WO1999000496A1 - Novel osteoclast differentiation promoting factor and its gene - Google Patents
Novel osteoclast differentiation promoting factor and its gene Download PDFInfo
- Publication number
- WO1999000496A1 WO1999000496A1 PCT/JP1998/002869 JP9802869W WO9900496A1 WO 1999000496 A1 WO1999000496 A1 WO 1999000496A1 JP 9802869 W JP9802869 W JP 9802869W WO 9900496 A1 WO9900496 A1 WO 9900496A1
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- Prior art keywords
- protein
- dna
- osteoclast differentiation
- differentiation promoting
- promoting activity
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/51—Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
Definitions
- the present invention relates to a novel osteoclast differentiation promoting factor designated as PF8 and its gene. More specifically, OPF-8 having an osteoclast differentiation promoting activity, a protein similar to OPF-8, a peptide fragment of these proteins, an antibody, a medicament containing these proteins as active ingredients, or these proteins A gene to be encoded, 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 related to the introduction and deletion of the gene, The present invention relates to use of the protein in screening for an osteoclast differentiation promoting activity inhibitor.
- Bone tissue is replaced by new bone without changing its basic shape due to bone formation by osteoblasts and bone resorption by osteoclasts. This process is called bone remodeling (bone remodeling).
- Osteoblasts which play an important role in maintaining the function of living organisms, play a central role in bone formation, and are derived from mesenchymal-derived undifferentiated cells. Differentiate and form bone matrix.
- osteoclasts which play a central role in bone resorption, are derived from bone marrow cells and are formed by differentiation through cell-to-cell contact with osteoblasts. Mature osteoclasts are multinucleated, poorly proliferating giant cells that express the calcitonin receptor and tartrate-resistant acid phosphatase (TRAP). It also has the activity of absorbing calcified tissues such as bone and dentin. -
- osteoclasts and osteoblasts The balance between bone remodeling by osteoclasts and osteoblasts is balanced by repeated bone resorption by osteoclasts and bone formation by osteoblasts. However, once this imbalance is lost, bone tissue becomes abnormal and presents various diseases.
- Osteoporosis is known as a typical disease caused by abnormal bone remodeling. Osteoporosis is classified into high-turn osteoporosis, which is observed in early menopause or hyperthyroidism, and low-turn osteoporosis, which is observed in senile, steroid, and diabetic osteoporosis, based on the turnover rate of bone remodeling. ing. Other than these, diseases such as hypercalcemia and bone destruction associated with bone metastasis of cancer or bone destruction associated with rheumatoid arthritis are known as diseases caused by abnormal bone remodeling. Bone metastasis of these cancers and bone destruction associated with rheumatoid arthritis are diseases resulting from abnormally enhanced functions of osteoclasts.
- a factor that promotes the differentiation of bone marrow cells into osteoclasts or an inhibitor thereof may be a therapeutic agent for the above-mentioned diseases.
- the factor that promotes osteoclast differentiation may restore the low turnover of bone remodeling seen in low turnover osteoporosis as described above (J. Bone Miner Res., 7, p65 (1992)).
- this will be an effective therapeutic agent for low-rotation osteoporosis.
- an inhibitor of an osteoclast differentiation promoting factor is considered to restore the normal rotation of bone remodeling in high-rotation osteoporosis as described above (Am.J.Med.Sci., 305).
- p40 (1993) and Mebio., 11 (2), p24 (1994)) are considered to be effective treatments for this high-speed osteoporosis.
- the inhibitor of osteoclast differentiation promoting factor may also be effective for bone destruction in patients with bone damage.
- the osteoclast differentiation promoting factor is also called a bone resorbing factor, and active forms of biminmin D 3, parathyroid hormone (PTH), interleukin 1, and prosthetic grandein have been known so far.
- PTH parathyroid hormone
- interleukin 1 interleukin 1
- prosthetic grandein active forms of biminmin D 3, parathyroid hormone (PTH), interleukin 1, and prosthetic grandein
- An object of the present invention is to provide a novel protein OPF8 having an activity to promote osteoclast differentiation and its gene. More specifically, OPF8 having an osteoclast differentiation promoting activity, a similar protein of OPF8, a peptide fragment of these proteins, an antibody, a medicament containing these proteins as an active ingredient, or these proteins A gene encoding the same, 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 related to the introduction and deletion of the gene, The present invention provides a use of the protein in screening for an inhibitor of osteoclast differentiation promoting activity.
- the present inventors searched for an osteoclast differentiation promoting factor from the BW514 cells, which are bone metastatic cells, by the expression cloning method, and found that ⁇ TRAP staining '', a marker enzyme for osteoclasts, and We succeeded in discovering a factor that induces bone marrow cells to differentiate into osteoclasts, which have two well-known properties, “ivory degradation activity”.
- this proteinaceous factor having osteoclast differentiation promoting activity OPF8 was completed further studies to complete the present invention. That is, the gist of the present invention is:
- a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted and / or added from the amino acid sequence of SEQ ID NO: 2, and which has an activity of promoting osteoclast differentiation;
- DNA comprising the nucleotide sequence of SEQ ID NO: 1, or a DNA which hybridizes with the DNA under stringent conditions and encodes a protein having osteoclast differentiation promoting activity
- 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).
- the protein according to (3) or (4) is used.
- a method for screening for an osteoclast differentiation promoting activity inhibitor is used.
- the DNA is not particularly limited as long as it is DNA of OPF8 or DNA similar to the OPF8 DNA and encoding a protein having osteoclast differentiation promoting activity.
- DNA encoding the DNA comprising the nucleotide sequence of SEQ ID NO: 1 or the DNA encoding the protein comprising the amino acid sequence of SEQ ID NO: 2 is exemplified.
- DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted and / or added in the amino acid sequence described in SEQ ID NO: 2, or the base described in SEQ ID NO: 1 DNAs that hybridize under stringent conditions to DNAs consisting of sequences are also included in the DNA of the present invention as long as they encode proteins having osteoclast differentiation promoting activity.
- these DNAs will be sequentially described.
- a DNA comprising the nucleotide sequence shown in SEQ ID NO: 1 or a DNA encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 refers to the DNA encoding the OPF 8 of the present invention.
- DNA that The DNA can be obtained, for example, by the “expression cloning method” described below. That is, first, bone metastatic cells are used as cells that produce an osteoclast differentiation promoting factor. Therefore, total RNA is prepared from the bone metastatic cells, and then mRNA is prepared from the total RNA.
- the bone metastasis cell used here includes, for example, a mouse BW5147 cell line.
- Total RNA can be prepared by a conventional method such as the AGPC method (acid guamdium thiocyanate-phenol-chloroform method; Tuji, Nakamura: Experimental Medicine 9, p99 (1991)).
- the mRNA can be prepared using an oligo dT cellulose column, for example, by the method described in Molecular Cloning 2nd Ed. Cold Spring Harbor Laboratory Press (1989) and the like.
- a cDNA library is prepared based on the obtained mRNA.
- the cDNA library may be prepared based on total mRNA or
- This mRNA fraction is obtained by collecting only the fraction having the osteoclast differentiation promoting activity and the fractions before and after it, among the mRNA fractionated by a conventional method such as sucrose density gradient centrifugation. It can also be prepared (the method of measuring the osteoclast differentiation promoting activity will be described later).
- a cDNA library is
- the above cDNA library is divided into a plurality of pools.
- the number of cDNA clones per pool can be arbitrarily determined according to the number of independent clones constituting the cDNA library.For example, it is composed of about 6 ⁇ 10 5 independent clones. In the case of the cDNA library, it is appropriate to divide it into about 60 pools with lxlO 4 clones as one pool for the subsequent operation.
- DNA is prepared from each pool by a conventional method, and cRNA is prepared using this DNA as a template.
- cRNA is, for example, commercially available mRNA It can be easily prepared using the capping kit (Stratagene).
- translation of cRNA into protein is performed by injecting the cDNA of each pool into the oocytes of Africa frog. The injection into the oocyte can be performed, for example, by the following method. In other words, an egg mass of oocytes was removed from a female African Megafrog, about 10 cm in length, and the oocytes were cut off one by one under a microscope. Stage V or VI intact living cells were selected, and the eggs were selected.
- the injection volume per oocyte is preferably 50 nl or less. After culturing for several days, collect the culture supernatant. Since the translation product (protein) translated from cRNA is present in the culture supernatant, this culture supernatant can be used as an assay sample.
- Atsushi Since osteoclasts are considered to be cells induced and differentiated from myeloid cells, it is desirable to use these myeloid cells as Atsushi cells. Specifically, for example, the epiphyses of femurs and tibias of 6- to 12-week-old mice were cut off, and bone marrows were collected in a 1 ml Hi-MEM medium using a syringe with a 26 G needle once from each end. The extruded cells are beveled, and the supernatant portion excluding the precipitated bone debris 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 (eg, 2 ⁇ 10 6 cells / ml), and spread on a plate (eg, a 96-well plate). Then, the above-mentioned Africa Megafell culture supernatant sample (sample for Atsey) was added thereto, and subjected to TRAP staining, which is a known method for identifying osteoclasts, and a method for forming a resorption pit (pit) using ivory, and Pools that show positive results in any of the identification methods are selected.
- TRAP staining is carried out, for example, according to Endocrinology, 122, pl373 (1988), etc.
- bone marrow cells treated with the sample for Atsusei as described above are fixed with acetonitrile buffer, and then the substrate (Naphtholol) is added in the presence of tartaric acid.
- the reaction can be detected by reacting AS-Mxphosphate) with a dye (Fastredviolet LB salt) at 37 ° C for about 1 hour.
- a dentin slice of about 6 thighs and about 1 thigh in thickness was laid on the bottom of a 96-well el-plate, and the above-mentioned assay was performed on this well.
- the cells on the dentin slice are stained with TRAP, treated with 0.25% trypsin-0.02% EDTA, overnight, and the cells on the slice are siliconized. After scraping with a scraper, the pits (absorbent pits) on the dentin slice can be observed under a microscope and counted.
- a pool that shows a positive result in the above identification method is further divided into subpools, and the same procedure is repeated to finally obtain a clone encoding the 0PF8 of the present invention.
- the nucleotide sequence of the 0PF DNA cloned by the expression cloning method as described above can be determined by a sequencer using, for example, an Auto Read Sequencin kit (Pharmacia), or BcaBEST Sequencing using the dideoxy method. With a kit (TAKARA), the nucleotide sequence can also be determined using RI.
- the 0PF DNA of the present invention can be cloned.
- the cloning is performed, for example, by Molecular Cloning 2nd Edt. Cold Spring Harbor Laboratory According to Press (1989) and the like, it can be easily performed by those skilled in the art.
- DNA encoding protein refers to a DNA that encodes a protein having an osteoclast differentiation promoting activity among artificially produced so-called modified proteins and allelic variants present in vivo.
- the DNA encoding the protein can be obtained by techniques such as site-directed mutagenesis (Methods in Enzymology, 100, p468 (1983)) and PCR (Molecular Cloning 2nd Edt. Chapter 15, Cold Spring Harbor Laboratory Press (1989)).
- deletion the here c Note that can be easily fabricated by those skilled in the art, deletions, number of amino acid residues to be substituted and / or added, by a known method such as the site-directed mutagenesis, Refers to the number that can be replaced and / or added.
- the “osteoclastic differentiation promoting activity” refers to, for example, the action of the modified protein of the present invention (the expression product of DNA encoding the modified protein of the present invention) on bone marrow cells prepared as described above. After that, it can be easily measured by examining the known action of osteoclasts—that is, TRAP staining and ivory degradation activity. Specifically, in item 1) above or 2.
- modified proteins having osteoclast differentiation promoting activity can be easily selected.
- DNA that hybridizes under stringent conditions with 0PF DNA that hybridizes under stringent conditions with 0PF
- DNA that hybridizes with DNA consisting of the nucleotide sequence of SEQ ID NO: 1 under stringent conditions and encodes a protein having osteoclast differentiation promoting activity This refers to DNA that hybridizes under stringent conditions to mouse OPF8 DNA consisting of the nucleotide sequence of SEQ ID NO: 1 such as OPF8 cDNA of all vertebrates such as human and rat.
- DNA that hybridizes under stringent conditions refers to, for example, the sequence number under standard hybridization conditions (formamide concentration: 50%, salt concentration: 5 XSSC, temperature: 42 ° C). : Refers to DNA that hybridizes with the DNA of 1.
- DNAs are cloned by, for example, hybridization with the DNA of SEQ ID NO: 1, but specific cDNA library preparation, hybridization, selection of positive colonies, and base sequence All operations such as determination are known and can be easily performed with reference to, for example, Molecular Cloning 2nd Edt. Cold Spring Harbor Laboratory Press (1989).
- a cDNA library is a cDNA library derived from human bone tissue or kidney.
- the probe used for hybridization include, for example, DNA having the nucleotide sequence of SEQ ID NO: 1.
- the protein is a protein encoded by the above-described various DNAs of the present invention, and has an activity of promoting osteoclast differentiation.
- the protein of the present invention having an amino acid sequence, such as OPF8, is mentioned.
- proteins are expressed and produced by, for example, ligating the DNA of the present invention to a known expression vector such as pBK-CMV, and then introducing the protein into an appropriate host. By doing so, it can be obtained.
- the host may be a prokaryotic or eukaryotic cell.
- Escherichia coli strains and animal cell strains are already widely spread and readily available unless otherwise specified.
- animal cell hosts include COS-1, COS-7, and CHO cells.
- a known method such as the LIPOFECTIN method (Feigner PL, et al, Proc. Natl. Acad. Sci. USA, 84, p7413 (1987)) is used.
- the osteoclast differentiation promoting activity should be measured using the culture supernatant. (See above for activity assay).
- the 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.
- the protein of the present invention can also be used as an active ingredient of a medicament. That is, a “medicine” containing the protein of the present invention as an active ingredient can be used, for example, as an osteoclast differentiation promoting agent. More specifically, as described in the section of “Prior Art”, it can be an effective therapeutic agent for low-rotation osteoporosis and the like observed in senile, steroid, and diabetic osteoporosis.
- intravenous injection is preferable, but oral administration, administration as a suppository, subcutaneous injection, intramuscular injection, local injection, intraperitoneal administration and the like can be performed.
- the osteoclast differentiation promoting agent of the present invention can be administered in various unit dosage forms depending on the above-mentioned administration method.
- the protein of the present invention is used by dissolving or suspending it in a pharmaceutically acceptable carrier, preferably an aqueous carrier. be able to.
- aqueous carrier for example, water, buffered water, 0.4% physiological saline, and the like can be used.
- the aqueous solution thus prepared can be packaged as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous solution before administration.
- the above preparations may contain pharmaceutically acceptable auxiliaries, such as ph regulators or buffers, tonicity regulators, infiltrants, and more specifically, for example, sodium acetate, sodium lactate, chloride It can contain sodium, potassium chloride, calcium chloride, sorbine monolaurate, triethanolamine oleate and the like.
- auxiliaries such as ph regulators or buffers, tonicity regulators, infiltrants, and more specifically, for example, sodium acetate, sodium lactate, chloride It can contain sodium, potassium chloride, calcium chloride, sorbine monolaurate, triethanolamine oleate and the like.
- the proteins of the present invention can be used in unit dosage forms of powders, tablets, pills, capsules and sugars.
- a unit dosage form containing, for example, azoles may be used.
- the protein of the present invention can be used by dissolving or suspending it in an aqueous or oil carrier. Alternatively, it can be administered as a sustained-release preparation using a biocompatible material such as collagen.
- the dose of the above-mentioned osteoclast differentiation promoting agent can be administered at a daily dose of about 0.0001 to 100 mg until the symptoms are improved.
- the peptide fragment refers to a peptide fragment comprising at least 6 amino acids or more in the amino acid sequence of the protein of the present invention.
- 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 preferably a peptide having a length of 8 amino acids or more, more preferably 1 amino acid or more.
- Peptides having a length of about 0 to 20 amino acids are exemplified. Note that the peptide is 10 to If it is as short as about 20 pieces, it can be synthesized with a peptide synthesizer.
- DNA prepared by ordinary genetic engineering techniques for example, by treatment with restriction enzymes
- DNA prepared by ordinary genetic engineering techniques can be used for animal cells, etc. It can be obtained by expressing the protein in E. coli. It should be noted that the peptide thus produced can be modified by a usual method.
- peptide fragments can be applied to pharmaceuticals as described below, and can also be used for producing antibodies.
- an antibody is an antibody against any of the protein of the present invention or a peptide fragment thereof.
- the antibody can be easily prepared, for example, by immunizing rabbits and the like using the method described in New Cell Engineering Experimental Protocol p210 Shujunsha (1993). Monoclonal antibodies can be easily produced, for example, by using the method described in Protein Experimental Methods for Molecular Biology Research, Chapter 4, Yodosha (1994). Furthermore, it can be a humanized antibody according to Japanese Patent Application No. 62-296890. Examples of the use of the antibody include affinity chromatography, screening of a cDNA library, diagnostic agents and experimental reagents. Furthermore, as described later, it can be used as a medicine.
- the protein of the present invention can also be used for screening of an osteoclast differentiation promoting activity inhibitor.
- the “inhibitor of osteoclast differentiation promoting activity” refers to an agent that inhibits the osteoclast differentiation promoting activity of the protein of the present invention.
- the “screening method for an osteoclast differentiation promoting activity” can be carried out by adding a candidate inhibitor, which is a test substance, to the above-described system for measuring osteoclast differentiation promoting activity.
- the osteoclast differentiation promoting activity inhibitor is found by the above screening method, and as described above, means an agent that inhibits the osteoclast differentiation promoting activity of the protein of the present invention.
- an osteoclast differentiation promoting activity inhibitor include, for example, those having an inhibitory effect among the peptide fragments of the present invention prepared above and those having a neutralizing activity among the antibodies of the present invention. Is mentioned.
- such an inhibitor of osteoclast differentiation promoting activity is used as a therapeutic agent for high-rotation osteoporosis observed in early menopause or hyperthyroidism, etc.
- the administration method and administration form of the osteoclast differentiation promoting agent may be the same as the administration method and administration form of the osteoclast differentiation promoting agent.
- the daily dose can be about 0.0001 to 100 mg until the symptom is improved.
- 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 has been deleted from the chromosome.
- transgenic animals can be easily prepared by those skilled in the art based on, for example, disease model mice: Molecular Medicine Extra Edition, Nakayama Shoten (1994).
- the transgenic animal can be used, for example, as a model animal for the development of a drug such as osteoporosis, or as an animal for screening the drug. Very useful as an object. .
- FIG. 1 is an electrophoretogram of the results of Northern blot analysis of the expression distribution of 8 mRNA of OPF in each mouse tissue.
- FIG. 2 is an electrophoresis photograph showing the results of Northern blot analysis of the expression distribution of OPF mRNA from mouse 7-day embryo to mouse 17-day embryo.
- FIG. 3 is an electrophoretic photograph of the result of examining the tissue distribution of 8 mRNA of human OPF by dot blot analysis of human RNA.
- FIG. 4 is an electrophoretic photograph showing the results of RT-PCR analysis of the expression of 8 mRNA of OPF in an osteoblast cell line (osteoblast-like cell line).
- the OPF 8 is indicated as OPFu 8.
- Fig. 5 (upper) is a micrograph of the activity of the culture supernatant in which the OPF8 cDNA was expressed in COS cells, as determined by TRAP staining and the formation of pit.
