Protein, specifically communicating with inhibiting osteoclastogenic factor (ocif) (options), his dna encoding (options), dna as a probe (options), the method of obtaining protein (options), the method of screening substances (options), antibody (options), the method of obtaining polyclonal antibodies, hybridoma (options), the method of obtaining monoclonal antibodies, a method of measuring ocif - binding protein, a pharmaceutical composition (options) and drug (options)

 

The invention relates to biotechnology, in particular genetic engineering, and medicine, and can be used to obtain a protein specifically binding to inhibiting osteoclastogenic factor (OCIF). OCIF-binding protein specifically binds to inhibiting osteoclastogenic factor and shows high affinity against OCIF (dissociation constant on the cell membrane Kd=10-9M or less), has a molecular mass of approximately 30000-40000 when measured by electrophoresis in SDS-polyacrylamide with non conditions, active maintaining or enhancing differentiation and maturation of osteoclasts. Recombinant OCIF-binding protein is produced by culturing cells transformed by a vector containing a DNA encoding OCIF - binding protein. The antibody recognizes OCIF-binding protein, obtained by immunization of an animal or by cultivation hybridoma BP-6264 or BP-6265, or BP-6266. The antibody used to produce pharmaceutical compositions for prevention or treatment of disorders or abnormalities of bone metabolism. The invention allows to develop the means for prevention or treatment of diseases related is hnologie

This invention relates to a new protein (molecule OCIF-binding protein, then the protein can be called RBM), which is associated with inhibiting osteoclastogenic factor (which hereinafter may be referred to as OCIF) and a method for production of this protein.

This invention relates to DNA encoding this protein, proteins containing the amino acid sequence encoded by this DNA, a method for production of this protein using methods of genetic engineering and pharmaceutical compositions containing this protein.

This invention also relates to methods of screening, with the use of this protein and the DNA, substances for the regulation of expression of this protein, substances inhibiting or regulating the biological activity of this protein, or receptor, transferring the signal of this protein through interaction with this protein substances obtained by means of this screening, and to pharmaceutical compositions that contain obtained substances.

This invention also relates to antibodies against this protein, methods of producing these antibodies and pharmaceutical compositions containing these antibodies.

Prior art Bone metabolism depends on the total activity of the human bone (resorption). It is believed that the disturbance of bone metabolism caused by violation of the balance between osteogenesis and bone resorption. Osteoporosis, hypercalcemia, Paget's disease (deformirujuschij osteodystrophy, renal osteodystrophy, chronic ramakant, osteoarthritis, etc., known as diseases associated with the disturbance of bone metabolism. Osteoporosis is a common disease caused such a disturbance of bone metabolism. This disease occurs when bone resorption by osteoclasts prevails over osteogenesis performed by osteoblasts. This disease is characterized by reduced subjected to calcification of bone material and bone matrix. Although the mechanism of this disease has not been elucidated completely, the disease causes pain in the bones, making them brittle and can lead to fractures. This disease is becoming a social problem because it increases the number of bedridden persons, as increasing population of older people. Obtaining a therapeutic agent for this disease is highly desirable. It is expected that the disease is caused by decrease in bone mass, must be cured by the suppression of bone resorption, acceleration of bone formation (OST the Xia acceleration of cell proliferation, differentiation or activation of osteoblasts, which form bone, or suppression of cell proliferation, differentiation or activation of osteoclasts, which resolves the bone. In recent years, great interest was caused by hormones, low molecular weight substance or a physiologically active proteins exhibiting such activity, and these substances are the subject of intensive fundamental research and development carried out at the present time.

Medicines such as calcitonine agents, agents active forms of vitamin D3, hormonal agents containing estradiol, ipriflavone, vitamin K2and biphosphonate compounds already known as medicines for the treatment and reduction treatment of diseases associated with bone. Developed clinical tests for derivatives of active forms of vitamin D3derivatives of estradiol and bisphosphonate connections of the second and third generation for the development of therapeutic agents with excellent efficacy and minimal side effects.

However, methods of treatment using these agents were not always satisfactory in efficiency and therapeutic results. On the effective. Some agents used for the treatment of diseases associated with bone metabolism, used only sparingly because of their side effects. In addition, methods of treatment using two or more agents in combination are currently the main focus in the treatment of diseases associated with bone metabolism such as osteoporosis. From this point of view, desirable to develop drugs with a mechanism of action different from conventional medicines, and exhibiting higher efficiency and minimal side effects.

As mentioned above, the cells that control bone metabolism are osteoblasts and osteoclasts. It is known that these cells have close interaction, called "mate". In particular, it is known that cytokines, such as Interleukin 1 (IL-1), 3 (IL-3), 6 (IL-6) and 11 (IL-11), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), interferon-(IFN-), tumor necrosis factor(TNF-) and transforming growth factor(TGF-), the Secretary is of theoklitos (Raisz: Disorders of Bone and Mineral Metabolism, 287-311, 1992; Suda et al.: Principles of Bone Biology, 87-102, 1996; Suda et al.: Endocrine Reviews, 4, 266-270, 1995, Lacey et al.: Endocrinology, 186, 2369-2376, 1995). It was reported that osteoblastic stromal cells play an important role in the differentiation and maturation of osteoclasts, as well as such functions of osteoclasts, as bone resorption by Mature osteoclasts through cell-cell contacts with immature precursors of osteoclasts or Mature osteoclasts.

Considered that factor, called factor in the differentiation of osteoclasts (ODF, Suda et al: Endocrine Rev., 13, 66-80, 1992; Suda et al.: Bone, 17, 87S-91S, 1995), is expressed on the membrane osteoblastic stromal cells and is involved in the formation of osteoclasts by contact of cells. According to this hypothesis on the cells-the precursors of osteoclasts present receptor ODF. However, still not been cleaned or identified neither ODF nor its receptor. No reports concerning their properties, mechanism or structure. Thus, the mechanism involved in the differentiation and maturation of osteoclasts, has not yet been clarified sufficiently. Elucidation of this mechanism will be a great contribution not only in fundamental medicine, but also in the development of new drugs for the treatment of sabedoria in connection with this situation and found inhibitory osteoclastogenic factor (OCIF) in the culture broth of lung fibroblasts of the human embryo, IMR-90 (ATCC Deposition No. CCL186) (WO 96/26217).

The authors of this invention have managed to clone DNA encoding OCIF, to obtain recombinant OCIF in animal cells and to confirm in vivo pharmaceutical effects (improving effects on bone metabolism, and so on) recombinant OCIF. It is expected that OCIF can be used as an agent for the prevention or treatment of diseases associated with impaired bone metabolism, with greater efficiency than the efficiency of conventional drugs, with fewer side effects.

Description of the invention

The authors of this invention have conducted a search of a protein that is associated with inhibiting osteoclastogenic factor (OCIF), and found that the OCIF-binding protein is expressed on osteoblastic stromal cells cultured in the presence of the factor of bone resorption, such as the active form of vitamin D3and parathyroid noise (RTN). In addition, the authors of the present invention investigated the characteristics and physiological functions OCIF-binding protein and found that this protein exhibits the biological activity of factor that supports or enhances differentiation and maturation of osteoclasts from immature precursors of osteoclasts. it represents an important protein, regulating the differentiation and maturation of osteoclasts from immature precursors of osteoclasts in the system cocultivation osteoblastic stromal cells and spleen cells. Success in identifying and allocating protein, acts as a factor that supports or enhances differentiation and maturation of osteoclasts, the present invention has made possible the screening of new drugs, applicable for treatment of diseases of bone metabolism, based on the mechanism of bone metabolism using the protein of the present invention.

In accordance with this object of this invention to provide a new protein (OCIF-binding molecule or RBM), which is associated with inhibiting osteoclastogenic factor (OCIF), and the method of obtaining this protein.

Another purpose of this invention to provide a DNA that encodes this protein, a protein containing the amino acid sequence encoded by this DNA, and the method of obtaining this protein using methods of genetic engineering and pharmaceutical compositions containing this protein.

The next objective of this invention is the development of methods for screening substances that control the expression of this protein, with Pimenov, transducers action of this protein by binding to this protein, substances obtained by screening, and pharmaceutical compositions containing these substances.

Another purpose of this invention to provide antibodies against this protein, methods of obtaining such antibodies and pharmaceutical compositions containing these antibodies.

The protein of the present invention has the following physico-chemical properties and biological activity:

(a) affinity: specifically binds to inhibiting osteoclastogenic factor (OCIF) and exhibits high affinity against OCIF (dissociation constant on the cell membrane Kd=10-9M or less);

(b) molecular weight: the molecular weight of about 30000-40000 in the determination by electrophoresis in SDS-polyacrylamide gel (SDS-page-electrophoresis) when non conditions and the average molecular weight of approximately 90000-110000 when sewing with the Monomeric form of OCIF and

(c) biological activity: active maintaining or enhancing differentiation and maturation in the system cocultivation mouse osteoblastic stromal cells and mouse spleen cells in the presence of factors of bone resorption, such as the active form in the fine lines and murine spleen cells in the presence of the active form of vitamin D3or PTH is well known as a typical system in vitro culture for the formation of osteoclasts. Cells expressing the protein of the present invention, can be determined by testing the binding of OCIF mouse osteoblastlike stromal cells or mouse spleen cells in the presence or in the absence of the active form of vitamin D3. The protein of the present invention is characterized as a protein that is specifically induced on osteoblastic stromal cells cultured in the presence of osteo factors, such as the active form of vitamin D3or PTH. In addition, the protein of the present invention may be characterized as a protein exhibiting biological activity that supports or enhances differentiation and maturation of osteoclasts, on the basis of the following results. Namely, the formation of osteoclasts is inhibited dependent dose-dependent manner by adding 1-40 ng/ml OCIF to the above system coculture in the presence of the active form of vitamin D3, the time course of expression of the protein of the present invention on the ST2 cells in the presence of the active form of vitamin D3correlates well with the time course of the formation of osteoclasts in this coculture. For these cells to maintain the formation of osteoclasts and OCIF binding protein of the present invention on the ST2 cells completely inhibits the formation of osteoclasts.

The affinity of the protein of the present invention in respect of OCIF can be estimated by tagging OCIF and test the binding of labeled OCIF with the surface membrane of animal cells. OCIF may be labeled by standard way of tagging, such as tagging radioactive isotope or fluorescent tagging. Tagging tyrosine residues125I may be described as a typical example of tagging OCIF radioactive isotope. Can be used such methods of tagging as a way to Iodophenol, with chloramine T and enzymatic method. The binding of labeled OCIF with the surface membrane of animal cells may be tested by a common way. The addition of unlabeled OCIF to the environment used to test binding in a concentration of 100-400 times higher than the concentration of labeled OCIF, provides a measure of nonspecific binding. The amount of specific binding OCIF can be calculated by subtracting the magnitude of nonspecific binding from total binding labeled OCIF. The affinity of the protein of the present invention expressed on the cell membrane, in respect of OCIF can be measured by the change in the value labeled OCIF and analysis of specific binding by using the curve of Scatchard. the th invention is characterized by a high affinity (dissociation constant on the cell membrane d=10-9M or less) in relation to enzyme inhibition osteoclastogenic factor (OCIF). The molecular mass of the RBM can be measured by means of gel filtration, electrophoresis in SDS-page or so on, To determine the exact molecular weight is preferable electrophoresis in SDS-page. RBM is a protein having a molecular weight of about 40,000 (40000±4000) in reducing conditions.

The protein of this invention can be derived from murine osteoblastic stromal cell line ST2, a murine cell line preadipocytes RA, osteoblastic cell lines of human or other osteoblastic cells selected from mammalian cells, such as human, mouse or rat. As substances that induce the expression of the protein of the present invention may be provided osteo factors, such as the active form of vitamin D3(calcitriol), parathyroid hormone (PTH), interleukin (IL-1, IL-6, IL-11), Oncostatin M and leukemia inhibitory factor (LIF). These substances can be added at a concentration of 10-8M (the active form of vitamin D3and PTH), 10 ng/ml (IL-11), or 1 ng/ml (Oncostatin M). IL-6 is preferably used at a concentration of 20 ng/ml 500 ng/ml of soluble receptor of IL-6. Preferably can be used /chr/945.gif">MEME, to which was added 10-8M active form of vitamin D3, 10-7M dexamethasone, and 10% fetal calf serum. Cultured cells can be collected by scratching "scraper" for cells. The collected cells can be stored at -80To use.

The protein of this invention can be effectively purified from the membrane fractions of the collected cells. The membrane fraction can be obtained by the conventional method, which is used to produce intracellular organelles. Different types of protease inhibitors can be added to the buffer used to obtain the membrane fraction. Examples of protease inhibitors include inhibitors of serine protease inhibitors of thiol proteases and inhibitors Metaprocess. As specific examples are PMSF, APMSF, EDTA, o-phenanthroline, leupeptin, pepstatin And, Aprotinin, soybean trypsin inhibitor. The homogenizer of the downs, polychrony homogenizer or ultrasonic processor can be used for homogenization of the cells. The homogenate of cells suspended in a buffer solution containing 0.5 M sucrose, and centrifuged for 10 minutes at 600g to separate nuclei and n the early fractions in the form of sediment. The obtained membrane fraction is treated with different types of detergents for effective solubilization and extraction of the protein of the present invention from the cell membrane. Can be used detergents that are commonly used to solubilize proteins of cell membranes, such as CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), Triton X-100, Nikkol and n-octylglucoside. It is preferable for the membrane fraction added 0.5% CHAPS, and the mixture is stirred for 2 hours at 4For solubilization of the protein of the present invention. Thus obtained sample centrifuged at 150000g for 60 minutes to obtain a fraction solubilizing membranes in the supernatant.

The protein of this invention can be purified from fraction solubilizing membranes using columns, gel or resin associated with OCIF. Immobilized OCIF can be OCIF selected from the culture broth of lung fibroblasts human embryo IMR-90, described in WO 96/26217, or rOCIF obtained using methods of genetic engineering. rOCIF can be obtained by introducing the cDNA of human, mouse cDNA or cDNA of the rat in expressing the vector in accordance with generally accepted way, moved the key insect to get rOCIF and cleaning rOCIF. Received OCIF has a molecular mass of approximately 60 kDa (Monomeric form) or 120 kDa (dimeric form). OCIF dimeric form is preferred for immobilization. Examples of gels and resins, which immobilized OCIF are ECH-Sepharose 4B, EAH-Sepharose 4B, Thiopropyl Sepharose 6B, CNBr-activated Sepharose 4B, activated CH Sepharose 4B, epoxy-activated Sepharose 6B, activated thiol Sepharose 4B (manufactured Pharmacia Co.), TSKgel AF-Epoxy Toyopal 650, TSKgel AF-Amino Toyopal 650, TSKgel AF-Formyl Toyopal 650, TSKgel AF-Carboxy Toyopal 650, TSKgel AF-Tresyl Toyopal 650 (manufactured Tosoh Corporation), Amino-Cellulofine, Carboxy-Cellulofine, FMP activated Cellulofine, Formyl-Cellulofine (manufactured Seikagaku Kogyo Co.), Affigel 10, Affigel 15 and Affiprep 10 (manufactured BioRad Co.). As speakers, which may be immobilized OCIF can be given HiTrap NHS-activated column (Pharmacia Co.), TSKgel AF-Tresyl 5PW (Tosoh Corporation), etc. as a specific example of the method of immobilization OCIF with HiTrap NHS-activated column (1 ml, Pharmacia Co.) can be described the following way. In particular, 1 ml of 0.2 M solution Panso3/0.5 M NaCl (pH 8.3) containing 13,0 mg OCIF, is injected into this column for carrying out binding assays at room temperature for 30 minutes. The column is applied sequentially with 0.5 M ethanolamine/0.5 M NaCl (pH 8,3) and 0.1 M acetic acid, 0.5 M room temperature to block excess active groups. The column is sequentially washed twice with 0.5 M ethanolamine/0.5 M NaCl (pH 8,3) and 0.1 M acetic acid/0.5 M NaCl (pH 4,0) and then washed with a solution of 50 mm Tris/1 M NaCl/0,l% CHAPS (pH 7.5), receiving a column with immobilized OCIF. The protein of this invention can be effectively purified by a column with immobilized OCIF, thus prepared, or gel or resin with immobilized OCIF.

For the inhibition of the degradation of the protein of the present invention, it is desirable to add the above-mentioned protease inhibitors to the buffer solutions used for the purification of this protein. The protein of this invention can be purified by applying the above-mentioned fractions solubilizing membranes on a column with immobilized OCIF or mixing with the gel or resin with immobilized OCIF and elution of the protein from the column, gel or resin acid, various protein denaturing agents, cacodylate buffer, or etc., it is Desirable to use for elution acid and neutralized immediately after elution to reduce the denaturation of the protein of the present invention. As the acid solution used for elution may be given a solution of 0.1 M glycine-hydrochloric acid (pH 3.0), a solution of 0.1 M glycine-hydrochloric acid (pH of 2.0), a solution of 0.1 M citrate notresponsible for purification of various proteins from biological materials and various cleaning methods, using physico-chemical properties of this protein. For the concentration of solutions containing the protein of the present invention can be used generally accepted methods used in the cleaning process for proteins, such as ultrafiltration, lyophilization and salting out. Preferably use, for example, ultrafiltration with the use of Method-10 (BioRad Co.). As the cleaning methods used in the combination of different methods commonly used for protein purification such as ion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, chromatography with reversed-phase (reverse-phase chromatography and preparative electrophoresis. More specifically, it is possible to purify the protein of the present invention by a combination of gel filtration on a column of Superose 12 (Pharmacia Co.) and reversed-phase chromatography. For detection of the protein of the present invention determine the activity of the binding protein of the present invention with immobilized OCIF or material, contacting the immobilized OCIF, immunoprecipitation antibodies against OCIF and analyzed by electrophoresis in SDS-polyacrylamide gel electrophoresis SDS-page).

Received this protein izopet as congenital systemic osteoporosis (a disease Albers-Schoenberg), or as a reagent for research or diagnosis of such diseases.

This invention also provides DNA encoding a new protein (OCIF-binding molecule or RBM), which is associated with inhibiting osteoclastogenic factor proteins containing the amino acid sequence encoded by this DNA, the method of obtaining this protein by methods of genetic engineering and pharmaceutical compositions containing such protein. In addition, this invention provides methods of screening substances for regulating the expression of RBM, the method of screening substances, inhibiting or modifying the biological activity of RBM, or a method of screening for receptors that transmit the RBM action by binding with RBM, and pharmaceutical compositions containing the compounds, obtained as a result of this screening.

New RBM protein encoded by the DNA of the present invention has the following physico-chemical properties and biological activity:

(a) binds specifically to inhibiting osteoclastogenic factor (OCIF),

(b) has a molecular weight of about 40,000 (±4000) in determining by means of electrophoresis in SDS-page under reducing conditions and an average molecular weight 90000-110000 when stitching with monomer>gebarowski osteoclastogenic factor (OCIF) of a person who is used as a probe to identify the DNA coding for the RBM, OCIF-binding molecule of the present invention, and to evaluate properties RBM, can be isolated from the culture broth fibroblastoid cell lines the lungs of a human embryo IMR-90 according to WO 96/26217. Recombinant OCIF human, recombinant OCIF mouse, recombinant OCIF rats, etc. can be also used for separation and identification of DNA coding for the RBM. These recombinant proteins OCIF can be obtained by embedding the DNA fragments encoding these proteins, in expressing the vectors in accordance with conventional methods, the expression in animal cells, such as cells Cho cells KSS or Namalwa cells, or insect cells, and their cleanup.

As a way of marking the cDNA that encodes a protein target (clone cDNA) can be used a method comprising the determination of a partial amino acid sequence of this protein and isolation of cDNA targets using hybridization using the nucleotide sequence corresponding to this amino acid sequence. Another available method, even when the amino acid after the introduction of this cDNA into cells and screened for expression of the protein target for selection of the target cDNA (method of expression cloning, D'andrea et al.: Cell, 57, 277-285, 1989; FuKunaga et al.: Cell, 61, 341-350, 1990). In the method of expression cloning suitable cell host, such as bacteria, yeast, animal cells, etc., that is selected depending on the purpose. In many cases choose animal cells as host cells for cloning cDNA that encodes a protein such as a protein of the present invention, which presumably is present on the membrane surface of animal cells. Usually select cells-owners, having a high efficiency of transfection of DNA and reaching high levels of expression of the introduced DNA. One example of such cells animal cells are monkey kidney COS-7 used in this invention. Because of the large T-antigen SV40 is expressed in the cell COS-7, plasmid, which has a Replicator SV40 may be present in the form of an episome of multiple copies in the cell, so it is expected a high level of expression. In addition, because the expression of the protein target cells COS-7 reaches its maximum within a few days after the introduction of DNA, this cell is suitable for rapid screening of zwyczajna high level. The factor that has the greatest influence on the expression of a gene located on a plasmid, is the promoter. As promoters, providing a high expression, using such promoters as the promoter SRand derived from the cytomegalovirus promoters. For screening of cDNA coding for a membrane protein, according to the strategy expression cloning using screening procedures, such as the method of binding the method of panning or film emulsion method.

This invention relates to a DNA that encodes a protein (RBM), which specifically binds to OCIF, the selected combination of strategy expression cloning and screening by way of binding to expressed protein, and screening of physiologically active substances using this DNA or expressed protein. RBM encoded by the DNA of the present invention, can be detected by tagging OCIF and testing the binding of labeled OCIF with the surface membrane of animal cells. OCIF can be marked accepted way of tagging, such as a method of tagging a radioactive isotope or a method for fluorescent labeling, which is used for labeling of normal proteins. As a specific example of tagging OCIF is their ways of tagging, as a way to Iodophenol, with chloramine T and enzymatic method. The binding of labeled OCIF with the surface membrane of animal cells may be tested in a common manner. The addition of unlabeled OCIF to the environment used to test binding in a concentration of 100-400 times higher than the concentration of labeled OCIF, provides a measure of nonspecific binding. The amount of specific binding OCIF can be calculated by subtracting the magnitude of nonspecific binding from total binding labeled OCIF.

The authors of this invention have assumed that there is an interaction between a factor involved in the differentiation of osteoclasts, and OCIF. Based on this assumption, the authors of the invention for separation of a protein, which binds to OCIF, was subjected to screening the expression library derived from mRNA of mouse osteoblastic stromal cell line ST2 in accordance with the following method. In particular, DNA is synthesized using mRNA ST2, was built in expressing the vector in animal cells and the vector with this insert was introduced into the cells of monkey kidney COS-7. The target protein is expressed on the cell COS-7, were subjected to screening using OCIF labeledThe OCIF. Then determined the nucleotide sequence of the DNA coding this OCIF-binding molecule (OS IF-binding molecule; RBM). In addition, it was found that the RBM encoded by this DNA, binds specifically and strongly to OCIF on the cell membrane.

Relatively mild conditions for hybridization of the DNA of the present invention are, for example, when DNA is transferred to nylon membrane and immobilized on it in accordance with generally accepted methods and hybridized in buffer solution for hybridization with probe DNA labeled with an isotope at a temperature of 40-70C for from about 2 hours to night followed by washing in 0.5SSC (0,075 M sodium chloride and 0,0075 M sodium citrate) at 45C for 10 minutes.

In particular, as a nylon membrane for transferring at her and immobilization of the DNA used Highbond N (mersham Co.). Then DNA hybridized with probe DNA labeled with32P in the buffer for rapid hybridization (Amersham, Co) at 65C for 2 hours followed by washing with 0.5SSC (0,075 M sodium chloride and 0,0075 M sodium citrate) at 45C for 10 minutes.

The system with the vitamin D3or PTH is well known as a typical system of in vitro culture for the formation of osteoclasts. The protein of the present invention is characterized as a protein that is induced specifically on osteoblastic stromal cells cultured in the presence of an agent that increases bone resorption, such as the active form of vitamin D3or PTH. In addition, due to the fact that the formation of osteoclasts stimulated by the addition of a protein encoded by a DNA of the present invention, to mouse spleen cells cultivated even in the absence of the active form of vitamin D3or PTH, believe that the RBM, which is encoded by a DNA of the present invention, is involved in the differentiation and maturation of osteoclasts.

