JP2000229879A - Osteoplasty accelerating agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To prepare an osteoplasty-accelerating agent having excellent propagation accelerating activity for osteoblast and useful as a prophylaxis and/or therapeutic agent for an osteopenia such as osteoporosis by including an adipocellular development inhibitory factor as an active ingredient. SOLUTION: This osteoplasty-accelerating agent includes an adipocellular development inhibitory factor as an active ingredient. The adipocellular development inhibitory factor can efficiently be isolated at high yield in a purified state from a culture solution of human fibroblast. For example, the adipocellular development inhibitory factor can be prepared by isolating and purifying itself from a culture solution obtained by making human fibroblast [e.g. human fetal lung fibroblast IMR-90 (ATCC deposit-CCL186)] attach to a piece of alumina ceramic, followed by the static culture using a Dulbecco-modified Eagle medium added with a bovine neonate serum as a culture solution for about 1 week to 10 days in a T-flask or a roller bottle.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an osteogenesis promoter containing an adipocyte formation inhibitory factor (ADIF) as an active ingredient.

[0002]

BACKGROUND OF THE INVENTION Bone metabolism depends on the combined activity of osteoblasts, which are responsible for bone formation, and osteoclasts, which are responsible for bone resorption. It is believed that an imbalance in bone formation and resorption results in abnormal bone metabolism. Known diseases due to abnormal bone metabolism include osteoporosis, hypercalcemia, Paget's disease of bone, renal osteodystrophy, rheumatoid arthritis, and osteoarthritis. Osteoporosis, known as a representative of these disorders of bone metabolism, is a disease that occurs when bone resorption by osteoclasts exceeds bone formation by osteoblasts, and the bone calcareous and bone matrix are equally reduced. It is characterized by. The mechanism of development of this disease has not yet been fully elucidated. In addition, this disease is a disease in which bone pain occurs and a fracture occurs due to weakening of the bone. As the elderly population grows, the disease, which causes bedridden elderly people due to fractures, has also become a social issue,
The development of such therapeutics is urgently needed. It is expected that bone loss caused by such bone metabolic disorders can be treated by suppressing bone resorption, promoting bone formation, or improving the balance between them. That is, bone formation promotes the proliferation and differentiation function of osteoblasts responsible for bone formation, suppresses the differentiation and maturation of osteoclast precursor cells into osteoclasts,
Alternatively, it is expected to be promoted by suppressing functions such as bone resorption activity of osteoclasts. Currently, vigorous search and development research is being conducted on hormones, low-molecular substances, or bioactive proteins having such activities.

Pharmaceuticals for treating bone-related diseases and shortening the treatment period include calcitonin preparations, active vitamin D3 preparations, hormone preparations containing estradiol, ipriflavone, vitamin K2, and bisphosphonate compounds. In addition, clinical trials have been conducted on active vitamin D3 derivatives, estradiol derivatives, and second or third generation bisphosphonate compounds with the aim of developing therapeutic agents with fewer side effects and superior efficacy. Have been. However, therapeutic methods using these drugs are not always satisfactory in their effects and therapeutic results, and development of new safer and more effective therapeutic agents is expected. In addition, some of the drugs used for treating bone metabolic diseases have limited curable diseases due to their side effects. Furthermore, at present, multi-drug combination therapy using a plurality of drugs at the same time is becoming mainstream for the treatment of bone metabolic diseases such as osteoporosis.
From such a viewpoint, development of a drug having an action mechanism different from that of a conventional drug, and having higher efficacy and fewer side effects is expected.

[0004]

DISCLOSURE OF THE INVENTION The present inventors have conducted intensive studies in view of the above situation, and have found that the adipocyte formation inhibitor ADIF, which has been newly found by the present inventors, has excellent osteoblast proliferation. Promoting activity was found. That is, an object of the present invention is to provide an osteogenesis promoter containing an adipocyte formation inhibitory factor (ADIF) as an active ingredient.