- Figure 5 (bottom) is a photomicrograph of the results of a similar experiment with the introduction of Vector-1.
- Example 1
- Mouse BW5147 cell line (ATCC CRL 1588) 1 10 8 cells were subjected to AGPC method, acid guanidium thiocyanate-phenol-chloroform method; Experimental RNA 9, 15, p99 (1991)).
- 10 ml of 4 M guanidine isothionate was added to the cell pellet, immediately shaken vigorously, and the solution was partially sheared by passing the solution five times back and forth through 21 dollars of 18 G. .
- To this solution was added 2M sodium acetate lm1, water-saturated phenol 10ml and black form-isoamyl alcohol
- RNA was eluted with 4 ml of elution buffer.
- the eluate was heated at 65 ° C for 2 minutes, then cooled, adjusted to 0.5 M NaCl 1, added to the re-equilibration column again, and the elution procedure was performed in the same manner. So The mRNA was recovered from the eluate by ethanol precipitation and washed with 75% ethanol.
- Density gradient fraction (one day; DGF-U) and centrifuge tube treated with getylbirokaone, two different concentrations of RNase-free sucrose solution (5% and 20% (w / v) sucrose) ), 0.1 mM NaCl, 10 mM Tris-HC1 (pH 7.5), lmMEDTA, 0.5% SDS, and make a sucrose gradient in a tube for Beckman SW41 Ti in a density gradient fractionator overnight. Leave at room temperature above to eliminate the discontinuity of the gradient.
- the mRNA was dissolved in a 200/1 TE solution (99% dimethyl sulfoxide, 10 mM Tris-HC1 (pH 7.5), lmMEDTA, 0.1% SDS) and treated at 37 ° C for 5 minutes.
- the non-specific association was dissociated by adding 400 1 of 5 mM Tris-HC1 (pH 7.5), lmMEDTA, and 0.5% SDS and heat-treating at 65 ° C. for 10 minutes. Then quench, place on a sucrose density gradient, Beckman SW4
- Centrifugation was performed at 20,000 rpm and 14 ° C. for 1 hour at 25 ° C. overnight. After centrifugation, 0.5 ml was fractionated from the tube by fractionation overnight and precipitated with ethanol. mRNA was washed at least three times with 75% ethanol.
- a part of the mRNA fractionated into 50 fractions was injected into oocytes of African frogs according to the method described later in 2.1.3, and was translated into protein.
- the culture supernatant containing this translation product is added to mouse bone marrow cells for use in the assay according to the method described in 2.2.2 described below, and cultured, and then the osteoclasts are differentiated by the TRAP staining method described in 2.3.1. Whether it was formed (ie, which mRNA fraction Whether a factor having osteoclast differentiation-promoting activity is contained therein). As a result, peaks of activity were present in the 27th and 32nd fractions.
- the 27th to 33rd peaks of the activity peak fraction were collected as the active fraction, and a cDNA library for this fraction was prepared by a modified method of the Gubler & Hoffman method (Gene, 25, p263 (1983)). That is, a first strand was synthesized from M-MuLV reverse transcriptase using an oligo dT primer having an XhoI site based on 2 g of the mRNA of the active fraction. Subsequently, a second strand was synthesized with DNA Polymerase I, and ligated with EcoRI adapter 1 and XhoI digested. Thereafter, the adapter and the primer were removed by gel filtration (Sephacryl Spin Column; Pharmacia). The above cDNA synthesis step was performed using Stratagene's ZAP cDNA synthesis kit, and the reverse transcriptase was performed using BRL's Superscript II.
- the cDNA library prepared in 1. Were divided into a total of 63 pools, and the cDNA of each pool was injected into oocytes of Africa megafrog by the method described in 2.1.2 to 2.1.3 described below, and translated into protein. .
- the culture supernatant containing this translation product was added to mouse bone marrow cells for Atsey according to the method described in 2.2.2 described below, and subjected to each of the Assay methods described below, and a pool determined to be positive was selected.
- 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. I got a clone.
- the osteoclast differentiation promoting activity was determined by the TRAP staining method described in 2.3.1 described later, and three positive pools were selected from 63 pools. From the second screening onwards, positive reactions were observed in both the two types of osteoclast differentiation-promoting activity measurement methods described later in 2.3.1, TRAP staining method, and 2.3.2, pit formation method using ivory. The pools shown were selected. First, each of the three pools described above was divided into 10 subpools (1000 clones Z pool) and subjected to each assay.
- the top 3 pools were selected in the order of the strength of the positive reaction, and these 3 pools were further divided into 10 subpools (200 clones / pool), and a third screening was performed.
- three positive pools were selected in the order of the strength of the positive reaction, which were divided into 10 subpools (24 clones / pool), and further subjected to a fourth screening.
- the top 2 pools were selected in the order of TRAP staining and intense it-forming activity, and the 2 pools were further divided into 36 individual clones and subjected to fifth screening.
- the top three clones were selected in descending order of TRAP staining and pit formation activity.
- One of the three clones was designated OPF8. The results of each of the OPF 8 tests are described in (Results) below. 2.1 Preparation of samples for Atsushi
- E. coli XL1-B1ue was infected with 1 ⁇ 10 4 pfu of lambda phage from each pool and spread on a 15-cm petri dish to form plaques.
- a plate lysate was prepared by adding 13 ml of SM buffer to this plate.
- DE52 DEAE cellulose; Petman
- This DNA was extracted once each with phenol and phenol-chloroform (1: 1), and recovered by ethanol precipitation to obtain phage DNA.
- the prepared DNA was digested with a restriction enzyme NotI, and 1/50 of the DNA was quantified by 1% agarose electrophoresis.
- cDNA was synthesized according to the mRNAcapping kit (Stratagene). This was subjected to phenol-chloroform treatment and ethanol precipitation to recover cRNA, and 1/10 of the cRNA was quantified by 1% agarose gel electrophoresis. Then, it was adjusted to a concentration of ⁇ 1 to obtain cRNA for microinjection.
- the oocytes were dissected out one at a time and live cells without stage V or VI damage were selected. Using a 10-1 digital microdispenser (Drummond), these oocytes were injected with 5 On 1 cRNA per oocyte per capillaries. After that, cells that had died or were damaged were removed, and cultured at 20 ° C. for 3 days in MBS containing 2% FCS. The culture supernatant was centrifuged and further passed through a 0.22 m filter to remove the residue and at the same time to remove bacteria. The supernatant was used as an Atsushi sample.
- the femur and tibia of a 6-12 week old mouse (C3H / He J; CLEA Japan) were aseptically removed, their epiphyses were cut off, and lml syringes with a 26G needle were applied once from both ends.
- MEM medium (10% fetal calf serum, 1
- Bone marrow cells were extruded with 00 units / ml 1-nicillin G and 100 ⁇ g / ml 1 streptomycin), pipetted well, waited until the bone residue precipitated, and the supernatant was collected. It was further washed once or twice with fresh medium to prepare bone marrow cells for Atsushi.
- Trap (tartrate-resistant acid phosphatase), a marker enzyme of osteoclasts, was stained with a substrate. That is, after fixation of the cultured bone marrow cells of 2.2.2 with acetone-citrate buffer, the substrate (Naphthol AS-MXphosphate) and the dye (Fastredviolet LB salt) are reacted at 37 ° C for 1 hour in the presence of tartaric acid. Thus, it was stained (Endocrinology, 122, ⁇ 1373, (1988)). (Result)
- OPF8 is a known osteoclast differentiation factor IL-11? Bone marrow cells were treated with (50 ⁇ g / ml) or LIF (25 U / ml) and compared to the TRAP staining of a positive control in which osteoclasts were differentiated and formed.
- a dentin slice 6 mm in diameter and 1 mm thick from ivory was prepared and
- each slice was transferred to the bottom of a 96-well plate, and then osteoclasts were induced from bone marrow cells according to the method described in 2.2.2. After one or two weeks, the osteoclasts on the dentin slice are stained using the TRAP staining method described in 2.3.1, treated with 0.25% trypsin and 0.02% EDTA overnight, and The cells were scraped off with a silicon scraper. The pits (resorption pits) on the dentin slices are observed under a microscope, and the number of the pits or the number of meshes per pit is measured. Activity).
- Cells differentiated from bone marrow cells by 8 were osteoclasts, and OPF8 was determined to be a factor having osteoclast differentiation promoting activity.
- ZAP Express vectors can be subcloned by ex vivo insertion of the insert DNA into pBK-CMV.
- XL1-BlueMRF 'Escherichia coli was infected with ZAP Express phage and ExAssist helper phage to produce pBK-CMV phagemid, killed by heat treatment of the original E. coli, and newly infected with XL0LR E. coli. .
- the medium was added thereto, cultured for 45 minutes, plated on an LB plate, and cultured.
- the nucleotide sequence of the OPF cDNA 8 obtained in Example 4 was determined by Sang This was done by the dideoxy method proposed by Er.
- RNA from various tissues such as mouse bone, bone marrow cells and muscle was prepared according to 1.1.1. Then, 10-20 ⁇ g of total RNA was dissolved in sample buffer, denatured by heating at 65 ° C for 5 minutes, and subjected to 6% formaldehyde agarose electrophoresis. Then, a nylon filter (Hybond N +, Amersham) ). In addition, MTN blot membrane and human RNA mass spectroscopy (Clontech), in which poly-A + RNA from various tissues had already been blocked, were used. As the probe, the full-length OPF ⁇ 8 cDNA (about 1.5 kbp) was labeled with 32 P and used.
- the above probe was applied to RNA fixed at 50% (v / v) formaldehyde Z5 XSSC / 5 X Denhardt / 1% (w / v) SDS / 0.01% (w / v) denatured salmon sperm DNA. Hybridized at 42 ° C. and washed in 2 ⁇ SSC / 0.1% SDS, 50 ° C., then in 0.1% SSC / 0.1% SDS, 50 ° C. After draining, 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.
- FIG. 1 shows the results of Northern plot analysis of each mouse tissue (10 total RNAs).
- OPF 8 band (arrow) is slightly in all tissues However, it was expressed.
- BW5147 cells, a cloning source of OPF8, and osteoblast-like ST2 cells showed higher expression.
- FIG. 1B shows the results of Northern blot analysis of mouse and rat bone tissue (20 / g mRNA).
- OPF mRNA 8 mRNA is expressed in bone tissues of normal mice, collagen arthritis (CIA) mice, osteopetrosis (op / op) mice, and normal rats and ovariectomized (ovx) rats.
- Figure 1 shows that OPF8 is widely expressed in bone tissue.
- Figure 2 shows that OPF8 was expressed in mouse 7-day to 17-day embryos (2 / g mRNA). The result of Xan blot analysis is shown. As a result, the expression level was reduced in the 11-day embryo, but was high in other days.
- Figure 3 shows the results of RNA dot plots for human tissues (100-500 ng mRNA).
- human OPF mRNA was strongly expressed in kidney and weakly expressed in ovary, liver and lung. From the results of FIG. 3 and FIG. 1, it is preferable to use a cDNA library derived from a kidney or a cDNA library derived from a bone tissue to clone the human OPF8 gene.
- Example 7
- RNA From total RNA (l ⁇ g) prepared from various cells and tissues according to 1.1.1: First, according to RT-PCR kit (manufactured by PERKIN ELMER), first synthesize double-stranded DNA and use it as template. To perform a PCR reaction. The primer sequence for amplification of this gene is 5, Primer (5'-
- TGTGGATACGCCTACTATCA-3 ' was synthesized.
- the DNA size amplified with these primer combinations is 252 base pairs.
- G 3 PDH (glyceraldehyde-3-phosphate dehydrogenase) Imaichi (5 'primer is (5' — TGAAGGT CGGTGTGAA
- the reaction composition and the like are determined according to standard procedures using a DNA cycler under the conditions of heat denaturation at 94 ° C for 1 minute, annealing at 60 ° C for 1 minute, and chain elongation at 72 ° C for 2 minutes. The reaction was performed in cycles. The 1Z10 amount of the reaction mixture was electrophoresed on a 1% agarose gel, and a band was confirmed.
- FIG. 4 shows the results of using all MAI ⁇ g of each tissue as a template.
- OPF8 was expressed in four types of osteoblasts and osteoblast-like cells (in FIG. 4, the arrow in the photograph labeled OPFu8). These results were not inconsistent with the results of the Northern blot analysis in FIG. 1, confirming that OPF8 was widely expressed in bone tissue and osteoblasts.
- control G3PDH RT-PC the same RNA, control G3PDH RT-PC
- R was also performed, and this was used as an index of the degree of degradation of each RNA.
- the OPF ⁇ 8 cDNA obtained in Example 2 was subcloned in pBK-CMV vector and transformed into E. coli JM109 strain. Alkali—S
- DNA was prepared by the DS method and purified by two ultracentrifugation methods.
- the purified DNA was transfected into COS-7 cells using LIPOFECTAMINE (GIBCO BRL). Thereafter, the cells were cultured in a serum-free medium for 5 days, and the culture supernatant was collected to obtain a 0 P F 8 culture supernatant sample.
- the activity of the above culture supernatant preparation was determined by the TRAP staining property described in 2.3.1 and 2.3.3. Pit formation activity using two dentin slices was measured by two different identification methods. As a result, bone marrow cells cultured on dentin slices in the presence of the culture supernatant preparation were stained red-brown and showed TRAP positive. Next, as a result of physically scraping the TRAP-positive cells on the ivory slice, pits were formed although TRAP-positive cell residues remained as shown in Fig. 5 (top). As shown in (bottom), when a similar experiment was performed using the culture supernatant of cells into which only one vector had been introduced, neither TRAP-positive cells nor pits were formed.
- OPF8 having osteoclast differentiation promoting activity a protein similar to OPF8, a peptide fragment of these proteins, an antibody, a medicament containing these proteins as an active ingredient, or encoding these proteins Gene, an expression vector of 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 related to the introduction and deletion of the gene, The use of the protein in screening for an inhibitor of osteoclast differentiation promoting activity is provided.
- Sequence type nucleic acid
- T OST ⁇ 1 on s ⁇ is ⁇ dsy an dsy ⁇ ⁇ 9 ⁇ ⁇ ⁇ ⁇ J3 ⁇ 4 ⁇ 3 ⁇ 4 ⁇ 8 II J9S ⁇ g OJJ
- GCT CCC AGT TCT GAG GAG AGG AAT ATC ATA CAT GAA CTG TTC CTT ACC 877 Ala Pro Ser Ser Glu Glu Glu Arg Asn lie lie His Glu Leu Phe Leu Thr
- Gly Arg lie Leu Glu Asp Leu Asp Ser Leu Gly Val Leu Val Cys Tyr
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Abstract
OPF-U-8 having the activity of promoting osteclast differentiation and proteins similar thereto; peptide fragments of these proteins; the antibodies thereof; drugs containing as the active ingredient these proteins; genes encoding these proteins; expression vectors of these genes; transformants having these expression vectors transferred thereinto; a process for producing recombinant proteins with the use of these transformants; transgenic animals relating to the transfer or defect of these genes; and use of the above proteins in screening osteclast differentiation promoting activity inhibitors. Because of having the activity of promoting osteoclast differentiation, the above novel protein OPF-U-8 is usable as an osteoclast differentiation promoter. Further, OPF-U-8 is useful as a material in screening osteoclast differentiation promoting activity inhibitors. The osteoclast differentiation promoting activity inhibitors thus screened are also usable as drugs.
Description
明 細 新規破骨細胞分化促進因子及びその遺伝子 技術分野 Description New osteoclast differentiation promoting factor and its gene
本発明は、 ◦ P Fう 8と命名された新規破骨細胞分化促進因子及びその 遺伝子に関する。 さらに詳しくは、 破骨細胞分化促進活性を有する O P F う 8及び該 O P Fう 8の類似タンパク質、 またはこれらのタンパク質のぺ プチド断片、 抗体、 これらのタンパク質を有効成分とする医薬、 あるいは これらのタンパク質をコードする遺伝子、 該遺伝子の発現ベクター、 該発 現ベクターを導入した形質転換体、 該形質転換体を用いる組換えタンパク 質の生産方法、 前記遺伝子の導入及び欠損に係るトランスジエニック動物、 さらには前記タンパク質の、 破骨細胞分化促進活性阻害剤のスクリーニン グにおける用途に関する。 TECHNICAL FIELD The present invention relates to a novel osteoclast differentiation promoting factor designated as PF8 and its gene. More specifically, OPF-8 having an osteoclast differentiation promoting activity, a protein similar to OPF-8, a peptide fragment of these proteins, an antibody, a medicament containing these proteins as active ingredients, or these proteins A gene to be encoded, 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 related to the introduction and deletion of the gene, The present invention relates to use of the protein in screening for an osteoclast differentiation promoting activity inhibitor.
背景技術 Background art
骨組織は、 骨芽細胞による骨形成と破骨細胞による骨吸収とによって基 本的形状を変えることなく、 新生骨に置換される。 この過程は骨リモデリ ング (骨再造形) と呼ばれ、 生体の機能維持に重要な役割を果たしている 骨形成の中心的な役割を果たしている骨芽細胞は、 間葉系由来の未分化 細胞から分化し、 骨基質を形成する。 Bone tissue is replaced by new bone without changing its basic shape due to bone formation by osteoblasts and bone resorption by osteoclasts. This process is called bone remodeling (bone remodeling). Osteoblasts, which play an important role in maintaining the function of living organisms, play a central role in bone formation, and are derived from mesenchymal-derived undifferentiated cells. Differentiate and form bone matrix.
一方、 骨吸収の中心的な役割を果たしている破骨細胞は骨髄細胞に由来 し、 骨芽細胞との細胞間接触を介して分化することにより形成される。 成 熟破骨細胞は多核で増殖性の乏しい巨細胞であり、 カルシトニン受容体や 酒石酸抵抗性酸性ホスファタ一ゼ (T R A P ) を発現する。 また、 骨や象 牙質等の石灰化組織を吸収する活性を有している。
- On the other hand, osteoclasts, which play a central role in bone resorption, are derived from bone marrow cells and are formed by differentiation through cell-to-cell contact with osteoblasts. Mature osteoclasts are multinucleated, poorly proliferating giant cells that express the calcitonin receptor and tartrate-resistant acid phosphatase (TRAP). It also has the activity of absorbing calcified tissues such as bone and dentin. -
これら破骨細胞と骨芽細胞による骨リモデリングのバランスは、 破骨細 胞による骨吸収と骨芽細胞による骨形成の繰り返しにより平衡に保たれて いる。 しかし、 ひとたびこの平衡バランスが崩れると骨組織は異常をきた し、 種々の疾患を呈することになる。 The balance between bone remodeling by osteoclasts and osteoblasts is balanced by repeated bone resorption by osteoclasts and bone formation by osteoblasts. However, once this imbalance is lost, bone tissue becomes abnormal and presents various diseases.