Recombinant RBM can be obtained by embedding the DNA of the present invention in expressing vector to construct the plasmid and the introduction of this plasmid in different cells or organisms for the expression of recombinant RBM. As the owner, which can be expressed recombinant OBM can be used mammalian cells such as COS-7, CHO, Namalwa, or bacteria, such as Escherichia coli. OBM can expressionate in the form of a membrane-bound form of the protein with ispolzovaniya transmembrane domain. Produced recombinant OBM can be effectively cleaned using a suitable combination of conventional purification methods used for normal proteins, such as affinity chromatography using a column with immobilized OCIF, ion-exchange chromatography and gel filtration chromatography. Derived protein of the present invention is applicable as an agent for the treatment of diseases caused by disturbance of bone metabolism, such as congenital systemic osteoporosis, or as a reagent for research and diagnosis of such diseases.

Using protein OBM encoded by the DNA of the present invention, can be carried out the following operations screening:

(1) the screening of substances that regulate the expression of OBM,

(2) screening of substances that are specifically associated with OBM and inhibit the biological activity of OBM, and

(3) screening of proteins that are present in the cells-the precursors of osteoclasts and transmit (transducer) biological activity of OBM (receptor OBM). You can also create antagonists and agonists to this receptor OBM. In combinatorial chemistry using the above OBM or receptor RBM peptide library used for screening and the fast way of splitting (Lam et al.: Nature, 354, 82-84, 1991). According to this method, synthetic media (granules), each of which contains a specific amino acid (unit) associated with it, are prepared separately for all units. The synthesized media mix together and divide into portions equal to the number of these units. Then connect these units. This procedure is repeated n times to obtain library containing media for which there are n units. According to the method of synthesis of each storage pool has one type of sequence. Thus, it is possible to identify a peptide that specifically bind to the protein of the present invention by selection pool, which gives the signal, the positive in this screening method, using the protein of the present invention, and by determining the amino acid sequence of a peptide linked to this pool. Another method is a method ragovoy display that uses phage carrying the synthetic DNA that encodes a peptide with random amino acid sequences. This method has the advantage that the number of molecules in the library in comparison with the above method, a library of synthetic peptides, but has the disadvantage that honor, which are not available in this library, if the phage is unable to Express these sequences. In the way ragovoy display screening system using the protein of the present invention can also be applied to determine the nucleotide sequence that encodes the peptide, i.e., the phage that specifically bind to the protein of the present invention, concentrate penninga selected phage amplified in Escherichia coil and determine the nucleotide sequence encoding this peptide. In addition, the peptide having a high specificity and high affinity in relation to RBM or receptor RBM can be siniawan of peptide libraries using screening systems mentioned above in (2) and (3) screening in the presence of RBM OCIF or by increasing the concentration of RBM or OCIF. Thus select only positive media pools or phages. For example, low-molecular peptide agonists exhibiting EPO (erythropoietin-like) activity, were subjected to the screening of peptide libraries using erythropoietin receptor (EPO), which is a hematopoietic hormone, tertiary structure of this substance was analyzed and based on the tertiary structure of sintesio).

The authors of this invention have found previously using enzyme inhibition osteoclastogenic factor OCIF that OCIF-binding protein specifically expressed on osteoblastic stromal cell line ST2, cultured in the presence of the active form of vitamin D3or parathyroid hormone (PTH). Further, the authors of this invention have found that this protein exhibits a biological activity to maintain or stimulate the differentiation or maturation of osteoclasts from immature precursor cells of osteoclasts, and found a variety of physico-chemical properties and biological activity of this protein by cleaning it. For comparison recombinant RBM expressed by the DNA of the present invention, and the above-mentioned purified natural protein type, which specifically binds to OCIF, the authors of this invention have investigated the physico-chemical properties and biological activity of these two proteins. In the result, it was confirmed that these two proteins (1) are both membrane-bound proteins specifically binding to OCIF, (2) have a molecular weight of about 40,000 in the determination by electrophoresis in SDS-page and (3) have an average molecular weight in the identical but both detect protein biological activity to maintain or stimulate the differentiation or maturation of osteoclasts, suggesting that they represent one and the same protein. In addition, it was confirmed that the rabbit polyclonal antibodies against RBM obtained using purified protein produced by expression of the DNA of the present invention with the aid of genetic engineering methods (recombinant RBM), cross-reacted with the above-described purified natural protein type and inhibited the specific binding of this purified protein natural type OCIF as these antibodies inhibit the specific binding of the RBM OCIF. Based on these results it is clear that recombinant RBM expressed by the DNA of the present invention, identical protein natural type, which specifically binds to OCIF.

For isolation of the gene (cDNA) encoding OCIF-binding protein of the person (hereinafter called RBM person), which specifically binds to OCIF and active support and promote the differentiation and maturation of osteoclasts from mouse spleen cells as well as the RBM dose natural type or recombinant murine RBM, Bibliotheek cDNA fragment RBM person as a probe. cDNA-fragment RBM person received polymerase chain reaction (PCR) in accordance with the method mentioned above, using cDNA derived from a lymph node of a person, as a matrix, and primers derived from the cDNA of murine RBM. The result was isolated cDNA encoding a human protein that specifically binds to OCIF, and determined the nucleotide sequence of cDNA coding this OCIF-binding protein molecule (i.e., cDNA, coding for the RBM person). Like murine this RBM RBM human encoded by this cDNA has the property of strong and specific binding to OCIF on the cell membrane and actively maintain, and enhance the differentiation and maturation of osteoclasts from the spleen cells of the mouse. In particular, this invention provides DNA encoding a new protein RBM person that is associated with inhibiting osteoclastogenic factor (OCIF), a protein that has the amino acid sequence encoded by this DNA, a method of obtaining a protein exhibiting the property of specific binding to OCIF and activity maintain, and enhance the differentiation and maturation of osteoclasts from the spleen cells of the mouse by means of genetic engineering, pharmaceutical to the screening of substances, regulating the expression of RBM person, the method for screening substances that inhibit or modulate the activity of RBM person and transmit the action of the RBM, and pharmaceutical compositions containing the compounds, obtained these screenings.

This invention also provides DNA encoding a new protein RBM person, which specifically binds to OCIF and exhibits biological activity of maintaining and stimulating the differentiation and maturation of osteoclasts, a protein that has the amino acid sequence encoded by this DNA, a method of obtaining a protein exhibiting the property of specific binding to OCIF and activity maintain, and enhance the differentiation and maturation of osteoclasts from the spleen cells of the mouse by means of genetic engineering, and pharmaceutical compositions containing this protein, for the treatment of diseases that cause disturbance of bone metabolism. In addition, this invention provides a method of screening substances that regulate the expression of RBM person, the method of screening substances that inhibit or modulate the activity of RBM person by associating with him, the method of screening of receptors, binding with RBM person and transmitting the action RBM prevention or treatment of diseases, causing disturbance of bone metabolism.

New OCIF-binding protein molecule man (RBM), which is encoded by a DNA of the present invention has the following physico-chemical properties and biological activity:

(a) binds specifically to inhibiting osteoclastogenic factor (OCIF) (WO 96/26217),

(b) has a molecular weight of about 40,000 (±5000) in determining by means of electrophoresis in SDS-page under reducing conditions and an average molecular weight of approximately 90000-110000 when sewing with the Monomeric form of OCIF and

(c) active support and promote the differentiation and maturation of osteoclasts.

cDNA RBM mouse, which encodes murine OCIF-binding protein and is used as a probe for isolation and identification of cDNA coding for the RBM person of this invention may be selected in accordance with the above method, the cDNA library of mouse osteoblastic stromal cell line ST2. Ingibiruyushee osteoclastogenic the human factor (OCIF), which is necessary for the evaluation of properties and biological activity of the protein obtained by expression of the cDNA for the RBM person, may be received in accordance with the method described in WO 96/26217, the selection of cultural drowsey OCIF. Recombinant OCIF human, recombinant OCIF mouse, recombinant OCIF rats, etc. can be used to evaluate properties and biological activity RBM person. These recombinant OCIF can be obtained in accordance with generally accepted ways of embedding expressing the cDNA in the vector, expression of this cDNA in animal cells, such as cells Cho cells KSS or Namalwa cells, or insect cells and purification of expressed proteins.

The following methods can be used to identify cDNA that encodes a protein target (clone cDNA). (1) a Method comprising the purification of this protein, determination of partial amino acid sequence of this protein and isolation of cDNA targets using hybridization using a DNA fragment containing the nucleotide sequence corresponding to this amino acid sequence as a probe, (2) the method is applicable even in the case when the amino acid sequence of the protein is unknown, providing for the construction of cDNA library in expressing vector, the introduction of this cDNA library into cells and screened for expression of the protein target for selection of the target cDNA (method expression cloning), ywaniem cells or human tissue, using hybridization or polymerase chain reaction (PCR) using cDNA that encodes a protein derived from a mammal (not a person) that has the same characteristics and biological activity of that protein is the target of human origin, as a probe, under the assumption that this cDNA probe has a high homology with the cDNA of human origin, which should be cloned. Based on the assumption that the cDNA RBM human origin has a high homology with cDNA RBM mouse, you can determine which cells or tissues produce RBM person, by way of Northern hybridization using cDNA RBM mouse as a probe. cDNA RBM person can be obtained in the following way using the mouse primer RBM obtained from cDNA RBM mouse. The cDNA fragments RBM person can be obtained by the method of PCR using cDNA derived from producing RBM human tissues, such as lymph nodes of a person, as a matrix. These cDNA fragments RBM person used as probes for screening cDNA library producing RBM human cells or tissues that have been identified according to either the IKI specific binding to OCIF and active support and promote the differentiation and maturation of osteoclasts. Since RBM encoded by the DNA of the present invention is a membrane-bound protein containing the transmembrane domain, this protein can be detected by labeling OCIF and testing the binding of labeled OCIF with the surface of animal cells, which is expressed cDNA of the present invention. The above-described method of labeling with a radioactive isotope or fluorescein commonly used for labeling of proteins, can be used for tagging OCIF.

The molecular mass of the protein expressed cDNA RBM person of this invention may be evaluated by gel-filtration chromatography, electrophoresis in SDS-page or etc., For accurate determination of molecular weight, it is desirable to use the method of electrophoresis in SDS-page, in which RBM man was characterized as a protein having a molecular weight of about 40,000 (40000±5000), under reducing conditions.

Relatively mild conditions for hybridization of the DNA of the present invention are, for example, when DNA is transferred to nylon membrane and immobilized on it in accordance with the generally accepted method and hybridized with probe DNA labeled with the isotope, in a buffer solution for hybridization with temperd src="https://img.russianpatents.com/chr/215.gif">SSC (0,075 M sodium chloride and 0,0075 M sodium citrate) at 45C for 10 minutes. In particular, as a nylon membrane for transferring at her and immobilization of the DNA used Highbond N (Amersham, Co.). Then DNA hybridized with probe DNA labeled with32P in the buffer for rapid hybridization (Amersham, Co) at 65C for 2 hours followed by washing with 0.5SSC at 45C for 10 minutes.

System coculture mouse osteoblastic stromal cells and mouse spleen cells in the presence of the active form of vitamin D3or PTH is well known as a typical system of in vitro culture for the formation of osteoclasts. Interactions through adhesion osteoblastic stromal cells and cells of the spleen and the presence of osteo factors, such as the active form of vitamin D3or PTH are required in this culture system in vitro. In this system, the in vitro culture of COS cells, kidney cells of monkeys, not capable of supporting the formation of osteoclasts, acquire the ability to maintain the formation of osteoclasts from spleen cells in the absence of an osteo-factor in the expression of this cDNA isnc of the present invention encodes a protein, containing the transmembrane domain, this cDNA can be expressed in the form Sekretareva form or a soluble form by removing the part that encodes this transmembrane domain. It was confirmed that the osteoclasts could be formed by adding Sekretareva form RBM person in the above system of in vitro culture in the absence of osteo factors. Based on these results RBM human encoded by cDNA of the present invention, is characterized as a factor involved in the differentiation and maturation of osteoclasts.

Recombinant RBM person can be obtained by embedding the DNA of the present invention in expressing vector, obtaining the expression plasmids RBM man, the introduction of this plasmid in different cell strains and expression of RBM in the cells. As host for the expression of RBM, can be used mammalian cells such as COS-7, CHO, Namalwa, or bacteria, such as Escherichia coli. In this case, the RBM can be expressed in the form of a membrane-bound form of the protein using the full-length DNA or in the form of Sekretareva form or a soluble form of the protein by removing a segment that encodes a transmembrane domain. Produced thus recombinant RBM's for normal protein, such as affinity chromatography using a column with immobilized OCIF, ion-exchange chromatography and gel filtration chromatography. Thus obtained RBM person present invention is applicable as an agent for the treatment of diseases caused by disturbance of bone metabolism, such as congenital systemic osteoporosis, or as a reagent for research and diagnosis of such diseases.

Using RBM protein encoded by the DNA of the present invention can be carried out the following operations screening: (1) screening of substances that can regulate the expression of RBM person, (2) screening of substances that are specifically associated with RBM person and inhibit or modify the biological activity of RBM, and (3) screening of human proteins that are present in the cells-the precursors of osteoclasts and transmit (transducer) biological activity RBM human (receptor RBM person). You can also create antagonists and agonists to this receptor RBM person. In combinatorial chemistry using the above RBM person or receptor RBM human peptide libraries for screening of antagonists or agonists, can byteam RBM human instead of mouse RBM can be derived peptide with extremely high specificity and affinity.

Although this RBM is very valuable, as described above, and antibodies that specifically recognize the RBM, and enzyme-linked immunosorbent assay with the use of these antibodies to determine the concentration of RBM, still were not available antibodies used for estimating the concentration of RBM. In addition, it is assumed that antibodies against RBM or antibodies against sOBM inhibit the biological activity of the RBM or sOBM, in particular the activity of inducing the formation of osteoclasts. It is expected that they will be applicable as therapeutic agents for the treatment of disorders of bone metabolism. However, until now such antibodies were not available.

In connection with this situation, the authors of this invention have conducted intensive studies. The authors of this invention have found antibodies (antibodies against RBM/s), which recognize both forms of RBM, membrane-bound protein that specifically binds to inhibiting osteoclastogenic factor (OCIF), and soluble RBM (s), which has no transmembrane domain. Thus, the invention provides antibodies (antibodies against RBM/s), which recognize both forms of RBM, membrane-bound protein that specifically binds to inhibiting osteoclastoma determination of the concentration of the RBM s using these antibodies and agents for the prevention or treatment of diseases, caused by impairment of bone metabolism.

This invention relates to antibodies (antibodies against RBM/s), which recognize both forms of RBM, membrane-bound protein that specifically binds to inhibiting osteoclastogenic factor (OCIF), and soluble RBM (s), which has no transmembrane domain; the way they are received; the method of quantitative determination of the RBM s using these antibodies and agents for preventing or treating diseases caused by a disturbance of bone metabolism.

Antibodies of this invention are active neutralization reinforcing osteoclastogenic activity, which is the biological activity of the RBM soum, and include antibodies having the following characteristics:

(a) polyclonal antibodies that recognize both mouse RBM and mouse s (polyclonal antibodies against mouse RBM/s,

(b) polyclonal antibodies that recognize how RBM person and s human (polyclonal antibodies against RBM/s person,

(c) monoclonal antibodies that recognize both mouse RBM and mouse s (monoclonal antibodies against murine RBM/s),

(d) monoclonal antibodies that recognize how RBM person and s coveka, which cross-react with mouse RBM and mouse s.

Polyclonal antibodies that recognize both mouse RBM and mouse s (hereinafter referred to as polyclonal antibodies against mouse RBM/s), and polyclonal antibodies that recognize how RBM person and s person (hereinafter referred to as polyclonal antibodies against RBM/s person), obtained according to the following method. Purified mouse RBM used as antigen for immunization can be obtained by the above method. In particular, mouse osteoblast stromal cell line ST2 processed active form of vitamin D3and RBM on the cell membrane was purified using a column with immobilized OCIF and gel-filtration chromatography with obtaining natural RBM mouse (native RBM). The above-mentioned cDNA mouse RBM (table sequences SEQ ID No. 15) or cDNA RBM person (table of the sequences SEQ ID No. 12) was built in expressing the vector in a common manner. Recombinant murine RBM (table sequences SEQ ID No. 1) and recombinant RBM person (table of the sequences SEQ ID No. 11) can be obtained by expression of the cDNA in animal cells, that the above-mentioned cleaning methods. In this case, the cleaning of the large number and high level mouse RBM or RBM person, which are membrane-bound proteins, is a task that requires great effort. On the other hand, as mentioned above, it is known that the RBM, which is a membrane-bound protein, and soluble RBM (s), which is produced by the delegation of the transmembrane domain of RBM's are almost identical in their activity in the differentiation and maturation of osteoclasts. For immunization can be used as antigens mouse s and s person, which are relatively easily expressed and purified to a high level.

Mouse s (table sequences SEQ ID No. 16) and s person (table of the sequences SEQ ID No. 17) can be obtained by attaching a nucleotide sequence that encodes a known signal sequence derived from another secreted protein, against the course of transcription from the 5'-end of cDNA mouse s (table sequences SEQ ID No. 18) and cDNA s person (table of the sequences SEQ ID No. 19), by embedding them in expressing vector using methods of genetic engineering, the creation of conditions for the expression of these Belknap products. Antigens for immunization, thus obtained, was dissolved in phosphate buffered saline (PBS), mixed with the same volume of complete adjuvant's adjuvant for the emulsification solution, if necessary, and injected subcutaneously to the animals about once a week for immunization of these animals several times. Booster injection given at the maximum antibody titer. Bleeding spend 10 days after the booster injection. The resulting anticigarette treated by precipitation with ammonium sulfate. The IgG fraction purified using anion-exchange chromatography or purified column chromatography on a column of protein A - or protein G-separate after dilution of antisera twice buffer for binding (Binding Buffer, BioRad Co.) obtaining polyclonal antibodies against RBM/s mouse or human.

Monoclonal antibodies of this invention can be obtained by the following method. As in the case of polyclonal antibodies, natural mouse RBM (native RBM), recombinant murine or human RBM or murine or human s can be used as immunogens to obtain monoclonal antibodies. Hybridoma p what their lymphocytes in vitro and merge immunized cells with myeloma cells. Using the analysis of culture supernatant hybridomas thus obtained, with the use of solid-phase ELISA method selected producing antibodies of hybridoma, recognizing highly purified antigen. Received hybridoma clone and install as stable producing antibodies hybridoma clones. These hybridoma cultivated to generate antibodies. To obtain hybridomas typically use small animals such as mice or rats. Animals subjected to immunization by intravenous or intraperitoneal injection of antigen, diluted to a suitable concentration with a suitable solvent such as physiological saline. The antigen can be entered together with complete adjuvant's adjuvant. Injections usually spend 3-4 times, once a week or every two weeks. Immunized animals reveal three days after the last immunization. As the immunized cells using splenocytes from a remote spleen. As a mouse myeloma to merge with the immunized cells use p3/x63-Ag8, 3XUl, NS-1, MPC-11, SP-2/0, FO, P363 Ad.653 and S194. Cell line, such as R-210, are used as cells of rat origin. Human antibodies produces the ka or cell line, transformed by EB virus. The fusion of immunized cells and myeloma cells can be performed in accordance with generally accepted method such as the method of Kohler and Milstein (Koehler et al.: Nature, 256, 495-497 (1975)). Also suitable way of electrical impulses. Immunized cells and myeloma cells are mixed with the conventional ratio and poured in not containing fetal calf serum (FCS) in the culture medium with the addition of poliatilenglikola and cultured in containing FCS selectivity environment GAT (environment gipoksantin-aminopterin-thymidine) for selection of fused cells (hybridomas). Then hybridoma producing antibodies, were selected using the common method of detection antibodies, such as ELISA, method plaques way of Ochterlony or method of aggregation, to establish a stable hybrid. Hybridoma installed in this way can be subculturally conventional method of cultivation or can be stored in frozen form, if necessary. Hybridoma can be cultivated accepted way to collect the culture supernatant or may be implanted into the abdominal cavity of a mammal to obtain antibodies from ascitic fluid. Antibodies in the culture is modern and gel-filtration chromatography, or protein A - or protein-affinity chromatography. Almost all of the monoclonal antibodies obtained using s as antigen, can specifically learn not only sCBM, but also the RBM (these antibodies are further monoclonal antibodies against OBM/sOBM). These antibodies can be used to quantify the RBM or s. The number of RBM and s can be determined by labeling the antibody with radioactive isotope and the use of labeled antibodies in the system for the quantitative determination known as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Using these systems the quantitative determination of the number s in a biological sample, such as blood or urine, can be determined easily with high sensitivity. In addition, the number of RBM, communicating with the tissue or the surface of the cells, can be easily measured with high sensitivity by using the test link using these antibodies.

When using antibodies as drugs for humans it is preferable to use human antibodies against OBM/sOBM person. Human antibodies against OBM/sOBM person can be obtained in accordance with sleduet, were immunized with antigen RBM person or s human in vitro in the presence of IL-4. Received immunized human lymphocytes was merged with hybridomas KN/B5 (ATSS CRL1823), which is heterohybridomas mouse and man, and were subjected to screening to obtain producing the target antibody hybridoma. Antibodies produced obtained producing antibodies by hybridomas are human monoclonal antibodies against OBM/sOBM person. These antibodies are selected antibodies that neutralize the activity of OBM/sOBM person. However, it is usually difficult to obtain antibodies having a high affinity towards the antigen, the method of immunization of human lymphocytes in vitro. Therefore, to obtain monoclonal antibodies with high affinity against the RBM s person you want to increase the affinity human monoclonal antibodies against OBM/sOBM person, obtained as described above. First, introducing a random mutation in the region CDR (in particular, in the region CDR3) of human monoclonal antibodies against OBM/sOBM person, which neutralizes the RBM, but has a low affinity, and allow the phage to Express the protein. Phages, which can strongly connect with OBM/sOBM person, selected by way ragovoy indicate in Escherichia coli. Amino acid sequence of CDR, which shows high affinity, determined on the basis of the nucleotide sequence of the DNA, cloned in the phage. Thus obtained DNA encoding the human monoclonal antibodies against RBM/s person, enter commonly used in expressing vector for mammalian cells to obtain human monoclonal antibodies against RBM/s person. Of these monoclonal antibodies can be selected to target human monoclonal antibodies having a high affinity and is able to neutralize the biological activity of the RBM/s person. In method (2) monoclonal mouse antibodies against type RBM/s person will receive according to the method of this invention using BALB/c mice (Koehler et al.: Nature, 256, 495-49, 1975) and select monoclonal antibodies that can neutralize the biological activity of the RBM/s person and exhibit high affinity. These high-affinity murine antibodies against RBM/s person can be turned into a human-type antibody using the method of CDR-grafting (Winter and Milstein: Nature, 349, 293-299, 1991) by implantation of their CDR regions (CDR 1, 2 and 3) in the CDR regions of human IgG. In Method (3) limpii is ICU implanted with SCID mouse can produce human antibodies (sier D. E. et al.: Nature, 335, 256-259, 1988; M. A. Duchosal et al.: Nature, 355, 258-262, 1992), lymphocytes that can produce human monoclonal antibodies specific against RBM/s person may be collected by screening with SCID mice immunized with RBM or s person. The resulting lymphocytes merge with hybridomas KN/B5 (ATSS CRL 1823), which is heterohybridomas mouse and human, in accordance with the procedure described above for antibodies person in method (1). Received hybridoma subjected to screening to obtain a hybrid that can produce the target human monoclonal antibodies. Thus obtained hybridoma cultivated with obtaining large quantities of the target human monoclonal antibodies. These antibodies can be purified as described above cleanup. In addition, it is possible to produce recombinant human monoclonal antibodies in large quantities by constructing a cDNA library from hybridoma, which can produce target human monoclonal antibody, to obtain a gene (cDNA) encoding the target human monoclonal antibody, clone, integration of this gene into a suitable expressing vector methods Genn is s, cells insects or Escherlchia coli. Large quantities of purified human monoclonal antibodies can be obtained by purification from the resulting culture supernatant purification methods described above.

