JPH10210982A - New protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new protein, which is one kind of matrix metalloproteinase(MMP), having an ability to activate a latent type gelatinse A and natural murine MT-MMP activities and useful for diagnosis, etc., of the presence or absence of cancer cells and the malignancy of cancers. SOLUTION: This new protein is a new protein (salt) derived from a mouse which is one kind of matrix metalloproteinase(MMP) having an ability to activate a latent type gelatinase A and activities of natural murine MT-MMP which is a latent type gelatinase A activating factor other than murine MT 1-MMP or activities substantially equal thereto. The protein is useful for diagnosis, treatment, etc., of cancers such as diagnosis, etc., of the malignancy of the cancers. Furthermore, the protein is obtained by screening a murine pulmonary cDNA library with a human MT1-MMP cDNA fragment as a probe, integrating the resultant gene into a vector, transferring the resultant gene into a host cell such as Escherichia coli and expressing the gene.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention is useful as a diagnostic means useful for research relating to the diagnostic treatment of cancer, such as diagnosis of the presence or absence of cancer cells and the degree of malignancy of cancer, or useful for Alzheimer's disease and other medical and physiological uses. , A novel protein and a gene encoding the same. In particular, the present invention
Matrix metalloproteinase (MMP) capable of activating latent gelatinase A
Latent gelatinases other than mouse MT1-MMP
The present invention relates to a mouse-derived novel membrane-bound protein that is an A activator and a gene encoding the same. More specifically, the present invention relates to a novel membrane-bound matrix metalloprotease cloned from a mouse lung cDNA library [the novel membrane-bound matrix metalloprotease disclosed in the present invention is used for mouse MT2-MMP (Membrane-Typ
e 2 Matrix metalloproteinase), a DNA containing a nucleotide sequence encoding the same, a host cell transformed with the DNA, a method for producing the membrane-bound matrix metalloprotease using the host cell, and the matrix The present invention relates to monoclonal antibodies that bind to metalloprotease proteins, and to uses of those proteins and antibodies and nucleic acid oligomers.

[0002]

2. Description of the Related Art In order for cancer cells existing in primary tissues to infiltrate and metastasize, extracellular matrices around the cells hinder the migration of cancer cells. Therefore, in order for cancer cells to invade tissues and metastasize, they need to be released from the primary focus and destroy the surrounding extracellular matrix. The metastasis of cancer cells is established through the subsequent steps such as destruction of the basement membrane, invasion and invasion into blood vessels, engraftment into secondary organs, and proliferation. The extracellular matrix, which is a barrier to metastasis of cancer cells, is composed of complex components such as type IV collagen, proteoglycan, elastin, fibronectin, laminin, and heparan sulfate. Refers to matrix metalloproteases with different substrate specificities (MM
(Abbreviated as P) are involved.

[0003] Stromal collagenase (MMP-1), gelatinase A (MMP-2), gelatinase
B (MMP-9), stromlysin 1 (MMP-3), matrilysin (MMP-7), neutrophil collagenase (MMP-8),
Stromlysin 2 (MMP-10), Stromlysin 3
(MMP-11), metalloelastase (MMP-12), collagenase 3 (MMP-13) and the like have been reported. Of these MMPs
Homology is recognized in the amino acid sequence deduced from the cDNA sequence, and it is known that the amino acid sequence forms an MMP family. These MMPs consist of a N-terminal signal peptide, which is basically removed during secretory production, followed by a propeptide domain, a Zn-binding catalytic domain, a proline-rich hinge domain of 5 to 50 amino acids, and a C-terminal hemopexin It is composed of a clotting enzyme-like domain. There is no hemopexin clotting enzyme-like domain in MMP-7. Gelatinase A and gelatinase B contain an additional gelatin binding domain.

Among these MMPs, gelatinases using type IV collagen, which is the main structure of the basement membrane, as a main substrate
A and gelatinase B have been reported to be highly expressed in highly metastatic cancer cells, and their involvement in invasion of basement membrane by cancer cells has been proposed (Cell, 64: 327-336, 1991). MMP
The activity expression regulation of at least the transcription level, the stage of activation from a latent enzyme that does not show enzyme activity to an active enzyme,
It is thought that the activity is controlled at the stage of controlling the activity by tissue inhibitors of metalloproteases (TIMPs), which are specific inhibitors of MMP (Trends G
enet., 6: 121-125, 1990). All MMPs are secreted as inactive latent forms, but in vitro experiments have shown that MMP-
1) It has been shown that gelatinase B activation is caused by serine proteases such as plasmin, trypsin, and cathepsin G, and that the activation of gelatinase B is also caused by the action of activated MMP-3. (J. Biol. Chem., 267: 3581-358).
4, 1992).

However, since gelatinase A has no cleavage site for the above-mentioned protease, gelatinase A
Is not believed to occur by these (Curr. Opin. Cell Biol., 5: 891-897, 1993).
). This gelatinase A is expressed in fibroblasts at various sites with alterations in tissue architecture, but when compared to normal tissue and cancer tissue gelatinase A, its activation occurs specifically in cancer tissue. Has been reported in cases of lung cancer (Clin. Exp-Metastasis, 11: 183-189,1993
). In vitro experimental systems have shown that activated gelatinase A localizes at the tip of cancer cell invasion (invadopodia), suggesting the importance of gelatinase A in cancer cell invasion (Cancer Res., 53: 3159-3164, 19
93. Breast Cancer Res. Treat. 31: 217-226, 1994.
). Against this background, the mechanism of activation of gelatinase A, which is deeply involved in cancer invasion and metastasis, has attracted attention, but as described above, the mechanism of activation of gelatinase A is unknown.
In particular, no activator has been identified.

[0006] Recently, a novel MMP gene has been cloned by genetic engineering techniques, and has a typical transmembrane domain at the C-terminus and encodes a novel MMP that activates latent gelatinase A. The gene was cloned (Nature, 370: 61-65, 19
94). When this gene was expressed in cultured cells, the gene product was localized on the cell membrane without being secreted.
This MMP was named MT1-MMP (Membrane-Type 1 MMP). Furthermore, to date, similar to MT1-MMP, MMP with a typical transmembrane domain at the C-terminus
MT2-MMP (Eur. J. Biochem., 231: 602-608, 1995
), MT3-MMP (J. Biol. Chem., 270: 23013-23020,
1995) and MT4-MMP (Cancer Res., 56: 944-949,
1996) has been cloned. These MT-MMPs show high homology to each other and have the domain structure characteristic of the aforementioned MMP family, the pro-form highly conserved in the MMP family and the sequence PRCGVPD near the active cleavage site ( The homologous site of MT4-MMP is the sequence PR
CSLPD). In addition, these MT-MMPs had characteristic insert sequences in addition to the transmembrane domain as compared to other MMP families.

That is, an insertion sequence of 9 to 13 amino acid residues including the sequence of RXR / KR (X is an arbitrary amino acid residue) existing between the propeptide domain and the catalytic domain, and an 8 amino acid residue in the catalytic domain Insertion (not present in MT4-MMP) is common, and these MT-MMPs
It is thought to form a subfamily of the family. Activation of latent gelatinase A by the MT-MMP family was confirmed in the culture supernatant of COS-1 cells co-transfected with human MT1-MMP or MT3-MMP and the latent gelatinase A gene (J. Biol. Chem. ., 270: 23013-23
020, 1995), at least human MT of the MT-MMP family
1-MMP and MT3-MMP were shown to be activators of latent gelatinase A. Human MT2-MMP and MT4-
There are currently no reports on MMP features. As mentioned above, gelatinase A plays an important role in the process of invasive metastasis of cancer,
A has been found to degrade β-amyloid protein involved in the development of Alzheimer's disease (Nature, 362: 839-
841, 1993), there is great hope for elucidation of the mechanism of gelatinase A activation. MT-MMP family human identified as an activator of latent gelatinase A
Analysis of MT1-MMP and MT3-MMP and novel gelatinase A
Searching and identifying activators can be said to be extremely important for the diagnosis of these diseases and the development of pharmaceuticals.

[0008]

In the field of biochemistry including medicine and pharmacy, experimental animals are provided as experimental materials for the isolation and purification of proteins having various physiological activities, and their biological materials are used today. It is widely used for analysis of functional function, treatment of various diseases and diseases, basic and applied research on pharmacology and efficacy. There is no experimental study using humans, and the results of research using experimental animals have been extrapolated to humans and applied to human disease treatment and drug development. Non-mammals such as nematodes and Drosophila, birds such as chickens and quail as experimental animals used for such purposes,
Examples include small rodents such as mice, rats, rabbits, guinea pigs, and hamsters, and large mammals such as cats, dogs, pigs, cows, and monkeys, which are selected and used according to the purpose of experimental research. Among them, mice have been bred for a long time as experimental animals, have abundant accumulated genetic research, are large in size, and are easy to breed, and are the most widely used animal species.

[0009] In recent years, studies at the molecular level of genes have been remarkably developed, and the genes of humans and non-human mammals, particularly mice, have been compared, and homology has been confirmed in the genetic background. As a result, it has been recognized that proteins with high homology exist in both humans and mice, and that they have common structures and functions. This genetic homology is
It can be said that the knowledge obtained from biological function models and disease models in mice can be extrapolated to humans.
In addition, with technological advances in genetic and embryo manipulation,
Transgenic mice have been developed in which any gene is introduced and expressed during development and growth. In addition, by deleting only a specific gene or replacing it with another gene (knockout mouse), a mutant mouse that could not exist conventionally can be produced. With these techniques, various disease model mice have become available, and more complex biological function analysis has become possible. Thus, the importance of mice as laboratory animals is increasing.

[0010] Against this background, gene search for animal species other than humans, particularly mice, is important for establishing human disease model mice and analyzing the functions of human and mouse proteins. As described above, human and mouse genes and proteins have homology, and some antibodies and nucleic acid probes widely used in various analyzes can be used across animal species. However, strictly speaking, proteins and genes are unique to each animal species, and it is experimentally desirable to use antibodies and nucleic acid probes tailored to each animal species. The significance of producing a nucleic acid probe that hybridizes to a derived gene is significant. The antibodies and nucleic acid oligomers thus obtained have applications as important reagents in experimental medicine for the purpose of detecting or quantifying various proteins and genes derived from mice. Further, among these nucleic acid oligomers and antibodies, antisense oligomers and neutralizing antibodies also have uses as therapeutic agents, and provide an effective clue not only to experimental medicine in mice but also to research and development of pharmaceuticals.

By the way, it is localized on the cell membrane as a factor capable of activating latent gelatinase A in humans.
Following the discovery of human MT1-MMP, a type of MMP, human MT2-MMP, human MT3-MMP and human MT4-MMP having a domain structure similar to this human MT1-MMP were discovered. MT-MMPs have become known to form subfamilies within the MMP family. To date, latent gelatinases have been identified in non-human animal species.
Rat MT1-MMP and mouse MT1-MMP as MT-MMPs capable of activating A (Proc. Natl. Acad. Sci. USA, 92:
2730-2734, 1995), but no other latent gelatinase A activator has been identified.

[0012]

DISCLOSURE OF THE INVENTION In view of the above, the present inventors have determined that a plurality of gelatinases A can be used in mice as in humans.
There is a possibility that MT-MMP may act as an activator of the protein and may form a family.As a result of various studies using genetic engineering techniques, a new latent type of gelatinase A activation other than mouse MT1-MMP was found. Succeeded in isolating a mouse gene encoding MT-MMP having the above, and clarified all of the gene base sequence and amino acid sequence, thereby completing the present invention. Therefore, the present invention relates to a novel mouse-derived protein which is a kind of MMP having an ability to activate latent gelatinase A and is a latent gelatinase A activator other than mouse MT1-MMP, a method for producing the same, and a use thereof. It is an object of the present invention to provide a gene encoding the protein and its use, an antibody against the protein, a method for producing the antibody, a use of the antibody, and the like. The novel cryptic gelatinase A activator mouse MMP gene cloned according to the present invention was used for mouse MT2-MMP
The gene and mouse-derived protein encoded by the mouse MT2-MMP gene were designated as mouse MT2-MMP.

That is, the present invention provides a novel mouse MT2-MMP
It is related to the gene and mouse-derived protein mouse MT2-MMP and its analogs. Furthermore, the present invention provides a DNA encoding the whole or a part of the novel mouse MT2-MMP.
It also relates to sequences, vectors having such DNA sequences and host cells transformed or transfected with such vectors. Furthermore, recombinant mouse MT2-MM
It also covers the production of P and its use. Also mouse
It also relates to antibodies that bind to MT2-MMP, especially monoclonal antibodies and hybridoma cells producing the antibodies. The present invention also relates to a nucleic acid oligomer that hybridizes to the mouse MT2-MMP gene, for example, a DNA or RNA probe.
The present invention further provides a reagent for detecting and measuring mouse MT2-MMP using the above product, and a method for detecting and measuring using the reagent.

Furthermore, latent MM of mouse MT2-MMP
P-2 Provides a method for detecting a substance that inhibits the ability to activate and a substance that inhibits the activity of degrading extracellular matrix. Neutralizing antibodies that inhibit the ability to
The present invention also relates to a monoclonal antibody that binds to mouse MT2-MMP and a partial peptide of mouse MT2-MMP that binds to latent gelatinase A and does not have latent MMP-2 activating ability, a derivative thereof, or a salt thereof. Also, mouse MT2-MM
The present invention relates to an antisense oligomer and a derivative thereof that block the transcription or translation of P 2, and an anticancer agent and a cancer metastasis inhibitor containing the same as an active ingredient.

That is, the present invention relates to the following: [1] An MMP capable of activating latent gelatinase A
A mouse-derived protein characterized in that it has a natural mouse MT-MMP activity which is a latent gelatinase A activator other than mouse MT1-MMP or has an activity substantially equivalent thereto, or A salt thereof; [2] the protein described above, wherein the protein has substantially the same activity as mouse MT2-MMP or a salt thereof, or has substantially the same primary structure conformation; [3] Val ⁶¹⁴ of SEQ ID NO: 2 in the C-terminal region
A protein according to the above [1] or [2], which has an amino acid sequence represented by -Val ⁶³⁵ or an amino acid sequence substantially equivalent thereto; [4] represented by SEQ ID NO: 2 in the sequence listing A mouse MT2-MMP having an amino acid sequence substantially equivalent thereto or a salt thereof, wherein the protein according to any one of the above [1] to [3]; [5] exogenous DNA The protein according to any one of the above [1] to [4], wherein the protein is obtained by expressing the sequence in a prokaryote, or obtained by expressing the sequence in a eukaryote;

[6] a partial peptide of the protein according to any one of the above [1] to [5] or a salt thereof; [7] a protein according to any one of the above [1] to [6] or a partial peptide thereof [8] The mouse according to any one of [2] to [4], which has a nucleotide sequence encoding
The nucleic acid according to the above-mentioned [7], which is a DNA gene having a base sequence encoding MT2-MMP;

[9] The above-mentioned [7] or [8], which has an open reading frame portion or a nucleotide sequence having substantially the same activity as the open reading frame portion of the nucleotide sequence represented by SEQ ID NO: 1 in the sequence listing. [10] The above [7] to

[9] a vector comprising the nucleic acid of any one of [9]; [11] the above [7] to

[9] a transformant characterized by having the nucleic acid of any one of [9] or the vector of [10]; and [12] a nutrient medium capable of growing the transformant of [11]. And cultured as mouse MT2-
The above-mentioned [1] to [6], which comprises a mouse MT2-MMP or a salt thereof, wherein the protein or the partial peptide thereof according to any one of the above [1] to [6] including MMP or a salt thereof is produced. 6] A method for producing the protein or partial peptide thereof according to any one of [6].