骨リモデリングの異常により起こる代表的な疾患として、 骨粗鬆症が知 られている。 骨粗鬆症は、 骨リモデリングの代謝回転速度からみて、 閉経 早期あるいは甲状腺機能亢進症などにおいて認められる高回転型骨粗鬆症 と、 老人性、 ステロイ ド性、 糖尿病性骨粗鬆症において認められる低回転 型骨粗鬆症に分類されている。 それ以外では、 例えば癌の骨転移に伴う高 カルシウム血症や骨破壊、 あるいは慢性関節リウマチに伴う骨破壊等の疾 患が、 上記骨リモデリングの異常により起こる疾患として知られている。 これら癌の骨転移や慢性関節リゥマチに伴う骨破壊等は、 破骨細胞の機能 が異常に亢進した結果生じる疾患である。 Osteoporosis is known as a typical disease caused by abnormal bone remodeling. Osteoporosis is classified into high-turn osteoporosis, which is observed in early menopause or hyperthyroidism, and low-turn osteoporosis, which is observed in senile, steroid, and diabetic osteoporosis, based on the turnover rate of bone remodeling. ing. Other than these, diseases such as hypercalcemia and bone destruction associated with bone metastasis of cancer or bone destruction associated with rheumatoid arthritis are known as diseases caused by abnormal bone remodeling. Bone metastasis of these cancers and bone destruction associated with rheumatoid arthritis are diseases resulting from abnormally enhanced functions of osteoclasts.
以上のような疾患に対する有効な治療薬の探索が種々なされているが、 未だ決定的な治療薬は見出されていない。 アブローチの一つとして、 骨髄 細胞から破骨細胞への分化を促進する因子、 あるいはその阻害剤が、 上記 疾患に対する治療薬となることが考えられる。 すなわち破骨細胞の分化促 進因子は、 上述の如き低回転型骨粗鬆症においてみられる骨リモデリング の低回転を正常に戻すことが考えられるため (J. Bone Miner Res. , 7, p65 ( 1992) ) 、 この低回転型骨粗鬆症の有効な治療薬となることが考えられ る。 一方、 破骨細胞分化促進因子の阻害剤は、 上述の如き高回転型骨粗鬆 症における骨リモデリングの高回転を正常に戻すことが考えられるため (Am.J.Med. Sci . , 305( 1 ) , p40( 1993 )及び Mebio.,11(2 ), p24 ( 1994) )、 この高回転型骨粗鬆症の有効な治療薬となることが考えられる。 また、 前 記の如き破骨細胞の機能の亢進に伴う癌患者の骨破壊や、 慢性間接リウマ
- There have been various searches for effective therapeutic agents for the above diseases, but no definitive therapeutic agent has yet been found. As one of the approaches, a factor that promotes the differentiation of bone marrow cells into osteoclasts or an inhibitor thereof may be a therapeutic agent for the above-mentioned diseases. In other words, the factor that promotes osteoclast differentiation may restore the low turnover of bone remodeling seen in low turnover osteoporosis as described above (J. Bone Miner Res., 7, p65 (1992)). ) However, it is considered that this will be an effective therapeutic agent for low-rotation osteoporosis. On the other hand, an inhibitor of an osteoclast differentiation promoting factor is considered to restore the normal rotation of bone remodeling in high-rotation osteoporosis as described above (Am.J.Med.Sci., 305). (1), p40 (1993) and Mebio., 11 (2), p24 (1994)), are considered to be effective treatments for this high-speed osteoporosis. In addition, bone destruction of cancer patients due to the enhancement of osteoclast function as described above, -
チ患者の骨破壊等にも、 この破骨細胞分化促進因子の阻害剤が有効に働く ことが考えられる。 It is conceivable that the inhibitor of osteoclast differentiation promoting factor may also be effective for bone destruction in patients with bone damage.
破骨細胞分化促進因子は骨吸収因子とも呼ばれ、 これまでに活性型のビ 夕ミン D 3 、副甲状腺ホルモン (P T H ) 、 インターロイキン 1、 プロス 夕グランデイン等が知られている。 しかしこれらの因子は種々の問題点が あり、 未だ治療薬とはなっておらず、 新たな破骨細胞分化促進因子の出現 が望まれている。 The osteoclast differentiation promoting factor is also called a bone resorbing factor, and active forms of biminmin D 3, parathyroid hormone (PTH), interleukin 1, and prosthetic grandein have been known so far. However, these factors have various problems, and have not yet become therapeutic agents, and the emergence of new osteoclast differentiation promoting factors is desired.
発明の開示 Disclosure of the invention
本発明が解決しょうとする課題は、 破骨細胞分化促進活性を有する新規 なタンパク質 O P Fう 8及びその遺伝子を提供することにある。 さらに詳 しくは、 破骨細胞分化促進活性を有する O P Fう 8及び該 O P Fう 8の類 似タンパク質、 またはこれらのタンパク質のペプチド断片、 抗体、 これら のタンパク質を有効成分とする医薬、 あるいはこれらのタンパク質をコー ドする遺伝子、 該遺伝子の発現ベクター、 該発現ベクターを導入した形質 転換体、 該形質転換体を用いる組換えタンパク質の生産方法、 前記遺伝子 の導入及び欠損に係るトランスジヱニック動物、 さらには前記タンパク質 の、 破骨細胞分化促進活性阻害剤のスクリーニングにおける用途を提供す しとに ¾)る。 An object of the present invention is to provide a novel protein OPF8 having an activity to promote osteoclast differentiation and its gene. More specifically, OPF8 having an osteoclast differentiation promoting activity, a similar protein of OPF8, a peptide fragment of these proteins, an antibody, a medicament containing these proteins as an active ingredient, or these proteins A gene encoding the same, 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 related to the introduction and deletion of the gene, The present invention provides a use of the protein in screening for an inhibitor of osteoclast differentiation promoting activity.
本発明者らは、 骨転移細胞である B W 5 1 4 7細胞から発現クロ一ニン グ法により破骨細胞分化促進因子を探索したところ、 破骨細胞のマーカー 酵素である 「T R A P染色性」 及び 「象牙の分解活性」 という、 公知の 2 つの特性を有する 「破骨細胞」 へと骨髄細胞を分化誘導する因子を発見す ることに成功した。 我々は、 この破骨細胞分化促進活性を有するタンパク 性因子を O P Fう 8と命名し、 さらに研究を重ねて本発明を完成するに至 つた。
即ち本発明の要旨は、 . The present inventors searched for an osteoclast differentiation promoting factor from the BW514 cells, which are bone metastatic cells, by the expression cloning method, and found that `` TRAP staining '', a marker enzyme for osteoclasts, and We succeeded in discovering a factor that induces bone marrow cells to differentiate into osteoclasts, which have two well-known properties, “ivory degradation activity”. We named this proteinaceous factor having osteoclast differentiation promoting activity OPF8, and completed further studies to complete the present invention. That is, the gist of the present invention is:
(1) 以下の (a) 又は (b) のタンパク質をコードする DNA、 (1) DNA encoding the following protein (a) or (b),
(a)配列番号: 2に記載のアミノ酸配列からなるタンパク質 (a) a protein consisting of the amino acid sequence of SEQ ID NO: 2
(b)配列番号: 2に記載のアミノ酸配列のうち 1若しくは複数のァミノ 酸が欠失、 置換及び/又は付加されたアミノ酸配列からなり、 かつ破骨細 胞分化促進活性を有するタンパク質、 (b) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted and / or added from the amino acid sequence of SEQ ID NO: 2, and which has an activity of promoting osteoclast differentiation;
(2) 配列番号: 1に記載の塩基配列からなる DNA、 又はその DNA とストリンジヱン卜な条件下でハイブリダィズし、 かつ破骨細胞分化促進 活性を有するタンパク質をコ一ドする DNA、 (2) DNA comprising the nucleotide sequence of SEQ ID NO: 1, or a DNA which hybridizes with the DNA under stringent conditions and encodes a protein having osteoclast differentiation promoting activity;
(3) 前記 (1) 又は (2) 記載の DNAがコードするタンパク質、 (3) a protein encoded by the DNA according to (1) or (2),
( 4 ) 配列番号: 2に記載のアミノ酸配列からなる、 前記 ( 3 ) 記載の タンパク質、 (4) the protein of (3), which comprises the amino acid sequence of SEQ ID NO: 2;
(5) 前記 (1) 又は (2) 記載の DNAを含有する発現べクタ一、 (5) An expression vector containing the DNA according to (1) or (2),
(6) 前記 (5)記載の発現べクタ一によって形質転換された形質転換 体、 (6) a transformant transformed by the expression vector according to (5),
(7) 前記 (6) 記載の形質転換体を、 前記 (5)記載の発現ベクター の発現可能な条件下で培養することを特徴とする、 組換えタンパク質の生 産方法、 (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).
(8) 前記 (3) 又は (4) 記載のタンパク質を有効成分として含有す る医薬、 (8) a medicine containing the protein according to (3) or (4) as an active ingredient;
(9) 前記 (3) 又は (4) 記載のタンパク質の、 少なくとも 6ァミノ 酸以上の部分よりなるぺプチド断片、 (9) a peptide fragment of the protein according to the above (3) or (4), comprising a portion of at least 6 amino acids or more;
(10) 前記 (3) 又は (4)記載のタンパク質、 あるいは前記 (9) 記載のペプチド断片の、 いずれかに対する抗体、 (10) an antibody against any one of the protein according to (3) or (4) or the peptide fragment according to (9),
(11) 前記 (3) 又は (4) 記載のタンパク質を用いることを特徴と
する、 破骨細胞分化促進活性阻害剤のスクリ一ニング方法、 (11) The protein according to (3) or (4) is used. A method for screening for an osteoclast differentiation promoting activity inhibitor,
(12) 前記 (11)記載のスクリーニング方法により得られる、 破骨 細胞分化促進活性阻害剤、 (12) an osteoclast differentiation promoting activity inhibitor obtained by the screening method according to (11),
(13) 前記 ( 9 )記載のベプチド断片又は前記 (10)記載の抗体か らなる、 前記 (12)記載の破骨細胞分化促進活性阻害剤、 並びに (13) The osteoclast differentiation promoting activity inhibitor according to (12), comprising the peptide fragment according to (9) or the antibody according to (10); and
( 14) 前記 (1) 又は (2)記載の DN Aを人為的に染色体中に導入 するか、 あるいはいずれかを染色体中から欠損させたトランスジエニック 動物、 に関する。 (14) A transgenic animal in which the DNA of the above (1) or (2) is artificially introduced into a chromosome, or any of which is deleted from the chromosome.
本発明において DN Aとは、 OPFう 8の DNA、 あるいは該 OP Fう 8 DNAに類似の DNAであって破骨細胞分化促進活性を有するタンパク 質をコードする DNAであれば特に限定されないが、 例えば、 配列番号: 1に記載の塩基配列からなる D N A、 又は配列番号: 2に記載のァミノ酸 配列からなるタンパク質をコードする DN Aが挙げられる。 さらに、 配列 番号: 2に記載のアミノ酸配列のうち 1若しくは複数のァミノ酸が欠失、 置換及び/又は付加されたァミノ酸配列からなるタンパク質をコードする DNA、 あるいは配列番号: 1に記載の塩基配列からなる DNAとストリ ンジェン卜な条件下でハイブリダィズする DN Aも、 破骨細胞分化促進活 性を有するタンパク質をコードする限り、 本発明の DNAに含まれる。 以 下、 これらの DNAにっき順次説明する。 In the present invention, the DNA is not particularly limited as long as it is DNA of OPF8 or DNA similar to the OPF8 DNA and encoding a protein having osteoclast differentiation promoting activity. For example, DNA encoding the DNA comprising the nucleotide sequence of SEQ ID NO: 1 or the DNA encoding the protein comprising the amino acid sequence of SEQ ID NO: 2 is exemplified. Furthermore, DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted and / or added in the amino acid sequence described in SEQ ID NO: 2, or the base described in SEQ ID NO: 1 DNAs that hybridize under stringent conditions to DNAs consisting of sequences are also included in the DNA of the present invention as long as they encode proteins having osteoclast differentiation promoting activity. Hereinafter, these DNAs will be sequentially described.
1) OPFう 8をコードする DNA 1) DNA encoding OPF8
上記 DN Aのうち、 「配列番号: 1に記載の塩基配列からなる DNA、 又は配列番号: 2に記載のァミノ酸配列からなるタンパク質をコードする DNA」 とは、 本発明の OPFう 8をコードする DNAである。 該 DNA は、 例えば以下に示す 「発現クローニング法」 により得ることができる。 すなわち、 まず破骨細胞分化促進因子を産生する細胞として骨転移細胞
が考えられるため、 この骨転移細胞から全 RN Aを調製し、 続いて全 RN Aから mRNAを調製する。 ここで使用する骨転移細胞としては、 例えば マウス BW5147細胞株が挙げられる。 全 RNAは、 AGPC法( acid guamdium thiocyanate- phenol - chloroform method ; Tuji, Nakamura: 実験医学 9, p99 (1991)) 等の常法により調製することができる。 また、 mRNAはオリゴ d Tセルロースカラムを用い、 例えば Molecular Cloning 2nd Edt. Cold Spring Harbor Laboratory Press(1989)等に記 載の方法により調製することができる。 Among the above DNAs, “a DNA comprising the nucleotide sequence shown in SEQ ID NO: 1 or a DNA encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2” refers to the DNA encoding the OPF 8 of the present invention. DNA that The DNA can be obtained, for example, by the “expression cloning method” described below. That is, first, bone metastatic cells are used as cells that produce an osteoclast differentiation promoting factor. Therefore, total RNA is prepared from the bone metastatic cells, and then mRNA is prepared from the total RNA. The bone metastasis cell used here includes, for example, a mouse BW5147 cell line. Total RNA can be prepared by a conventional method such as the AGPC method (acid guamdium thiocyanate-phenol-chloroform method; Tuji, Nakamura: Experimental Medicine 9, p99 (1991)). The mRNA can be prepared using an oligo dT cellulose column, for example, by the method described in Molecular Cloning 2nd Ed. Cold Spring Harbor Laboratory Press (1989) and the like.
次に、 得られた mRNAをもとに cDNAライブラリ一を調製する。 c DNAライブラリ一は、 全 mRNAをもとに調製してもよいし、 全 mRN Next, a cDNA library is prepared based on the obtained mRNA. The cDNA library may be prepared based on total mRNA or
Aの一部からなる mRNA画分をもとに調製してもよい。 この mRN A画 分は、 たとえばショ糖密度勾配遠心法等の常法により分画された mR N A のうち、 破骨細胞分化促進活性の存在する画分およびその前後の画分のみ を集めることにより調製することもできる (破骨細胞分化促進活性の測定 法については後述する) 。 cDNAライブラリ一は、 たとえば It may be prepared based on the mRNA fraction comprising a part of A. This mRNA fraction is obtained by collecting only the fraction having the osteoclast differentiation promoting activity and the fractions before and after it, among the mRNA fractionated by a conventional method such as sucrose density gradient centrifugation. It can also be prepared (the method of measuring the osteoclast differentiation promoting activity will be described later). One example of a cDNA library is
Gubler&Hoffman法(Gene, 25, p263 (1983)) 等の常法により調製するこ とができる。 It can be prepared by a conventional method such as the Gubler & Hoffman method (Gene, 25, p263 (1983)).
次に、 上記 cDN Aライブラリーを複数のプールに分ける。 ここで、 1 プール当たりの cDNAクローンの数は、 cDNAライブラリ一を構成す るインディペンデントクローン数によって任意に決定することができるが、 たとえば 6X105個程度のィンディペンデントクロ一ンからなる cDN A ライブラリ一の場合、 lxlO4 クロ一ンを 1プールとする 60プール程度 に分けるのが、 後の操作上適当であろう。 Next, the above cDNA library is divided into a plurality of pools. Here, the number of cDNA clones per pool can be arbitrarily determined according to the number of independent clones constituting the cDNA library.For example, it is composed of about 6 × 10 5 independent clones. In the case of the cDNA library, it is appropriate to divide it into about 60 pools with lxlO 4 clones as one pool for the subsequent operation.
続いて各プールより常法により DNAを調製し、 この DN Aをテンプレ —トに用いて cRNAを調製する。 cRNAは、 たとえば市販の mRNA
cappingキット(Stratagene 社) を用いて容易に調製することができる。 次に、 各プール毎の c R NAをァフリカヅメガエル卵母細胞に注入する ことにより、 c R N Aからタンパク質への翻訳を行う。 卵母細胞への注入 は、 たとえば以下の方法により行うことができる。 すなわち、 体長 10cm程 度のメスのアフリカヅメガエルから卵母細胞の卵塊を取り出し、 顕微鏡下 で卵母細胞を一つずつ切り離し、 ステージ Vか V Iの傷のない生細胞を選 別し、 この卵母細胞にデジタルマイクロディスペンザ一等を用いてキヤピ ラリーより c R N Aを注入する。 卵母細胞 1個当たりの注入量は 50nl以下 が望ましい。 その後数日間培養した後、 培養上清を採取する。 c R N Aか ら翻訳された翻訳産物 (タンパク質) は培養上清中に存在するため、 この 培養上清をアツセィ用のサンプルとすることができる。 Subsequently, DNA is prepared from each pool by a conventional method, and cRNA is prepared using this DNA as a template. cRNA is, for example, commercially available mRNA It can be easily prepared using the capping kit (Stratagene). Next, translation of cRNA into protein is performed by injecting the cDNA of each pool into the oocytes of Africa frog. The injection into the oocyte can be performed, for example, by the following method. In other words, an egg mass of oocytes was removed from a female African Megafrog, about 10 cm in length, and the oocytes were cut off one by one under a microscope. Stage V or VI intact living cells were selected, and the eggs were selected. Inject cRNA from the capillary into the mother cells using a digital microdispenser or the like. The injection volume per oocyte is preferably 50 nl or less. After culturing for several days, collect the culture supernatant. Since the translation product (protein) translated from cRNA is present in the culture supernatant, this culture supernatant can be used as an assay sample.
次に、 この培養上清サンプルを用い、 以下のアツセィを行う。 Next, the following assay is performed using the culture supernatant sample.
