST-cathepsin Z fusion protein. Table 4 shows the reactivity of the obtained hybridoma and its culture supernatant. The number of + in Table 4 indicates the reactivity. Further,-in Table 4 indicates that no reactivity was observed. A series of hybridomas obtained by immunization with the GST-cathepsin Z fusion protein were designated with the identification number 237 at the beginning of each hybridoma name.

[0136]

[Table 4]

(O) Cross-Reactivity of Antibody Cross-reactivity of the hybridoma 237-13 selected in the above section (n) with cathepsin L2 was examined by Western blotting. Activated cathepsin Z and 32 on amino acid sequence
% Active cathepsin L2 having a homology of 2% was subjected to SDS-PAGE, and Western blotting was performed according to the method described in the above section (n). The culture supernatant of hybridoma 237-13 did not show any reaction with activated cathepsin L2.
The amino acid sequence homology between active cathepsin Z and other active cathepsins is as follows: cathepsin C, H, S and L are 32% equivalent to cathepsin L2, cathepsin O is 30%, cathepsin O
K is 29%, cathepsin W is 27%, and cathepsin B is 26%. Therefore, it was suggested that the hybridoma 237-13 produces a specific antibody which does not react with these other active cathepsins but reacts only with the active cathepsin Z similarly to the active cathepsin L2.

(P) Culture of Hybridoma and Purification of Monoclonal Antibody The obtained hybridoma cells are cultured in NS-1 medium, and a monoclonal antibody having a concentration of 10 to 100 μg / ml can be obtained from the supernatant. Moreover, mice with pristane before advance one week hybridoma ¹⁰ cells obtained was administered intraperitoneally (Balb / c system, female, 6 weeks old) was also administered intraperitoneally, 1
After 2 weeks, ascites containing 4-7 mg / ml of monoclonal antibody can be obtained from ascites. 40% of ascites obtained
After salting out with saturated ammonium sulfate, an IgG class antibody is adsorbed to Protein A Affigel (Bio-Rad) and purified by elution with 0.1 M citrate buffer, pH 5.0.

Example 7: Sandwich EIA According to the following method, a sandwich EIA for specifically detecting and measuring human cathepsin Z from the anti-human cathepsin Z antibody prepared in Example 6 using a combination of two suitable antibodies. The system can be configured. The EIA system can be either a one-step method or a two-step method, and the labeled antibody is not limited to Fab'-HRP. The composition and reaction conditions of each reaction buffer can be adjusted, for example, shortened or extended, according to the purpose of measurement. In addition, human cathepsin Z as a standard can be purified from a tissue culture supernatant, a cell culture supernatant, or a recombinant expressed by the method described in Example 3 or other methods. Purification can be achieved by ion exchange, gel filtration, affinity chromatography using an anti-human cathepsin Z monoclonal antibody, or a combination of various other types of affinity chromatography.

(A) Preparation of labeled antibody Anti-human cathepsin Z monoclonal antibody was added to 0.1 M acetate buffer containing 0.1 M NaCl, pH 4.2, and 2% (W / W) of pepsin was added to the antibody at 37 ° C. Digest for 24 hours. 3M Tris- for digest
The reaction is stopped by adding HCl, pH 7.5. The F (ab ') ₂ fraction is collected by gel filtration using an Ultrogel AcA54 column equilibrated with 0.1 M phosphate buffer, pH 7.0. This F (ab ') ₂
Cysteinamine hydrochloride was added to the fraction to a final concentration of 0.01 M, reduced at 37 ° C for 1.5 hours, and subjected to gel filtration using an Ultrogel AcA54 column equilibrated with 0.1 M phosphate buffer containing 5 mM EDTA, pH 6.0. Collect the Fab 'fraction. Separately from the above operation, dissolve HRP in 0.1 M phosphate buffer, pH 7.0,
Add 25 times the molar amount of EMCS as a DMF solution and react at 30 ° C for 30 minutes. This is gel-filtered through a NICK-5 column (Pharmacia) equilibrated with 0.1 M phosphate buffer, pH 6.0, and a maleimide-labeled HRP fraction is collected. Fab 'fraction and maleimide labeling
After mixing both fractions so that HRP becomes equimolar, and reacting at 4 ° C. for 20 hours, unreacted thiol groups are blocked with N-ethylmaleimide in a 10-fold molar amount of Fab ′. This is subjected to gel filtration through an Ultrogel AcA54 column equilibrated with a 0.1 M phosphate buffer, pH 6.5, and a Fab'-HRP-labeled antibody is collected. to this
Add 0.1% BSA and 0.001% chlorhexidine at 4 ° C
To save.