In another embodiment, the present invention provides: [13] an antibody against the protein of the above [1] or a salt thereof or a partial peptide thereof or a salt thereof; [14] substantially equivalent to mouse MT2-MMP or a salt thereof The antibody according to the above [13], which is an antibody against a protein that has a high activity or a substantially equivalent primary structure conformation; [15] the antibody of SEQ ID NO: 2 in the sequence listing; The antibody of the above-mentioned [13] or [14], which is an antibody against a mouse MT2-MMP having a represented amino acid sequence or an amino acid sequence substantially equivalent thereto or a protein which is a salt thereof; [16] An antibody against a protein that is obtained by expressing an exogenous DNA sequence in prokaryotes or by eukaryotic expression. [13] The antibody according to any one of [13] to [15], wherein the antibody is an antibody against a partial peptide of a protein or a salt thereof. [18] the antibody described in [17], wherein the partial peptide or the salt thereof is Asp ^{281 to} Gly ²⁹⁴ of SEQ ID NO: 2 in the sequence listing;
The antibody according to the above [17], which has an amino acid sequence represented by:

[19] the antibody according to any one of the above [13] to [18], which is an antiserum; [20] the antibody [13] to [18], which is a monoclonal antibody [21] a monoclonal antibody against mouse MT2-MMP or a salt thereof; [13] to [1]
[22] production of an antibody against the above-mentioned [1] protein or a salt thereof, or a partial peptide thereof or a salt thereof, wherein the antibody of the above-mentioned [13] is obtained; [23] Producing an antibody against the protein [1] or a salt thereof or a partial peptide or a salt thereof obtained from an animal immunized with the protein or a salt thereof or a partial peptide or a salt thereof of the above [1]. The cells to be subcultured, and selecting hybrid cells that can be subcultured and produce an antibody against a protein including mouse MT2-MMP. ] A method for producing the antibody described above;

[24] Use of the protein of the above [1] or a salt thereof or a partial peptide thereof or a salt thereof as a reagent, or use of the antibody of any one of the above [13] to [21] as a reagent. Characteristic MT2-
[25] Antibody to labeled MT2-MMP used in the method for detecting and measuring MT2-MMP in [24] above; [26] Detection and measurement of MT2-MMP in [24] above A labeled protein or a salt thereof or a partial peptide or a salt thereof characterized by being the protein or a salt thereof or a partial peptide or a salt thereof according to the above [1]; [27] a labeled above-mentioned [ [7] The nucleic acid according to [27], which is a hybridization probe.

In still another aspect, the present invention relates to a method for detecting a compound or a protein or a salt thereof, which inhibits the latent gelatinase A activating ability of the protein or a salt thereof of the above-mentioned [1]; [1] a compound or a protein or a salt thereof that inhibits the latent gelatinase A activating ability of the protein or a salt thereof; [31] a compound and a protein or a salt thereof according to the above [30] as an active ingredient [32] a neutralizing antibody for mouse MT2-MMP, wherein the protein of the above-mentioned [31] or a salt thereof is selected from the antibodies of the above-mentioned [13] to [18] as an active ingredient; [33] an anticancer agent and a cancer metastasis inhibitor, which comprises: [33] the antibody according to [20], wherein the neutralizing antibody of [32] is an active ingredient; [34] an anticancer agent and a cancer metastasis inhibitor characterized by containing a monoclonal antibody or a derivative thereof selected from the group consisting of: [34] the protein of the above-mentioned [31] or a salt thereof having an activity of activating latent gelatinase A as an active ingredient; An anticancer agent and a cancer metastasis inhibitor comprising a partial peptide and derivative of the protein of the above [1] or a salt thereof;

[35] A method for detecting a compound or a protein or a salt thereof that inhibits the extracellular matrix degrading activity of the protein or a salt thereof of the above [1]; [36] A method of detecting the protein or a salt thereof of the above [1] [37] An anticancer agent and a cancer metastasis inhibitor, characterized by containing a compound and a protein or a salt thereof that inhibits extracellular matrix degrading activity; [38] an anticancer agent, wherein the protein of the above-mentioned [37] or a salt thereof contains a neutralizing mouse MT2-MMP antibody selected from the antibodies of the above-mentioned [13] to [18] as an active ingredient; And a cancer metastasis inhibitor; [39] the neutralizing antibody of [38] is selected from the antibodies of [20] as an active ingredient [40] An anticancer agent or a cancer metastasis inhibitor characterized by containing a noclonal antibody or a derivative thereof; An anticancer agent and a cancer metastasis inhibitor characterized by containing a partial peptide and a derivative of a protein or a salt thereof; [41] hybridizing to a DNA and RNA sequence containing the base sequence represented by SEQ ID NO: 1 in the sequence listing [42] An antisense oligomer and a derivative thereof, which inhibit transcription or translation of mouse MT2-MMP gene and block mouse MT2-MMP expression with the antisense oligomer and derivative thereof of the above-mentioned [41]. A derivative; and [43] the antisense oligomer of the above-mentioned [41] as an active ingredient; Providing anticancer and cancer metastasis inhibitor characterized by containing the derivatives.

[0022]

BEST MODE FOR CARRYING OUT THE INVENTION A natural MT-MMP which is a kind of MMP having an ability to activate latent gelatinase A but is a latent gelatinase A activator other than MT1-MMP (for example,
Mouse MT2-MMP), a protein having substantially the same activity as that of a protein or a salt thereof, a characteristic partial peptide of the protein or a salt thereof, a gene encoding them, for example,
A vector or plasmid containing the gene such as DNA or RNA so that it can be manipulated by genetic recombination technology, a host cell transformed with such a vector, or a transgenic mouse containing the gene A method for producing the protein or a salt thereof by culturing a transgenic animal such as a transgenic animal, a knockout mouse such as a knockout mouse in which the gene is specifically deleted (deleted), or a host cell thereof; An antibody obtained using the salt or a characteristic partial peptide of the protein or the salt thereof, in particular, a monoclonal antibody, a hybridoma cell producing the antibody, the isolated gene, for example, DNA, RNA or the like as a probe Or a diagnostic means for measurement using the antibody.

More specifically, the present invention provides a mouse MT2-MMP or a salt thereof having the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing. The mouse MT2-MMP of the present invention is a kind of MMP having the activity of activating latent gelatinase A, and is a non-mouse MT1-MMP and has the activity of activating latent gelatinase A. Any activator may be used as long as it has a novel amino acid sequence. More preferably, as the mouse MT2-MMP of the present invention, SEQ ID NO:
All those having the amino acid sequence represented by 2 or an amino acid sequence substantially equivalent thereto are included. Further, as the mouse MT2-MMP of the present invention, as a prepro part, the Tet at position 128 from the Met at position 1
It may have part or all of the amino acid sequence up to yr. Any having such an arrangement may be included. The mouse MT2-MMP of the present invention is represented by SEQ ID NO:
2 from the ATG at positions 29 to 31 of the nucleotide sequence represented by 1
It may be one encoded by a nucleotide sequence composed of TGA at positions 000 to 2002 (the stop codon TGA at positions 2000 to 2002 may be TAA or TAG), and may have homology to the nucleotide sequence. However, it may be a DNA sequence having a sequence other than mouse MT1-MMP and having a nucleotide sequence having the same effect as that of having a capability of activating latent gelatinase A. The nucleotide sequence of the mouse MT2-MMP can be modified (eg, added, removed, substituted, etc.), and such modified ones may be included.

The DNA of the present invention containing the base sequence represented by SEQ ID NO: 1 in the sequence listing or a base sequence having the same effect as the base sequence
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 Labora
tory, Cold Spring Harbor, NT (1989); edited by The Biochemical Society of Japan, “Seizoku Chemistry Laboratory Course 1, Genetic Research Method II”, Tokyo Kagaku Dojin (1986); III (recombinant DNA technology) ", Tokyo Chemical Dojin (1992); R. Wed.," Methods in Enzymol
ogy ", Vol. 68, Academic Press, New York (1980);
Wu et al. Ed., "Methods in Enzymology", Vol. 100
& 101, Academic Press, New York (1983); R. Wu et a
l. ed., "Methods in Enzymology", Vol. 153, 154 & 1
55, Academic Press, New York (1987) and the like, or the method described in the literature cited therein, or a method substantially similar to or a modification thereof.

First, a known MMP family (in particular, MM
Various mouse tissues (lung, liver, brain, kidney, intestine, etc.) were probed with DNA fragments obtained from MT-MMP which is a subfamily of P family
A clone that screens a cDNA library constructed from stomach, uterus, ovary, pancreas, placenta, oral cancer, lung cancer, etc. or cultured cells (mouse fibrosarcoma cells, mouse monocytic leukemia cells, etc.) and hybridizes to the probe Is selected, the nucleotide sequence of the cDNA insertion sequence in the clone is determined, and a DNA fragment having a novel MMP gene sequence is obtained. If necessary, the inserted sequence of the cDNA in the clone can be subcloned. The nucleotide sequence is determined by the Maxa method such as the dideoxy method such as the M13 dideoxy method.
It can be performed using the m-Gilbert method, etc. Can be done.

Thus, the novel MMP gene sequence has
DNA fragments can be obtained,
If necessary, for example, to obtain a DNA fragment containing a reading frame, the DNA fragment having the novel MMP gene sequence thus obtained can be used as a probe to construct DNA fragments from various mouse tissues or cultured cells as described above. The cloned cDNA library is screened, and a clone that hybridizes to the probe is selected.
The base sequence of the inserted sequence of the cDNA is determined, and a DNA fragment having a novel MMP gene sequence is obtained. Needless to say, the insert sequence of the cDNA in the clone can be subcloned if necessary. Based on the determined base sequence,
The desired DNA can be isolated. Preferably, a mouse lung cDNA library is screened, the nucleotide sequence is determined, and the desired DNA is isolated. The labeling of the probe or the like with a radioisotope or the like can be performed using a commercially available labeling kit, for example, a random primed DNA labeling kit (Boehringer Mannhaim).

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 mouse tissues or cultured cells as described above. Particularly preferably, mRNA can be isolated from mouse lung cells. Isolation of mRNA can be carried out by a method known in the art or a method substantially similar to or a modification thereof.
"Molecular Cloning", 2nd ed., Chapter 7, Cold Sprin
g Harbor Laboratory, Cold Spring Harbor, NT (19
89); L. Grossman et al. Ed., "Methods in Enzymolog
y ", Vol. 12, Part A & B, Academic Press, New York
(1968); SL Berger et al. Ed., "Methods in Enzym
ology ", Vol. 152, p. 33 & p. 215, Academic Press, Ne
w York (1987); Biochemistry, 18: 5294-5299, 1979, etc., such as the guanidine-cesium chloride method,
It can be carried out by a method such as a guanidine thiocyanate method or a phenol method. If necessary, the obtained total RNA can be purified using an oligo (dT) -cellulose column or the like to obtain poly (A) ⁺ mRNA. CDNA is prepared using this mRNA and reverse transcriptase. cDNA synthesis using mRNA and reverse transcriptase can be performed by a method known in the art or a method substantially similar thereto or a modified method, but H. Land et al., Nucleic Acids Res., 9: 2251,
1981; U. Gubler et al., Gene, 25: 263-269, 1983;
SL Berger et al. Ed., "Methodsin Enzymology",
Vol. 152, p. 307, Academic Press, New York (1987)
And the like.

Based on the cDNA thus prepared, a cDNA library can be constructed by using a phage vector or the like. In addition to using a phage vector, transformation of host cells such as Escherichia coli can be performed by a method known in the art such as a calcium method, a rubidium / calcium method, or a method substantially similar thereto. (D. Hanahan, J. Mol. Biol., 166: 557, 1983)
Such). Furthermore, commercially available cDNA libraries derived from various tissues can also be directly used. For example, cDNA libraries commercially available from Clontech, USA and the like can be used. Another method is also possible by performing a PCR amplification reaction using the thus prepared cDNA as a template. Typically, the known MMP family (in particular, MM
A highly conserved amino acid sequence (eg, pro- and active cleavage sites) selected from an amino acid sequence obtained from a subfamily of the P family, MT-MMP, more preferably human MT1-MMP Neighbor array) and MT
-The amino acid sequence of the region specifically present in MMP (for example,
Based on the amino acid sequence of the transmembrane domain region on the C-terminal side, the amino acid sequence of the region existing between the propeptide domain and the catalytic domain, etc., a degenerated primer is prepared. The primer can be prepared by a method known in the art, for example, using an automatic DNA synthesizer by a phosphodiester method, a phosphophotoester method, a phosphoramidite method, or the like. PCR is performed using this primer and the cDNA prepared above. The PCR reaction is
It can be carried out by a method known in the art or a method substantially similar to or a modification thereof, for example, R. Saik
i, et al., Science, 230: 1350, 1985; R. Saiki, et
al., Science, 239: 487, 1985; PCR technology
(PCR Technology), Stockton Pre
ss) and the like.
The resulting PCR product was cloned and the resulting PCR
The nucleotide sequence of the product can be determined to obtain a DNA fragment having a novel MMP gene sequence. Also, this DNA
Various cDNA libraries can be screened in the same manner using the fragment as a probe to isolate a target DNA.

The details will be described below. Probe DN
Using A, a novel MT2-MMP gene is screened from the cDNA library, and the nucleotide sequence of the obtained DNA fragment encoding the novel MT2-MMP gene is determined. Probe DN
As A, for example, a human MT1-MMP cDNA fragment (Gene, 15
5: 293-298, 1995), which can be labeled and used as a probe. For labeling, a DNA labeling kit can be used. For example, using a random primed DNA labeling kit (Boehringer Mannhaim) or the like, the probe DNA is converted to [α- ³² P] dCTP (Amersha
m) or the like to obtain a probe having radioactivity. Plaque hybridization is performed using the labeled cDNA fragment and a cDNA library prepared from mouse tissue cells, such as a mouse lung cDNA library (available from Clontech). The mouse lung 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 and forming plaques. In the hybridization, the plaque formed as described above is transferred to a membrane such as a nylon filter, denaturation treatment, immobilization treatment, washing treatment, etc., if necessary, and then transferred to the membrane, The reaction is performed by reacting with a denatured labeled probe cDNA fragment in a hybridization buffer as necessary.

[0030] The hybridization process is usually carried out at about 40
C. to about 80C, more preferably about 55C to about 65C, for about 15 minutes to
It is carried out for about 24 hours, more preferably for about 1 hour to about 4 hours, but it can be carried out by selecting optimal conditions as appropriate. For example, the hybridization treatment is performed at about 60 ° C. for about 2 hours. The hybridization buffer can be selected from those commonly used in the art, for example, Rapid hybridization buffer
(Amersham) can be used. Examples of the denaturation treatment of the transferred film include a method using an alkali denaturing solution, and after the treatment, treatment with a neutralizing solution or a buffer solution is preferred. The immobilization of the membrane is usually performed at about 40 ° C to about 1 ° C.
Baking is performed at 00 ° C, more preferably at about 70 ° C to about 90 ° C, for about 15 minutes to about 24 hours, more preferably for about 1 hour to about 4 hours. It can be carried out. For example, the immobilization is performed by baking the filter at about 80 ° C. for about 2 hours. Washing of the transferred membrane includes washing solutions commonly used in the art, such as 1 M NaCl, 1 mM EDTA and 0.1% Sodium D
50mM Tris-HC1 buffer containing odecyl sulfate (SDS), pH
It can be done by washing with 8.0. As a membrane such as a nylon filter, a membrane commonly used in the art can be selected and used. For example, a nylon filter [Hybond-N, Amsham]
And the like.