Antibodies that can neutralize the biological activity of the RBM/s, can be obtained from monoclonal antibodies against RBM/s obtained according to this method. It is expected that antibodies that neutralize the biological activity of the RBM/s applicable as a means for treating or preventing disorders of bone metabolism due to their ability to inhibit in vivo biological activity RBM/s, in particular the ability to prevent the induction of the formation of osteoclasts. The activity of the antibodies against RBM/s to neutralize the biological activity of the RBM or s can be measured by determining the activity of inhibiting the formation of osteoclasts in vitro. In particular, can be given the following system of culture for osteoclastogenesis in vitro: (1) system coculture strain mouse osteoblastic stromal cells, ST2, and mouse spleen cells in the presence of the active form of vitamin D3and dexamethasone, (2) system coculture, soderjaschiesya and cultivation of mouse spleen cells to these cells in the presence of M-CSF and (3) culture system of mouse spleen cells in the presence of recombinant s and M-CSF. Inhibiting osteoclastogenesis activity of the antibodies against RBM/s can be measured by adding antibodies against RBM/s at various concentrations to these cultural systems and study their action on the formation of osteoclasts. Inhibiting osteoclastogenesis activity of the antibodies against RBM/s can also be estimated by measuring their inhibitory bone resorption activity using experimental animals in vivo. In particular, the model animal subjected oophorectomy, is given as a model animal with progressive formation of osteoclasts. Inhibiting osteoclastogenesis activity of the antibodies against RBM/s can be defined antibodies against RBM/s this experimental animals and evaluation of inhibition of bone resorption (increasing bone density activity).

Thus obtained antibodies capable of neutralizing the biological activity of the RBM/s applicable, in particular in pharmaceutical compositions for the prevention or treatment of disorders of bone metabolism or as antibodies for immunological diagnosis of such diseases. Drugs, containing antibodies of this invention can be administered orally Il the way, which contain antibodies recognizing RBM and/or s, as an active ingredient. As forms of the pharmaceutical compositions are agents for injection, including intravenous drip infusions, suppositories, sublingual agents, percutaneous absorption agents, etc., Because monoclonal antibodies are macromolecular proteins, not only are they easily stick to the glass container such as a vial or syringe, but also easily denature physico-chemical factors such as heat, pH or humidity. Therefore, drugs should be stabilized by the addition of stabilizers, pH correctors, bafarawa agents, solubilizing agents or detergents. As stabilizers can be given amino acids such as glycine and alanine, saccharides such as dextran 40 and mannose, and polyalcohols, xylytol, such as sorbitol, mannitol and xylitol. These stabilizers can be used individually or in combinations of two or more. The number of added stabilizers preferably 0.01 to 100 times, particularly preferably 0.1 to 10 times more than the number of antibodies. The addition of these stabilizers increases the storage stability of liquid medication or atnie buffers. Tabularasa agents not only adjust the pH of liquid drugs or aqueous solutions obtained by their reconstitution of lyophilised products, but also increase the stability and solubility of antibodies. It is desirable to add sauterelle agent in a quantity to obtain 1 - 10 mm final concentration in the liquid or aqueous solution prepared from a lyophilized product. Examples of detergent are Polysolbate 20, Pulluronic F-68 and polyethylene glycol. Especially preferred example is Polysolbate 80. These detergents can be used alone or in combinations of two or more detergents. Macromolecular proteins, such as antibodies, easily stick to glass containers. Adhesion to containers of antibodies in the liquid or in aqueous solution, prepared by re-dissolving the lyophilized product can be prevented by adding such detergents at a concentration of from 0.001 to 1%. Drugs, containing antibodies of this invention can be obtained by the addition of stabilizers, sautereau agents or agents that prevent adsorption. When using these drugs as injectable agents for drug cured the t osmotic pressure can be adjusted by increasing or decreasing the amount of sodium chloride in the preparation of drugs. The number of antibodies in the drug may appropriately be adjusted depending on the disease, route of administration, etc. Dose of antibodies for men may vary depending on the affinity of antibodies against OBM/sOBM person, in particular from the dissociation constants (Kd values) for OBM/sOBM person. The higher the affinity (or the lower the Kd value), the smaller should be the dose, introduce the person to get a specific healing effect. Because antibodies to human type have a long half-life in blood (approximately 20 days), it is enough to introduce them to the people at a dose of ~0.1 to 100 mg/kg once or several times in 1-30-day period of time.

BRIEF DESCRIPTION of DRAWINGS

Fig.1 shows the result of electrophoresis in SDS-PAG mouse RBM protein of the present invention obtained in Example 3.

Explanation of symbols

(A): Lane 1: molecular mass markers.

Track 2: partially purified sample (allerona fraction Gly-HCl (pH 2,0)), obtained from ST2 cells cultured in the presence of the active form of vitamin D3and dexamethasone.

Track 3: partially purified sample (allerona fraction Gly-HCl (pH 2,0)), obtained in the absence of the active form of vitamin D3and dexamethasone.

(In): Dor the chromatography high-resolution reversed-phase (Example 3).

Fig.2 shows the result of analysis of binding125I-labeled OCIF with osteoblastlike stromal ST2 cells in Example 4.

Fig.3 shows the ability of the formation of osteoclasts osteoblastic stromal ST2 cells from different generations in Example 5(1).

Explanation of symbols

1: the ability of ST2 cells from ~10th subculture to maintain the formation of osteoclasts.

2: the ability of ST2 cells from ~40-th subculture to maintain the formation of osteoclasts.

Fig.4 shows the change with passage of time in the expression of the protein of the present invention on the cell membrane osteoblastic stromal cells cultured in the presence of the active form of vitamin D3and dexamethasone in Example 5(2).

Fig.5 shows the change with passage of time in the formation of osteoclasts in the system coculture of Example 5(2).

Fig.6 shows the inhibitory effect of the formation of osteoclasts in the processing of OCIF during different periods of culture during the period of cocultivation in Example 5(3).

Fig.7 shows the results of the test stapling125I-labeled OCIF protein of the present invention in Example 6.

Explanation of symbols

Track 1:125I-labeled OCIF-CDD1.

Track 2:125I-labeled OCIF-CDD1, stitched with ST2 cells.

Value electrophoresis in SDS-page in Example 9.

Explanation of symbols

Lane 1: protein transfected with plasmid ROM cells COS-7, immunoprecipitation in the absence of OCIF.

Lane 2: proteins transfected with plasmid ROM cells COS-7, immunoprecipitation in the presence of OCIF.

Fig.9 shows the results of the analysis of the ability of binding125I-labeled OCIF with cells COS-7, transfected ROM in Example 10.

Explanation of symbols

Tracks 1 and 2: number125I-labeled OCIF binding to cells COS-7, transfected ROM.

Tracks 3 and 4: number125I-labeled OCIF binding to cells COS-7, transfected ROM, in the presence of 400-fold excess of unlabeled OCIF.

Fig.10 shows the result of the test stapling using OCIF, labeled125I, in Example 11.

Explanation of symbols

Track 1:125I-labeled OCIF.

Track 2:125I-labeled OCIF made with cells COS-7, transfected ROM.

Track 3:125I-labeled OCIF made with cells COS-7, transfected ROM, in the presence of 400-fold excess of unlabeled OCIF.

Fig.11 shows the result of Northern blotting in Example 12.

Oyesanya characters

Lane 1: RNA originating from ST2 cells cultured without added vitamin D and dexamethasone.

Dorval OCIF-binding ability of the proteins in the conditioned medium at different concentrations of OCIF in Example 14 (2).

Explanation of symbols

about: rser.

•: rser sOBM.

Fig.13 shows the OCIF-binding ability of protein in the conditioned medium at different ratios, air-conditioned environment in Example 14 (2).

about: rser.

•: rser sOBM.

Fig.14 shows the result of electrophoresis in SDS-PAG fused protein consisting of thioredoxin and mouse RBM expressed in Escherichia coli in Example 15(2).

Explanation of symbols

Lane 1: molecular mass markers.

Lane 2: soluble protein fraction originating from G1724/pTrxFus.

Lane 3: soluble protein fraction originating from G1724/pTrxOBM25.

Fig.15 shows the OCIF-binding capacity at different ratios of soluble protein fractions in Example 15(3).

Explanation of symbols

: GI724/pTrxFus.

about: GI724/pTrxOBM25.

Fig.16 shows the OCIF-binding ability of the soluble protein fractions (1%) at different concentrations of OCIF in Example 14(3).

Explanation of symbols

: GI724/pTrxFus.

about: GI724/pTrxOBM25.

Fig.17 shows the results of inhibition of specific binding to OCIF mouse RBM obtained by expression of the cDNA of murine RBM of this invention and the treatment of natural OCIF-binding protein using rabbit antibodies against mouse RBM.

O the 2: natural protein in the presence of antibodies +125I-OCIF,

3: mouse RBM obtained by expression of the cDNA in the absence of antibodies, murine RBM +125I-OCIF,

4: natural protein in the absence of antibodies +125I-OCIF,

5: 3 + unlabeled OCIF (400 times more than125I-OCIF),

6: 4 + unlabeled OCIF (400 times more than125I CCIF).

Fig.18 shows the result of electrophoresis in SDS-page protein RBM person expressed cDNA of the present invention.

Explanation of symbols

Lane 1: molecular mass markers.

Lane 2: protein cells COS-7, transfected phOBM (expressing the vector containing the cDNA of the present invention), immunoprecipitation rabbit polyclonal antibody against OCIF, in the absence of OCIF.

Lane 3: proteins cells COS-7, transfected phOBM (expressing the vector containing the cDNA of the present invention), immunoprecipitation rabbit polyclonal antibody against OCIF, in the presence of OCIF.

Fig.19 shows the result of analysis of the OCIF-binding cells COS-7, transfected phOBM expressing vector containing the cDNA of the present invention.

Explanation of symbols

Lane 1: cells COS-7, transfected phOBM, and add125I-OCIF.

Lane 2: cells COS-7, transfected phOBM, and add125I-OCIF, in the presence of 400-cartogram cDNA of the present invention, with125I-OCIF (monomer type).

Explanation of symbols

Track 1:125I-OCIF.

Track 2: sewn products125I-OCIF protein on the membrane of cells COS-7, transfected phOBM.

Track 3: sewn products125I-OCIF protein on the membrane of cells COS-7, transfected phOBM, in the presence of 400-fold excess of unlabeled OCIF.

Fig.21 shows the OCIF-binding ability of the protein (Sekretareva form RBM person (hOBM)) in the conditioned medium at different concentrations of OCIF in Example 24 (2).

Explanation of symbols

about: this air-conditioned environment of the cells 293-EBNA, transfected rser, which contains cDNA coding secreterial form RBM person.

•: this air-conditioned environment of the cells 293-EBNA, transfected pCEPshOBM, which contains cDNA encoding secreterial form RBM person.

Fig.22 shows the OCIF-binding ability of the protein (Sekretareva form RBM person) in an air-conditioned environment at a certain concentration of OCIF and when changing the amount of added air-conditioned environment in Example 24 (2).

Explanation of symbols

about: this air-conditioned environment of the cells 293-EBNA, transfected rser, which contains cDNA coding secreterial form RBM person.

•: this air-conditioned Wed is

Fig.23 shows the result of electrophoresis in SDS-PAG fused protein consisting of thioredoxin and RBM person, expressed in Escherichia coli.

Explanation of symbols

Lane 1: molecular mass markers.

Lane 2: soluble protein fraction originating from Escherichia coli GI724/pTrxFus.

Lane 3: soluble protein fraction originating from Escherichia coli GI724/pTrxhOBM.

Fig.24 shows the OCIF-binding ability of the fused protein consisting of thioredoxin and RBM person with OCIF by varying the amount of soluble protein fraction originating from Escherichia coli, containing added protein in Example 25 (3).

Explanation of symbols

a: the fraction of soluble protein derived from Escherichia coli GI724/pTrxFus.

•: soluble protein fraction originating from Escherichia coli GI724/pTrxshOBM.

Fig.25 shows OCIF-binding ability of the fused protein consisting of thioredoxin and RBM in the soluble protein fractions originating from Escherichia coli, with OCIF in various concentrations in Example 25(3).

Explanation of Smolov

a: the fraction of soluble protein derived from Escherichia coli GI724/pTrxFus.

•: soluble protein fraction originating from Escherichia coli GI724/pTrxshOBM.

Fig.26 shows the result of quantitative determination of RBM human and human sOBM PR is subramania.

Explanation of symbols

: RBM person.

•: sOBM person.

Fig.27 shows the result of quantitative determination of RBM human and human sOBM using the sandwich ELISA method using rabbit monoclonal antibodies against OBM/sOBM person of this invention.

Explanation of symbols

: RBM person.

•: sOBM person.

Fig.28 shows the result of quantitative determination of mouse RBM and sOBM using the sandwich ELISA method using monoclonal antibodies against OBM/sOBM person present invention, which cross-react with mouse RBM and sOBM.

Explanation of symbols

: Mouse RBM.

•: Mouse sOBM.

Fig.29 shows the activity of the fused protein consisting of thioredoxin and mouse RBM, stimulation of education osteoclastogenic human cells.

Fig.30 shows the inhibition antibodies against OBM/sOBM activity of bone resorption stimulated by vitamin D3.

Fig.31 shows the inhibition antibodies against OBM/sOBM activity of bone resorption stimulated by prostaglandin2(PGE2).

Fig.32 shows the inhibition antibodies against OBM/sOBM activity of bone resorption, timetable bones, stimulated by interleukin 1(IL-1).

The BEST WAY of CARRYING out the INVENTION

The invention will be described in more detail by means of examples, which are given to illustrate the present invention and not limit it in any way in the rest of the description.

Example 1

Obtaining the protein of the present invention

(1) large-Scale cultivation of cells ST2

Mouse line osteoblastic stromal ST2 cells (RIKEN CELL BANK RCB0224) were cultured by using a-MEM containing 10% fetal calf serum. The ST2 cells, cultured to confluently in T-225 flask cm2for culture attached cells were treated with trypsin and collected from the T-flask. After washing the cells was transferred into five T-225 flasks cm2. After adding 60 ml-MEM containing 10-8M active Form of vitamin D3(Calcitriol), 10-7M dexamethasone, and 10% fetal calf serum, the cells in each flask were cultured for 7-10 days in CO2thermostat. The cultured cells ST2 collected cell "scraper" and kept at -80To use.

(2) Obtain the membrane fraction and solubilization of medicati T-225 flasks cm2added three volumes (36 ml), 10 mm Tris-model HC1 buffer (pH of 7.2) containing protease inhibitors (2 mm APMSFP, 2 mm EDTA, 2 mm o-phenanthroline, 1 mm leupeptin, 1 μg/ml of pepstatin a and 100 units/ml Aprotinin). After vigorous mixing for 30 seconds using a voltex mixer cells were left for 10 minutes on ice. Cells are homogenized using a homogenizer (DOUNCE TISSUE GRINDER, A syringe, WHEATON SCIENTIFIC Co.). Equal volume (48 ml) of 10 mm Tris-Hcl buffer (pH of 7.2), containing the above protease inhibitors, 0.5 M sucrose, 0.1 M potassium chloride, 10 mm magnesium chloride and 2 mm calcium chloride was added to gomogenizirovannykh cells. After mixing, the mixture was centrifuged at 600g for 10 minutes at 4With the separation of the nuclei and dehomogenization cells in the form of sediment. The supernatant obtained by centrifugation was centrifuged at 150000g for 90 minutes at 4With obtaining fractions of cell membranes ST2 in the form of sediment. 8 ml of 10 mm Tris-Hcl buffer (pH of 7.2), containing the same protease inhibitors, 150 mm sodium chloride and 0.1 M sucrose was added to this fraction of the membranes. After adding 200 µl of 20% CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, Sigma Co.) ttp://img.russianpatents.com/chr/215.gif">g for 60 minutes at 4With obtaining supernatant as solubilizing membrane fraction.

Example 2

Purification of the protein of the present invention

(1) Preparation of affinity column with immobilized OCIF

After replacing isopropanol in HiTrap NHS-activated column (1 ml, manufactured by Pharmacia Co.) 1 mm hydrochloric acid in a column was added by syringe (5 ml, manufactured Agimo Corp.) 1 ml of 0.2 M Panso3/0.5 M NaCl (pH 8,3), containing 13,0 mg recombinant OCIF obtained according to the method of WO 96/26217 to perform binding assays at room temperature for 30 minutes. In column gave 3 ml of a mixture of 0.5 M ethanolamine/0.5 M NaCl (pH 8,3) and 3 ml of a mixture of 0.1 M acetic acid/0.5 M NaCl (pH 4.0) is three times alternately to inactivate excess active groups, then the solution was replaced with a mixture of 0.5 M ethanolamine/0.5 M NaCl (pH 8,3). After leaving at room temperature for 1 hour the column was washed twice alternately with a mixture of 0.5 M ethanolamine/0.5 M NaCl (pH 8,3), and a mixture of 0.1 M acetic acid/0.5 M NaCl (pH 4,0) and this solution was then replaced by a buffer of 50 mm Tris/1 M NaCl/a 0.1% CHAPS (pH 7.5).

(2) Purification of the protein of the present invention using affinity column with immobilized OCIF

Cleaning OCIF-binding protein was carried out at 4(3) Purification of the protein of the present invention, the gel-filtration

Concentrated OCIF-binding protein (fractions obtained by elution with 0.1 M glycine-cleaners containing hydrochloride buffer (pH 3,3) containing 0.1 M sodium citrate buffer (pH 2.0), obtained in Example 2(2), was applied to a column of Superose 12 HR10/30 (1,030 cm produced Phamacia Co.), which was equilibrated with 10 mm Tris-HCl, 0.5 M NaCl, and 0.5% CHAPS (pH 7.0), and was used as mobile phase buffer for equilibration at the speed of a current of 0.5 ml/min, and collected fractions of 0.5 ml Fractions containing the protein of the present invention (non fractions 27-32), identified in accordance with the method described above. Each of the fractions were concentrated using Method-10 (produced by Amicon).

(4) Purification of high-performance liquid chromatography with reversed phase

The above OCIF-binding protein, purified by gel-filtration was applied on the column With a4(2,1250 mm, Vydac, USA), which was equilibrated with 0.1% triperoxonane acid (TFA) and 30% acetonitrile. Proteins associated with the column, was suirable linear gradient of acetonitrile from 30 to 55% within the first 50 minutes and 55 to 80% in the next 10 minutes the Proteins in the different peaks were analyzed to identify fractions, containing the protein of the present invention, and obtained highly purified protein of the present invention.

Example 3

Electrophoresis in SDS-page the purified protein of the present invention

Fraction solubilizing membranes obtained from ST2 cells, which were cultured in the presence or in the absence of the active form of vitamin D3was subjected to purification using affinity column with immobilized OCIF. Purified products were subjected to electrophoresis in SDS-page. As shown in Fig.1 (A), a major protein band with molecular weight (MW) -30000-40000 were detected only in a purified preparation of ST2 cells, which were cultured in the presence of the active form of vitamin D3that confirms that the protein, which specifically binds to OCIF (i.e., the protein of this invention) can be selectively purified by affinity column with immobilized OCIF. However, bands of several proteins other than the protein of this invention), which are nonspecific contacted by the media or spacers column with immobilized OCIF were found in both treated preparations. Proteins other than the protein of the present invention removed in accordance with the above-described method of gel filtration and chromatography with reversed phase Colo is detected, that highly purified protein of the present invention is an electrophoretic homogeneous and has a molecular mass of approximately 30000-40000.

Example 4

Test OCIF binding to osteoblasts

(1) Obtaining125I-labeled OCIF

OCIF were labeled125I use Imogen method. Specifically, 20 μl of 2.5 mg/ml solution of Imogen-chloroform was transferred to a test tube Eppendorf 1.5 ml) and the chloroform evaporated at 40With receiving tubes, covered with Iodophenol. The tube was washed three times with 400 µl containing 0.5 M sodium phosphate buffer (Na-Pi, pH 7.0). 5 μl of 0.5 M Na-Pi (pH 7.0) was added to each tube. Immediately after this, add in a test tube was added 1,3 ál (of 18.5 MBq) solution of Na125I (NEZ-033H20 manufactured by Amersham Co.), 10 μl of 1 mg/ml solution rOCIF (Monomeric or dimeric). After mixing by vortex mixer and the mixture was allowed to stand at room temperature for 30 seconds. The solution was transferred into a test tube containing 80 μl of a solution of 10 mg/ml of potassium iodide in 0.5 M Na-Pi (pH 7.0) and 5 μl of phosphate buffered saline containing 5% bovine serum albumin, and stirred. The mixture was applied on a rotating column (1 ml, G-25 fine, produced by Pharmacia Co.), which was equilibrated phosphate buffered saline phosphate buffered saline, containing 0.25% bovine serum albumin, was added to the fraction, elyuirovaniya from this column, and the mixture was stirred. An aliquot of 2 µl was taken for measurement of radioactivity using a gamma counter. Radiochemical purity125I-labeled OCIF was determined by measuring the radioactivity precipitated with 10% THU. Biological activity125I-labeled OCIF was measured in accordance with the method described in WO 96/26217. Concentration125I-labeled OCIF was measured using ELISA in accordance with the following procedure.

(2) Measurement of concentrations125I-labeled OCIF using ELISA

100 μl of 50 mm Panso3(pH 9,6), which were dissolved rabbit polyclonal antibodies against OCIF described in WO 96/26217, to a concentration of 2 μg/ml was added into each well of 96-well immunoplate (MaxiSorpmanufactured by Nunc Co.). The plate was allowed to stand overnight at 4C. After removal of the solution by suction to each well was added 300 μl of Block ACE(Snow Brand Milk Products Co., Ltd.), buffered phosphate saline (25/75). Then the plate was allowed to stand for 2 hours at room temperature. After removal of the solution by suction, the wells were washed three times ="https://img.russianpatents.com/chr/8482.gif">, phosphate buffered saline (25/73), which was mixed125I-labeled OCIF or standard drug OCIF. Then the plate was allowed to stand for 2 hours at room temperature. After removal of the solution by suction each well was washed six times with 200 μl of P-PBS.

100 μl of Block ACE(Snow Brand Milk Products Co., Ltd.), buffered phosphate saline (25/75) containing peroxidase labeled rabbit polyclonal antibodies against OCIF was added to each well. The plate was allowed to stand for 2 hours at room temperature. After removal of the solution by suction, the wells were washed six times with 200 μl of P-PBS. Then to each well was added 100 μl of TMB solution (TMB Soluble Reagent, High Sensitivity, Scytek Co.). After incubation at room temperature for 2-3 minutes in each well was added 100 μl of stop solution (stop solution) (Stopping Reagent, Scytek Co.). The absorption of each well was measured at 490 nm using a microplate reader. Concentration125I-labeled OCIF was determined from a calibration curve constructed using the standard drug OCIF.

(3) Test OCIF binding to osteoblasts or cells of the spleen

Line mouse osteoblastic stromal ST2 cells or cells ser> M active form of vitamin D3(Calcitriol) and 10-7M dexamethasone or without them, to a concentration of 4104cells/ml (ST2 cells) or 2106cells/ml (spleen cells), respectively. Each cell suspension was inoculable in 24-well microplate. Cells were cultured for 4 days in CO2thermostat. After washing cells-MEM to each well was added 200 μl of a test environment for binding (MEME, to which were added with 0.2% bovine serum albumin, 20 mm HEPES buffer, and 0.2% NaN3) containing 20 ng/ml above125I-labeled OCIF (monomer form or dimeric form). The holes for nonspecific binding was added 200 μl of medium for the test link containing 8 µg/ml rOCIF (400-fold concentration), in addition to 20 ng/ml125I-labeled OCIF. Cells were cultured for 1 hour in CO2-thermostat and washed 3 times with 1 ml phosphate buffered saline. In this procedure, the spleen cells were washed by centrifugation 24-hole tablet in each stage of the washing cycle as cells of the spleen were not attached. After washing each well was added 500 μl in RI in each well was measured in a gamma counter.

As shown in Fig.2,125I-labeled OCIF was not contacted by cultured spleen cells, but specifically associated only with osteoblastlike stromal cells, which were cultured in the presence of the active form of vitamin D3. These results suggest that the protein of the present invention is a membrane-bound protein, induced by the active form of vitamin D3and dexamethasone on osteoblastic stromal cells.