[0005]

Means for Solving the Problems The present invention relates to an osteogenesis promoter containing an adipocyte formation inhibitory factor (ADIF) as an active ingredient. According to the present invention, a preparation having excellent osteoblast proliferation promoting activity is provided, and is useful as an agent for preventing and / or treating a bone loss disease such as osteoporosis.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION ADIF as an active ingredient of the present invention
Can be efficiently isolated and purified from a culture of human fibroblasts at a high yield. Human fibroblasts used as production cells are not limited, but are particularly preferably human fetal lung fibroblasts IMR-90 (ATCC Deposit-Accession No. C
CL186) is used. Human fibroblasts were attached to alumina ceramic pieces, and Dulbecco's modified Eagle's medium (DMEM) supplemented with neonatal bovine serum was used as a culture medium in T-flasks and roller bottles for one week to one week.
Incubate statically for about 0 days. The protein A of the present invention is
DIF is isolated and purified. At this time, the isolation and purification method used is carried out by using various purification procedures utilizing the physical or chemical properties of the target protein ADIF according to a conventional method generally used for purification of a protein from a biological sample. can do. As the concentration means, for example, a conventional method such as ultrafiltration, freeze-drying and salting out is used. Purification means include, for example, ion exchange chromatography, affinity chromatography, gel filtration chromatography, hydrophobic chromatography, reverse phase chromatography, preparative electrophoresis, and various methods used for protein purification, and combinations thereof. Can be used. In addition, a surfactant may be added when the protein of the present invention is purified from the culture solution. At this time, the surfactant used is not limited, but is preferably 0.1% C
HAPS (3-[(3-cholamidopropyl) -dimethylammonio]-
1-propanesulfonate), 0.01% polysorbate 8
0 or 0.01% polysorbate 20 can be used. The protein ADIF of the present invention particularly preferably treats a culture solution in the order of a heparin column, SP / cation exchange column, Q / anion exchange column, gel filtration column, heparin column, weak cation exchange column, and reverse phase column. Thus, it can be isolated and purified. ADIF of the protein of the present invention thus obtained is specified by the following biological and physicochemical properties. (a) activity: having activity to suppress adipocyte differentiation and / or maturation (b) molecular weight (by SDS-PAGE): about 45 kDa (under non-reducing conditions) about 28 kDa and / or about 23 kDa (under reducing conditions) (c) Affinity: has affinity for heparin. (d) It contains a sugar chain. (e) N-terminal amino acid sequence: Xaa Ser Gln Gln Lys Gly Arg Leu Ser Leu Gln Asn Thr Ala Glu Ile 1 5 10 15 Gln His Cys Leu Val Asn Ala Gly Asp Val Gly Cys Gly Val Phe Glu 20 25 30 Cys Phe Glu Asn Asn Xaa Xaa Glu 35 40 (However, Xaa indicates an unidentified amino acid.)

Further, the protein of the present invention can be produced by genetic engineering techniques. That is, a cDNA encoding this protein is cloned based on the amino acid sequence information of natural ADIF obtained according to the method described above, and the cDNA is expressed by a genetic engineering technique to obtain a recombinant ADIF. Can be. Specifically, N of the natural ADIF obtained in the present invention is
-Analyze the terminal amino acid sequence and produce a mixture of oligonucleotides that can encode this sequence. Next, PCR was performed using the prepared oligonucleotide mixture as a primer.
Method (preferably RT-PCR method)
Obtain a cDNA fragment. By cloning this cDNA fragment as a probe using a cDNA library prepared from IMR-90 cells, a full-length ADIF cDNA can be obtained (SEQ ID NO: 1 in the Sequence Listing). Further, the obtained full-length cDNA is inserted into an appropriate expression vector to prepare an ADIF expression plasmid, which is introduced into various bacteria or animal cells to express the plasmid, whereby a recombinant ADIF can be obtained.
The recombinant ADIF thus obtained is
It has the same physicochemical properties as the above-mentioned natural ADIF, and its N-terminal amino acid sequence is shown below. N-terminal amino acid sequence (guessed from cDNA; SEQ ID NO. 3 in Sequence Listing): Ser Ser Gln Gln Lys Gly Arg Leu Ser Leu Gln Asn Thr Ala Glu Ile 1 5 10 15 Gln His Cys Leu Val Asn Ala Gly Asp Val Gly Cys Gly Val Phe Glu 20 25 30 Cys Phe Glu Asn Asn Ser Cys Glu 35 40

[0008] ADIF activity was determined by the method of Kodama H. et al. (Journal of Cellular Physiology, Vol. 112, p83 (19
82)) That is, the suppression of adipocyte formation in the presence of dexamethasone using mouse preadipocyte cell lines as target cells can be confirmed by evaluating the inhibition of triglyceride accumulation.

