JPH1118781A - Novel protein c9 and its gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide the subject novel gene that comprises the DNA encoding a novel protein C9 having a specific amino acid sequence and is useful for manufacturing the protein that has an activity of inducing the differentiation from bone marrow cells to the cells having the hydroxyapatite-decomposing function. SOLUTION: This novel DNA encodes a protein that comprises a protein C9 having the amino acid sequence given by the formula or comprises an amino acid sequence formed by deleting, substituting or adding one or more amino acids from, in or to the amino acid sequence described in the formula, and has the activity of inducing the differentiation from bone marrow cells to those having the function to decompose hydroxyapatite and is used to produce the protein C9 that is useful for screening an inhibitor against the induction of differentiation from the bone marrow cells to those that can decompose hydroxyapatite. This DNA is prepared by screening the cDNA library obtained by using the mRNA from the mouse BW5147 strain through the hydroxyapatite decomposition activity in the presence of bone marrow cells.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a novel protein C9 and its gene. More specifically, C9 having a differentiation-inducing activity from bone marrow cells to cells having hydroxyapatite decomposability and analogous proteins of C9, peptide fragments of these proteins, antibodies, medicaments containing these proteins as active ingredients, or A gene encoding the protein, an expression vector for the gene, a transformant into which the expression vector has been introduced, a method for producing a recombinant protein using the transformant, a transgenic animal having the gene introduced and deleted, and The present invention relates to the use of the protein in screening for an inhibitor for the activity of inducing differentiation from bone marrow cells to cells having hydroxyapatite degradability.

[0002]

2. Description of the Related Art The regulation of blood calcium metabolism is very important for forming a bone skeleton of life support, and its concentration is kept very strictly at a constant value. When blood calcium concentration increases for some reason, various diseases such as hypertension, arteriosclerosis, diabetes, myocardial infarction, and hypercalcemia are caused. Conversely, a decrease in blood calcium concentration causes diseases such as hypocalcemia.
In addition, massive blood loss due to massive bleeding or radiation exposure may cause death. Osteoclasts are currently known as cells that regulate blood calcium concentration which is extremely important in living bodies. Osteoclasts are thought to directly regulate blood calcium levels by dissolving bone (bone matrix) and releasing calcium into the blood. However, osteoclasts only 50,000 or so only without in vivo in mammals, whereas, bone cells embedded in calcified hard tissue accumulated calcium is about 25,000 units, even in the bone of 1 mm ³ Therefore, it is thought that osteoclasts alone are too few to regulate blood calcium concentration (Kumegawa et al., Molecula
r Medicine, 30, p1254 (1993) and Ozawa et al., Japanese Clinic
vol.52, No.9, p2246 (1994)).

Further, op / o, a model animal of osteopetrosis,
Since osteoclasts are absent in p mice, bone remodeling, which is an intrinsic effect of osteoclasts, hardly occurs.
However, despite the absence of osteoclasts, the calcium concentration in the blood remains normal (Molecular Me
dicine Vol.30, No.10, p1240 (1993)). This suggests that the regulation of blood calcium levels by osteoclasts is only ancillary, and that there are other cells that mainly regulate blood calcium levels other than osteoclasts. Suggests that Although the cell has not yet been identified, if a factor that proliferates the cell or a factor that induces differentiation into the cell is discovered, the factor itself or an inhibitor thereof may cause abnormalities in blood calcium concentration as described above. Can be a therapeutic agent for various diseases caused by From such a viewpoint, identification of the factor is desired, but there is no report that it has been cloned yet.

[0004]

An object of the present invention is to provide a novel protein C9 having an activity of inducing differentiation of bone marrow cells into cells having hydroxyapatite decomposability, and a gene thereof. More specifically, C9 having a differentiation-inducing activity from bone marrow cells to cells having hydroxyapatite decomposability and analogous proteins of C9, or peptide fragments of these proteins,
Antibodies, drugs containing these proteins as active ingredients, or genes encoding these proteins, expression vectors for the genes, transformants into which the expression vectors have been introduced, methods for producing recombinant proteins using the transformants, An object of the present invention is to provide a use of the protein in screening for an inhibitor for the activity of inducing the differentiation of bone marrow cells into cells having the ability to degrade hydroxyapatite.

[0005]

Means for Solving the Problems The present inventors have conventionally proposed:
An osteoclast differentiation promoting factor was being searched for from BW5147 cells, which are bone metastatic cells, by the expression cloning method. That is, mRNA isolated from BW5147 cells is injected into Xenopus laevis oocytes for translation, the resulting translation product (protein) is allowed to act on mouse bone marrow cells, and osteoclast differentiation is induced from bone marrow cells. The search for the factor was performed using the index as an index. Specifically, (1) TRAP
A cell having three well-known characteristics of osteoclasts, which has staining properties, (2) ivory degradation activity, and (3) hydroxyapatite, which is a crystal of calcium in bone, is identified as an “osteoclastic cell”. A factor that has the effect of inducing differentiation from bone marrow cells into the osteoclasts has been identified as an "osteoclastic differentiation promoting factor".

[0006] In the course of this expression cloning, we discovered a novel proteinaceous factor having properties different from those of the above-mentioned osteoclast differentiation promoting factor. That is, the cells induced to differentiate by the factor are the above (1) to (3)
Among them, (3) has the hydroxyapatite decomposition activity, but (1) does not have the activity of (2).
Thus, because it has only the activity of (3),
The factor is another factor different from the osteoclast differentiation promoting factor, and has the activity of (3), ie, a cell that degrades hydroxyapatite, which is a crystal of calcium in bone (more specifically, degrades hydroxyapatite). It has the activity of inducing bone marrow cells to differentiate into cells that release calcium by activation. Therefore, a differentiation inducing factor into blood calcium concentration regulator cells that has not yet been identified (hereinafter simply referred to as a blood calcium concentration regulator) May be abbreviated). We call this novel proteinaceous factor C9
The present inventors completed further studies and completed the present invention.