The above-mentioned alkali denaturing solution, neutralizing solution and buffer solution can be selected from those commonly used in the art, and examples of the alkali denaturing solution include:
A solution containing 0.5 M NaOH and 1.5 M NaCl can be mentioned. As the neutralizing solution, for example, a solution containing 1.5 M NaCl
0.5M Tris-HCl buffer, pH 8.0, etc., and the buffer may be, 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% polyvinylpyroldone), 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 cDNA used for hybridization
Denaturation of the fragments can be 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 cDNA fragment that has undergone the specific hybridization reaction. The washing treatment of the filter can be performed by selecting from those commonly used in the art. For example, 0.5 × SSC containing 0.1% SDS (O.15M NaCl,
It can be carried out by washing with a 15 mM citric acid) solution or the like. 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.
5), and then appropriately dilute the phage suspension, infect Escherichia coli, culture the obtained Escherichia coli, and obtain the desired recombinant phage from the cultured Escherichia coli. The process of screening for a target recombinant phage from a cDNA library by hybridization using the above-described 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). DNA can be purified and separated from the phage particles by a method commonly used in the art.
O ₄ containing 50 mM Tris-HCl buffer, pH 7.8) were suspended in such,
After treatment with DNase I and RNase A, 20mM EDTA, 50
Add a mixture of μg / ml Proteinase K and 0.5% SDS, incubate at about 65 ° C for about 1 hour, extract with phenol and diethyl ether, and precipitate with DNA by ethanol precipitation.
Then, the obtained DNA is washed with 70% ethanol, dried, and dissolved in a TE solution (10 mM Tris-HC1 buffer containing 10 mM EDTA, 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. The DNA obtained by such subcloning can be purified and separated by methods such as centrifugation, phenol extraction, and ethanol precipitation in the same manner as described above.

The nucleotide sequence of the DNA thus obtained, for example, the nucleotide sequence of a single-stranded DNA, is determined by using a fluorescent DNA sequencer Model 373.
The sequence can be determined using A (Applied Biosystems), Taq Dye Primer Cycle Sequencing Kit (Applied Biosystems), or the like. Regarding the determined nucleotide sequence of the DNA, for example, out of 8 clones containing the target DNA thus obtained, 6 clones are Proc. Natl. Acad. Sci. USA., 92: 2730-2734, 199
5 and a part of the mouse MT1-MMP gene. Using the DNA insert of this clone as a probe,
Again, by screening the mouse lung cDNA library etc. in the same manner as above, it is necessary to make a clone containing the complete open reading frame, a clone containing the missing gene part, a complete open reading frame. It is possible to obtain a clone or the like containing a portion that is not allowed. When screening such a re-cDNA library, it is preferable to select and use hybridization conditions with higher specificity. In addition, primers are designed based on the determined base 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 mouse cells). ,
Similarly, a desired clone can be obtained. In addition, PCR amplification is performed using these primers to obtain a target gene, a novel gene, a fragment thereof, and the like. Thus, a PCR product having homology to mouse MT2-MMP but having a novel sequence can be searched.

Thus, according to the present invention, a positive clone can be searched and obtained by screening using the DNA insert of the clone containing the target DNA obtained above as a probe. For example, one clone of the recombinant phage can be isolated as a clone containing the desired DNA. When the nucleotide sequence of the DNA of the gene isolated from the cloned recombinant phage was determined, the determined total length of the nucleotide sequence was 3339 bp,
It is recognized that the sequence shown in SEQ ID NO: 1 in the sequence listing was obtained. GENBANK / EMBL DNA Data Ba
When se was searched for the base sequence described in SEQ ID NO: 1 in the sequence listing, no identical sequence was found. The isolated and identified DNA sequence of approximately 3.3 kb contains an estimated 65
The presence of an open reading frame encoding 7 amino acids was confirmed, and the deduced amino acid sequence was confirmed to be as shown in SEQ ID NO: 2 in the sequence listing. This putative protein was named "mouse MT2-MMP". The mouse MT2-MMP gene is a novel M
It is clear that they encode the MP protein, and the recombinant plasmids produced using the mouse MT2-MMP gene are all novel recombinants, and the traits obtained by transformation or transfection with the plasmids were obtained. Transformants or transfectants are also novel. The obtained DNA fragment is inserted into an appropriate vector as described in detail below, for example, a vector such as plasmid pEX, pMAMneo, pKG5, etc., and a suitable host cell as described in detail below, for example, Escherichia coli, CHO cell And the like. The DNA fragment may be used as it is or as a DNA fragment to which an appropriate control sequence has been added, or may be incorporated into an appropriate vector and introduced into an animal to obtain MT2-
Transgenic animals expressing the MMP gene, for example, mouse MT2-MMP, can be created. Animals include warm-blooded animals, such as mice, rats, rabbits, guinea pigs, pigs, and the like. Preferably, the transgenic animal can be prepared by introducing the DNA fragment into a fertilized egg of an animal such as a mouse.

The amino acid sequence described in SEQ ID NO: 2, which is deduced from the nucleotide sequence of mouse MT2-MMP described in SEQ ID NO: 1 in the sequence listing obtained by the present invention, has a known human MT-M
When compared with the amino acid sequence of MPs, the amino acid sequence shown in SEQ ID NO: 2 has high homology with human MT-MMPs, and has a domain structure characteristic of human MT-MMPs, that is, it is removed during secretory production. Signal peptide, propeptide domain, catalytic domain, hinge domain,
The hemopexin clotting enzyme-like domain is well conserved. Therefore, those having such characteristics may be included in the mouse-derived protein of the present invention. In particular, human MT
The sequence PRCGVPD near the pro-form and the active cleavage site, which is highly conserved in the MMP family, including -MMPs,
It is completely conserved also in mouse MT2-MMP (Pro ^{105 to} Asp ¹¹¹ of SEQ ID NO: 2 in the sequence listing). Mouse MT2-MMP
Has three insertion sequences, which are characteristic points in the amino acid sequence of human MT-MMPs compared to other MMP families. That is, the insertion sequence of 13 amino acid residues including the sequence of RRKR existing between the propeptide domain and the catalytic domain-1 (IS-1; Val ¹¹⁵ to SEQ ID NO: 2 in the sequence listing)
Arg ¹²⁷ ), consisting of the sequence of SYDDIRLR in the catalytic domain
Sequence of amino acid residues 2 (IS-2; Ser ^{179 to} Arg ¹⁸⁶ of SEQ ID NO: 2 in the sequence listing) and a continuous sequence of 22 hydrophobic amino acids like transmembrane domain VVMVLVPLLL
Insertion sequence of 102 amino acid residues including LLCILGLAFALV-3
(IS-3; the sequence listing SEQ ID NO: 2 of Gln ⁵⁵⁶ ~Val ⁶⁵⁷⁾ is present.

The first inserted sequence IS-1 is exceptionally present in MMP-11 in the human MMP family, but the sequence RXKR (X is any amino acid residue) conserved in IS-1. ) Is the sequence of the cleavage site for subtilisin-like protease, and the amino acid sequence RXKR is known to be the cleavage site for many eukaryotic secretory proteins by subtilisin-like protease (J. Biol. Chem., 266). : 12127 〜
12130, 1991). The continuous sequence of hydrophobic amino acids in IS-3 is considered to be a transmembrane domain, and human MT-MMPs
(Cancer Res., 56: 944-
949, 1996), and the continuous sequence of hydrophobic amino acids present in IS-3 of mouse MT2-MMP is also considered as a transmembrane domain. Therefore, those having all or any or any combination of these characteristics may be included in the mouse-derived protein of the present invention. mouse
When the amino acid sequence of MT2-MMP was compared with other known human MT-MMPs, the homology to human MT2-MMP was highest at 87%, and the homology to other MT-MMPs was human MT1-MM.
52% for P, 50% for human MT3-MMP, human MT4-
Since it shows 29% homology to MMP, the mouse-derived protein of the present invention includes SEQ ID NO: 2 in the sequence listing.
Over 87% are included in that.

The confirmation of the mouse MT2-MMP gene product was performed by using the COS-transfected mouse MT2-MMP gene.
It 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), the DEAE-dextran method (for example, D. Warden et al., J. Gen. Virol., 3:
371, 1968), an electroporation method (for example, E. Neumann et al., EMBO J, 1: 841, 1982 and the like), a microinjection method, a ribosome method, a virus infection method, a phage particle method, and the like. The gene product produced by the animal cells transfected with the mouse MT2-MMP gene in this manner is an anti-mouse MT2-MMP
It can also be analyzed by an immunoprecipitation experiment using a monoclonal antibody. In such an analysis, for example, a specific transfected gene product protein is immunoprecipitated from a transfected animal cell lysate, while the corresponding protein is not detected in the culture supernatant. You can also. That is, the mouse MT2-MMP gene product is not secreted,
It can be done by showing that it is expressed on the cell surface.

As a plasmid incorporating the mouse MT2-MMP gene, host cells commonly used in genetic engineering (for example,
Prokaryotic host such as Escherichia coli and Bacillus subtilis, eukaryotic host such as yeast and CHO cell, and insect cell host such as Sf21).
Any plasmid can be used as long as it can express A. In such a sequence, for example, a codon suitable for expression in a selected host cell can be introduced, a restriction enzyme site can be provided, and expression of a target gene can be easily performed. Linkers and adapters that are useful for binding the target gene, such as regulatory sequences and promoter sequences, and sequences that are useful for controlling antibiotic resistance, metabolism, selecting, etc. Etc. can be included. Preferably,
For a suitable promoter, for example, a plasmid using Escherichia coli as a host, tryptophan (trp) promoter, lactose (lac) promoter, tryptophan lactose (tac) promoter, lipoprotein (l
pp) promoter, λ phage PL promoter, etc., in plasmids using animal cells as hosts, SV40 rate promoter, MMTV LTR promoter, R
SV LTR promoter, CMV promoter, SR
GAL1, GAL10 promoters and the like can be used for plasmids using an α promoter or the like as a yeast host.

As a plasmid using Escherichia coli as a host,
For example, pBR322, pUC18, pUC19, pUC
118, pUC119, pSP64, pSP65, pT
Z-18R / -18U, pTZ-19R / -19U, p
GEM-3, pGEM-4, pGEM-3Z, pGEM
-4Z, pGEM-5Zf (-), pBluecri
pt KS ^™ (Stratagene) and the like. Plasmid vectors suitable for expression in E. coli include pA
S, pKK223 (Pharmacia), pMC1403, pM
C931, pKC30 and the like are also included. Examples of plasmids using animal cells as a host include SV40 vectors, polyoma virus vectors, vaccinia virus vectors, retrovirus vectors, and the like. For example, pcD, pcD-SRα, CDM8, pCEV4, pM
E18S, pBC12BI, pSG5 (Stratagene) and the like. Examples of plasmids using yeast as a host include YIp-type vectors, YEp-type vectors, YRp-type vectors, and YCp-type vectors.
PD-2 and the like. When the host cell is Escherichia coli, examples of the host cell include those derived from Escherichia coli K12 strain, for example, NM533 XL1-Blue,
C600, DH1, HB101, JM109 and the like. When the host cell is an animal cell, for example, COS7 cell, COS-1 cell, CV-1 cell derived from African green monkey fibroblast, COP cell derived from mouse fibroblast, MOP cell, WOP cell, derived from Chinese hamster cell 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 the silkworm nuclear polyhedrosis virus (Bombyx mori)
nuclear polyhedrosis virus) as a vector, and using silkworm larvae or silkworm culture cells, such as BM-N cells.

In the genetic engineering technique of the present invention, restriction enzymes, reverse transcriptases, and DNA fragments known or widely used in the art are modified or converted into a structure suitable for cloning. DNA modifying / degrading enzymes, DNA polymerase, terminal nucleotidyltransferase, DNA ligase, 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, etc., and particularly, RNase H-deficient ones and the like can be preferably used. As a DNA polymerase, for example, E. coli DN
A polymerase, its derivative Klenow fragment, Escherichia coli phage T4 DNA polymerase, Escherichia coli phage T7 DNA polymerase, thermostable DNA polymerase and the like. Examples of the terminal nucleotidyl transferase include R. Wu et al. Ed., "Methods
in Enzymology ", Vol. 100, p. 96, Academic Press,
New York (1983), such as TdTase for adding a deoxynucleotide (dNMP) to the 3'-OH end. Examples of DNA modifying / degrading enzymes include exonucleases and endonucleases.
II, λ exonuclease, DNase I, nuclease S1, Micrococcus nuclease and the like. Examples of the DNA ligase include Escherichia coli DNA ligase and T4 DNA ligase. Suitable vectors for cloning DNA genes and constructing DNA libraries include plasmids, λ
Phage, cosmid, P1 phage, factor F, YAC, etc., and preferably a vector derived from λ phage, such as Charon 4A, Charon
21A, λgt10, λgt11, λDASHII, λ
FIXII, λEMBL3, λZAPII ^™ (Stratage
ne).

Furthermore, by using a method commonly used in genetic engineering based on the gene base sequence of mouse MT2-MMP according to the present invention, one or more amino acids can be appropriately contained in the amino acid sequence of mouse MT2-MMP. Amino acid substitution, deletion,
Corresponding proteins into which mutations such as insertion, transposition or addition have been introduced can be produced. Such mutation, conversion, and modification methods are described in, for example, The Biochemical Society of Japan, “Seismological Chemistry Laboratory Course 1, Gene Research Method II”, p105 (Susumu Hirose), Tokyo Kagaku Dojin (1986);
"New Course on Experimental Chemistry 2, Nucleic Acid III (Recombinant DNA Technology)", p233 (Susumu Hirose), Tokyo Chemical Dojin (199)
2); R. Wu, L. Grossman, ed., "Methods in Enzymol
ogy ", Vol. 154, p. 350 & p. 367, Academic Press, N
ew York (1987); R. Wu, L. Grossman, ed., "Methods i
n Enzymology ", Vol. 100, p. 457 & p. 468, Academic
Press, New York (1983); JA Wells et al., Gen
e, 34: 315, 1985; T. Grundstroem et al., Nucleic A
cids Res., 13: 3305, 1985; J. Tayloret al., Nucle.
ic Acids Res., 13: 8765, 1985; R. Wu ed., "Methods
in Enzymology ", Vol. 155, p. 568, Academic Press,
New York (1987); AR Oliphant et al., Gene, 4
4: 177, 1986 and the like. For example, position-directed mutagenesis using synthetic oligonucleotides (site-directed mutagenesis), Kunkel method, dNTP [αS]
Method (Eckstein), a region-directed mutagenesis method using sulfurous acid, nitrous acid, or the like. Furthermore, the obtained protein of the present invention can modify the amino acid residues contained therein by a chemical method, and can also use enzymes such as peptidases such as pepsin, chymotrypsin, papain, bromelain, endopeptidase, and exopeptidase. It can be used to modify or partially decompose to 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 mouse MT2-MMP in vivo or in vitro. Good. 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. For example, modification and alteration of the structure of a protein is described in, for example,
VII, Protein Engineering ", Tokyo Kagaku Dojin (1993), and can be carried out by the method described therein, or the method described in the literature cited therein, or a method substantially similar thereto. As described below, the biological activity may include having an immunological activity, for example, having an antigenicity.