まずアツセィ用の細胞であるが、 破骨細胞は、 骨髄系細胞から分化 -誘 導される細胞であると考えられているため、 アツセィ用細胞として、 この 骨髄系細胞を使用するのが望ましい。 具体的には、 たとえば 6〜; 12週令の マウスの大腿骨および脛骨の骨端を切り落とし、 両端から 1回づっ 2 6 G の針を付けたシリンジで 1 m 1のひ一 M E M培地で骨髄細胞を押し出した ものをビベッティングし、 沈殿した骨残渣を除いた上清部分を骨髄細胞と して使用することができる。 この調製された骨髄細胞を、 活性型ビタミン Dを含む培養液中に懸濁させ、 適当な濃度( 例えば 2 X 106 個細胞/ ml ) に調製してプレート( 例えば 96穴プレート) 上にまき、 そこへ上述のァフ リカヅメガエル培養上清サンプル (アツセィ用サンプル) を加え、 破骨細 胞の公知の同定法である T R A P染色法及び象牙を用いた吸収窩 (p i t ) 形成法に供し、 これら同定法のいずれにおいても陽性を示したプール を選別する。
ここで TRAP染色は、 例えば Endocrinology, 122, pl373(1988)等に 従い、 まず上記の如くアツセィ用サンプルで処理された骨髄細胞をァセト ンークェン酸緩衝液で固定した後、 酒石酸存在下で基質 (Naphthol AS- Mxphosphate)と色素 (Fastredviolet LB salt) を 37°Cで 1時間程度反 応させることにより、 検出することができる。 また pi t形成測定は、 例 えば、 あらかじめ直径 6匪、 1腿厚程度の象牙質スライスを 96穴ゥエルプレ 一トのゥエル底に敷いたものを用意し、 このゥエル上で、 上記の如きアツ セィ用サンプルによる骨髄細胞の処理を行い、 適当な期間の後、 象牙質ス ライス上の細胞を先の TRAP染色し、 0.25%トリブシン一 0.02%EDT Aで一晩処理し、 スライス上の細胞をシリコンスクレイパ一で削り取った 後、 象牙質スライス上の p it (吸収窩) を顕微鏡下で観察し、 その数を 数えることにより測定することができる。 First of all, cells for Atsushi. Since osteoclasts are considered to be cells induced and differentiated from myeloid cells, it is desirable to use these myeloid cells as Atsushi cells. Specifically, for example, the epiphyses of femurs and tibias of 6- to 12-week-old mice were cut off, and bone marrows were collected in a 1 ml Hi-MEM medium using a syringe with a 26 G needle once from each end. The extruded cells are beveled, and the supernatant portion excluding the precipitated bone debris 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 (eg, 2 × 10 6 cells / ml), and spread on a plate (eg, a 96-well plate). Then, the above-mentioned Africa Megafell culture supernatant sample (sample for Atsey) was added thereto, and subjected to TRAP staining, which is a known method for identifying osteoclasts, and a method for forming a resorption pit (pit) using ivory, and Pools that show positive results in any of the identification methods are selected. Here, TRAP staining is carried out, for example, according to Endocrinology, 122, pl373 (1988), etc. First, bone marrow cells treated with the sample for Atsusei as described above are fixed with acetonitrile buffer, and then the substrate (Naphtholol) is added in the presence of tartaric acid. The reaction can be detected by reacting AS-Mxphosphate) with a dye (Fastredviolet LB salt) at 37 ° C for about 1 hour. For the pit formation measurement, for example, a dentin slice of about 6 thighs and about 1 thigh in thickness was laid on the bottom of a 96-well el-plate, and the above-mentioned assay was performed on this well. After the bone marrow cells have been treated with the sample for the appropriate period, the cells on the dentin slice are stained with TRAP, treated with 0.25% trypsin-0.02% EDTA, overnight, and the cells on the slice are siliconized. After scraping with a scraper, the pits (absorbent pits) on the dentin slice can be observed under a microscope and counted.
上記同定法にて陽性を示したプールをさらにサブプールに分け、 同様の 操作を繰り返すことにより、 最終的に、 本発明の 0PFう 8をコードする クローンを得ることができる。 A pool that shows a positive result in the above identification method is further divided into subpools, and the same procedure is repeated to finally obtain a clone encoding the 0PF8 of the present invention.
以上の如き発現クローニング法によりクローニングされた 0PFう 8 c DNAは、 例えば Auto Read Sequencin キット( フアルマシア社製) を 用いてシークェンサ一により塩基配列を決定することもできるし、 ジデォ キシ法を用いる BcaBEST Sequencingキット(TAKARA社製) により、 RIを 使用して塩基配列を決定することもできる。 The nucleotide sequence of the 0PF DNA cloned by the expression cloning method as described above can be determined by a sequencer using, for example, an Auto Read Sequencin kit (Pharmacia), or BcaBEST Sequencing using the dideoxy method. With a kit (TAKARA), the nucleotide sequence can also be determined using RI.
なお、 このような 「発現クロ一ニング法」 によらなくとも、 本発明の 0 PFう 8 cDNAの塩基配列の公開に伴い、 該 cDNAの全部あるいは一 部をプローブあるいは P CRプライマーに用いて、 本発明の 0PFう 8 c DNAをクローニングすることができる。 該クロ一ニングは、 例えば Molecular Cloning 2nd Edt. Cold Spring Harbor Laboratory
Press(1989)等に従い、 当業者ならば容易に行うことができる。 Even without using such an "expression-cloning method", with the release of the nucleotide sequence of the 0PF8 cDNA of the present invention, all or a part of the cDNA can be used as a probe or PCR primer, The 0PF DNA of the present invention can be cloned. The cloning is performed, for example, by Molecular Cloning 2nd Edt. Cold Spring Harbor Laboratory According to Press (1989) and the like, it can be easily performed by those skilled in the art.
2) OPFう 8の改変タンパク質あるいはアレル変異体等をコードする D NA 2) DNA encoding a modified protein or allelic variant of OPF8
前記 DNAのうち、 「配列番号: 2に記載のアミノ酸配列のうち 1若し くは複数のアミノ酸が欠失、 置換及び/又は付加されたアミノ酸配列から なり、 かつ破骨細胞分化促進活性を有するタンパク質をコードする DN A」 とは、 人為的に作製したいわゆる改変タンパク質や、 生体内に存在す るアレル変異体等のうち、 破骨細胞分化促進活性を有するタンパク質をコ —ドする DNAを指す。 該タンパク質をコードする DNAは、 例えば部位 特異的変異誘発 ( Methods in Enzymology, 100, p468 (1983))や PCR 法 (Molecular Cloning 2nd Edt. 15章、 Cold Spring Harbor Laboratory Press( 1989) )等の手法により、 当業者ならば容易に作製することができる c なおここで、 欠失、 置換及び/又は付加されるアミノ酸残基の数は、 上記 部位特異的変異誘発等の周知の方法により欠失、 置換及び/又は付加でき る程度の数を指す。 Among the above DNAs, “consisting of an amino acid sequence in which one or more amino acids are deleted, substituted and / or added from the amino acid sequence described in SEQ ID NO: 2, and having osteoclast differentiation promoting activity "DNA encoding protein" refers to a DNA that encodes a protein having an osteoclast differentiation promoting activity among artificially produced so-called modified proteins and allelic variants present in vivo. . The DNA encoding the protein can be obtained by techniques such as site-directed mutagenesis (Methods in Enzymology, 100, p468 (1983)) and PCR (Molecular Cloning 2nd Edt. Chapter 15, Cold Spring Harbor Laboratory Press (1989)). deletion, the here c Note that can be easily fabricated by those skilled in the art, deletions, number of amino acid residues to be substituted and / or added, by a known method such as the site-directed mutagenesis, Refers to the number that can be replaced and / or added.
ここで 「破骨細胞分化促進活性」 は、 例えば前述のようにして調製した 骨髄細胞に対し、 本発明の改変タンパク質 (本発明の改変タンパク質をコ —ドする DN Aの発現産物) 等を作用させた後、 破骨細胞の公知の作用— すなわち TRAP染色性、 及び象牙の分解活性を調べることにより、 容易 に測定することができる。 具体的には上記 1) の項あるいは実施例の 2. Here, the “osteoclastic differentiation promoting activity” refers to, for example, the action of the modified protein of the present invention (the expression product of DNA encoding the modified protein of the present invention) on bone marrow cells prepared as described above. After that, it can be easily measured by examining the known action of osteoclasts—that is, TRAP staining and ivory degradation activity. Specifically, in item 1) above or 2.
3. 1〜2. 3. 2を参照されたい。 このような破骨細胞分化促進活性の 測定法に、 種々の改変タンパク質等を供することにより、 破骨細胞分化促 進活性を有する改変タンパク質等を、 容易に選別することができる。 Please refer to 3.1-2.3.2.2. By providing various modified proteins and the like to such a method for measuring osteoclast differentiation promoting activity, modified proteins having osteoclast differentiation promoting activity can be easily selected.
3) 0PFう 8DNAとストリンジェントな条件下でハイブリダィズする DNA
前記 DNAのうち、 「配列番号: 1に記載の塩基配列からなる DN Aと ストリンジェントな条件下でハイプリダイズし、 かつ破骨細胞分化促進活 性を有するタンパク質をコードする DNA」 とは、 すなわちヒト、 ラット 等の脊椎動物全ての OPFう 8 cDNAのような、 配列番号: 1に記載の 塩基配列からなるマウス OPFう 8DNAにストリンジェントな条件下で ハイプリダイズする D N Aを指す。 3) DNA that hybridizes under stringent conditions with 0PF Among the above-mentioned DNAs, “DNA that hybridizes with DNA consisting of the nucleotide sequence of SEQ ID NO: 1 under stringent conditions and encodes a protein having osteoclast differentiation promoting activity” This refers to DNA that hybridizes under stringent conditions to mouse OPF8 DNA consisting of the nucleotide sequence of SEQ ID NO: 1 such as OPF8 cDNA of all vertebrates such as human and rat.
ここで、 「ストリンジェン卜な条件下でハイブリダィズする DNA」 と は、 例えば標準的なハイブリダィゼ一シヨンの条件 (ホルムアミ ド濃度: 50%、 塩濃度: 5 X S S C、 温度: 42 °C) において配列番号: 1の D N Aとハイブリダィズする DN Aを指す。 Here, “DNA that hybridizes under stringent conditions” refers to, for example, the sequence number under standard hybridization conditions (formamide concentration: 50%, salt concentration: 5 XSSC, temperature: 42 ° C). : Refers to DNA that hybridizes with the DNA of 1.
これら DNAは、 例えば配列番号: 1に記載の DNAとのハイプリダイ ゼーシヨン等によりクローニングされるものであるが、 具体的な c DNA ライブラリ一の作製、 ハイブリダィゼ一シヨン、 ポジティブコロニーの選 択、 塩基配列の決定等の操作はいずれも公知であり、 例えば Molecular Cloning 2nd Edt. Cold Spring Harbor Laboratory Press(1989)等を参照 して容易に行うことができる。 cDNAライブラリ一としては、 例えばヒ トの骨組織、 あるいは腎臓由来の cDNAライプラリーが挙げられる。 ま たハイブリダィゼ一シヨンに用いるプローブとしては、 例えば配列番号: 1に記載の塩基配列を有する D N Aが挙げられる。 These DNAs are cloned by, for example, hybridization with the DNA of SEQ ID NO: 1, but specific cDNA library preparation, hybridization, selection of positive colonies, and base sequence All operations such as determination are known and can be easily performed with reference to, for example, Molecular Cloning 2nd Edt. Cold Spring Harbor Laboratory Press (1989). One example of a cDNA library is a cDNA library derived from human bone tissue or kidney. Examples of the probe used for hybridization include, for example, DNA having the nucleotide sequence of SEQ ID NO: 1.
本発明においてタンパク質とは、 上記した本発明の種々の DN Aがコ一 ドするタンパク質であり、 かつ破骨細胞分化促進活性を有するものである 具体例としては、 例えば配列番号: 2に記載のアミノ酸配列を有する本発 明のタンパク質、 OPFう 8が挙げられる。 In the present invention, the protein is a protein encoded by the above-described various DNAs of the present invention, and has an activity of promoting osteoclast differentiation. The protein of the present invention having an amino acid sequence, such as OPF8, is mentioned.
これらタンパク質は、 たとえば本発明の DNAを、 pBK— CMV等の 公知の発現ベクターに連結した後、 適当な宿主に導入して発現 ·生産する
ことにより、 得ることができる。 宿主としては、 原核性生物細胞または真 核性生物細胞のいずれでもよく、 例えば大腸菌株や動物細胞株は、 とくに 記載のない限り既に広く普及しており入手は容易である。 例えば動物細胞 宿主としては、 C O S— 1、 C O S— 7、 C H O細胞が挙げられる。 発現 プラスミ ドを用い適当な動物細胞宿主を形質転換するには、 LIPOFECTIN法 (Feigner P.L., et al, Proc. Natl . Acad. Sci. USA, 84, p7413 (1987) )等の公知の方法を用いればよい。 形質転換された細胞の培養上清 は、 適当な希釈によりそのまま種々のアツセィに使用され得る程度の発現 タンパク質を含んでいるため、 該培養上清を用いて破骨細胞分化促進活性 を測定することができる (活性測定法については前記参照) 。 培養上清中 に産生された上記発現タンパク質は、 亜鉛キレートァガロース、 コンカナ バリン Aァガロース、 セフアデックス G— 1 5 0等を用いる公知の方法に よって、 容易に精製することができる。 These proteins are expressed and produced by, for example, ligating the DNA of the present invention to a known expression vector such as pBK-CMV, and then introducing the protein into an appropriate host. By doing so, it can be obtained. The host may be a prokaryotic or 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, and CHO cells. To transform an appropriate animal cell host using the expression plasmid, a known method such as the LIPOFECTIN method (Feigner PL, et al, Proc. Natl. Acad. Sci. USA, 84, p7413 (1987)) is used. I just need. Since the culture supernatant of the transformed cells contains the expressed protein to the extent that it can be directly used in various assays by appropriate dilution, the osteoclast differentiation promoting activity should be measured using the culture supernatant. (See above for activity assay). The 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.
本発明のタンパク質はまた、 医薬の有効成分として使用することができ る。 即ち本発明のタンパク質を有効成分として含有する 「医薬」 は、 例え ば破骨細胞分化促進剤として使用することができる。 より具体的には、 「従来の技術」 の項にも記載したように、 老人性、 ステロイ ド性、 糖尿病 性骨粗鬆症において認められる低回転型骨粗鬆症等に対する有効な治療薬 となることができる。 The protein of the present invention can also be used as an active ingredient of a medicament. That is, a “medicine” containing the protein of the present invention as an active ingredient can be used, for example, as an osteoclast differentiation promoting agent. More specifically, as described in the section of “Prior Art”, it can be an effective therapeutic agent for low-rotation osteoporosis and the like observed in senile, steroid, and diabetic osteoporosis.
このような破骨細胞分化促進剤の患者への投与方法としては、 静脈注射 による投与が好ましいが、 経口投与、 坐薬としての投与、 皮下注射、 筋肉 注射、 局所注入、 腹腔内投与などが行える。 本発明の破骨細胞分化促進剤 は、 上記の投与方法に依存して、 種々の単位投与形態で投与することがで きる。 例えば静脈投与のためには、 本発明のタンパク質を、 医薬として許 容され得る担体、 好ましくは水性担体の中に溶解または懸濁させて用いる
ことができる。 水性担体としては、 例えば、 水、 緩衝化水、 0 . 4 %の生 理的食塩水などを使用することができる。 このようにして作製された水溶 液は、 そのまま包装するか、 あるいは凍結乾燥することができ、 凍結乾燥 した調製物は投与前に無菌の水溶液と組み合わせる。 As a method of administering such an osteoclast differentiation promoting agent to a patient, intravenous injection is preferable, but oral administration, administration as a suppository, subcutaneous injection, intramuscular injection, local injection, intraperitoneal administration and the like can be performed. The osteoclast differentiation promoting agent of the present invention can be administered in various unit dosage forms depending on the above-mentioned administration method. For example, for intravenous administration, the protein of the present invention is used by dissolving or suspending it in a pharmaceutically acceptable carrier, preferably an aqueous carrier. be able to. As the aqueous carrier, for example, water, buffered water, 0.4% physiological saline, and the like can be used. The aqueous solution thus prepared can be packaged as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous solution before administration.
以上の調製物は、 医薬として許容される補助剤、 例えば、 ph調節剤ある いは緩衝剤、 張度調節剤、 浸潤剤などを、 より具体的には、 例えば酢酸ナ トリウム、 乳酸ナトリウム、 塩化ナトリウム、 塩化カリウム、 塩化カルシ ゥム、 ソルビ夕ンモノラウレート、 トリエタノールアミンォレエ一トなど を含有することができる。 The above preparations may contain pharmaceutically acceptable auxiliaries, such as ph regulators or buffers, tonicity regulators, infiltrants, and more specifically, for example, sodium acetate, sodium lactate, chloride It can contain sodium, potassium chloride, calcium chloride, sorbine monolaurate, triethanolamine oleate and the like.
経口的投与のためには、 本発明のタンパク質を、 粉末、 錠剤、 ピル、 力 プセル剤及び糖剤の単位投与形態にして用いることができる。 また局所投 与のためには、 例えばェァゾ一ルを包含する単位投与形態をとることがで さる。 For oral administration, the proteins of the present invention can be used in unit dosage forms of powders, tablets, pills, capsules and sugars. For local administration, a unit dosage form containing, for example, azoles may be used.
皮下注射、 筋肉注射、 局所注入、 腹腔内投与のためには、 本発明のタン パク質を水性または油担体の中に溶解または懸濁させて用いることができ る。 あるいは、 コラーゲン等の生体親和性の材料を用いて、 徐放性製剤と して投与することもできる。 For subcutaneous injection, intramuscular injection, local injection, and intraperitoneal administration, the protein of the present invention can be used by dissolving or suspending it in an aqueous or oil carrier. Alternatively, it can be administered as a sustained-release preparation using a biocompatible material such as collagen.
以上のような破骨細胞分化促進剤の投与量は、 一日量 0.0001 〜100mg 程度を症状が改善されるまで投与することが可能である。 The dose of the above-mentioned osteoclast differentiation promoting agent can be administered at a daily dose of about 0.0001 to 100 mg until the symptoms are improved.
本発明においてペプチド断片とは、 本発明のタンパク質の有するァミノ 酸配列のうち、 少なくとも 6アミノ酸以上の部分よりなるぺプチド断片を 指す。 ここで、 「少なくとも 6アミノ酸以上」 との限定は、 安定な構造を とり得る最小のサイズが 6アミノ酸であることによるが、 好ましくは 8ァ ミノ酸以上の長さよりなるペプチドが、 より好ましくは 1 0〜2 0アミノ 酸程度の長さよりなるペプチドが挙げられる。 なお該ペプチドは、 1 0〜
2 0個程度の短いものであればベプチド合成装置により合成することがで きるし、 長いものであれば通常の遺伝子工学的手法により (たとえば制限 酵素処理等により) 調製された D N Aを、 動物細胞等に発現させることに より得ることができる。 なお、 このようにして作製されたペプチドを、 通 常の方法により修飾することも可能である。 In the present invention, the peptide fragment refers to a peptide fragment comprising at least 6 amino acids or more 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 preferably a peptide having a length of 8 amino acids or more, more preferably 1 amino acid or more. Peptides having a length of about 0 to 20 amino acids are exemplified. Note that the peptide is 10 to If it is as short as about 20 pieces, it can be synthesized with a peptide synthesizer. If it is long, DNA prepared by ordinary genetic engineering techniques (for example, by treatment with restriction enzymes) can be used for animal cells, etc. It can be obtained by expressing the protein in E. coli. It should be noted that the peptide thus produced can be modified by a usual method.
これらペプチド断片は、 後述のように医薬への応用が可能である他、 抗 体作製のためにも使用することができる。 These peptide fragments can be applied to pharmaceuticals as described below, and can also be used for producing antibodies.
本発明において抗体とは、 本発明のタンパク質又はそのべプチド断片の いずれかに対する抗体である。 該抗体は、 例えば新細胞工学実験プロトコ —ル p210 秀潤社(1993)に記載された方法を用いてゥサギ等を免疫するこ とにより、 容易に作製することができる。 また、 例えば分子生物学研究の ためのタンパク実験法 第 4章 羊土社(1994)に述べられている手法を用 いることで、 容易にモノクロ一ナル抗体を作製することができる。 さらに は、 特閧昭 62-296890により擬人化抗体とすることもできる。 該抗体の用 途としては、 ァフィ二ティークロマトグラフィー、 c D N Aライブラリ一 のスクリーニング、 診断薬 ·実験用試薬等が挙げられる。 さらには後述の ように、 医薬として使用することもできる。 In the present invention, an antibody is an antibody against any of the protein of the present invention or a peptide fragment thereof. The antibody can be easily prepared, for example, by immunizing rabbits and the like using the method described in New Cell Engineering Experimental Protocol p210 Shujunsha (1993). Monoclonal antibodies can be easily produced, for example, by using the method described in Protein Experimental Methods for Molecular Biology Research, Chapter 4, Yodosha (1994). Furthermore, it can be a humanized antibody according to Japanese Patent Application No. 62-296890. Examples of the use of the antibody include affinity chromatography, screening of a cDNA library, diagnostic agents and experimental reagents. Furthermore, as described later, it can be used as a medicine.