That is, the gist of the present invention is to provide (1) a DNA encoding the following protein (a) or (b): (a) a protein comprising the amino acid sequence of SEQ ID NO: 2; (2) a protein comprising an amino acid sequence in which one or more amino acids of the amino acid sequence described in (2) have been deleted, substituted and / or added, and having an activity of inducing differentiation of bone marrow cells into cells having hydroxyapatite decomposability; DN consisting of the nucleotide sequence of SEQ ID NO: 1
A or a DNA that hybridizes with the DNA thereof under a stringent condition and encodes a protein having an activity of inducing differentiation of bone marrow cells into cells having hydroxyapatite decomposability; (3) the above (1) or (2) A protein encoded by the DNA according to the above (4) a protein according to the above (3), which comprises the amino acid sequence according to SEQ ID NO: 2, (5) a D according to the above (1) or (2),
An expression vector containing NA, (6) a transformant transformed by the expression vector according to (5),
(7) A method for producing a recombinant protein, comprising culturing the transformant according to (6) under conditions that allow expression of the expression vector according to (5). Or (4) a pharmaceutical comprising the protein of (4) as an active ingredient; (9) a peptide fragment of the protein of (3) or (4) comprising at least 6 amino acids; Or an antibody against any one of the protein according to (4) or the peptide fragment according to (9), (11) a method for preparing a protein from (3) or (4), wherein A method for screening an inhibitor for the activity of inducing differentiation into cells having apatite decomposability,
(12) an inhibitor for the activity of inducing differentiation of bone marrow cells into cells having hydroxyapatite degradability, obtained by the screening method according to (11), (13)
The inhibitor according to (12), which comprises the peptide fragment according to (9) or the antibody according to (10), and (14) the DNA according to (1) or (2) is artificially incorporated into a chromosome. A transgenic animal, which has been introduced or one of which has been deleted from the chromosome.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, DNA is C9
Or a DNA similar to the C9 DNA, which encodes a protein having an activity of inducing differentiation from bone marrow cells to cells having hydroxyapatite degradability
Although it is not particularly limited as long as it is A, for example, SEQ ID NO: 1
Or a protein encoding the protein consisting of the amino acid sequence of SEQ ID NO: 2
NA. Furthermore, a DNA encoding a protein consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted and / or added in the amino acid sequence of SEQ ID NO: 2, or the nucleotide sequence of SEQ ID NO: 1 DNA that hybridizes with other DNA under stringent conditions is also included in the DNA of the present invention as long as it encodes a protein having an activity of inducing differentiation from bone marrow cells to cells having hydroxyapatite decomposability. Hereinafter, these DNAs will be sequentially described.

1) DNA encoding C9 Among the above-mentioned DNAs, “DNA having the nucleotide sequence of SEQ ID NO: 1 or DNA encoding the protein having the amino acid sequence of SEQ ID NO: 2” It is DNA encoding C9 of the present invention. The DNA can be obtained, for example, by the “expression cloning method” described below. That is, first, total RNA is prepared from bone metastatic cells, and then mRNA is prepared from total RNA. The bone metastasis cells used herein include, for example, mouse BW5147
Cell lines. Total RNA was analyzed by the AGPC method (acid guani
dium thiocyanate-phenol-chloroform method; Tuji,
Nakamura: Experimental Medicine 9, p99 (1991)). For mRNA, an oligo dT cellulose column is used, for example, Molecular Cloning 2nd E
dt. Cold Spring Harbor Laboratory Press (1989). Next, a cDNA library is prepared based on the obtained mRNA.
The cDNA library may be prepared based on total mRNA, or may be prepared based on an mRNA fraction consisting of a part of total mRNA. This mRNA fraction is obtained by fractionating mRNA by a conventional method such as sucrose density gradient centrifugation.
Among them, it can also be prepared by collecting only the fraction having the activity of inducing differentiation from bone marrow cells to cells having the ability to degrade hydroxyapatite and the fractions before and after the activity (the method for measuring the activity will be described later). The cDNA library is prepared, for example, by the Gubler & Hoffman method (Gene, 25, p.
263 (1983)).

Next, the cDNA library is divided into a plurality of pools. Here, the number of cDNA clones per pool can be arbitrarily determined by the number of independent clones constituting a cDNA library. For example, a cDNA library consisting of about 6 × 10 ⁵ independent clones can be used. For rally, 1 ×
10 ⁴ clones that divide about 60 Pool 1 Pool, after would be the operation proper. Subsequently, DNA is prepared from each pool by a conventional method, and cRNA is prepared using this DNA as a template. cRNA can be easily prepared, for example, using a commercially available mRNA capping kit (Stratagene). Next, cRNA for each pool
By injecting into Xenopus oocytes
Translate cRNA to protein. The injection into the oocyte can be performed, for example, by the following method.
That is, an egg mass of an oocyte is taken out from a female Xenopus with a body length of about 10 cm, the oocytes are cut off one by one under a microscope, and live cells without damage of stage V or VI are selected. Inject cRNA from the capillary using a digital microdispenser or the like. The injection volume per oocyte is preferably 50 nl or less. After culturing for several days thereafter, the culture supernatant is collected. Since the translation product (protein) translated from cRNA is present in the culture supernatant, this culture supernatant can be used as a sample for the assay. Next, using this culture supernatant sample,
Perform the following assays.

First, bone marrow cells are used as cells for the assay. Specifically, for example, the epiphysis of the femur and the tibia of a mouse of 6 to 12 weeks of age are cut off, and 1 ml of α-MEM is syringed with a 26G needle once from each end.
Pipette the bone marrow cells extruded with the medium,
The supernatant portion excluding the precipitated bone residue can be used as bone marrow cells. The prepared bone marrow cells are suspended in a culture solution containing active vitamin D, adjusted to an appropriate concentration (for example, 2 × 10 ⁶ cells / ml), and plated (for example,
96-well plate) and add the above Xenopus culture supernatant sample (sample for assay) to it,
The assay is performed as shown below. That is, as methods for identifying osteoclasts, (1) TRAP staining method, (2) ivory decomposition activity measurement method (hereinafter, also referred to as pit formation method using ivory), (3) hydroxyapatite decomposition activity measurement method (hereinafter, referred to as pit formation method) , Also called pit formation method using Osteologic well)
Among these identification methods, a pool having only the hydroxyapatite decomposition activity of 3) is selected. Here, the TRAP staining of (1) is performed, for example, using Endocrinol.
ogy, 122, p1373 (1988), etc., the bone marrow cells treated with the assay sample as described above are first fixed with an acetone-citrate buffer, and then the substrate (Naphth
ol AS-Mxphosphate) and pigment (Fastredviolet LB salt)
By reacting at 37 ° C. for about 1 hour. In the pit formation measurement using ivory in (2), for example, a dentin slice having a diameter of about 6 mm and a thickness of about 1 mm was prepared in advance and laid on the bottom of a well of a 96-well plate. The bone marrow cells are treated with an assay sample such as
The cells on the dentin slice were stained with TRAP and 0.25%
After treatment with trypsin-0.02% EDTA overnight, the cells on the slices are scraped off with a silicon scraper, and the pits (absorbent pits) on the dentin slices are observed under a microscope and the number thereof is measured by counting. it can. Also,
The pit formation measurement using the Osteologic well of (3)
For example, in a well coated with hydroxyapatite (trade name: Osteologic: MILLENIUMBIOLOGIX), the bone marrow cells are treated with the sample for assay as described above, and after an appropriate period, treated with 20% hypochlorous acid for 5 minutes. It can be measured by removing the cells and converting the pits on the wells as the number of meshes per pit.