ADIF, an adipocyte formation inhibitory factor, which is an active ingredient of the present invention, can be formulated into humans and animals and orally or parenterally administered safely. Examples of the form of the pharmaceutical composition include an injection composition, an infusion composition, a suppository, a nasal agent, a sublingual agent, a transdermal absorbent, and the like. These preparations are prepared according to known pharmaceutical manufacturing methods, and pharmacologically acceptable carriers, excipients, stabilizers,
By using a coloring agent, a surfactant, and other additives, the desired preparation can be obtained. In the case of an injectable composition, a mixture of a pharmacologically effective amount of the adipocyte formation inhibitory factor of the present invention and a pharmaceutically acceptable excipient / activator is contained therein, and amino acids, saccharides, and cellulose derivatives are contained therein. And other excipients / activators commonly added to injectable compositions, such as organic / inorganic compounds.
When preparing an injection using the adipocyte formation inhibitor ADIF of the present invention and these excipients / activators, a pH adjuster, a buffer, a stabilizer, a solubilizing agent, and the like may be used, if necessary. Various injections can be prepared by the addition thereof in a conventional manner.

[0010]

The present invention will be described in more detail with reference to the following examples, which are merely illustrative, and do not limit the present invention.

[0011]

Example 1 Preparation of Human Fibroblast IMR-90 Culture Solution Human embryonic lung fibroblast IMR-90 (ATCC deposition-accession number CL186) was prepared using a roller bottle (490c).
80 g in m ² , 110 × 171 mm, Corning)
Was adhered to an alumina ceramic piece (alumina 99.5%, Toshiba Ceramic Co., Ltd.) and cultured. Use 40 roller bottles for culture, 5% per roller bottle
Using 500 ml of DMEM (Gibco BRL) supplemented with newborn bovine serum and 10 mM HEPES buffer, 37
The culture was allowed to stand for 7 to 10 days at 5 ° C. in the presence of 5% CO ₂ . After the culture, the culture was collected and fresh medium was added to obtain a 20 L IMR-90 culture per culture cycle. The obtained culture solution was used as Sample 1.

[0012]

Example 2 Method for Measuring Adipocyte Formation Inhibitory Activity The activity of ADIF, which is the protein of the present invention, was measured by Kodama H.
et al.'s method (Journal of Cellular Physiology, Vol.
112, p83, 1982). That is, mouse preadipocyte cell line MC3T3-G2 / PA6 cells (RIKEN GENE BANK, RCB112
Using 7) as a target cell, adipocyte formation in the presence of dexamethasone was examined by examining the accumulation of triglyceride as an index, and measuring its inhibitory activity.
That is, 50 μl of a sample diluted with α-MEM (Gibco BRL) containing 10% fetal bovine serum was placed in a 96-well microplate, and then the mouse preadipocyte cell line MC3T3-
G2 / PA6 cells were suspended in α-MEM medium containing 2 × 10 ⁻⁷ M dexamethasone and 10% fetal bovine serum, and seeded at a cell density of 3 × 10 ³ cells / 50 μl / well.
The cells were cultured for one week at 5% CO2, 37 ° C and 100% humidity. After 7 days of culture, the medium was removed by suction, air-dried, and the amount of triglyceride accumulated in the fat cells was measured using a triglyceride measurement kit (Triglyceride G-Test Wako, Wako Pure Chemical Industries, Ltd.). OD510nm decrease AD
IF activity.

[0013]

Example 3 Purification of ADIF i) Purification by Heparin Sepharose CL-6B About 60 liters of an IMR-90 culture solution (sample 1) was used.
0.22 μm filter (Hydrophilic Millidisk, 2,
000 cm ² , Millipore) and divided into three portions of 10 mM Tris-HC containing 0.3 M NaCl.
Heparin-Sepharose CL-6B column (5 x 4.1 cm, gel volume 8) equilibrated with 1 buffer, pH 7.5
0 ml). 10 mM at a flow rate of 500 ml / hr
Tris-HCl buffer, pH 7.5 (hereinafter referred to as Tri
s-HCl), elution was performed with 10 mM Tris-HCl containing 2 M NaCl, pH 7.5, and heparin-Sepharose CL-6B adsorbed fraction 1.5
One liter was obtained and designated as Sample 2.

Ii) Purified heparin-Sepharose-adsorbed fraction by HiLoad-SP / HP (sample 2) was 10 mM
After dialysis against Tris-HCl, pH 7.5,
CHAPS was added to a concentration of 0.1%, and the mixture was allowed to stand at 4 ° C. overnight. The cation exchange column (6 parts) was equilibrated with 50 mM Tris-HCl containing 0.1% CHAPS, pH 7.5, and equilibrated (6 times). 1. HiLoad-SP / HP;
(6 × 10 cm, Pharmacia). 0.1%
50 mM Tris-HCl containing CHAPS, pH
After washing with 7.5, the NaCl concentration was reduced to 0.1 for 120 minutes.
Elution was carried out with a linear gradient of 5 M and a flow rate of 6 ml / min.
Fractionation was performed at 2 ml / fraction. ADIF was performed according to the method described in Example 2 using 30 μl of each fraction.
The activity was measured, and 936 ml of an ADIF active fraction eluted with about 0.15 to 0.25 M NaCl was obtained and used as sample 3.