(B) Preparation of Monoclonal Antibody-Binding Carrier The anti-human cathepsin Z monoclonal antibody was diluted with 0.1 M phosphate buffer.
Dissolve in buffer solution, pH 7.5, and adjust to a concentration of 50 μg / mL. This
Of the monoclonal antibody solution into a 96-well microplate
Add 100 μL per well and incubate at 4 ℃ for 18 hours.
Remove the monoclonal antibody solution and once with saline,
0.05% Tween20, 0.1M NaCl, 5mM CaCl _TwoTris-HCl
After washing 3 times with buffer, pH 8.0, 1% BSA, 0.1M NaCl, 5m
M CaCl_TwoAdd Tris-HCl buffer, pH 8.0 and blockin
To

(C) One-step sandwich EIA method A standard curve for quantification of human cathepsin Z is prepared using the purified human cathepsin Z fraction as a standard antigen. 1% BSA, 0.05%
Brij35, 0.05% Tween20, 0.1M NaCl, 5mM CaCl ₂
Dispense 60 μL of standard human cathepsin Z serially diluted with Tris-HCl buffer, pH 8.0, 1% BSA and 0.05% Bri respectively.
j35,0.05% Tween 20,0.1M NaCl, 5mM CaCl 2 containing Tri
Add labeled antibody Fab'-HRP prepared at 100 ng / 50 μL with s-HCl buffer, pH 8.0, add 60 μL each, and mix well. Prepare the prepared antibody binding microplate with 0.05% Tween20, 0.1M NaC
l, 5 mM CaCl ₂ containing Tris-HCl buffer, washed 3 times with pH 8.0, the standard antigen and the labeled antibody mixture is added at 100 [mu] L / well. After reacting for 1 hour at room temperature, 0.05% Tween20, 0.1M
NaCl, 5 mM CaCl ₂ containing Tris-HCl buffer, washed three times with pH 8.0. Next, 0.1M citric acid containing 0.02% hydrogen peroxide-
Add 100 μL per well of 1.2 mg / mL o-phenylenediamine dissolved in phosphate buffer, pH 4.9, and after reacting at room temperature for 20 minutes, add 100 μL of 2N sulfuric acid to stop the reaction. The 492 nm of this reaction mixture is measured using a microplate reader to determine a standard curve. The measurement sample is human serum,
It is prepared from body fluid components derived from humans such as plasma, synovial fluid, urine and saliva, extracts of various human tissues, cell extracts of various cultured cells such as human-derived or recombinant, and culture supernatants. Each measurement sample is standard human cathepsin Z
Instead, subject it to the one-step sandwich EIA described above to allow the reaction to proceed simultaneously with standard human cathepsin Z. The amount of human cathepsin Z contained in the measurement sample is calculated by fitting to a standard curve obtained from the measurement sample.

Example 8: Searching for an antibody that inhibits the enzyme activity of cathepsin Z According to the method described in Example 4, an antibody that inhibits the enzyme activity of cathepsin Z is obtained from the anti-cathepsin Z antibody prepared in Example 6. Can be selected. The selected antibody is
It has the property of specifically binding only to cathepsin Z, and its ability to inhibit enzyme activity is also specific to cathepsin Z. Such properties are features not found in inhibitors common to cysteine proteases, such as E-64 described in Example 4. Recombinant cathepsin Z and antibody
After incubation at 30 ° C for 15 minutes, 20 μM Z-Phe-Arg-AMC (Bube
ndorf, Switzerland) and 8 mM dithiothreitol, 2 mM
EDTA, 100 mM sodium acetate buffer containing 0.05% Brij35 p
By reacting in H5.5 and measuring the remaining activity of cathepsin Z, the inhibitory activity of the added antibody can be evaluated. The antibody to be added is desirably a purified antibody described in item (p) of Example 6 or by another method. The antibody was prepared in Example 7
F (a) prepared and purified as described in paragraph (a) of
b ') It can be used even if it is fragmented like ₂ or Fab' fraction. Various conditions such as the amount of the antibody to be added, the type and amount of the substrate, the composition and amount of the reaction solution, the reaction time and the temperature can be appropriately changed. The antibody that inhibits the enzymatic activity of cathepsin Z selected as described above is itself cathepsin Z.
In addition to functioning as an inhibitor of Z 2, it is extremely important for elucidation of the pathogenesis of diseases caused by excessive activity of cathepsin Z, treatment and development of therapeutic drugs.