[0012] Among the above identification methods, the pool showing a positive result only in (3) is further divided into subpools, and by repeating the same operation, a clone encoding C9 of the present invention can be finally obtained. The C9 cDNA cloned by the expression cloning method as described above can be obtained, for example, by using an Auto Read Sequencing kit (Pharmacia)
The nucleotide sequence can be determined by a sequencer using BcaBEST Sequencin using the dideoxy method.
The nucleotide sequence can also be determined using RI using a g kit (TAKARA). Even without such an “expression cloning method”, the C9 cDNA of the present invention is cloned using the whole or a part of the C9 cDNA as a probe or a PCR primer with the publication of the nucleotide sequence of the C9 cDNA of the present invention. be able to. The cloning is performed, for example, by Molecular Cloning 2nd Edt. Col.
d According to Spring Harbor Laboratory Press (1989) and the like, it can be easily performed by those skilled in the art.

2) Modified protein or allele of C9
Among the DNAs encoding the mutants or the like, one or more amino acids in the amino acid sequence of SEQ ID NO: 2 are deleted, substituted, and / or
Or `` DNA encoding a protein comprising an added amino acid sequence and having an activity of inducing differentiation from bone marrow cells to cells having hydroxyapatite decomposability, '' which is an artificially produced so-called modified protein or present in vivo. It refers to a DNA encoding a protein having an activity of inducing differentiation of bone marrow cells into cells having hydroxyapatite decomposability among allele mutants and the like. DNA encoding the protein may be, for example, site-directed mutagenesis (Met
hods in Enzymology, 100, p468 (1983)) and PCR (Mo
lecular Cloning 2nd Edt. Chapter 15, Cold Spring Harbor L
aboratory Press (1989)) and others can easily produce it. Here, the number of amino acid residues to be deleted, substituted and / or added refers to the number of amino acids that can be deleted, substituted and / or added by a well-known method such as site-directed mutagenesis.

Here, “the activity of inducing the differentiation of bone marrow cells into cells having hydroxyapatite degradability” refers to the modified protein of the present invention (encoding the modified protein of the present invention) against the bone marrow cells prepared as described above, for example. DNA
Is ligated to a well-known expression vector, and introduced into an appropriate host. The resulting product is allowed to act, and then the pit formation activity using the Osteologic well of the above-mentioned assay method (3) is examined. Can be easily measured. At this time, it is also necessary to confirm that the modified protein of the present invention shows negative results when subjected to other measurement methods, namely, the TRAP staining method of (1) and the pit formation method using ivory of (2). is there. About these measuring methods, specifically, the above item 1) or 2.3.1 to 2
See 2.3.3. By providing various modified proteins and the like to these measurement methods, it is possible to easily select modified proteins and the like having an activity of inducing differentiation of bone marrow cells into cells having the ability to degrade hydroxyapatite.

3) C9 DNA and stringent conditions
Of the hybridizing DNA the DNA under, "SEQ ID NO: hybridizes with DNA under stringent conditions comprising the nucleotide sequence described in 1, and the differentiation-inducing activity of bone marrow cells into cells with hydroxyapatite resolution "A DNA encoding a protein having the following sequence" is a DNA that hybridizes under stringent conditions to mouse C9 DNA consisting of the nucleotide sequence of SEQ ID NO: 1, such as C9 cDNA of all vertebrates such as humans and rats. Point to.

Here, “DNA that hybridizes under stringent conditions” refers to, for example, standard hybridization conditions (formamide concentration: 50%,
At a salt concentration of 5 × SSC and a temperature of 42 ° C.). These DNAs are cloned by, for example, hybridization with the DNA described in SEQ ID NO: 1, and specific DNA library preparation, hybridization, selection of positive colonies, determination of base sequence, etc. All operations are known, for example, Mo
lecular Cloning 2nd Edt.Cold Spring Harbor Labora
This can be easily performed with reference to tory Press (1989) and the like. Examples of the cDNA library include a human testis or kidney-derived cDNA library. In addition, as a probe used for hybridization, for example, a DNA having the base sequence of SEQ ID NO: 1 can be mentioned.

In the present invention, the protein is a protein encoded by the various DNAs of the present invention described above.
In addition, it has an activity of inducing differentiation from bone marrow cells to cells having hydroxyapatite decomposability. A specific example is the protein of the present invention, which has the amino acid sequence of SEQ ID NO: 2, C9. These proteins can be obtained, for example, by ligating the DNA of the present invention to a known expression vector such as pBK-CMV, introducing the resulting DNA into an appropriate host, and expressing and producing it. The host may be either a prokaryotic cell or a eukaryotic cell. For example, Escherichia coli strains and animal cell strains are already widely spread and readily available unless otherwise specified. For example, animal cell hosts include COS-1, COS
-7, CHO cells. To transform an appropriate animal cell host using the expression plasmid, the LIPOFECTIN method (Felgner PL, et al, Proc. Natl. Acad. Sci. USA,
84, p7413 (1987)). Since the culture supernatant of the transformed cells contains an expressed protein that can be directly used in various assays by appropriate dilution, the culture supernatant is used to convert bone marrow cells to cells having hydroxyapatite decomposability. Can be measured for the activity of inducing differentiation (see the above for the method of measuring the activity). The above expressed protein produced in the culture supernatant can be easily purified by a known method using zinc chelate agarose, concanavalin A agarose, Sephadex G-150, or the like.

[0018] The protein of the present invention can also be used as an active ingredient of a medicament. As described above, the protein of the present invention has the activity of decomposing hydroxyapatite, which is a crystal of calcium in bone, into cells that release calcium to induce the differentiation of bone marrow cells. Can increase the blood calcium concentration. Therefore, a "medicine" containing the protein of the present invention as an active ingredient is, for example, a disease such as hypocalcemia, or blood associated with massive bleeding or radiation exposure, as described in the section of "prior art". It can be an effective remedy for the large decrease in calcium.

As a method for administering such an osteoclast differentiation promoting agent to a patient, intravenous injection is preferred.
Oral administration, administration as a suppository, subcutaneous injection, intramuscular injection, local injection, intraperitoneal administration and the like can be performed. The agent for promoting osteoclast differentiation of the present invention can be administered in various unit dosage forms depending on the administration method described above. For example, for intravenous administration, the protein of the present invention can be used dissolved or suspended in a pharmaceutically acceptable carrier, preferably an aqueous carrier. As the aqueous carrier, for example, water,
Buffered water, 0.4% saline, or the like can be used. The aqueous solutions thus prepared can be packaged as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration. The preparations described above contain pharmaceutically acceptable auxiliaries, such as ph regulators or buffers, tonicity regulators, infiltrants, and more specifically, for example, sodium acetate, sodium lactate, sodium chloride, chloride, etc. Potassium, calcium chloride, sorbitan monolaurate, teriethanolamine oleate and the like can be included. For oral administration, the proteins of the invention can be used in unit dosage forms of powders, tablets, pills, capsules and dragees. For topical administration, unit dosage forms including, for example, aerosols can be employed. For subcutaneous injection, intramuscular injection, local injection, or intraperitoneal administration, the protein of the present invention can be used by dissolving or suspending it in an aqueous or oily carrier. Alternatively, it can be administered as a sustained-release preparation using a biocompatible material such as collagen. The dosage of the above-mentioned osteoclast differentiation promoting agent can be administered at a daily dose of about 0.0001 to 100 mg until symptoms are improved.

