New proteins and methods for producing such proteins

 

(57) Abstract:

The invention relates to a protein characterized by the following properties: (a) molecular weight during electrophoresis in polyacrylamide gel in the presence of LTOs (SDS-PAGE), which constitutes approximately 60 KD in terms of recovery, approximately 60 KD and 120 KD in non conditions; (b) high affinity for the cation-exchange column and a column of heparin; (b) biological activity of inhibiting the differentiation and/or maturation of osteoclasts, and this activity is reduced by heating at 70oC for 10 min or 56oC for 30 min, the activity is lost when heated at 90oC for 10 min; (g) internal amino acid sequences presented in SEQ ID NO: 1, 2, and 3, and (d) with optional N-terminal amino acid sequence represented in SEQ ID NO: 7; a method for production of such proteins by culturing human fibroblasts; protein purification by a combination of ion-exchange column chromatography, affinity column chromatography and column chromatography with reversed phase. Proteins with amino acid sequence represented in SEQ ID NO: 4, and K is lnasty, presented in SEQ ID NO: 6, encoding OCIF protein with a signal peptide having the amino acid sequence represented in SEQ ID NO: 5, protein expressed from the cDNA that encodes the amino acid sequence represented in SEQ ID NO: 4; a protein with the biological activity of inhibiting the differentiation and/or maturation of osteoclasts, which is obtained as the amino acid sequence of the expressed cDNA having at least 80% identity with the amino acid sequence presented in SEQ ID NO: 4; the method of obtaining a protein that inhibits the development of osteoclasts, by means of genetic engineering using cDNA that encodes the amino acid sequence represented in SEQ ID NO: 4: (a) molecular weight during electrophoresis in polyacrylamide gel in the presence of LTOs (SDS-PAGE) of approximately 60 KD in terms of recovery, approximately 60 KD and 120 KD in non conditions; (b) high affinity for the cation-exchange column and a column of heparin; (b) activity of inhibiting the differentiation and/or maturation of osteoclasts, decreasing by heating at 70oC for 10 min or 56oC for 30 min, and the activity is lost sing in SEQ ID NO: 1-3; cDNA encoding OCIF protein or its variants; genomic DNA with the nucleotide sequence represented in SEQ N: 104 or 105; polyclonal antibody with specific affinity towards the OCIF; monoclonal antibody specific against OCIF obtained by hybridoma technology using OCIF as antigen and characterized by a molecular weight of about 150000; the method of determining the concentration of OCIF protein. 14 C. and 5 C.p. f-crystals, 16 tab., 15 Il.

The invention relates to a new protein factor inhibiting osteoclastogenesis (OCIF) and to methods of producing such a protein.

Human bones are constantly transformed by repeating the processes of resorption and recreation. I believe that when these processes osteoblasts and osteoclasts are the cells responsible mainly for bone formation and bone resorption, respectively. A typical example of a disease caused by the development of abnormal bone metabolism is osteoporosis. It is known that this disease is triggered by a condition in which bone resorption by osteoclasts exceeds bone formation by osteoblasts, but the mechanism of osteoporosis until completely outstanding. Osteoporosis causes bone pain and the logo of the age, he becomes a social problem with increasing numbers of older people. It is therefore expected that will develop effective drugs for the treatment of this disease. The decline in bone mass caused by abnormal bone metabolism, as it is considered, can be prevented by inhibition of bone resorption, improve bone formation or improvement of a balanced metabolism.

It is assumed that bone formation promoterwise by stimulating growth, differentiation or activation of osteoblasts. It is reported that many cytokines stimulate the growth or differentiation of osteoblasts, i.e., fibroblast growth factor (FGF) (Rodan, S. B. et al., Endocrinology, vol.121, R. 1917, 1987), insulin-like growth factor I (IGF-I) (Hock J. M. et al., Endocrinolory, vol. 122, p. 254, 1988), insulin-like growth factor II (IGF-II) (T. McCarthy et al. , Endocrinology, vol. 124, p.301, 1989), activin A (Centrella M. et al., Mol. Cell. Biol., vol.11, p.250, 1991), vasculotropic (Varonique M. et al., Biochem. Biophys. Res. Commun., vol.199, p.380, 1994) and bone morphogenetic protein (BMP) (A. Yamaguchi et al., J. Cell Biol., vol.113, p.682, 1991, Sampath T. K. et al., J. Biol. Chem., vol.267, p.20532, 1992, and Knutsen R. et al., Biochem. Biophys. Res. Commun., vol. 194, p.1352, 1993).

On the other hand, we paid attention and has been studied intensively cytokines that inhibit differentiated is. 5, p.5683, 1988) and interleukin-4 (Kasano K. et al., Bone Miner. vol.21, p.179, 1993) inhibit the differentiation of osteoclasts. Found that calcitonin (Bone Miner. vol.17, R. 347, 1992), macrophage colony-stimulating factor (G. Hattersley et al., J. Cell. Physiol. vol. 137, p.199, 1988), interleukin-4 (K. Watanabe et al., Biochem. Biophys. Res. Commun., vol.172, p.1035, 1990) and interferon (Cowen M. et al., J. Bone Miner. Res., vol.1, p. 469, 1986) inhibit bone resorption by osteoclasts.

It is assumed that these cytokines are effective medicines to improve the recovery of bone mass by stimulating bone formation and/or by inhibition of bone resorption. Cytokines, such as insulin-like growth factor I and bone morphogenetic proteins, currently have been clinically tested for their effect in the treatment of patients with bone diseases. Calcitonin is already used as a drug for the treatment of osteoporosis and to reduce the pain in osteoporosis.

Examples of drugs currently used clinically for the treatment of bone diseases and to reduce the treatment period, are dihydroxyvitamin3vitamin K2, calcitonin and its derivatives, hormones such as estradiol, ipriflavon, what about the actions and, as expected, the development of new medicines. As already mentioned, bone metabolism is regulated by a balance between bone resorption and osteogenesis. Therefore, it is assumed that the quality of medicines for the treatment of bone diseases such as osteoporosis, will be developed cytokines, which inhibit the differentiation and/or maturation of osteoclasts.

The present invention is made taking into account the above considerations. The aim of the present invention is as a new factor, called factor inhibiting osteoclastogenesis (OCIF) and effective way of obtaining the specified factor.

The authors of the present invention have conducted intensive searches factors inhibiting osteoclastogenesis air-conditioned human embryonic fibroblasts IMR-90 (ATSS CCL186) environment and found a new factor inhibiting osteoclastogenesis (OCIF), which inhibits the differentiation and/or maturation of osteoclasts.

The authors present invention has created the method of accumulation of the protein to a high concentration by culturing cells IMR-90 using pieces of alumina ceramics as the basis for adhesion glue iodirovannoi IMR-90 environment to use the serial number column chromatography - ion exchange, heparin-affine, affine to cibachrome blue and reversed-phase.

The authors of the present invention, based on the amino acid sequence of purified OCIF, successfully cloned cDNA encoding this protein. The authors present invention also created a procedure for obtaining this protein, which inhibits the differentiation of osteoclasts. The present invention relates to a protein, which is produced with the help of fibroblast cells of the human lung has a molecular weight in the determination by the method of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) 60 KD in terms of recovery and 120 KD under non conditions, has affinity as cation exchange resins, and heparin, reduces its activity of inhibiting the differentiation and maturation of osteoclasts in the processing for 10 minutes at 70oWith or for 30 minutes at 56oAnd loses its activity of inhibiting the differentiation and maturation of osteoclasts in the processing for 10 minutes at 90oC. amino acid sequence of OCIF protein, which is described in the present invention is clearly different from any known factors ingibiruet (1) cultivation of human fibroblasts (2) the introduction of air-conditioned environment in a column of heparin to obtain the adsorbed fraction, (3) the OCIF protein purification using cation-exchange column, (4) the OCIF protein purification using heparin-affinity column, (5) the OCIF protein purification using affinity columns with cibachrome blue, (6) the allocation of OCIF protein using columns for reversed-phase chromatography. Zibarro blue, coupled with media made of synthetic hydrophilic polymers, is an example of material used to produce columns with cibachrome blue. These speakers are usually called "blue columns".

The present invention includes a method of accumulation of OCIF protein to high concentrations by culturing human fibroblasts using pieces of alumina ceramics as the basis for adhesion of cells.

In addition, the authors present invention has determined the amino acid sequences of peptides derived from OCIF, constructed priming on the basis of these amino acid sequences and obtained cDNA fragments encoding OCIF, from cDNA libraries of cells, IMR-90.

OCIF protein of the present invention can be distinguished from conditioninga methods protein purification from biomaterials in accordance with the physical and chemical properties of the OCIF protein. For example, the method of concentration includes conventional biochemical methods, such as ultrafiltration, lyophilization and dialysis. Cleaning method includes a combination of several techniques of chromatography for purification of proteins, such as ion-exchange column chromatography, affinity column chromatography, column gel filtration, hydrophobic column chromatography, column chromatography with reversed phase, and preparatively gel electrophoresis. Human fibroblasts were used to obtain the OCIF protein, are preferably IMR-90. The method of obtaining the air-conditioned IMR-90 environment is a preferable method comprising the adhesion of human embryonic cells IMR-90 to bits of alumina ceramics in roller bottles, the use of modified according to the method of Dulbecco eagle medium (DMEM) supplemented with 5% newborn calf serum for cell culture and culturing the cells in roller bottles for 7-10 days method poster cultivation. To the buffer as a detergent stages of protein purification OCIF add preferably CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate).

OCIF protein of the present invention can at first what arinam (heparinase CL-6B, Pharmacia), elution with 10 mm buffer Tris-Hcl, pH 7.5, containing 2 M NaCl, and subsequent fractions of OCIF in Q-anyoneeven column (HiLoad Q/FF, Pharmacia) and collect readsorbing faction. OCIF protein can be cleared by subjecting the obtained fraction OCIF cleaning S cation-exchange column (HiLoad-S/FF, Pharmacia), the column of heparin (heparin-5PW, TOSOH), column cibachrome blue (Blue-5PW, TOSOH) and a column for reversed-phase chromatography (BU-300 C4, Perkin Elmer), and the substance is determined by the previously described properties.

The present invention relates to a method for cloning cDNA encoding OCIF protein, based on the amino acid sequence of natural OCIF, and to a method for producing recombinant OCIF protein, which inhibits the differentiation and/or maturation of osteoclasts. OCIF protein purified by the method described in the present invention, and is treated with endopeptidase (for example, leilajapanese). Determine the amino acid sequences of peptides obtained by hydrolysis, and synthesize a mixture of oligonucleotides that can encode each internal amino acid sequence. Fragment of OCIF cDNA PCR receive (preferably RT-PCR RT - PCR reverse transcriptase), using as a nucleating OPI using as a probe the obtained DNA fragment OCIF. OCIF cDNA containing the complete coding region are inserted into the expression vector. Recombinant OCIF can be obtained by expression in mammalian cells or in bacteria OCIF cDNA containing the complete coding region.

The present invention relates to new proteins OCIF2, OCIF3, OCIF4 and OCIF5, which are variations of OCIF and have the above activity. These OCIF variants derived from cDNA libraries made from IMR-90 poly(A)+RNA by hybridization using as a probe a fragment OCIF cDNA. Each of the cDNA of OCIF variants containing the full coding region are inserted into the expression vector. Each variant recombinant OCIF can be obtained by expression of each cDNA OCIF variants, encoding the complete coding region, in normal hosts. Each variant recombinant OCIF can be cleaned by the method described in this invention. Each variant recombinant OCIF has the ability to inhibit osteoclastogenesis.

The present invention also includes mutants of OCIF. There are substitution mutants in which one cysteine residue, probably involved in dimer formation, replaced by a serine residue, and various deletion mutants of OCIF. Timesensitivity enzymes. Each of these mutated OCIF cDNA insert in the vector containing the appropriate promoter for gene expression. The resulting expression vector for each of the OCIF mutants transfection in eukaryotic cells such as mammalian cells. Each of the OCIF mutants can be obtained and cleaned the air-conditioned environment transfection cells.

The present invention relates to polyclonal antibodies and to the method of quantitative determination of the concentration of OCIF using these polyclonal antibodies.

As antigens (immunogens) you can use natural OCIF obtained from air-conditioned IMR-90 environment, recombinant OCIF produced by such hosts as microorganisms and eukaryotes, using OCIF cDNA, synthetic peptides based on the amino acid sequence of OCIF or peptides derived from OCIF by partial digestion. Polyclonal antibodies against OCIF obtained by immunization of appropriate mammalian antigens in combination with adjuvants for immunization, if necessary, and purification from serum by ordinary cleaning methods. Polyclonal antibodies against OCIF, which have been labelled with radioisotopes of inalize (ELISA). Using these analysis systems can easily determine the concentration of OCIF in biological materials such as blood and ascites, and in the medium for cell culture.

Antibodies in the present invention can be used when radioimmune analysis (RIA) or enzyme-linked immunosorbent analysis (ELISA). Using these analysis system, you can easily determine the concentration of OCIF in biological materials such as blood and ascites.

The present invention relates to novel monoclonal antibodies and to the method of quantitative determination of the concentration of OCIF using these monoclonal antibodies.

Monoclonal antibodies against OCIF can be obtained using a conventional method using OCIF as antigen. As antigens can use native OCIF obtained from culture medium of cells IMR-90, and recombinant OCIF produced by such hosts as microorganisms and eukaryotes, using OCIF cDNA. On the other hand, as the antigens can also be used synthesized peptides designed on the basis of amino acid sequence of OCIF, and peptides derived from OCIF by partial digestion. Immunized lymphocytes obtained put the t hybridoma. From hybridomas obtained by cell fusion, selected hybridoma clones secreting antibody that recognizes OCIF. The desired antibody can be obtained by growing the cells selected hybridoma clones. Upon receipt of hybridoma especially useful for immunization small animals such as mice or rats. For immunization OCIF was diluted to the required degree of saline (0.15 M NaCl) and injected with adjuvant animal 2-5 times every 2-20 days intravenously or administered intraperitoneally. Immunized animal killed three days after the last immunization, remove the spleen and use splenocytes immunized as b-lymphocytes.

Cell lines are murine myeloma to merge cells from the immunized B-lymphocytes include, for example, P3/h-Ag8, p3-Ul, NS-1, MPC-11, SP-2/0, F0, RH Ag8.653 and S194. You can also use a rat cell line R-210. Human b-lymphocytes are subjected to immunization method of immunization in vitro and merge with cell lines of human myeloma or human B-lymphocytes transformed by EB virus, which is used as a parent cell line to merge cells, and produce human antibody type.