本発明のタンパク質はまた、 破骨細胞分化促進活性阻害剤のスクリー二 ングのためにも使用できる。 ここで、 「破骨細胞分化促進活性阻害剤」 と は、 本発明のタンパク質の有する破骨細胞分化促進活性を、 阻害する薬剤 を指す。 そして 「破骨細胞分化促進活性阻害剤のスクリーニング方法」 は、 先に述べた破骨細胞分化促進活性の測定系に、 被験物質である阻害剤候補 物質を添加することによって実施することができる。 The protein of the present invention can also be used for screening of an osteoclast differentiation promoting activity inhibitor. Here, the “inhibitor of osteoclast differentiation promoting activity” refers to an agent that inhibits the osteoclast differentiation promoting activity of the protein of the present invention. The “screening method for an osteoclast differentiation promoting activity” can be carried out by adding a candidate inhibitor, which is a test substance, to the above-described system for measuring osteoclast differentiation promoting activity.
例えば上記の破骨細胞分化促進活性測定法のうち、 象牙を用いた吸収窩 ( p i t ) 形成法により阻害剤のスクリーニングを行う際は、 まず前記の
如く象牙質スライス上で、 マウス骨髄細胞に対し、 本発明のタンパク質及 び阻害剤候補物質を添加、 作用させる。 その際、 阻害剤候補物質に阻害作 用があれば、 象牙質に吸収窩が形成されない。 この吸収窩の形成を指標に、 容易に阻害剤をスクリーニングすることができる。 For example, among the above-described methods for measuring osteoclast differentiation promoting activity, when screening for an inhibitor by the method of forming resorption pits (pits) using ivory, As described above, the protein of the present invention and the candidate inhibitor are allowed to act on mouse bone marrow cells on the dentin slice. At that time, if the inhibitor candidate substance has an inhibitory action, no resorption pit is formed in the dentin. Inhibitors can be easily screened using the formation of the resorption pits as an indicator.
本発明において破骨細胞分化促進活性阻害剤とは、 上記スクリーニング 方法により見出されるものであり、 前記したように、 本発明のタンパク質 の有する破骨細胞分化促進活性を阻害する薬剤を意味する。 このような破 骨細胞分化促進活性阻害剤の具体例としては、 例えば上記で作製された本 発明のぺプチド断片のうち阻害効果を有するもの、 あるいは本発明の抗体 のうち中和活性を有するものが挙げられる。 このような破骨細胞分化促進 活性阻害剤は、 「従来の技術」 にも記載したように、 閉経早期あるいは甲 状腺機能亢進症などにおいて認められる高回転型骨粗鬆症に対する治療薬、 また癌の骨転移に伴う高カルシゥム血症や骨破壊、 あるいは慢性関節リゥ マチに伴う骨破壊等の疾患に対する有効な治療薬となることが考えられる このような本発明の破骨細胞分化促進活性阻害剤の患者への投与方法及 び投与形態としては、 前記破骨細胞分化促進剤と同様の投与法及び投与形 態が考えられる。 また投与量は、 一日量 0.0001 〜100mg程度を症状が改 善されるまで投与することが可能である。 In the present invention, the osteoclast differentiation promoting activity inhibitor is found by the above screening method, and as described above, means an agent that inhibits the osteoclast differentiation promoting activity of the protein of the present invention. Specific examples of such an osteoclast differentiation promoting activity inhibitor include, for example, those having an inhibitory effect among the peptide fragments of the present invention prepared above and those having a neutralizing activity among the antibodies of the present invention. Is mentioned. As described in the “prior art”, such an inhibitor of osteoclast differentiation promoting activity is used as a therapeutic agent for high-rotation osteoporosis observed in early menopause or hyperthyroidism, etc. It is considered to be an effective therapeutic agent for diseases such as hypercalcemia and bone destruction associated with metastasis, or bone destruction associated with chronic rheumatoid arthritis. The administration method and administration form of the osteoclast differentiation promoting agent may be the same as the administration method and administration form of the osteoclast differentiation promoting agent. The daily dose can be about 0.0001 to 100 mg until the symptom is improved.
本発明においてトランスジエニック動物とは、 本発明の D N Aを人為的 に染色体中に導入した、 いわゆるトランスジヱニック動物や、 染色体中か ら該 D N Aを欠損させた、 いわゆるノックアウト動物を指す。 これらトラ ンスジエニック動物は、 例えば疾患モデルマウス: Molecular Medicine臨 時増刊号 中山書店 (1994) 等に基づき、 当業者ならば容易に作製するこ とができる。 該トランスジエニック動物は、 例えば骨粗鬆症等の医薬品開 発のためのモデル動物として、 あるいは該医薬品のスクリーニング用の動
物として、 非常に有用である。 . 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 has been deleted from the chromosome. These transgenic animals can be easily prepared by those skilled in the art based on, for example, disease model mice: Molecular Medicine Extra Edition, Nakayama Shoten (1994). The transgenic animal can be used, for example, as a model animal for the development of a drug such as osteoporosis, or as an animal for screening the drug. Very useful as an object. .
図面の簡単な説明 BRIEF DESCRIPTION OF THE FIGURES
図 1は、 マウス各組織における OPFう 8mRNAの発現分布を、 ノー ザンブロット解析により調べた結果の電気泳動写真である。 FIG. 1 is an electrophoretogram of the results of Northern blot analysis of the expression distribution of 8 mRNA of OPF in each mouse tissue.
図 2は、 マウス 7日胚から 17日胚までにおける OPFう 8mRNAの 発現分布を、 ノーザンブロット解析により調べた結果の電気泳動写真であ る。 FIG. 2 is an electrophoresis photograph showing the results of Northern blot analysis of the expression distribution of OPF mRNA from mouse 7-day embryo to mouse 17-day embryo.
図 3は、 ヒト型 OPFう 8 mRN Aの組織分布をヒト RNAのドットブ ロット解析により調べた結果の電気泳動写真である。 FIG. 3 is an electrophoretic photograph of the result of examining the tissue distribution of 8 mRNA of human OPF by dot blot analysis of human RNA.
図 4は、 骨芽細胞株 (骨芽細胞様細胞株) における OPFう 8mRNA の発現を、 RT— PC R解析により調べた結果の電気泳動写真である。 図 FIG. 4 is an electrophoretic photograph showing the results of RT-PCR analysis of the expression of 8 mRNA of OPF in an osteoblast cell line (osteoblast-like cell line). Figure
4中では、 OPFう 8を OPFu 8と表示してある。 In 4, the OPF 8 is indicated as OPFu 8.
図 5 (上) は、 OPFう 8 cDNAを COS細胞で発現させた培養上清 中の活性を、 TRAP染色及び p i t形成法により調べた結果の顕微鏡写 真である。 図 5 (下) は、 ベクタ一を導入して同様の実験を行った結果の 顕微鏡写真である。 Fig. 5 (upper) is a micrograph of the activity of the culture supernatant in which the OPF8 cDNA was expressed in COS cells, as determined by TRAP staining and the formation of pit. Figure 5 (bottom) is a photomicrograph of the results of a similar experiment with the introduction of Vector-1.
発明を実施するための最良の形態 BEST MODE FOR CARRYING OUT THE INVENTION
以下、 本発明の一例として実施例により本発明をさらに詳しく説明する が、 本発明はこれらの実施例によりなんら限定されるものではない。 実施例 1 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
マウス cDNAライブラリーの構築 Construction of mouse cDNA library
1. 1 マウス BW5147細胞からの mRNAの単離 1.1 Isolation of mRNA from mouse BW5147 cells
1. 1. 1 全 RNAの単離 1.1.1 Isolation of total RNA
マウス BW5147細胞株 (ATCC CRL 1588) 1 108 個を A G P C法 、 acid guanidium thiocyanate-phenol-chloroform
method;実験医学 9, 15, p99 (1991)) に従い全 R N Aを分離した。 即ち、 まず、 細胞のペレットに 4Mのグァニジンィソチオシァネ一ト 10mlを 加え、 直ちに激しく振とうし、 その溶液を 18 G二一ドル中を 5往復通過 させることで DNAを部分剪断した。 この溶液に 2M酢酸ナトリウム lm 1、 水飽和フエノール 10ml及びクロ口ホルム一イソアミルアルコールMouse BW5147 cell line (ATCC CRL 1588) 1 10 8 cells were subjected to AGPC method, acid guanidium thiocyanate-phenol-chloroform method; Experimental RNA 9, 15, p99 (1991)). First, 10 ml of 4 M guanidine isothionate was added to the cell pellet, immediately shaken vigorously, and the solution was partially sheared by passing the solution five times back and forth through 21 dollars of 18 G. . To this solution was added 2M sodium acetate lm1, water-saturated phenol 10ml and black form-isoamyl alcohol
(49 : 1) 2mlを順次加え、 添加ごとに混和した。 その後激しく振と うし、 15分間氷冷した後、 4°Cで 10, 000 g、 20分間遠心した。 その水層を分取し、 等量のイソプロパノールを加えて良く混和した。 これ を— 20°Cに 1時間置いた後、 4°Cで 10, 000 g、 10分間遠心した c 遠心後、 RN Aの沈殿に 4Mグァニジンチオシァネート 3mlを加えて完 全に溶解させ、 等量のイソプロパノールを加え、 — 20°Cで 1時間放置し た。 その後、 4°Cで 10, 000 g、 15分間遠心した後、 上清を捨て、 RNAの沈殿を 75%エタノールで洗浄することにより全 RNAを得た。 1. 1. 2 mRNAの単離 (49: 1) 2 ml were added sequentially and mixed after each addition. Then, the mixture was shaken vigorously, cooled on ice for 15 minutes, and 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 at --20 ° C for 1 hour, centrifuge at 10,000g for 10 minutes at 4 ° C. C After centrifugation, add 3 ml of 4M guanidine thiocyanate to the RNA precipitate to completely dissolve. Then, an equal volume of isopropanol was added, and the mixture was left at —20 ° C. for 1 hour. Then, 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
上記の方法を数回繰り返すことで全 RNAを 15mg集め、 溶離緩衝液 Repeat the above procedure several times to collect 15 mg of total RNA and use elution buffer.
(10mMトリス— HC1 (pH 7. 5) 、 ImMEDTA及び 0. 2% SDS) 5mlに溶解し、 65°Cで 2分間加熱し、 直ちに室温まで急冷し た。 5MNaClを 0. 55ml添加後、 その溶液を洗浄緩衝液 ( 0. 5 MNaCl、 10mMトリス— HC1 (pH 7. 5) 、 ImMEDTA及 び 0. 2%SDS) で平衡化したオリゴ dTセルロース (タイプ 7、 ファ ルマシアバイオテク) 0. 5gのカラムに添加し、 通過液をさらに 2回、 カラムに添加することにより mRNAをカラムに結合させた。 カラムを洗 浄緩衝液 15mlで洗浄した後、 結合した RNAを溶離緩衝液 4mlで溶 出した。 溶出液を 65°Cで 2分間加熱し、 その後冷却し、 0. 5MNaC 1に調節し、 再平衡カラムに再度添加して、 同様に溶出操作を行った。 そ
の溶出液からエタノール沈殿により mRNAを回収し、 75%エタノール で洗浄した。 (10 mM Tris-HCl (pH 7.5), ImMEDTA and 0.2% SDS) was dissolved in 5 ml, heated at 65 ° C for 2 minutes, and immediately cooled to room temperature. After adding 0.55 ml of 5M NaCl, the solution was washed with a washing buffer (0.5 M NaCl, 10 mM Tris-HC1 (pH 7.5), ImMEDTA, and 0.2% SDS) and oligo dT cellulose (type 7). , Pharmacia Biotech). The mRNA was bound to the column by adding 0.5 g of the column to the column and adding the permeate to the column twice more. After washing the column with 15 ml of wash buffer, the bound RNA was eluted with 4 ml of elution buffer. The eluate was heated at 65 ° C for 2 minutes, then cooled, adjusted to 0.5 M NaCl 1, added to the re-equilibration column again, and the elution procedure was performed in the same manner. So The mRNA was recovered from the eluate by ethanol precipitation and washed with 75% ethanol.
1. 1. 3 ショ糖密度勾配遠心による mRNAの分画 1.1.3 Fractionation of mRNA by sucrose gradient centrifugation
ジェチルビロカ一ボネィ 卜で処理した密度勾配フラクシヨネ一夕 (日 立; DGF— U) と遠心チューブ、 2種類の濃度の RNa s eフリーのシ ョ糖溶液 (5%と 20% (w/v) ショ糖) 、 0. lMNaCl、 10m Mトリスー HC1 (pH 7. 5) 、 lmMEDTA、 0. 5%SDSを用 意し、 Beckman SW41 T i用チューブに密度勾配フラクシヨネ 一夕でショ糖勾配を作り、 2時間以上室温に放置して、 勾配の不連続性を なくした。 次に、 mRNAを 200 /1の TE溶液 (99%ジメチルスル ホキサイ ド、 10mMトリス— HC1 (pH 7. 5) 、 lmMEDTA、 0. 1%SDS) に溶解し、 37 °Cで 5分間処理し、 400 1の 5mM トリスー HC1 (pH 7. 5) 、 lmMEDTA、 0. 5%SDSを加え て 65°Cで 10分間熱処理をすることにより、 その非特異的な会合を解離 させた。 その後急冷し、 ショ糖密度勾配にのせ、 Beckman SW4 Density gradient fraction (one day; DGF-U) and centrifuge tube treated with getylbirokaone, two different concentrations of RNase-free sucrose solution (5% and 20% (w / v) sucrose) ), 0.1 mM NaCl, 10 mM Tris-HC1 (pH 7.5), lmMEDTA, 0.5% SDS, and make a sucrose gradient in a tube for Beckman SW41 Ti in a density gradient fractionator overnight. Leave at room temperature above to eliminate the discontinuity of the gradient. Next, the mRNA was dissolved in a 200/1 TE solution (99% dimethyl sulfoxide, 10 mM Tris-HC1 (pH 7.5), lmMEDTA, 0.1% SDS) and treated at 37 ° C for 5 minutes. The non-specific association was dissociated by adding 400 1 of 5 mM Tris-HC1 (pH 7.5), lmMEDTA, and 0.5% SDS and heat-treating at 65 ° C. for 10 minutes. Then quench, place on a sucrose density gradient, Beckman SW4
1 T i口一夕一で 25°C、 20, 000 rpm、 14時間遠心を行った。 遠心後、 チューブより 0. 5mlずつ密度勾配フラクシヨネ一夕で分画し、 エタノール沈殿した。 mRNAの沈殿は最低 3回、 75%エタノールで洗 浄した。 Centrifugation was performed at 20,000 rpm and 14 ° C. for 1 hour at 25 ° C. overnight. After centrifugation, 0.5 ml was fractionated from the tube by fractionation overnight and precipitated with ethanol. mRNA was washed at least three times with 75% ethanol.
1. 1. 4 mRNAの同定 1.1.4 Identification of mRNA
50画分に分画した mRNAは、 その一部を後述の 2. 1. 3の方法に 従いァフリカツメガエルの卵母細胞に注入し、 タンパク質に翻訳させた。 この翻訳産物を含む培養上清を、 後述の 2. 2. 2の方法によりアツセィ 用のマウス骨髄細胞に添加して培養した後、 2. 3. 1の TRAP染色法 により、 破骨細胞が分化形成されたか否か (すなわち、 どの mRNA画分
中に破骨細胞分化促進活性を有する因子が含まれているか) を同定した。 その結果、 活性のピークは、 27番目の分画と 32番目の画分に存在して いた。 A part of the mRNA fractionated into 50 fractions was injected into oocytes of African frogs according to the method described later in 2.1.3, and was translated into protein. The culture supernatant containing this translation product is added to mouse bone marrow cells for use in the assay according to the method described in 2.2.2 described below, and cultured, and then the osteoclasts are differentiated by the TRAP staining method described in 2.3.1. Whether it was formed (ie, which mRNA fraction Whether a factor having osteoclast differentiation-promoting activity is contained therein). As a result, peaks of activity were present in the 27th and 32nd fractions.
1. 2 cDNAライブラリ一の作製 1.2 Preparation of cDNA library
活性のピーク画分の 27番目から 33番目を活性画分として集め、 この 画分に対する cDN Aライブラリ一を、 Gubl er&Hof fman法 (Gene, 25, p263 (1983) ) の変法にて調製した。 即ち、 この活性画分の mRNA2〃gをもとに、 Xho Iサイ トを持つオリゴ d Tプライマーを 用いて、 M— MuLVの逆転写酵素によりファーストストランドを合成し た。 続いて DNA Polymerase I によりセカンドストランドを合成し、 Ec oRIアダプタ一とのライゲ一シヨンおよび Xho I消化を行った。 その 後、 アダプターとプライマ一をゲル濾過 (Sephacryl Spin Column; ファ ルマシア社) により除いた。 以上の cDNA合成ステ ヅプは Stratagene 社の Z AP cDNA合成キットを用い、 逆転写酵素は BRL社のスーパ一 スクリプト I Iを用いて行った。 The 27th to 33rd peaks of the activity peak fraction were collected as the active fraction, and a cDNA library for this fraction was prepared by a modified method of the Gubler & Hoffman method (Gene, 25, p263 (1983)). That is, a first strand was synthesized from M-MuLV reverse transcriptase using an oligo dT primer having an XhoI site based on 2 g of the mRNA of the active fraction. Subsequently, a second strand was synthesized with DNA Polymerase I, and ligated with EcoRI adapter 1 and XhoI digested. Thereafter, the adapter and the primer were removed by gel filtration (Sephacryl Spin Column; Pharmacia). The above cDNA synthesis step was performed using Stratagene's ZAP cDNA synthesis kit, and the reverse transcriptase was performed using BRL's Superscript II.
次に、 EcoRI、 Xho l切断済み ZAP Expres sTMベクタ —を先に作製した c DNAとライゲ一シヨンした後、 Gigapack II Gold packing extract (m c r A ヽ mcrB mmr ; Stratagene 社) を用いてパッケージングを行い、 大腸菌 PLK— F' 株に感染させ た。 その結果、 平均長 2. 26kb、 インディペンデントクローン数 6.Next, EcoRI and Xhol-cut ZAP Express ™ vector were ligated with the previously prepared cDNA, and then packaged using Gigapack II Gold packing extract (mcrA ヽ mcrBmmr; Stratagene). And infected E. coli strain PLK-F '. As a result, the average length was 2.26 kb, and the number of independent clones was 6.
3 X 105 個の cDNAライブラリ一が得られた。 One 3 × 10 5 cDNA library was obtained.
実施例 2 Example 2
発現クロ一ニング Expression cloning
概要 Overview
1. 2で作製した cDNAライブラリ一を、 10, 000個/プールと
して計 63プールに分け、 後述の 2. 1. 2〜2. 1. 3の方法にて各プ ールの cRN Aをァフリカヅメガエルの卵母細胞に注入し、 タンパク質に 翻訳させた。 この翻訳産物を含む培養上清を、 後述の 2. 2. 2の方法に よりアツセィ用のマウス骨髄細胞に添加して後述の各ァッセィ法に供し、 陽性と判断されたプールを選別した。 更に、 その陽性プールを 10のサブ プールに分け、 同様にして cRNAを調製し、 卵母細胞中で発現させ、 そ の活性を測定して陽性プールを選別することを繰り返し、 最終的に単一ク ローンを得た。 The cDNA library prepared in 1. Were divided into a total of 63 pools, and the cDNA of each pool was injected into oocytes of Africa megafrog by the method described in 2.1.2 to 2.1.3 described below, and translated into protein. . The culture supernatant containing this translation product was added to mouse bone marrow cells for Atsey according to the method described in 2.2.2 described below, and subjected to each of the Assay methods described below, and a pool determined to be positive was selected. Furthermore, 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. I got a clone.