Thus, in the present invention, one or more amino acid residues may be different from the natural one in terms of identity, and the position of one or more amino acid residues may be different from the natural one. The present invention relates to a mouse MT2-MMP having one or more amino acid residues (for example, 1 to 80, preferably 1 to 6).
0, more preferably 1 to 40, more preferably 1 to 20, especially 1 to 10 deletion analogs, one or more of the unique amino acid residues (eg 1 to 8
0, preferably 1 to 60, more preferably 1 to 4
0, more preferably 1 to 20, especially 1 to 10, etc. substituted analogues substituted with other residues, 1 or more (eg 1 to 80, preferably 1 to 60) , More preferably 1 to 40, more preferably 1 to 20
(Especially 1 to 10, etc.) amino acid residues. As long as the domain structure and the C-terminal transmembrane main structure, which are common features of MMPs, are maintained, all of the above mutants are included in the present invention. Further, it is considered that the mouse MT2-MMP of the present invention may also include those having a primary structure conformation substantially equivalent to the natural mouse MT2-MMP or a part thereof, and furthermore, the natural mouse MT2-MMP. It is contemplated that those having a biological activity substantially equivalent to that of MMP may be included. It can also be one of the naturally occurring variants. Such a mouse MT2-MMP of the present invention,
Separation and purification can be performed as described below. Natural MT-MMP, which is a kind of MMP having the ability to activate latent gelatinase A of the present invention thus obtained and which is a latent gelatinase A activator other than MT1-MMP
(Especially mouse MT2-MMP), a protein or a salt thereof or a partial peptide thereof having substantially the same activity as that of a mouse MT2-MMP. Development research, mouse MT2-MMP
Can be used to study biological phenomena and reactions that may be involved, can be used to generate antibodies against it, and can be used to study specific analytes or analytes. You can also. On the one hand, the invention thus also encompasses DNA sequences coding for the above-mentioned polypeptides, and mouse MT2-MMP polypeptides having all or part of the natural properties, as well as DNA sequences coding for their analogs or derivatives. .

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 applications include, for example, the design of probes for the isolation and detection of genomic DNA and cDNA from mammals, particularly preferably humans, encoding mouse MT2-MMP 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 mouse and human, encoding mouse MT2-MMP and related proteins. When isolating genes,
PCR, and PCR using reverse transcriptase (RT)
Method (RT-PCR) can be used. Mouse MT
The 2-MMP cDNA and its related DNA were obtained by selecting a characteristic sequence region based on the amino acid sequence deduced from the cloned and sequenced mouse MT2-MMP cDNA sequence, designing DNA primers and chemically synthesizing them. The obtained DNA primers can be used for the isolation and detection of mouse MT2-MMP-related genes using PCR, RT-PCR, and other methods. For example, expression of mouse MT2-MMP mRNA in mouse tissues is determined by poly (A) ⁺ RNA derived from various tissues.
Can be examined by Northern blot analysis. When the cDNA of the present invention is used as a probe, for example, Northern blotting, Southern blotting, ins
Itu hybridization can be used to detect and measure mouse MT2-MMP mRNA expression in mouse tissues and the mouse MT2-MMP gene itself.Thus, based on studies using mice, the presence or absence of cancer cells in human tissues, And the development of research such as diagnosis and treatment of cancer such as diagnosis of malignancy of Alzheimer's disease.

The DNA obtained in the present invention (for example, mouse MT
When the DNA encoding 2-MMP) is transferred to a target animal, it is generally advantageous to use it as a DNA fragment or by binding the DNA downstream of a promoter capable of expressing the DNA in animal cells. For example, mouse MT
When introducing 2-MMP DNA, a gene construct in which mouse MT2-MMP DNA derived from an animal having high homology to the gene is linked to the downstream of various promoters capable of expressing the same in animal cells is used as a fertilized egg of a target animal, for example, a mouse fertilized mouse. By microinjection into eggs, transgenic mice can be produced that produce high levels of mouse MT2-MMP. Although the mouse is not particularly limited to a pure mouse, for example, C57BL / 6, Balb / C, C3H, (C57BL
/ 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 mouse MT2-MMP 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. DNA obtained by the present invention at the fertilized egg cell stage (for example, mouse MT2-MMP
Is ensured to be present in all germinal and somatic cells of the subject animal. Encodes MT2-MMP in germ cells of produced animals after DNA transfer
The presence of DNA indicates that the offspring of the animal produced will have the MT2-MMP encoding DNA in all of its germinal and somatic cells.
Has the following meaning. The offspring of such animals that have inherited the gene have the potential to express the MT2-MMP in all of their germinal and somatic cells.

The MT2-MMP DNA-introduced animal can be bred in a normal breeding environment as an animal having the DNA after confirming that the gene is stably maintained by the cross.
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. The animal into which the MT2-MMP DNA has been introduced is
Since the MT2-MMP protein is highly expressed, the MT2
It is useful as an animal for screening for inhibitors (inhibitors) against 2-MMP protein. Also MT2-
It is useful as an animal for screening an antisense oligonucleotide capable of inhibiting MMP gene replication, for example, antisense DNA. This transgenic animal can also be used as a cell source for tissue culture. For example, by directly analyzing DNA or RNA in the tissue of a transgenic mouse, or by analyzing the protein tissue expressed by the gene,
MMPs-related proteins can be analyzed.
The cells of the tissue having the MT2-MMP can be cultured by standard tissue culture techniques and used to study the function of cells from generally difficult-to-culture tissues, for example, from lungs and other tissues. In addition, by using the cells, it is possible to contribute to the development of a drug that enhances the functions of various tissues, for example. In addition, if there is a high expression cell line, MT2-MMP can be isolated and purified therefrom. Techniques related to transgenic mice and the like are described, for example, in Brinster, RL, et al.,; Proc.
Acad. Sci. USA, 82: 4438, 1985; Costantini, F. & Ja
enisch, R. (eds): Genetic manipulation of the early
mammalian embryo, Cold Spring Habor Laboratory, 1
The method can be carried out according to the method described in a document such as 985 or the method described in the document cited therein, or a modified method thereof.

The gene obtained by the present invention (for example, mouse
DNA encoding MT2-MMP) has a mutation in mouse MT2-
Mutant mice that do not express MMP at all (knockout mice) can be created. For example, genomic DNA of about 8 kb including 4 kb before and after the translation initiation codon of the gene
In the exon near the center of the translation start codon
o A targeting vector having a mutant gene into which a gene cassette comprising a resistance gene and a polyA addition signal is inserted can be constructed. The gene cassette to be inserted is ne
o In addition to the resistance gene cassette, DT-A cassette, tk cassette, lacZ cassette and the like can be mentioned. The targeting vector is opened linearly and the established mouse embryonic stem cells (em
The cells are introduced into bryonic stem cells (ES cells) by electroporation and further cultured to select the ES cells that have acquired neo resistance. ES cells are 129, C57BL / 6, F1 (C57BL / 6 × CBA)
It can be prepared by selecting from mouse strains such as mice. It is assumed that the ES cells that have acquired neo resistance have undergone homologous recombination with the targeting vector into which the gene cassette has been inserted in the MT2-MMP gene region, and at least one of the MT2-MMP gene alleles is disrupted and
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.

Mutant-introduced ES cells are C57BL / 6, BALB
/ c, injected into 8-cell stage embryos taken from ICR mice, etc.
An individual can be grown by culturing it for one day and developing the blastocyst into 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 thus obtained has only the MT2-MMP gene disrupted in all germ cells and somatic cells and does not express MT2-MMP at all. Have. This knockout mouse is useful for analyzing the role of MT2-MMP in the life cycle of an individual, such as development, growth, reproduction, aging, and death, and the function of MT2-MMP in various organs and tissues, compared to normal mice. . In addition, by analyzing the susceptibility to carcinogens, primary cancers and transplanted cancers, MT2-MMP is useful for analyzing the involvement of cancer invasion and metastasis. Can be applied. 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 mouse MT2-MMP at the cell level. Technologies related to knockout mice and the like include, for example,
Mansour, SL, et al.,; Nature, 336: 348-352, 198
8; Joyner, AL, ed .; Gene targeting, IRL Press,
1993; Aizawa, Shin-ichi, Generation of mutant mice using gene-targeting ES cells, methods described in references such as Yodosha, 1995, or methods cited therein, and modifications thereof. Can be.

According to the present invention, an antisense oligonucleotide (nucleic acid) capable of inhibiting the replication or expression of the MT2-MMP gene is cloned or determined. The nucleotide sequence of the DNA encoding MT2-MMP is determined. It can be designed and synthesized based on information. Such an oligonucleotide (nucleic acid) can hybridize with the RNA (or DNA) of the MT2-MMP gene and inhibit the synthesis or function of the RNA, or interact with the MT2-MMP-related RNA. The expression of the MT2-MMP gene can be regulated and controlled through such methods. Oligonucleotides complementary to the selected sequence of the MT2-MMP-related gene and oligonucleotides capable of specifically hybridizing with the MT2-MMP-related gene are capable of in vitro and in vitro expression of the MT2-MMP gene. It is useful for regulation and control, and 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 a nucleotide, base sequence or nucleic acid and a peptide (protein) usually refers to the amino acid of the 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 (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special linkages, such as base pairing or bases as found in DNA or RNA Containing nucleotides having a configuration permitting the attachment of). 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- -Those having a side chain group such as lysine or sugar (e.g., monosaccharide), those having an interactive compound (e.g., acridine, psoralen, etc.), chelating compounds (e.g., metal, 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, crude water-based substances such as phospholipid and cholesterol can be used. 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. Other groups include cap groups specifically arranged at the 3 'end or 5' end of nucleic acids, which are 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 the MT2-MMP translation system in vivo or in vitro. The nucleic acid can be applied to cells by various methods known per se. As described above, based on the research results of the present inventors, the mouse MT2-MMP gene and the recombinant DNA molecule were transferred to a host, and the mouse MT2-MMP was expressed.
-A method for obtaining MMP is provided. Thus, according to the present invention, a recombinant or transfectant which substantially expresses the mouse MT2-MMP gene, a method for producing the same, and uses thereof are also provided. In another aspect, the present invention relates to a type of MMP capable of activating latent gelatinase A and an activity substantially equivalent to that of natural MT-MMP which is a latent gelatinase A activator other than MT1-MMP. Or a salt thereof, more preferably a mouse MT2-MMP or a salt thereof, having at least substantially the same activity, or at least one of the proteins having substantially the same primary structure conformation. The present invention can relate to a nucleic acid such as DNA or RNA that enables a polypeptide having a part or all thereof to be expressed in a prokaryote such as Escherichia coli or a eukaryote such as a mammalian cell. Further, such a nucleic acid, particularly a DNA, may be (a) a sequence capable of encoding the amino acid sequence represented by SEQ ID NO: 2 or a sequence complementary thereto, and (b) the DN of (a).
It may be a sequence capable of hybridizing with the A sequence or a fragment thereof, and (c) a sequence having a degenerate code capable of hybridizing with the sequence of (a) or (b). 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.

Further, the present invention provides an antibody such as a monoclonal antibody which specifically reacts with the mouse MT2-MMP of the present invention. Antibodies such as the monoclonal antibody according to the present invention provide useful research means for research relating to cancer invasion and metastasis as well as cancer diagnosis, as well as research related to the mechanism of onset of Alzheimer's disease and diagnostic methods. Provided. Antibodies such as the monoclonal antibody according to the present invention can be used to immunize animals by a known method using the mouse MT2-MMP or a fragment thereof obtained according to the present invention as an immunogen, a method known or widely used in the art, For example, the method of Milstein et al. (Nature, 256: 495-
97, 1975). In this method, the immunogen has an amino acid sequence selected from a region having low homology with other MT-MMP families among native mouse MT2-MMP, recombinant mouse MT2-MMP and mouse MT2-MMP. Fragments obtained from synthetic peptides or proteins, for example, a synthetic peptide having a partial amino acid sequence of mouse MT2-MMP consisting of at least 8 consecutive amino acids, for example, a synthetic peptide having an amino acid sequence having 14 amino acid residues Preferably, any peptide having a sequence portion characteristic of mouse MT2-MMP can be used.

The mouse MT2-MMP can be obtained from in vivo and external production cells, for example, cultured cells, excised tissues, cultured tissues, etc., for example, cells such as lung cells and cancer cells. In addition, mouse MT2-MMP
Can be obtained as recombinant mouse MT2-MMP,
For example, it can be obtained from mouse MT2-MMP-producing cells such as lung cells by utilizing the technique of gene recombination. Mouse MT2-MMP prepared in the present invention or one derived therefrom can be suitably used as an immunizing antigen. These mouse MT
2-MMP is used for various raw materials, such as cultured cells and cultured tissues.
Conventionally known methods from antigen-producing materials such as transformed cells, such as salting out such as ammonium sulfate precipitation, gel filtration using Sephadex, etc., for example, ion exchange using a carrier having a diethylaminoethyl group or a carboxymethyl group, etc. Chromatography method, for example, hydrophobic chromatography method using a carrier having a hydrophobic group such as butyl group, octyl group and phenyl group, dye gel chromatography method, electrophoresis method, dialysis, ultrafiltration method, affinity chromatography It can be obtained by purification by a chromatography method, a high performance liquid chromatography method or 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. The purified recombinant mouse MT2-MMP can be suitably used as an immunizing antigen for producing a monoclonal antibody.

For example, gelatin-agarose affinity chromatography, heparin-agarose
Chromatography. Further, a peptide is synthesized based on the information of the mouse MT2-MMP gene obtained in the present invention, and the synthesized peptide can be suitably used as an immunizing antigen for producing a monoclonal antibody. Further, the monoclonal antibody 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, pigment substances, biotin and the like can be used. Hereinafter, production of the antibody will be described in detail.

It goes without saying that the monoclonal antibody of the present invention may be a monoclonal antibody obtained by cell fusion technology 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 and monocloning of hybridomas (fused cells) Production of monoclonal antibodies

1. Preparation of Immunogenic Antigen Recombinant mouse pro-MT2-MMP prepared according to the method described above can be used as an antigen, but an appropriate oligopeptide is chemically synthesized based on the determined MT2-MMP information. It can be used as an antigen.
As the mouse MT2-MMP, latent mouse MT2-MMP and active mouse MT2-MMP 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, mouse MT2-MMP is obtained by fragmenting it, or selecting a characteristic sequence region based on the amino acid sequence deduced from the cloned and sequenced cDNA sequence, designing the polypeptide, and chemically synthesizing it. The resulting synthetic polypeptide fragment may be combined with various carrier proteins via a suitable condensing agent to form an immunogenic conjugate such as a hapten-protein, Can be used to design a monoclonal antibody that can react only with the sequence (or can recognize only a specific sequence). 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 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. Carrier proteins include 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 antigens To immunize animals, for example, Shigeru Muramatsu, et al., Experimental Biology Course 14, Immunobiology, Maruzen Co., Ltd., 1985, Ed. 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 subcutaneously, intravenously or intramuscularly
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 measurement system is prepared, and the antibody titer is measured to confirm the degree of animal immunity.