Merge cells immunized with the Ute, generally, the method of Koehler G. with TCS. (Nature, 256, 495-497, 1975), as well as to merge cells, you can use the method of electrical pulse. Immunized b cells and transformed b-cells are mixed in the usual proportions, and to merge cells using, as a rule, the culture medium without serum fetal cow (FBS) containing polyethylene glycol. Fused with the myeloma cell line b-lymphocytes for the selection of hybridoma grown in selective medium GAT containing FBS.

For selection of hybridoma producing antibody against OCIF, you can use mainly ELISA analysis belascoaran, Ouchterlony or the agglutination reaction. Among these methods are simple and easy to implement with sufficient accuracy is ELISA, and it mainly use it for. Using ELISA using purified OCIF can easily and accurately select the desired antibody. Thus obtained hybridoma can be grown by conventional method of growing cells and freeze to have on hand when needed. The antibody can be obtained by culturing hybridoma with the conventional method of growing cells or by intraperitoneally transplantation hybridoma animals. Integrate. The obtained antibody specifically reacts with OCIF and can be used to determine the concentration of OCIF and cleaning OCIF. Antibodies of the present invention recognize epitopes OCIF and have a high affinity to the OCIF. Therefore, you can use them to create ELISA. Using (use) this system of analysis can easily determine the concentration of OCIF in biological materials such as blood and ascites.

The present invention relates to the means used for the treatment of bone diseases, who as an active ingredient contain OCIF. Rats subjected to denervation of the left front limb. The test compound is administered daily after surgery within 14 days. After a 2-week treatment of animals kill and dissect their forelimbs. After this experience bone mechanical strength by a method of bending at three points. OCIF improves the mechanical strength of the bone depending on the dose.

OCIF protein of the present invention is suitable as a pharmaceutical ingredient for treating or reducing the loss of bone mass in osteoporosis, bone diseases, such as rheumatoid arthritis, osteoarthritis, and abnormal bone metabolism in multiple myeloma who go pharmaceuticals, containing OCIF protein as an active ingredient, and can be administered orally or parenterally. The preparations contain OCIF protein of the present invention as an active ingredient and safe when administered to humans or animals. Examples of pharmaceutical compositions are for injection or intravenous drip infusions, suppositories, nasal preparations, sublingual preparations and plasters for percutaneous absorption. Pharmaceutical preparations for injection can be obtained by mixing a pharmacologically effective amount of OCIF protein and pharmaceutically acceptable carriers. Carriers are fillers and/or activators, i.e., amino acids, sugars, cellulose derivatives, and other organic and inorganic compounds, which, as a rule, are added to the active ingredients. When OCIF protein is mixed with fillers and/or activators for injections may be added, if necessary, pH regulators, buffers, stabilizers, soljubilizatory etc.

In Fig.1 provides a picture of the elution OCIF protein (fraction passes through a Hiload Q/FF, sample 3) from the column with Hilaod-S/HP.

In Fig.2 illustrates the pattern of elution of Slovenia OCIF protein (fraction 49-50, blue-5W) from the column for reversed-phase chromatography.

In Fig. 4 data SDS-PAGE of isolated proteins OCIF in terms of recovery and non conditions.

Description bands:

strip 1,4 - marker proteins of molecular weight;

band of 2.5 - OCIF protein peak 6 in Fig.3;

lanes 3,6 - OCIF protein peak 7 in Fig.3.

Fig. 5 gives a picture of the elution of peptides obtained by digestion pyridinedimethanol OCIF protein by listendownload on the column for chromatography with reversed phase.

In Fig. 6 provides data SDS-PAGE of isolated natural (n) OCIF protein and recombinant (r) OCIF protein in non conditions. rOCIF(E) and rOCIF(C) produced in the cells 293/EBNA and in Cho cells, respectively.

Description bands:

lane 1 - marker proteins of molecular weight;

band 2 - protein nOCIF Monomeric type;

band 3 protein nOCIF dimeric type;

band 4 - protein rOCIF(E) Monomeric type;

band 5 - protein rOCIF(E) dimeric type;

band 6 protein rOCIF(C) Monomeric type;

band 7 protein rOCIF(C) dimeric type.

In Fig. 7 provides data SDS-PAGE of isolated natural (n) OCIF protein and recombinant (r) OCIF protein in vos is>Description bands:

band 8 - marker proteins of molecular weight;

band 9 - protein nOCIF Monomeric type;

band 10 - protein nOCIF dimeric type;

band 11 - protein rOCIF(E) Monomeric type;

band 12 - protein rOCIF(E) dimeric type;

band 13 - protein rOCIF(C) Monomeric type;

the strip 14 protein rOCIF(C) dimeric type.

In Fig. 8 presents data SDS-PAGE of isolated natural (n) OCIF protein and recombinant (r) OCIF protein, of which in terms of recovery of deleted N-linked chains of sugars. rOCIF(E) and rOCIF(C) are protein rOCIF produced in the cells 293/EBNA and in Cho cells, respectively.

Description bands:

band 15 - marker proteins of molecular weight;

band 16 - protein nOCIF Monomeric type;

the band 17 is a protein nOCIF dimeric type;

band 18 - protein rOCIF(E) Monomeric type;

the strip 19 is a protein rOCIF(E) dimeric type;

band 20 - protein rOCIF(C) Monomeric type;

band 21 - protein rOCIF(C) dimeric type.

In Fig.9 compares the amino acid sequence of OCIF and OCIF2.

Fig. 10 shows comparison of the amino acid sequences of OCIF and OCIF3.

Fig. 11 shows comparison of the amino acid sequences of OCIF and OCIF4.

In Fig.14 is a standard curve for determining the concentration of OCIF protein using ELISA using monoclonal antibodies against OCIF.

In Fig.15 shows the effect OCIF protein in osteoporosis.

The present invention will be further explained using the following examples, but the invention is not limited to these examples.

EXAMPLE 1

Receiving the conditioned medium of human fibroblasts IMR-90.

Human embryonic lung fibroblast cells IMR-90 (ATCC-CCL186) grown on pieces of alumina ceramics (80 g) (99.5% dioxide aluminum, manufacturer Toshiba Ceramic K. K.), in DMEM (manufactured by Gibco BRL Co.) with the addition of 5% CS (calf serum) and 10 mm HEPES buffer (500 ml per roller bottle) at 37oIn the presence of 5% CO2within 7-10 days using 60 roller-bottles (490 cm2, 110171 mm, manufacturer Coning Co.) in stationary culture. The conditioned medium is collected and in roller bottles add a fresh environment. Get about 30 l air-conditioned IMR-90 culture on the download culture. Air-conditioned environment is designated as sample 1.

EXAMPLE 2

Method of analysis activity ingibiruemaya activity tetratriacontane acid phosphatase (TRAP) methods M Kumegava with TCS. (Protein. Nucleic Acid. Enzyme, vol.34, p.999, 1989) and N. Takahashi with TCS. (Endocrynology, vol.122, p.1373, 1988) with some modifications. Briefly, bone marrow cells obtained from mice at the age of 17 days, suspended in a-MEM (minimal supportive environment) (manufactured by GIBCO BRL Co.), containing 10% FBS, 210-8M of activated vitamin D3and each test sample, and inoculant in each well of 96-well plate at the density of cells 310 cells/0.2 ml/well. Tablets incubated for 7 days at 37oC in humidified atmosphere with 5% CO2. Culture continue to grow further, replacing 0.16 ml of the old medium with the same volume of fresh medium at 3 and 5 days after the start of cultivation. On day 7, after washing tablets phosphate buffered saline, the cells are fixed with ethanol-acetone (1:1) for 1 min at room temperature, and then examine the development of osteoclasts by determining the activity of phosphatase using a kit (acid phosphatase, leucocyte, catalog 387-A, production of Sigma Co.). The reduction in the number of positive to TRAP cells take over the activity indicator OCIF.

EXAMPLE 3

Cleaning OCIF.

I) Column chromatography on heparin-sepharose CL-6B.

To mallidis, 2000 cm2, Millipore Co.) and divide the filtrate into three parts. Each part (30 l) make a column of heparin-separate CL-6B (54.1 cm, Pharmacia Co.), balanced 10 mm Tris-HCl containing 0.3 M NaCl, pH 7.5. After washing the column with 10 mm Tris-HCl, pH 7.5, at a flow rate of 500 ml/hour protein fraction adsorbed to heparin-sepharose CL-6B, elute 10 mm Tris-HCl, pH 7.5, containing 2 M NaCl. The fraction is designated as sample 2.

II) Column chromatography on HiLoad Q/FF

The faction with the heparin-Safronova adsorbent (sample 2) cialiswhat against 10 mm Tris-HCl, pH 7.5, with the addition of HOPS to a final concentration of 0.1%, incubated at 4oWith overnight and divided into two parts. Then each part contribute to anion-exchange column (HiLaod-Q/FF 2.610 cm, Pharmacia Co.), which balance 50 mm Tris-HCl, 0.1% CHAPS, pH 7.5, and get readsorbing fraction (1000 ml). This fraction is designated as sample 3.

III) Column chromatography on HiLoad-S/HP.

Readsorbing on HiLoad Q fraction (sample 3) is loaded into cation-exchange column (HiLoad-S/HP, 2.610 cm, Pharmacia Co.), which balance 50 mm Tris-HCl, 0.1% CHAPS, pH 7.5. After washing the column with 50 mm Tris-HCl, 0.1% CHAPS, pH 7.5, the adsorbed protein elute with a linear gradient from 0 to 1 M NaCl at a speed pot is Oia one portion. 100 μl of each of the four portions have OCIF activity. The OCIF activity observed in the fractions from 11 to 30 (as shown in Fig. 1). Fractions 21 to 30, which have a higher specific activity, collect and designated as sample 4.

IV) Affinity column chromatography with heparin 5PW.

Bred 120 ml fractions with HiLoad from 21 to 30 (sample 4) 240 ml of 50 mm Tris-HCl, 0.1% CHAPS, pH 7.5, and applied to a column for affinity chromatography with heparin 5PW (0.87.5 cm, Tosoh Co.), which balance 50 mm Tris-HCl, 0.1% CHAPS, pH 7.5. After washing the column with 50 mm Tris-HCl, 0.1% CHAPS, pH 7.5, the adsorbed protein elute with a linear gradient from 0 to 2 M NaCl at a flow rate of 0.5 ml/min for 60 min and collect fractions (0.5 ml). From each fraction are selected based on 50 µl testing OCIF activity. Active fractions, erwerbende 0.7-1.3 M NaCl, combined and designated as sample 5.

V) Affinity column chromatography on blue 5PW.

5 sample (10 ml), diluted with 190 ml of 50 mm Tris-HCl, 0.1% CHAPS, pH 7.5, and applied to a column of blue 5PW for affinity chromatography (0.55 cm, Tosoh Co. ), which balance 50 mm Tris-HCl, 0.1% CHAPS, pH 7.5. After washing the column with 50 mm Tris-HCl, 0.1% CHAPS, pH 7.5, the adsorbed protein elute with 30 ml of a linear , cenvat OCIF activity. Fractions with numbers from 49, 70, erwerbende 0.1-1.6 M NaCl, have OCIF activity.

VI) Column chromatography with reversed phase.

Fraction with blue 5PW obtained when collecting fractions 49-50, acidified with 10 μl of 25% TFA (triperoxonane acid) and bring in C4 column for chromatography with reversed phase (BU-300, 2.1220 mm, production Perkin-Elmer), which balance 0.1% TFA and 25% acetonitrile. Adsorbed protein elute with a gradient of acetonitrile from 25 to 55% at a flow rate of 0.2 ml/min for 60 min and each protein peak is collected (Fig.3). The fraction of each peak (100 μl) experience on the OCIF activity, and the activity of OCIF have peak 6 and peak 7. The results are given in table.1.

EXAMPLE 4

The molecular mass of the protein OCIF.

Two protein peaks (6 and 7) with OCIF activity is subjected to polyacrylamide gel electrophoresis in the presence of LTOs in terms of recovery and non conditions. Briefly, 20 µl fractions of each peak was concentrated in vacuo, dissolved in 1.5 μl of 10 mm Tris-HCl, pH 8, 1 mm etc, 2.5% LTOs, 0.01% bromophenol blue and incubated at 37oWith during the night in non conditions or restoration conditions (5% 2-mercaptoethanol). Then the Penta gel 10-15% acrylamide (Pharmacia Co.) and device for electrophoresis (Fast System, Pharmacia Co.). To calculate the molecular weight using the following marker proteins of molecular weight: phosphorylase b (94 KD), bovine serum albumin (67 KD), ovalbumin (43 KD), carbonic anhydrase (30 KD), tripeny inhibitor (20.0 KD) and lactalbumin (14.4 KD). After electrophoresis, protein bands visualize a silver dye using a set of Phast with a silver dye. The results are shown in Fig.4.

The protein band is clearly in the 60 KD is detected in the protein peak 6 in terms of recovery and non conditions. In the protein peak 7 protein band with an apparent 60 KD is detected in terms of recovery and the protein band with apparent 120 KD is detected in non conditions. Therefore, the protein peak 7 is considered as a glycosilated protein peak 6.

EXAMPLE 5

Thermostability OCIF.

Samples of 20 µl fractions 51 and 52 of the blue 5PW diluted to 30 μl of 10 mm phosphate buffered saline, pH 7.2, and incubated for 10 min at 70oWith the 90oWith or within 30 min at 56oC. After heat treatment, the samples tested for OCIF activity. The results are given in table.2.

EXAMPLE 6

Internal amino acid sequence of OCIF protein.