Example 5

The biological activity of the protein of the present invention

(1) the Formation of osteoclasts, supported osteoblastlike stromal cells

The ability to maintain the formation of osteoclasts osteoblastic stromal cells was assessed by measurement of resistant tartaric acid phosphatase activity (TRAP activity) formed osteoclasts. In particular, cells of the spleen (2105cells/100 µl/well) from mice ddy (8-12 weeks of age) and mouse osteoblastic stromal ST2 cells (5103cells/100 μl/well) suspended inMEME, to which were added 10-8M active form of vitamin D3, 10-7M dexamethason and 10% fetal calf serum.wash each well phosphate buffered saline to each well was added 100 μl of a mixture of ethanol/acetone (1:1) and the cells were immobilized at room temperature for 1 minute. After immobilization in each well was added 100 μl of 50 mm citrate buffer (pH 4.5) containing 5.5 mm p-nitrophenylphosphate, and 10 mm sodium tartrate. After 15 minutes of reaction at room temperature, each well was added 0.1 N. NaOH solution and measured the absorbance at 405 nm using a microplate reader. The results of the formation of osteoclasts cells ST2 with the number of passages about 10 or 40 upon receipt of these cells from the RIKEN CELL BANK shown in Fig.3. These results suggest that ST2 cells with a higher number of passages show a higher ability to support the formation of osteoclasts.

(2) the Time course of changes in the expression of the protein of the present invention on the membrane osteoblastic stromal cells in a culture system comprising an active form of vitamin D3and dexamethasone, and the time course of changes in the formation of osteoclasts in the system coculture

In the same way as in Example 4(3), osteoblast stromal ST2 cells were cultured for 7 days in the presence of the active form of vitamin D3and dexamethasone. Test OCIF binding was performed using125I-labeled OCIF (monomer type), as described in the experiment of Example 4(1). Nonspecific binding was measured jumping ridino, the amount of specific binding125I-labeled OCIF increases with each culture period in the presence of the active form of vitamin D3and dexamethasone. In particular, as shown in Fig.4 and 5, the protein of the present invention expressively on the surface of ST2 cells under the action of the active form of vitamin D3accordingly, the increase of the culture period and the expression reached a maximum on the 4th day of cultivation. On the other hand, when the co-cultivation of murine spleen cells and ST2 cells in the presence of the active form of vitamin D3formed osteoclastogenic cells. TRAP (a marker enzyme of osteoclasts)-positive mononuclear preosteoclasts cells are formed on the third or fourth day of culture. More differentiated and Mature TRAP-positive multinucleated cells formed in the fifth or sixth day of culture. Thus, it was demonstrated good correlation between the time course of expression of the protein of this invention and the formation of osteoclasts.

(3) Inhibiting the formation of osteoclasts by treatment OCIF in different periods of coculture

In order to clearly show that the protein of the present invention is a factor in the and during the six day period securityservice, described in the Example above, 5(2) (two consecutive days in a six-day period, except for 5-th day, for which you have applied a one-day period). As a result, as shown in Fig.6, it was found that the processing of OCIF when lasts for 48-96 hours after the start of cultivation, when expression of the protein of the present invention on the cells ST2 is maximum, was the most effective for inhibiting the formation of osteoclasts. In particular, it was confirmed that the OCIF regulates the formation of osteoclasts by binding to ST2 cells through a protein of the present invention.

Based on the results of the above experiments, it was confirmed that the protein of the present invention is induced on the cell membrane osteoblastic stromal cells active form of vitamin D3and dexamethasone and exhibits biological activity maintain or accelerate the differentiation or maturation of osteoclasts.

Example 6

Test stitching for125I-labeled OCIF and the protein of the present invention

For a clearer identification of the protein of the present invention, this protein was made with125I-labeled OCIF. Line mouse osteoblastic stromal ST2 cells were cultured for 4 days in the presence or in the absence of arenoso phosphate salt solution, added 200 μl of a test environment for binding (MEME, to which were added with 0.2% bovine serum albumin, 20 mm S buffer, and 0.2% NN3and 100 μg/ml heparin) containing 25 ng/ml125I-labeled OCIF (monomer type) or 40 ng/ml125I-labeled OCIF-CDD1, which was obtained by expression of the protein of SEQ ID No. 76 (WO 96/26217) in the cells of the animal. The above culture medium to test the binding of complement is also a 400-fold concentration of OCIF and added to other wells to assess nonspecific binding. After culturing for 1 hour in CO2-thermostat each well was washed three times with 1 ml phosphate buffered saline containing 100 μg/ml heparin. 500 μl of phosphate buffered saline containing 100 μg/ml cross-linking agent, DSS (disuccinimidyl, Pierce Co.), was added to each well and the plate was kept for 10 minutes at 0C. the Wells were washed twice with 1 ml phosphate buffered saline with 0C. Then the wells were added with 100 μl of 20 mm HEPES buffer containing 1% Triton X-100, 10 μm pepstatin, 10 μm leupeptin, 2 mm PMSF (phenylmethylsulfonyl), 10 μm antipain and 2 mm EDTA. The plate was allowed to stand in during ivalsa conditions in accordance with the conventional method and subjected to electrophoresis in SDS-page (4-20% gradient polyacrylamide, produced by Daiichi Chemical Co., Ltd.). After electrophoresis the gels were dried and exposed to BioMax film MS (manufactured by Kodak) for 24 hours at -80With using intensifying screens BioMax MS (manufactured by Kodak). After exposure the film showed an accepted way. When using125I-labeled OCIF (monomer type, 60 kDa) was detected lane cross-linked product with the molecular weight 90000-110000. When using125I-labeled OCIF-CDD1 (31 kDa) was detected strip sewn product ~70-80 kDa (average 78 kDa), as shown in Fig.7.

Example 7

Analysis of the protein of the present invention expressed by the cells of the ST, using the curve of Scatchard

The above125I-labeled OCIF (monomer type) was added to a concentration of 1000 PM to culture medium to test binding (MEME, to which were added with 0.2% bovine serum albumin, 20 mm S buffer and 0.2% NaN3), and this culture medium was serially diluted 1:2 culture medium not containing125I-labeled OCIF. Solutions to measure nonspecific binding was prepared by further adding a 400-fold concentration of the Monomeric form OCIF to these solutions. 200 ál of the prepared solutions of DOI 10-8M active form of vitamin D3(Calcitriol) and 10-7M dexamethasone, to assess binding125I-labeled OCIF by the method described in Example 4(3). These results were subjected to Scatchard Plot analysis to determine the dissociation constants OCIF and OCIF-binding protein and the number (sites) OCIF-binding protein on the cell ST2. In the result it was found that the dissociation constant OCIF and the protein of the present invention is equal to 280 PM, and the number of sites OCIF-binding protein on the cell ST2 ~33000/cell. On the basis of the detection in Example 5(1) that the formation of osteoclasts, supported by ST2 cells, with the number of passages ~40 was more intense than with the number of passages of ~10, estimated the number (sites) of the protein of the present invention expressed on the cell ST2, with the number of passages of ~40. This number (sites) was 58000/cell and was clearly larger than in the case of ST2 cells with the number of passages of ~10, which indicated that the protein of the present invention expressed on the cell ST2, due to their ability to support the formation of osteoclasts. These results showed that the protein of the present invention is a factor supporting or inducing differentiation or maturation of osteoclasts.

Example 8

Cloning of cDNA was Ovuliferous-MEM (Gibco BRL Co.), containing 10% fetal calf serum. The ST2 cells cultured in confluently in T-flask (225 cm2for attached cells were treated with trypsin to collect cells from the T-flask. Cells were washed and transferred in five T-flasks (225 cm2). To each flask was added 60 ml (-MEM containing 10 M active form of vitamin D3(Calcitriol, Waco Pure Chemicals Co., Ltd.), 10-7M dexamethasone, and 10% fetal calf serum, and cells were cultured for 5 days in CO2thermostat. Total RNA was extracted from cultured cells ST2 using ISOGEN (Wako Pure Chemicals Co., Ltd.). Poly A+PHK was obtained from ~600 µg total RNA using columns of Oligo(dt)-cellulose (5'-3' Prime Co.). Got ~8 µg Poly+The RNA.

(2) Construction of expression libraries

Docebocore cDNA was synthesized from 2 μg of poly A+PHK obtained in Example 8(1), with a set of Great Length cDNA Synthesis (Clontech Co.) in accordance with the instructions in the manual. Specifically, 2 μg poly And+RNA and Oligo(dt) 25 (dN) -primer were mixed and added to the mixture of distilled water to a final volume of 6.25 μl. After incubation for ~3 minutes at 70°C. the mixture was cooled on ice for 2 minutes. To the mixture was added 2,2 NC-basics PRIME (1 u/ml) (5'-3' Prime Co.), 0,5 µl [-32P]dCTP (Amersham Co., 3000 CI/mmol) diluted with distilled water to obtain 2 µci/µl, of 0.65 μl dNTP (20 mm each) and 1.25 μl (250 units) MMLV reverse transcriptase (RNA-Aza N-). The mixture is incubated for 90 minutes at 42With, then added another 62,25 µl of distilled water, 20 μl of 5x buffer for the second circuit, of 0.75 μl dNTP (20 mm each) and 5 μl of enzyme mixture for the second circuit. The resulting mixture was kept at 16C for two hours. Then to this reaction mixture was added with 7.5 units of DNA polymerase T4. After incubation at 16C for 30 minutes the reaction was stopped by adding 5 ál of 0.2 M EDTA. After treatment with phenol-chloroform, the product precipitated with ethanol. EcoRI-SalI-NotI-linker (Clontech Co.) attached to the ends obtained by docebocore cDNA. Then the ends were fosforilirovanii and the product was applied to a fractionation by size (gel filtration column to obtain cDNA with a length of more than 500 p. N. DNA precipitated with ethanol, dissolved in water and ligated with pcDL-SR296 (Molecular and Cellular Biology, Vol. 8, PP. 466-472, 1988), which was cut by the restriction enzyme EcoRI (Takara Shuzo Co.), and was treated SAR (alkaline phosphatase hia coli XL2 Blue MRF' (Toyobo Co., Ltd.) transformed DNA obtained in Example 8(2), and were cultured on L-Carbenisilin-agar (1% tripton, 0,5% yeast extract, 1% NaCl, 60 µg/ml carbenicillin, 1.5% agar prepared in 24-hole plastic tablets with obtaining approximately 100 colonies per well. Transformants in each well is suspended in 3 ml of culture media Terrific Broth ampicillin (1.2% tripton, a 2.4% yeast extract, 0.4% glycerol, of 0.017 M KH2PO4, 0,072 M2NRA4, 100 μg/ml ampicillin) and cultured at 37With over night with shaking. The cells were collected by centrifugation to obtain plasmid DNA with a set QIAwell kit (QIAGEN Co.). The DNA concentration was determined by absorption measurement at 260 nm. DNA was concentrated by precipitation with ethanol and dissolved in distilled water to a concentration of 200 ng/ál. Received 500 DNA pools, each of which was obtained from ~100 colonies, and used for transfection into cells COS-7 (RIKEN CELL BANK, RCB0539). Cells COS-7 were sown in DMEM containing 10% fetal calf serum in each well of 24-well plates at a density of 8 cells104cells/well and cultured overnight at 37With CO2thermostat. On sledno plasmid DNA, which is pre-diluted culture medium OPTI-MEM (Gibco BRL Co.) and mixed with Lipofectamine (reagent for tranfection manufactured by Gibco BRL Co.), under the Protocol, supplemented by Lipofectamine. After 15 minutes the mixture was added to cells in each well. The number of Lipofectamine and DNA were respectively 1 and 4 µg µl per well. After 5 hours the culture medium was removed and to each well was added 1 ml of culture medium DMEM (Gibco BRL Co.), containing 10% fetal calf serum. The plates were incubated for 2-3 days at 37°C in CO2-thermostat (5% CO2). Cells COS-7, transfected and cultured for 2-3 days so were washed in serum-free culture medium DMEM. Then 200 ál of culture medium to test binding (serum-free culture medium DMEM containing 0.2% calf serum albumin, 20 mm HEPES buffer, 0.1 mg/ml heparin and 0.02% NaN3) with 20 ng/ml125I-labeled OCIF, added thereto, was added to each well. After culturing for 1 hour at 37With CO2-thermostat (5% CO2cells were washed twice with 500 µl phosphate buffered saline containing 0.1 mg/ml heparin. After washing is the temperature for lysis of the cells. The number of125I in each well was measured using a gamma counter (Packard Co.). By screening a total of 500 pools was detected in one pool of DNA containing cDNA encoding a protein that specifically binds to OCIF. This pool of DNA containing the cDNA, were separated by the above-described operations transfection and screening were repeated for the selection of cDNA that encodes a protein, binding to OCIF. A plasmid containing this cDNA was named ROM. Escherichia coli containing this plasmid, were deposited in The National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, Biotechnology Laboratory, as ROM may 23, 1997 under the Deposit number FERM BP-5953.

Methods tagging OCIF125I and quantitative analysis125I-labeled OCIF using ELISA shown below. Tagging OCIF125I have spent Imogen-way. 20 μl of 25 mg/ml solution of Imogen-chloroform was added to the Eppendorf tube of 1.5 ml and the chloroform evaporated by heating at 40With to get covered Iodophenol tubes. This test tube was washed three times with 400 µl containing 0.5 M sodium phosphate buffer (Na-Pi, pH 7.0) and 5 ál of 0.5 M Na-Pi (pH 7.0). Immediately after this, add in a test tube was added 1,3 ál (of 18.5 MBq) solution of Na125I (NEZ-033H20, Amersham Co.), 10 μl of 1 mg/ml solution rOCIF (the Monomeric terature within 30 seconds. The solution was transferred into a test tube, to which was previously added 80 μl of a solution of 10 mg/ml of potassium iodide in 0.5 M Na-Pi (pH 7.0) and 5 μl of phosphate buffered saline containing 5% bovine serum albumin (BSA-PBS). After mixing, the mixture was applied on a rotating column (1 ml, G-25 fine, produced by Pharmacia Co.), which was balanced by BSA-PBS, and the column was centrifuged for 5 minutes at 2000 rpm/min 400 ál BSA-PBS was added to the fraction, elyuirovaniya from this column. After mixing, an aliquot of 2 µl was taken for measurement of radioactivity using a gamma counter. Radiochemical purity125I-labeled OCIF was determined by measurement of the radioactivity deposited 10% of TDU. Biological activity125I-labeled OCIF was measured in accordance with the method described in WO 96/26217. Concentration125I-labeled OCIF was measured using ELISA as follows. Specifically, 100 μl of 50 mm Panso3(pH 9,6), which were dissolved rabbit polyclonal antibodies against OCIF described in WO 96/26217, to a concentration of 2 μg/ml was added into each well of 96-well immunoplate (Max-iSorpmanufactured by Nunc Co.). The plate was allowed to stand overnight at 4°C. After removal of the solution by suction in each of LStore (25:75) (B-PBS). Then the plate was allowed to stand for 2 hours at room temperature. After removal of the solution by suction, the wells were washed three times in phosphate buffered saline containing 0.01% of Polysorbate 80 (P-PBS). Then to each well was added 100 μl of B-PBS containing125I-labeled OCIF or standard drug OCIF. Then the plate was allowed to stand for 2 hours at room temperature. After removal of the solution by suction each well was washed six times with 200 μl of P-PBS. 100 μl of peroxidase labeled rabbit polyclonal antibodies against OCIF, diluted B-PBS, was added to each well. The plate was allowed to stand for 2 hours at room temperature. After removal of the solution by suction, the wells were washed six times with 200 μl of P-PBS. Then to each well was added 100 μl of TMB solution (TMB Soluble Reagent, High Sensitivity, Scytek Co.). After incubating the plate at room temperature for 2-3 minutes in each well was added 100 μl of stop solution (stop solution) (Stopping Reagent, Scytek Co.). The absorption of each well was measured at 490 nm using a microplate reader. Concentration125I-labeled OCIF was determined on the basis of the calibration curve, constructed using the standard drug OCIF.

(4) Determination of n is the selected cDNA RBM, obtained in Example 8(3), was determined with a set of Taq DyeDeoxy Terminator Cycle Sequencing kit (Perkin Elmer Co.). In particular, the nucleotide sequence of the fragment insert was determined directly using ROM as a matrix. Fragments with a length of ~1,0, etc., N. and 0.7, etc. ad, which was obtained by splitting ROM the restriction enzyme EcoRI, was built into the EcoRI site of plasmid pUC19 (Takara Shuzo Co.). The nucleotide sequences of these fragments were also identified. Used the following primers: primer SRR2, which is used to determine the nucleotide sequences of DNA fragments built into pcDL-SR296, primers M13PrimerM3 and M13PrimerRV (both produced by Takara Shuzo Co.), used to determine the nucleotide sequences of DNA fragments embedded in plasmid pUC19, and synthesized primer OBM#8, is designed based on the nucleotide sequence of the cDNA for the RBM. Sequences of these primers are shown as SEQ ID No. 3 to SEQ ID No. 6 in the table of sequences.

In addition, a certain nucleotide sequence of the cDNA RBM shown as SEQ ID No. 2, and some of her amino acid sequence shown as SEQ ID No. 1.

Example 9

Expression of the protein encoded by use of Lipofectamine, and transfected cells COS-7 were cultured for 2 days in DMEM containing 10% fetal bovine serum. The medium was replaced not containing cysteine-methionine DMEM (Dainippon Seiyaku Co. Ltd.) (800 μl/well) containing 5% cialisbuynow fetal bovine serum. Cells were cultured for 15 minutes followed by the addition of 14 μl of the mixture for rapid tagging Express Protein Labeling Mix (10 MCI/ml, produced NEN Co.). After culturing for 4 hours was added 200 μl of DMEM containing 10% fetal bovine serum. After 1 hour of cultivation, the cells were twice washed in PBS. Then add 0.5 ml of TSA-buffer (10 mm Tris-Hcl (pH 8.0) containing 0,14 M such as NaCl, 0.025% Of NaN3) containing 1% Triton X-100, 1% bovine hemoglobin, 10 μg/ml leupeptin, of 0.2 TIU/ml Aprotinin, 1 mm PMSF, and the mixture was allowed to stand for 1 hour on ice. Cells were destroyed by pipetting and centrifuged at 3000g for 10 minutes at 4With obtaining the supernatant. 200 ál of dilution buffer (TSA-buffer containing 1% Triton X-100, 1% bovine hemoglobin, 10 μg/ml leupeptin, of 0.2 TIU/ml Aprotinin, 1 mm PMSF) was added to 100 μl of this supernatant. The mixture was shaken for 1 hour at 4With Protein a-Separate (50 μl). Received cm is and supernatant and thereby removed the faction (faction), which nonspecific adsorbed on Protein a-Sepharose. To this supernatant was added OCIF (1 g) and the mixture was shaken at 4C for 1 hour to achieve binding OCIF with RBM. Added polyclonal antibodies against OCIF (50 mg) and the mixture was shaken for 1 hour at 4C. Then was added to Protein a-Sepharose (10 μl) and the mixture was shaken for 1 hour at 4C, followed by centrifugation at 1500g for 1 minute at 4(C) to collect sediment. The precipitate was washed twice with dilution buffer, twice with dilution buffer without bovine hemoglobin, once TSA-buffer and once with 50 mm Tris-Hcl (pH 6.5). After washing to the precipitate was added to SDS-buffer (0.125 M Tris-HCl, 4% sodium dodecyl sulphate, 20% glycerol, of 0.002% bromophenol blue, pH 6.8) containing 10%-mercaptoethanol. The mixture was heated for 5 minutes at 100C and subjected to electrophoresis in SDS-page with 12.5% polyacrylamide gel, Daiichi Chemical Co., Ltd.). The gel was fixed in accordance with generally accepted way. The isotope signals amplified by using the Amplify(Amersham Co.) and the sample exhibited n what about the protein, encoded by cDNA of the present invention, has a molecular mass of -40000.

Example 10

Binding of the protein encoded by cDNA of the present invention, with OCIF

Plasmid ROM was transferrable in COS cells in each well of a 24-hole tablet using Lipofectamine. After culturing for 2-3 days, the cells were washed in serum-free culture medium DMEM. To the wells was added 200 μl of culture medium to test binding (serum-free culture medium DMEM containing 0.2% calf serum albumin, 20 mm HEPES, 0.1 mg/ml heparin and 0.2% NaN3), supplemented with 20 ng/ml125I-labeled OCIF. In other wells were added 200 μl of culture medium to test the binding to which was added unlabeled OCIF (8 µg/ml), in addition to 20 ng/ml125I-labeled OCIF. After culturing for 1 hour at 37With CO2-thermostat (5% CO2cells were washed twice with 500 µl phosphate buffered saline containing 0.1 mg/ml heparin. Then to each well was added 500 μl of 0.1 n NaOH solution and the plate was allowed to stand for 10 minutes at room temperature to dissolve the cells. The number of125I in each well was measured by gamma counter. As a result, as shown in Fig.9, was obname, it was confirmed that this binding is markedly inhibited by addition of (unlabeled) OCIF 400-fold concentration. These results demonstrated that RBM protein encoded by cDNA in the plasmid ROM, specifically binds to OCIF on the surface of transfected cells COS-7.

Example 11

The stitching125I-labeled OCIF and the protein encoded by cDNA of the present invention

The stitching125I-labeled OCIF monomer type and the proteins encoded by cDNA of the present invention, carried out a more detailed study of the characteristics of the proteins encoded by cDNA of the present invention. After transfection with plasmids ROM cells COS-7 according to the method used in Example 8(3), in the wells was added 200 μl of culture medium to test the binding described above, containing125I-labeled OCIF (25 ng/ml). In other wells were added to the culture medium to test the binding to which was added unlabeled OCIF 400-fold concentration, except for the125I-labeled OCIF. After culturing for 1 hour at 37With CO2-thermostat (5% CO2cells were washed twice with 500 µl phosphate buffered saline containing 0.1 mg/ml heparin. These cells were added to 500 µl of buffered FOS is rce Co.), followed by reaction for 10 minutes at 0C. the Cells in these wells were washed twice with 1 ml of cold phosphate buffered saline solution (0C). After adding 100 μl of 20 mm HEPES buffer containing 1% Triton X-100 (Wako Pure Chemicals Co., Ltd.), 2 mm PMSF (phenylmethylsulfonyl, Sigma Co.), 10 µm Pepstatin (Wako Pure Chemicals Co., Ltd.), 10 μm Leupeptin (Wako Pure Chemicals Co., Ltd.), 10 μm antipain (Wako Pure Chemicals Co., Ltd.) and 2 mm EDTA (Wako Pure Chemicals Co., Ltd.), the hole was allowed to stand for 30 minutes at room temperature to dissolve the cells. Aliquots of 15 μl of these samples were heated in the presence of SDS under reducing conditions in accordance with the conventional method and subjected to electrophoresis in SDS-page using 4-20% polyacrylamide gradient gel (Daiichi Pure Chemical Co., Ltd.). After electrophoresis the gels were dried and exposed to BioMax film MS (Kodak Co.) within 24 hours at -80With using intensifying screens BioMax MS (Kodak Co.). The exposed film showed an accepted way. The result is a band with a molecular mass in the range 90000-110000 shown in Fig.10, were detected by binding125I-labeled OCIF monomer type and the protein encoded by this cDNA izobreteny the tour of attached cells, were treated with trypsin and extracted from the T-flask. After washing the cells were sown in T-flask (225 cm2) and were cultured for 4 days in CO2-thermostat with 60 ml of culture medium-MEM containing 10-8M active form of vitamin D3, 10-7M dexamethasone, and 10% fetal bovine serum. Total RNA was extracted from cultured cells ST2 using ISOGEN (Wako Pure Chemicals Co., Ltd.). Total RNA was also extracted in the same way from ST2 cells, which were cultured in the absence of the active form of vitamin D3and dexamethasone. After addition of 2.0 ál 5x buffer solution for gel electrophoresis (0.2 M morpholinepropanesulfonic acid, pH 7.0, 50 mm sodium acetate, 5 mm EDTA), and 3.5 μl of formaldehyde and 10.0 ál of formamide to 20 µg (4,5 µl) of each of the total RNA mixtures were incubated for 15 minutes at 55C and subjected to electrophoresis. Gel electrophoresis was prepared in accordance with the formula: 1.0% agarose, 2.2 M deionized formaldehyde, 40 mm morpholinepropanesulfonic acid (pH 7.0), 10 mm sodium acetate and 1 mm EDTA. Electrophoresis was performed in a buffer solution consisting of 40 mm morpholinepropanesulfonic acid, pH 7.0, 10 mm sodium acetate and 1 is started splitting ROM the restriction enzyme EcoRI. Hybridization was performed using this DNA fragment labeled with a set for tagging Megaprime DNA labeling kit (Amersham Co.) and-32P-dCTP (Amersham Co.), as a probe. As a result, as shown in Fig.11, it was confirmed that when the cultivation of ST2 cells in the presence of the active form of vitamin D3and dexamethasone, the gene expression of the proteins encoded by cDNA of the present invention (RBM), is strongly induced.