3. Preparation of myeloma cells (myeloma cells) Infinitely proliferative strains (tumor cell strains) used for cell fusion can be selected from cell lines that do not produce immunoglobulin. For example, P3-NS-1-Ag4-1 ( NS-
1, Eur. J. Immunol., 6: 511-519, 1976), SP2 /
0-Ag14 (SP2, Nature, 276: 269-270, 1978
), P3-X63-Ag8-U1 derived from the mouse myeloma MOPC-21 cell line (P3U1, Curr.
cs Microbiol. Immunol., 81: 1-7, 1978), P3-
X63-Ag8 (X63, Nature, 256: 495-497, 197
5), P3-X63-Ag8-653 (653, J. Imm
unol., 123: 1548-1550, 1979). The 8-azaguanine-resistant mouse myeloma cell line is a Dulbecco MEM medium (DMEM medium), RPMI-
For example, an antibiotic such as penicillin or amikacin, fetal calf serum (FCS) or the like is added to a cell medium such as 1640 medium, and 8-azaguanine (for example, 5-45 μg /
ml) in the medium supplemented with
The required number of cell lines can be prepared by passage in a normal medium one day before. The cell strain used was a cryopreserved strain at about 37 ° C.
After completely thawed in step (1), the cells may be washed three times or more with a normal medium such as an RPMI-1640 medium, and then cultured in a normal medium to prepare a required number of cell lines.

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 step is placed in a cell medium such as a minimum essential medium (MEM medium), a DMEM medium, and an RPMI-1640 medium, and a cell fusion agent such as polyethylene glycol is added. As the cell fusion agent, other various known in the art can be used,
Examples of such a virus include an inactivated Sendai virus (HVJ: Hemagglutinating Vir).
us of Japan). Preferably, for example, 0.5 to 2 ml of 30 to 60% polyethylene glycol can be added, and the molecular weight is 1,000.
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 to
It is preferable to set it to 60%. As needed,
For example, a small amount of dimethyl sulfoxide or the like can be added to promote fusion. The ratio of splenocytes (lymphocytes) to myeloma cell line used for fusion is, for example, 1: 1 to 2
0: 1, more preferably 4:
It can be 1-7: 1. 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, the cells are separated by centrifugation or the like, and then transferred to a selection medium.

5. Selection and Monocloning of Hybridomas (Fused Cells) As a selection medium, for example, an FCS-containing MEM medium containing hypoxanthine, aminopterin and thymidine, R
A medium such as a PMI-1640 medium, a so-called HAT medium can be used. The method of replacing the selective medium is generally that the same volume as the volume dispensed to the culture plate is added the next day, and then 1 to 3
The treatment can be carried out such that half of the HAT medium is replaced every day, but it can be carried out with appropriate changes. On the 8th to 16th days after the fusion, the medium can be exchanged every 1 to 4 days with a so-called HT medium excluding aminopterin. As a feeder, for example, mouse thymocytes can be used, which may be preferred. The culture supernatant of the culture well in which the growth of the hybridoma is active can be analyzed using a measurement system such as radioimmunoassay (RIA), enzyme immunoassay (ELISA), or fluorescence immunoassay (FIA), or a fluorescence-induced cell separation device (FACS). Screening is performed by using mouse MT2-MMP 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 a suitable 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 were treated with pristane (2,6,10,14-) prior to hybridoma transplantation.
Mineral oil such as tetramethylpentadecane) can be administered intraperitoneally. After the treatment, hybridomas can be grown and ascites can be collected. 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 can be purified and separated by fractionating with ammonium sulfate and then treating it with an anion exchange gel such as DEAE-Sepharose and an affinity column such as a protein A column. Particularly preferred are affinity chromatography in which an antigen or antigen fragment (eg, a synthetic peptide, a recombinant antigen protein or peptide, a site specifically recognized by an antibody, etc.) is immobilized, and affinity chromatography in which protein A is immobilized. No.

It is also possible to determine the sequence of the antibody obtained in such a large amount, and to prepare the antibody by a gene recombination technique using a nucleic acid sequence encoding the antibody obtained from the hybridoma strain. Further, these antibodies are treated with an enzyme such as trypsin, papain, pepsin and the like, and optionally reduced to obtain Fab, Fab ', F
It may be used as an antibody fragment such as (ab ') ₂ . The antibody to be labeled may be an IgG fraction, or a specific binding site Fa obtained by digestion and reduction after pepsin digestion.
b ′ can be used. Examples of labeled substances in these cases include enzymes (peroxidase, alkaline phosphatase, β-D-galactosidase, etc.), chemical substances, fluorescent substances, radioisotopes, etc., as described below. Detection and measurement in the present invention can be performed by immunostaining, for example, tissue or cell staining, immunoassay, for example, competitive immunoassay or non-competitive immunoassay, and radioimmunoassay, ELISA, etc. can be used. May be performed or the measurement may be performed without the measurement. 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 mouse MT2-MMP 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 unbound antibody, the labeled substance is measured. The amount of label measured is proportional to the amount of antigen, ie, mouse MT2-MMP. 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, pre-extraction of antigen, etc. are appropriately adopted in these measurement steps under specific circumstances. Other measurement conditions such as the concentration of a specific reagent, buffer, etc., temperature, or incubation time can be changed according to factors such as the concentration of the antigen in the sample and the properties of the sample. Those skilled in the art can appropriately select the optimum conditions effective for each measurement and perform the measurement using ordinary experimental methods.

Many carriers on which an antigen or an antibody can be immobilized are known. In the present invention, they can be appropriately selected and used. As the carrier, various carriers used for an antigen-antibody reaction 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 modified albumin, collagen, gelatin, dextran, agarose, crosslinked agarose, cellulose, microcrystalline cellulose, carboxymethylcellulose, cellulose acetate, polyamide such as crosslinked dextran, nylon, and organic polymers such as polyurethane and polyepoxy resin Substances, and those obtained by emulsion polymerization of these substances, cells, erythrocytes, etc., where necessary, functional groups such as silane coupling agents Which are introduced are mentioned. In addition, 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 substance (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 the mouse MT2-MMP 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 the like, or a chemical method using an activated one or the like. It can be performed by a method utilizing a chemical binding reaction or the like. As a sign,
Enzymes, enzyme substrates, enzyme inhibitors, prosthetic molecules, coenzymes, zymogens, apoenzymes, fluorescent substances, dye substances, chemiluminescent compounds, luminescent substances, chromogenic substances, magnetic substances, metal particles such as gold colloid Substances and the like can be mentioned. Examples of enzymes include dehydrogenases, reductases, oxidoreductases such as oxidases, and transferases that catalyze transfer of amino groups, carboxyl groups, methyl groups, acyl groups, phosphate groups, and the like, such as ester bonds, Examples thereof include hydrolases that hydrolyze glycoside bonds, ether bonds, peptide bonds, and the like, lyases, isomerases, and ligases. 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, substrates such as umbelliferone derivatives such as 4-methylumbelliferyl phosphate, phosphorylated phenol derivatives such as nitrophenyl phosphate, enzymatic cycling systems using NADP, luciferin derivatives, and dioxetane derivatives are used. It can be measured by the fluorescence, luminescence, etc. generated by use. 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). A plurality of different types of labels may be used. 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, etc., horseradish peroxidase, umbelliferyl galactoside, nitrophenyl galactoside, etc. 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 equolin, imidazole, oxalate, rare earth chelate compounds, coumarin derivatives and the like. Labeling can be carried out 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 methods that can be used, and methods modified from those methods. In addition, a condensing agent that can be used for the preparation of 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, ethylene glycol bissuccinimidyl succinate , Bisdiazobenzidine, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, succinimidyl 3- (2-pyridyldithio) propionate (S
PDP), N-succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (S
MCC), 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.

According to the measuring 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 and the like and an antibody bound to a carrier can be sequentially reacted, or simultaneously. You can also. The order in which the reagents are added depends on the type of carrier system chosen. When sensitized plastic beads or the like are used, a labeled antibody reagent such as a monoclonal antibody labeled with an enzyme or the like is first put together with an analyte sample containing a substance to be measured in an appropriate test tube, and then, 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 carried out 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 antibody-antigen reaction is about 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 point in time when the equilibrium of the antibody-antigen 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 antibody-antigen 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 antibody-antigen reaction itself. In addition, proteins, stabilizers, surfactants, chelating agents, etc. should be added to the incubation solution to eliminate 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 the 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 biological sample, such as blood, serum, plasma, synovial fluid, etc. is preferable. , Cerebrospinal fluid, saliva, amniotic fluid,
Urine, other body fluids, cell cultures, tissue cultures, tissue homogenates, biopsy samples, tissues, cells and the like.
In applying these individual immunological measurement methods to the measurement method of the present invention, no special conditions, operations, and the like need to be set. In addition to the ordinary conditions and procedures of each method, with the ordinary technical considerations of those skilled in the art, they have the natural mouse MT-MMP activity, which is the latent gelatinase A activator of the present invention, or have substantially the same activity. What is necessary is just to construct a measurement system related to a mouse-derived protein having the same activity. For more information on these common technical means,
Review articles, books, etc. can be referred to [for example, edited by Hiroshi Irie, "Radio Immunoassay", Kodansha, Showa 49
Published by Hiroshi Irie, "Continuing Radio Immunoassay", Kodansha, published in 1979; Eiji Ishikawa et al., "Enzyme Immunoassay", Medical Shoin, published in 1978; Eiji Ishikawa, et al., "Enzyme Immunoassay" (2nd edition), Medical Shoin, published in 1982; Eiji Ishikawa et al., “Enzyme Immunoassay” (3rd edition), Medical Shoin, published in 1987; “Methods in Enzymology”.
Vol. 70 (Immunochemical Techniques (Part A)); same book
Vol. 73 (Immunochemical Techniques (Part B)); Ibid. Vol. 74 (Immunochemical Techniques (Part C));
Ibid.Vol. 84 (Immunochemical Techniques (Part D: S
electedImmunoassays)); Ibid. Vol. 92 (Immunochemica
l Techniques (Part E: Monoclonal Antibodies and Ge
neral Immunoassay Methods)); Ibid. Vol. 121 (Immuno
chemical Techniques (Part I: Hybridoma Technology
and Monoclonal Antibodies)) (Academic Press
(USA)).

Thus, typically, it is an object of the present invention to use a monoclonal antibody against mouse MT2-MMP and a monoclonal antibody against mouse MT2-MMP or TIMPs for use as a solid phase carrier, and to provide free active mouse MT in a test sample.
An object of the present invention is to provide an excellent method for separating and quantifying 2-MMP and a reagent kit therefor. It is understood that the present invention includes, in its embodiment, all the reagents of the reagent kit capable of differentially quantifying such free active mouse MT2-MMP. It is a further object of the present invention to provide a method, a reagent or a diagnostic agent capable of monitoring a disease state such as tissue destruction or cancer metastasis by differentially quantifying free active mouse MT2-MMP using the above-mentioned quantification method. It is in. Therefore, the various uses of the above reagents in the medical and physiological fields, the determination of the degree of tissue destruction or cancer metastasis, the degree of tissue repair, or the use of the above reagents as indicators of physiological effects such as promotion of cell proliferation are all essential. It is understood to be included within that embodiment of the invention.

By utilizing the above-mentioned various aspects of the present invention, the present invention can be used as a diagnostic means useful for research relating to the diagnostic treatment of cancer, such as the presence or absence of cancer cells, the diagnosis of cancer malignancy, or other medical physiology. It is possible to provide various technical means applied to a typical application. Hereinafter, the present invention will be described in detail with reference to Examples, but it should be understood that the present invention is not limited to these Examples, and various embodiments based on the concept of the present specification are possible. is there. In the description and drawings, the terms refer to the IUPAC-IUB Commissionon Bioch
emical Nomenclature or based on the meaning of terms commonly used in the art. Mouse-derived monoclonal anti-mouse MT2-MMP antibody-producing hybridoma 162-4E3 described in Example 2f) below.
Was commissioned to the Institute of Biotechnology and Industrial Technology (NIBH) of the Ministry of International Trade and Industry of 1-3-1 Higashi, Tsukuba City, Ibaraki Prefecture (zip code 305) from December 13, 1996.
Deposited as 16003. Similarly, Escherichia coli JM109 carrying a vector (pSG5 ^™ (Stratagene)) into which the nucleotide sequence encoding mouse MT2-MMP described in Example 1 has been inserted.
(pSGMMT2 / JM109) has accession number FERM P-160 to NIBH.
Deposited as No. 10 (December 18, 1996)
.

[0075]

EXAMPLES The present invention will be described in more detail with reference to the following examples, but it should be understood that the present invention is not limited to the examples and includes various embodiments. Example 1 Screening of novel MT2-MMP gene from cDNA library and determination of nucleotide sequence (a) Screening of cDNA library Human MT1-MMP cDNA fragment (Gene, 155: 293-298, 1995) 2
5 ng of random primed DNA labeling kit (Boeh
[α- ³² P] dCTP (Amersha
m) to obtain a probe having a specific activity of 2 to 5.0 CPM / μg. This was used as a probe to screen a mouse lung cDNA library. λgt10
The thus constructed mouse lung cDNA library (Clontech) was infected with the host E. coli C600hfl strain at a concentration of 4 × 10 ⁴ plaque forming units / 15 cm diameter plate to form plaques. First, E. coli C600hfl strain contains 0.02% maltose
After overnight culture in L medium, the cells were collected and suspended in 10 mM MgSO ₄ .
This cell suspension and the phage solution were mixed and kept at 37 ° C. for 15 minutes to adsorb the phage to the host cells. Soft agar was added thereto, and spread on an L plate having a diameter of 15 cm prepared in advance. Plates were incubated overnight at 42 ° C. to form plaques.

A nylon filter [Hybond
d) -N, Amsham] was placed on the plate on which the plaque was formed, and left for about 30 seconds. Gently peel off the membrane, immerse the membrane in an alkaline denaturing solution (0.5 M NaOH and 1.5 M NaCl) for 1 minute, and then neutralize the solution (0.5 M Tris-HCl buffer containing 1.5 M NaCl,
pH 8.0) for 15 minutes. This filter is 2 × SSPE
(0.36 M NaCl, 20 mM NaH ₂ PO ₄ and 2 mM EDTA) and then air-dried. Repeat the transfer of the plaque to the filter as described above to prepare at least two filters.
However, the contact time between the second and subsequent filters and the plate is
Extended to about 2 minutes. The filter was baked at 80 ° C for 2 hours to fix the DNA. At least two filters prepared from a single plate are each
Time wash solution (1M NaCl, 1mM EDTA and 0.1% Sodium
50mM Tris-HC1 buffer containing Dodecyl sulfate (SDS), pH
After washing with 8.0), put the filter in the hybridization bag, pre-hybridization solution [50%
formamide, 5 × Denhardt's solution (0.2% bovine serum albumin (BSA), 0.2% polyvinylpyroldone), 5 × SSP
E, 0.1% SDS, 100 μg / ml heat-denatured salmon sperm DNA], and prehybridized at 42 ° C. for 6 to 8 hours.

The filter was transferred to a new hybridization bag, and the above-mentioned ³² P-labeled probe denatured by heating at 100 ° C. for 5 minutes was added to a Rapid hybridization buffer (Am
ersham) and hybridized at 60 ° C. for 2 hours. After the hybridization was completed, the filter was washed at 50 ° C with a large amount of 0.5% SSC containing 0.1% SDS (0.15M NaC
1, 15 mM citric acid) solution. After repeating this operation twice, the filter is air-dried.
(Kodak XR) and autoradiography was performed at -80 ° C for 12 hours. The X-ray film was developed and two films from one plate were overlaid to mark overlapping signals. Plaques corresponding to the marked signals
M solution (50 mM Tris-H containing 100 mM NaCl and 10 mM MgSO ₄
Cl buffer, pH 7.5). This phage suspension is diluted appropriately, preferably diluted to a concentration of 10 to 100 plaque forming units / 9 cm diameter plate, plated on a 9 cm diameter plate in which E. coli has been cultured, and subjected to the same screening as described above. As a result, eight clones of the recombinant phage were obtained.