In the present invention, the peptide fragment refers to a peptide fragment comprising at least 6 amino acids in the amino acid sequence of the protein of the present invention. Here, the limitation of "at least 6 amino acids or more" is based on the fact that the minimum size that can take a stable structure is 6 amino acids, but a peptide having a length of 8 amino acids or more is more preferably 10 to 20 amino acids. Peptides having a length of about an amino acid are exemplified. The peptide is
If it is as short as about 10 to 20, it can be synthesized by a peptide synthesizer, and if it is long, DNA prepared by ordinary genetic engineering techniques (for example, by treatment with a restriction enzyme) can be used for animal cells or the like. Can be obtained. In addition, it is also possible to modify the peptide produced in this way by an ordinary method. These peptide fragments can be applied to medicine as described later, and can also be used for antibody production.

In the present invention, the term "antibody" refers to an antibody against either the protein of the present invention or a peptide fragment that can constitute an epitope. The antibody can be easily prepared, for example, by immunizing a rabbit or the like using the method described in New Cell Engineering Experiment Protocol p210 Shujunsha (1993). In addition, for example, a monoclonal antibody can be easily produced by using the method described in Protein Experimental Methods for Molecular Biology Research, Chapter 4, Yodosha (1994). Furthermore, a humanized antibody can be used according to JP-A-62-296890. Uses of the antibody include affinity chromatography, screening of a cDNA library, diagnostic reagents and experimental reagents. Further, as described later, it can be used as a medicine.

The protein of the present invention can also be used for screening for an inhibitor of the activity of inducing differentiation of bone marrow cells into cells having hydroxyapatite decomposability. Here, "inhibitor of differentiation induction activity from bone marrow cells to cells having hydroxyapatite degradability"
It refers to an agent that inhibits the activity of the protein of the present invention to induce differentiation of bone marrow cells into cells having the ability to degrade hydroxyapatite. The “screening method for an inhibitor of the activity of inducing differentiation from bone marrow cells to cells having hydroxyapatite degradability” is based on the aforementioned system for measuring the activity of inducing differentiation from bone marrow cells to cells having hydroxyapatite degradability (Osteologic well). To the used pit formation measurement system) by adding an inhibitor candidate substance, which is a test substance. That is, as described above, Os
In a teologic well, a protein of the present invention and an inhibitor candidate substance are added to and act on mouse bone marrow cells. At this time, if the inhibitor candidate substance has an inhibitory action, no pit is formed. Inhibitors can be easily screened using the pit formation as an index.

In the present invention, the inhibitor for the activity of inducing differentiation of bone marrow cells into cells having hydroxyapatite degradability is found by the above-mentioned screening method. It refers to an agent that inhibits the activity of inducing differentiation into cells having hydroxyapatite decomposability. Specific examples of such inhibitors for differentiation-inducing activity from bone marrow cells to cells having hydroxyapatite decomposability include:
For example, among the peptide fragments of the present invention prepared above, those having an inhibitory effect, and those of the present invention having neutralizing activity can be mentioned. As described in the “prior art”, such inhibitors for differentiation-inducing activity from bone marrow cells to cells having hydroxyapatite decomposability include hypertension and arterial diseases, which are associated with an increase in blood calcium concentration. It is considered to be an effective therapeutic agent for sclerosis, diabetes, myocardial infarction, hypercalcemia and the like. Since the protein of the present invention has an activity of inducing differentiation into cells that degrade hydroxyapatite, which is a crystal of calcium in bone, it simultaneously increases the calcium concentration in blood as described above and simultaneously Can also be said to have an action of dissolving (hydroxyapatite), which is an inorganic component of the above. Therefore, such an inhibitor of the protein of the present invention may be an effective therapeutic agent for osteoporosis, a disease associated with bone dissolution, rheumatoid arthritis, bone destruction accompanying bone metastasis of cancer, or Paget's disease. Can be considered.

The method and mode of administration of such an inhibitor of the present invention for inducing the differentiation-inducing activity of bone marrow cells into cells having hydroxyapatite decomposability into a patient include the aforementioned protein of the present invention as an active ingredient. The same administration method and administration form as the contained medicine can be considered. The daily dose can be about 0.0001 to 100 mg until the symptoms are improved.

In the present invention, a transgenic animal refers to a so-called transgenic animal in which the DNA of the present invention has been artificially introduced into a chromosome, or a so-called knockout animal in which the DNA of the present invention has been deleted from the chromosome. These transgenic animals are, for example, disease model mice: Molecu
A person skilled in the art can easily produce it based on lar Medicine special issue Nakayama Shoten (1994) and the like. The transgenic animal is very useful as a model animal for drug development such as hypertension and osteoporosis, or as an animal for screening the drug.

[0026]

EXAMPLES Hereinafter, the present invention will be described in more detail by way of examples as examples of the present invention, but the present invention is not limited to these examples.

Example 1 Construction of mouse cDNA library 1.1 Isolation of mRNA from mouse BW5147 cells 1.1.1 Isolation of total RNA Mouse BW5147 cell line (ATCC CRL 158)
8) 1 × 10 ⁸ pieces were subjected to AGPC method (acid guanidium thio
cyanate-phenol-chloroform method ； Experimental medicine 9, 15 ,
Total RNA was isolated according to p99 (1991)). That is, first, 10 ml of 4M guanidine isothiocyanate was added to the cell pellet, immediately shaken vigorously, and the solution was partially sheared by passing the solution through an 18G needle 5 times. 1 ml of 2M sodium acetate in this solution,
10 ml of water-saturated phenol and 2 ml of chloroform-isoamyl alcohol (49: 1) were sequentially added and mixed every time. Thereafter, the mixture was vigorously shaken, cooled on ice for 15 minutes, and then centrifuged at 10,000 g for 20 minutes at 4 ° C. The aqueous layer was separated, added with an equal amount of isopropanol, and mixed well. After placing this at -20 ° C for 1 hour,
Centrifugation was performed at 000 g for 10 minutes. After centrifugation, 3 ml of 4M guanidine thiocyanate was added to the RNA precipitate to completely dissolve it, and an equal volume of isopropanol was added.
Left for hours. Thereafter, after centrifugation at 10,000 g for 15 minutes at 4 ° C., the supernatant was discarded, and the RNA precipitate was washed with 75% ethanol to obtain total RNA.