Example 13

Supporting the formation of osteoclasts, the ability of the proteins encoded by cDNA of the present invention

ROM was transfusional in COS cells by the method described in Example 8(3). After three days trypsinization cells were washed once with phosphate buffered saline solution using centrifugation, and then recorded PBS containing 1% paraformaldehyde at room temperature for 5 minutes with PBS followed by washing six times with the use of centrifugation. 700 ál 1106/ml of mouse spleen cells and 350 ál 4104/ml of ST2 cells, which were suspended in-MEM culture medium containing 10-8M active formula of vitamin D3, 10-7DEXA. The above-described fixed cells COS (350 μl) diluted to various concentrations of the above-mentioned culture medium, and the OCIF solution (50 ml) was added to the TC-box and were cultured for 6 days at 37C. In the result, it was confirmed that inhibiting the formation of osteoclasts OCIF activity can also be inhibited by the protein encoded by cDNA of the present invention.

Example 14

Expression Sekretareva form RBM

(1) Construction of plasmids for expression Sekretareva form RBM

The PCR reaction was carried out using the RBM HF (table sequences SEQ ID No. 7) and RBM XR (table sequences SEQ ID No. 8) as primers and ROM as a matrix. After purification by agarose gel electrophoresis, the product was digested with restrictase HindIII and EcoRI and was additionally purified by agarose gel electrophoresis. The purified fragment (0,6, etc., ad), HindIII/EcoRI fragment (5,2, etc., ad) pSec TagA (Invitrogen Co.) and EcoRI/PmacI fragment (0,32, etc., ad) cDNA RBM ligated with a set for ligating PERC. 2 (Takara Shuzo Co.). Escherichia coli DH5transformed by the reaction product. Plasmids were purified by alkaline-SDS method from the obtained resistant to ampicillin strains and digested with restrictase for selection is whether in the form of plasmids, having a sequence encoding secreterial form RBM (nucleotide sequence: 338-1355) in SEQ ID no 2, amino acid sequence: 72-316 in SEQ ID No. 1), by sequencing through a set Dyeter-minator cycle Sequencing FS kit (Perkin Elmer Co.). This plasmid was digested with restrictase NheI and XhoI for marquee (1,0, etc., ad), containing the cDNA Sekretareva form RBM, agarose gel-electrophoresis. This fragment was embedded in a fragment NheI/XhoI (10,4, etc., ad) expressing vector, rser (Invitrogen Co.), using set for ligating, and Escherichia coil DH5a was transformed using the reaction product. Plasmids were purified alkaline-SDS method from the obtained resistant to ampicillin strains and digested with restrictase for selection of an Escherichia coli strain having expressing secreterial form RBM plasmid (s) with the exact structure. The Escherichia coli strain containing rser-sOBM, cultivated and s was purified using a set of QIA filter plasmid midi kit (QIAGEN Co.).

(2) the Expression Sekretareva form RBM

Cells 293-EBNA suspended in IMDM containing 10% FCS (IMDM-10% FCS) and were sown in a 24-well plate coated with collagen (produced by Sumitomo Bakelite Co., Ltd.), at a density of 2 cells105/2 ml/well and the cult of the(Gibco Co.). After culturing for 2 days in 0.5 ml serum-free IMDM or IMDM-10% FCS conditioned medium were collected. Expression Sekretareva form RBM in air-conditioned environment was confirmed as follows. Sodium bicarbonate was added to the conditioned medium to a final concentration of 0.1 M and the solution was added to the 96-well plate. The plate was allowed to stand overnight at 4For immobilization thereby RBM in air-conditioned environment on 96-well pad. The tablet was filled with a solution of Block ACE(Snow Brand Milk Products Co., Ltd.), diluted 4 times with PBS (B-PBS), and allowed to stand for 2 hours at room temperature to block residual binding sites of the tablet. After adding to each well 100 ál of 3-100 ng/ml OCIF, diluted B-PBS, the plate was allowed to stand for 2 hours at 37With subsequent washing with PBS containing 0.05% tween-20 (PBS-T). Then to each well was added 100 μl of peroxidase labeled rabbit polyclonal antibodies against OCIF described in WO 96/26217, which were diluted B-PBS. After leaving for 2 hours at 37With the wells were washed six times in PBS-T. Then add TMB solution (TMB Sol is ut, then the reaction was stopped by adding 100 µl of stop solution (stop solution) (Stopping Reagent, Scytek Co.) in each well. The absorption of each well at 450 nm was measured by microplate reader. These results are presented in Fig.12, which shows that the absorption at 450 nm was increased according to the concentration added OCIF in the tablet, which was immobilized conditioned medium of cells transfected s. On the other hand, did not observe increase the absorption of the tablet, which was immobilized conditioned medium of cells transfected with a vector rser. Fig.13 shows the results of an experiment in which the ratio of the air-conditioned environment, which is used for immobilization was changed in the range of 5-90% and added some OCIF concentration (50 ng/ml). You can see that the absorption at 450 nm was increased in proportion to the increase in air-conditioned environment in the tablet, which was immobilized conditioned medium of cells transfected s, whereas such an increase was not observed in the tablet, which was immobilized conditioned medium of cells transfected with a vector rser. These results confirmed that the CR 15

Expression flushed thioredoxin-RBM protein (TGH-RBM)

(1) Constructing expressing vector flushed thioredoxin-RBM protein (TGH-RBM)

10 ál of 10x ExTaq buffer (Takara Shuzo Co.), 8 μl of 10 mm dNTP (Takara Shuzo Co.), 77,5 μl of sterilized distilled water, 2 μl of an aqueous solution rovm (10 ng/ml), 1 μl primer of OWS (100 pmol/μl, the table of the sequences SEQ ID No. 9), 1 μl primer OBMSalR2 (100 pmol/μl, the table of the sequences SEQ ID No. 10) and 0.5 μl of ExTaq (5 u/μl) (Takara Shuzo Co.) mixed and subjected to reaction (PCR reaction) in a microcentrifuge tube. After the reaction at 95C for 5 min, at 50C for 1 sec, at 55C for 1 min, with 74C for 1 sec and at 72C for 5 min cyclic reaction, consisting of reactions at 96C for 1 min, at 50C for 1 sec, at 55C for 1 min, with 74C for 1 sec and 72C for 3 minutes was repeated 25 times. Of all the reaction liquid DNA fragment of ~750 p. N. was purified by electrophoresis in 1% agarose gel using QIAEX II gel extraction kit (QIAGEN Co.). All the number cleaned up the purification of the DNA fragment is ~160 p. N. (Fragment 1), which was dissolved in 20 μl of sterilized distilled water. In the same way DNA fragment of ~580 p. N. (Fragment 2) obtained by cleavage of 4 µg ROM restrictase BamHI and EcoRI (Takara Shuzo Co.) and a DNA fragment of ~3.6V, etc., N. (Fragment 3), obtained by cleavage of 2 µg pTrXFus (Invitrogen Co.) restrictase BamHI and SaII (Takara Shuzo Co.), accordingly, purified and dissolved in 20 μl of sterilized distilled water. QIAEXII gel extraction kit was used for purification of DNA fragments. Fragments 1-3 ligated by incubation at 16C for 2.5 hours using a set of ligation of DNA DNA ligation kit ver.2 (Takara Shuzo Co.). Using the reaction liquid ligating the Escherichia coil G1724 (Invitrogen) were transformed according to the method described in the Instruction Manual of ThioFusion Expression System (Invitrogen Co.). The microorganism strain with a plasmid in which the cDNA fragment RBM (nucleotide sequence: 350-1111 in SEQ ID no 2, amino acid sequence: 76-316 in SEQ ID No. 1) fused in reading frame with the gene of thioredoxin, were selected from the obtained resistant to ampicillin transformed by analysis of restriction maps obtained by splitting restrictase, and DNA sequencing. Thus obtained strain of microorganisms which have thiverval respectively six hours with shaking at 30With in 2 ml of culture medium RMG-Amp (0.6% Of Na2NRA4for 0.3% KN2RHO4, of 0.05% NaCl, 0.1% NH4Cl, 1.2% of Casinocity (Difco Co.), 1% glycerol, 1 mm MgCl2and 100 μg/ml of ampicillin (Sigma Co.), a pH of 7.4). 0.5 ml of broth was added to 50 ml of Induction culture medium (0.6% of Na2HPO4for 0.3% KH2PO4, of 0.05% NaCl, 0.1% NH4Cl, 0.2% Casinocity, 0.5% of glucose, 1 mm MgCl2and 100 μg/ml ampicillin, pH 7,4) and cultured with shaking at 30C. When OD600 nmreached ~0.5, and added L-tryptophan to a final concentration of 0.1 mg/ml and then were shaken culture at 30Even within 6 hours. The culture broth was centrifuged at 3000g to collect the cells, which are suspended in 12.5 ml of PBS (10 mm phosphate buffer, 0.15 M NaCl, pH 7,4). The suspension was subjected to sonication (Ultrasonics Co.) for cell disruption. Damaged cells were centrifuged at 7000 x g for 30 minutes to obtain a supernatant as a soluble protein fraction. 10 ml of this soluble protein fraction was subjected to electrophoresis in SDS-page (10% SDS page) under reducing conditions. The result is a band with a molecular mass of 40 kDa, which was not detected in the soluble fraction is about protein (TGH-RBM) thioredoxin and RBM expressively in Escherichia coli.

(3) the Ability of binding TGH-RBM with OCIF

Binding is expressed TGH-RBM with OCIF was confirmed in the following experiment. Antibodies against thioredoxin (Invitrogen Co.), which was diluted 5000 times 10 mm sodium bicarbonate solution was added to 96-well immunoplate (Nunc Co.) in the amount of 100 μl per well. After standing overnight at 4With the liquid in the wells was discarded. 200 μl of a solution prepared by dilution twice Block ACE(Snow Brand Milk Products Co., Ltd.) using PBS (BA-PBS) was added to each well. After standing for 1 hour at room temperature the solution was discarded and each well was added 100 μl of soluble protein fractions originating from the above plasmids G1724/pTrxOBM25 or G1724/pTrxFus, diluted (each) BA-PBS at various concentrations. After standing for 2 hours at room temperature, each well was washed three times with PBS-T and loaded with 100 μl of OCIF (100 ng/ml), which was diluted with BA-PBS. After standing for 2 hours at room temperature, each well was washed three times with PBS-T and loaded with 100 μl of peroxidase labeled rabbit polyclonal antibodies against OCIF (described in WO 96/26217) diluted 2000 times BA-PBS. After standing in the course is luble Reagent, High Sensitivity, Scytek Co.). After standing for about 10 minutes at room temperature, each well was added 100 μl of stop solution (Stopping Reagent, Scytek Co.). The absorption of each well at 450 nm was measured by microplate reader. The results are shown in Fig.15. There was no difference in absorption between the sample with added soluble protein fraction originating from G1724/pTrxFus, and break without adding the soluble protein fraction. On the other hand, the absorption was increased in the samples to which was added the soluble protein fraction originating from G1724/pTrxOBM25, proportional to the concentration of the soluble protein fraction. The results of another experiment in which the rate of dilution of the soluble protein fraction was kept constant (1%), while the added OCIF, diluted with BA-PBS at various concentrations (0-100 ng/ml), shown in Fig.16. You can see that the absorption remained low at all OCIF concentration in the samples when using soluble protein fraction originating from G1724/pTrxFus, whereas absorption was increased proportional to the concentration of OCIF in the samples to which were added the soluble protein fraction originating from G1724/pTrxOBM25. Based on these results, it was confirmed that she Escherichia coil, producing TGH-RBM

Cells G1724/pTrxOBM25 distributed on RMG-Amp-agar (0.6% of Na2PO4for 0.3% KH2PO4, of 0.05% NaCl, 0.1% NH4Cl, 2% Casinocity, 1% glycerol, 1 mm MgCl2, 100 μg/ml ampicillin and 1.5% agar, pH 7.4) platinum loop. Cells were cultured overnight at 30C. the Cultured cells suspended in 10 ml of Induction medium. 5 ml of the suspension was added to each of two flasks Erlenmeyer 2 l containing 500 ml of Induction medium and cultured at 30With shaking. Upon reaching D600 nmof ~0.5 was added L-tryptophan to a final concentration of 0.1 mg/ml Cultivation with shaking was continued for 6 hours at 30C. the Culture broth was centrifuged for 20 minutes at 3000 x g to collect the cells, which are suspended in 160 ml of PBS. The suspension was subjected to processing by the ultrasonic generator (Ultrasonics Co.) for cell disruption. The supernatant was centrifuged for 30 minutes at 7000g to obtain the soluble protein fraction.

(5) Preparation of affinity column with immobilized OCIF

2 g TSKgel AF-TolesyI Toyopal 650 (Tosoh Corp.) and 40 ml containing 1.0 M potassium phosphate buffer (pH 7.5) containing 35,0 mg re what and when 4For performing the reaction combinations. The reaction mixture was centrifuged to remove the supernatant. For inactivation of surplus balances at the besieged media was added 40 ml of 0.1 M Tris-Hcl buffer (pH 7.5) and the mixture was gently rocked at room temperature for 1 hour. The media in the column was washed with buffer 0.1 M glycine-Hcl (pH 3,3) containing 0.01% of Polysorbate 80 and 0.2 M NaCl, and buffer with 0.1 M sodium citrate (pH 2.0) containing 0.01% of Polysorbate 80 and 0.2 M NaCl. The media in the column was balanced by the introduction of the double-buffer with 10 mm sodium phosphate (pH 7.4) containing 0.01% of Polysorbate 80.

(6) Cleaning TGH-RBM using affinity column with immobilized IF

If there are no other indications, treatment TGH-RBM was carried out at 4C. the Above-mentioned affinity media with immobilized OCIF (10 ml) and the soluble protein fraction (120 ml) obtained in Example 15(4), was mixed. The mixture was gently stirred overnight at 4With four centrifuge tubes 50 ml with the aid of the rotor. Column Econo-column(inner diameter 1.5 cm, length 15 cm, manufactured by BioRad Co.) filled carrier in the mixture. The column was loaded with 300 ml of PBS containing 0.01% of Polysorbate 80, 100 ml containing 10 mm sodium phosphate buffer (the second sequence. The proteins adsorbed to the column was suirable 0.1 M sodium citrate-buffer (pH 2.0) containing 0.01% of Polysorbate 80 and 0.2 M NaCl. The eluate was collected in fractions of 5 ml Each fraction collected in this way, immediately neutralized by adding 10% volume of 2 M Tris buffer (pH 8.0). The presence or absence of TGH-RBM in buervenich fractions was determined according to the method previously described in Example 15(3) (the ability of binding to OCIF). The fractions containing TGH-RBM, collected and optionally purified.

(7) Cleaning TGH-RBM gel-filtration

~ 25 ml fractions TGH-RBM obtained in Example 15(6), concentrated to ~0.5 ml by centrifugation using a Centriplus 10 and Method 10 (Amicon Co.). This sample was applied to a column of Superose 12 HR 10/30 (1,030 cm, Pharmacia Co.), pre-equilibrated PBS containing 0.01% of Polysorbate 80. To separate as the mobile phase used PBS containing 0.01% of Polysorbate 80, when the speed of the current of 0.25 ml/min, the Eluate from the column was collected portions of 0.25 ml TGH-RBM in the collection of such fractions were detected by the method previously described in Example 15(3), and by electrophoresis in SDS-page (10-15% polyacrylamide gel, Pharmacia Co.) using system Phast System (Pharmacia Co.) and silver staining. Fractions (Fractions 20-23), steadily with bovine serum albumin as a standard substance, using the test set DC-Protein (BioRad Co.).

Example 16

Inducing the formation of osteoclasts activity RBM

ROM and pcDL-SR296, respectively, were transferrable cells COS-7 using Lipofectamine (Gibco Co.). Cells were cultured in DMEM containing 10% FCS for 1 day, was trypsinization, were sown on top of the glass (15 mm, round shape, produced by Matsunami Co.) in 24-hole tablets when 5104cells per well and were cultured for 2 days. Cultural tablet were washed once with PBS. The cells were fixed with PBS containing 1% paraformaldehyde at room temperature for 8 minutes. The tablet, on which were attached the fixed cells were washed 6 times with PBS, then to each well was added 700 μl of mouse spleen cells, suspended in 1106/ml-MEM containing 10-8M active form of vitamin D3, 10-7M dexamethasone, and 10% fetal bovine serum. Millicell PCF (Millipore Co.) was installed in each well and added to cell suspension ST2 in the above-mentioned culture medium (4104/ml), 700 μl per well, in Millicell PCF, followed by incubation at 37C for 6 days. After ku is within 1 minute. Then cells that detect resistant tartaric acid activity of acid phosphatase (TRAP), which is a specific marker for osteoclast selectively stained using a set of LEUKOCYTE ACID PHOSPHATASE kit (Sigma Co.). Microscopic observation showed that TRAP-positive cells were not detected in the wells in which cells were fixed COS-7, transfected pcDL-SR296. In contrast, 45±18 (average value±standard deviation, n=3) trap-positive cells were observed in the wells, which were fixed cells COS-7, transfected ROM. In addition, it was also confirmed that calcitonin was associated with these trap-positive cells. These discoveries proved that the RBM has induce the formation of osteoclasts activity.

Example 17

Inducing the formation of osteoclasts activity TGH-RBM Sekretareva form RBM

Mouse spleen cells suspended in-MEM containing 10-8M active form of vitamin D3, 10-7M dexamethasone, and 10% fetal bovine serum, at a concentration of 2106/ml. of This suspension was added to 24-well plate in the amount of 350 µl per well. Then each well Sagl), 350 μl of the solution obtained with 10-fold dilutions of conditioned medium, which was obtained by culturing cells 293-EBNA, which were transfected pCEPsOBM or RER in IMDM-10% FGS, the above culture medium, or only above 350 cells / ml of culture medium. Millicell PCF (Millipore Co.) was placed in each well, to which was added 600 μl of ST2 cells, suspended in the above culture medium (4104/ml). After culturing for six days Millicell PCF was removed. The tablet was washed once with PBS and cells were fixed with a solution of acetone-ethanol (50:50) for 1 minute. Then cells exhibiting resistant tartaric acid activity of acid phosphatase (TRAP activity), selectively stained using a set of LEUKOCYTE ACID PHOSPHATASE kit (Sigma Co.). Microscopic observation showed that in the wells, which were not added TGH-RBM) were not found cells expressing TRAP-activity, while 106±21 (mean±standard deviation, n=3) TRAP-positive cells were observed in the wells, to which was added TGH-RBM. Similarly while in the holes, to which was added air-conditioned environment of the cells 293-EBNA, transfected rser, did not detect cells that are showing to which was added air-conditioned environment of the cells 293-EBNA, transfected s. In addition, it was confirmed that calcitonin is associated with these TRAP-positive cells. These results proved that TGH-RBM secretiruema form RBM are inhibitory to the formation of osteoclasts activity.

Example 18

Identity RBM protein expressed by the cDNA of the present invention, and OCIF-binding protein natural type of this invention

(1) preparation of rabbit polyclonal antibodies against RBM

Three male Japanese white rabbits (weight 2.5-3.0 kg, supplied Kitayama Labes Co.) were immunized with purified RBM (thioredoxin-RBM protein) obtained in accordance with the method of Example 14(6) 14(7), subcutaneous injection of 1 ml/dose of the emulsion obtained by mixing 200 mg/ml purified RBM with an equal volume of complete adjuvant's adjuvant (DIFCO Co.), six times, once a week. 10 days after the last immunization, the rabbits were bled. Antibodies were purified from serum as follows. Ammonium sulfate was added to anticigarette, which was diluted 2 times with PBS to a final concentration of 40% (wt./vol.). After standing for 1 hour at 4The mixture was centrifuged for 20 minutes at 8000g to obtain a precipitate. The residue RA is G-Separato column (produced by Pharmacia Co.). After washing PBS adsorbed immunoglobulin G was suirable 0.1 M glycine-HCl buffer solution (pH 3.0). The eluate was immediately neutralized with 1.5 M Tris-Hcl-buffer (pH 8,7). After dialysis buervenich protein fractions against PBS was measured by absorption at 280 nm to determine the concentration of protein (E1%13,5). Antibodies against RBM, labeled with horseradish peroxidase was obtained using a set of maleimide-activated peroxidase (Pierce Co.) in the following way. 80 µg of N-succinimide-S-acetylthiocholine acid was added to 1 mg of purified antibodies and the reaction was carried out at room temperature for 30 minutes. To the mixture was added 5 mg of hydroxylamine to deacetylation of antibodies. The modified antibodies were fractionally using polyacrylamide desalting column. Protein fractions were mixed with 1 mg maleimide-activated peroxidase and gave them to react for 1 hour at room temperature to obtain enzyme labeled antibodies.

(2) the Ability of rabbit polyclonal antibodies against RBM to inhibit specific binding of the protein (RBM), expressed by the cDNA of the present invention or the protein of the natural type of the present invention with OCIF

Purified RBM (thioredoxin-RBM fused protein of Example 2(4) was dissolved respectively in a containing 0.1 M sodium carbonate buffer to a concentration of 2 µg/ml An aliquot of each solution was added at 100 μl per well, respectively, in 96-well immunoplate (produced by Nunc Co.). The plate was allowed to stand overnight at 4C. To each well was added 200 μl of 50% Block ACE and the plate was allowed to stand at room temperature for 1 hour. After washing each well three times with PBS containing 0.1% Polysolbate 20 (P20-PBS), 100 μl of a solution of rabbit antibodies against RBM, which were dissolved in 25% Block ACE, cooked with P20-PBS to a concentration of 200 μg/ml, or 100 μl of 25% Block ACE (containing antibodies) were added to each well followed by incubation at 37C for 1 hour. Each well was washed three times P20-PBS and loaded with 100 μl/well test solution for binding (P20-PBS containing 0.2% serum albumin calf, 20 mm HEPES and 0.1 mg/ml heparin), to which was added 20 ng/ml125I-labeled OCIF described in Example 8(3). Alternate each well was loaded with 100 μl/well of another test solution for binding, containing 8 μg/ml of unlabeled OCIF, in addition to 20 ng/ml125I-labeled OCIF. After incubation of these immunopositive at 37C for 1 hour, the wells were washed six times P20-PBS. The number of125I in every hole by cDNA of the present invention, and a protein that specifically binds to OCIF-binding protein natural type of this invention, are not associated with a125I-labeled OCIF in General, when they are treated with rabbit polyclonal antibodies against RBM, while both protein bound125I-labeled OCIF, when they were not treated with antibodies. It was confirmed that the binding of both proteins with125I-labeled OCIF was clearly specific, because this binding is significantly inhibited by the addition of 400-fold concentration of unlabeled OCIF (8 µg/ml). The above results showed that rabbit polyclonal antibodies against RBM learn how RBM, which is a protein expressed by the cDNA of the present invention, and OCIF-binding protein natural type of the present invention and inhibit the specific binding of these proteins with OCIF.

Example 19

Cloning of cDNA RBM man

(1) preparation of primer mouse RBM

Primers mouse RBM obtained according to the method of Examples (RBM#3 and RBM#8), described above, was used for screening cDNA RBM person. These sequences are shown in Table sequences SEQ ID No. 9 and SEQ ID No. 6, respectively.