[0144]

As described above, the full length of the gene encoding human cathepsin Z belonging to the papain family of cysteine proteases was cloned from a human cDNA library, and its nucleotide sequence was analyzed, whereby intracellular protein metabolism and activation of hormone precursors were obtained. And many other normal cellular processes, such as bone modification, and many other diseases such as Alzheimer's disease, emphysema, rheumatoid arthritis, muscular dystrophy, osteoporosis, neurodegenerative diseases and cancer invasion and metastasis. Now available. Furthermore, expression of this gene in Escherichia coli and other hosts can be used to clarify the enzymatic properties of the recombinant protein, and it is also possible to study substances that inhibit its activity. In addition, the chromosomal locus of the human cathepsin Z gene can be determined, and the expression in human tissues can be investigated. An antibody specifically reacting with human cathepsin Z, in particular, a monoclonal antibody can be prepared to provide a means for detecting human cathepsin Z, such as immunological tissue staining, and a reagent therefor. In addition, detecting human cathepsin Z,
Provide a method to measure, for example, EIA system. The reagent has a possibility for use in detection and diagnosis of cancer, elucidation of the pathogenesis of the above-mentioned diseases, development of therapeutics and therapeutic drugs, and the like. Obviously, the present invention can be practiced other than as specifically described in the foregoing description and examples. Many modifications and variations of the present invention are possible in light of the above teachings,
Accordingly, they are also within the scope of the appended claims.

[0145]