Protein peak 6 or peak 7, purified C4-HPLC, concentrated by centrifugation and pyridinethione in terms of recovery. Briefly, 50 µl of 0.5 M Tris-HCl, pH 8.5, containing 100 μg of dithiothreitol, 10 mm etc, 7 M guanidine hydrochloride and 1% CHAPS add to each sample and the mixture incubated overnight in the dark at room temperature. Each mixture was acidified with 25% TFA (final concentration 0.1%) and bring in C4 column for chromatography with reversed phase (BU-300, 2.130 mm, Perkin-Elmer Co.), balanced 20% acetonitrile containing 0.1% TFA. Pyridylmethylamine OCIF elute 9 ml linear gradient of acetonitrile from 20 dgme and dissolved in 25 μl of 0.1 M Tris-HCl, pH 9, containing 8 M urea and 0.1% tween 80. Add in a test tube 73 μl of 0.1 M Tris-HCl, pH 9, and 0.02 μg of lisianthius (Wako Pure Chemical, Japan) and incubated at 37oC for 15 hours. Each hydrolysate acidified with 1 μl of 25% TFA and applied to C8 column for chromatography with reversed phase (RP-300, 2.1220 mm, Perkin-Elmer Co.), balanced 0.1% TFA.

Peptide fragments elute from the column with a linear gradient of acetonitrile from 0 to 50% at a flow rate of 0.2 ml/min for 70 min and collect each peptide peak. Each peptide fragment (R1-RZ) contribute to protein sequencing machine. The sequence of the peptides shown in sequence No. 1-3, respectively.

EXAMPLE 7

Determination of the nucleotide sequence of OCIF cDNA.

I) Isolation of poly(A)+RNA from cells IMR-90.

1108cells IMR-90 emit approximately 10 μg of poly(A)+RNA using the dial to highlight the Fast Track mRNA (Invitrogen), in accordance with the manufacturer's recommendations.

II) production of mixed seed

The two shown below mixed seed synthesized based on the amino acid sequences of two peptides (peptide P2 and peptide P3, sequence 2 and 3, respectively). All oligonucleotides in Gln) to the twelfth residue - leucine (Leu). All oligonucleotides in mixed seed 3R can encode the amino acid sequence of the peptide 3 from the sixth residue is histidine (His) to the twelfth residue is lysine (Lys). Sequence of mixed seed 2F and 3R are shown in table.3.

III) Amplification of a fragment of OCIF cDNA by PCR (polymerase chain reaction).

First strand cDNA generated using the kit for cDNA synthesis Superscript II (Gibco BRL) and 1 μg of poly(A)+RNA obtained in example 7-I), in accordance with the manufacturer's recommendations. The DNA fragment encoding OCIF, is obtained by PCR using cDNA-matrix and nucleating described in example 7-11).

PCR carried out under the following conditions:

10X buffer Ex Taq (Takara Shuzo), ál - 5

2.5 mm dNTP solution*, µl - 4

The cDNA solution, ál - 1

Ex Taq (Takara Shuzo), ál - 0.25

Sterile distilled water - 29.75

40 µm solution nucleating 2F, ál - 5

40 µm solution nucleating 3R, ál - 5

*dNTP - deoxynucleoside-5'-triphosphate. -

Components of the reaction are mixed in a microcentrifuge tube. For the initial stage of denaturation at 95oC for 3 min followed by 30 cycles of denaturation at 95oC for 30 s and annealing at 50oC for 30 s, and is SUP>C for 5 min. Size of PCR products is determined at the electrophoresis in 1.5% agarose gel. Get a DNA fragment OCIF 400 p. O.

EXAMPLE 8

Cloning of the fragment of the OCIF cDNA, amplified by PCR and determination of its DNA sequence.

Fragment of OCIF cDNA, amplificatory PCR in example 7-(III), inserted into plasmid pBluescript II SK-using the set for ligating DNA, var.2 (Takara Shuzo) according to the method of D. Marchuk with TCS. (Nucleic Acids Res., vol.19, p. 1154, 1991). E. coli DH5 (Gibco BRL), transform the ligation mixture. Grown transformants and purified plasmid containing the OCIF cDNA (about 400 p. O.), using the usual methods. This plasmid is called pBSOCIF. The cDNA sequence of OCIF in pBSOCIF determined using a kit for sequencing Taq Dye Deoxy Terminater Cycle (Perkin-Elmer). The amount of OCIF cDNA is 397 p. O. OCIF cDNA encodes the amino acid sequence containing 132 balance. Amino acid sequences of internal peptides (peptide P2 and peptide P3, sequence 2 and 3, respectively), which are used to create the seed, found in the N - or C-terminal region in the amino acid sequence of the polypeptide of 132 amino acids, predicted OCIF cDNA in 397 p. O. furthermore, the amino acid is the amino acid sequence of the polypeptide. These data show that OCIF cDNA in 397 p. O. is part of the OCIF cDNA full length.

EXAMPLE 9

Obtaining a DNA probe.

Get OCIF cDNA in 397 p. O. under the conditions described in example 7-(III). OCIF cDNA is subjected to preparative agarose gel electrophoresis. From gel OCIF cDNA purified using a kit for extraction from gel with QIAEX (QIAGEN), mark [32P]dCTP (cytogeneticist-5'-triphosphate), using a system for tagging Megaprime DNA (Amersham) and used for selection of phage containing OCIF cDNA full length.

EXAMPLE 10

Obtaining a cDNA library.

Generate cDNA using the kit for the synthesis of large length cDNA (Clontech), the priming oligo(dt), [32P]dCTP and 2.5 μg of poly(A)+RNA obtained in example 7-1), using the manufacturer's instructions. Adapter EcoRI-SalI-NotI are ligated with cDNA. Separate cDNA from free adapter, free and unbound [32P]dCTP. The purified cDNA is then precipitated with ethanol and dissolved in 10 μl of TE buffer (10 mm Tris-Hcl, pH 8.0, 1 mm etc). Insert cDNA with adaptor in vector ZAP EXPRESS (Stratagene) at the EcoRI site. DNA recombinant ZAP EXPRESS phage containing cDNA, packaged in vitro using extracts for packaging Gigapack gold II (Stratagene) and receive recombinant ZAP EXPRESS phage library the example 10, infect E. coli XL1-Blue MRF' (Stratagene) at 37oWith over 15 minutes of Infected E. coli cells, add to the NZY medium containing 0.7% agar, 50oWith and sown on NZY-agar plates. After incubation of the plates overnight at 37oWith the surface of plates containing plaques placed Hybond N (Amersham). Membranes are denatured in alkaline solution, neutralized and washed in 2SSC in the usual way. Phage DNA immobilized on the membranes using

UV-crosslinking (Stratagene). Membranes incubated in the buffer for hybridization (Amersham) containing 100 μg/ml DNA, salmon sperm, when 65oC for 4 hours and then incubated at 65oWith overnight in the same buffer containing 2105Chim/ml DNA probe was denatured OCIF. The membrane was washed at 65oWith double-2SSC and twice with a solution containing 0.1 SSC and 0.1% LTOs, each time for 10 minutes of Positive clones purified by twice repeating the screening. The purified clone ZAP EXPRESS phage containing the insert DNA of the order of 1.6 KB, used in the following experiments. This phage is called OCIF. Purified OCIF and infect them with E. coli XL1-Blue MRF' (Stratagene) according to the scheme set to clone ZAP EXPRESS (Stratagene). Get the culture fluid of infected XL1-Blue MPF'. Purified OCIF and turlow liquid condicionado XL-1 blue MRF' add to culture E. coli, strain XLOR (Stratagene) to transform them. So get resistant to kanamycin, the transformant containing a plasmid designated pBKOCIF, which is a vector pBKCMV (Stratagene) containing the fragment-insertion of 1.6 KB. The transformant comprising a plasmid containing cDNA OCIF approximately 1.6 KB, obtained by selecting resistant to kanamycin colonies. Plasmid called pBKOCIF. The transformant was deposited in National Institute of Bioscience and Human-Tecnology (NIBH), Department of engineering science and technology, under index FERM BP-5267 as pBK/01F10. National Deposit (inventory number FERM P-14998) transferred to international Deposit of 25 October 1995 in accordance with the Budapest Treaty. Grown transformant pBK/01F10 and purify plasmid pBKOCIF according to the standard scheme.

EXAMPLE 12

Determination of the nucleotide sequence of OCIF cDNA containing the complete coding region.

The nucleotide sequence of OCIF cDNA obtained in example 11, are determined with the use of the kit for sequencing Taq Dye Deoxy Terminator Cycle (Perkin-Elmer). Use of seed T3, T7 (Stratagene) and synthetic seed, established in accordance with the cDNA sequence of OCIF. The sequence of these basic structures are shown in the list of sequences on etelnost, the predicted cDNA sequence presented in the sequence 5.

EXAMPLE 13

Production of recombinant OCIF cells 293/EBNA.

I) Construction of plasmids for the expression of OCIF cDNA.

Get pBKOCIF containing cDNA OCIF about 1.6 KB, as described in example 11, and digested by restriction enzymes BamHI and XhoI. Cut out the insert OCIF cDNA, separated by agarose gel electrophoresis and purified using the kit for extraction from gel with QIAEX (QIAGEN). Purified insert OCIF cDNA are ligated using a set for ligating DNA, var.2 (Takara Shuzo), with expression vector rser (Invitrogen), digested with restriction enzymes BamHI and XhoI. E. coli DH5 (Gibco BRL), transform the ligation mixture. Grow the transformants, and a plasmid containing the OCIF cDNA (about 1.6 KB), purified using QIAGEN columns (QIAGEN). Expression plasmid pCEPOCIF precipitated with ethanol and used in the following experiments in the form of a solution in sterile distilled water.

II) an intermediate expression of OCIF cDNA and analysis of biological activity.

Recombinant OCIF is obtained using expression plasmids pCEPOCIF obtained in example 13-(I) by the method described numdivisions according to the method of Claims), containing 10% fetal calf serum (Gibco BRL). After incubation of cells for 24 hours, the culture medium is removed and cells are washed with IMDM without serum. Expression plasmid pCEPOCIF and lipofectamine (Gibco BRL) diluted with OPTI-MEM (Gibco BRL) and mixed, and added to cells in each well in accordance with the manufacturer's instructions. For each transfection using 3 µg pCEPOCIF and 12 ál of lipofectamine. After incubation of the cells with pCEPOCIF and lipofectamine 38 hours Wednesday substituted with 1 ml OPTI-MEM. After incubation transfection cells for 30 hours air-conditioned environment are collected and used for biological analysis. The biological activity of OCIF analyzed by the method described below. Obtained from mice at the age of 17 days, the bone marrow is suspended in the medium-MEM (manufactured by Gibco BRL) containing 10% FBS, 210-8M of activated vitamin D3and each of the test samples, and inoculant and cultured for 7 days at 37oC in humidified atmosphere with 5% CO2as described in example 2. During incubation, 3 and 5 day, 160 μl of the old medium in each well is replaced with the same volume of fresh medium containing the test sample, diluted 110-8M of activated vitamin D3

III) Isolation of recombinant OCIF air-conditioned 293/EBNA environment.

The air-conditioned 293/EBNA environment (1.8 l), obtained by culturing cells described in example 13-(II) add 0.1% CHAPS and filtered through a membrane filter of 0.22 μm (Steribecs GS, Millipore Co.). Air-conditioned environment contribute in 50 ml of heparin-separato column CL-6B (2.610 cm, Pharmacia Co.), balanced 10 mm Tris-HCl, pH 7.5. After washing the column with 10 mm Tris-HCl, pH 7.5, the adsorbed protein elute from the column with a linear gradient of NaCl from 0 to 2 M at a flow rate of 4 ml/min for 100 min and collect fractions (8 ml). Using 150 μl of each fraction, analyze the OCIF activity according to the method described in example 2. The active fraction OCIF (112 ml) obtained when the elution approximately 0.6-1.2 M NaCl.

The active fraction (112 ml) was diluted to 1000 ml of 10 mm Tris-HCl, 0.1% CHAPS, pH 7.5, and applied to japonki 10 mm Tris-HCl, 0.1% CHAPS, pH 7.5, the adsorbed protein elute from the column with a linear gradient of NaCl from 0 to 2 M at a flow rate of 0.5 ml/min for 60 min and collect fractions (0.5 ml). 4 μl of each fraction analyzed by electrophoresis in polyacrylamide gel in the presence of LTOs in terms of recovery and non conditions as described in example 4. When SDS-PAGE in terms of recovery detects a single band of protein rOCIF with an average weight of 60 KD in the fractions from 30 to 32, in non conditions also detect protein bands rOCIF with an average weight of 60 KD and 120 KD in the fractions from 30 to 32. Selected rOCIF-fraction from 30 to 32 is designated as recombinant OCIF obtained from 293/EBNA (rOCIF(E)). Get 1.5 ml rOCIF(E) (535 µg/ml), when the determination carried out by the method of Lowry using as a standard protein, bovine serum albumin.

EXAMPLE 14

Production of recombinant OCIF using Cho cells.

I) Construction of plasmids for the expression of OCIF.

Get pBKOCIF containing cDNA OCIF about 1.6 KB, as described in example 11, and digested with restriction enzymes SalI and EcoRV. Insert OCIF cDNA of about 1.4 KB was separated using agarose gel electrophoresis and purified using the ut restriction enzymes > PST and Cloned. Cut out a fragment of the expression vector about 3.4 KB, separated by electrophoresis in agarose gel and purified using the kit for the extraction of the gel QIAEX (QIAGEN). The ends of the purified insert OCIF cDNA or fragment expression vector "dull", using the set for removal of phosphate groups at the ends of the DNA (Takara Shuzo). Purified cDNA insert OCIF and a fragment of the expression vector are ligated using a set for ligating DNA, var.2 (Takara Shuzo). E. coli DH5 (Gibco BRL), transform the ligation mixture. Get transformant containing OCIF-expression plasmid pSROCIF.

II) Obtaining expression plasmids.

The transformant containing OCIF-expression plasmid pSROCIF obtained in example 13-I), and the transformant containing the murine DHFR-gene-expression plasmid pBAdDSV described in WO 92/01053 grow in the usual way. Both plasmids purified alkaline treatment, precipitation with polyethylene glycol and ultracentrifugation with a density gradient of cesium chloride according to the method of Maniatis with TCS. (Molecular cloning, 2nd edition).

III) the Adaptation of cells CHOdhFr - to protein-free environment.

Cells CHOdhFr- (ATCC, CRL9096) grown in IMDM containing 10% fetal calf serum. Cells adapted to EX-CELL 301 (JRH Bioscience)CIF-expression plasmids and the mouse DHFR-gene-expression plasmids into cells CHOdhFr-.