Iii) Purification by HiLoad-Q / HP The obtained sample 3 was treated with 50 mM containing 0.1% CHAPS.
After diluting with Tris-HCl, pH 7.5, 1900 ml, the mixture was divided into six portions and the 50 m containing 0.1% CHAPS was added.
Anion exchange column (HiLoad-Q / HP, 2.6 × 10 6) equilibrated with M Tris-HCl, pH 7.5
cm, Pharmacia). 0.1% CHAP
After washing with 50 mM Tris-HCl containing S, pH 7.5 containing S, elution was carried out at a linear gradient of NaCl concentration of 0.5 M for 120 minutes, at a flow rate of 6 ml / min, and fractionation was carried out at 12 ml / fraction. . Using 30 μl of each fraction, ADIF activity was measured according to the method described in Example 2, and ADI eluted with about 0.1 to 0.18 M NaCl.
936 ml of the F-active fraction was obtained and used as sample 4.

Iv) Purification by HiLoad-SP / HP The obtained sample 4 was subjected to 50 mM containing 0.1% CHAPS.
BisTris-HCl buffer, pH 6.0 (hereinafter, referred to as BisTris-HCl buffer)
After dilution with 1900 ml of BisTris-HCl, 50 mM containing 0.1% CHAPS was divided into three portions.
Cation exchange column (HiLoad-SP / HP, 2.6 ×) equilibrated with BisTris-HCl, pH 6.0
10 cm, Pharmacia). 0.1% CH
50 mM BisTris-HCl containing APS, pH
After washing with 6.0, the NaCl concentration was reduced to 0.1 for 100 minutes.
Elution was performed at a linear gradient from 1M to 0.6M, at a flow rate of 6 ml / min, and fractionation was performed at 12 ml / fraction. Using 30 μl of each fraction, the ADIF activity was measured according to the method described in Example 2, and about 0.3 to 0.45 M Na
360 ml of the ADIF active fraction eluted with Cl was obtained and used as sample 5.

V) Purification by Resource S The obtained sample 5 was subjected to 50 mM containing 0.1% CHAPS.
BisTris-HCl, pH 6.0, 1080 ml
Cation exchange column (Resource S, equilibrated with 50 mM BisTris-HCl, pH 6.0 containing 0.1% CHAPS, divided into three portions)
0.5 × 5 cm, Pharmacia). 0.1
50 mM BisTris-HC with% CHAPS
After washing with 1, pH 6.0, elution was performed at a linear gradient of NaCl concentration of 0.6 M in 40 minutes, at a flow rate of 1 ml / min, and fractionation was performed at 1 ml / fraction. Using 10 μl of each fraction, AD was prepared according to the method described in Example 2.
The IF activity was measured, and 30 ml of an ADIF active fraction eluted with about 0.2 to 0.3 M NaCl was obtained, which was designated as Sample 6.

Vi) Purification by Superose 12 The obtained sample 6 is transferred to a centrifugal concentrator (Centricon 10,
After concentrating to about 600 μl with Amicon, the solution was divided into three portions and containing 50% containing 0.1% CHAPS and 0.5 M NaCl.
Gel filtration column (Superose 12, 1.0 × 30c) equilibrated with mM Tris-HCl, pH 7.5
m, Pharmacia) and 50 mM Tris-HC containing 0.1% CHAPS and 0.5 M NaCl.
1, developing at a flow rate of 0.5 ml / min at pH 7.5,
The fraction was collected in ml / fraction. Each fraction 1
Using 0 μl, the ADIF activity was measured according to the method described in Example 2, and AD eluted at an elution time of about 25 to 30 minutes.
9 ml of an IF-active fraction was obtained and used as Sample 7.

Vii) Purification by Heparin 5PW The obtained sample 7 was subjected to 50 mM containing 0.1% CHAPS.
After dilution with 41 ml of BisTris-HCl, pH 6.0, 50 mM BisTr containing 0.1% CHAPS was used.
Heparin affinity column (Heparin 5PW, 0.5 × 5) equilibrated with is-HCl, pH 6.0
cm, Tosoh Corporation). After washing with 50 mM BisTris-HCl containing 0.1% CHAPS, pH 6.0, elution was performed at a linear gradient of NaCl concentration from 0.2 M to 0.8 M in 120 minutes, at a flow rate of 0.5 ml / min. Fractionation was performed at 1 ml / fraction (FIG. 1).
Using 10 μl of each fraction, ADIF activity was measured according to the method described in Example 2, and about 0.2 to 0.3 M Na
16 ml of an ADIF active fraction eluted with Cl was obtained and used as sample 8.