[Sequence List] SEQUENCE LISTING <110> Fuji Yakuhin Kogyo Kabushiki Kaisha <120> Antibodies against cathepsin and use there <130> P-99NF300 <140> <141> <150> 10-167778 <151> 1998-06-02 < 160> 18 <170> PatentIn Ver. 2.0 <210> 1 <211> 303 <212> PRT <213> Human <400> 1 Met Ala Arg Arg Gly Pro Gly Trp Arg Pro Leu Leu Leu Leu Val Leu 1 5 10 15 Leu Ala Gly Ala Ala Gln Gly Gly Leu Tyr Phe Arg Arg Gly Gln Thr 20 25 30 Cys Tyr Arg Pro Leu Arg Gly Asp Gly Leu Ala Pro Leu Gly Arg Thr 35 40 45 Thr Tyr Pro Arg Pro His Glu Tyr Leu Ser Pro Ala Asp Leu Pro Lys 50 55 60 Ser Trp Asp Trp Arg Asn Val Asp Gly Val Asn Tyr Ala Ser Ile Thr 65 70 75 80 Arg Asn Gln His Ile Pro Gln Tyr Cys Gly Ser Cys Trp Ala His Ala 85 90 95 Ser Thr Ser Ala Met Ala Asp Arg Ile Asn Ile Lys Arg Lys Gly Ala 100 105 110 Trp Pro Ser Thr Leu Leu Ser Val Gln Asn Val Ile Asp Cys Gly Asn 115 120 125 Ala Gly Ser Cys Glu Gly Gly Asn Asp Leu Ser Val Trp Asp Tyr Ala 130 135 140 His Gln His Gly Ile Pro Asp Glu Thr Cys Asn Asn Tyr Gln Ala L ys 145 150 155 160 Asp Gln Glu Cys Asp Lys Phe Asn Gln Cys Gly Thr Cys Asn Glu Phe 165 170 175 Lys Glu Cys His Ala Ile Arg Asn Tyr Thr Leu Trp Arg Val Gly Asp 180 185 190 Tyr Gly Ser Leu Ser Gly Arg Glu Lys Met Met Ala Glu Ile Tyr Ala 195 200 205 Asn Gly Pro Ile Ser Cys Gly Ile Met Ala Thr Glu Arg Leu Ala Asn 210 215 220 Tyr Thr Gly Gly Ily Tyr Ala Glu Tyr Gln Asp Thr Thr Tyr Ile Asn 225 230 235 240 His Val Val Ser Val Ala Gly Trp Gly Ile Ser Asp Gly Thr Glu Tyr 245 250 255 Trp Ile Val Arg Asn Ser Trp Gly Glu Pro Trp Gly Glu Arg Gly Trp 260 265 270 270 Leu Arg Ile Val Thr Ser Thr Tyr Lys Asp Gly Lys Gly Ala Arg Tyr 275 280 285 Asn Leu Ala Ile Glu Glu His Cys Thr Phe Gly Asp Pro Ile Val 290 295 300 <210> 2 <211> 1541 <212> DNA <213> Human <400> 2 aaatggtgaa agggggggag gggagaaaag aaaaaaaaaa aaagaaaggg gaaagggggg 60 aaaaataaga aaaagcgaga cagaggcgct gccgcgtccg ctcgcgggga aggctggggt 120 cggccgggtg ctaggccggg gccgaggccg aggccggggc gggatccaga gcgggagccg 180 gcgggatggg tgg cgggac ccaggagccg gcgcggggcc 240 atggcgaggc gcgggccagg gtggcggccg cttctgctgc tcgtgctgct ggcgggcgcg 300 gcgcagggcg gcctctactt ccgccgggga cagacctgct accggcctct gcggggggac 360 gggctggctc cgctggggcg cacgacatac ccccggcctc atgagtacct gtccccagcg 420 gatctgccca agagctggga ctggcgcaat gtggatggtg tcaactatgc cagcatcacc 480 cggaaccagc acatccccca atactgcggc tcctgctggg cccacgccag caccagcgct 540 atggcggatc ggatcaacat caagaggaag ggagcgtggc cctccaccct cctgtccgtg 600 cagaacgtca tcgactgcgg taacgctggc tcctgtgaag ggggtaatga cctgtccgtg 660 tgggactacg cccaccagca cggcatccct gacgagacct gcaacaacta ccaggccaag 720 gaccaggagt gtgacaagtt taaccaatgt gggacatgca atgaattcaa agagtgccac 780 gccatccgga actacaccct ctggagggtg ggagactacg gctccctctc tgggagggag 840 aagatgatgg cagaaatcta cgcaaatggt cccatcagct gtggaataat ggcaacagaa 900 agactggcta actacaccgg aggcatctat gccgaatacc aggacaccac atatataaac 960 catgtcgttt ccgtggctgg gtggggcatc agtgatggga ctgagtactg gattgtccgg 1020 aattcatggg gtgaaccatg gggcgagaga ggctggctga ggatcgtgac c agcacctat 1080 aaggatggga agggcgccag atacaacctt gccatcgagg agcactgtac atttggggac 1140 cccatcgttt aaggccatgt cactagaagc gcagtttaag aaaaggcatg gtgacccatg 1200 accagagggg atcctatggt tatgtgtgcc aggctggctg gcaggaactg gggtggctat 1260 caatattgga tggcgaggac agcgtggtac tggctgcgag tgttcctgag agttgaaagt 1320 gggatgactt atgacacttg cacagcatgc tctgcctcac aatgatgcag tcagccacct 1380 ggtgaagaag tgacctgcaa cacaggaaac gatgggacct cagtcttctt cagcagagga 1440 cttgatattt tgtatttggc aactgtgggc aataatatgg catttaagag gtgaaagagt 1500 tcagacttat caccattctt atgtcacttt agaatcaagg g 1541 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide to act as a primer for PCR <400> 3 cacccggaac cagcacat 18 <210> 4 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide to act as a primer for PCR <400> 4 cgatggggtc