すなわち具体的アツセィとしては、 1次スクリーニングは後述の 2. 3. 1の TRAP染色法にて破骨細胞分化促進活性を判定し、 63プールから 陽性プールを 3プール選別した。 2次スクリーニング以降は、 後述の 2. 3. 1の TRAP染色法、 2. 3. 2の象牙を用いた p i t形成法の、 2 種類の破骨細胞分化促進活性測定法の両方に陽性反応を示すプールを選別 することにし、 まず、 上述の 3プールをそれぞれ 10のサブプール (10 00クローン Zプール) に分けて各アツセィを行った。 That is, as a specific assay, in the primary screening, the osteoclast differentiation promoting activity was determined by the TRAP staining method described in 2.3.1 described later, and three positive pools were selected from 63 pools. From the second screening onwards, positive reactions were observed in both the two types of osteoclast differentiation-promoting activity measurement methods described later in 2.3.1, TRAP staining method, and 2.3.2, pit formation method using ivory. The pools shown were selected. First, each of the three pools described above was divided into 10 subpools (1000 clones Z pool) and subjected to each assay.
その結果、 陽性反応の強さの順に上位 3プールを選び、 更にこの 3プ一 ルをそれぞれ 10のサブプール (200クローン/プール) に分けて 3次 スクリーニングを行った。 その結果、 陽性反応の強さの順に 3つの陽性プ —ルを選択し、 これを各々 10のサブプール (24クローン/プール) に 分けて、 さらに 4次スクリーニングを行った。 その結果、 TRAP染色及 び i t形成活性の強い順に上位 2プール選択し、 更にこの 2プールを全 36個の個々のクローンに分けて 5次スクリ一ニングを行った。 5次スク リーニングの結果、 TRAP染色及び p i t形成活性の強い順に上位 3ク ローンを選別した。 この 3つのクローンのうちの一つを OP Fう 8と命名 した。 なお OPFう 8の各アツセィ結果は、 後述の (結果) の項に記した。
2. 1 アツセィ用サンプルの調製 As a result, the top 3 pools were selected in the order of the strength of the positive reaction, and these 3 pools were further divided into 10 subpools (200 clones / pool), and a third screening was performed. As a result, three positive pools were selected in the order of the strength of the positive reaction, which were divided into 10 subpools (24 clones / pool), and further subjected to a fourth screening. As a result, the top 2 pools were selected in the order of TRAP staining and intense it-forming activity, and the 2 pools were further divided into 36 individual clones and subjected to fifth screening. As a result of the fifth screening, the top three clones were selected in descending order of TRAP staining and pit formation activity. One of the three clones was designated OPF8. The results of each of the OPF 8 tests are described in (Results) below. 2.1 Preparation of samples for Atsushi
2. 1. 1 DNAの調製 2.1.1 DNA preparation
大腸菌 XL 1— B 1 u eに各プールのラムダファージ 1 X 104 pfuを 感染させ、 15 cmシャーレにまき、 プラークを形成させた。 このプレー トに 13mlの SM緩衝液を加え、 プレートライセ一トを調製した。 この ファ一ジライセ一卜に DE 52 (DEAEセルロース ; ヮットマン社) を 加えてファージ DNA以外を吸着させ、 遠心後の上清に再度 DE 52を加 え、 その上清中のファージ DNAを回収した。 この DNAをフエノールと フエノ一ルークロロホルム (1 : 1) で 1回ずつ抽出し、 エタノール沈殿 にて回収し、 ファージ DNAとした。 調製した DNAを制限酵素 No t I で切断し、 1/50量を 1 %ァガロース電気泳動にて定量した。 E. coli XL1-B1ue was infected with 1 × 10 4 pfu of lambda phage from each pool and spread on a 15-cm petri dish to form plaques. A plate lysate was prepared by adding 13 ml of SM buffer to this plate. To this phage lysate, DE52 (DEAE cellulose; Petman) was added to adsorb other than phage DNA, and the centrifuged supernatant was re-added with DE52, and the phage DNA in the supernatant was recovered. This DNA was extracted once each with phenol and phenol-chloroform (1: 1), and recovered by ethanol precipitation to obtain phage DNA. The prepared DNA was digested with a restriction enzyme NotI, and 1/50 of the DNA was quantified by 1% agarose electrophoresis.
2. 1. 2 cRN Aの合成 2.1.2 Synthesis of cRNA
2. 1. 1で調製した各プールのファ一ジ DNAの少なくとも 1〃gを プロティナ一ゼ K (Stratagene社) で 37°C1時間処理し、 フエノ一ルー クロ口ホルム処理後、 エタノール沈殿により回収することによりテンプレ 2. At least 1 μg of phage DNA from each pool prepared in 1.1.1 is treated with proteinase K (Stratagene) for 1 hour at 37 ° C, treated with phenolic-monochrome form, and recovered by ethanol precipitation. By doing the template
—ト DNAを調製した。 この DNAを用いて、 mRNAcappingキット (Stratagene社) に従い c RN Aを合成した。 これをフエノールークロロ ホルム処理、 エタノール沈殿に供することにより cRNAを回収し、 1/ 10量を 1%ァガロースゲル電気泳動により定量した。 その後、 〃 1の濃度に調製してマイクロインジェクション用 cRN Aとした。-The DNA was prepared. Using this DNA, cDNA was synthesized according to the mRNAcapping kit (Stratagene). This was subjected to phenol-chloroform treatment and ethanol precipitation to recover cRNA, and 1/10 of the cRNA was quantified by 1% agarose gel electrophoresis. Then, it was adjusted to a concentration of 〃1 to obtain cRNA for microinjection.
2. 1. 3 アフリカッメガエル卵母細胞による発現 2.1.3 Expression in African oocytes
体長 10 cm程度のメスのァフリカツメガエルから卵母細胞の卵塊を取 り出し、 MBS ( + Ca2+ ; 88. OmM NaCl、 1. OmM KC 1、 2. 4mM Na2S03、 0. 3 mM C a (N03 ) 2 4 H20、 0. 4 ImM CaC 12 4H20、 0.82 mM MgS04 7 H20、
10〃g/mlペニシリン、 10〃g/ml ストレプトマイシン、 50 U/mlニス夕チン、 15mMトリス一 HC 1 (pH 7. 6) ) を入れ たシャーレに移し、 実体顕微鏡下精密用鉄とピンセットで卵母細胞を一つ ずつ切り放し、 ステージ Vか V Iの傷のない生きている細胞を選別した。 これらの卵母細胞に 10〃 1デジタルマイクロディスペンサー (Drummond 社) を用いて、 キヤビラリ一より、 卵母細胞 1個当たり 5 On 1の cRN Aを注入した。 その後、 死んだり傷ついた細胞を除き、 2%FCSを含む MB Sにて 3日間、 20°Cで培養した。 その培養上清を遠心し、 更に 0. 22 mのフィル夕一を通し、 残査を除くと同時に除菌した。 その上清を アツセィ用サンプルとした。 Out of the § African clawed frog body length 10 cm approximately female Ri preparative egg masses of oocytes, MBS (+ Ca 2+; 88. OmM NaCl, 1. OmM KC 1, 2. 4mM Na 2 S0 3, 0. 3 mM C a (N0 3) 2 4 H 2 0, 0. 4 ImM CaC 1 2 4H 2 0, 0.82 mM MgS0 4 7 H 2 0, Transfer to a Petri dish containing 10 μg / ml penicillin, 10 μg / ml streptomycin, 50 U / ml varnish, 15 mM Tris-HC1 (pH 7.6)), and use a precision iron and tweezers under a stereomicroscope. The oocytes were dissected out one at a time and live cells without stage V or VI damage were selected. Using a 10-1 digital microdispenser (Drummond), these oocytes were injected with 5 On 1 cRNA per oocyte per capillaries. After that, cells that had died or were damaged were removed, and cultured at 20 ° C. for 3 days in MBS containing 2% FCS. The culture supernatant was centrifuged and further passed through a 0.22 m filter to remove the residue and at the same time to remove bacteria. The supernatant was used as an Atsushi sample.
2. 2 アツセィ 2.2 Atsushi
2. 2. 1 マウス骨髄細胞の調製 2.2.1 Preparation of mouse bone marrow cells
6〜12週令のマウス (C3H/He J;日本クレア) の大腿骨及び脛 骨を無菌的に取り出し、 その骨端を切り落とし、 両端から 1回づっ 26G の針を付けたシリンジで lmlのひ— MEM培地 (10%牛胎児血清、 1 The femur and tibia of a 6-12 week old mouse (C3H / He J; CLEA Japan) were aseptically removed, their epiphyses were cut off, and lml syringes with a 26G needle were applied once from both ends. — MEM medium (10% fetal calf serum, 1
00単位/ m 1ぺニ シリン G、 100〃 g/m 1ストレプトマイシンを含 む) で骨髄細胞を押し出し 、 良くピペッティングした後骨残査が沈殿す るまで待ち、 その上清を回収した。 それを更に新鮮な培地で 1〜2回洗い、 アツセィ用の骨髄細胞を調製した。 Bone marrow cells were extruded with 00 units / ml 1-nicillin G and 100 μg / ml 1 streptomycin), pipetted well, waited until the bone residue precipitated, and the supernatant was collected. It was further washed once or twice with fresh medium to prepare bone marrow cells for Atsushi.
2. 2. 2 破骨細胞分化形成法 2.2.2 Osteoclast differentiation and formation method
上記の骨髄細胞を 10— 8 Mの活性型ビタミン D 〔1、 25 (OH) 2 D3〕 を 含むひ— MEM培地中にけん濁させ、 2 X 106 個細胞/ ml の濃度に調製し、 96穴プレートに 180〃1と 2. 1. 3で調製したァ ヅセィ用サンプルを 20〃1加え、 37。 5%COz 下、 1または 2 週間培養した。 その間、 3— 4日間隔で培地の 3Z4を新しい培地と交換
し、 新たにァヅセィ用サンプルを同量添加した。 . Additional active vitamin D in bone marrow cells 10- 8 M a [1, 25 (OH) 2 D 3 ] Fukumuhi - allowed tendon Nigosa in MEM medium, adjusted to a concentration of 2 X 10 6 cells / ml Then, add 20 を 1 of the assay sample prepared in 180〃1 and 2.1.3 to a 96-well plate, and 37. Under 5% CO z, and cultured for one or two weeks. During that time, replace the 3Z4 medium with new medium every 3-4 days Then, the same amount of a case sample was newly added. .
2. 3 破骨細胞の同定法 2.3 Identification of osteoclasts
2. 3. 1 TRAP染色法 2.3.1 TRAP staining method
破骨細胞のマーカ一酵素である T R A P (酒石酸抵抗性酸性フォスファ 夕ーゼ) を基質で染色した。 即ち 2. 2. 2の培養骨髄細胞をアセ トン一 クェン酸緩衝液で固定した後、 酒石酸存在下で基質 (Naphthol AS一 MXphosphate) と色素 ( Fastredviolet LB salt) を 37°Cで 1時間反応 させることにより、 染色した ( Endocrinology, 122, ρ1373, (1988)) 。 (結果) Trap (tartrate-resistant acid phosphatase), a marker enzyme of osteoclasts, was stained with a substrate. That is, after fixation of the cultured bone marrow cells of 2.2.2 with acetone-citrate buffer, the substrate (Naphthol AS-MXphosphate) and the dye (Fastredviolet LB salt) are reacted at 37 ° C for 1 hour in the presence of tartaric acid. Thus, it was stained (Endocrinology, 122, ρ1373, (1988)). (Result)
OP Fう 8は、 既知の破骨細胞分化形成因子である IL一 1 ? (50η g/ml) や LIF (25U/ml) で骨髄細胞を処理し、 破骨細胞を分 化形成させたポジティブコントロールの TRAP染色性と比較して、 陽性 と判断した。 OPF8 is a known osteoclast differentiation factor IL-11? Bone marrow cells were treated with (50 ηg / ml) or LIF (25 U / ml) and compared to the TRAP staining of a positive control in which osteoclasts were differentiated and formed.
2. 3. 2 象牙を用いた pit形成法 2.3.2 Pit formation method using ivory
象牙より直径 6mm、 1 mm厚の象牙質スライスを作製し、 それを 8 A dentin slice 6 mm in diameter and 1 mm thick from ivory was prepared and
0%アルコール中で超音波処理することにより滅菌した。 ひ一 MEM培地 で洗浄した後、 各スライスを 96ゥエルプレートのゥエル底に移し、 その 上で 2. 2. 2の方法に従って骨髄細胞から破骨細胞を分化誘導した。 1 または 2週間後、 象牙質スライス上の破骨細胞を 2. 3. 1の TRAP染 色法にて染色し、 0. 25%トリプシン一 0. 02%EDTAで一晩処理 し、 スライス上の細胞をシリコンスクレイパーで削り取った。 象牙質スラ イス上の pi t (吸収窩) を顕微鏡下で観察し、 その数または pi tあた りのメッシュ数を測定することにより骨髄細胞より分化誘導された細胞の 骨吸収活性 (骨分解活性) を調べた。 Sterilized by sonication in 0% alcohol. After washing with Hi-MEM, each slice was transferred to the bottom of a 96-well plate, and then osteoclasts were induced from bone marrow cells according to the method described in 2.2.2. After one or two weeks, the osteoclasts on the dentin slice are stained using the TRAP staining method described in 2.3.1, treated with 0.25% trypsin and 0.02% EDTA overnight, and The cells were scraped off with a silicon scraper. The pits (resorption pits) on the dentin slices are observed under a microscope, and the number of the pits or the number of meshes per pit is measured. Activity).
(結果)
OPFう 8により分化形成された細胞の象牙質スライス上の p i t形成 数は 100個であり、 ポジティブコントロールである L I F ( 19 U/m 1) の 35個に比較して、 活性は同等かそれ以上であることが判明した。 (結論) (result) The number of pits formed on the dentin slice of the cells differentiated and formed by OPF8 was 100, and the activity was equal to or higher than that of 35 cells of the positive control LIF (19 U / m1). Turned out to be. (Conclusion)
以上 2通りの同定法いずれにおいても陽性を示したことから、 OPFう Since the positive results were obtained in both of the above two identification methods, the OPF
8により骨髄細胞から分化誘導された細胞は破骨細胞であり、 OP Fう 8 は破骨細胞分化促進活性を有している因子であると判断した。 Cells differentiated from bone marrow cells by 8 were osteoclasts, and OPF8 was determined to be a factor having osteoclast differentiation promoting activity.
実施例 3 Example 3
組換えファージ DN Aのファ一ジミ ド DN Aへの変換 Conversion of recombinant phage DNA to phagemid DNA
ZAP Expressベクタ一はインサート DNAを pBK— CMVへ in vivo excisionすることでサブクロ一ニングすることができる。 XL 1— BlueMRF' 大腸菌に ZAP Expressファージと ExAssistヘルパー ファージを感染させることで、 pBK— CMVファージミ ドを産生させ、 元の大腸菌を熱処理することで死滅させ、 新たに X L 0 L R大腸菌に感染 させた。 これに培地を加え、 45分間培養後、 LBプレートにプレーティ ングし、 培養した。 ZAP Express vectors can be subcloned by ex vivo insertion of the insert DNA into pBK-CMV. XL1-BlueMRF 'Escherichia coli was infected with ZAP Express phage and ExAssist helper phage to produce pBK-CMV phagemid, killed by heat treatment of the original E. coli, and newly infected with XL0LR E. coli. . The medium was added thereto, cultured for 45 minutes, plated on an LB plate, and cultured.
実施例 4 Example 4
プラスミ ド DNAの調製 Preparation of plasmid DNA
陽性コロニーを爪楊枝で拾い 2mlの LB (カナマイシン 100〃g/ ml) で一晩培養後、 アルカリ— SDS法によりプラスミ ドを調製した。 このプラスミ ド DN Aを適当な制限酵素で切断し、 1%ァガロースゲル中 で電気泳動し、 OP Fう 8 cDNAのべクタ一への挿入を確認した。 実施例 5 Positive colonies were picked with a toothpick, cultured overnight in 2 ml of LB (kanamycin 100 µg / ml), and then plasmid was prepared by the alkali-SDS method. This plasmid DNA was cleaved with an appropriate restriction enzyme and subjected to electrophoresis in a 1% agarose gel to confirm insertion of the OPF8 cDNA into the vector. Example 5
〇PFう 8 cDNAの塩基配列決定 決定 Base sequence determination of PF8 cDNA
実施例 4で得られた OPFう 8 cDNAの塩基配列の決定は、 Sang
e rらによって閧発されたジデォキシ法によって行った。 The nucleotide sequence of the OPF cDNA 8 obtained in Example 4 was determined by Sang This was done by the dideoxy method proposed by Er.
その結果、 配列表の配列番号: 1に記載の 1400bpからなる cDN Aが得られ、 また 439アミノ酸からなる配列番号: 2のアミノ酸配列が 決定された。 As a result, a cDNA comprising 1400 bp described in SEQ ID NO: 1 in the sequence listing was obtained, and the amino acid sequence of SEQ ID NO: 2 comprising 439 amino acids was determined.
実施例 6 Example 6
OPFう 8の臓器特異的遺伝子発現のノーザンブロット及びドットプロッ 卜解析 Northern blot and dot plot analysis of organ-specific gene expression of OPF8
マウスの骨、 骨髄細胞や筋肉等の各組織由来の全 RNAを、 1. 1. 1 に従い調製した。 その後、 10— 20〃gの全 RNAをサンプル緩衝液に 溶解し、 65°Cで 5分間加熱変性させて 6%ホルムアルデヒドァガロース 電気泳動を行った後、 ナイロンフィルター (ハイボンド N+ 、 アマシャ ム社) に移した。 また、 種々の組織由来のポリ A+RN Aが既にブロッテ ィングされた、 MTNブロットメンブレンとヒト RN Aマス夕一ブロヅト (クローンテック社) も用いた。 プローブは、 OPFぅ8 cDNA全長 (約 1. 5kbp) を32 Pで標識して用いた。 Total RNA from various tissues such as mouse bone, bone marrow cells and muscle was prepared according to 1.1.1. Then, 10-20 µg of total RNA was dissolved in sample buffer, denatured by heating at 65 ° C for 5 minutes, and subjected to 6% formaldehyde agarose electrophoresis. Then, a nylon filter (Hybond N +, Amersham) ). In addition, MTN blot membrane and human RNA mass spectroscopy (Clontech), in which poly-A + RNA from various tissues had already been blocked, were used. As the probe, the full-length OPF ぅ 8 cDNA (about 1.5 kbp) was labeled with 32 P and used.