1.1.2 Isolation of mRNA By repeating the above method several times, 15 mg of total RNA was collected, and an elution buffer (10 mM Tris-HCl (pH 7.
5) Dissolved in 5 ml of 1 mM EDTA and 0.2% SDS, heated at 65 ° C. for 2 minutes, and immediately cooled to room temperature. After adding 0.55 ml of 5 M NaCl, the solution was washed with a washing buffer (0.5 M NaCl, 10 mM Tris-HCl).
(PH 7.5), 1 mM EDTA and 0.2% SDS
Was added to a column of 0.5 g of oligo dT cellulose (Type 7, Pharmacia Biotech) equilibrated in step 2, and the passed solution was further added to the column twice to bind the mRNA to the column. After washing the column with 15 ml of wash buffer, the bound RNA was eluted with 4 ml of elution buffer. Heating the eluate at 65 ° C. for 2 minutes, then cooling,
It was adjusted to 0.5 M NaCl, added again to the re-equilibration column, and the elution operation was performed in the same manner. MRNA was recovered from the eluate by ethanol precipitation and washed with 75% ethanol.

1.1.3 m by sucrose density gradient centrifugation
Fractionation of RNA A density gradient fractionator (Hitachi; DGF-U) treated with diethylpyrocarbonate and a centrifuge tube, two concentrations of RNase-free sucrose solutions (5% and 20%)
(W / v) sucrose), 0.1 M NaCl, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.5%
Prepare SDS, make a sucrose gradient in a tube for Beckman SW41Ti with a density gradient fractionator,
Leave at room temperature for more than 2 hours to eliminate gradient discontinuities. Next, the mRNA was added to a 200 μl TE solution (99% dimethyl sulfoxide, 10 mM Tris-HCl (pH
7.5) dissolved in 1 mM EDTA, 0.1% SDS), treated at 37 ° C. for 5 minutes, and 400 μl of 5 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.5% SDS.
The non-specific association was dissociated by adding DS and heat treating at 65 ° C. for 10 minutes. Then quench,
Place on a sucrose density gradient, Beckman SW41Ti
Centrifugation was performed at 20,000 rpm for 14 hours at 25 ° C. with a rotor. After centrifugation, the mixture was fractionated from the tube by 0.5 ml each using a density gradient fractionator, and precipitated with ethanol. m
The RNA precipitate was washed at least three times with 75% ethanol.

1.1.4 Identification of mRNA A part of the mRNA fractionated into 50 fractions is described in 2.
The cells were injected into Xenopus oocytes according to the method described in 1.3 and translated into proteins. The culture supernatant containing this translation product was added to mouse bone marrow cells for assay according to the method described in 2.2.2 described below, followed by culturing.
By RAP staining, it was identified whether or not osteoclasts were differentiated (that is, which mRNA fraction contains a factor having osteoclast differentiation promoting activity). As a result, peaks of activity were present in the 27th and 32nd fractions.

1.2 Preparation of cDNA Library The 27th to 33rd positions of the peak activity fraction were collected as the active fraction, and a cDNA library for this fraction was prepared.
Gubler & Hoffman method (Gene, 25, p263 (198
It was prepared by a modification of 3)). That is, mR of this active fraction
Oligo dT with XhoI site based on 2 μg NA
Using the primers, a first strand was synthesized with M-MuLV reverse transcriptase. Then DNA Polymer
Second strand is synthesized by ase I and EcoRI
Ligation with the adapter and XhoI digestion were performed. Then, the adapter and the primer were subjected to gel filtration (Se
(Phacryl Spin Column; Pharmacia). The above cDNA synthesis step is performed by Stratagene ZA.
Reverse transcriptase was performed using Superscript II from BRL using a pcDNA synthesis kit. Next, Eco
After ligation of the RI and XhoI-cut ZAP Express ^™ vector with the previously prepared cDNA,
^{Gigapack II Gold packing extract (mcrA -} , mc
Packaging was performed using rB ⁻ , mmr ⁻ ; Stratagene) to infect Escherichia coli strain PLK-F ^′ . As a result, a cDNA library having an average length of 2.26 kb and the number of independent clones of 6.3 × 10 ⁵ was obtained.

Example 2 Outline of Expression Cloning The cDNA library prepared in 1.2 was
Divided into a total of 63 pools as 0 / pool and described in 2.
CRNA from each pool was injected into Xenopus oocytes by the method described in 1.2 to 2.1.3 and translated into protein. The culture supernatant containing the translation product is used in 2.
The mixture was added to mouse bone marrow cells for assay according to the method described in 2.2 and subjected to each assay described below, and a pool determined to be positive was selected. Further, the positive pool was divided into 10 subpools, cRNA was prepared in the same manner, expressed in oocytes, its activity was measured, and the positive pool was selected. Obtained.

That is, as a specific assay, in the primary screening, the osteoclast differentiation promoting activity was determined by the TRAP staining method described later in 2.3.1, and three positive pools were selected from 63 pools. After the second screening,
Promotion of three types of osteoclast differentiation by the TRAP staining method described later in 2.3.1, the pit formation method using ivory in 2.3.2, and the pit formation method using osteologic well in 2.3.3 A pool showing a positive reaction in all the activity measurement methods was selected. First, each of the above three pools was divided into 10 subpools (1000 clones / pool), and each assay was performed. As a result, the top three pools were selected in the order of the strength of the positive reaction, and the three pools were further divided into 10 subpools (200 clones / pool) and subjected to tertiary screening. As a result, three positive pools were selected in the order of the intensity of the positive reaction, and each of them was divided into 10 subpools (24
(Clone / pool) and further performed a fourth screening. As a result of the screening, a pool was shown to be positive in all three assays, and a pool was shown to be positive only for the pit formation activity using Osteologic wells and was not shown to be positive in the other two assays. Among these pools, of the pools that showed only pit formation activity using Osteologic wells, two pools with higher activity were single-cloned and subjected to fifth screening, and the upper eight clones were defined as positive clones. The above three assays were performed on these eight clones, and Osteol
One of the clones that showed only pit formation using ogic wells was named C9. In addition, each assay result of C9 was described in the section of (results) described later.

2.1 Preparation of Sample for Assay 2.1.1 Preparation of DNA Lambda phage 1 of each pool was added to Escherichia coli XL1-Blue.
× 10 ⁴ pfu was infected and spread on a 15 cm petri dish to form plaque. 13 ml of SM buffer was added to this plate to prepare a plate lysate. DE52 (DEAE cellulose; Whatman) was added to this phage lysate to adsorb other than phage DNA, and DE52 was added again to the supernatant after centrifugation, and the phage DNA in the supernatant was recovered. This DNA was extracted once with phenol and phenol-chloroform (1: 1), and recovered by ethanol precipitation to obtain phage DNA. The prepared DNA was cut with the restriction enzyme NotI, and 1/50 amount was
% Agarose electrophoresis.