Cells CHOdhFr-, obtained in example 14-(III), transferout using electroporation pSROCIF and pBAdDSV obtained in example 14-(II). Under sterile conditions dissolve 200 mcg pSROCIF and 20 µg pBAdDSV in 0.8 ml of IMDM (Gibco BRL) containing 10% fetal calf serum CG. Suspended 210 cells HOdhFr - 0.8 ml of the specified environment. The cell suspension is transferred into a cuvette (Bio Rad) and cells transferout method of electroporation using gene pulser (Bio Rad) at 360 V and 960 μf. Suspension elektroborudovaniya cells transferred into T-flasks (Sumitomo Bakelite), containing 10 ml of EX-CELL PF-CHO, and incubated in CO2-the incubator for 2 days. Then transfetsirovannyh cells inoculant in each well of 96-hole tablet (Sumitomo Bakelite) at a density of 5000 cells per well and cultured for 2 weeks. Selected transformants expressing DHFR, as EX-CELL PF-CHO does not contain nucleotides, and the parent cell line Cho dhFr - can't grow in this environment. Most transformative expressing DHFR, Express OCIF as OCIF-expressing plasmid take ten times more than a mouse DHFR-expressing plasmids. Among the transformants expressing DHFR selected transformants whose air-conditioned environment has a high activity O is here limited cultivation. Clones whose air-conditioned environment has a high OCIF activity, selected as described above, and obtain transformants 5561 expressing a large number of OCIF.

V) Production of recombinant OCIF.

For producing recombinant OCIF (rOCIF) medium EX-CELL 301 (3 l) in a 3-liter rotating flask inoculant clone (5561) at a density of cells 1105cells/ml Cells 5561 grown in a rotating flask at 37oC for 4-5 days. When the cell concentration 5561 reaches 1106cells/ml, harvested approximately 2.7 l air-conditioned environment. Then in a rotating flask add 2.7 l EX-CELL 301 and re-cultured cells 5561. Using 3 of the rotating flask, collect about 20 l air-conditioned environment.

VI) Isolation of recombinant OCIF protein from conditioned medium of Cho cells.

The air-conditioned environment SNO-cells (1.0 l), described in example 14-(V), add 1.0 g CHAPS and filtered through a membrane (0.22 μm) filter (Steribecks GS, Millipore Co.). Air-conditioned environment contribute to the heparin-separato (FF) column (2.610 cm, Pharmacia Co.), balanced 10 mm Tris-HCl, pH 7.5. After washing the column with 10 mm Tris-model HC1, 0.1% CHAPS, pH 7.5, the adsorbed protein elute from the column with a linear the CL of each fraction, analysiert OCIF activity according to the method described in example 2. Get active fraction (112 ml), which eluted at approximately 0.6-1.2 M NaCl.

The active fraction (112 ml) was diluted to 1200 ml of 10 mm Tris-HCl, 0.1% CHAPS, pH 7.5, and applied to affinity column (blue 5PW, 0.55.0 cm, Tosoh Co.), balanced 10 mm Tris-HCl, 0.1% CHAPS, pH 7.5. After washing the column with 10 mm Tris-HCl, 0.1% CHAPS, pH 7.5, the adsorbed protein elute from the column with a linear gradient of NaCl from 0 to 3 M at a flow rate of 0.5 ml/min for 60 min and collect fractions (0.5 ml). 4 μl of each fraction is subjected to polyacrylamide gel electrophoresis in the presence of LTOs in regenerating and non conditions as described in example 4. When SDS-PAGE in terms of recovery detects a single band of protein rOCIF with an average molecular weight of 60 KD in fractions 30-38, when non conditions also detect protein bands rOCIF with an average weight of 60 KD and 120 KD in fractions 30-38. The selected fraction of rOCIF - 30-38 - defined as purified recombinant OCIF derived from Cho cells (rOCIF(C)). Receive 4.5 ml rOCIF(C) (113 μg/ml), when the determination carried out by the method of Lowry using as a standard protein, bovine serum albumin.

WHEN Alannah rOCIF(E) and rOCIF(C) on polyvinylidenedifluoride (PVDF) membranes with Prospin (PERKIN ELMER Co.). The membrane was washed with 20% ethanol and analyze the N-terminal amino acid sequence of adsorbed proteins by protein sequencing machine (PROCISE 492, PERKIN ELMER Co. ). Installed N-terminal amino acid sequence shown in sequence 7.

N-Terminal amino acid rOCIF (E) and rOCIF(C) is the 22nd amino acid glutamine from the Met as the starting point of the broadcast, as shown in sequence 5. 21 from amino acid Met up Gln identified as signal peptide. N-terminal amino acid sequence of OCIF, isolated from air-conditioned IMR-90 environment, not detected. Accordingly, the N-terminal glutamine OCIF can be blocked by transformation of glutamine in pyroglutamic during cultivation or treatment.

EXAMPLE 16

Biological activity of recombinant (r)OCIF and natural (n)OCIF.

I) Inhibition-induced vitamin D3the formation of osteoclasts cells from murine bone marrow.

Samples rOCIF(E) and nOCIF bred-MEM (GIBCO BRL Co.), containing 10% FBS and 210-8M of activated vitamin D3(final concentration of 250 ng/ml). Each sample serially diluted with the same medium and 100 µl of each diluted the image is, inoculant at a density of cells 3105cells/100 µl per well in each well of 96-well plates and cultured for 7 days at 37oC in humidified atmosphere with 5% CO2. On day 7, cells are fixed and stained using a kit for measuring acid phosphatase (acid phosphatase, leucocyte, 387-A, Sigma) according to the method described in example 2. The decrease in activity of acid phosphatase (TRAP), take a OCIF activity. Fewer positive for acid phosphatase cells assessed by solubilize the pigment dye and measuring the absorption. More, 100 μl of a mixture of 0.1 N. NaOH and DMSO (1: 1) is added to each well and tablets shake to dissolve the dye. After complete solubilization of the dye in each well measure the absorbance at 590 nm, subtracting the absorbance at 490 nm, using a spectrophotometer to read the microplate (Immunoreader NJ-2000, InterMed). The apparatus is installed on the absorption Of using the hole with monocline the bone marrow cells that were cultured in the medium without activated vitamin D3. The decrease in TRAP activity is expressed as percentage of control value absorption (= 100%) dye, solubilising from bone marrow cells, which were grown in UTS is depending on the dose at a concentration of 16 ng/ml or higher.

II) Inhibition-induced vitamin D3the formation of osteoclasts in coculture stromal cells and murine splenic cells.

Action OCIF on the formation of osteoclasts induced by vitamin D3in coculture stromal cells and murine splenic cells test method N. Udagava with TCS. (Endocrinology, vol.125, p.1805-1813, 1989). Specifically, samples of rOCIF(E) and rOCIF(C) and nOCIF diluted serially-MEM (GIBCO BRL Co. ) containing 10% FBS, 210-8M of activated vitamin D3and 210-7M dexamethasone, and 100 µl of each diluted sample added to each well of 96-well titration microplate. Derived from murine bone marrow stromal ST2 cells (RIKEN Cell Bank RCB0224), 5103cells in 100 ál-MEM containing 10% FBS, and splenic cells from 8-week-old ddy mice, 1105cells per 100 μl in the same environment, inoculant in each well of 96-well plates and cultured for 5 days at 37oC in humidified atmosphere with 5% CO2. After 5 days the cells fixed and stained using the kit for acid phosphatase (acid phosphatase, leucocyte, 387-A, Sigma). The decrease in the number positive for acid phosphatase cells evaluated by the method described in example 16-I). Results for rOCIF(E) and rOCIF is depending on the dose at a concentration of 6 to 16 ng/ml or higher.

III) Inhibition of PTH induced the formation of osteoclasts cells from murine bone marrow.

Action OCIF on the formation of osteoclasts induced by PTH, check according to the method of N. Takahashi with TCS. (Endocrinology, vol.122, p.1373-1382, 1988). Specifically, samples of rOCIF(E) and nOCIF (125 ng/ml) was diluted serially-MEM (production GIBCO BRL Co.), containing 10% FBS and 210-8M PTH, and 100 μl each of the diluted samples are added to 96-well plates. Cells mouse bone marrow 17-day-old ddy mice at a density of 3105cells in 100 ál-MEM containing 10% FBS, inoculant in each well of 96-well plates and cultured for 5 days at 37oC in humidified atmosphere with 5% CO2. After 5 days the cells fixed with ethanol-acetone (1:1) for 1 min at room temperature and stained using the kit for acid phosphatase (acid phosphatase, leucocyte, 387-A, Sigma) according to the method described in example 2. The decrease in the number positive for acid phosphatase cells evaluated by the method described in example 16-I). The results are given in table.8.

nOCIF and rOCIF(E) inhibit the formation of osteoclasts, depending on the dose at a concentration of 16 ng/ml and above.

IV) Inhibiting the formation of osteoclasts induced IL. The OEWG. Natl. Acad. Sci., USA, vol.90, p. 11924-11928, 1993). Specifically, samples of rOCIF(E) and nOCIF diluted serially-MEM (production GIBCO BRL Co.), containing 10% FBS and 20 ng/ml IL-11, and 100 µl of each diluted sample is added to each well in 96-well plates. Derived from newborn mouse calvaria preadipocyte cells MC3T3-G2/PA6 (RIKEN Cell Bank RCB1127), 5103cells in 100 ál-MEM containing 10% FBS, and splenic cells from ddy mice aged 8 weeks, 1105cells per 100 μl in the same environment, inoculant in each well of 96-well plates and cultured for 5 days at 37oC in humidified atmosphere with 5% CO2. After 5 days the cells fixed and stained using the kit for acid phosphatase (acid phosphatase, leucocyte, 387-A, Sigma). Cells positive for acid phosphatase, counted under a microscope, and a decrease in the number of cells taken for the OCIF activity. The results are given in table.9.

As nOCIF, and rOCIF(E) inhibit the formation of osteoclasts, depending on the dose at a concentration of 2 ng/ml and above.

The results are shown in tables 4-8 show that OCIF inhibits the formation of osteoclasts in all cases, induction - vitamin D3, PTH and IL-11, almost at the same doses. Accordingly, OCIF could b slishnimi substances, which induce bone resorption.

EXAMPLE 17

The allocation of OCIF monomer type and OCIF dimeric type.

In each sample rOCIF(E) and rOCIF(C) containing 100 µg protein OCIF, add 1/100 volume of 25% triperoxonane acid, and applied to column chromatography with reversed phase (PROTEIN-RP, 2.0250 mm, YMC Co.), balanced 30% acetonitrile containing 0.1% triperoxonane acid. OCIF protein elute from the column with a linear gradient of acetonitrile from 30 to 55% at a flow rate of 0.2 ml/min for 50 min and collect each peak OCIF. Each peak fraction OCIF monomer type and the fraction of the peak OCIF dimeric type lyophilized respectively.

EXAMPLE 18

Determination of molecular weight of recombinant OCIF.

1 µg selected nOCIF Monomeric and dimeric type purified using column chromatography with reversed phase, as described in example 3-IV), and 1 µg rOCIF monomer and dimer of the type described in example 17, was concentrated in vacuo. Each sample is incubated in the buffer for SDS-PAGE, subjected to polyacrylamide gel electrophoresis in the presence of LTOs and protein bands on the gel stained with silver dye according to the method described in example 4. The results of the electrophoresis when nevosstanovlenie mass of 60 KD detected in each sample OCIF monomer type and the protein band with an average molecular mass of 120 KD detected in each sample OCIF dimeric type when non conditions. In terms of recovery of the protein band with an average molecular weight of 60 KD detected in each sample OCIF monomer type. Accordingly, the molecular weight nOCIF Monomeric type of cells, IMR-90, rOCIF of cells 293/EBNA and rOCIF from Cho-cells are almost the same. Molecular weight nOCIF dimeric type of cells, IMR-90, rOCIF of cells 293/EBNA and rOCIF from Cho-cells are almost the same.

EXAMPLE 19

Removal of N-linked oligosaccharide chains and measurement of molecular weight natural and recombinant OCIF.

Each sample containing 5 μg selected nOCIF Monomeric and dimeric type, purified using column chromatography with reversed phase example 3-IV), and each sample containing 5 μg rOCIF monomer and dimer of the type described in example 17, was concentrated in vacuo. Each sample was dissolved in 9.5 μl of 50 mm nutrifaster buffer, pH 8.6, containing 100 mm 2-mercaptoethanol, 0.5 ál 250 u/ml N-glycanase (Seikagaku kogyo Co.) and incubated overnight at 37oC. To each sample, add 10 ál of 20 mm Tris-HCl, pH 8.0, containing 2 mm etc, 5% LTOs and 0.02% bromophenol blue) and heated for 5 min at 100oC. 1 µl of each sample is subjected to electrophoresis 4. Electrophoresis painting, is shown in Fig.8.

The average molecular weight of each deglycosylation nOCIF of cells, IMR-90, rOCIF from Cho cells and rOCIF of cells 293/EBNA is 40 KD in terms of recovery. The average molecular mass of raw nOCIF of cells, IMR-90, rOCIF of cells 293/EBNA and rOCIF from Cho-cells in the recovery of 60 KD. Accordingly, the results show that proteins OCIF are glycoproteins with N-connected chains of sugars.

EXAMPLE 20

Cloning of cDNA of OCIF variants and determination of their DNA sequences

Plasmid pBKOCIF, which is the insertion of OCIF cDNA into pBKCMV (Stratagene), is obtained from one of the treated positive phages as in example 10 and 11. In addition, during the screening of a cDNA library with a probe OCIF cDNA in 397 p. O. get transformants containing plasmids, cotour the insert sizes different from the size pBKOCIF. Grow these transformants containing plasmids, and plasmids purified in a standard way. The sequence of the inserted DNA in each plasmid was determined by using a kit for sequencing Taq Dye Deoxy Terminater Cycle (Perkin Elmer). Use of seed T3, T7 (Stratagene) and synthetic seed derived from nucleotide the CIF2 is contained in the sequence 8, and amino acid sequence OCIF2 predicted nucleotide sequence shown in sequence 9. The nucleotide sequence OCIF3 is contained in the sequence 10, and the amino acid sequence OCIF3 predicted nucleotide sequence shown in sequence 11. The nucleotide sequence OCIF4 is contained in the sequence 12, and amino acid sequence OCIF4 predicted nucleotide sequence shown in the sequence 13. The nucleotide sequence OCIF5 is contained in the sequence 14, and the amino acid sequence OCIF5 predicted nucleotide sequence shown in the sequence 15. The structure of the OCIF variants shown in Fig. 9-12 and briefly described below.