上記プローブを 50% (v/v) ホルムアルデヒド Z5 X S S C/5 X デンハルト /1% (w/v) SDS/0. 01% (w/v) 変性サケ精子 DNA中でフィル夕一に固定した RNAに 42°Cでハイブリダィズさせ、 2 X S SC/0. 1%SDS、 50°C中で、 次に 0. l%SSC/0. 1%SDS、 50°C中で洗浄した。 水気を除いた後、 —80°Cで 1— 3日 間ォートラジォグラフィ一を行った。 使用した X線フィルムはコダヅク S B 5またはフジ A I F,RXを増感スクリーンの存在下で用いた。 The above probe was applied to RNA fixed at 50% (v / v) formaldehyde Z5 XSSC / 5 X Denhardt / 1% (w / v) SDS / 0.01% (w / v) denatured salmon sperm DNA. Hybridized at 42 ° C. and washed in 2 × SSC / 0.1% SDS, 50 ° C., then in 0.1% SSC / 0.1% SDS, 50 ° C. After draining, 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.
(結果) (Result)
図 1の Aには、 マウス各組織 ( 10 全 RNA) のノーザンプロット 解析の結果を示す。 OPFう 8のバンド (矢印) は、 全ての組織で僅かに
ではあるが発現していた。 また、 OPFう 8のクローニング源の BW51 47細胞、 及び骨芽細胞様 ST 2細胞では、 より高発現していた。 A of FIG. 1 shows the results of Northern plot analysis of each mouse tissue (10 total RNAs). OPF 8 band (arrow) is slightly in all tissues However, it was expressed. BW5147 cells, a cloning source of OPF8, and osteoblast-like ST2 cells showed higher expression.
図 1の Bには、 マウスとラヅトの骨組織 (20 / gmRNA) のノーザンブ ロット解析の結果を示す。 その結果、 OPFう 8mRNAは、 正常マウス、 コ ラ一ゲン関節炎 (C IA) マウス、 骨大理石病 (op/op) マウス、 及 び正常ラヅト、 卵巣摘出 (ovx) ラヅ トの骨組織において発現していた c 以上、 図 1より、 OPFう 8は骨組織で広く発現していることが分かった c 図 2には、 マウス 7日胚から 17日胚まで (2/ g mRNA) のノ一ザ ンブロット解析の結果を示す。 その結果、 11日胚で発現量が低下している が、 それ以外では高発現していた。 FIG. 1B shows the results of Northern blot analysis of mouse and rat bone tissue (20 / g mRNA). As a result, OPF mRNA 8 mRNA is expressed in bone tissues of normal mice, collagen arthritis (CIA) mice, osteopetrosis (op / op) mice, and normal rats and ovariectomized (ovx) rats. Figure 1 shows that OPF8 is widely expressed in bone tissue. Figure 2 shows that OPF8 was expressed in mouse 7-day to 17-day embryos (2 / g mRNA). The result of Xan blot analysis is shown. As a result, the expression level was reduced in the 11-day embryo, but was high in other days.
図 3には、 ヒトの各組織 (100-500ng mRNA) の RNAドットプロヅトの結 果を示す。 その結果、 ヒト型 OPFう 8mRNAは、 腎臓で強く、 卵巣、 肝臓 と肺で弱く発現していた。 この図 3の結果、 あるいは図 1の結果より、 ヒ ト型 OPFう 8遺伝子のクロ一ニングには、 腎臓由来の cDN Aライブラ リ一あるいは骨組織由来の cDNAライブラリーを用いることが好ましい c 実施例 7 Figure 3 shows the results of RNA dot plots for human tissues (100-500 ng mRNA). As a result, human OPF mRNA was strongly expressed in kidney and weakly expressed in ovary, liver and lung. From the results of FIG. 3 and FIG. 1, it is preferable to use a cDNA library derived from a kidney or a cDNA library derived from a bone tissue to clone the human OPF8 gene. Example 7
0 P Fう 8の臓器特異的遺伝子発現の R T— P C R解析 RT-PCR analysis of organ-specific gene expression in 0 P F 8
1. 1. 1に従い種々の細胞や組織から調製した全 RNA ( l〃g) よ り、 : RT— PCRキット (PERKIN ELMER社製) に従い、 まず 2本鎖 DN A を合成し、 それをテンプレートにして PCR反応を行った。 本遺伝子増幅 のためのプライマ一配列として、 5,プライマ一 (5'— From total RNA (l〃g) prepared from various cells and tissues according to 1.1.1: First, according to RT-PCR kit (manufactured by PERKIN ELMER), first synthesize double-stranded DNA and use it as template. To perform a PCR reaction. The primer sequence for amplification of this gene is 5, Primer (5'-
TGTCCTCACGCTCACGCTCT —3') および 3, プライマ一 (5,一 TGTCCTCACGCTCACGCTCT —3 ') and 3, Primer (5, 1
TGTGGATACGCCTACTATCA—3') を合成した。 これらのプライマ一の組み合 わせで増幅される DNAサイズは 252塩基対である。 また、 コント口一 レとして G 3 PD H (glyceraldehyde-3-phosphatedehydrogenase) ブラ
イマ一 (5' プライマーが (5' — TGAAGGT CGGTGTGAATGTGGATACGCCTACTATCA-3 ') was synthesized. The DNA size amplified with these primer combinations is 252 base pairs. G 3 PDH (glyceraldehyde-3-phosphate dehydrogenase) Imaichi (5 'primer is (5' — TGAAGGT CGGTGTGAA
CGGATTTGGC— 3' )、 3, プライマーが (5' — CATGT AGGCCATGAGGTCCACCAC-35 ) 、 増幅される DNAサ ィズが 983塩基対) を用いた。 反応組成等は標準的手法に従い DN Aサ —マルサイクラ一を用いて、 熱変性 94°Cで 1分間、 アニーリング 60°C で 1分間、 鎖伸張反応 72 °Cで 2分間の条件で 30— 40サイクルで反応 を行った。 反応混合物の 1Z10量を 1 %ァガロースゲルで電気泳動し、 バンドを確認した。 CGGATTTGGC- 3 '), 3, primer (5' - CATGT AGGCCATGAGGTCCACCAC-3 5), DNA Sa I's to be amplified using a 983 bp). The reaction composition and the like are determined according to standard procedures using a DNA cycler under the conditions of heat denaturation at 94 ° C for 1 minute, annealing at 60 ° C for 1 minute, and chain elongation at 72 ° C for 2 minutes. The reaction was performed in cycles. The 1Z10 amount of the reaction mixture was electrophoresed on a 1% agarose gel, and a band was confirmed.
(結果) (Result)
図 4に、 各,組織の全 MAI〃gをテンプレートにして行った結果を示す。 その結果、 4種類の骨芽細胞及び骨芽細胞様細胞において、 OPFう 8が 発現していた (図 4中、 OPFu 8と表示した写真の矢印部分) 。 これら の結果は、 図 1のノーザンブロット解析の結果と矛盾するものではなく、 OPFう 8は骨組織、 骨芽細胞において広く発現していることが確認され た。 なお、 同一の RNAを用いてコントロールの G3PDHの RT— PC Figure 4 shows the results of using all MAI〃g of each tissue as a template. As a result, OPF8 was expressed in four types of osteoblasts and osteoblast-like cells (in FIG. 4, the arrow in the photograph labeled OPFu8). These results were not inconsistent with the results of the Northern blot analysis in FIG. 1, confirming that OPF8 was widely expressed in bone tissue and osteoblasts. In addition, using the same RNA, control G3PDH RT-PC
Rも行い、 これを、 各 RN Aの分解度の指標とした。 R was also performed, and this was used as an index of the degree of degradation of each RNA.
実施例 8 Example 8
OPFう 8 cDN Aの哺乳動物培養細胞による発現 Expression of OPF8 cDNA in cultured mammalian cells.
pBK— CMVべク夕一に実施例2で得られたOPFぅ 8 cDNAをサ ブクローニングし、 大腸菌 JM 109株に形質転換させた。 アルカリ— S The OPF ぅ 8 cDNA obtained in Example 2 was subcloned in pBK-CMV vector and transformed into E. coli JM109 strain. Alkali—S
DS法により DNAを調製し、 2回の超遠心分離法により精製した。 この 精製 DNAを COS— 7細胞に LIPOFECTAMINE (GIBCO BRL) を用いて トランスフエクトした。 その後、 細胞を無血清培地で 5日間培養し、 培 養上清を回収して 0 P Fう 8培養上清標品とした。 DNA was prepared by the DS method and purified by two ultracentrifugation methods. The purified DNA was transfected into COS-7 cells using LIPOFECTAMINE (GIBCO BRL). Thereafter, the cells were cultured in a serum-free medium for 5 days, and the culture supernatant was collected to obtain a 0 P F 8 culture supernatant sample.
上記培養上清標品の活性は、 2. 3. 1の TRAP染色性及び 2. 3.
2の象牙質スライスを用いた p i t形成活性の、 2種類の同定法で測定し た。 その結果、 前記培養上清標品存在下、 象牙質スライス上で培養した骨 髄細胞は、 赤褐色に染色され、 TRAP陽性を示した。 次に、 象牙スライ ス上の TRAP陽性細胞を物理的に削り取った結果、 図 5 (上) に示すよ うに TRAP陽性細胞残渣が残っているものの、 pitが形成されていた c 一方、 図 5 (下) に示すように、 ベクタ一のみを導入した細胞の培養上清 を用いて同様の実験を行った場合は、 TRAP陽性細胞、 pit共に形成 されなかった。 The activity of the above culture supernatant preparation was determined by the TRAP staining property described in 2.3.1 and 2.3.3. Pit formation activity using two dentin slices was measured by two different identification methods. As a result, bone marrow cells cultured on dentin slices in the presence of the culture supernatant preparation were stained red-brown and showed TRAP positive. Next, as a result of physically scraping the TRAP-positive cells on the ivory slice, pits were formed although TRAP-positive cell residues remained as shown in Fig. 5 (top). As shown in (bottom), when a similar experiment was performed using the culture supernatant of cells into which only one vector had been introduced, neither TRAP-positive cells nor pits were formed.
以上の結果より、 OPFう 8 cDNAを哺乳動物培養細胞に導入し得ら れた培養上清は、 破骨細胞分化促進活性を有することが明らかとなった。 発明の効果 From the above results, it was clarified that the culture supernatant obtained by introducing OPF8 cDNA into cultured mammalian cells has osteoclast differentiation promoting activity. The invention's effect
本発明により、 破骨細胞分化促進活性を有する OPFう 8及び該 OPF う 8の類似タンパク質、 またはこれらのタンパク質のペプチド断片、 抗体、 これらのタンパク質を有効成分とする医薬、 あるいはこれらのタンパク質 をコードする遺伝子、 該遺伝子の発現ベクター、 該発現ベクターを導入し た形質転換体、 該形質転換体を用いる組換えタンパク質の生産方法、 前記 遺伝子の導入及び欠損に係るトランスジヱニック動物、 さらには前記タン パク質の、 破骨細胞分化促進活性阻害剤のスクリーニングにおける用途が 提供される。
According to the present invention, OPF8 having osteoclast differentiation promoting activity, a protein similar to OPF8, a peptide fragment of these proteins, an antibody, a medicament containing these proteins as an active ingredient, or encoding these proteins Gene, an expression vector of 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 related to the introduction and deletion of the gene, The use of the protein in screening for an inhibitor of osteoclast differentiation promoting activity is provided.
配 列 表 配列番号: 1 Sequence list SEQ ID NO: 1
配列の長さ: 1 4 0 0 Array length: 1 4 0 0
配列の型:核酸 Sequence type: nucleic acid
鎖の数:二本鎖 Number of chains: double strand
トポロジー:直鎖状 Topology: linear
配列の種類: c D N A Sequence type: c D N A
配列の特徴 Array features
特徴を決定した方法: E How the features were determined: E
配列 Array
TCACGTTTTT TTTGTACGGT TTGTCTCTAC CCGGGAGGAC CTTGGTTGTC CACCCGTGGG 60 TAGGCAGTGG AGTGCC ATG AAG CGG GCA GCG ATA CGG CTT TGG ACC TTG 109 TCACGTTTTT TTTGTACGGT TTGTCTCTAC CCGGGAGGAC CTTGGTTGTC CACCCGTGGG 60 TAGGCAGTGG AGTGCC ATG AAG CGG GCA GCG ATA CGG CTT TGG ACC TTG 109
Met Lys Arg Ala Ala lie Arg Leu Trp Thr Leu Met Lys Arg Ala Ala lie Arg Leu Trp Thr Leu
5 10 5 10
AAC AAG GGG CTT CTT ACT CAT GGC AGA GGA CTG TCT CAA GGA TCC CAA 157 Asn Lys Gly Leu Leu Thr His Gly Arg Gly Leu Ser Gin Gly Ser Gin AAC AAG GGG CTT CTT ACT CAT GGC AGA GGA CTG TCT CAA GGA TCC CAA 157 Asn Lys Gly Leu Leu Thr His Gly Arg Gly Leu Ser Gin Gly Ser Gin
15 20 25 15 20 25
TAC AAA ATA AGT GAA CCC TTA CAC ATC CAT CAA GTT CGA GAC AAG CTG 205 Tyr Lys lie Ser Glu Pro Leu His l ie His Gin Val Arg Asp Lys Leu TAC AAA ATA AGT GAA CCC TTA CAC ATC CAT CAA GTT CGA GAC AAG CTG 205 Tyr Lys lie Ser Glu Pro Leu His lie His Gin Val Arg Asp Lys Leu
30 35 40 30 35 40
CGG GAG ATA GTA GGC GTA TCC ACA GTC TGG AGA GAC CAT GTA AAA GCA 253 Arg Glu He Val Gly Val Ser Thr Val Trp Arg Asp His Val Lys Ala CGG GAG ATA GTA GGC GTA TCC ACA GTC TGG AGA GAC CAT GTA AAA GCA 253 Arg Glu He Val Gly Val Ser Thr Val Trp Arg Asp His Val Lys Ala
45 50 55 45 50 55
ATG GAG GAA AGG AAG TTA CTT CAT AGT TTT TTG CCT AAA TCA CAG AAA 301
6Z 3W A 9 J¾ ¾iv dsy n9i ΐ¾Λ ojj dJi α^χ nt9 dsy usy STH Z 989 9IV VIS III 00V V39 IVO 911 XIO 100 991 XVI VW 3V0 IVD IW OVD ATG GAG GAA AGG AAG TTA CTT CAT AGT TTT TTG CCT AAA TCA CAG AAA 301 6Z 3W A 9 J¾ ¾iv dsy n9i ΐ¾Λ ojj dJi α ^ χ nt9 dsy usy STH Z 989 9IV VIS III 00V V39 IVO 911 XIO 100 991 XVI VW 3V0 IVD IW OVD
S8T 08 T I S8T 08 T I
Π9Ί ui9 9i s s^i Ι¾Λ nig J usy Λ dJI S9 010 OVO Oil 3IV VW 9IV 9W 919 WO 9IV DDI VOV IW V90 119 991 Π9Ί ui9 9i s s ^ i Ι¾Λ nig J usy Λ dJI S9 010 OVO Oil 3IV VW 9IV 9W 919 WO 9IV DDI VOV IW V90 119 991
0 Ϊ 991 09Ϊ Z 0 Ϊ 991 09Ϊ Z
J9S ΐ¾Λ STH ^ΐθ J¾ 9Hd 3Π dsy uig nig oaj J9S ngq s STHJ9S ΐ¾Λ STH ^ ΐθ J¾ 9Hd 3Π dsy uig nig oaj J9S ngq s STH
689 DDV 1X9 IVD 09D IDV Oil 9W XXV 0V9 OVD W9 103 09V VXI 39V DVD689 DDV 1X9 IVD 09D IDV Oil 9W XXV 0V9 OVD W9 103 09V VXI 39V DVD
T OST ^1 on s^i s ^ dsy an dsy Ι¾Λ η9Ί ΐ^Λ J¾ ΐ¾Λ 8I I J9S ^g OJJT OST ^ 1 on s ^ is ^ dsy an dsy η η9 ΐ ΐ ^ Λ J¾ ΐ¾Λ 8 II J9S ^ g OJJ
ItS WV XOX 9XV IV9 I1V 9W OVD 9X9 9X0 019 VOV 119 VXV VOX VII 130 9T ItS WV XOX 9XV IV9 I1V 9W OVD 9X9 9X0 019 VOV 119 VXV VOX VII 130 9T
9 I OCT Z\ 9 I OCT Z \
J9S s q J¾ s SIH usy STH usy SIH J s m J9S s q J¾ s SIH usy STH usy SIH J s m
131 9IV 9W 00V XOI IVO IW OVO OW OVO 9XV OVl 001 XI9 113 XIO 131 9IV 9W 00V XOI IVO IW OVO OW OVO 9XV OVl 001 XI9 113 XIO
ozi on ozi on
^ΐΰ ΠΘΊ J9S dsy Π97 dsy naq 9 Π Say ^ΐ9 a Say ΐ¾Λ J¾ usy 0Ϊ V09 VIO DDV 0V9 1X0 3V9 9V9 XIO XXV 99V 099 IIX V9V VIO VOV IW ^ ΐΰ ΠΘΊ J9S dsy Π97 dsy naq 9 Π Say ^ ΐ9 a Say ΐ¾Λ J¾ usy 0Ϊ V09 VIO DDV 0V9 1X0 3V9 9V9 XIO XXV 99V 099 IIX V9V VIO VOV IW
90Ϊ 001 36 90Ϊ 001 36
UI9 ΐ¾Λ J Ι¾Λ J s dsy Say n^g OJJ dsy J¾ OJJ 6S WO XI9 I0V 919 IVX VW 3V9 V9D VI3 WD 133 OVO I3V 199 911 X03 UI9 ΐ¾Λ J Ι¾Λ J s dsy Say n ^ g OJJ dsy J¾ OJJ 6S WO XI9 I0V 919 IVX VW 3V9 V9D VI3 WD 133 OVO I3V 199 911 X03
06 98 08 9 06 98 08 9
Ti9q ΙΙΘΊ A nig d\ \ αΑ J9S dsy Say ^Θ^ S^I Say OJJ OJJ ΓΙΘΙ Ti9q ΙΙΘΊ A nig d \ \ αΑ J9S dsy Say ^ Θ ^ S ^ I Say OJJ OJJ ΓΙΘΙ
m VIO 310 119 W9 OIV OVl I9V IV9 99V 9IV WV 99V ODD V03 VXD 319 m VIO 310 119 W9 OIV OVl I9V IV9 99V 9IV WV 99V ODD V03 VXD 319
9A 99 09 sXq u^g J9S sX^ OJJ na 9 J8S STH sXq S y n|0 n^g 9A 99 09 sXq u ^ g J9S sX ^ OJJ na 9 J8S STH sXq S y n | 0 n ^ g
698Z0/86df/XDd 96W0/66 OW
190 195 200 698Z0 / 86df / XDd 96W0 / 66 OW 190 195 200
GTG GCT CGA GAT TCT GAA AAT AAG GGG CCG GCA TTT GTA AAT CCA CTC 733 Val Ala Arg Asp Ser Glu Asn Lys Gly Pro Ala Phe Val Asn Pro Leu GTG GCT CGA GAT TCT GAA AAT AAG GGG CCG GCA TTT GTA AAT CCA CTC 733 Val Ala Arg Asp Ser Glu Asn Lys Gly Pro Ala Phe Val Asn Pro Leu
205 210 215 205 210 215