(B) Preparation of recombinant phage-inserted DNA Eight recombinant phage clones cloned in the previous section (a) were plated in the same manner as described in the previous section (a).
Incubate at 42 ° C for 3 hours, then at 37 ° C overnight, then SM
Several drops of chloroform were added to the solution and left at room temperature for 30 minutes. The upper soft agar was scraped off together with the SM solution and centrifuged. Polyethylene glycol-6000 (PEG-6000) was added to the supernatant after centrifugation to a final concentration of 10%, and the mixture was stirred.
It was left at ℃ for 1 hour. This was centrifuged, the supernatant was discarded, and phage particles were collected. The phage particles are suspended in an SM solution, and glycerol gradient ultracentrifugation (Molecule
ar cloning, a laboratory manual, ed.T. Maniatis,
Cold Spring Harbor Laboratory, 2nd ed. 78, 1989)
And purified. The phage obtained was transferred to a TM solution (10 mM MgS
O ₄ containing 50 mM Tris-HCl buffer, were suspended in pH 7.8), DNase
After treatment with I and RNase A, 20 mM EDTA, 50 μg / ml Pro
A mixed solution of teinase K and 0.5% SDS was added, and the mixture was kept at 65 ° C for 1 hour. After extracting this with phenol and diethyl ether, DNA was precipitated by ethanol precipitation. Got
The DNA is washed with 70% ethanol, dried, and TE solution (10 mM E
DTA containing 10 mM Tris-HC1 buffer, pH 8.0).

[0079] (c) a .lambda.gt10 DNA prepared in sequencing preceding insert (b) was digested with EcoRI (Takara Shuzo), after isolated and purified insert, the vector pBluescript ^TM
(Stratagene) at the EcoRI site.
Escherichia coli NM533 XL1-Blue was transformed with this recombinant pBluescript. After F 'selection of the transformed cells, the cells were infected with helper phage VCSM13 (Stratagene) and cultured overnight. The culture was centrifuged to remove the cells, and PEG / NaCl was added to precipitate the phage. After suspending the precipitate in TE solution, single-stranded DN
A was recovered by phenol extraction and ethanol precipitation.
The base sequence of this single-stranded DNA is converted to a fluorescent DNA sequencer Model
The sequence was determined using 373A (Applied Biosystems) and Taq Dye Primer Cycle Sequencing Kit (Applied Biosystems). Of the 8 clones whose nucleotide sequences were determined, 6 clones were Proc. Natl. Acad. Sci. USA.,
92: 2730-2734, mouse MT1-MM described in 1995.
It corresponded to the full length or part of the P gene. Other than these
The two clones had the same insert fragment different from the mouse MT1-MMP gene, and the fragment contained 62% of the human MT1-MMP gene used as the probe and 88% of the mouse MT1-MMP gene. Showed homology. However, since this clone did not contain the complete open reading frame, this insert was used as a probe,
Again, the mouse lung cDNA library was screened.

(D) Rescreening of mouse lung cDNA library The inserted fragment obtained in the previous section (c) was purified, and a ³² P-labeled probe was prepared in the same manner as in the procedure described in the section (a). It was added as a hybridization probe for filters prepared according to the method described. For hybridization, select conditions with higher specificity, hybridization at 65 ° C for 2 hours, and washing at 65 ° C with 0.1% SDS containing 0.1 x SSC.
I went in. After the washing was completed, positive clones were searched according to the method described in (a), and as a result, one clone of recombinant phage was isolated.

(E) Analysis of mouse MT2-MMP gene From the recombinant phage cloned in the preceding section (d), subcloning, preparation of an inserted fragment and determination of the nucleotide sequence according to the methods described in the sections (b) and (c) Was done. The determined total length of the base sequence was 3339 bp, and the sequence was described in SEQ ID NO: 1 in the sequence listing. Using GENBANK / EMBL DNA Data Base, the base sequence described in SEQ ID NO: 1 in the sequence listing was searched, but no identical sequence was found. This about 3.3kb
The presence of an open reading frame encoding a putative 657 amino acids was confirmed in the DNA sequence of No. 5, and the deduced amino acid sequence was described in SEQ ID NO: 2 in the sequence listing. This putative protein was named "mouse MT2-MMP". The obtained DNA fragment was converted into plasmids pEX and pMAMne.
o, pKG5, etc., and can be expressed in E. coli, CHO cells, etc. Vector was inserted nucleotide sequence encoding the murine MT2-MMP (pSG5 ^TM (Strata
gene)), E. coli JM109 (pSGMMT2 / JM109) has accession number FERM P-160
Deposited as 10 (December 18, 1996)
.

(F) Amino acid sequence analysis of mouse MT2-MMP Sequence number deduced from the nucleotide sequence of mouse MT2-MMP described in SEQ ID NO: 1 in the sequence listing The amino acid sequence described in SEQ ID NO: 2 is known human The alignment in comparison with the amino acid sequence of MT-MMPs is shown in FIGS. That is, FIGS.
In the mouse MT2-MMP of the present invention (mMT2-MMP) amino acid sequence and known human MT-MMP family (hMT1-MMP, hMT2-MMP,
The homology with the amino acid sequences of hMT3-MMP and hMT4-MMP) is compared. Amino acids are represented by a general one-letter code, and the amino acids at the N-terminal are numbered 1st. The amino acid sequence shown in SEQ ID NO: 2 in the sequence listing shows high homology with human MT-MMPs, and has a domain structure characteristic of human MT-MMPs, that is, a signal peptide and a propeptide domain which are removed during secretory production. , The catalytic domain, the hinge domain, and the hemopexin clotting enzyme-like domain were well conserved. In particular, the sequence PRCGVPD near the pro-form and the active cleavage site, which is highly conserved in the MMP family including human MT-MMPs, is
P was completely conserved (SEQ ID NO: 2 in the sequence listing).
Pro ¹⁰⁵ ~ Asp ¹¹¹ ). In addition, mouse MT2-MMP contains human MT
-There were three insertion sequences that are characteristic points in the amino acid sequence of -MMPs as compared with other MMP families. That is, an insertion sequence of 13 amino acid residues including the sequence of RRKR existing between the propeptide domain and the catalytic domain-1
(IS-1; Val ^{115 to} Arg ¹²⁷ of SEQ ID NO: 2 in the sequence listing), an insertion sequence-2 of 8 amino acid residues consisting of the sequence of SYDDIRLR in the catalytic domain (IS-2; SEQ ID NO: 2 in the sequence listing) : 2 Ser
^179- Arg ¹⁸⁶ ) and transmembrane-like 2
Contiguous sequence of two hydrophobic amino acids VVMVLVPLLLLLCILGLAFAL
There was an insertion sequence-3 (IS-3; Gln ^{556 to} Val ⁶⁵⁷ of SEQ ID NO: 2 in the sequence listing) of 102 amino acid residues including V.

The first inserted sequence IS-1 is exceptionally present in MMP-11 in the human MMP family, but the sequence RXKR (X is any amino acid residue) conserved in IS-1. ) Is the sequence of the cleavage site of a subtilisin-like protease, and the amino acid sequence RXKR is known to be the cleavage site of many eukaryotic secretory proteins by a subtilisin-like protease (J. Biol. Chem., 266). : 12127
-12130, 1991). The continuous sequence of hydrophobic amino acids in IS-3 is considered to be a transmembrane domain, and human MT-MMPs
(Cancer Res., 56: 944-
949, 1996), the continuous sequence of hydrophobic amino acids present in IS-3 of mouse MT2-MMP was also considered as a transmembrane domain. When the amino acid sequence of mouse MT2-MMP was compared with other known human MT-MMPs, the homology to human MT2-MMP was the highest at 87%, and the homology to other MT-MMPs was also higher than that of human MT1-MMP. 52%, 50% to human MT3-MMP and 29% to human MT4-MMP.
The amino acid sequence of the protein encoded by the mouse MT2-MMP cDNA isolated according to the present invention may be other human MT-M
It is highly homologous to MP and similar to mouse MT1-MMP, but clearly different in detail. These sequence features suggest that mouse MT2-MMP together with mouse MT1-MMP constitute a family similar to human MT-MMPs.

Example 2 Preparation of Monoclonal Antibody (a) Preparation of Antigen Polypeptide A mouse having lower homology with other MT-MMP family than the amino acid sequence of mouse MT2-MMP shown in SEQ ID NO: 2 in the sequence listing The following sequences were selected as sequences characteristic of MT2-MMP and synthesized.

[SEQ ID NO: 3] Asp Thr Asp Asn Phe Gln Leu Pro Glu Asp Asp Leu Arg Gly (sequence of Asp ^{281 to} Gly ^{294 in} SEQ ID NO: 2 in Sequence Listing; abbreviated as “polypeptide A”)

The polypeptide A was synthesized by the Fmoc-bop method using a peptide synthesizer (Peptide Synthesizer 9600, MilliGen / Biosearch). Cysteine was introduced at the N-terminus of the polypeptide. The synthesized peptide is μBond
Purification was performed by high performance liquid chromatography using asphere, C18 column (Waters).

(B) Preparation of a complex of polypeptide and BSA An antigen conjugate was obtained by binding to BSA via a cysteine residue. 18.2mg BSA in 2ml 0.1M phosphate buffer,
21.1mg N- (6-maleimidcaproylo
xy) succinimide was mixed with a solution prepared by dissolving 200 μl of dimethylformamide, and reacted at 30 ° C. for 30 minutes. Next, the above mixture was subjected to gel filtration with PD-10 (Pharmacia) equilibrated with 0.1 M phosphate buffer, pH 7.0. The maleimide-bound BSA was separated and concentrated to 1.5 ml or less. A 50-fold molar amount of the polypeptide synthesized in the above (a) with respect to the maleimide-bound BSA was added to 1 ml of 0.1 M phosphate buffer, pH 7.0.
, And incubated at 4 ° C for 20 hours to prepare a BSA-polypeptide complex.

(C) Preparation of antibody-producing cells A complex of polypeptide A and BSA prepared in (b) above,
200 μg with complete Freund's adjuvant for 8 weeks
lb / c female mice were intraperitoneally administered and immunized for the first time. On the 19th day
Complex dissolved in 0.1 M phosphate buffer, pH 7.5 on day 34
200 μg of the mice that had been initially immunized were intraperitoneally administered and boosted. Further, on the 69th day, 200 μg of the conjugate was intravenously administered to obtain a final immunization. Three days later, the spleen was removed to prepare a spleen cell suspension.

(D) Cell fusion (1) The following materials and methods were used. RPMI-1640 medium: RPMI-1640 (Flow Lab.) With sodium bicarbonate (24 mM), sodium pyruvate (1 mM), potassium penicillin G (50 U / ml), amikacin sulfate (100 μg /
ml), the mixture was adjusted to pH 7.2 with dry ice, and sterilized and filtered through a 0.2 μm Toyo membrane filter. NS-1 medium: Fetal bovine serum (FCS, MA Bioproducts) that had been sterilized and filtered was added to the above RPMI-1640 medium to a concentration of 15% (v / v). PEG-4000 solution: A serum-free medium was prepared by adding polyethylene glycol-4000 (PEG-4000, Merk & Co.) to a RPMI-1640 medium to a concentration of 50% (w / w). Fusion with 8-azaguanine resistant myeloma cell SP2 (SP2 / 0-Ag14)
edMethod in culture immunology p. 351-372 (ed.
B. Mishell and SN Shiigi), WH Freeman and C
The method of Oi et al. described in ompany (1980) was slightly modified.

(2) The nucleated splenocytes (100% viable cell) and myeloma cells (100% viable cell) prepared in (c)
Fused in the following procedure at a ratio of 5: 1. Polypeptide A
Immunized splenocyte suspension and myeloma cells were added to RPMI1640
Washed with medium. Then suspended in the same medium, Yukaku脾cells 4.8 to fuse × 10 ⁸ cells and myeloma cells 9.6 × 10
^Seven were mixed. Next, precipitate the cells by centrifugation,
The supernatant was completely aspirated off. To the precipitated cells, 3.2 ml of a PEG-4000 solution heated to 37 ° C. was added dropwise over 1 minute, followed by stirring for 1 minute to resuspend and disperse the cells. Next, RP heated to 37 ° C
After dropping 6.4 ml of MI1640 medium for 2 minutes, 22.4 ml of the same medium
Was added dropwise for 2-3 minutes with constant stirring to disperse the cells. This was centrifuged, and the supernatant was completely removed by suction.
Next, 31 ml of NS-1 medium heated to 37 ° C. was added to the precipitated cells.
Was quickly added and the large cell mass was carefully dispersed by pipetting. Further diluted by adding the same medium 64 ml, per well 6.0 × 10 ⁵ in 96-well polystyrene microwell
Cells / 0.1 ml of cells were added. The microwells containing cells were placed in 7% carbon dioxide / 93% air at 37 ° C and 100% humidity.
%.

(E) Selective growth of hybridoma on selective medium (1) The medium to be used is as follows. HAT medium: The hypoxanthine (100 μM), aminopterin (0.4 μM) and thymidine (16 μM) were further added to the NS-1 medium described in the above section (d) (1). HT medium: The same composition as the above HAT medium except that aminopterin was removed.

(2) The day after the start of the culture in the above (d) (the first day)
2 drops of HAT medium with Pasteur pipette to cells (approx.0.1 ml)
Was added. Half of the medium (about 0.1 ml) on days 2, 3, 5, and 8
Was replaced with fresh HAT medium, and on day 11 half of the medium was replaced with fresh HT medium. On day 14, positive wells were examined by solid phase-antibody binding test (ELISA) in all the wells where hybridoma growth was visually observed. That is, a polystyrene 96-well plate was coated with polypeptide A as an antigen, and then washed with PBS for washing (containing 0.05% Tween 20) to remove unadsorbed peptides. In addition, the uncoated portion of each well was blocked with 1% BSA.
To each of the wells, 0.1 ml of the supernatant of the well in which the growth of the hybridoma was confirmed was added, and the mixture was allowed to stand at room temperature for about 1 hour. Two
Horseradish peroxidase (HRP) -labeled goat anti-mouse immunoglobulin (Cappele Lab.) Was added as a secondary antibody, and the mixture was allowed to stand at room temperature for about 1 hour. Next, hydrogen peroxide and o-phenylenediamine as substrates were added, and the degree of color development was measured at 492 nm using a microplate absorbance meter (MRP-A4, Tosoh).

(F) Cloning of hybridoma The hybridoma in the positive well for the antigenic peptide obtained in the above section (e) was monocloned by the limiting dilution method. That is, the cloning medium containing 10 ⁷ mouse thymocytes were prepared as NS-1 medium 1ml per feeder, five wells per hybridomas in 96 microwells, one, diluted to 0.5 or, respectively 36
Hole, 36 holes, added to 24 holes. On days 5 and 12, about 0.1 ml of NS-1 medium was added to all wells. About 2 after the start of cloning
After a week, the hybridomas were visually observed to grow sufficiently, and the ELISA described in (e) was performed for the group in which colony formation was negative or 50% or more. When all the wells examined were not positive, 4 to 6 wells each having one colony in the antibody-positive well were selected and recloned.
Finally, as shown in Table 1, 16 hybridomas producing a monoclonal antibody against polypeptide A were obtained.