2.1.2 Synthesis of cRNA At least 1 μg of the phage DNA of each pool prepared in 2.1.1 was digested with proteinase K (Stratagene).
After treatment at 7 ° C for 1 hour, phenol-chloroform treatment,
The template DNA was prepared by collecting by ethanol precipitation. Using this DNA, mRNAcapp
cRNA was synthesized according to an ing kit (Stratagene). This was subjected to phenol-chloroform treatment and ethanol precipitation to recover cRNA, and 1/10 was quantified by 1% agarose gel electrophoresis. Thereafter, the concentration was adjusted to 1 μg / μl to obtain cRNA for microinjection.

2.1.3 Expression by Xenopus oocytes Oocytes from female Xenopus laevis with a body length of about 10 cm were taken out and subjected to MBS (+ Ca ²⁺ ; 88.0 m).
M NaCl, 1.0 mM KCl, 2.4 mM Na
SO ₃ , 0.3 mM Ca (NO ₃ ) ₂ 4H ₂ O, 0.
41 mM CaCl ₂ 4H _20, 0.82 mM MgS
O ₄ 7H ₂ 0, 10 μg / ml penicillin, 10 μg / ml
ml streptomycin, 50 U / ml nystatin,
Transfer to a Petri dish containing 15 mM Tris-HCl (pH 7.6), cut out the oocytes one by one with precision scissors and tweezers under a stereoscopic microscope, and select live cells without stage V or VI damage. did. These oocytes have 10
50 n per oocyte from capillary using μl digital micro dispenser (Drummond)
1 cRNA was injected. Thereafter, the cells were removed at 20 ° C. for 3 days in MBS containing 2% FCS except for dead or damaged cells.
And cultured. The culture supernatant is centrifuged, and 0.22 μm
And the bacteria were removed at the same time as removing the residue. The supernatant was used as an assay sample.

2.2 Assay 2.2.1 Preparation of Mouse Bone Marrow Cells 6-12 week old mice (C3H / HeJ; CLEA Japan)
Of the femur and tibia were aseptically removed, their epiphyses were cut off, and 1 ml of α-MEM medium (10% fetal bovine serum, 100%
Bone marrow cells were extruded with a unit / ml penicillin G and 100 μg / ml streptomycin), pipetted well, and waited until the bone residue precipitated, and the supernatant was collected. It was further washed 1-2 times with fresh medium to prepare bone marrow cells for the assay.

2.2.2 Osteoclast differentiation and formation method The above bone marrow cells were ^treated with 10 ^-8 M of active vitamin D [1,2].
5 (OH) ₂ D ₃ ], suspended in an α-MEM medium containing 2 × 10 ⁶ cells / ml, and prepared in a 96-well plate with 180 μl and 2.1.3 for the assay. 20 μl of the sample was added and cultured at 37 ° C. under 5% CO ₂ for 1 or 2 weeks. Meanwhile, 3-4 days of medium at 3-4 day intervals
/ 4 was replaced with a fresh medium, and the same amount of a sample for assay was newly added.

2.3 Identification method of osteoclasts 2.3.1 TRAP staining method TRAP (tartrate-resistant acid phosphatase), a marker enzyme of osteoclasts, was stained with a substrate. That is, 2.
After fixing the cultured bone marrow cells of 2.2 with acetone / citrate buffer, the substrate (Naphthol AS @MXphos) was added in the presence of tartaric acid.
phate) and dye (Fastredviolet LB salt) at 37 ° C for 1
After reacting for hours, staining was performed (Endocrinolog
y, 122, p1373, (1988)). (Results) C9 is a known osteoclast differentiation-forming factor I
L─1β (50 ng / ml) or LIF (25 U / ml)
The bone marrow cells were treated with, and compared to the TRAP staining of a positive control in which osteoclasts were differentiated and formed.

2.3.2 Pit formation method using ivory Dentin slices having a diameter of 6 mm and a thickness of 1 mm were prepared from ivory and sterilized by ultrasonic treatment in 80% alcohol. After washing with α-MEM medium, each slice was transferred to the bottom of a well of a 96-well plate, and osteoclasts were induced to differentiate from bone marrow cells according to the method described in 2.2.2. One or two weeks later, the osteoclasts on the dentin slice were stained with the TRAP staining method of 2.3.1, and 0.2% or less.
The cells were treated with 5% trypsin / 0.02% EDTA overnight, and the cells on the slices were scraped off with a silicon scraper.
The pits (resorption pits) on the dentin slice were observed under a microscope, and the number or the number of meshes per pit was measured to examine the bone resorption activity (osteolytic activity) of cells induced to differentiate from bone marrow cells. . (Results) The number of pits formed on dentin slices of cells formed by C9 was 1/10 of LIF (25 U / ml).
It was found that the pit-forming activity was very small, that is, １ ／.

2.3.3 p using Osteologic wells
It forming method 2.2.2 In a well coated with hydroxyapatite (trade name: Osteologic: MILLENIUMBIOLOGIX)
Osteoclasts were induced from bone marrow cells according to the method described above.
One week later, the cells were treated with 20% hypochlorous acid for 5 minutes to remove the cells, and the pits on the wells were converted to the number of meshes per pit to convert the bone resorption of cells induced to differentiate from bone marrow cells ( Osteolytic activity) was measured. (Results) The number of pits on Osteologic wells of cells formed by C9 was an average of 200 or more per well. This is the result of the negative control (distilled water introduced into Xenopus laevis oocytes instead of cRNA and the culture supernatant was added to bone marrow cells) (= average 1).
5 to 20) and a pit result (5 U) using the positive control LIF (25 U / ml).
0), the pit formation activity was determined to be positive. (Conclusion) In all of the above three identification methods, all were not positive, and only the pit formation activity using Osteologic well was positive. Therefore, the cells differentiated and formed by C9 were not osteoclasts. The cells were found to be cells having hydroxyapatite decomposition activity.

Example 3 Conversion of Recombinant Phage DNA to Phagemid DNA The ZAP Express vector uses pBK
-Subcloning can be performed by in vivo excision into CMV. XL1-BlueMRF 'E. coli was infected with ZAP Express phage and ExAssist helper phage to produce pBK-CMV phagemid, and the original E. coli was killed by heat treatment and newly infected with XLOLR E. coli. A medium was added thereto, and after culturing for 45 minutes, the cells were plated on an LB plate and cultured.

Example 4 Preparation of Plasmid DNA A positive colony was picked up with a toothpick, cultured overnight in 2 ml of LB (kanamycin 100 μg / ml), and then subjected to alkaline-SDS.
The plasmid was prepared by the method. This plasmid DNA
Was digested with an appropriate restriction enzyme, and electrophoresed in a 1% agarose gel to confirm insertion of the C9 cDNA into the vector.