OCIF2

cDNA OCIF2 has a deletion of 21 p. O. from under guanine nucleotide number 265 to guanine number 285 in OCIF cDNA (sequence 6).

Accordingly, OCIF2 has a deletion of 7 amino acid from glutamic acid (Glu) under (amino acid) 68 to glutamine (Gln) under 74 in OCIF (sequence 5).

OCIF3

cDNA OCIF3 has a point mutation at nucleotide 9 in OCIF cDNA (sequence 6), where citizen samanlaisina (Lys). It turns out that the mutation is located in the signal sequence and has no significant effect on secreted OCIF3. cDNA OCIF3 has a deletion 117 p. O. from guanine under 872 to cytidine under 988 in OCIF cDNA (sequence 6).

Accordingly, OCIF3 has a deletion of 39 amino acid from threonine (Thr) at 270 to leucine (Leu) to amino acid 308 in OCIF (sequence 5).

OCIF4

cDNA OCIF4 has two point mutations in the OCIF cDNA (sequence 6). Citizen at 9 in the OCIF cDNA is replaced by a guanine and guanine under 22 is replaced by a thymidine (sequence 6).

Accordingly, OCIF4 has two mutations. Asparagine (Asn) under 19 in OCIF (sequence 5) is replaced by lysine (Lys), and alanine (Ala) under 14 is replaced by a serine (Ser). It turns out that these mutations are located in the signal sequence and does not have a significant effect on secreted OCIF4.

cDNA OCIF4 contains DNA of about 4 KB, which is the intron 2 of the gene OCIF inserted between nucleotide under 400 and nucleotide under 401 in OCIF cDNA (sequence 6). The open reading frame ends in intron 2.

Accordingly, OCIF4 has additional a new amino acid sequence containing 21 amino acids is cleotide 9 in OCIF cDNA (sequence 6), where citizen is replaced by a guanine. Accordingly, OCIF5 has the mutation, and asparagine (Asn) under 19 in OCIF (sequence 5) is replaced by lysine (Lys). It turns out that the mutation is located in the signal sequence and has no significant effect on secreted OCIF5. cDNA OCIF5 is the last part (about 1.8 KB) intron 2 between 400 nucleotide and nucleotide 401 in OCIF cDNA (sequence 6). The open reading frame ends at the last part of intron 2.

Accordingly, OCIF5 has additional a new amino acid sequence containing 12 amino acids, after alanine (Ala) at 112 in OCIF (sequence 5).

EXAMPLE 21

The production of OCIF variants.

I) Construction of plasmids for expression of OCIF variants.

The plasmids containing OCIF2 or OCIF3 receive as described in example 20, and called pBKOCIF2 and pBKOCIF3 respectively. pBKOCIF2 and pBKOCIF3 digested with restriction enzymes BamHI and XhoI. Insert cDNA OCIF2 and OCIF3 separated using agarose gel electrophoresis and purified from the gel using a set of extraction from gel with QIAEX (OIAGEN). Purified cDNA insert OCIF2 and OCIF3 are ligated individually using the kit for DNA ligation, var. 2 (Takara Shuzo), expr ansformer E. coli, DH5 (Gibco BRL).

The plasmids containing cDNA OCIF4 receive as described in example 20, and called pBKOCIF4. Digest pBKOCIF4 restriction enzymes SpeI and XhoI (Takara Shuzo). The cDNA insert OCIF4 separated by agarose gel electrophoresis and purified from the gel using a set of extraction from gel with QIAEX (QIAGEN). Purified cDNA insert OCIF4 are ligated using the kit for DNA ligation, var. 2 (Takara Shuzo), with expression vector rser (Invitrogen), digested with restriction enzymes NheI and XhoI (Takara Shuzo). The ligation mixture to transform E. coli DH5 (Gibco BRL).

The plasmids containing cDNA OCIF5 receive as described in example 20, and called pBKOCIF5. Digest PBKOCIF5 restriction enzyme HindIII (Takara Shuzo). Part of the 5'-coding region of the cDNA insert OCIF5 separated by agarose gel electrophoresis and purified from the gel using a set of extraction from gel with QIAEX (QIAGEN). Plasmid expression OCIF pCEPOCIF obtained in example 13-(I), digested with restriction enzymes HindIII (Tagara Shuzo). Part of the 5'-coding region cDNA OCIF removed. The remainder of the plasmid, which contains the vector rser and part of the 3'-coding region OCIF cDNA, called pCEPOCIF-3'. Separate pCEPOCIF-3' by agarose gel electrophoresis and purified from the gel using Naberevnye, var.2 (Takara Shuzo). The ligation mixture to transform E. coli DH5 (Gibco BRL).

The resulting transformants are grown at 37oWith during the night and expression plasmids OCIF variants (pCEPOCIF2, pCEPOCIF3, pCEPOCIF4 and pCEPOCIF5) purified using column QIAEX (QIAGEN). These plasmids for the expression of OCIF variants precipitated with ethanol, dissolved in sterile distilled water and used in the following experiments.

II) an intermediate expression of the cDNA of OCIF variants and analysis of the biological activity of variants of recombinant OCIF.

Variants of recombinant OCIF produce using expression plasmids pCEPOCIF2, pCEPOCIF3, pCEPOCIF4 and pCEPOCIF5, obtained as described in example 21-(I) by the method described in example 13-(II). Analyze the biological activity of variants of recombinant OCIF. The results are that these options OCIF (OCIF2, OCIF3, OCIF4 and OCIF5) have weak activity.

EXAMPLE 22

Getting OCIF mutants.

I) Construction of plasmid vectors for sublimirovanny cDNA encoding OCIF mutants.

Plasmid vector (5 μg) as described in example 11, digested with restriction enzymes BamHI and XhoI (Takara Shuzo). Digested DNA is subjected to preparative electrophoresis in drowsey sequence for OCIF, purified from the gel using a set of extraction from gel with QIAEX (QIAGEN). Purified DNA was dissolved in 20 μl of sterile distilled water. The solution is called DNA solution 1. Digest Bluescript II SK + (3 µg) (Stratagene) with restriction enzymes BamHI and XhoI (Takara Shuzo). Digested DNA is subjected to preparative agarose gel electrophoresis. The DNA fragment size of approximately 3.0 KB was purified from gel using the set for extraction from gel with QIAEX (QIAGEN). Purified DNA was dissolved in 20 μl of sterile distilled water. The solution is called DNA solution 2. Mix 1 ál of DNA solution of 2, 4 ál of DNA solution of 1 and 5 μl of buffer for ligating I set for DNA ligation, var.2 (Takara Shuzo), and incubated at 16oC for 30 minutes (the ligation Mixture used to transform E. coli in this way, as described below.) Conditions for transformation of E. coli following. Mix 100 ál of competent cells E. coli DH5 (GIBCO BRL) and 5 ál of ligation mixture in a sterile 15-ml tube (IWAKI glass). The tube kept on ice for 30 minutes After incubation for 45 s at 42oWith the cells add 250 μl of L-broth (1% tryptone, 0.5% yeast extract, 1% NaCl). The cell suspension is incubated for 1 hour at 37oWith plastinka incubated overnight at 37oC.

Six colonies grown on the plate, incubated separately overnight at 37oWith shaking, each in 2 ml of L-broth containing 50 μg/ml ampicillin. Analyze the structure of the plasmids in the colonies. Receive a plasmid in which the DNA fragment of 1.6 KB containing the complete OCIF cDNA inserted between the sites of digestion of BamHI and XhoI pBluescript II SK+, and denote its pSK+ -OCIF.

II) Obtaining mutants, in which one of the Cys residues in OCIF replaced by a Ser residue.

1) Introduction of mutations in the OCIF cDNA.

Get the OCIF mutants in which one of the five Cys residues present at positions 174, 181, 256, 298 and 379 (sequence 4) is replaced by a Ser residue, and denote them OCIF-C19S (174s replaced by Ser), OCIF-C20S (181Cys replaced by Ser), OCIF-C21S (256Cys replaced by Ser), OCIF-C22S (298Cys replaced by Ser) and OCIF-C23S (379Cys replaced by Ser), respectively.

To obtain mutant nucleotides encoding the corresponding Cys-residues that are replaced by nucleotides encoding Ser. The mutagenesis is carried out by two-step polymerase chain reaction (PCR). The first stage PCR consists of two reactions: PCR 1 and PCR 2.

PCR 1, µl:

10X buffer Ex Taq (Takara Shuzo) - 10

2.5 M solution of dNTP - 8

Plasmid vector, described the M solution is diluted 2 (mutagenesis) - 1

Ex Taq (Takara Shuzo) - 0.5

PCR 2, ál:

10X buffer Ex Taq (Takara Shuzo) - 10

2.5 M solution of dNTP - 8

The plasmid vector described in example 11 (8 ng/ml) - 2

Sterile distilled water - 73.5

20 μm solution diluted 3 - 5

a 100 μm solution of the seed 4 (mutagenesis) - 1

Ex Taq (Takara Shuzo) - 0.5

Specific sets of blades are used for each mutation, and the other components are not changed. The seed used for the reactions are given in table. 10. The nucleotide sequence of the seed contained in the placenta. 20, 23, 27 and 30-40. PCR carried out under the following conditions. After initial denaturation at 97oC for 3 min followed by 25 cycles of denaturation at 95oC for 1 min, annealing at 55oC for 1 min and elongation at 72oC for 3 minutes After these amplication cycles last elongation is carried out at 70oC for 5 min. Size of PCR products confirmed by agarose gel electrophoresis using the reaction solution. After the first PCR excess salt is removed using microcontroller and ADI high Amicon (Amicon). The final volume of the solutions that contain the PCR products, brought to 50 μl with sterile distilled water. Purified PCR products using voderady DNA fragment, obtained by PCR 1 - 5

The solution containing the DNA fragment, obtained by PCR 2 - 5

Sterile distilled water - 61.5

20 μm solution diluted 1 to 5

20 μm solution diluted 3 - 0.5

Ex Taq (Takara Shuzo) - 0.5

Reaction conditions are the same as for PCR 1 or PCR 2. The size of PCR products confirmed by electrophoresis in 1.0% or 1.5% agarose gel. DNA fragments precipitated with ethanol, dried in vacuum and dissolved in 40 ál of sterile distilled water. Solutions containing DNA fragments with mutations C19S, C20S, C21S, C22S and C23S, is designated as the DNA solution, the DNA solution, the DNA solution, the DNA solution D and the DNA solution E, respectively.

The DNA fragment, which contains in solution A (20 μl), digested with restriction enzymes NdeI and SphI (Takara Shuzo). A DNA fragment of approximately 400 base pairs (p. O.) extracted from a preparative agarose gel and dissolved in 20 μl of sterile distilled water. This DNA solution called DNA solution 3. Digest 2 μg pSK+ -OCIF restriction enzymes NdeI and SphI. The DNA fragment size of approximately 4.2 KB purified from preparative agarose gel using a kit for the extraction of QIAEX gel and dissolved in 20 μl of sterile distilled water. is Yerevani I set for DNA ligation, var. 2, and perform the ligation reaction. Competent cells of E. coli DH5 transformed with 5 μl of ligation mixture. Resistant to ampicillin transformants sceneroot on the clone containing plasmid DNA. The structure of the DNA analyzed by restricting mapping and DNA sequencing. Thus obtained plasmid is called pSK-OCIF-C19S.

The DNA fragment which is contained in the solution (20 μl), digested with restriction enzymes NdeI and SphI. A DNA fragment of approximately 400 p. O. extracted from a preparative agarose gel using the kit for the extraction of QIAEX gel and dissolved in 20 μl of sterile distilled water. This DNA solution called DNA solution 5. Mix 2 μl of DNA solution 5, 3 µl of DNA solution 4 and 5 μl of buffer for ligating I set for DNA ligation, var.2, and perform the ligation reaction. Competent cells of E. coli DH5 transformed with 5 μl of ligation mixture. Resistant to ampicillin transformants sceneroot on the clone containing plasmid DNA. The structure of the DNA analyzed by restricting mapping and DNA sequencing. Thus obtained plasmid is called pSK-OCIF-C20S.

The DNA fragment which is contained in the solution (20 μl), re is operativnogo agarose gel using a kit for the extraction of QIAEX gel and dissolved in 20 μl of sterile distilled water. This DNA solution called DNA solution 6. Mix 2 μl of DNA solution 6, 3 µl of DNA solution 4 and 5 μl of buffer for ligating I set for DNA ligation, var. 2, and perform the ligation reaction. Competent cells of E. coli DH5 transformed with 5 μl of ligation mixture. Resistant to ampicillin transformants sceneroot on the clone containing plasmid DNA. The structure of the DNA analyzed by restricting mapping and DNA sequencing. Thus obtained plasmid is called pSK-OCIF-C21S.

The DNA fragment which is contained in solution D (20 μl), digested with restriction enzymes NdeI and BstPI. A DNA fragment of approximately 600 p. O. extracted from a preparative agarose gel using the kit for the extraction of QIAEX gel and dissolved in 20 μl of sterile distilled water. This DNA solution called DNA solution 7. Digest 2 μg pSK+ -OCIF restriction enzymes NdeI and BstPI. The DNA fragment size of approximately 4.0 KB extracted from a preparative agarose gel using the kit for the extraction of QIAEX gel and dissolved in 20 μl of sterile distilled water. This DNA solution called DNA solution 8. Mix 2 μl of DNA solution 7, 3 µl of DNA solution of 8 and 5 μl of buffer for l is oli transform 5 ál of ligation mixture. Resistant to ampicillin transformants, through analysis of the structure of DNA, sceneroot a clone containing a plasmid DNA in which the NdeI fragment-BstI 600 p. O. mutation (mutation C22S) replaced by NdeI fragment-BstI 600 p. O. pSK+ -OCIF. The structure of the DNA analyzed by restricting mapping and DNA sequencing. Thus obtained plasmid is called pSK-OCIF-C22S.