(2) Isolation of cDNA RBM person using PCR

cDNA-fragments of RBM human Lymph Node Marathon (Clontech Co.) as the matrix. Conditions for PCR were as follows:

10 x EX Taq buffer (Takara Shuzo Co.) 2 ál

2.5 mm dNTP I,6 ál

Solution 1 µl cDNA

EX Taq (Takara Shuzo Co.) 0.2 µl

Distilled water of 14.8 ál

40 μm primer RBM#3 0.2 µl

40 μm primer RBM#8 0.2 µl

These solutions were mixed in a centrifuge tube and was preincubate at 95C for 2 minutes followed by 40 cycles of three-stage reaction, consisting of reactions at 95C for 30 seconds, with 57C for 30 seconds and at 72C for 2.5 minutes. After this reaction solution was incubated for 5 minutes at 72And part of the solution was subjected to electrophoresis on agarose gel. Was discovered DNA fragment (~690 p. N.), amplificatory cDNA primers mouse RBM described above.

(3) Purification of cDNA-fragment RBM person, the amplified PCR and determination of nucleotide sequence of

cDNA-fragment RBM person obtained in Example 19(2), were separated by electrophoresis on agarose gel and optionally purified using the kit for the extraction of gels QIAEX (Qiagen Co.). Again, PCR was performed using purified cDNA fragment RBM person as a matrix and cDNA primers machinely with the use of the kit for the extraction of QIAEX gel, as explained above. The nucleotide sequence of the purified DNA fragment RBM man was determined with a set of Taq Dye Deoxy Terminator Cycle Sequencing FS (Perkin Elmer Co.) using RBM#3 or RBM#8 (SEQ ID No. 9 and SEQ ID No. 6, respectively) as a primer. When comparing this sequence with the corresponding plot cDNA mouse RBM nucleotide sequence of the cDNA fragment RBM person found 80,7% homology with the cDNA sequence of murine RBM.

(4) Screening the full-size cDNA RBM person using hybridization using cDNA fragment (approximately 690 p. N.) RBM person as probe

Screening the full-size cDNA RBM were performed using cDNA fragment (~690 p. N.) RBM person who has been purified in Example 19(3) and marked [32P]-dCTP using the kit for labeling Megaprime DNA Labeling kit (Amersham Co.). A cDNA library from Human Lymph Node 5'-STRETCH PLUS (Clontech Co., the USA) were subjected to screening using a DNA probe. In accordance with the Protocol of the manufacturer Escherichia coil C600 Hfl infected with recombinant phage for 15 minutes at 37C. Infected Escherichia coil was added to LB-agar (1% tripton, 0,5% yeast extract, 1% NaCl, 0.7% agar), which was Nanya overnight at 37With HyBond-N(Amersham Co.) was placed on the Cup, which was producirovanie sterile spot, and was sustained for ~3 minutes. In accordance with the generally accepted method filter treated with an alkaline solution, neutralized and were immersed into the solution of 2 x SSC. Then DNA was immobilized on the filter using a UV CROSSLINKER (Stratagene Co.). The resulting filter was immersed in a buffer for rapid hybridization (Rapidhyb buffer, Amersham Co.). After preprocessing for 15 minutes at 65The filter was placed in Rapid-hyb buffer containing the denatured by heating cDNA fragment RBM person (~690 p. N., 5105pulse/min/ml), as described above. After hybridization overnight at 65With the filter was washed in 2 x SSC, 1 x SSC and 0.1 xSSC, each of which contained 0.1% of SDS, in sequence, respectively, for 15 minutes at 65C. received Several positive clones was further purified by repeating twice this screening. The clone having the insert (~2,2, etc., ad), was collected from the purified clones and used in subsequent experiments. This purified phage was namedhOBM. Approximately 10 μg of DNA was obtained from oceanog the NC were digested with restriction enzyme SalI and subjected to electrophoresis on agarose gel to separate the cDNA inserthOBM (~2,2, etc., ad). This DNA fragment is purified using a kit for the extraction of gels QIAEX (Qiagen Co.), were digested with restriction enzyme SalI, and was built in plasmid pUC19 (MBI Co.), which was pre-digested with restriction enzyme SalI and dephosphorylated, using a set of DNA ligation kit ver. 2 (Takara Shuzo Co.). Escherichia coli DH5a (Gibco BRL Co.) transformed pUC19 containing the DNA fragment. The obtained transformant was named pUC19hOBM. The transformant was grown and pUC19hOBM, which was built cDNA RBM person (~2,2, etc., ad), was purified in a conventional manner.

(5) determining the nucleotide sequence of the cDNA encoding the complete amino acid sequence of RBM man

The nucleotide sequence obtained in Example 19(4) cDNA RBM man was determined with a set of Taq Dye Deoxy Terminator Cycle Sequencing FS (Perkin Elmer Co.). Specifically, the nucleotide sequence of the embedded fragment was determined using pUC19hOBM as a matrix. As primers the primers used were to determine the nucleotide sequence of embedded DNA fragment in pUC19hOBM, M13 Primer M3 and M13 Primer RV (manufactured by Takara Shuzo Co.), and a synthetic primer, RBM#8 person designed based on the nucleotide sequence of the cDNA fragment RBM people who estwenno as SEQ ID No. 4 and SEQ ID No. 5. Amino acid sequence RBM person, which is decoded from the nucleotide sequence of the cDNA RBM person shown in the Table of sequences as SEQ ID No. 11. The nucleotide sequence of cDNA RBM person shown as SEQ ID No. 12.

Escherichia coli, which was transformed pUC19hOBM, which is a plasmid containing the cDNA obtained RBM man, was deposited in National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, August 13, 1997 under the Deposit number FERM BP-6058.

Example 20

Radioiodinated OCIF125I and quantitative analysis125I-labeled OCIF using ELISA

OCIF were labeled125I using IODO-GEN method. 20 μl of 2.5 mg/ml solution of IODO-GEN-chloroform was transferred to a test tube Eppendorf 1.5 ml) and the chloroform evaporated at 40With receiving tubes, covered with IODO-GEN. The tube was washed three times with 400 µl containing 0.5 M sodium phosphate buffer solution (Na-Pi, pH 7.0), followed by adding 5 μl of 0.5 M Na-Pi (pH 7.0). In this test tube was added 1,3 ál (of 18.5 MBq) solution of Na125I (Amersham Co., NEZ-033H) and immediately after that, 10 μl of 1 mg/ml solution OCIF (monomer type or dimeric type). The mixture was stirred voltex-mixer and gave it to stand at room temperature for 30 secondage 10 mg/ml KI, and 5 μl of phosphate buffered saline containing 5% bovine serum albumin (BSA-PBS). This solution was stirred, were applied to the rotary column (1 ml, G-25 fine Sephadex produced by Pharmacia Co.), which had been equilibrated in advance BSA-PBS, and centrifuged for 5 minutes at 2000 rpm To fractions, elyuirovaniya from this column were added 400 μl of BSA-PBS. After mixing 2 µl of this solution was used for measuring radioactivity in a gamma counter. Radiochemical purity of the obtained solution125I-labeled OCIF was measured by the radioactivity of the fractions precipitated with 10% trichloroacetic acid (TCA).

Biological activity125I-labeled OCIF was measured by the method described in WO 96/26217. Concentration125I-labeled OCIF was measured using ELISA as follows. Specifically, 50 mm Panso3(pH 9,6), which were dissolved rabbit polyclonal antibodies against OCIF described in WO 96/26217, to a concentration of 2 μg/ml was added into each well of 96-well immunoplate (MaxiSorpmanufactured by Nunc Co.) in the amount of 100 μl per well. After standing these wells overnight at 4To the solution was removed. Then the wells were loaded with a mixture of aqueous solution of Block ACEExample 21

Expression of the proteins encoded by cDNA of the present invention

(1) To the HT (~2,2, etc., ad) was purified by electrophoresis on 1% agarose gel. The ends of the DNA fragments was a small mistake with a set for blunting DNA (Takara Shuzo Co.) (hOBMcDNA-fragment with blunt ends). Expression plasmid pcDL-SR296 (Molecular and Cellular Biology, Vol. 8, pp. 466-472 (1988)) was digested with restriction enzyme EcoRI, was a small mistake with a set for blunting the ends and ligated with cDNA-fragment hOBM with blunt ends with a set for ligating (ver.2). Escherichia coli DHtransformed reaction mixture for ligation. The plasmids obtained are resistant to ampicillin transformants was subjected to cleavage by restriction enzyme analysis restriction maps of DNA and determination of DNA sequence. The result was selected strain with a plasmid in which the cDNA hOBM embedded in the same direction of transcription, the promoter SR. This microorganism was named DH5/phOBM.

(2) the Expression of RBM human cells COS-7

Escherichia coil DH5/phOBM were cultured and plasmid phOBM was purified using the kit Qiafilter Plasmid Midi kit (Qiagen Co.). phOBM was respecitvely using Lipofectamine cells COS-7 hole 6-hole tablet and were cultured for 2 days in DMEM containing 10% fetal the n Seiyaku Co., Ltd.), to which was added 5% cialisavandia fetal bovine serum (88 μl/well). Cells were incubated for 15 minutes followed by the addition of 14 μl of the mixture for rapid labeling of proteins Express Protein Labeling Mix (NEN Co., 10 MCI/ml). After 4 hours incubation each well was added 200 μl of DMEM containing 10% fetal bovine serum. Cells were cultured for 1 hour and washed twice in PBS. Then to each well was added 0.5 ml of TSA-buffer (10 mm Tris-HCl, containing about 0.14 M NaCl and 0.025% NaN3, pH 8.0) containing 1% Triton X-100, 1% bovine hemoglobin, 10 μg/ml leupeptin, of 0.2 TIU/ml Aprotinin and 1 mm PMSF, and the mixtures were allowed to stand for 1 hour on ice. The cells were mixed by pipetting and centrifuged at 3000g for 10 minutes at 4With obtaining supernatant. 200 ál of dilution buffer (TSA-buffer containing 0.1% Triton X-100, 0.1% of bovine hemoglobin, 10 μg/ml leupeptin, of 0.2 TIU/ml Aprotinin and 1 mm PMSF) was added to 100 μl of supernatant from each well. The resulting mixture was stirred at 4C for 1 hour with Protein a-Separate (50 ál) and centrifuged at 1500g for 1 minute at 4With a collection of supernatants,Lyali OCIF (1 g) and the mixture was stirred for 1 hour at 4For binding RBM person and OCIF. Then add the rabbit polyclonal antibodies against OCIF (50 mg) and stirred at 4C for 1 hour. To the resulting solution was added to Protein a-Sepharose (10 μl), followed by stirring at 4Even within one hour. Thus obtained mixture was centrifuged for 1 minute at 1500g if 4With to collect precipitation. Precipitates were washed twice with buffer for cultivation, double-buffer for cultivation without bovine hemoglobin, once S-buffer and once with 50 mm Tris-Hcl (pH 6.5). After adding SDS buffer containing 10%-mercaptoethanol (0.125 M Tris-HCl, 4% sodium dodecyl sulphate, 20% glycerol, of 0.002% bromophenol blue, pH 6.8), the mixture was heated for 5 minutes at 100C and subjected to electrophoresis in SDS-page with 12.5% polyacrylamide gel (Daiichi Pure Chemical Co.,). The gel was fixed and dried in accordance with generally accepted way. After the signal amplification isotope using Amplify(Amersham Co.) the dried gel was subjected to autoradiography at -80With using film Bio Max MR (Kodak Co.). The results are presented>the example 22

Binding of the protein encoded by cDNA of the present invention, and OCIF

Plasmid phOBM, which was purified according to the method described in Example 21(2), transferrable cells COS-7 in each well of a 24-hole tablet using Lipofectamine. After culturing for 2-3 days, the cells were washed in serum-free DMEM. To the wells was added 200 μl of culture medium for the test-environment link (serum-free DMEM to which were added with 0.2% bovine serum albumin, a solution of 20 mm S-buffer, 0.1 mg/ml heparin and 0.2% NaN3) containing 20 ng/ml125I-labeled OCIF. In other wells were added 200 μl of culture medium for the test-environment linkage, containing 8 μg/ml of unlabeled OCIF, in addition to 20 ng/ml125I-labeled OCIF. After incubation for 1 hour at 37With CO2-thermostat (5% CO2cells were washed twice with 500 µl phosphate buffered saline containing 0.1 mg/ml heparin. Then to each well was added 500 μl of 0.1 n NaOH solution and the plate was allowed to stand for 10 minutes at room temperature to dissolve the cells. Radioactivity125I in the wells was measured by gamma counter. As a result, as shown in Fig.19, it was confirmed that the125Example 23

The stitching125I-labeled OCIF and the protein encoded by cDNA of the present invention

For additional studies of the characteristics of the proteins encoded by cDNA of the present invention conducted stitching125I-labeled OCIF monomer type and the proteins encoded by cDNA of the present invention. After constructing expressing vector phOBM and transfection in his cell COS-7 according to the method used in Example 21(1) and 21(2), was added 200 μl of a test environment for binding, containing125I-labeled OCIF (25 ng/ml) described above. For the other wells used test environment for binding, to which was added unlabeled OCIF 400-fold concentration. After culturing for 1 hour at 37With CO2-thermostat (5% CO2cells were washed twice with 500 µl phosphate buffered saline containing 0.1 mg/ml heparin. 500 μl of phosphate buffered saline solution in which was dissolved a crosslinking agent (DSS: disuccinimidyl produced by Pierce Co.) (100 μg/ml), was added to the cells followed by incubation for frame phosphate salt solution. After adding 100 μl of 20 mm NON-PES-buffer containing 1% Triton X-100 (Wako Pure Chemicals Co., Ltd.), 2 mm PMSF (phenylmethylsulfonyl, Sigma Co.), 10 µm Pepstatin (Wako Pure Chemicals Co., Ltd.), 10 μm Leupeptin (Wako Pure Chemicals Co., Ltd.), 10 μm antipain (Wako Pure Chemicals Co., Ltd.) and 2 mm EDTA (Wako Pure Chemicals Co., Ltd.), the hole was allowed to stand for 30 minutes at room temperature to dissolve the cells. Aliquots of 15 μl of these samples were treated with SDS under reducing conditions in accordance with the conventional method and subjected to SDS-electrophoresis using 4-20% polyacrylamide gradient gel (Daiichi Pure Chemical Co., Ltd.). After electrophoresis the gel was dried and subjected autoradiography within 24 hours at -80With using film BioMax MS (Kodak Co.) and intensifying screen BioMax MS (Kodak Co.). The film subjected to autoradiography, showed an accepted way. The result is a band with a molecular mass in the range 90000-110000 shown in Fig.20, were detected by binding125I-labeled OCIF monomer type and the proteins encoded by cDNA of the present invention.

Example 24

Expression Sekretareva form RBM man

(1) Construction of plasmids expressing secreterial form RBM man

The PCR reaction was carried out using the RBM S primers and pUC19hOBM as a matrix. After purification by agarose gel electrophoresis, the product was digested with restrictase SplI and HindIII and was additionally purified by agarose gel electrophoresis to obtain a purified fragment (0,27, etc., ad). cDNA RBM man was partially digested with restriction enzyme DraI and DNA fragments, split DraI in the same site, was purified by electrophoresis on agarose gel. The purified fragment was further purified by restriction enzyme HindIII. Fragment of 0.53, etc., N. DraI/HindIII, purified by electrophoresis on agarose gel. The purified fragment is ligated with a fragment of SplI/HindIII 0,27 T. p. N., obtained from PCR, as described above, using the set for ligating PERC.2 (Takara Shuzo Co.) together with a fragment of HindIII/EcoRI (5,2, etc., ad) pSec TagA (Invitrogen Co.). Escherichia coil DH5transformed by the reaction product. Plasmids were purified by alkaline-SDS-cnoco6y from the obtained resistant to ampicillin of transformants and digested with restrictase for selection of plasmids containing fragments with a length of 0.27, etc., N. and 0,53 etc., N. in the form of inserts in pSec TagA. It was confirmed that this plasmid was sequence encoding secreterial form RBM person, by sequencing through a set of Tag Dyedeoxyterminator cycle Sequencing FS kit (Perkin Elmer Co.). This plasmid was digested with restrictase NheI and XhoI to obtain fra is. the same fragment was embedded in a fragment NheI/XhoI (10,4, etc., ad) expressing vector, rser (Invitrogen Co.), using set for ligating, and scherichia coli DH5transformed using the reaction product. Plasmids were purified alkaline SDS method from the obtained resistant to ampicillin of transformants and digested with restrictase for selection of an Escherichia coli strain with a plasmid expressing secreterial form RBM person (pCEPshOBM). The Escherichia coli strain containing pCEPshOBM, cultivated and pCEPshOBM was purified using a kit Qiafilter plasmid midi kit (QIAGEN Co.).

(2) the Expression Sekretareva form RBM

Cells 293-EBNA suspended in IMDM containing 10% FCS (IMDM-10% FCS) were added to 24-well plate coated with collagen (produced by Sumitomo Bakelite Co. Ltd.), at a density of 2 cells105/2 ml/well and cultured overnight. The cells were transferrable 1 µg pCEPshOBM or rser using 4 µl of Lipofectamine (Gibco Co.). After culturing for 2 days in 0.5 ml serum-free IMDM or IMDM-10% FCS were collected cultural supernatant. The expression Sekretareva form RBM person in the culture supernatant was found in the following way. Sodium bicarbonate was added to the culture soup is the significance of the night when 4For immobilization thereby RBM person in the culture supernatant on 96-well pad. The tablet is blocked with a solution of Block ACE(Snow Brand Milk Products Co., Ltd.), diluted 4 times with PBS (B-PBS), and allowed to stand for 2 hours at room temperature. After adding to each well 3-100 ng/ml OCIF, diluted B-PBS, the plate was allowed to stand for 2 hours at 37With subsequent washing with PBS containing 0.05% Polysolvate 20 (P-PBS). Then to each well was added 100 μl of peroxidase labeled rabbit polyclonal antibodies against OCIF described in WO 96/26217, which were diluted B-PBS. After standing for 2 hours at 37With the wells were washed six times P-PBS. Then add TMB solution (TMB Soluble Reagent, High Sensitivity, ScyTek Co.) in the amount of 100 μl per well and allowed to stand at room temperature for ~10 minutes. The reaction was stopped by adding 100 µl of stop solution (stop solution) (Stopping Reagent, ScyTek Co.) in each well. The absorption at 450 nm for each well was measured by a microplate reader. These results are presented in Fig.21, which shows that the absorption at 450 nm was increased according to the concentration added OCIF in the tablet kotowali increase the absorption in the wells, which was immobilized conditioned medium of cells transfected with a vector rser. Fig.22 shows the results of an experiment in which the ratio of the air-conditioned environment, used for immobilization was changed in the range of 5-90% in the presence of a constant concentration of OCIF (50 ng/ml). The absorption at 450 nm was increased in proportion to the increase in air-conditioned environment in the tablet, which was immobilized conditioned medium of cells transfected pCEPshOBM, whereas this increase in absorption was not observed in the tablet, which was immobilized conditioned medium of cells transfected with a vector rser. These results confirmed that secretiruema form RBM person is produced in the conditioned medium of cells transfected pCBPshOBM.

Example 25

The expression of the fused protein thioredoxin-RBM person (Trx-hOBM)

(1) Konstruirovanie vector expressing the protein thioredoxin-RBM person (Trx-hOBM)

10 ál of 10x ExTaq buffer (Takara Shuzo Co.), 8 μl of 10 mm dNTP (Takara Shuzo Co.), 77,5 μl of sterilized distilled water, 2 μl of an aqueous solution pUC19hOBM (10 ng/ml), 1 μl of primer, mouse RBM#3 (100 pmol/μl, the table of the sequences SEQ ID No. 9), 1 μl of primer, hOBMSalR2 (100 is(PCR reaction) in a microcentrifuge tube. After the reaction at 95C for 5 min, at 50C for 1 sec, at 55C for 1 min, with 74C for 1 sec and at 72C for 5 min cyclic reaction, consisting of reactions at 96C for 1 min, at 50C for 1 sec, at 55C for 1 min, with 74C for 1 sec and at 72C for 3 minutes was repeated 25 times. Of the entire reaction mixture was purified DNA fragment (750 p. N.). All the amount of purified DNA fragment was digested with restrictase SalI (Takara Shuzo Co.) and BspHI (New England Biolabs Co.) and were subjected to electrophoresis on 1% agarose gel to obtain a purified DNA fragment (Fragment 1, ~320 p. N.). This fragment was dissolved in 20 μl of sterilized distilled water. In the same way DNA fragment (Fragment 2, ~450 p. N.) obtained by cleavage of 4 µg pUC19hOBM restrictase BamHI and BspHI (Takara Shuzo Co.), and a DNA fragment (Fragment 3, ~3.6V, etc., ad), obtained by cleavage of 2 µg pTrXFus (Invitrogen Co.) restrictase BamHI and SalI (Takara Shuzo Co.), accordingly, purified and dissolved in 20 μl of sterilized distilled water. QIAEXII gel extraction kit IP for 2.5 hours using a set of ligation of DNA DNA ligation kit ver.2 (Takara Shuzo Co.). Using the reaction liquid ligating the Escherichia coil G1724 (Invitrogen) were transformed according to the method described in the Instruction Manual of ThioFusion Expression System (Invitrogen Co.). The microorganism strain with a plasmid in which the cDNA fragment hOBM fused in reading frame with the gene of thioredoxin, were selected from the obtained resistant to ampicillin transformed by analysis of restriction maps of the DNA obtained by splitting restrictase, and DNA sequencing. Thus obtained strain of microorganism was named G1724/pTrxhOBM.

(2) the Expression of Trx-hOBM in Escherichia coli

Plasmids G1724/pTrxhOBM and G1724 containing pTrxFus (G1724/pTrxFus), were cultured respectively six hours with shaking at 30With in 2 ml of culture medium RMG-Amp (0.6% Of Na2HPO4for 0.3% KN2RHO4, of 0.05% NaCl, 0.1% NH4Cl, 1.2% of Casinocity (Difco Co.), 1% glycerol, 1 mm MgCl2and 100 μg/ml ampicillin, pH 7.4). 0.5 ml of broth was added to 50 ml of Induction culture medium (0.6% of Na2HPO4for 0.3% KN2RHO4, of 0.05% NaCl, 0.1% NH4Cl, and 0.2% kasuminome acid, 0.5% of glucose, 1 mm MgCl2and 100 μg/ml ampicillin, pH 7.4) and cultured with shaking at 30C. When D600 nmreached ~0.5, and added L-tryptoph the f within 6 hours. The culture broth was centrifuged at 3000g to collect the cells, which are suspended in 12.5 ml PBS. The suspension was subjected to sonication (Ultrasonics Co.) for cell disruption. Damaged cells were centrifuged at 7000 x g for 30 minutes to obtain a supernatant as a soluble protein fraction. 10 ml of this soluble protein fraction was subjected to electrophoresis in SDS-page (10% SDS page) under reducing conditions. As a result, as shown in Fig.23, the band with the molecular weight of 40,000, which was not detected in the soluble protein fraction G1724/pTrxFus, was observed in the fraction of soluble protein G1724/pTrxhOBM (Fig.14). Thus, it was confirmed that the protein (Trx-hOBM) thioredoxin and RBM person expressively in Escherichia coli.

(3) the binding Ability of Trx-hOBM with OCIF

Binding is expressed Trx-hOBM with OCIF was confirmed in the following experiment. Antibodies against thioredoxin (Invitrogen Co.), which was diluted 5000 times 10 mm sodium bicarbonate solution was added to 96-well immunoplate (Nunc Co.) in the amount of 100 μl per well. After standing overnight at 4With the liquid in the wells was discarded. 200 μl of a solution prepared by dilution in two Rania for 1 hour at room temperature, the wells were washed three times P-PBS. To each well was added 100 μl of soluble protein fractions originating from the above G1724/pTrxhOBM25 or G1724/pTrxFus, diluted (each) BA-PBS at various concentrations. After standing for 2 hours at room temperature, each well was washed three times P-PBS and add 100 ál of OCIF (100 ng/ml), which was diluted with BA-PBS. After standing for 2 hours at room temperature, each well was washed three times P-PBS and add 100 ál of peroxidase labeled antibodies against OCIF (described in WO 96/26217) diluted 2000 times BA-PBS. After standing for 2 hours at room temperature, each well was washed six times P-PBS and add 100 ál of TMB solution. After standing for about 10 minutes at room temperature, each well was added 100 μl of stop solution (Stopping Reagent, ScyTek Co.). The absorption of each well at 450 nm was measured by microplate reader. The results are shown in Fig.24. There was no difference in absorption between the sample with added soluble protein fraction originating from G1724/pTrxFus, and break without adding the soluble protein fraction. On the other hand, the absorption was increased in the samples to which was added the soluble protein fraction originating from G1724/pTrxhOBM proportion constructuring protein was held constant (1%), while added OCIF, diluted with BA-PBS at various concentrations (0-100 ng/ml), shown in Fig.25. You can see that the absorption remained low at all OCIF concentration in the samples when using soluble protein fraction originating from G1724/pTrxFus, whereas absorption was increased proportional to the concentration of OCIF in the samples to which were added the soluble protein fraction originating from G1724/pTrxhOBM. On the basis of these results, it was confirmed that the Trx-hOBM obtained from G1724/pTrxhOBM, has the ability of binding to OCIF.