Example 5 Determination of Nucleotide Sequence of C9 cDNA Determination of the nucleotide sequence of C9 cDNA obtained in Example 4
It was performed by the dideoxy method developed by Sanger et al. (AutoRead Sequencing kit, Pharmacia Bio
tech). As a result, a cDNA consisting of 1708 bp shown in SEQ ID NO: 1 of the Sequence Listing was obtained, and the amino acid sequence of SEQ ID NO: 2 consisting of 251 amino acids was determined. After the above-described nucleotide sequence determination, a plasmid in which the cDNA of the novel protein C9 of the present invention was incorporated into an expression vector pBK-CMV was used to construct a plasmid. E. coli JM109 strain, which is a transformant for storage, E. coli JM109. coli JM
109 (C9) was prepared. E. FIG. coli JM109
(C9) has been deposited at the Institute of Biotechnology and Industrial Technology, 1-3-1 Higashi, Tsukuba, Ibaraki Prefecture (Microbial indication: E. coli JM109 (C9); date of receipt:
July 4, 1997; Accession number: FERM P-1630
5).

Example 6 Northern Blot Analysis of C9 Gene Expression Northern blot analysis was performed using an MTN blot membrane on which poly A ⁺ RNA from various tissues had been blotted. As a probe, the full-length C9 cDNA (about 1.7 Kbp) was labeled with ³² P.

The above probe was prepared by adding 50% (v / v) formaldehyde / 5 × SSC / 5 × Denhardt / 1% (w / v
v) SDS / 0.01% (w / v) denatured salmon sperm DNA
Hybridized at 42 ° C. to RNA fixed to a filter in 2 × SSC / 0.1% SDS at 50 ° C.
Next, the substrate was washed in 0.1% SSC / 0.1% SDS at 50 ° C. After removing water, autoradiography was performed at -80 ° C for 1-3 days. The X-ray film used was Kodak SB5 or Fuji AIF, RX in the presence of an intensifying screen.

(Results) FIG. 1 shows that each mouse tissue (2 μg
2 shows the results of Northern blot of RNA). C9cDN
The mRNA band using A as a probe was strongly expressed in the kidney in the range examined. 3 different for C9 mRNA
There were different sizes, and mRNA corresponding to the size of the C9 cDNA showed strong expression in testis.

[0048]

Industrial Applicability According to the present invention, C9 having a differentiation-inducing activity from bone marrow cells to cells having hydroxyapatite decomposability, a similar protein of C9, a peptide fragment of these proteins, an antibody, and these proteins as active ingredients Or a gene encoding these proteins, an expression vector for the gene, a transformant into which the expression vector has been introduced, a method for producing a recombinant protein using the transformant, and a transgene for transfection or deletion of the gene. Use of the protein in screening of an inhibitor for an activity to induce differentiation of a transgenic animal, and further from a bone marrow cell to a cell having hydroxyapatite decomposability is provided.

[0049]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 1708 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA Sequence characteristics Characteristic determination method: E sequence CTCGTGCCGCAGACTGGAAG CAGCCT ATG TAC TTG GGA TTT GAG AAG GAT GTC 53 Met Tyr Leu Gly Phe Glu Lys Asp Val 5 TTT AAG ACT ATA GCA GAT TAC TAT GGT CAC CTG AAG GAG CCA CTG CTC 101 Phe Lys Thr Ile Ala Asp Tyr Tyr Gly His Leu Lys Glu Pro Leu Leu 10 15 20 25 ACA TTT CAT CTC TTT GAT GCT TTC GTC AGC GTC TTA GGT TTG CTG CCG 149 Thr Phe His Leu Phe Asp Ala Phe Val Ser Val Leu Gly Leu Leu Pro 30 35 40 AAG GAG AAA ACG GCC ATT GAA GCA TTT CAG CTT TGC TGT CTC CTC CTG 197 Lys Glu Lys Thr Ala Ile Glu Ala Phe Gln Leu Cys Cys Leu Leu Leu 45 50 55 CCT CCT GAA AAC AGG AGA AAG TTA CAG CTG TTG ATG AGG ATG ATG GCG 245 Pro Pro Glu Asn Arg Arg Lys Leu Gln Leu Leu Met Arg Met Met Ala 60 65 70 AGG ATC TGC CTA AAC AAG GAG ATG CCC CCA CTG AGT GAT GGC TTC GGC 293 Arg Ile Cys Leu Asn Lys Glu Met Pro Pro Leu Ser A sp Gly Phe Gly 75 80 85 ACC CGC ACA CTG ATG GTT CAG ACC TTT TCC CGG TGC ATC CTG TGC TCT 341 Thr Arg Thr Leu Met Val Gln Thr Phe Ser Arg Cys Ile Leu Cys Ser 90 95 100 105 AAG GAT GAA GTA GAC TTA GAT GAG TTA CTA GCT GCT CGA TTG GTG ACA 389 Lys Asp Glu Val Asp Leu Asp Glu Leu Leu Ala Ala Arg Leu Val Thr 110 115 120 TTC CTG ATG GAC AAT TAC CAG GAG ATT CTG AAA GTG CCT CTG GCC CTG 437 Phe Leu Met Asp Asn Tyr Gln Glu Ile Leu Lys Val Pro Leu Ala Leu 125 130 135 CAG ACC TCC ATA GAA GAG CGT GTG GCT CAT CTG CGA AGA GTC CAG ATA 485 Gln Thr Ser Ile Glu Glu Arg Val Ala His Leu Arg Arg Val Gln Ile 140 145 150 AAA TAC CCA GGA GCT GAT ATG GAT ATC ACT CTG TCT GCA CCT TCG TTT 533 Lys Tyr Pro Gly Ala Asp Met Asp Ile Thr Leu Ser Ala Pro Ser Phe 155 160 165 TGC CGT CAA ATT AGT CCC GAG GAG TTT GAG TAC CAA AGG GCG TAT GGC 581 Cys Arg Gln Ile Ser Pro Glu Glu Phe Glu Tyr Gln Arg Ala Tyr Gly 170 175 180 180 185 TCC CAG GAG CCC CTG GCA GCC TTG TTG GAA GAA GTC ATA GCC GAT GAC 629 Ser Gln Glu Glu Pro Leu Ala Ala Leu Leu Gl u Glu Val Ile Ala Asp Asp 190 195 200 AAA CTC TCC AGC AAG GAG AAG AAG AAG AAG CTG AAG CAG TTT CAG AAA 677 Lys Leu Ser Ser Lys Glu Lys Lys Lys Lys Leu Lys Gln Phe Gln Lys 205 210 215 TCT TAC CCT GAG GTC TAC CAG GAA CGA TTT CCT ACC CCA GAG AGC GAA 725 Ser Tyr Pro Glu Val Tyr Gln Glu Arg Phe Pro Thr Pro Glu Ser Glu 220 225 230 GAC TTC TCT TTC CTG AAA AAC CCA AGG CCA AGC CGC AGC TGT TCA TGT 773 Asp Phe Ser Phe Leu Lys Asn Pro Arg Pro Ser Arg Ser Cys Ser Cys 235 240 245 GGG CAC TGAGGAAGCC CTTCCAGCCG TTTCAGAGAA CTAGAAGTTT CCGGATGTGA 829 Gly His 250 TGCAGTGATG GAGCTTGAGA GACCTCTGCG GCAGAGACAG TTTGAGTCAG CGGAGGTTCT 889 GGAAGGCTGG TGTAAACTAC TGCAATATGG AGTCTCGGAG CATCCAGCCG ATCGCCCGAC 949 TCCTAGTGCT GAGCCCTTGT CAGTATTCTG ATAAATCTCG ATGCTGTTTG TAGAGATGTA 1009 AATGAGTGGG TGACTGTGAG TCTCGTGGGT AGGGGTGGCG TGAACAGGCT TGGGATGGTT 1069 ACAAAATCTA AAGGTAAAAC ATTCGTTCCC CTCCTTCTGT TTGTTGTTTT CCACGGTGTG 1129 TAAAGTCCTT GTGTGTGCCC CCTGCTCTCG TGTGCGTCTG TGAAAACCAACCTGTCGATGACTGAG CCCTG TGTGTGCTGT TACTGTGGTA GCCCTGACCA 1249 GTGCATATTC TGATTTCTCC TGTGTAAGTC CCACTGGTTG TTGTTTGTTT GTTTGTTTGT 1309 TTTTATTTCT TAATTTCTTG TTTGTTTGGT TAGTGTGAAA AGTCCAACTG TTTTGCACTT 1369 TGATTGCCGT GTCTTGTATT GCAGATTTGC CTGTCGCTCT TTGATTACCC GTATGCCGGG 1429 CTGGGTCTGC GCGTGGCTGG ATGTGGAATG GTGTGCATCT GCCCCCGGGC CCGCAATGCG 1489 GGTCTCCGCT AAAGACCGCA GGTTGAGCTG GATGAAGTCT TTGGTTCCCT TAGACCTGCA 1549 AACCTGAATT CAGTCTGGTT TTATGAAAAG TATAAAGGGG TTGAGTGAGA TGAATTACGT 1609 CCTTTATGAA TATATTTTCT CAGCTTCTTC ATTGTTTTGA AAGTACATGG AATGTAAATA 1669 AATACTGAGT TGTTTATATA TAAAAAAAAA AAAAAAAAA 1708