The DNA fragment which is contained in the solution E (20 μl), digested with restriction enzymes BstPI and EcoRV. The DNA fragment size of approximately 120 p. O. extracted from a preparative agarose gel using the kit for the extraction of QIAEX gel and dissolved in 20 μl of sterile distilled water. This DNA solution called DNA solution 9. Digest 2 μg pSK+ -OCIF restriction enzymes BstEII and EcoRV. The DNA fragment size of approximately 4.5 KB extracted from a preparative agarose gel using the kit for the extraction of QIAEX gel and dissolved in 20 μl of sterile distilled water. This DNA solution called DNA solution 10. Mix 2 μl of DNA solution 9, 3 ál of DNA solution, 10 and 5 μl of buffer for ligating I set for DNA ligation, var.2, and perform the ligation reaction. Competent cells of E. coli DH5 transform 5 the Structure of DNA analyzed by restricting mapping and DNA sequencing. Thus obtained plasmid is called pSK-OCIF-C23S.

2) Construction of vectors for expression of OCIF mutants.

Digested pSK-OCIF-Cl9S, pSK-OCIF-C20S, pSK-OCIF-C21S, pSK-OCIF-C22S and pSK-OCIF-C23S restriction enzymes BamHI and XhoI. The DNA fragment BamHI-XhoI 1.6 KB, encoding each OCIF mutant, isolated and dissolved in 20 μl of sterile distilled water. Solutions of DNA that contain fragments of cDNA 1.6 KB derived from pSK-OCIF-C19S, pSK-OCIF-C20S, pSK-OCIF-C21S, pSK-OCIF-C22S and pSK-OCIF-C23S, known as DNA-solution C19S, DNA-solution C20S, DNA-solution C21S, DNA-solution C22S and DNA solution C23S, respectively. Digest 5 μg expression vector rser (Invitrogen) with restriction enzymes BamHI and XhoI. The DNA fragment size of approximately 10 KB clean and dissolved in 40 ál of sterile distilled water. This DNA solution was designated as DNA solution rser. Mix independently 1 ál of DNA solution rser and 6 ál of DNA solution C19S or DNA solution C20S, or DNA solution C21S, or DNA solution C22S, or DNA solution C23S with 7 μl of buffer for ligating I set for DNA ligation, var.2, and perform the ligation reaction. Competent cells of E. coli DH5 (100 μl) transform 7 μl of each ligation mixture. Resistant to ampicillin transformants by analyzing the situation recognition BamHI and XhoI pCEP4. The resulting plasmid containing cDNA encoding OCIF-C19S, OCIF-C20S, OCIF-C21S, OCIF-C22S and OCIF-C23S, referred to as pCEP4-OCIF-C19S, pCEP4-OCIF-C20S, pCEP4-OCIF-C21S, pCEP4-OCIF-C22S and pCEP4-OCIF-C23S, respectively.

II) Obtaining OCIF mutants domain deletion.

(1) Deletion mutagenesis OCIF cDNA.

Get a number of OCIF mutants with deletions from Thr 2 to Ala 42, from Pro 43 to Cys 84, from Glu Lys 85 to 122, Arg 123 to Cys 164, Asp 177 to Gln 251 and Il 252 to His 326 (position amino acid residues are indicated in sequence 4). These mutants known as OCIF-DCR1, OCIF-DCR2, OCIF-DCR3, OCIF-DCR4, OCIF-DDD1, OCIF-DDD2, respectively.

Mutagenesis carry out two-stage PCR, as described in example 22-II). Sets the seed for the reaction are shown in table. 11, and nucleotide sequence of the nucleating sequences shown in 19, 25, 40-53 and 54.

The final PCR products are precipitated with ethanol, dried in vacuum and dissolved in 40 ál of sterile distilled water. Solutions of DNA fragment encoding part of the OCIF-DCR1, OCIF-DCR2, OCIF-DCR3, OCIF-DCR4, OCIF-DDD1, OCIF-DDD2, indicate how the solutions F,G, H, I, J and K, respectively.

The DNA fragment which is contained in the solution F (20 μl), digested with restriction enzymes NdeI and XhoI. A DNA fragment of approximately 500 p. O. extracted from preparative agda. This DNA solution called DNA solution 11. Digest 2 μg pSK+ -OCIF restriction enzymes NdeI and XhoI. The DNA fragment size of approximately 4.0 KB extracted from a preparative agarose gel using the kit for the extraction of QIAEX gel and dissolved in 20 μl of sterile distilled water. This DNA solution called DNA solution 12. Mix 2 μl of DNA solution 11, 3 µl of DNA solution 12 and 5 μl of buffer for ligating I set for DNA ligation, var.2, and perform the ligation reaction. Competent cells of E. coli DH5 transformed with 5 μl of ligation mixture. Resistant to ampicillin transformants sceneroot on the clone containing plasmid DNA. The structure of the DNA analyzed by restricting mapping and DNA sequencing. Thus obtained plasmid is called pSK-OCIF-DCR1.

The DNA fragment which is contained in the solution G (20 μl), digested with restriction enzymes NdeI and XhoI. A DNA fragment of approximately 500 p. O. extracted from a preparative agarose gel using the kit for the extraction of QIAEX gel and dissolved in 20 μl of sterile distilled water. This DNA solution called DNA solution 13. Mix 2 μl of DNA solution 13, 3 µl of DNA solution 12 and 5 μl of buffer for E. coli transformed with 5 μl of ligation mixture. Resistant to ampicillin transformants sceneroot on the clone containing plasmid DNA. The structure of the DNA analyzed by restricting mapping and DNA sequencing. Thus obtained plasmid is called pSK-OCIF-DCR2.

The DNA fragment which is contained in the solution N (20 μl), digested with restriction enzymes NdeI and XhoI. A DNA fragment of approximately 500 p. O. extracted from a preparative agarose gel using the kit for the extraction of QIAEX gel and dissolved in 20 μl of sterile distilled water. This DNA solution called DNA solution 14. Mix 2 μl of DNA solution 14, 3 µl of DNA solution 12 and 5 μl of buffer for ligating I set for DNA ligation, var.2, and perform the ligation reaction. Competent cells of E. coli DH5 transformed with 5 μl of ligation mixture. Resistant to ampicillin transformants sceneroot on the clone containing plasmid DNA. The structure of the DNA analyzed by restricting mapping and DNA sequencing. Thus obtained plasmid is called pSK-OCIF-DCR3.

The DNA fragment which is contained in solution I (20 μl), digested with restriction enzymes XhoI and SphI. A DNA fragment of approximately 900 p. O. extracted from philliopines water. This DNA solution called DNA solution 15. Digest 2 μg pSK+ -OCIF restriction enzymes XhoI and SphI. The DNA fragment size of approximately 3.6 KB exteriour from preparative agarose gel using the kit for the extraction of QIAEX gel and dissolved in 20 μl of sterile distilled water. This DNA solution called DNA solution 16. Mix 2 μl of DNA solution 15, 3 µl of DNA solution 16 and 5 μl of buffer for ligating I set for DNA ligation, var.2, and perform the ligation reaction. Competent cells of E. coli DH5 transformed with 5 μl of ligation mixture. Resistant to ampicillin transformants sceneroot on the clone containing plasmid DNA. The structure of the DNA analyzed by restricting mapping and DNA sequencing. Thus obtained plasmid is called pSK-OCIF-DCR4.

The DNA fragment which is contained in the solution J (20 μl), digested with restriction enzymes BstPI and NdeI. A DNA fragment of approximately 400 p. O. extracted from a preparative agarose gel using the kit for the extraction of QIAEX gel and dissolved in 20 μl of sterile distilled water. This DNA solution called DNA solution 17. Mix 2 μl of DNA solution 17, 3 µl of DNA solution of 8 and 5 μl of buffer for E. coli transformed with 5 μl of ligation mixture. Resistant to ampicillin transformants sceneroot on the clone containing plasmid DNA. The structure of the DNA analyzed by restricting mapping and DNA sequencing. Thus obtained plasmid is called pSK-OCIF-DDD1.

The DNA fragment which is contained in the solution To (20 μl), digested with restriction enzymes NdeI and BstPI. A DNA fragment of approximately 400 p. O. extracted from a preparative agarose gel using the kit for the extraction of QIAEX gel and dissolved in 20 μl of sterile distilled water. This DNA solution called DNA solution 18. Mix 2 μl of DNA solution 18, 3 µl of DNA solution of 8 and 5 μl of buffer for ligating I set for DNA ligation, var.2, and perform the ligation reaction. Competent cells of E. coli DH5 transformed with 5 μl of ligation mixture. Resistant to ampicillin transformants sceneroot on the clone containing plasmid DNA. The structure of the DNA analyzed by restricting mapping and DNA sequencing. Thus obtained plasmid is called pSK-OCIF-DDD2.

(2) Construction of vectors for expression of OCIF mutants.

Digested pSK-OCIF-DCR1, pSK-OCIF-DCR2, pSK-OCIF-DCR3, pSK-OCIF-DCR4, pSK-OCIF-DDD1 and pSK-OCIF-DDD2 restriction enzymes BamHI and Astonaut in 20 ál of sterile distilled water. These solutions of DNA that contain fragments BamHI-XhoI derived from pSK-OCIF-DCR1, pSK-OCIF-DCR2, pSK-OCIF-DCR3, pSK-OCIF-DCR4, pSK-OCIF-DDD1 and pSK-OCIF-DDD2, known as DNA-solution DCR1, DNA-solution DCR2, DNA-solution DCR3, a DNA solution DCR4, DNA-solution DDD1 and DNA solution DDD2 respectively. Mix independently 1 ál of DNA solution rser and 6 ál of DNA solution DCR1 or DNA solution DCR2, or DNA solution DCR3, or DNA solution DCR4, or DNA solution DDD1, or DNA solution DDD2 with 7 μl of buffer for ligating I set for DNA ligation, var.2, and perform the ligation reaction. Competent cells of E. coli DH5 (100 μl) transform 7 μl of each ligation mixture. Resistant to ampicillin transformants, by analyzing the structure of DNA, sceneroot on clones containing plasmid DNA in which a DNA fragment with deletion inserted between the recognition sites BamHI and XhoI pCEP4. Plasmids containing cDNA encoding OCIF-DCR1, OCIF-DCR2, OCIF-DCR3, OCIF-DCR4, OCIF-DDD1 and OCIF-DDD2, referred to as pCEP4-OCIF-DCR1, pCEP4-OCIF-DCR2, pCEP4-OCIF-DCR3, pCEP4-OCIF-DCR4, pCEP4-OCIF-DDD1 and pCEP4-OCIF-DDD2, respectively.

III) Obtaining OCIF with truncation of C-terminal domain.

(1) Mutagenesis OCIF cDNA.

Get a number of OCIF mutants with deletions from the amino acid residue Cys 379 to 380 Leu, Ser 331 to 380 Leu, Asp 252 to 380 Leu, Asp 177 to 380 Leu, Arg 123 to 380 Leu and Cys 86 to Leu R4 and OCIF-CCR3, respectively.

Mutagenesis OCIF-CL carry out two-stage PCR, as described in example 22-II). Set the seed for the reaction are given in table.12. The nucleotide sequence of the blades shown in the sequence 23, 40, 55, and 56. The final PCR products are precipitated with ethanol, dried in vacuum and dissolved in 40 ál of sterile distilled water. This DNA solution is designated as solution L.

The DNA fragment which is contained in the solution L (20 μl), digested with restriction enzymes BstPI and EcoRV. The DNA fragment size of approximately 100 p. O. extracted from a preparative agarose gel using the kit for the extraction of QIAEX gel and dissolved in 20 μl of sterile distilled water. This DNA solution called DNA solution 19. Mix 2 μl of DNA solution (19, 3 µl of DNA solution 10 (described in example 22-II) and 5 μl of buffer for ligating I set for DNA ligation, var.2, and perform the ligation reaction. Competent cells of E. coli DH5 transformed with 5 μl of ligation mixture. Resistant to ampicillin transformants sceneroot on the clone containing plasmid DNA. The structure of the DNA analyzed by restricting mapping and DNA sequencing. Thus obtained plasmid is called pSK-OCIF-CL. Mutagenesis of cDNA OCIF mutagenesis to obtain OCIF-CC, OCIF-CDD2, OCIF-CDD1, OCIF-CCR4 and OCIF-CCR3, µl:

10X buffer Ex Taq (Takara Shuzo) - 10

2.5 M solution of dNTP - 8

The plasmid vector containing the full OCIF cDNA described in example 11 (8 ng/ml) - 2

Sterile distilled water - 73.5

20 μm solution of the seed OCIF Xho F - 5

a 100 μm solution of the seed (mutagenesis) - 1

Ex Taq (Takara Shuzo) - 0.5

Specific seed is used for each mutagenesis, and other components are not changed.

The seed used for mutagenesis are listed in table.13. Their nucleotide sequences shown in the sequences 57-61. Components of each PCR are mixed in a microcentrifuge tube and perform PCR as follows. Microcentrifuge tubes treated for 3 minutes at 97oC, and then incubated sequentially for 30 seconds at 95oC, 30 seconds at 50oWith and for 3 minutes at 70oC. This three-stage procedure incubation repeat 25 times and then the tubes incubated for 5 minutes at 70oC. From each tube take an aliquot of the reaction mixture and analyzed by electrophoresis in agarose gel to confirm the size of each product.

The size of PCR products was confirmed by agarose gel.with ethanol, dried in vacuum and dissolved in 40 ál of sterile distilled water. The DNA fragment in each solution digested with restriction enzymes XhoI and BamHI. After reaction the DNA precipitated with ethanol, dried in vacuum and dissolved in 20 μl of sterile distilled water.

Solutions containing fragments with a deletion of the SS, a deletion of the CDD2, deletion CDD1, a deletion of CCR4 and deletion CCR3, is designated as the DNA solution SS DNA solution CDD2, DNA-solution CDD1, DNA-solution CDR4 and DNA solution CDR3 respectively.

(2) Construction of vectors for expression of OCIF mutants.

Digested pSK-OCIF-CL restriction enzymes BamHI and XhoI. The DNA fragment BamHI-XhoI containing the full coding sequence for OCIF-CL, is isolated and dissolved in 20 μl of sterile distilled water. This solution-DNA is designated as solution CL DNA. Mix independently 1 ál of DNA solution rser and 6 MCP DNA solution CL or DNA solution SS, or CDD2, or DNA solution CDD1, or DNA solution CCR4 or DNA solution CCR3 with 7 μl of buffer for ligating I set for DNA ligation, var.2, and perform the ligation reaction. Competent cells of E. coli DH5 (100 μl) transform 7 μl of each ligation mixture. Resistant to ampicillin transformants, by analyzing strelitzia inserted between the recognition sites XhoI and BamHI rser. Plasmids containing cDNA encoding OCIF-CL, OCIF-CC, OCIF-CDD1, OCIF-CDD2, OCIF-CCR4 and OCIF-CCR3, referred to as pCEP4-OCIF-CL, pCEP4-OCIF-CC, pCEP4-OCIF-CDD1, rser-OCIF-CDD2, rser-OCIF-CCR4 and pCEP4-OCIF-CCR3, respectively.