cccaatg 17 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artific ial Sequence: Oligonucleotide to act as a primer for PCR <400> 5 atgctgccca agagctggga c 21 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide to act as a primer for PCR <400> 6 cgatggggtc cccaaatg 18 <210> 7 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed peptide to act as an immunogen <400> 7 Ala Gln Gly Gly Leu Tyr Phe Arg Arg Gly Gln Thr Cys Tyr Arg Pro 1 5 10 15 Leu Arg Gly Asp Gly Leu Ala Pro Leu Gly Arg Thr Thr Tyr Pro Arg 20 25 30 Pro His Glu Tyr Leu Ser Pro Ala Asp 35 40 <210> 8 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed peptide to act as an immunogen <400> 8 Asn Val Asp Gly Val Asn Tyr Ala Ser Ile Thr Arg Asn Gln His Ile 1 5 10 15 Pro Gln Tyr Cys 20 <210> 9 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed peptide to act as an immunogen <400> 9 His Ala Ser Thr Ser Ala Met Ala Asp Arg Ile Asn Ile Lys Arg Lys 1 5 10 15 Gly Ala Trp Pro Ser Thr Leu Leu Ser Val Gln Asn Val Ile Asp Cys 20 25 30 <210> 10 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed peptide to act as an immunogen <400> 10 Asn Asp Leu Ser Val Trp Asp Tyr Ala His Gln His Gly Ile Pro Asp 1 5 10 15 Glu Thr Cys <210> 11 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed peptide to act as an immunogen <400> 11 Asp Lys Phe Asn Gln Cys Gly Thr Cys Asn Glu Phe Lys Glu 1 5 10 <210> 12 <211> 68 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed peptide to act as an immunogen <400> 12 Cys His Ala Ile Arg Asn Tyr Thr Leu Trp Arg Val Gly Asp Tyr Gly 1 5 10 15 Ser Leu Ser Gly Arg Glu Lys Met Met Ala Glu Ile Tyr Ala Asn Gly 20 25 30 Pro Ile Ser Cys Gly Ile Met Ala Thr Glu Arg Leu Ala Asn Tyr Thr 35 40 45 Gly Gly Ile Tyr Ala Glu Tyr Gln Asp Thr Thr Tyr Ile Asn His Val 50 55 60 Val Ser Val Ala 65 <210> 13 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed peptide to act as an immunogen <400> 13 Leu Arg Ile Val Thr Ser Thr Tyr Lys Asp Gly Lys Gly Ala Arg Tyr 1 5 10 15 Asn Leu Ala Ile Glu Glu His Cys 20 <210> 14 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed peptide to act as an immunogen <400> 14 Asn Tyr Ala Ser Ile Thr Arg Asn Gln His Ile Pro Gln Tyr Cys 1 5 10 15 <210> 15 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed peptide to act as an immunogen <400> 15 Trp Arg Val Gly Asp Tyr Gly Ser Leu Ser Gly Arg Glu Lys Met 1 5 10 15 <210> 16 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed peptide to act as an immunogen <400> 16 Thr Tyr Lys Asp Gly Lys Gly Ala Arg Tyr Asn Leu Ala Ile Glu Glu 1 5 10 15 His Cys <210> 17 <211> 1541 <212> DNA <213> Human <220> <221> CDS <222> (241) .. (1149) <400> 17 aaatggtgaa agggggggggag gggagaaaag aaaaaaaaaa aaagaaaggg gaaagggggg 60 aaaaataaga aaaagcgaga cagaggcggccgccg ctcgcgggga aggctggggt 120 cggccgggtg ctaggccggg gccgaggccg aggccggggc gggatccaga gcgggagccg 180 gcgcgggatc tgggactcgg agcgggatcc ggagcgggac ccaggagccg gcgcggggcc 240 atg gcg agg cgc ggg cca ggg tgg cgg ccg ctt ctg ctg ctc gtg ctg 288 Met Ala Arg Arg Gly Pro Gly Trp Arg Pro Leu Leu Leu Leu Val Leu 1 5 10 15 ctg gcg ggc gcg gcg cag ggc ggc ctc tac ttc cgc cgg gga cag acc 336 Leu Ala Gly Ala Ala Gln Gly Gly Leu Tyr Phe Arg Arg Gly Gln Thr 20 25 30 tgc tac cgg cct ctg cgg ggg gct ccg ctg ggg cgc acg 384 Cys Tyr Arg Pro Leu Arg Gly Asp Gly Leu Ala Pro Leu Gly Arg Thr 35 40 45 aca tac ccc cgg cct cat gag tac ctg tcc cca gcg gat ctg ccc aag 432 Thr Tyr Pro Arg Pro His Glu Tyr Leu Ser Pro Ala Asp Leu Pro Lys 50 55 60 agc tgg gac tgg cgc aat gtg gat ggt gtc aac tat gcc agc atc acc 480 Ser Trp Asp Trp Arg Asn Val Asp Gly Val Asn Tyr Ala Ser Ile Thr 65 70 75 80 cgg aac cag cac atc ccc caa tac tgc ggc tcc tgc tgg gcc cac gcc 528 Arg Asn Gln His Ile Pro Gln Tyr Cys Gly Ser Cys Trp Ala His Ala 85 90 95 agc acc