(4) large-Scale cultivation of Escherichia coli producing Trx-hOBM

Cells G1724/pTrxhOBM distributed on RMG-Amp-agar (0.6% PA2HPO4for 0.3% KN2RHO4, of 0.05% NaCl, 0.1% NH4Cl, 2% Casinocity, 1.5% agar, pH 7.4) platinum loop 100 RUB. Cells were cultured overnight at 30C. the Cultured cells suspended in 10 ml of Induction medium. 5 ml of the suspension was added to each of two flasks Erlenmeyer 2 l containing 500 ml of Induction medium and cultured at 30With shaking. Upon reaching D600 nmof ~0.5 was added L-tryptophan to a final concentration of 0.1 mg/ml Cultivation with shaking cont is at 3000g to collect the cells, which are suspended in 160 ml of PBS. The suspension was subjected to processing by the ultrasonic generator (With Ultrasonics.) for cell disruption. The supernatant was centrifuged for 30 minutes at 7000 x g to obtain the soluble protein fraction.

(5) Preparation of affinity column with immobilized OCIF

2 g TSKgel AF-TolesyI Toyopal 650 (Tosoh Corp.) and 40 ml containing 1.0 M potassium phosphate buffer (pH 7.5) containing 35,0 mg recombinant OCIF obtained by the method described in WO 96/26217, mixed. The mixture was gently rocked overnight at 4°C for performing binding assays. The reaction mixture was centrifuged to remove the supernatant. For inactivation of surplus balances at the besieged media was added 40 ml of 0.1 M Tris-Hcl buffer (pH 7.5) and the mixture was gently rocked at room temperature for 1 hour. The media in the column was washed with buffer 0.1 M glycine-Hcl (pH 3,3) containing 0.01% of Polysorbate 80 and 0.2 M such as NaCl, and buffer with 0.1 M sodium citrate (pH 2.0) containing 0.01% of Polysorbate 80 and 0.2 M NaCl. The media in the column was balanced by the introduction of the double-buffer with 10 mm sodium phosphate (pH 7.4) containing 0.01% of Polysorbate 80.

(6) Purification of Trx-hOBM using affinity column with immobilized OCIF

If there are no other indications, treatment of Trx-hOBM was carried out at 4With four centrifuge tubes 50 ml with the aid of the rotor. Column Econo-column(inner diameter 1.5 cm, length 15 cm, manufactured by BioRad Co.) filled carrier in the mixture. The column was loaded with 300 ml of PBS containing 0.01% of Polysorbate 80, 100 ml containing 10 mm sodium phosphate buffer (pH 7.0) containing 0.01% of Polysorbate 80 and 0.2 M NaCl, and 100 ml of 0.1 M glycine-Hcl buffer (pH 3,3) containing 0.01% of Polysorbate 80 and 0.2 M NaCl, in this sequence. The proteins adsorbed to the column was suirable 0.1 M sodium citrate-buffer (pH 2.0) containing 0.01% of Polysorbate 80 and 0.2 M NaCl. The eluate was collected in fractions of 5 ml Each fraction collected in this way, immediately neutralized by adding 10% volume of 2 M Tris buffer (pH 8.0). The presence or absence of Trx-hOBM in buervenich fractions was determined according to the method previously described in Example 25(3) (the ability of binding to OCIF). The fractions containing Trx-hOBM, collected and optionally purified.

(7) Purification of Trx-hOBM gel-filtration

~25 ml fractions Trx-hOBM obtained in Example 25(6), concentrated to ~0.5 ml by centrifugation using a Centriplus 10 and Method 10 (Amicon Co.). This sample was applied to a Superose column 1 is selenia as the mobile phase used PBS, containing a 0.01% Polysorbate 80, at a speed of current of 0.25 ml/min, the Eluate from the column was collected portions of 0.25 ml Trx-hOBM in the collection of such fractions were detected by the method previously described in Example 25(3), and by electrophoresis in SDS-page. The fractions containing the purified Trx-hOBM, collected and determined the concentration of protein Trx-hOBM. Measurement of protein concentration was performed with bovine serum albumin as a standard substance, using the test set DC-Protein (BioRad Co.).

Example 26

Inducing the formation of osteoclasts activity hOBM

phOBM and pcDL-SR296, respectively, were transfusional cells COS-7 using Lipofectamine (Gibco Co.). Cells were cultured in DMEM containing 10% FCS for 1 day, was trypsinization, were sown on top of the glass (15 mm, round shape, produced by Matsunami Co.) in 24-hole tablets when 5104cells per well and were cultured for 2 days. Cultural tablet were washed once with PBS. The cells were fixed with PBS containing 1% paraformaldehyde at room temperature for 8 minutes. The tablet, on which were attached the fixed cells were washed 6 times with PBS, then to each well was added 700 μl of mouse spleen cells, suspisous form of vitamin D3, 10-7M dexamethasone, and 10% fetal bovine serum. Minicell PCF (Millipore Co.) was installed in each well and added to cell suspension ST2 in the above-mentioned culture medium (4104/ml), 700 μl per well, in Millicell PCF, followed by incubation at 37C for 6 days. After cultivation Millicell PCF was removed, the tablet was washed once with PBS and cells were fixed with a solution of acetone-ethanol (50:50) for 1 minute. Then cells that detect resistant tartaric acid activity of acid phosphatase (TRAP), which is a specific marker for osteoclast selectively stained using a set of LEUKOCYTE ACID PHOSPHATASE kit (Sigma Co.). Microscopic observation showed that TRAP-positive cells were not detected in the wells in which cells were fixed COS-7, transfected pcDL-SR296. In contrast, 65±18 (average value±standard deviation, n=3) TRAP-positive cells were observed in the wells, which were fixed cells COS-7, transfetsirovannyh phOBM. In addition, the expression of the calcitonin receptor was confirmed by the fact that125I-labeled calcitonin salmon (Amersham Co.) specifically associated with these TRAP-positive who this invention, has to induce the formation of osteoclasts activity.

Example 27

Inducing the formation of osteoclasts activity of Trx-hOBM and decretorum form RBM man

Mouse spleen cells suspended in-MEM containing 10-8M active form of vitamin D3, 10-7M dexamethasone, and 10% fetal bovine serum, at a concentration of 2106/ml. of This suspension was added to 24-well plate in the amount of 350 µl per well. Then each well was loaded with 350 μl of a solution obtained by diluting purified Trx-hOBM the above culture medium (40 ng/ml), 350 μl of the solution obtained with 10-fold dilutions of conditioned medium, which was obtained by culturing cells 293-EBNA, which were transfected pCEPshOBM or RER in culture medium IMDM-10% FGS, the above culture medium, or only above 350 cells / ml of culture medium. To each well was placed Millicell PCF (Millipore Co.), to which was added 600 μl of ST2 cells, suspended in the above culture medium (4104/ml). After culturing for six days Millicell PCF was removed. The tablet was washed once PDS and the cells were fixed with a solution of apostasy (TRAP activity), selectively stained using a set of LEUKOCYTE ACID PHOSPHATASE kit (Sigma Co.). Microscopic observation showed that in the wells, which were not added Trx-hOBM were not found cells expressing TRAP-activity, while 115±19 (mean±standard deviation, n=3) TRAP-positive cells were observed in the wells, to which was added Trx-hOBM. Similarly while in the holes, to which was added air-conditioned environment of the cells 293-EBNA, transfected rser, did not detect cells expressing TRAP activity, 125±23 (mean±standard deviation, n=3) TRAP-positive cells were observed in the wells, to which was added air-conditioned environment of the cells 293-EBNA, transfected pCEPshOBM. In addition, the expression of the calcitonin receptor was confirmed by the fact that125I-labeled calcitonin salmon (Amersham Co.) associated with these TRAP-positive cells. These results proved that TGH-ROM and secretiruema form h show inhibitory formation of osteoclasts activity.

Example 28

Obtaining polyclonal antibodies

Mouse s or human sOBM, used as immunogen, obtained according to the method described above. In particular, cDNA mouse s (cDNA, SEQ ID No. 18), Cody the amino acids from N-Terminus to the 72 amino acids) cDNA or human sOBM (cDNA, SEQ ID No. 19) encoding human sOBM (SEQ ID No. 17), which does not have a district associate with the membrane RBM person due to the lack of amino acids from N-Terminus to the 71st amino acids), ligated with a fragment HindIII/EcoRV (5,2, etc., ad) expressing vector pSec TagA (Invitrogen Co.), comprising a nucleotide sequence encoding a signal peptide-chain immunoglobulin, together with a fragment of EcoRI/PmaCI (0,32, etc., ad) cDNA RBM, using a set of ligation (ver. 2, Takara Shuzo Co.). Escherichia coli DH5transformed by the reaction product. Plasmids obtained from the resulting resistant to ampicillin strains was purified alkaline SDS method and digested with restriction enzyme for selection of plasmids with fragments of 0.6 T. p. N. and 0.32, etc., ad, built-in pSec TagA. The sequence of this plasmid was identified with a set Dyedeoxyterminator Cycle Sequencing FS kit (product of Perkin Elmer Co.). In the result, it was confirmed that this plasmid has a sequence encoding a mouse sOBM or human sOBM. After cleavage of the plasmid with restrictase NheI/XhoI fragment (1,0, etc., ad), the corresponding cDNA Sekretareva form RBM, was recovered by agarose gel electrophoresis. This fragment was embedded in a fragment NheI/XhoI (10,4, etc., ad) expressing vector rser (Invitrogen Co.) the. lamidi was purified alkaline SDS method from the obtained resistant to ampicillin strains. The analysis of this plasmid by cleavage with restriction enzyme was selected strain of Escherichia coli possessing expressing a plasmid RBM secreted type (rser sOBM), with the target structure. This strain of Escherichia coli that has rser sOBM, cultivated and rser sOBM was purified using the kit Qiafilter plasmid midy kit (Qiagen Co.). Then cells 293-EBNA suspended in IMDM medium (IMDM-10% FCS) containing 10% FCS, and were sown on 24-well plate coated with collagen (product of Sumitomo Bakelite Co., Ltd.) at a density of 2 cells105/2 ml/well. After culturing overnight, the cells were transformed with 1 mg of rser sOBM or rser using 4 µl of Lipofectamine (Gibco Co.) and further cultured for 2 days in 0.5 ml serum-free IMDM or IMDM-10% FCS. The culture supernatant was recovered. Cell line with high productivity of recombinant murine soluble RBM (msOBM) or human soluble RBM (hsOBM) were subjected to screening as follows. Sodium bicarbonate was added to culture supernatant, which presumably contained msOBM or hsOBM, to a final concentration of 0.1 M, 100 μl of culture supernatant was added to each what redstem what msOBM or hsOBM in the culture supernatant was immobilized on each well. To each well was added 200 μl of a solution of Block ACE(Snow Brand Milk Products Co., Ltd.), diluted 4 times with PBS (B-PBS), and the plates were allowed to stand for 2 hours at room temperature. After washing each well in tablets three times with PBS (P-PBS) containing 0.1% Polysorbate 20, 100 μl each of a solution of recombinant OCIF (rOCIF) (3-100 ng/ml), serially diluted P-PBS, was added to each well in the plates. The tablets were allowed to stand for 2 hours at 37C. After washing tablets three times in PBS to each well was added 100 μl of peroxidase labeled polyclonal antibodies against OCIF (WO 96/26217) diluted B-PBS. After standing for 2 hours at 37With the wells were washed six times P-PBS. Then 100 μl of TMB solution (TMB Soluble Reagent, High Sensitivity ScyTek Co.) was added to each well in the tablets and the tablets were allowed to stand at room temperature for about 10 minutes, then the reaction was stopped by adding 100 μl of stop solution (Stopping Reagent, ScyTek Co.) in each well. The absorption at 450 nm of each well was measured using a microplate reader. It was confirmed that the absorption was significantly increased proportional to the concentration of added OCIF in tablets, which was immobilizes the traveler found a high rate of absorption, he was selected as a strain with high productivity. Thus obtained cells 293-EBNA with high productivity msOBM or hsOBM cultivated in large-scale environment IMDM containing 5% FCS, with application of 25 T-flasks (T-225). After reaching confluently cells in each of the T-225 flasks was added to fresh culture medium in the amount of 100 ml per flask, and the cells were cultured for 3-4 days to collect the culture supernatant. These procedures were repeated 4 times to obtain 10 liters of culture supernatant containing msOBM or hsOBM. Purified msOBM (10 mg) or hsOBM (12 mg) detecting a homogeneous band (molecular weight 32 kDa) during electrophoresis in SDS-polyacrylamide gel, obtained from the culture supernatant using affinity chromatography on a column with immobilized OCIF and gel-filtration chromatography according to the method described in Examples 25(6) 25(7). Each thus obtained purified product was used as antigen for immunization. Each of the obtained protein antigen was dissolved in phosphate buffered saline (PBS) to a concentration of 200 μg/ml and was emulsiable with an equivalent volume of complete adjuvant's adjuvant. 1 ml of this emulsion was injected subcutaneously to immunizing maximum antibody titer. Whole blood was collected 10 days after that. Serum was diluted 2-fold with buffer for binding to chromatography on Protein A-Sepharose column (BioRad Co.) and inflicted on Protein A-column, equilibrated with the same buffer. After extensive washing of the column with the same buffer, antibodies against sOBM adsorbed on the column was suirable buffer for elution (BioRad Co.) or 0.1 M glycine-HCl-buffer, pH 3.0. For immediate neutralization of the eluate, the eluate was fractionally using a test-tube containing a small amount of 1.0 M Tris-Hcl (pH 8.0). The eluate were dialyzed against PBS overnight at 4C. the antibody in the antibody solution was measured by the method of Lowry using bovine IgG as a standard protein. So get ~10 mg of purified immunoglobulin (IgG), including polyclonal antibodies of the present invention, in 1 ml of rabbit antisera.

Example 29

Measuring the RBM sOBM using ELISA using polyclonal antibodies

Sandwich ELISA was performed using rabbit polyclonal antibodies against human sOBM obtained in Example 28 as a solid phase antibody and enzyme labeled antibodies. Labeled with peroxidase (POD) antibodies were obtained according to d 28, was dissolved in 0.1 M Panso3up to a concentration of 2 μg/ml 100 ál of the resulting solution was added to each well in 96-well immunoplate (Nunc Co.), which then was allowed to stand at room temperature overnight. Then 200 ál of 50% Block ACE(Snow Brand Milk Products Co., Ltd.) was added to each well and the plates were allowed to stand for 1 hour at room temperature. The wells were washed 3 times with PBS containing 0.1% Polysorbate 20 (buffer for washing).

RBM person expressed according to the method of Example 26 and purified according to the method of Example 2. Purified RBM person and purified s person obtained in Example 28, serially diluted first reaction buffer (0.2 M Tris-Hcl buffer, pH of 7.2, containing 40% Block ACE and 0.1% Polysorbate 20), respectively, and 100 μl of the diluted solution was added to each well in the plates. The tablets were allowed to stand at room temperature for 2 hours and washed 3 times with the above buffer for washing. Then to each well on tablets was added 100 μl of POD-labeled polyclonal antibodies against s person, diluted 1000 times a second reaction buffer (0.1 M Tris-Hcl buffer, pH of 7.2, containing 25% Block ACE and 0.1% Polysorbate 20). After standing tablets at room temperature during the CL of the substrate solution, enzyme (TVM ScyTek Co.) and the tablets were allowed to stand for 10 minutes, followed by adding 100 ál of stopping the reaction solution (Stopping Reagent, ScyTek Co.) to stop the enzymatic reaction. The absorption at 450 nm of each well was measured using a microplate reader. The results are shown in Fig.26. Sandwich ELISA using rabbit polyclonal antibodies against s man knew almost equally human sOBM (molecular weight ~32 kDa) and RBM person (molecular weight ~40 kDa) with a sensitivity measure ~12,510-3pmol/ml (RBM person ~500 PG/ml, human sOBM ~400 PG/ml). Measurement of mouse and mouse sOBM RBM using ELISA using rabbit polyclonal antibodies against mouse sOBM obtained in Example 28, could be performed in a similar manner. It was confirmed that an extremely small number of mouse sOBM or mouse RBM can be measured with almost the same sensitivity as described above.

As mentioned above, polyclonal antibodies against human sOBM of the present invention obtained in Example 28, can equally recognize the antigens human sOBM and RBM person. Thus, these antibodies were named polyclonal antibodies against RBM/sOBM person. Podobn the RBM mice. These antibodies are, therefore, called polyclonal antibodies against mouse RBM/sOBM.

Example 30

Obtaining monoclonal antibodies

Purified human sOBM obtained in Example 28 was used as antigen for immunization. Purified human sOBM was dissolved in physiological saline to a concentration of 10 μg/ml and was emulsiable by mixing with an equal volume of complete adjuvant's adjuvant. This emulsion was injected intraperitoneally to BALB/c mice at a dose of 200 μl three times, once a week, for immunization of mice. Then equal volume of complete adjuvant's adjuvant was added to physiological saline solution containing 5 μg/ml s person and mixtures gave sufficiently emulgirovanija. This emulsion was injected with intraperitoneally to BALB/c mice at a dose of 200 μl, once a week for 4 weeks for immunization. After 1 week after the fourth immunization, 100 μl of physiological saline containing 10 μg/ml s man, was injected intravenously to BALB/c mice as a booster injection. After three days I removed the spleen was isolated cells of the spleen. Cells of the spleen was merged with cells of the mouse myeloma P363-Ad.653 in accordance with generally accepted method (Koehler and G. Milstei, aminopterin and thymidine. After the death of myeloma cells and the emergence of a hybrid environment GAT replaced not containing aminopterin environment GAT and continued cultivation of cells.

Example 31

The selection of hybridoma and cloning

The emergence of hybrid celebrated 10 days after the merge cells in Example 30. Monoclonal antibody binding with high affinity s person, and hybridoma producing these antibodies, selected in accordance with the following procedure using advanced solid-phase ELISA described below. In addition, for the selection of monoclonal antibodies against RBM, who know how s person and s mouse, except s person, as an antigen to a solid phase ELISA used a mouse s obtained in Example 27. s person and s mouse, respectively, was dissolved in 0.1 M sodium bicarbonate solution at a concentration of 5 μg/ml to 50 ml of each antigen was added to each well in 96-well immunoplate (Nunc Co.). The tablets were allowed to stand at 4With overnight for immobilization of antigens. The solution of the antigen in each well was discarded. Then each well was filled with 200 µl of 50% Block ACE(Snow Brand Milk Products Co., Ltd.) and allowed to stand a thief (PBS-P), containing 0.1% Polysorbate 20, to each well was added 40 μl of calf serum (Hiclone Inc.). Then to each well was added 10 μl of each hybridoma culture supernatant and each well was incubated at room temperature for 2 hours in the presence of 80% calf serum. Order solid-phase ELISA in the presence of 80% calf serum is the selection of hybridoma, which produces antibodies that can detect very small amounts of s person or s mouse even in a solution containing a high concentration of protein, and in the presence of interfering with the immune response to substances originating from serum, i.e. hybridoma, which can produce antibodies with high affinity against s human or murine s. After reaction at room temperature for 2 hours the tablets were washed in PBS-P and then to each well was added 50 μl of peroxidase labeled antibodies against mouse IgG (KPL.), diluted 5000 times with physiological saline containing 25% Block ACE. After reaction at room temperature for 2 hours the tablets were washed 3 times with PBS-P. After adding 50 μl of a solution of the enzyme's substrate (TMB, ScyTek Co.) in each well, the reaction was continued at room temperature for 5 is hybridoma, producing antibodies that recognize s human or murine s, was selected by measuring the absorption at 450 nm of each well using a microplate reader (Immune Reader NJ2000, Nippon InterMed. Co.). Taking hybridoma producing antibodies that detect particularly high absorption (D450 nm). The cloning of the hybridomas according to the method of serial dilution was repeated 3-5 times to get a stable hybrid. Hybridoma exhibiting particularly high productivity antibodies, were selected among the installed producing antibodies hybridoma clones.

Example 32

Production and purification of monoclonal antibodies

Producing antibodies of hybridoma obtained in Example 31, i.e. producing high-affinity antibodies hybridoma, recognizing s person, and hybridoma producing antibodies that detect cross-reactivity with s mouse were cultured respectively. Each hybridoma implanted intraperitoneally to BALB/c mice (1106cells per mouse), which last week introduced the Wharf (Aldorich.). After ~2-3 weeks collected accumulated in the ascites. Monoclonal antibodies that recognize s man of the present invention or as s human and murine s m Protein A-column, described in Example 28. Purified monoclonal antibodies were obtained thus from ascites using chromatography on Protein A-column (Pharmacia Co.).

Example 33

Antigenic specificity of monoclonal antibodies

Antigenic specificity of monoclonal antibodies that specifically recognize s person, and monoclonal antibodies exhibiting cross-reactivity with s human and murine s, investigated using s person, intact RBM man, having the area of the binding membrane, mouse s and mouse intact RBM with district associate with the membrane. Received more than 30 types of monoclonal antibodies. The results of several typical (representative) of the antibodies shown in Table 1. In the result, it was found that the majority of monoclonal antibodies against s person that specifically recognize s person, learn also intact RBM person with district associate with the membrane, but does not recognize mouse RBM and mouse intact RBM, which is the area of the binding membrane. On the other hand, it was found that it had obtained only a small number of antibodies capable of recognizing how s human and mouse sOBM, and that these antibodies detect cross type which are common sites of antigen recognition, namely common epitopes in RBM human and murine RBM. On the basis of the fact that monoclonal antibodies against human sOBM obtained using s person as an immune antigen, equally also learn RBM man, having the area of the binding membrane, monoclonal antibodies against human sOBM were called monoclonal antibodies against OBM/sOBM person.

Example 34

The definition of the class and subclass of monoclonal antibodies

The class and subclass of the monoclonal antibodies of this invention were determined by means of a set of analysis classes and subclasses of immunoglobulins (Amersham Co.) in accordance with the specified Protocol. The results for the representative monoclonal antibodies shown in Table 2. As shown in Table 2, the majority of monoclonal antibodies against OBM/sOBM people were IgG1others were IgG2aand IqG2b. Light chain for all antibodies were K-chains.

Example 35

Measurement of dissociation constants (valued) monoclonal antibodies

The dissociation constant of the monoclonal antibodies was measured in accordance with the known method (Betrand Friguet et al.: Journal of Immunological Methods, 77, 305-319, 1986). Namely, the purified antibody obtained in Example 32 was dissolved 0.4 to This solution was mixed with an equal volume of diluted solution of purified soluble RBM person (hsOBM), obtained in Example 28, in the primary buffer with a concentration range of 6.25-10 µg/ml of the Mixture was allowed to stand for 15 minutes at 4For binding hsOBM with a monoclonal antibody. After 15 hours of antibodies is not associated with hsOBM (10 μg/ml, 100 µl/well), were measured using immobilized solid-phase ELISA for the calculation of the dissociation constants of monoclonal antibodies with hsOBM. In addition, the affinity against msOBM antibodies are monoclonal antibodies for hsOBM and also showing the cross-reactivity against mouse soluble RBM (msOBM), was measured in the same way, but using msOBM instead hsOBM. Dissociation constants of antibodies that exhibit high affinity for each antigen and is applicable for enzyme immunoassay and analysis of the binding shown in Table 3.