IV) Obtaining OCIF mutants with truncation of the C-end.

(1) the introduction of the C-terminal clipping in OCIF.

Get a number of mutants with truncation of the C-end. The OCIF mutant, in which 10 residues Gln 371 to 380 Leu replaced by 2 residues Leu-Val indicate OCIF-CBst. The OCIF mutant, in which 83 Cys residue from 298 to Leu 380 replaced by 3 residues Ser-Leu-Asp denote OCIF-CSph. The OCIF mutant, in which deleted 214 residues Asn Leu 167 to 380 denote OCIF-CBsp. The OCIF mutant, in which 319 residues from Asp 62 to Leu 380 replaced by 2 residues Leu-Val indicate OCIF-CPst. The position of the amino acid residues shown in sequence 4.

Digest 2 μg each of pSK+ -OCIF with one of the restriction enzymes BstPI, SphI, PST (Takara Shuzo) and EspEI (New England Biolabs), and then extracted with phenol and precipitated with ethanol. Precipitated DNA was dissolved in 10 μl of sterile distilled water. The ends of the DNA in 2 μl of each solution dull, using the set for dephosphorylation of DNA in a final volume of 5 ál. To the reaction mixture was added 1 μg (1 μl) containing an amber codon XbaI-linker (5'-HUNDRED-GTCTAGACTAG-3') and 6 ál of buffer for ligation Noi mixture for transformation of E. coli DH5. Resistant to ampicillin transformants sceneroot on clones containing plasmids. The structure of the DNA analyzed by restricting mapping and DNA sequencing. The resulting plasmid is called pSK-OCIF-CBst, pSK-OCIF-CSph, pSK-OCIF-CBsp and pSK-OCIF-CPst, respectively.

(2) Construction of vectors for expression of OCIF mutants.

Digested pSK-OCIF-CBst, pSK-OCIF-CSph, pSK-OCIF-CBsp and pSK-OCIF-CPst restriction enzymes BamHI and XhoI. The DNA fragment of 1.5 KB containing the complete coding sequence for each of OCIF mutant, isolated and dissolved in 20 μl of sterile distilled water. These solutions of DNA that contain the fragment BamHI-XhoI derived from pSK-OCIF-CBst, pSK-OCIF-CSph, pSK-OCIF-CBsp and pSK-OCIF-CPst, denoted respectively as DNA solution CBst, the DNA solution CSph, DNA-solution CBsp and DNA solution CPst. Mix independently 1 ál of DNA solution rser (described in example 22-II) and 6 ál of DNA solution CBst or DNA solution CSph, or DNA solution CBsp, or DNA solution CPst with 7 μl of buffer for ligating I set for DNA ligation, var. 2, and perform the ligation reaction. Competent cells of E. coli DH5 (100 μl) transform 7 μl of each ligation mixture. Resistant to ampicillin transformants, by analyzing the structure of DNA, sceneroot on containing cDNA, encoding OCIF-CBst, OCIF-CSph, OCIF-CBsp and OCIF-CPst, is designated as pCEP4-OCIF-CBst, pCEP4-OCIF-CSph, pCEP4-OCIF-CBsp and pCEP4-OCIF-CPst, respectively.

V) Obtaining vectors for the expression of OCIF mutants.

Grow E. coli clones carrying expression vectors for OCIF mutants (21 clone), and the vector purified by QIAGEN column (QIAGEN). All expression vectors precipitated with ethanol and dissolved in appropriate volume of sterile distilled water and used for further discussed below manipulations.

VI) intermediate expression of cDNA for human OCIF mutants and biological activity of the mutants.

The OCIF mutants produced using expression vectors obtained in example 22-V). The method is essentially the same as described in example 13. Enter only the following modifications. For transfection of DNA using a 24-well plate. In each well Planeta sow 2105cells 293/EBNA suspended in IMDM containing 10% serum fetal cows. For each transfection using 1 μg of purified vector DNA and 4 ál of lipofectamine. To the cells in the well add the mixture expression vector and lipofectamine in OPTI-MEM (GIBCO BRL) in a final volume of 0.5 ml After incubation of cells at 37oC for 24 hours WITH2
-incubator. Conditioned medium collected and used for analysis of biological activity in vitro. Nucleotide sequence of the cDNA of OCIF mutants are sequences 83-103. The decoded amino acid sequence of OCIF mutants are sequences 62-82. Tests of biological activity in vitro carried out as described in example 13. The concentration of antigen in each air-conditioned environment is determined by the ELISA method (enzyme-linked immunosorbent assay), as described in example 24. In table.14 shows the specific activity of the mutants relative to the activity of the unmodified OCIF.

VII) Western-blot analysis.

For Western blotting using 10 µl of the final air-conditioned environment. Mix 10 μl of the sample with 10 μl of sample buffer for SDS-PAGE (0.5 M Tris-HCl, 20% glycerol, 4% LTOs, 20 μg/ml bromophenol blue, pH 6.8), boiled for 3 min and subjected to electrophoresis in 10% polyacrylamide gel in the presence of LTOs in non conditions. After electrophoresis, separated proteins bitteroot on PVDF membrane (ProBlottR, Perkin Elmer) using semi-dry electroblotting (BIO-RAD). The membrane is incubated at 37oC for 2 hours with the sword which react with the labeled antibody, using the ECL system (Amersham). In the case of constant OCIF detects two protein bands with molecular mass of about 60 KD and 120 KD. On the other hand, in the case of OCIF-C23S, OCIF-CL and OCIF-CC detects almost exclusively protein band at 60 KD. Bands of proteins with mass of the order of 40-50 KD and 30-40 KD are the main if OCIF-CDD2 and OCIF-CDD1, respectively. These results indicate that Cys 379 is responsible for dimer formation; as monomers and dimers retain biological activity, and deletion of residues from Asp Leu 177 to 380 does not destroy the biological activity of OCIF (the position of the amino acid residues shown in sequence 4).

EXAMPLE 23

Selection of human genomic gene OCIF.

I) Screening a human genomic library.

Sceneroot library of amplified genes in human placenta in the vector lambda FIX II purchased from STRATAGENE, to the gene encoding human OCIF, using as a probe cDNA of human OCIF. Essentially, the screening is done according to the instructions that accompany the genomic library. The basic scheme described in Molecular Cloning: A Laboratory Manual, also used to work with phage, E. coli and DNA.

The library is titrated, 1106the battle of phage are mixed and incubated overnight at 37oC. raising the plaques on the filter gain, using a nylon membrane Hybond-N (Amersham). Membrane ProcessInput by denaturing solution containing 1.5 M NaCl and 0.5 M NaOH for 1 minute at room temperature. Then the membrane is neutralized by placing them sequentially for 1 minute every once in 1 M Tris-Hcl (pH 7.5) and in a solution containing 1.5 M NaCl and 0.5 M Tris-Hcl (pH 7.5). Then the membrane is transferred to filter paper, moistened 2SSC. Phage DNA is fixed on the membranes 1200 µj UV light UV cross-linker STRATALINKER (STRATAGENE) and dried membrane in the air. The membrane is immersed in a buffer for rapid hybridization (Amersham) and incubated for 1 hour at 65oWith before hybridization with32P-labeled cDNA probe in the same buffer overnight at 65oC. Skanirujushie probe obtained by labeling OCIF cDNA32P using the Megaprime labeling DNA (Amersham). For each ml of buffer for hybridization using the probe approximately 5105Chim. After hybridization the membranes are rinsed in 2SSC for 5 minutes at room temperature. Then the membrane is washed four times, each time for 20 minutes in 0.SSC containing 0.1% LTOs, when 65oC. After the last wash, the membrane is dried and subjected to AB is of the autoradiograms detects six positive signals. Choose Kosyachkov agar of the areas corresponding to these signals, for purification of phage. Each agar try for the extraction of phage soaked overnight in 0.5 ml of SM buffer containing 1% chloroform. Each extract containing phage diluted 1000 times SM-buffer and aliquot in 1 ml or 20 ml mixed with the above-described host E. coli. The mixture is plated on agar plates with top agarose as described above. Plates are incubated overnight at 37oWith and receive the bulging on the filter, prehybridization, hybridized, washed and Avtoreferat, as described above. This process of purification of phage applied to all six positive signals detected initially in the autoradiograms, and to repeat until all of phage plaques on agar plates do not hybridize with the cDNA probe. After cleaning agar Kosyachkov each phage isolate and soaked in SM buffer containing 1% chloroform, and stored at 4oC. Six individual phage isolates designated as 0IF3, 0IF8, 0IF9, 0IF11, 0IF12 and 0IF17 respectively.

II) Analysis of genomic clones by digestion with restriction enzymes and southern blot hybridization

Get DNA from each phage isolate method planetization, as described in Molecular from parivara, separated in agarose gels. Then the fragments are transferred to a nylon membrane and subjected to southern blot hybridization using OCIF cDNA as a probe. The results of the analysis show that the six phage isolates are separate clones. Among these fragments obtained by digestion with restriction enzymes, the fragments that hybridize with the probe OCIF cDNA, subcloning in plasmid vectors and subjected to the analysis of the nucleotide sequence, as described below.

III) Subclavian in plasmid vectors restriction fragments obtained from genomic clones, and determination of the nucleotide sequence.

Digested DNA 0IF8 restriction enzymes EcoRI and NotI and share the resulting DNA fragments on a 0.7% agarose gel. The fragment EcoRI/NotI in 5.8 thousand base pairs (KB) extracted from the gel using a set of extraction from gel with QIAEX II (QIAGEN), according to the method recommended by the manufacturer. The fragment EcoRI/NotI in 5.8 KB are ligated with the vector pBluescript II SK+ (STRATAGEN), which is linearized with restriction enzymes EcoRI and NotI, using DNA ligase Ready-To-Go T4 (Pharmacia), according to the methods recommended by the manufacturer. Competent cells of E. coLi DH5 (Amersham) transform the lawn carrying a recombinant plasmid containing the fragment EcoRI/NotI in 5.8 KB and this plasmid is called pBSG8-5.8. Digest pBSG8-5.8 with HindIII and separated from this parivara the obtained DNA fragment of 0.9 KB in the same way as described above. Then a fragment of 0.9 KB clone in pBluescript II SK - in HindIII site, as described above. This recombinant plasmid containing the HindIII-a fragment of 0.9 KB, indicate pBS8H0.9.

Digested DNA 0IF11 with EcoRI and EcoRI-fragments of 6 KB, 3.6 KB, 2.6 KB emit in the same way as described above, and clone into the vector pBluescript II SK+ EcoRI site, as described above. These recombinant plasmids are called pBSG11-6, pBSG11-3.6 and pBSG11-2.6, respectively. Digest pBSG11-6 with HindIII and prewar put on a 0.7% agarose gel. Extracted from the gel three fragment length 2.2 KB, 1.1 KB and 1.05 KB and clone independent in the vector pBluescript II SK - HindIII site in the same way described above. These recombinant plasmids are called pBS6H2.2, pBS6H1.1 and pBS6H1.05.

The nucleotide sequence of the cloned genomic DNA was determined by using a kit for rapid reactions for sequencing ABI Dyedeoxy Terminator Cycle (PERKIN ELMER) and DNA sequencing A (Applied Biosystems). Plasmids pBSGS-5.8, pBS8H0.9 and pBSG11-6, pBSG11-3.6, pBSG11-2.6 and pSG6H2.2, pBS6H1 and pBS6H1.05 get on metallicawelcome. The nucleotide sequence of the gene of human OCIF presented in sequence 104 and sequence 105. The DNA nucleotide sequence between exon 1 and exon 2 incompletely defined. Between sequences 104 and 105, there is a period of nucleotides approximately 17 KB.

EXAMPLE 24

Quantitative determination of OCIF by ELISA.

I) Obtaining antibodies against OCIF.

For immunization with the purpose of obtaining antisera used males JW rabbits (Kitayama LABES Co., LTD) weighing 2.5-3.0 kg is Used to immunize three males JW rabbits (Kitayama LABES Co., LTD) weighing 2.5-3.0 kg For immunization are emulsion by mixing equal volumes of rOCIF (200 μg/ml) and complete adjuvant's adjuvant (Difco, cat. 0638-60-7). Rabbits subjected to immunization subcutaneously six times at one week intervals, 1 ml emulsion for injection. Rabbits give six injections subcutaneously at intervals of seven days. Blood taken within 10 days after the last immunization and get the serum. Antibodies purified from the serum as follows. Diluted serum TFR twice. After addition of ammonium sulfate at a final concentration of 40% (m/o) whey stand at 4oC for 1 hour. The precipitate obtained is causesa the resulting solution is loaded into a column of protein G-separate (Pharmacia). After washing SFR absorbed immunoglobulin G elute with 0.1 M glycine-Hcl buffer (pH 3.0). Eluate immediately neutralized with 1.5 M Tris-Hcl-buffer (pH 8.7) and cialiswhat against SFR. The protein concentration is determined by absorption at 280 nm (E1%13.5).

Labeled with horseradish peroxidase antibody is obtained using the set of activated imide of maleic acid peroxidase from horseradish ImmunoPure (Pierce, cat. 31494). Briefly, incubated with 1 mg of IGD with 80 µg N-Succinimidyl-3-acetylcysteine within 30 minutes After deacetylation 5 mg of hydroxylamine hydrochloride modified IGD separated using polyacrylamide column for desalting. The protein pool was mixed with 1 mg of activated imide of maleic acid peroxidase from horseradish, incubated at room temperature for 1 hour.

II) Quantitative determination of OCIF using the dual ELISA.

Titration microplates (Nunc MaxiSorp Immunoplate) sensibiliser rabbit anti-OCIF IGD by incubating 0.2 µg in 100 µl of 50 mm attributableto buffer, pH 9.6, at 4oWith during the night. After blocking tablets by incubation for 1 hour at 37oWith 300 ál of 25% BlockAce/SFR (Snow Brand Milk Products), 100 ál of the samples incubated for 2 is billaut 100 ál diluted 1:10000 labeled with peroxidase from horseradish IGD anti-OCIF and incubated for 2 hours at room temperature. The amount of OCIF determined by incubation with 100 µl of substrate solution (TMB, ScyTek Lab., cat. TM) and measuring the absorption at 450 nm using ImmunoReader (Nunc NJ2000). Purified recombinant OCIF used as standard protein; a typical standard curve is shown in Fig.13.