agc gct atg gcg gat cgg atc aac atc aag agg aag gga gcg 576 Ser Thr Ser Ala Met Ala Asp Arg Ile Asn Ile Lys Arg Lys Gly Ala 100 105 110 tgg ccc tcc acc ctc ctg tcc gtg cag aac gtc atc gac tgc ggt aac 624 Trp Pro Ser Thr Leu Leu Ser Val Gln Asn Val Ile Asp Cys Gly Asn 115 120 125 gct ggc tcc tgt gaa ggg ggt aat gac ctg tcc gtg tgg gac tac gcc 672 Ala Gly Ser Cys Glu Gly Gly Asn Asp Leu Ser Val Trp Asp Tyr Ala 130 135 140 cac cag cac ggc atc cct gac gag acc tgc aac aac tac cag gcc aag 720 His Gln His Gly Ile Pro Asp Glu Thr Cys Asn Asn Tyr Gln Ala Lys 145 150 155 160 gac cag gag tgt gac aag ttt aac caa tgt ggg aca tgc aat gaa ttc 768 Asp Gln Glu Cys Asp Lys Phe Asn Gln Cys Gly Thr Cys Asn Glu Phe 165 170 175 aaa aaa gag tgc cac gcc atc cgg aac tac acc agg gt g gga gac 816 Lys Glu Cys His Ala Ile Arg Asn Tyr Thr Leu Trp Arg Val Gly Asp 180 185 190 tac ggc tcc ctc tct ggg agg gag aag atg atg gca gaa atc tac gca 864 Tyr Gly Ser Leu Ser Gly Arg Glu Lys Met Met Ala Glu Ile Tyr Ala 195 200 205 aat ggt ccc atc agc tgt gga ata atg gca aca gaa aga ctg gct aac 912 Asn Gly Pro Ile Ser Cys Gly Ile Met Ala Thr Glu Arg Leu Ala Asn 210 215 220 tac acc gga ggc atc tat gcc gaa tac cag gac acc aca tat ata aac 960 Tyr Thr Gly Gly Ile Tyr Ala Glu Tyr Gln Asp Thr Thr Tyr Ile Asn 225 230 235 240 cat gtc gtt tcc gtg gct ggg tgg ggc atc agt gat ggg act gag tac 1008 His Val Val Ser Val Ala Gly Trp Gly Ile Ser Asp Gly Thr Glu Tyr 245 250 255 tgg att gtc cgg aat tca tgg ggt gaa cca tgg ggc gag aga ggc tgg 1056 Trp Ile Val Arg Asn Ser Trp Gly Glu Pro Trp Gly Glu Arg Gly Trp 260 265 270 ctg agg atc gtg acc agc acc tat aag gat ggg aag ggc gcc aga tac 1104 Leu Arg Ile Val Thr Ser Thr Tyr Lys Asp Gly Lys Gly Ala Arg Tyr 275 280 285 285 aac ctt gcc atc gag gag cac tgt acatt t ggg gac ccc atc gtt 1149 Asn Leu Ala Ile Glu Glu His Cys Thr Phe Gly Asp Pro Ile Val 290 295 300 taaggccatg tcactagaag cgcagtttaa gaaaaggcat ggtgacccat gaccagaggg 1209 gatcctatgg ttatgtgtgc caggctggct ggcaggaact ggggtggcta tcaatattgg 1269 atggcgagga cagcgtggta ctggctgcga gtgttcctga gagttgaaag tgggatgact 1329 tatgacactt gcacagcatg ctctgcctca caatgatgca gtcagccacc tggtgaagaa 1389 gtgacctgca acacaggaaa cgatgggacc tcagtcttct tcagcagagg acttgatatt 1449 ttgtatttgg caactgtggg caataatatg gcatttaaga ggtgaaagag ttcagactta 1509 tcaccattg gg Tatcactg 1811 Tccattg gg ttcagacta 1811 Tccattg gg Arg Pro Leu Leu Leu Leu Val Leu 1 5 10 15 Leu Ala Gly Ala Ala Gln Gly Gly Leu Tyr Phe Arg Arg Gly Gln Thr 20 25 30 Cys Tyr Arg Pro Leu Arg Gly Asp Gly Leu Ala Pro Leu Gly Arg Thr 35 40 45 Thr Tyr Pro Arg Pro His Glu Tyr Leu Ser Pro Ala Asp Leu Pro Lys 50 55 60 Ser Trp Asp Trp Arg Asn Val Asp Gly Val Asn Tyr Ala Ser Ile Thr 65 70 75 80 Arg Asn Gln Hi s Ile Pro Gln Tyr Cys Gly Ser Cys Trp Ala His Ala 85 90 95 Ser Thr Ser Ala Met Ala Asp Arg Ile Asn Ile Lys Arg Lys Gly Ala 100 105 110 Trp Pro Ser Thr Leu Leu Ser Val Gln Asn Val Ile Asp Cys Gly Asn 115 120 125 Ala Gly Ser Cys Glu Gly Gly Asn Asp Leu Ser Val Trp Asp Tyr Ala 130 135 140 His Gln His Gly Ile Pro Asp Glu Thr Cys Asn Asn Tyr Gln Ala Lys 145 150 155 160 Asp Gln Glu Cys Asp Lys Phe Asn Gln Cys Gly Thr Cys Asn Glu Phe 165 170 175 Lys Glu Cys His Ala Ile Arg Asn Tyr Thr Leu Trp Arg Val Gly Asp 180 185 190 Tyr Gly Ser Leu Ser Gly Arg Glu Lys Met Met Ala Glu Ile Tyr Ala 195 200 205 Asn Gly Pro Ile Ser Cys Gly Ile Met Ala Thr Glu Arg Leu Ala Asn 210 215 220 Tyr Thr Gly Gly Ile Tyr Ala Glu Tyr Gln Asp Thr Thr Tyr Ile Asn 225 230 235 240 His Val Val Ser Val Ala Gly Trp Gly Ile Ser Asp Gly Thr Glu Tyr 245 250 255 Trp Ile Val Arg Asn Ser Trp Gly Glu Pro Trp Gly Glu Arg Gly Trp 260 265 270 Leu Arg Ile Val Thr Ser Thr Tyr Lys Asp Gly Lys Gly Ala Arg Tyr 275 280 285 Asn Leu Ala Ile Glu G lu His Cys Thr Phe Gly Asp Pro Ile Val 290 295 300