[0094]

[Table 1]

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

(H) Determination of Class and Subclass of Monoclonal Antibody According to the above-mentioned ELISA, the supernatant of the hybridoma obtained in section (f) was added to a 96-well polystyrene plate coated with polypeptide A. Next, after washing with PBS, an isotype-specific rabbit anti-mouse IgG antibody (Zymed Lab.) Was added. After washing with PBS, HRP-labeled goat anti-rabbit IgG (
H + L), and the class and subclass were determined using hydrogen peroxide and 2,2′-azino-di (3-ethylbenzothiazolic acid) as a substrate. Finally, as shown in Table 1, the class and subclass of the hybridoma producing a monoclonal antibody against mouse MT2-MMP were determined. The hybridoma of clone number 162-4E3 has been deposited and stored at the National Institute of Bioscience and Human Technology under the accession number FERM P-16003 (December 13, 1996).

Example 3 Expression and Identification of Gene Product In order to express mouse MT2-MMP using animal cells as a host, cDNA was ligated to an expression vector. In this example, the SV40 promoter, enhancer,
PSG5 ^™ (Stratgene) containing a poly A signal and an intervening sequence for the small T antigen gene was used. Recombinant pBlu obtained by cloning the mouse MT2-MMP gene constructed in Example 1 (e)
3339b by EcoRI digestion from escript ^TM (Stratagene)
Of the eukaryotic expression vector pSG5
Cloned into EcoRI site and used for expression plasmid pSGM
MT2 was prepared. The ligation reaction was performed according to the protocol attached to the ligation kit. 5% F
PSGM was added to African green monkey kidney-derived cells COS-1 cultured in Dulbecco's modified Eagle's medium (DMEM) containing CS and 2 mM glutamine.
MT2, pSGHMT1 (human MT1-MMP cDNA cloned into pSG5), pSGHMT2 (human MT2-MMP cDNA cloned into pSG5) and pSGHMT3 (human MT3-MMP cDNA
were transfected by the calcium phosphate method (FL Graham e
t al., Virology, 52: 456, 1973). As a control, pSG5
COS-1 was transfected alone.

That is, 2 μg of recombinant pSG5 in distilled water or 60 μl of 0.25 M CaCl ₂ in pSG5 alone was added, and then
× BBS solution (50 mM B containing 2.8 mM Na ₂ HPO ₄ and 280 mM NaCl
62.5 μl of ES buffer, pH 7.9) was added to the bottom of the tube.
After mixing, the mixture was allowed to stand at room temperature for about 30 minutes to sufficiently form a precipitate. The precipitate was dispersed by pipetting, dropped into COS-1 cells, and then incubated in a CO ₂ incubator for about 24 hours. Next, the medium was removed, the cells were washed with PBS, and fresh DMEM without fetal calf serum was added. The culture was continued for 24 hours, and the introduced gene was expressed. The cells and the condition medium are separated by centrifugation, and the cells are lysed in a lysis buffer [0.15M NaC1, 0.1% sodium deoxycholate, 0.1%
% SDS, 1mM Triton X-100, 1% NP-40, 1mM EDTA, 1m
M phenylmetanesulfonyl fluoride (PMSF) containing 10mM T
ris-HCl buffer, pH 7.5] at 4 ° C for 1 hour. The cell lysate was centrifuged and the supernatant was collected.
The cell lysate was added to a sample buffer for SDS polyacrylamide electrophoresis (10% glycerol, 2% SDS, 2% β-m
50mM Tr containing ercaptoethanol, 0.1% bromophenol blue
After adding is-HCl buffer solution (pH 6.5) and heating at 100 ° C for 3 minutes, 12% SDS polyacrylamide electrophoresis was performed.
After the electrophoresis, Western blotting was performed, and the anti-human MT1-MMP monoclonal antibody 113-15E7 (Nature, 370:
61-65, 1995) or anti-human MT3-MMP monoclonal antibody 117-4E1 (J. Biol. Chem., 270: 23013-23020,
1995) or after reacting with the anti-mouse MT2-MMP monoclonal antibody 162-4E3 obtained in Example 2, immunostaining was performed by an indirect method using an HRP-labeled goat anti-mouse immunoglobulin antibody (Cappel Lab.).

Anti-human MT1-MMP monoclonal antibody 113-15
Immunostaining with E7 showed that 63 kDa human MT1-MMP was obtained from COS-1 cell lysate transfected with pSGHMT1.
Was immunostained with the anti-human MT3-MMP monoclonal antibody 117-4E1 in COS transfected with pSGHMT3.
66 kDa human MT3-MMP was detected from the S-1 cell lysate. Immunostaining with anti-mouse MT2-MMP monoclonal antibody 162-4E3 detected 70 kDa human MT2-MMP and mouse MT2-MMP from both pSGHMT2 and pSGMMT2 transfected COS-1 cell lysates, respectively. COS-1 cell lysate transfected with the vector pSG5 did not show any protein that reacts with any of the antibodies. Anti-mouse MT2-MMP monoclonal antibody 162-
4E3 was confirmed to react with human MT2-MMP in addition to mouse MT2-MMP. Human MT1-MMP and human MT3-MMP
Anti-mouse MT2-MMP monoclonal antibody 162-4E3 against
No cross-reactivity was observed. Mouse MT used here
By transferring 2-MMP to an appropriate expression vector, mouse MT2-MMP can be mass-produced using Escherichia coli, Bacillus subtilis, yeast, animal cells and the like as hosts. In this example in which pSGMMT2 was introduced into COS-1, mouse MT2-MMP production was short-term (transient expr
ession) but appropriate selectable markers (neo gene,
A cell line that can be produced for a long period of time can also be obtained by using an expression vector having a dehydrofolate reductase gene or the like and introducing it into CHO cells or the like. Such a cell line that continuously expresses mouse MT2-MMP is useful for monitoring the state of inhibition of mouse MT2-MMP expression by antisense oligomers and derivatives thereof.

Example 4 Activation of Latent Gelatinase A by Expression of Mouse MT2-MMP pSGMMT2 used in Example 3, pSGHMT1, pSGHMT2, or plasmid pSGHMT3 obtained by cloning human MT3-MMP cDNA, or vector pSG5 And the plasmid pSGHGELA in which human gelatinase A cDNA was cloned were co-transfected into COS-1 by the calcium phosphate method described in Example 3. The obtained transfectants were cultured in serum-free DMEM for 24 hours, and the collected culture supernatant was subjected to zymography.
The culture supernatant was subjected to SDS polyacrylamide electrophoresis sample buffer (non-reducing; 10% glycerol, 2% SDS 0.1% brom
ophenol blue-containing 50 mM Tris-HCl buffer, pH 6.5), incubate at 37 ° C for 20 minutes, and add 0.1% gelat
In 20% current using 4% polyacrylamide gel containing 4 in
Electrophoresis was performed at ° C. After running, run the gel with 2.5% Tri
Wash with gentle shaking in ton X-100 solution for 1 hour, then buffer for gelatinase (10 mM CaCl ₂ , 0.15 M Na
Incubation was carried out in a 50 mM Tris-HCl buffer (pH 7.6) containing Cl and 0.02% NaN ₃ at 37 ° C. for 24 hours with gentle shaking. Discard buffer and reconstitute gel with 0.1% coomassie brill
iant blue R250 (dissolved in 50% methanol-10% acetic acid)
After 1 hour staining with destaining solution (5% methanol-7.5% acetic acid)
And decolorized. The results of the obtained zymography are shown in FIG. Lane 1 was transfected with pSGHGELA, lanes 2, 3, 4 and 5 were pSGHMT1,
pSGHMT2, pSGMMT2 and pSGHMT3 each and pSGHGE
The culture supernatant of COS-1 cells co-transfected with LA is developed.

In the culture supernatant of COS-1 co-transfected with the vectors pSG5 and pSGHGELA, only the 68 kDa band of human latent gelatinase A was detected, and no change in molecular weight upon activation was observed (FIG. 3, Lane 1)
. On the other hand, pSGHMT1 or pSGHMT2 or pSGHM
In the culture supernatant of COS-1 co-transfected with each of T3 and pSGHGELA, new 64 kDa and 62 kDa corresponding to gelatinase A active intermediate and active gelatinase A were newly added.
(Fig. 3, lanes 2, 3 and 5), and activation of human latent gelatinase A was observed. pSGMMT
In the culture supernatant of COS-1 co-transfected with pSGHGELA and pSGHGELA, as in the case of human MT-MMPs described above, it corresponds to the gelatinase A-active intermediate and gelatinase A-active form.
Bands of kDa and 62 kDa appeared (FIG. 3, lane 4), confirming activation of human latent gelatinase A. Using this method, mouse MT2-MMP and latent gelatinase A
It is possible to determine whether or not any substance added to the mixture of the above has the effect of inhibiting the latent gelatinase A activation ability of mouse MT2-MMP. By appropriately selecting a substrate for zymography, it is possible to detect a substance that inhibits the extracellular matrix degradation activity of mouse MT2-MMP.

[0102]

EFFECTS OF THE INVENTION The activity is substantially equivalent to that of mouse MT2-MMP, which is a kind of MMP having the ability to activate latent MMP-2 and is a latent gelatinase A activator derived from mice other than MT1-MMP. Can be obtained, and a nucleic acid encoding the protein can be obtained, which is useful for research related to cancer diagnostic treatment such as the presence or absence of cancer cells, diagnosis of cancer malignancy, etc. Was obtained. In particular, it is useful for experimental medical and physiological applications using mice. The present invention also provides a method for producing the protein by genetic engineering, a monoclonal antibody that binds to the protein and a method for producing the same, and uses of the protein, antibody, and nucleic acid oligomer. These are useful not only for detection and quantification of the protein and the gene, but also for detection of a substance that inhibits the latent gelatinase A activating ability of the protein or a substance that inhibits the extracellular matrix degradation activity. The substance is useful as a drug such as an anticancer agent or a cancer metastasis inhibitor and a drug raw material by its latent gelatinase A activation inhibition or extracellular matrix degradation activity inhibiting function. It also provides an effective means for studying those drugs. In particular, it can contribute to elucidation of the mechanism of activation of gelatinase A, which is thought to play an important role in the process of invasive metastasis of cancer. Analysis of human MT1-MMP and MT3-MMP and search and identification of novel gelatinase A activator will be possible. Furthermore, it is useful for diagnosis of diseases related to gelatinase A activation and development of pharmaceuticals. Gelatinase A has been found to have the activity of degrading β-amyloid protein involved in the onset of Alzheimer's disease, and thus is useful for diagnosing diseases related to such diseases and developing pharmaceuticals and the like.

[0103]

[Sequence list]

[SEQ ID NO: 1] Sequence length: 3339 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA Origin Organism: mouse Sequence CGGACTCCCC TTTCTCTGAC TCTGGAGCAT GGGCAGCGAC CGGAGCGCAC TCGGACGTCC 60 AGGCTGCACT GGCAGCTGCC TCAGGCCG AGCTTCGCTG CTCCCGCTGC TACTGGTGCT 120 TCTGGACTGT CTGGGCCATG GTACAGCGTC TAAAGACGCC GAAGTGTACG CCGCGGAGAA 180 CTGGCTGCGG CTCTATGGCT ACCTACCCCA GCCCAGCCGC CACATGTCCA CCATGCGCTC 240 TGCCCAGATC CTGGCCTCCG CCCTTGCCGA GATGCAGAGT TTCTATGGGA TCCCTGTCAC 300 GGGTGTGCTT GATGAAGAGA CGAAAACGTG GATGAAGCGG CCCCGATGTG GGGTTCCTGA 360 TCAGTTTGGG GTACATGTGA AAGCCAACCT GCGTCGACGG CGGAAACGTT ACACCCTGAC 420 AGGAAAGGCA TGGAACAATT ACCACCTGAC CTTCAGCATC CAGAACTACA CTGAGAAGCT 480 GGGCTGGTAC AACTCCATGG AGGCGGTGCG CAGGGCTTTC CAAGTGTGGG AGCAGGTCAC 540 ACCATTGGTC TTCCAGGAAG TATCCTATGA TGACATTCGG CTACGAAGGC GAGCGGAGGC 600 TGACATCATG GTACTCTTTG CCTCTGGCTT CCATGGCGAC AGCTCACCGT TTGATGGCGT 660 GGGTGGCTTT CTGGCCCACG CTTATTTCCCCGCCTG CTGGGTGGGG ACACCCATTT 720 CGACGCAGAT GAACCCTGGA CCTTCTCCAG CACTGACCTG CATGGAATCA GCCTCTTTCT 780 GGTGGCCGTG CATGAGCTGG GCCATGCCCT GGGGCTAGAA CACTCAAGTA ACCCCAGCGC 840 TATTATGGCA CCCTTCTACC AGTGGATGGA TACCGACAAC TTCCAGCTGC CCGAAGATGA 900 CCTTCGGGGC ATCCAGCAGC TGTATGGCTC CCCAGATGGT AAGCCACAGC CCACCCGGCC 960 TCTCCCCACT GTAAGGCCCC GGCGGCCAGG ACGGCCAGAT CACCAGCCAC CTCGGCCTCC 1020 CCAGCCACCA CATCCAGGTG GGAAGCCAGA AAGGCCCCCC AAACCAGGGC CCCCACCCCA 1080 GCCCCGAGCC ACAGAGAGGC CTGACCAGTA TGGCCCCAAC ATCTGCGATG GCAACTTCGA 1140 CACAGTGGCT GTGCTCCGCG GAGAGATGTT TGTGTTCAAG GGCCGCTGGT TCTGGCGAGT 1200 CAGGCACAAC CGCGTTCTAG ACAACTACCC CATGCCAATT GGCCACTTCT GGCGCGGTCT 1260 GCCGGGGAAC ATCAGTGCTG CCTACGAGCG CCAGGATGGA CATTTTGTCT TCTTCAAAGG 1320 TAACCGCTAC TGGCTTTTCC GAGAAGCCAA TCTGGAGCCC GGCTACCCAC AGCCGCTGAG 1380 CAGCTATGGC ACAGACATCC CCTATGACCG CATTGACACA GCCATCTGGT GGGAGCCCAC 1440 AGGTCACACC TTCTTCTTTC AAGCAGACAG GTACTGGCGC TTCAACGAGG AGACACAGCA 1500 TGGAGACCCT GGCTACCCCA AGCCCATCAG TGTCTGGCAG GGGATCCCCA CCTCTCCCAA 1560 AGGGGCCTTC CTCAGCAACG ATGCAGCCTA CACCTACTTC TACAAGGGCA CCAAGTACTG 1620 GAAATTCAAC AACGAACGCC TACGGATGGA ACCCGGCCAC CCCAAATCCA TCCTGCGGGA 1680 CTTCATGGGC TGCCAGGAGC ACGTGGAGCC CCGATCGCGA TGGCCCGATG TGGCTCGTCC 1740 ACCCTTCAAC CCCAACGGGG GTGCCGAGCC TGAGGCAGAT GGTGACAGCA AGGAAGAGAA 1800 CGCGGGTGAC AAGGATGAGG GCAGCCGCGT GGTGGTGCAG ATGGAGGAGG TGGTCCGGAC 1860 AGTGAACGTG GTGATGGTGT TGGTCCCCTT GCTGCTGTTG CTCTGTATTC TGGGCCTGGC 1920 CTTTGCTCTG GTGCAGATGC AGCGCAAGGG CGCCCCTCGC ATGCTGCTCT ACTGCAAGCG 1980 CTCACTGCAG GAGTGGGTGT GATCAGGTAG CACCCGCAGC ACCCGGTGCT CAGGTCTGCA 2040 GGGACAGGAG ACGTGTGGAT CCCAGATGCT CCCTCGGCGC GGCAGGATGC CTCCTAGCCA 2100 CACACATCCC CCTGCCAGCC TTGCCCCCCA CCCCCGTCCT CCTTTATTTA TGCCCAGGTG 2160 CCTCTTCTTT TTGGCACCCT CTCTCAGCCT TTGGTTCCGC TTTCCTGACT CGGGCCAGGA 2220 GATGCTTTGA GATCTCCCGG AGTGTAGTAC CGCCCCAGGC AGGAACGAAG GGACAGAGGC 2280 TCACACCTCT GGAAGATGGC AGAGGGCTGG GAAGCTTCTC TGGCTCTGGC TTTTCAGCCT 2340 GAACCTCTCC TCCCACGACA AGAACTGTGT TTGCCTTGAA GGAGCCCACC TCGGCT CTGC 2400 CCTCCTGAAT TTGCCTTAGG GAGAGTATAT GCTCCCCCCT GGGGGGGCAC CCTGATAACC 2460 TTGAGACTTG GTTCCTTCTT CTTAGGCTCC TAAGAAGCCC AGAAGCCTAA AAGCCCTCCC 2520 CCATCCTAGC ATTCTGCAGC TTCTTTCCTC TAGGAAGGGG CCCTGGACAT GTCTCACCTA 2580 GGACCAAAGG GAGCCTGCCA AGCTCCCCTC CCAATGCTAG TGACTAGCTC CTCCTTCCCT 2640 AGGCTGCCCA TATTCTCTTT CAGTGCCCCA TAGGTCTCCG GAGGCTGGCA CTCCCCGCCC 2700 GCCCCCACCC CCAGCTTCTG CAGTCCTTGT CCCAACTCTT AATGACATGG GGCAGACACA 2760 CCCACTAGGG TACCAGAGGT CCCTGGGCAG TGTGTGTGAT TGTGGATGCT CAGCAGGTCA 2820 CAGACCAAGC TTATGGGCTT CCTCCCTTCC AGATTCCTGT CCCCACACAA CACTACTAGG 2880 CTCAGGCCAG CAGCTCACCC CACACAGAGT CTGTGGAACC CCCAAAGGAA TCCACTAAGG 2940 CCTGGGAAGT CTGTCCTAAG GGTGAAAGGG GCCCTCCCTT CCTCTTCTCA TCCTGGAGTC 3000 AGCAGGAGGC TTCTGTGGGA AATGGTACTG TACAGCTGTG GCTCTGTCTC CTCTGCCTCC 3060 CTAAAGCCTT GAGCAGTAAA GGGCTGCCAG CCAGGATCCT CTGGGTCACC GCCGCTTTCC 3120 AAGTGGTGGA TTCTTATAGC CTGGCACTAG AGGTGACAGA TCGGAGGGGC ATGTCCTGCA 3180 GCACTCTAGA TGTCCCCTCT GCAGAACCTG AGAGTCAGCA AGGGGTGGGG CGTCCACCCC 3 240 CCCACTCCCC ATCGTTTAGT TCTGTAGATA ATCAGCACTG ATTAAATGTC CAGCCAGCGA 3300 GCGTGGCTGC ACTTGTACGC TCAAAAAAAA AAAAAACCG 3339