EXAMPLE 25

Monoclonal antibody against the OCIF.

I) Obtaining hybridoma producing monoclonal antibody against OCIF.

Purified OCIF until smooth from the culture medium of human fibroblasts IMR-90 using the purification method described in example 11. Purified OCIF dissolved in SFR at a concentration of 10 µg/100 ml in BALB/c Mice subjected to immunization by injecting this solution administered intraperitoneally three times every two weeks. When the first and second immunizations administered emulsion composed of equal amounts of OCIF and complete adjuvant's adjuvant. Three days after the last immunization, spleen extract, isolated lymphocytes and merge with cells of the mouse myeloma RH-Ag8.653 in the usual way using polyethylene glycol. Then merged cells cultivated in GAT-environment and select hybridoma. Then, to verify that produces whether selected hybridoma antibody against OCIF, OCIF antibody against the implementation of each well in 96-well tablets for immunoassay (immunoblastic) (Nunc) at 100 μl purified OCIF (10 μg/ml in 0.1 M NaHCO3and block each well 50% BlockAce (Snow Brand Milk Products Co., Ltd. ). Hybridoma clones secreting antibody against OCIF, create by cloning 3-5 times by limiting dilution and by screening using the above ELISA. From among the thus obtained hybridoma clones are selected several hybridoma clones with high production of antibodies against the OCIF.

II) Production of monoclonal antibodies against OCIF.

Each hybridoma clone secreting antibody against OCIF, which is obtained in example 25-(I), administered intraperitoneally transplanted mice from Pristane (Aldrich) at a density of cells 1106cells in the mouse. Accumulated ascites collected 10-14 days after transplantation and obtain ascites containing specific monoclonal antibody against OCIF of the present invention. Purified antibodies obtained using the affine gel chromatography on protein-a-sepharose (BioRad) according to the manufacturer's instructions. Briefly, ascites diluted in equal volume of buffer for binding (BioRad), and applied to a column of protein a-separate. The column was washed with a sufficient amount of buffer to bind and elute buffer for elution (BioRad). After neutralization of the eluate obtained cialiswhat in which to experience with a molecular mass of about 150000.

III) Screening monoclonal antibodies with high affinity to OCIF.

Each antibody obtained in example 25-II), dissolved in SFR, and determine the concentration of protein in solution by the method of Lowry. Prepare a solution of each of the antibodies of the same concentration and then diluted serially SFR. Monoclonal antibodies, which can recognize OCIF even in a highly diluted solution, selected solid phase ELISA as described in example 25-II). Thus it is possible to select three monoclonal antibodies - A1G5, EN and D2F4.

IV) determining the class and subclass of antibodies.

The class and subclass of the antibodies of the present invention obtained in example 25-III) analyze using the test kit of the class and subclass of immunoglobulins (Amersham). The procedure is carried out according to the scheme outlined in the instructions. The results are given in table.15. Antibodies of the present invention IN, A1G5 and D2F4 belong to IGD1, IgD2aand IGD2brespectively.

V) Determination of OCIF by ELISA.

Three types of monoclonal antibodies A1G5, ESN and D2F4 obtained in example 25-(IV), used as a solid phase antibody and enzyme labeled antibodies, respectively. Design dual ELISA by combining each t is activated imide of maleic acid peroxidase from horseradish (Pierce, cat. 31494). Each monoclonal antibody was dissolved in 0.1 M Panso3at a concentration of 10 μg/ml and 100 μl of solution was added to each well in 96-well immunoplate (Nunc, MaxiSorp, cat. 442404) and then stand at room temperature overnight. Then each well on tablets block 50% Blockace (Snow Brand Milk Products Co., Ltd.) at room temperature for 50 minutes, and then washed three times SFR containing 0.1% tween 20 (buffer flushing).

The number concentration of OCIF produced by breeding OCIF buffer for 1 reaction (0.2 M Tris-HCl, pH 7.4, containing 40% Blockace and 0.1% tween-20). Each well of 96-well immunoplates fill 100 μl of the solution prepared in each OCIF concentration, allowed to stand at 37oC for 3 hours, and then washed three times with buffer for washing. For cultivation labeled POD antibodies used buffer for the 2nd reaction (0.1 M Tris-HCl, pH 7.4, containing 25% Blocka and 0.1% tween-20). Labeled POD antibody diluted 400 times with buffer 2, the reaction in each well in immunoblastic add 100 μl of the diluted solution. Each immunoplate stand at 37oC for 2 hours, and then washed three times with buffer for washing. After washing each well in immunoblastic of daldinia and 0.006% of N2ABOUT2), and incubated immunoplate at 37oC for 15 minutes stop the Enzymatic reaction by adding to each well 50 µl of 6 N. N2SO4. Determine the optical density of each well at 492 nm, using immunizer (ImmunoReader NJ 2000, Nunc).

Using three kinds of monoclonal antibodies in the present invention each combination of antibodies in the solid phase and labeled POD antibodies leads to the precise definition of OCIF. Confirmed that each monoclonal antibody in the present invention recognizes a different epitope OCIF. Typical

a standard curve OCIF when using a combination of solid-phase antibody A1G5 and labeled POD antibody IN shown in Fig.14.

VI) Determination of OCIF in human serum.

The concentration of OCIF in five serum samples of healthy people determined using the ELISA described in example 25-V). Immunoplate sensibiliser A1G5 as described in example 25-V), and each well on immunoblastic add 50 ál of buffer for the 1st reaction. Then to each well in immunoblastic add 50 ál of each sample of human serum. Immunoplate incubated at 37oC for 3 hours, and then washed three times with buffer putera for the 2nd reaction, and incubated at 37oC for 2 hours. After triple washing buffer for washing each well in immunoblastic add 100 µl of substrate solution described in example 25-V), and incubated at 37oC for 15 minutes stop the Enzymatic reaction by adding to each well in immunoblastic 50 ál 6 N. N2SO4. Determine the optical density of each well at 492 nm, using immunizer (ImmunoReader NJ 2000, Nunc).

In the same way process buffer for 1 reaction containing a known amount of OCIF, and receive a standard curve of contents OCIF shown in Fig.2. Determine the amount of OCIF in the sample of human serum using the specified standard curve for OCIF. The results are given in table.16.

EXAMPLE 26

Therapeutic effect on osteoporosis.

(1) Method.

Male Fischer rats aged 6 weeks is subjected to denervation of the left front limb. These rats are placed into four groups (10 rats per group) and treated as follows: group a - rats with simulated operation without injection; group B - denervated rats with intravenous filler; group - denervated rats, which F at a dose of 50 mg/kg twice a day. After denervation OCIF prescribed daily for 14 days. After a 2-week treatment of animals put to death and cut off their forelimbs. Then the bones have mechanical strength.

(2) the Results.

Animals in the control groups see a decrease in bone strength compared with normal animals groups, while the animals in the groups receiving 50 mg OCIF per 1 kg of body mass, bone strength increases.

Industrial applicability

The present invention relates both to a new protein, which inhibits the formation of osteoclasts, and effective way to obtain this protein. The protein of the present invention has the activity of inhibiting the formation of osteoclasts. Protein will be useful for the treatment of many diseases associated with rarefaction of the bones, such as osteoporosis, and is useful as an antigen, which is used for immunological diagnosis of such diseases.

Help concerning deposited microorganism

Name and address of Depositary authority

Name: National Institute of Bioscience and Human-Technology

Agency of Industrial Science and Technology Ministry of International Trade and Industry

Address: 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken 305, Adachi: October 25, 1995)

Inventory number: FERM BP-5267. TA

1. Protein characterized by the following properties: (a) molecular weight during electrophoresis in polyacrylamide gel in the presence of LTOs (SDS-PAGE), which constitutes approximately 60 KD in terms of recovery, approximately 60 KD and 120 KD in non conditions; (b) high affinity for the cation-exchange column and a column of heparin; (b) biological activity of inhibiting the differentiation and/or maturation of osteoclasts, and this activity is reduced by heating at 70oC for 10 min or 56oC for 30 min, the activity is lost when heated at 90oC for 10 min; (g) internal amino acid sequences presented in SEQ ID NO : 1, 2, and 3, and (d) with optional N-terminal amino acid sequence represented in SEQ ID NO : 7.

2. Protein in p. 1 produced in human fibroblasts.

3. The method of obtaining protein under item 1 or 2 using a process comprising culturing human fibroblasts, protein purification by a combination of ion-exchange column chromatography, affinity column chromatography and column chromatography with reversed haematol ceramics.

5. OCIF protein with the amino acid sequence presented in SEQ ID NO : 4.

6. cDNA with the nucleotide sequence represented in SEQ ID NO : 6 encoding OCIF protein with the amino acid sequence presented in SEQ ID NO : 4.

7. cDNA with the nucleotide sequence represented in SEQ ID NO : 6 encoding OCIF protein with a signal peptide having the amino acid sequence represented in SEQ ID NO : 5.

8. OCIF protein expressed from the cDNA that encodes the amino acid sequence represented in SEQ ID NO : 4.

9. A protein with the biological activity of inhibiting the differentiation and/or maturation of osteoclasts, which is obtained as the amino acid sequence of the expressed cDNA having at least 80% identity in amino acid sequence represented in SEQ ID NO : 4.

10. A method of obtaining a protein that inhibits the development of osteoclasts, by genetic engineering methods using cDNA that encodes the amino acid sequence represented in SEQ ID NO : 4: (a) molecular weight during electrophoresis in polyacrylamide gel in the presence of LTOs (SDS-PAGE) component of approximately 60 KD in terms of bonneu column to a column of heparin; (in) activity of inhibiting the differentiation and/or maturation of osteoclasts, decreasing by heating at 70oC for 10 min or 56oC for 30 min, and the activity is lost when heated at 90oC for 10 min; (g) internal amino acid sequence represented in SEQ ID NO : 1-3.

11. The method of obtaining protein under item 8 methods of genetic engineering using mammalian cells as host cells.

12. A method of obtaining a protein by p. 11 by means of genetic engineering using as a master of mammalian cells 293/EBNA or cells SNO.

13. cDNA encoding OCIF protein or its variants, selected from

cDNA with the nucleotide sequence represented in SEQ ID NO : 8, amino acid coding sequence represented in SEQ ID NO : 9;

cDNA with the nucleotide sequence represented in SEQ ID NO : 10, amino acid coding sequence represented in SEQ ID NO : 11;

cDNA with the nucleotide sequence represented in SEQ ID NO : 12, amino acid coding sequence represented in SEQ ID NO : 13;

cDNA with the nucleotide sequence represented in SE, presented in SEQ ID NO : 83, amino acid coding sequence represented in SEQ ID NO : 62;

cDNA with the nucleotide sequence represented in SEQ ID NO : 84, amino acid coding sequence represented in SEQ ID NO : 63;

cDNA with the nucleotide sequence represented in SEQ ID NO : 85, amino acid coding sequence represented in SEQ ID NO : 64;

cDNA with the nucleotide sequence represented in SEQ ID NO : 86, amino acid coding sequence represented in SEQ ID NO : 65;

cDNA with the nucleotide sequence represented in SEQ ID NO : 87, amino acid coding sequence represented in SEQ ID NO : 66;

cDNA with the nucleotide sequence represented in SEQ ID NO : 88, amino acid coding sequence represented in SEQ ID NO : 67;

cDNA with the nucleotide sequence represented in SEQ ID NO : 89, amino acid coding sequence represented in SEQ ID NO : 68;

cDNA with the nucleotide sequence represented in SEQ ID NO : 90, amino acid coding sequence represented in SEQ ID NO : 69;

cDNA with the nucleotide sequence, as is Noah sequence, presented in SEQ ID NO : 92, amino acid coding sequence represented in SEQ ID NO : 71;

cDNA with the nucleotide sequence represented in SEQ ID NO : 93, encoding the amino acid sequence represented in SEQ ID NO : 72;

cDNA with the nucleotide sequence represented in SEQ ID NO : 94, amino acid coding sequence represented in SEQ ID NO : 73;

cDNA with the nucleotide sequence represented in SEQ ID NO : 95, amino acid coding sequence represented in SEQ ID NO : 74;

cDNA with the nucleotide sequence represented in SEQ ID NO : 96, amino acid coding sequence represented in SEQ ID NO : 75;

cDNA with the nucleotide sequence represented in SEQ ID NO : 97, encoding the amino acid sequence represented in SEQ ID NO : 76;

for DNA with the nucleotide sequence represented in SEQ ID NO : 98, encoding the amino acid sequence represented in SEQ ID NO : 77;

cDNA with the nucleotide sequence represented in SEQ ID NO : 99, amino acid coding sequence represented in SEQ ID NO : 78;

cDNA with the nucleotide sequence predstavlenie sequence, presented in SEQ ID NO : 101, the coding amino acid sequence represented in SEQ ID NO : 80;

cDNA with the nucleotide sequence represented in SEQ ID NO : 102, encoding the amino acid sequence represented in SEQ ID NO : 81;

cDNA with the nucleotide sequence represented in SEQ ID NO : 103, encoding the amino acid sequence represented in SEQ ID NO : 82.

14. OCIF protein encoded by cDNA selected from cDNA under item 13.

15. The genomic DNA with the nucleotide sequence represented in SEQ ID NO: 104 or 105 encoding OCIF protein with the amino acid sequence presented in SEQ ID NO: 4.

16. Genomic DNA under item 15 with the nucleotide sequence represented in SEQ ID NO: 104 or 105.

17. Polyclonal antibody with specific affinity to OCIF obtained by immunization of a suitable mammal using OCIF as antigen optionally in combination with adjuvants for immunization.

18. Monoclonal antibody specific against OCIF obtained by hybridoma technology using OCIF as antigen and characterized by the following properties: molecular weight on the Sabbath.

19. The method of determining the concentration of OCIF protein, including the introduction phase antibodies under item 17 or 18 in a solution containing OCIF protein and measuring the concentration of OCIF protein by forming a complex of the antibody/ OCIF.

Priority points:

20.02.1995 on PP. 1-4, 5, 8-10;

21.07.1995 on PP. 6, 13-19.

 

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