[Brief description of the drawings]

FIG. 1 shows the nucleic acid sequence and the predicted amino acid sequence of human cathepsin Z cloned from a brain cDNA library. Arrows indicate the cleavage sites of the signal peptide and propeptide, and of the propeptide and active enzyme. The structurally important glutamine residue at position 87, the cysteine residue at position 92, the histidine residue at position 241 and the asparagine residue at position 261 are underlined.

FIG. 2 shows the amino acid sequence alignment of cathepsin Z and human cysteine protease (continued in FIGS. 3 and 4). The amino acid sequence of a known human cysteine protease was retrieved from the SwissProt database and analyzed with the PILEUP program in the GCG package. The numbers correspond to cathepsin Z. The characteristic sequence on the N-terminal side of cathepsin C is not included in the alignment analysis. Amino acids common to cathepsin Z and the cathepsin family are boxed. Gaps are indicated by dots.

FIG. 3 shows the amino acid sequence alignment of cathepsin Z and human cysteine protease continued from FIG. 2.

FIG. 4 shows the amino acid sequence alignment of cathepsin Z and human cysteine protease continued from FIG.

FIG. 5 shows the results of in situ hybridization using a biotinylated probe specific for human cathepsin Z. A dot signal is seen at 13q on chromosome 20. The figure on the right is a chromosome 20 map.

FIG. 6 shows the results of purification of recombinant cathepsin Z expressed in E. coli. 5 μL of an extract of E. coli transformed with each of pGEX-3X and pGEX-3X CathZ was added per lane and analyzed by SDS-PAGE. To CathZ, 1 μL of the purified fusion protein was added per lane.
The arrow indicates GST (29 kDa) and the fusion protein (about 52 kDa). The molecular weight indicated by the molecular weight marker (MWM) is shown on the right side of the figure.

FIG. 7 shows the results of enzyme activity analysis of recombinant cathepsin Z expressed in Escherichia coli. Recombinant cathepsin Z and cathepsin O were reacted with 20 μM Z-Phe-Arg-AMC, and 20 μM E-64
1 shows the time-dependent hydrolysis of a substrate (30 ° C.) in the presence and absence of a.

FIG. 8. Cathepsin Z in human tissues and cultured cell lines
FIG. 4 is a diagram showing Northern blot analysis of mRNA. 2 μg poly (A) ⁺ RNA prepared from the indicated tissue was replaced with human cathepsin Z
And analyzed by hybridization with a 270 bp probe corresponding to the untranslated region at the 3'-end of the cDNA. RNA markers are shown beside the figure. After washing the probe, hybridization was repeated in the order of the human cathepsin Z probe and the human actin probe.

FIG. 9. Cathepsin Z expression in human tissues and cultured cell lines
FIG. 4 is a diagram showing Northern blot analysis of mRNA. Hybridization of 2 μg poly (A) ⁺ RNA prepared from the indicated cancer cell line and 20 μg of total RNA prepared from various cancer tissues with a 270 bp probe corresponding to the untranslated region at the 3′-end of human cathepsin Z cDNA. did. The filter finally hybridized with the human actin probe.

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (reference) A61K 31/00 625 A61K 31/00 625 626 626N 629 629A 635 635 635 635B 643 643D 643B 39/395 39/395 D P C07K 16 / 40 C07K 16/40 C12N 1/19 C12N 1/19 1/21 1/21 5/10 9/50 9/50 C12P 21/08 15/02 C12Q 1/68 A C12P 21/08 G01N 33/53 D C12Q 1/68 33/573 A G01N 33/53 33/577 B 33/573 C12N 5/00 B 33/577 15/00 C

Claims (11)