ATT CCG GAA AAT AAA GAG GAA GAA GAG CTC TTT AAA CAA GGA GAA TTG 781 lie Pro Glu Asn Lys Glu Glu Glu Glu Leu Phe Lys Gin Gly Glu Leu ATT CCG GAA AAT AAA GAG GAA GAA GAG CTC TTT AAA CAA GGA GAA TTG 781 lie Pro Glu Asn Lys Glu Glu Glu Glu Leu Phe Lys Gin Gly Glu Leu
220 225 230 235 220 225 230 235
AAC AAG AGC CGA AGG ATT GCC TTT AGC ACC AGT TCA TTA CTG AAA GTG 829 Asn Lys Ser Arg Arg lie Ala Phe Ser Thr Ser Ser Leu Leu Lys Val AAC AAG AGC CGA AGG ATT GCC TTT AGC ACC AGT TCA TTA CTG AAA GTG 829 Asn Lys Ser Arg Arg lie Ala Phe Ser Thr Ser Ser Leu Leu Lys Val
240 245 250 240 245 250
GCT CCC AGT TCT GAG GAG AGG AAT ATC ATA CAT GAA CTG TTC CTT ACC 877 Ala Pro Ser Ser Glu Glu Arg Asn l ie lie His Glu Leu Phe Leu Thr GCT CCC AGT TCT GAG GAG AGG AAT ATC ATA CAT GAA CTG TTC CTT ACC 877 Ala Pro Ser Ser Glu Glu Glu Arg Asn lie lie His Glu Leu Phe Leu Thr
255 260 265 255 260 265
ACA CTG GAT CCA AAG ACT ATA AGT TTT CAG AGT CGA ATT TTG CCT CCT 925 Thr Leu Asp Pro Lys Thr l ie Ser Phe Gin Ser Arg l ie Leu Pro Pro ACA CTG GAT CCA AAG ACT ATA AGT TTT CAG AGT CGA ATT TTG CCT CCT 925 Thr Leu Asp Pro Lys Thr lie Ser Phe Gin Ser Arg lie Leu Pro Pro
270 275 280 270 275 280
AAA GCA GTG TGG ATG GAG GAT ACA AAA CTC AAG AGC TTG GAT ATT TGT 973 Lys Ala Val Trp Met Glu Asp Thr Lys Leu Lys Ser Leu Asp l ie Cys AAA GCA GTG TGG ATG GAG GAT ACA AAA CTC AAG AGC TTG GAT ATT TGT 973 Lys Ala Val Trp Met Glu Asp Thr Lys Leu Lys Ser Leu Asp lie Cys
285 290 295 285 290 295
CAC CCT CAG GAA CGA AAC GTA TTC AAT CGG ATC TTT GGT GGT TTT CTT 1021 His Pro Gin Glu Arg Asn Val Phe Asn Arg l ie Phe Gly Gly Phe Leu CAC CCT CAG GAA CGA AAC GTA TTC AAT CGG ATC TTT GGT GGT TTT CTT 1021 His Pro Gin Glu Arg Asn Val Phe Asn Arg lie Phe Gly Gly Phe Leu
300 305 310 315 300 305 310 315
ATG AGA AAA GCA TAT GAA CTT GCA TGG GCT ACT GCT TGT AGC TTT GGC 1069 Met Arg Lys Ala Tyr Glu Leu Ala Trp Ala Thr Ala Cys Ser Phe Gly ATG AGA AAA GCA TAT GAA CTT GCA TGG GCT ACT GCT TGT AGC TTT GGC 1069 Met Arg Lys Ala Tyr Glu Leu Ala Trp Ala Thr Ala Cys Ser Phe Gly
320 325 330
GGT TCC CGG CCA TAT GTG GTA ACA GTG GAT GAT ATC ATG TTT CAG AAG 1117 Gly Ser Arg Pro Tyr Val Val Thr Val Asp Asp l ie Met Phe Gin Lys 320 325 330 GGT TCC CGG CCA TAT GTG GTA ACA GTG GAT GAT ATC ATG TTT CAG AAG 1117 Gly Ser Arg Pro Tyr Val Val Thr Val Asp Asp lie Met Phe Gin Lys
335 340 345 335 340 345
CCT GTT GAA GTT GGT TCA TTG CTC TTT CTT TCT TCA CAG GTA TGC TTC 1165 Pro Val Glu Val Gly Ser Leu Leu Phe Leu Ser Ser Gin Val Cys Phe CCT GTT GAA GTT GGT TCA TTG CTC TTT CTT TCT TCA CAG GTA TGC TTC 1165 Pro Val Glu Val Gly Ser Leu Leu Phe Leu Ser Ser Gin Val Cys Phe
350 355 360 350 355 360
ACC CAG GAC AAT TAC ATC C GTC AGA GTC CAT AGT GAA GTG TCT TCT 1213 Thr Gin Asp Asn Tyr l ie Gin Val Arg Val His Ser Glu Val Ser Ser ACC CAG GAC AAT TAC ATC C GTC AGA GTC CAT AGT GAA GTG TCT TCT 1213 Thr Gin Asp Asn Tyr lie Gin Val Arg Val His Ser Glu Val Ser Ser
365 370 375 365 370 375
CTT GAC AGT CGT GAG CAT ATG ACC ACC AAT GTC TTT CAT TTT ACA TTC 1261 Leu Asp Ser Arg Glu His Met Thr Thr Asn Val Phe His Phe Thr Phe CTT GAC AGT CGT GAG CAT ATG ACC ACC AAT GTC TTT CAT TTT ACA TTC 1261 Leu Asp Ser Arg Glu His Met Thr Thr Asn Val Phe His Phe Thr Phe
380 385 390 395 380 385 390 395
ATG TCG GAA AAA GAA GTA CCC TTG ATT TTC CCT AAA ACA TAT GGA GAG 1309 Met Ser Glu Lys Glu Val Pro Leu l ie Phe Pro Lys Thr Tyr Gly Glu ATG TCG GAA AAA GAA GTA CCC TTG ATT TTC CCT AAA ACA TAT GGA GAG 1309 Met Ser Glu Lys Glu Val Pro Leu lie Phe Pro Lys Thr Tyr Gly Glu
400 405 410 400 405 410
TCC ATG TTG TAT TTA GAT GGA CAG CGA CAT TTC AAG TCT ATG AGT ACC 1357 Ser Met Leu Tyr Leu Asp Gly Gin Arg His Phe Lys Ser Met Ser Thr TCC ATG TTG TAT TTA GAT GGA CAG CGA CAT TTC AAG TCT ATG AGT ACC 1357 Ser Met Leu Tyr Leu Asp Gly Gin Arg His Phe Lys Ser Met Ser Thr
415 420 425 415 420 425
CCA GTG ACC TTG AAA AAG GAC TAC CCT GTG GAG CCC TAGGATA 1400 Pro Val Thr Leu Lys Lys Asp Tyr Pro Val Glu Pro CCA GTG ACC TTG AAA AAG GAC TAC CCT GTG GAG CCC TAGGATA 1400 Pro Val Thr Leu Lys Lys Asp Tyr Pro Val Glu Pro
430 435 430 435
配列番号: 2 SEQ ID NO: 2
配列の長さ : 4 3 9 Array length: 4 3 9
配列の型:アミノ酸 Sequence type: amino acid
トポロジー:直鎖状
配列の種類:ぺプチド Topology: linear Sequence type: peptide
配列 Array
Met Lys Arg Ala Ala lie Arg Leu Trp Thr Leu Asn Lys Gly Leu Leu Met Lys Arg Ala Ala lie Arg Leu Trp Thr Leu Asn Lys Gly Leu Leu
5 10 15 Thr His Gly Arg Gly Leu Ser Gin Gly Ser Gin Tyr Lys lie Ser Glu 5 10 15 Thr His Gly Arg Gly Leu Ser Gin Gly Ser Gin Tyr Lys lie Ser Glu
20 25 30 20 25 30
Pro Leu His lie His Gin Val Arg Asp Lys Leu Arg Glu lie Val Gly Pro Leu His lie His Gin Val Arg Asp Lys Leu Arg Glu lie Val Gly
35 40 45 35 40 45
Val Ser Thr Val Trp Arg Asp His Val Lys Ala Met Glu Glu Arg Lys 50 55 60 Val Ser Thr Val Trp Arg Asp His Val Lys Ala Met Glu Glu Arg Lys 50 55 60
Leu Leu His Ser Phe Leu Pro Lys Ser Gin Lys Val Leu Pro Pro Arg 65 70 75 80 Leu Leu His Ser Phe Leu Pro Lys Ser Gin Lys Val Leu Pro Pro Arg 65 70 75 80
Lys Met Arg Asp Ser Tyr l ie Glu Val Leu Leu Pro Leu Gly Thr Asp Lys Met Arg Asp Ser Tyr lie Glu Val Leu Leu Pro Leu Gly Thr Asp
85 90 95 Pro Glu Leu Arg Asp Lys Tyr Val Thr Val Gin Asn Thr Val Arg Phe 85 90 95 Pro Glu Leu Arg Asp Lys Tyr Val Thr Val Gin Asn Thr Val Arg Phe
100 105 110 100 105 110
Gly Arg lie Leu Glu Asp Leu Asp Ser Leu Gly Val Leu Val Cys Tyr Gly Arg lie Leu Glu Asp Leu Asp Ser Leu Gly Val Leu Val Cys Tyr
115 120 125 115 120 125
Met His Asn His Asn His Ser Thr Lys Met Ser Pro Leu Ser lie Val 130 135 140 Met His Asn His Asn His Ser Thr Lys Met Ser Pro Leu Ser lie Val 130 135 140
Thr Val Leu Val Asp Lys l ie Asp Met Cys Lys His Ser Leu Ser Pro 145 150 155 160 Thr Val Leu Val Asp Lys lie Asp Met Cys Lys His Ser Leu Ser Pro 145 150 155 160
Glu Gin Asp lie Lys Phe Thr Gly His Val Ser Trp Val Gly Asn Thr Glu Gin Asp lie Lys Phe Thr Gly His Val Ser Trp Val Gly Asn Thr
165 170 175 Ser Met Glu Val Lys Met Lys Met Phe Gin Leu His Asn Asp Glu Lys
180 185 190 165 170 175 Ser Met Glu Val Lys Met Lys Met Phe Gin Leu His Asn Asp Glu Lys 180 185 190
Tyr Trp Pro Val Leu Asp Ala Thr Phe Val Met Val Ala Arg Asp Ser Tyr Trp Pro Val Leu Asp Ala Thr Phe Val Met Val Ala Arg Asp Ser
195 200 205 195 200 205
Glu Asn Lys Gly Pro Ala Phe Val Asn Pro Leu lie Pro Glu Asn Lys 210 215 220 Glu Asn Lys Gly Pro Ala Phe Val Asn Pro Leulie Pro Glu Asn Lys 210 215 220
Glu Glu Glu Glu Leu Phe Lys Gin Gly Glu Leu Asn Lys Ser Arg Arg 225 230 235 240 lie Ala Phe Ser Thr Ser Ser Leu Leu Lys Val Ala Pro Ser Ser Glu Glu Glu Glu Glu Leu Phe Lys Gin Gly Glu Leu Asn Lys Ser Arg Arg 225 230 235 240 lie Ala Phe Ser Thr Ser Ser Leu Leu Lys Val Ala Pro Ser Ser Glu
245 250 255 Glu Arg Asn l ie lie His Glu Leu Phe Leu Thr Thr Leu Asp Pro Lys 245 250 255 Glu Arg Asn lie lie His Glu Leu Phe Leu Thr Thr Leu Asp Pro Lys
260 265 270 260 265 270
Thr lie Ser Phe Gin Ser Arg lie Leu Pro Pro Lys Ala Val Trp Met Thr lie Ser Phe Gin Ser Arg lie Leu Pro Pro Lys Ala Val Trp Met
275 280 285 275 280 285
Glu Asp Thr Lys Leu Lys Ser Leu Asp lie Cys His Pro Gin Glu Arg 290 295 300 Glu Asp Thr Lys Leu Lys Ser Leu Asp lie Cys His Pro Gin Glu Arg 290 295 300
Asn Val Phe Asn Arg lie Phe Gly Gly Phe Leu Met Arg Lys Ala Tyr 305 310 315 320 Asn Val Phe Asn Arg lie Phe Gly Gly Phe Leu Met Arg Lys Ala Tyr 305 310 315 320
Glu Leu Ala Trp Ala Thr Ala Cys Ser Phe Gly Gly Ser Arg Pro Tyr Glu Leu Ala Trp Ala Thr Ala Cys Ser Phe Gly Gly Ser Arg Pro Tyr
325 330 335 Val Val Thr Val Asp Asp l ie Met Phe Gin Lys Pro Val Glu Val Gly 325 330 335 Val Val Thr Val Asp Asp lie Met Phe Gin Lys Pro Val Glu Val Gly
340 345 350 340 345 350
Ser Leu Leu Phe Leu Ser Ser Gin Val Cys Phe Thr Gin Asp Asn Tyr Ser Leu Leu Phe Leu Ser Ser Gin Val Cys Phe Thr Gin Asp Asn Tyr
355 360 365 355 360 365
lie Gin Val Arg Val His Ser Glu Val Ser Ser Leu Asp Ser Arg Glu 370 375 380
lie Gin Val Arg Val His Ser Glu Val Ser Ser Leu Asp Ser Arg Glu 370 375 380
01 01
OJd nig Ϊ¾Λ OJd J dsy s OJd nig Ϊ¾Λ OJd J dsy s
0 f Z OZf 0 f Z OZf
sXl ΙΙΘΊ J¾ ΐ¾Λ OJd J¾ s J9 sXq ai^ STH Say UT9 dsysXl ΙΙΘΊ J¾ ΐ¾Λ OJd J¾ s J9 sXq ai ^ STH Say UT9 dsy
1^ OTt- 90 1 ^ OTt- 90
J τΐθΐ ^9W J8S nig Xt9 J J¾ s οα^ 3¾ 311 ngq oaj J τΐθΐ ^ 9W J8S nig Xt9 J J¾ s οα ^ 3¾ 311 ngq oaj
00, S6C 06S 98G ητΰ s^i nig JQS J¾ 9¾d STH «sy J¾ J¾ STH 00, S6C 06S 98G ητΰ s ^ i nig JQS J¾ 9¾d STH «sy J¾ J¾ STH
8rO/86d /XDJ 96ΙΌ0/66 OAV
8rO / 86d / XDJ 96ΙΌ0 / 66 OAV
Claims
1. 以下の (a) 又は (b) のタンパク質をコードする DNA。 1. DNA encoding the following protein (a) or (b):
(a) 配列番号: 2に記載のアミノ酸配列からなるタンパク質 (a) a protein consisting of the amino acid sequence of SEQ ID NO: 2
( b )配列番号: 2に記載のァミノ酸配列のうち 1若しくは複数のァミノ 酸が欠失、 置換及びノ又は付加されたアミノ酸配列からなり、 かつ破骨細 胞分化促進活性を有するタンパク質 (b) a protein comprising an amino acid sequence in which one or more amino acids of the amino acid sequence described in SEQ ID NO: 2 have been deleted, substituted and / or added, and which has osteoclast differentiation promoting activity
2. 配列番号: 1に記載の塩基配列からなる DNA、 又はその DNAと ストリンジェントな条件下でハイブリダイズし、 かつ破骨細胞分化促進活 性を有するタンパク質をコードする DNA。 2. DNA comprising the nucleotide sequence of SEQ ID NO: 1, or a DNA which hybridizes with the DNA under stringent conditions and encodes a protein having osteoclast differentiation promoting activity.
3. 請求項 1又は 2記載の D N Aがコードするタンパク質。 3. A protein encoded by the DNA according to claim 1 or 2.
4. 配列番号: 2に記載のアミノ酸配列からなる、 請求項 3記載のタン パク質。 4. The protein according to claim 3, consisting of the amino acid sequence of SEQ ID NO: 2.
5. 請求項 1又は 2記載の DN Aを含有する発現べクタ一。 5. An expression vector containing the DNA of claim 1 or 2.
6. 請求項 5記載の発現べクタ一によって形質転換された形質転換体。 6. A transformant transformed by the expression vector according to claim 5.
7. 請求項 6記載の形質転換体を、 請求項 5記載の発現ベクターの発現 可能な条件下で培養することを特徴とする、 組換えタンパク質の生産方法7. A method for producing a recombinant protein, comprising culturing the transformant according to claim 6 under conditions that allow expression of the expression vector according to claim 5.
8. 請求項 3又は 4記載のタンパク質を有効成分として含有する医薬。8. A medicament comprising the protein according to claim 3 or 4 as an active ingredient.
9. 請求項 3又は 4記載のタンパク質の、 少なくとも 6アミノ酸以上の 部分よりなるペプチド断片。 9. A peptide fragment comprising at least 6 amino acids or more of the protein of claim 3 or 4.
10. 請求項 3又は 4記載のタンパク質、 あるいは請求項 9記載のぺプ チド断片の、 いずれかに対する抗体。 10. An antibody against any one of the protein according to claim 3 or 4, or the peptide fragment according to claim 9.
11. 請求項 3又は 4記載のタンパク質を用いることを特徴とする、 破 骨細胞分化促進活性阻害剤のスクリーニング方法。 11. A method for screening for an osteoclast differentiation promoting activity inhibitor, comprising using the protein according to claim 3 or 4.
12. 請求項 11記載のスクリーニング方法により得られる、 破骨細胞
分化促進活性阻害剤。 12. An osteoclast obtained by the screening method according to claim 11. Differentiation promoting activity inhibitor.
1 3 . 請求項 9記載のペプチド断片又は請求項 1 0記載の抗体からなる、 請求項 1 2記載の破骨細胞分化促進活性阻害剤。 13. The osteoclast differentiation promoting activity inhibitor according to claim 12, comprising the peptide fragment according to claim 9 or the antibody according to claim 10.
1 4 . 請求項 1又は 2記載の D N Aを人為的に染色体中に導入するか、 あるいはいずれかを染色体中から欠損させたトランスジエニック動物。
14. A transgenic animal in which the DNA according to claim 1 or 2 is artificially introduced into a chromosome, or one of which is deleted from the chromosome.
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP9/187548 | 1997-06-26 | ||
JP18754897 | 1997-06-26 | ||
JP19325597 | 1997-07-02 | ||
JP9/193255 | 1997-07-02 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1999000496A1 true WO1999000496A1 (en) | 1999-01-07 |
Family
ID=26504430
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP1998/002869 WO1999000496A1 (en) | 1997-06-26 | 1998-06-26 | Novel osteoclast differentiation promoting factor and its gene |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO1999000496A1 (en) |
-
1998
- 1998-06-26 WO PCT/JP1998/002869 patent/WO1999000496A1/en active Application Filing
Non-Patent Citations (3)
Title |
---|
BIOCHEM. BIOPHYS. RES. COMMUN., 196(3), 1993, AKIKO KUKITA et al., "Induction of Mononuclear Precursor Cells with Osteoclastic Phenotypes in a Rat Bone Marrow Culture System Depleted of Stromal Cells", p. 1383-1389. * |
CELL, 89, 18 April 1997, L. BENNETT et al., "Osteoprotegerin: A Novel Secreted Protein Involved in the Regulation of Bone Density", p. 309-319. * |
ENDOCRINOLOGY, 122(4), 1988, TATSUO SUDA et al., "Osteoclast-Like Cell Formation and Its Regulation by Osteotropic Hormones in Mouse Bone Marrow Culture", p. 1373-1382. * |
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