SEQ ID NO: 2 Sequence length: 251 Sequence type: amino acid Topology: Linear Sequence type: Peptide sequence Met Tyr Leu Gly Phe Glu Lys Asp Val Phe Lys Thr Ile Ala Asp Tyr 5 10 15 Tyr Gly His Leu Lys Glu Pro Leu Leu Thr Phe His Leu Phe Asp Ala 20 25 30 Phe Val Ser Val Leu Gly Leu Leu Pro Lys Glu Lys Thr Ala Ile Glu 35 40 45 Ala Phe Gln Leu Cys Cys Leu Leu Leu Pro Pro Glu Asn Arg Arg Lys 50 55 60 Leu Gln Leu Leu Met Arg Met Met Ala Arg Ile Cys Leu Asn Lys Glu 65 70 75 80 Met Pro Pro Leu Ser Asp Gly Phe Gly Thr Arg Thr Leu Met Val Gln 85 90 95 Thr Phe Ser Arg Cys Ile Leu Cys Ser Lys Asp Glu Val Asp Leu Asp 100 105 110 Glu Leu Leu Ala Ala Arg Leu Val Thr Phe Leu Met Asp Asn Tyr Gln 115 120 125 Glu Ile Leu Lys Val Pro Leu Ala Leu Gln Thr Ser Ile Glu Glu Arg 130 135 140 Val Ala His Leu Arg Arg Val Gln Ile Lys Tyr Pro Gly Ala Asp Met 145 150 155 160 Asp Ile Thr Leu Ser Ala Pro Ser Phe Cys Arg Gln Ile Ser Pro Glu 165 170 175 Glu Phe Glu Tyr G ln Arg Ala Tyr Gly Ser Gln Glu Pro Leu Ala Ala 180 185 190 Leu Leu Glu Glu Val Ile Ala Asp Asp Lys Leu Ser Ser Lys Glu Lys 195 200 205 Lys Lys Lys Leu Lys Gln Phe Gln Lys Ser Tyr Pro Glu Val Tyr Gln 210 215 220 Glu Arg Phe Pro Thr Pro Glu Ser Glu Asp Phe Ser Phe Leu Lys Asn 225 230 235 240 Pro Arg Pro Ser Arg Ser Cys Ser Cys Gly His 245 250

[Brief description of the drawings]

FIG. 1 shows C9-corresponding mR in each mouse tissue.
It is an electrophoresis photograph as a result of having investigated the expression distribution of NA by Northern blot analysis.

──────────────────────────────────────────────────の Continued on front page (51) Int.Cl. ⁶ Identification code FI C07K 14/475 C07K 16/22 16/22 C12N 1/21 C12N 1/21 C12P 21/02 C 5/10 A61K 37/02 ABJ C12P 21/02 C12N 5/00 B // (C12N 1/21 C12R 1:19) (C12P 21/02 C12R 1:19)

Claims (14)

[Claims] 1. A DNA encoding the following protein (a) or (b): (A) a protein consisting of the amino acid sequence of SEQ ID NO: 2; (b) a protein consisting of an amino acid sequence of SEQ ID NO: 2 in which one or more amino acids have been deleted, substituted and / or added, And a protein having an activity of inducing differentiation from bone marrow cells to cells having hydroxyapatite decomposability 2. A DNA comprising the nucleotide sequence of SEQ ID NO: 1, or a protein which hybridizes with the DNA under stringent conditions and has an activity of inducing differentiation from bone marrow cells to cells having hydroxyapatite decomposability. DNA encoding 3. A protein encoded by the DNA according to claim 1 or 2. 4. The protein according to claim 3, which comprises the amino acid sequence of SEQ ID NO: 2. 5. An expression vector containing the DNA according to claim 1 or 2. 6. A transformant transformed by the expression vector according to claim 5. 7. The transformant according to claim 6, wherein the transformant is
A method for producing a recombinant protein, comprising culturing under the conditions under which the expression vector described above can be expressed. 8. A pharmaceutical comprising the protein according to claim 3 as an active ingredient. 9. A peptide fragment comprising at least 6 amino acids or more of the protein according to claim 3 or 4. 10. An antibody against any one of the protein according to claim 3 or 4, or the peptide fragment according to claim 9. 11. A method for screening for an inhibitor for the activity of inducing differentiation of bone marrow cells into cells having hydroxyapatite degradability, comprising using the protein according to claim 3 or 4. 12. An inhibitor for the activity of inducing differentiation of bone marrow cells into cells having the ability to degrade hydroxyapatite, which is obtained by the screening method according to claim 11. 13. The inhibitor according to claim 12, comprising the peptide fragment according to claim 9 or the antibody according to claim 10. 14. A transgenic animal wherein the DNA according to claim 1 or 2 is artificially introduced into a chromosome, or either of the transgenic animals is deleted from the chromosome.