[Claims] 1. Antibodies against: (i) at least 40% of the cathepsin Z protein of human origin or the amino acid sequence of said cathepsin Z protein
A human-derived cathepsin protein or a salt thereof, which has the above homology and has cysteine protease activity or equivalent antigenicity, (ii)
Among the amino acid sequences represented by SEQ ID NO: 1 in the sequence listing, those having the amino acid sequence of positions 62 to 303, those having the amino acid sequences of positions 21 to 303, and those of the first
The amino acid sequence having the amino acid sequence of positions from position 303 to position 303, the amino acid sequence having an amino acid sequence substantially equivalent to any one of them, and the amino acid sequence having a biological activity substantially equivalent to any one of them. A protein or a salt thereof, which is a protein selected from the group consisting of those having the above, or (iii) a partial peptide of the protein according to the above (i) or (ii) or a salt thereof. 2. The antibody according to claim 1, wherein the antibody is a monoclonal antibody. (1) a propeptide region of human cathepsin Z (for example, positions 21 to 61 of SEQ ID NO: 1 in the sequence listing)
(2) a region containing a propeptide cleavage site of human cathepsin Z (for example, a region at positions 58 to 66 in SEQ ID NO: 1 in the sequence listing) , Or part of or near)
(3) Active human cathepsin Z region (for example, the region of positions 70 to 89, the positions 95 to 126, and the positions 136 to 154 of SEQ ID NO: 1 in the sequence listing) Rank 165
Region 178 to position 178, position 179 to position 246, or position 273 to position 296 or a part thereof or its vicinity). Item 3. The antibody according to Item 1 or 2. 4. The antibody according to claim 1, wherein the antibody is a solid-phased antibody. 5. The antibody according to claim 1, wherein the antibody is a labeled antibody. 6. An immunoassay for the following protein or a salt thereof, comprising using the antibody according to claim 1 as a measurement reagent: (i) Cathepsin Z protein of human origin Or at least 40% of the amino acid sequence of the cathepsin Z protein
A cathepsin protein of human origin or a salt thereof having the above homology and cysteine protease activity or equivalent antigenicity, or (ii) represented by SEQ ID NO: 1 in the sequence listing Among the amino acid sequences, those having the amino acid sequences of positions 62 to 303, those having the amino acid sequences of positions 21 to 303,
Having the amino acid sequence of positions 1 to 303, having substantially the same amino acid sequence as any one of them, and having substantially the same biological activity as any one of them A protein or a salt thereof, which is a protein selected from the group consisting of those having an amino acid sequence. 7. The method according to claim 6, wherein at least two of (a) a solid-phased antibody and (b) a labeled antibody are used. 8. An immunoassay reagent for the following protein or a salt thereof, which comprises the antibody according to any one of claims 1 to 5: (i) human-derived cathepsin Z protein or said cathepsin Z protein amino acid sequence and at least 40%
A cathepsin protein of human origin or a salt thereof having the above homology and cysteine protease activity or equivalent antigenicity, or (ii) represented by SEQ ID NO: 1 in the sequence listing Among the amino acid sequences, those having the amino acid sequences of positions 62 to 303, those having the amino acid sequences of positions 21 to 303,
Having the amino acid sequence of positions 1 to 303, having substantially the same amino acid sequence as any one of them, and having substantially the same biological activity as any one of them A protein or a salt thereof, which is a protein selected from the group consisting of those having an amino acid sequence. 9. A medicament comprising the antibody according to any one of claims 1 to 3. 10. The antibody according to any one of claims 1 to 5, wherein the antibody promotes or inhibits the biological activity of: (i) a human-derived cathepsin Z protein or an amino acid sequence of the cathepsin Z protein; And at least 40%
A human-derived cathepsin protein or a salt thereof, which has the above homology and has cysteine protease activity or equivalent antigenicity, (ii)
Among the amino acid sequences represented by SEQ ID NO: 1 in the sequence listing, those having the amino acid sequence of positions 62 to 303, those having the amino acid sequences of positions 21 to 303, and those of the first
The amino acid sequence having the amino acid sequence of positions from position 303 to position 303, the amino acid sequence having an amino acid sequence substantially equivalent to any one of them, and the amino acid sequence having a biological activity substantially equivalent to any one of them. A protein or a salt thereof, which is a protein selected from the group consisting of those having the above, or (iii) a partial peptide of the protein according to the above (i) or (ii) or a salt thereof. 11. A screening method and a screening kit for a compound that promotes or inhibits the biological activity of the following, characterized by containing the antibody according to any one of claims 1 to 5: (i At least 40% of the cathepsin Z protein of human origin or the amino acid sequence of the cathepsin Z protein
A human-derived cathepsin protein or a salt thereof, which has the above homology and has cysteine protease activity or equivalent antigenicity, (ii)
Among the amino acid sequences represented by SEQ ID NO: 1 in the sequence listing, those having the amino acid sequence of positions 62 to 303, those having the amino acid sequences of positions 21 to 303, and those of the first
The amino acid sequence having the amino acid sequence of positions from position 303 to position 303, the amino acid sequence having an amino acid sequence substantially equivalent to any one of them, and the amino acid sequence having a biological activity substantially equivalent to any one of them. A protein or a salt thereof, which is a protein selected from the group consisting of those having the above, or (iii) a partial peptide of the protein according to the above (i) or (ii) or a salt thereof.