[0104]

[SEQ ID NO: 2] Sequence length: 657 Sequence type: amino acid Sequence type: protein Origin Organism name: Mouse Sequence Met Gly Ser Asp Arg Ser Ala Leu Gly Arg Pro Gly Cys Thr Gly Ser 1 5 10 15 Cys Leu Ser Ser Arg Ala Ser Leu Leu Pro Leu Leu Leu Val Leu Leu 20 25 30 Asp Cys Leu Gly His Gly Thr Ala Ser Lys Asp Ala Glu Val Tyr Ala 35 40 45 Ala Glu Asn Trp Leu Arg Leu Tyr Gly Tyr Leu Pro Gln Pro Ser Arg 50 55 60 His Met Ser Thr Met Arg Ser Ala Gln Ile Leu Ala Ser Ala Leu Ala 65 70 75 80 Glu Met Gln Ser Phe Tyr Gly Ile Pro Val Thr Gly Val Leu Asp Glu 85 90 95 Glu Thr Lys Thr Trp Met Lys Arg Pro Arg Cys Gly Val Pro Asp Gln 100 105 110 Phe Gly Val His Val Lys Ala Asn Leu Arg Arg Arg Arg Lys Arg Tyr 115 120 125 Thr Leu Thr Gly Lys Ala Trp Asn Asn Tyr His Leu Thr Phe Ser Ile 130 135 140 Gln Asn Tyr Thr Glu Lys Leu Gly Trp Tyr Asn Ser Met Glu Ala Val 145 150 155 160 Arg Arg Ala Phe Gln Val Trp Glu Gln Val Thr Pro Leu Val Phe Gln 165 170 175 Glu Val Ser Tyr Asp Asp Ile Arg Leu Arg Arg Arg Ala Glu Ala Asp 180 185 190 Ile Met Val Leu Phe Ala Ser Gly Phe His Gly Asp Ser Ser Pro Phe 195 200 205 Asp Gly Val Gly Gly Phe Leu Ala His Ala Tyr Phe Pro Gly Pro Gly 210 215 220 Leu Gly Gly Asp Thr His Phe Asp Ala Asp Glu Pro Trp Thr Phe Ser 225 230 235 240 Ser Thr Asp Leu His Gly Ile Ser Leu Phe Leu Val Ala Val His Glu 245 250 255 Leu Gly His Ala Leu Gly Leu Glu His Ser Ser Asn Pro Ser Ala Ile 260 265 270 Met Ala Pro Phe Tyr Gln Trp Met Asp Thr Asp Asn Phe Gln Leu Pro 275 280 285 Glu Asp Asp Leu Arg Gly Ile Gln Gln Leu Tyr Gly Ser Pro Asp Gly 290 295 300 Lys Pro Gln Pro Thr Arg Pro Leu Pro Thr Val Arg Pro Arg Arg Pro 305 310 315 320 Gly Arg Pro Asp His Gln Pro Pro Arg Pro Pro Gln Pro Pro His Pro 325 330 335 Gly Gly Lys Pro Glu Arg Pro Pro Lys Pro Gly Pro Pro Pro Gln Pro 340 345 350 Arg Ala Thr Glu Arg Pro Asp Gln Tyr Gly Pro Asn Ile Cys Asp Gly 355 360 365 Asn Phe Asp Thr Val Ala Val Leu Arg Gly Glu Met Phe Val Phe Lys 370 375 380 Gly Arg Trp Phe Trp Arg Va l Arg His Asn Arg Val Leu Asp Asn Tyr 375 390 395 400 Pro Met Pro Ile Gly His Phe Trp Arg Gly Leu Pro Gly Asn Ile Ser 405 410 415 Ala Ala Tyr Glu Arg Gln Asp Gly His Phe Val Phe Phe Lys Gly Asn 420 425 430 Arg Tyr Trp Leu Phe Arg Glu Ala Asn Leu Glu Pro Gly Tyr Pro Gln 435 440 445 Pro Leu Ser Ser Tyr Gly Thr Asp Ile Pro Tyr Asp Arg Ile Asp Thr 450 455 460 Ala Ile Trp Trp Glu Pro Thr Gly His Thr Phe Phe Phe Gln Ala Asp 455 470 475 480 Arg Tyr Trp Arg Phe Asn Glu Glu Thr Gln His Gly Asp Pro Gly Tyr 485 490 495 Pro Lys Pro Ile Ser Val Trp Gln Gly Ile Pro Thrrl Ser Pro Lys Gly 500 505 510 Ala Phe Leu Ser Asn Asp Ala Ala Tyr Thr Tyr Phe Tyr Lys Gly Thr 515 520 525 Lys Tyr Trp Lys Phe Asn Asn Glu Arg Leu Arg Met Glu Pro Gly His 530 535 540 Pro Lys Ser Ile Leu Arg Asp Phe Met Gly Cys Gln Glu His Val Glu 545 550 555 560 Pro Arg Ser Arg Trp Pro Asp Val Ala Arg Pro Pro Phe Asn Pro Asn 565 570 575 Gly Gly Ala Glu Pro Glu Ala Asp Gly Asp Ser Lys Glu Glu Asn Ala 580 585 585 590 Gly Asp Lys Asp Glu Gly Se r Arg Val Val Val Gln Met Glu Glu Val 595 600 605 Val Arg Thr Val Asn Val Val Met Val Leu Val Pro Leu Leu Leu Leu 610 615 620 Leu Cys Ile Leu Gly Leu Ala Phe Ala Leu Val Gln Met Gln Arg Lys 625 630 635 640 Gly Ala Pro Arg Met Leu Leu Tyr Cys Lys Arg Ser Leu Gln Glu Trp 645 650 655 Val 657

[0105]

[SEQ ID NO: 3] Sequence length: 14 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Asp Thr Asp Asn Phe Gln Leu Pro Glu Asp Asp Leu Arg Gly 1 5 10 14

[Brief description of the drawings]

FIG. 1 is a diagram comparing the amino acid sequence of the mouse MT2-MMP (mMT2-MMP) of the present invention with the amino acid sequence of a known human MT-MMP (hMT-MMP) family. I have. The underlined portions indicate sequences corresponding to the respective IS-1 and IS-2 regions.

FIG. 2: Amino acid sequence of mMT2-MMP of the present invention and known hM
FIG. 2 is a diagram comparing the homology with the amino acid sequence of the T-MMP family, and is a continuation of FIG. 1. The underlined part shows the sequence corresponding to the IS-3 region.

Fig. 3 Human latent gelatinase by expression of MT-MMPs
The result of activation of A is shown in a schematic diagram of zymography.
Lane 1 shows COS- transfected with pSGHGELA.
1 Culture supernatant of cells and lanes 2, 3, 4 and 5
COS-1 co-transfected with pSGHMT1, pSGHMT2, pSGMMT2 and pSGHMT3 and pSGHGELA
The cell culture supernatant is unfolded.

Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12N 9/00 C12N 9/48 9/48 C12P 21/02 C 15/02 21/08 C12P 21/02 G01N 33/53 D 21/08 C12N 5/00 B G01N 33/53 15/00 C // (C12N 1/21 C12R 1:19) (C12P 21/02 C12R 1:91) (72) Inventor Shinichi Yoshida 530 Chokeiji Temple, Takaoka City, Toyama Prefecture Fuji Pharmaceutical Industrial Co., Ltd.

Claims (33)

[Claims] The present invention relates to an MMP capable of activating latent gelatinase A and having an activity of a natural mouse MT-MMP which is a latent gelatinase A activator other than mouse MT1-MMP. A mouse-derived protein or a salt thereof, which has an activity equivalent to that of a mouse. 2. The protein according to claim 1, wherein the protein has substantially the same activity as mouse MT2-MMP or a salt thereof, or has substantially the same primary structural conformation. The described protein. 3. The C-terminal region has SEQ ID NO: 2 in the sequence listing.
3. The protein according to claim 1, which has an amino acid sequence represented by Val ^{614 to} Val ⁶³⁵ or an amino acid sequence substantially equivalent thereto. 4. The mouse MT2-MMP having an amino acid sequence represented by SEQ ID NO: 2 in the sequence listing or an amino acid sequence substantially equivalent thereto, or a salt thereof. The protein according to any one of the above. 5. The method according to claim 1, wherein the exogenous DNA sequence is obtained by expression in a prokaryote or is obtained by expression in a eukaryote. The described protein. 6. A partial peptide of the protein according to any one of claims 1 to 5, or a salt thereof. 7. A nucleic acid having a base sequence encoding the protein according to claim 1 or a partial peptide thereof. 8. The nucleic acid according to claim 7, which is a DNA gene having a base sequence encoding the protein according to any one of claims 2 to 4. 9. The method according to claim 7, wherein the nucleotide sequence has an open reading frame portion or a nucleotide sequence having substantially the same activity as the nucleotide sequence represented by SEQ ID NO: 1 in the sequence listing. Nucleic acids. 10. A vector comprising the nucleic acid according to claim 7. 11. A transformant, comprising the nucleic acid according to any one of claims 7 to 9 or the vector according to claim 10. 12. The transformant according to claim 11, which is cultured in a nutrient medium capable of growing, and used as a recombinant protein in a mouse.
The mouse according to any one of claims 1 to 6, which comprises MT2-MMP or a salt thereof. A method for producing a protein or a partial peptide thereof according to any one of the above. 13. An antibody against the protein of claim 1 or a salt thereof or a partial peptide thereof or a salt thereof. 14. An antibody against a protein having substantially the same activity as mouse MT2-MMP or a salt thereof, or having substantially the same primary structure conformation. Item 14. The antibody according to Item 13. 15. An antibody against a mouse MT2-MMP having the amino acid sequence represented by SEQ ID NO: 2 or a substantially equivalent amino acid sequence thereof or a protein which is a salt thereof. 13. The antibody according to 13 or 14. 16. An antibody against a protein which is obtained by expressing an exogenous DNA sequence in a prokaryotic organism or expressing it in a eukaryotic organism. The antibody according to any one of the above. The antibody according to any one of claims 13 to 16, which is an antibody against a partial peptide of a protein or a salt thereof. 18. A partial peptide of the protein according to claim 17 or a salt thereof, wherein Asp ^{281 to} Gl of SEQ ID NO: 2 in the sequence listing.
claim 17, wherein the antibody is characterized by having the amino acid sequence represented by y ^294. 19. The method according to claim 19, which is an antiserum.
19. The antibody according to any one of 13 to 18. 20. The antibody according to any one of claims 13 to 18, which is a monoclonal antibody. 21. A monoclonal antibody against mouse MT2-MMP or a salt thereof, wherein the antibody is a monoclonal antibody against mouse MT2-MMP or a salt thereof.
21. The antibody according to any one of 18 and 20. 22. A method for producing an antibody against the protein according to claim 1 or a salt thereof, or a partial peptide thereof or a salt thereof, wherein the antibody according to claim 13 is obtained. 23. A cell producing an antibody against the protein or its salt or its partial peptide or its salt obtained from an animal immunized with the protein or its salt or its partial peptide or its salt of claim 1. Is fused with a cell that can be subcultured, and a hybrid cell that can be subcultured and produces an antibody against a protein including mouse MT2-MMP is selected. Production method. 24. MT2- characterized by using the protein of claim 1 or its salt or its partial peptide or its salt as a reagent, or using the antibody according to any one of claims 13 to 21 as a reagent. MMP detection and measurement method. 25. A labeled antibody against MT2-MMP used in the method for detecting and measuring MT2-MMP according to claim 24. 26. A labeled protein or a salt thereof, which is a protein or a salt thereof or a partial peptide or a salt thereof according to claim 24, which is used in the method for detecting and measuring MT2-MMP of claim 24. Partial peptide or a salt thereof. 27. The nucleic acid of claim 7, which is labeled. 28. The nucleic acid according to claim 27, which is a hybridization probe. 29. A method for detecting a compound which inhibits the latent gelatinase A activating ability of the protein or its salt according to claim 1, and a method for detecting the protein or its salt. 30. A compound, a protein or a salt thereof, which inhibits the latent gelatinase A activating ability of the protein of claim 1 or a salt thereof. 31. A method for detecting a compound or a protein or a salt thereof, which inhibits the extracellular matrix degrading activity of the protein or a salt thereof according to claim 1. 32. Compounds and proteins or salts thereof which inhibit the extracellular matrix degrading activity of the proteins or salts thereof according to claim 1. 33. An antisense oligomer which hybridizes to DNA and RNA sequences containing the base sequence represented by SEQ ID NO: 1 in the sequence listing.