As a result, dissociation constants (Kd) N-RBM 1 and N-RBM 4, which are antibodies specific for soluble RBM person (hsOBM), were of the order of 10-11M, which indicates a high affinity against hsOBM. On the other hand, the value of d antibodies N-RBM 9, which recognizes both hsOBM and murine soluble RBM (msOBM), was of the order of 10-8M for msOBM and order 10-9M for hsOBM. Cu the Association of the N-RBM 13 for each antigen, was equal to N-RBM 9, and these two antibodies belong to the same subclass. These facts suggest the possibility that they are identical antibodies that recognize the same epitope of each antigen.

Example 36

The method of measuring the RBM sOBM person using sandwich ELISA using monoclonal antibodies against OBM/sOBM man

Sandwich ELISA was performed using two high-affinity monoclonal antibodies obtained in Example 35 N-RBM 1 and N-RBM 4, respectively, in the form of solid-phase antibody and enzyme labeled antibodies. Tagging of antibodies was performed using the set of maleimide-activated peroxidase (Piers Co.). Antibody, N-RBM 1, was dissolved in 0.1 M sodium bicarbonate solution to a concentration of 10 μg/ml and 100 μl of this solution was added to each well in 96-well immunoplate (Nunc Co.). After standing overnight at 4For immobilization of the antibody solution was discarded and 300 μl of a solution of 50% Block ACE was added to each well in the plates. Each well in the plates were blocked by keeping the tablets at room temperature for 2 hours. After blocking the tablets were washed in phosphate buffered saline containing 0.1% Polysorbate 20 (PBS-P). OBM people ACE(Snow Brand Milk Products Co., Ltd.) and 0.1% Polysorbate 20 (Wako Pure Chemicals Co., Ltd.) (first reaction buffer), for the preparation of test samples with different concentrations. These test samples with different concentrations was added to each well in the amount of 100 μl per well and they reacted with the antibody N-RBM 1 immobilized on each well, incubation at room temperature for 2 hours. After 2 hours the tablets were washed in PBS-P. Then 100 μl of the solution POD-labeled antibodies N-RBM 4 in 0.2 M Tris-Hcl-buffer, pH 7.4, containing 25% Block ACEand 0.1% Polysorbate 20 (second reaction buffer) was added to each well followed by an additional incubation at room temperature for 2 hours. Then the tablets were washed in PBS-P and 100 μl of a solution of the enzyme's substrate (TMB, ScyTek Co.) was added to each well to initiate the reaction of the enzyme. The enzymatic reaction was stopped by adding 100 ál of stopping the reaction solution (Stopping Reagent, ScyTek Co.) in each well. The absorption of each well at 450 nm was measured using a microplate reader. The results are shown in Fig.27.

In the result, it was confirmed that the sandwich ELISA performed using two monoclonal antibodies against OBM/sOBM person, N-RBM 1 and N-RBM 4, high-affinity to measure a very small amount of the RBM s person in the quantitative limits of approximately 1.2510-3-2,510-3pmol/ml (~50-100 PG/ml for RBM person with a molecular mass of 40 kDa, ~40-80 PG/ml for s person with a molecular mass of 32 kDa). Hybridoma producing these two monoclonal antibodies against OBM/sOBM person, N-OVM and N-OVM were named as N-OVM and N-OVM respectively. Hybridoma producing monoclonal antibody against OBM/sOBM person (H-RBM 9), which recognizes mouse RBM and mouse s and also has inhibiting the formation of osteoclasts activity, was named N-OVM. These hybridoma have been deposited in National Institute of Bioscience and Human Technology on 5 November 1993, under the Deposit numbers FERM BP-6264 (N-RBM 1), FERM BP-6265 (N-RBM 4) and FERM BP-6266 (N-RBM 9).

Example 37

Measurement of mouse RBM and mouse s using monoclonal antibodies against OBM/sOBM person that recognizes murine RBM and mouse sOBM

Sandwich ELISA was performed using monoclonal antibodies against OBM/sOBM person, N-RBM 9, recognizing mouse RBM and mouse sOBM obtained as a solid-phase antibody in Examples 33 and 35, and polyclonal antibodies against mouse RBM/s as enzyme labeled antibody obtained in Example 28. Mouse RBM and mouse sOBM respectively diluted first reaction buffer as 36. The results are shown in Fig.28. It was found that murine RBM and mouse sOBM can be similarly measured using N-RBM 9, which are monoclonal antibodies against RBM/s person that recognizes murine RBM and mouse sOBM of the present invention. As the results of Example 35, a monoclonal antibody N-RBM 9 against RBM/s person has a high dissociation constant in relation to the mouse sOBM, i.e. it has a relatively low affinity against mouse sOBM. The sensitivity in the measurement of mouse RBM (molecular weight ~40 kDa) and mouse RBM (molecular weight ~32 kDa) this ELISA test was approximately 2510-3pmol/ml (~1 ng/ml for mouse RBM and ~0.8 ng/ml for mouse sOBM).

Example 38

Inhibiting osteoclastogenesis activity of the antibodies against RBM/s

It is known that osteoclastogenic cells (OCL) induced by co-cultivation of murine spleen cells and ST2 cells (derived from bone marrow of mice stromal cells; Endocrinology, 125, 1805-1813 (1989)). We studied the ability of antibodies against RBM/s to inhibit the formation of OCL adding system coculture. Because mouse RBM is expressed in this system coculture in this Paragraph is monoclonalny antibodies against RBM/s person (H-RBM 9), that recognize antigens as RBM person and RBM mouse. 700 µl per well of each antibody against RBM, diluted serially environment-MEM containing 10% fetal calf serum, and 350 ál/well of splenocytes of male mice (2106/ml), suspended in the same medium described above was added to each well in a 24-hole tablet (Nunc). Then cells ST2, trypsinization and suspended in the above culture medium containing 410-8M vitamin D3and 410-7M dexamethasone (8104cells/ml), was added to each well in the amount of 350 μl/well followed by cultivation for 6 days at 37C. After washing tablets 1 time in PBS, the cells in each well were fixed with a mixture of ethanol and acetone (50:50) for 1 hour at room temperature. The tablets were dried in air and 500 µl of substrate solution was added to each well in accordance with the Protocol set LEUKOCYTE ACID PHOSPHATASE kit (Sigma Co.) with further incubation for 55 minutes at 37C. Only cells exhibiting resistant tartaric acid activity sour focii. The tablets were washed 1 time with distilled water, air-dried and counted the number of TRAP-positive cells. The results are presented in Table 4. As shown in Table 4, as rabbit polyclonal antibodies against mouse RBM/s and monoclonal antibodies against RBM/s person, N-RBM 9, recognizing mouse RBM, inhibited the formation of OCL-dependent dose-dependent manner. It was found that these antibodies possess the inhibitory osteoclastogenic activity similar to the inhibitory osteoclastogenic factor, OCIF/OPG, and, therefore, are promising as a therapeutic agent for the treatment of symptoms of disorders of bone metabolism.

Example 39

Inducing the formation of osteoclasts human activity

TGH-RBM

Mononuclear cells were obtained from whole blood collected from a vein of a healthy adult using density gradient using Histopaque (Sigma Co.) in accordance with the attached Protocol. Mononuclear cells are suspended at a density of cells 1,3106/ml-MEM containing 10-7M dexamethasone, 200 ng/ml macrophage colony-stimulating factor (The Green Cross Corp.), 10% fetal bovine serum and purified TGH-Ovate 300 µl per well, and the cells were cultured at 37With in 3 days. After replacing the culture broth of the above culture medium, the cells were cultured at 37C for 4 days. Cultured cells with resistant tartaric acid activity of acid phosphatase (TRAP activity) selectively stained by the method described in Example 5. The number of colored multinucleated measured by observation under a microscope. The results are shown in Fig.29. It was confirmed that the TRAP-positive multinucleate were induced dependent dose-dependent manner by the addition TGH-RBM, while in the holes, to which was added TGH-RBM, did not TRAP-positive cells. In addition, it was found that these TRAP-positive multinucleate are positive in relation to the vitronectin receptor, which is a marker for osteoclasts. In addition, when conducting such culturing cells on the plates of ivory (ivory slices, placed on each well of a 48-hole tablet, the formation of pits were observed on these plates only in the presence of TGH-RBM. Based on these facts we can conclude that an educated TGH-RBM had an activity of inducing the formation of osteoclasts -solution (Amersham Co.) were subcutaneously injected with ddY mouse (Japan SLC Co.) on the 15th day of beremennosti at a dose of 25 µci mouse for labeling the bones of the fetus45Sa. The next day, the mouse was slaughtered to obtain the fetus. The front foot of the fruit was extracted and removed the skin and muscle to get long (tubular) bones. The cartilage was removed to obtain the body (shaft) of long bones. Body bones freely floated one by one in 0.5 ml of culture medium (BGJb-medium (GIBCO BRL company) containing 0.2% bovine serum albumin (Sigma Co.)) in each well in 24-hole tablets and they were cultured for 24 hours at 37With 5% CO2. After precultivation these bones was transferred into a variety of fresh culture medium (0.5 ml), each of which contained one of the various factors of bone resorption (vitamin D3, prostaglandin E2, parathyroid hormone, interleukin 1) and normal rabbit IgG (100 μg/ml, as control) or rabbit polyclonal antibodies against OBM/sOBM obtained in Example 28, followed by culturing for 72 hours. After culturing long (tubular) bones were placed in a 0.5 ml aqueous solution of 5% trichloroacetic acid (Wako Pure Chemicals Co., Ltd.) and IO.) was added to the culture broth and the extract solution of trichloroacetic acid (0.5 ml each) for measurement of radioactivity45Sa, whereby expected ratio45Sa, which was released into the culture broth as a result of bone resorption. The results are shown in Fig.30-33. In the result it was found that vitamin D3(10-8M) increases the activity of bone resorption, but the rabbit polyclonal antibodies against OBM/sOBM suppressed bone resorption stimulated by vitamin D3dependent on the concentration of the way, completely inhibiting increased bone resorption at a concentration of 100 μg/ml (Fig.30). Prostaglandin E2(10-6M) and parathyroid hormone (100 ng/ml) also increased the activity of bone resorption. However, the addition of 100 μg/ml rabbit polyclonal antibodies almost fully inhibited bone resorption stimulated by prostaglandin E2and parathyroid hormone (Fig.31 and 32). On the other hand, normal rabbit IgG (100 μg/ml), which was used as a positive control, did not affect the activity of bone resorption induced by prostaglandin E2and parathyroid hormone. The bone resorption was also increased interleukin 1(10 ng/ml), but significantly inhibited by the addition of rabbit polyclonal antibodies against OBM/s substance as an inhibitor of bone resorption. The results obtained in a similar experiment using N-RBM 9, which are murine antibodies against OBM/sOBM person confirmed that these antibodies detect almost equal inhibiting the bone resorption activity, and rabbit polyclonal antibodies against OBM/sOBM.

Industrial applicability

This invention provides a novel protein that specifically binds to inhibiting osteoclastogenic factor (OCIF), method of production of this protein, the method of screening for a substance that regulates the expression of this protein, using this protein, the method of screening for a substance that inhibits or modulates the activity of this protein, the method of screening for a receptor that transmits (transduces) the activity of this protein by binding with them, pharmaceutical composition, which contains a substance obtained by these methods of screening an antibody to this protein and the agent (the agent) for the treatment of disorders of bone metabolism using this antibody.

In addition, this invention provides DNA encoding a new protein (OCIF-binding molecule) that is associated with inhibiting osteoclastogenic factor (OCIF), a protein that has aminokislot what Itanium this DNA according to the genetic engineering method, and the agent containing this protein, for the treatment of akatastasia bone metabolism. In addition, this invention provides a method of screening for a substance that regulates the expression of the OCIF-binding molecule, the method of screening for a substance that inhibits or modulates the activity of the OCIF-binding molecule by linking to it, the method of screening for a receptor that transmits (transduces) activity OCIF-binding molecule by linking to it, and a pharmaceutical composition, which contains a substance obtained by these methods of screening.

In addition, this invention provides DNA encoding a new human protein, is able to communicate with inhibiting osteoclastogenic factor, OCIF (OCIF-binding molecule man, RBM person), a protein containing the amino acid sequence encoded by this DNA, a method of obtaining a protein having the characteristics of specific binding to OCIF and displaying biological activity maintain, and enhance the differentiation and maturation of osteoclasts using the method of genetic engineering, and agent for the treatment of disorders of bone metabolism using this protein. In addition, this invention provides a method of screening will ingibiruet or modulates the activity of the OCIF-binding molecule by linking to it, method of screening for a receptor that transmits (transduces) biological activity OCIF-binding molecule by linking to it, pharmaceutical composition, which contains a substance obtained by these methods of screening, antibody to IF-binding protein of a person and the agent for the blue and/or treatment of symptoms of disorders of bone metabolism using this antibody.

In addition, the invention provides antibodies that recognize both antigen (antibodies against RBM/s), one of which is a membrane-bound protein which specifically binds to OCIF (OCIF-binding molecule; RBM), and the other is soluble RBM (s), which does not have a district associate with the membrane, the method of obtaining this antigen, the method of measuring the RBM s using these antibodies and the agent for preventing and/or treating the symptoms of disorders of bone metabolism using these antibodies as an effective component.

Protein and antibody obtained by the method of the present invention, applicable as of drugs and/or reagents for the purposes of research and analysis.

Description of deposited microorganisms

(1) the Name and address of institution Depository for the code 305)

Date of Deposit in the institution-depositories

may 23, 1997

Room Deposit

FERM BP-5953

(2) the Name and address of institution Depository, which was deposited microorganism

Agency of Industrial Science and Technology 1-3,

Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken,

Japan (postal code 305)

Date of Deposit in the institution-depositories

August 13, 1997

Room Deposit

FERM BP-6058

(3) the Name and address of institution Depository, which was deposited microorganism

Agency of Industrial Science and Technology 1-3,

Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken,

Japan (postal code 305)

Date of Deposit in the institution-depositories

November 5, 1997 (initial date of Deposit)

Room Deposit

FERM BP-6264

(4) the Name and address of institution Depository, which was deposited microorganism

Agency of Industrial Science and Technology 1-3, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan (postal code 305)

Date of Deposit in the institution-depositories

November 5, 1997 (initial date of Deposit)

Room Deposit

FERM BP-6265

(5) the Name and address of institution Depository, which was deposited microorganism

Agency of Industrial Science and Technology 1-3, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan (postal code 305)

Date of Deposit in the institution-depositories

November 5, 1997 (initial date of Deposit)

Room Deposit

FERM BP-6266

Claims

1. A protein specifically binding to the inhibitory (a) affinity: specifically binds to inhibiting osteoclastogenic factor (OCIF) and exhibits high affinity against OCIF (dissociation constant on the cell membrane: Kd=10-9M or less); (b) molecular weight: the molecular weight of about 30000-40000 when measured by electrophoresis in SDS-polyacrylamide with non conditions and the molecular weight of approximately 90000-110000 when binding to OCIF Monomeric type; (c) biological activity: active maintaining or enhancing differentiation and maturation of osteoclasts in the system coculture osteoblastic stromal cells and spleen cells in the presence of the stimulating factors of bone resorption, as the active form of vitamin D3and parathyroid hormone (PTH).

2. DNA encoding a protein specifically binding to inhibiting osteoclastogenic factor comprising the nucleotide sequence corresponding to the amino acid sequence of SEQ ID NO:1, or a functional fragment of the specified protein.

3. DNA under item 2, which includes the nucleotide sequence encoding the amino acid sequence of SEQ ID NO:1.

4. DNA under item 2, comprising a nucleotide sequence encoding a protein comprising the amino acid sequence consisting of amino acid residues 76-316 SEQ ID NO:1.

5. DNA under item 2, comprising a nucleotide sequence coderush the DNA according to any one of paragraphs.2-5, characterized in that the protein is active suppression of the biological activity of inhibiting osteoclastogenesis factor.

7. DNA encoding a protein specifically binding to inhibiting osteoclastogenic factor, which consists of the nucleotide sequence of SEQ ID NO:2.

8. DNA used as a probe, which hybridizes with DNA that encodes a protein specifically binding to inhibiting osteoclastogenic factor comprising the nucleotide sequence corresponding to the amino acid sequence of SEQ ID NO:1, under relatively mild conditions, such as (a) the labeling of the cDNA fragment encoding the protein under item 1; (b) immobilization of the cDNA library on the filter; (c) the application of the specified labeled cDNA on the filter; (d) hybridization at 65With over night; wash filter 2xSSC solution containing 0.1% of SDS at 65C for 15 min, 1SSC solution containing 0.1% of SDS at 65C for 15 min, and 0.1 xSSC solution containing 0.1% of SDS at 65C for 15 minutes

9. DNA encoding a protein specifically binding to inhibiting osteoclastogenic factor with 80% or more homology with DNA, Cody is the sequence the corresponding amino acid sequence of SEQ ID NO:1.

10. DNA encoding a protein specifically binding to inhibiting osteoclastogenic factor comprising the nucleotide sequence corresponding to the amino acid sequence of SEQ ID NO:11, or a functional fragment of the specified protein.

11. DNA under item 10, characterized in that the protein is active suppression of the biological activity of inhibiting osteoclastogenesis factor.

12. DNA under item 10, characterized in that it has the nucleotide sequence SEQ ID NO:12.

13. DNA used as a probe, which hybridizes with DNA that encodes a protein specifically binding to inhibiting osteoclastogenic factor comprising the nucleotide sequence corresponding to the amino acid sequence of SEQ ID NO:11, under relatively mild conditions, such as (a) the labeling of the cDNA fragment encoding the protein under item 1; (b) immobilization of the cDNA library on the filter; (c) the application of the specified labeled cDNA on the filter; (d) hybridization at 65With over night; wash filter 2xSSC solution containing 0.1% of SDS at 65C for 15 min, 1xSSC solution containing 0.1% of SDS at 6515. Recombinant protein specifically binding to inhibiting osteoclastogenic factor encoded by the DNA according to any one of paragraphs.2-9.

16. Protein under item 15, wherein the active maintain, and enhance the differentiation or maturation of osteoclasts.

17. Protein under item 15 or 16, comprising the amino acid sequence of SEQ ID NO:1.

18. Recombinant protein specifically binding to inhibiting osteoclastogenic factor encoded by the DNA according to any one of paragraphs.10-14.

19. Protein under item 18, characterized in that it exhibits a biological activity maintain, and enhance the differentiation and maturation of osteoclasts.

20. Protein under item 18 or 19, comprising the amino acid sequence of SEQ ID NO:11.

21. A method of obtaining a protein by p. 1, comprising culturing cells of the osteoblast-like cell line or stromal cells derived from the bone marrow in the presence of the article is ka using detergent and the protein purification using affinity chromatography on a column with immobilized OCIF.

22. A method of obtaining a protein according to any one of paragraphs.15-20, including the stage of obtaining a vector comprising the DNA according to any one of paragraphs.2-9, the transformation of the specified vector into a suitable cell, culturing the specified cells expressing this protein, and the allocation of the specified protein.

23. A method of obtaining a protein according to any one of paragraphs.15-20, including the stage of obtaining a vector comprising the DNA according to any one of paragraphs.10-14, the transformation of the specified vector into a suitable cell, culturing the specified cells expressing this protein, and the allocation of the specified protein.

24. Method of screening a substance that specifically binds with the protein under item 1 and inhibits its biological activity, comprising contacting a specified substance with the specified protein in suitable conditions, and the measurement of its biological activity.

25. Method of screening a substance that specifically binds with the protein encoded by the DNA according to any one of paragraphs.2-9, and inhibits its biological activity, comprising contacting a specified substance with the specified protein encoded by the specified DNA, in suitable conditions, and the measurement of its biological activity.

26. Method of screening a substance that specifically binds to a protein encoded On the CTV with the specified protein, encode the specified DNA, in suitable conditions, and the measurement of its biological activity.

27. The antibody recognizes a protein on p. 1 induced using protein under item 1.

28. The antibody recognizes OCIF-binding protein of mouse and/or soluble OCIF-binding protein (sOCIF-binding protein), which contains no transmembrane plot induced with recombinant protein under item 15.

29. The antibody under item 28, which is a polyclonal antibody.

30. The antibody under item 28, which is a monoclonal antibody.

31. Antibody on p. 30, characterized in that it has cross-reactivity with OCIF-binding protein and sOCIF-binding protein of man.

32. Antibody on p. 30, characterized in that it has no cross-reactivity with OCIF-binding protein and sOCIF-binding protein of man.

33. Antibody on p. 30 that have the following characteristics: (a) subclass: IgG1or IgG2b) light chain: K-chain.

34. The antibody recognizes OCIF-binding protein of human and/or soluble OCIF-binding protein (sOCIF-binding protein), which contains no transmembrane plot induced with recombinant protein under item 18.

35. The antibody according to p. 34, which is polyclonal intraday cross-reactivity with OCIF-binding protein mouse and sOCIF-binding protein of the mouse.

38. The antibody according to p. 36, characterized in that it has no cross-reactivity with OCIF-binding protein mouse and sOCIF-binding protein of the mouse.

39. The antibody according to p. 36, having the following characteristics: (a) subclass: IgG1or IgG2b) light chain: K-chain.

40. The method of obtaining polyclonal antibodies to OCIF-binding protein that has an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 11, SEQ ID NO:16 and SEQ ID NO:17, including immunization of an animal specified in protein and purification of the antibodies from the blood of this animal.

41. Hybridoma FERM BP-6264 producing a monoclonal antibody that specifically binds to OCIF-binding protein/sOIF-binding protein of man.

42. Hybridoma FERM BP-6265 producing a monoclonal antibody that specifically binds to OCIF-binding protein/sIF-binding protein of man.

43. Hybridoma FERM BP-6266, producing a monoclonal antibody that specifically binds to OCIF-binding protein/sOIF-binding protein of man.

44. The method of obtaining monoclonal antibodies to OCIF-binding protein of a person having the amino acid sequence selected from the group consisting of SEQ ID NO:11 and SEQ ID NO:17 comprising College antibodies in the culture broth and purification and isolation of monoclonal antibodies from the culture broth.

45. The way of measuring OCIF-binding protein or sOCIF-binding protein, comprising using the antibody according to any one of paragraphs.28-39.

46. The method according to p. 45, characterized in that the antibody is immobilized on a solid phase, or observed by an enzyme.

47. Pharmaceutical composition for prevention or treatment of disorders or abnormalities of bone metabolism, comprising as active ingredient an effective amount of the antibody according to any one of paragraphs.28-39, and a pharmaceutically acceptable carrier.

48. Drug for prevention or treatment of disorders or abnormalities of bone metabolism, comprising as active ingredient an effective amount of the antibody according to any one of paragraphs.28-39.

49. Pharmaceutical composition for prevention or treatment of disorders or abnormalities of bone metabolism, comprising as active ingredient an effective amount of the antibody according to p. 27, and a pharmaceutically acceptable carrier.

50. Drug for prevention or treatment of disorders or abnormalities of bone metabolism, comprising as active ingredient an effective amount of the antibody on p. 27.

Priority points and attributes:

15.04.1997 - 9/97808 - PP.1, 21, 24, 27, 45, 49, 50;

09.06.1997 - 9/151434 - PP.2, 3, 7, 8, 9, 17, 22; �Telenesti SEQ IDNO:1;

12.08.1997 - 9/217897 - PP.4, 5, 28, 29, 30, 46; p. 6, in addition to the characteristic relating to the nucleotide sequence corresponding to the amino acid sequence of SEQ IDNO:1; PP.15, 16, 23, 25 - a sign relating to a nucleotide sequence that encodes a protein comprising the amino acid sequence consisting of amino acid residues 76-316 or 72-316 SEQ IDNO:1;

21.08.1997 - 9/224803 - PP.10, 11, 12, 13, 14, 18-20, 26, 34, 40; PP.15, 16, 23, 25 in addition to the characteristic relating to the nucleotide sequence corresponding to the amino acid sequence of SEQ IDNO:1, and a characteristic relating to a nucleotide sequence that encodes a protein comprising the amino acid sequence consisting of amino acid residues 76-316 or 72-316 SEQ ID NO:1;

02.12.1997-9/332241 - PP.31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 47,48.

 

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