Viii) Purification by Polycat A The obtained sample 8 is centrifuged (Centricon 10,
After concentrating to about 300 μl with Amicon, 600 μl
50 mM BisTris containing 0.1% CHAPS
-HCl, pH 6.0, and diluted with 50 mM BisTris-HCl, pH 6.0 containing 0.1% CHAPS, weak cation exchange column (Polycat A, 0.
46 × 20 cm, Polyersie). 0.1
50 mM BisTris-HC with% CHAPS
After washing with 1, pH 6.0, elution was performed at a flow rate of 1.0 ml / min with a linear gradient of 0.4 M NaCl concentration in 50 minutes, and fractionation was performed at 1 ml / fraction (FIG. 2). . Using 10 μl of each fraction, ADIF activity was measured according to the method described in Example 2, and about 0.3 to 0.4
ADIF fraction No. eluted with MNaCl. 3
7-46 were obtained.

Ix) Purification by Reverse Phase Column Polycat A fraction No. 1 ml of 43
Was added to a reverse phase column (C4, 2.1 × 250 mm, Baydac) equilibrated with 30% acetonitrile containing 0.1% TFA after adding 10 μl of 20% TFA (trifluoroacetic acid). 5 minutes of acetonitrile in 50 minutes
Elution was performed at a linear gradient of 5% and a flow rate of 0.2 ml / min, and each protein peak was collected (FIG. 3). Using 35 μl of each peak fraction, ADIF activity was measured according to the method described in Example 2, and peak 5 concentration-dependent activity was detected. FIG. 4 shows the results.

[0022]

Example 4 Measurement of ADIF Molecular Weight SDS-polyacrylamide gel electrophoresis was performed under reducing and non-reducing conditions using 100 μl of the peak 5 in which ADIF activity was recognized. That is, 50 μl of each of the peak 5 fractions was collected into two tubes and concentrated under reduced pressure, and then 1 mM EDTA, 2.5% SDS, and 0.
10 mM Tri containing 01% bromophenol blue
s-HCl, pH 8, dissolved in 1.5 µl, and left at 37 ° C overnight under non-reducing conditions and reducing conditions (in the presence of 5% 2-mercaptoethanol).
1 was loaded on SDS-polyacrylamide gel electrophoresis. The electrophoresis was performed using a 10-15% acrylamide gradient gel (Pharmacia) and an electrophoresis apparatus Phas
The measurement was performed using tSystem (Pharmacia). As molecular weight markers, phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43
kDa), carbonic anhydrase (30 kD
a), trypsin inhibitor (20.1 kDa), α
-Lactalbumin (14.4 kDa) was used. After completion of the electrophoresis, silver staining was performed using a Phast Gel Silver Stain Kit (Pharmacia). In addition, the molecular weight of the protein of the peak 5 was calculated as follows. That is, for each molecular weight marker, the migration distance from the upper end of the separation gel was measured, and the measured migration distance was plotted against the molecular weight on a semilogarithmic graph to prepare a standard straight line.
Furthermore, the migration distance of the protein at peak 5 was measured, and the molecular weight was calculated using the prepared standard straight line. FIG. 5 shows the results. As a result, peak 5 shows 4 under non-reducing conditions.
Only the 5 kDa protein band and about 2
Only 8 kDa and about 23 kDa protein bands were detected.

[0023]

Example 5 Determination of N-Terminal Amino Acid Sequence Polycat A fraction No. obtained in Example 3-viii) 37 to 46, a centrifugal concentrator (Centricon 1)
0, Amicon) and concentrated to about 500 μl.
1% 25% TFA, 30% with 0.1% TFA
Reversed phase column (C4, 2.1) equilibrated with acetonitrile
× 250 mm, Vydac), a linear gradient of 55% acetonitrile in 50 minutes, flow rate 0.2 ml
The elution was carried out at a rate of 5 min / min. After concentrating the obtained peak 5 under reduced pressure with a centrifugal concentrator, 1 mM EDTA,
10 mM Tris-HCl containing 2.5% SDS and 0.01% bromophenol blue, pH 8, 0.5
After dissolving in μl and leaving overnight at 37 ° C. under reducing conditions (in the presence of 5% 2-mercaptoethanol), 4 μl was loaded on SDS-polyacrylamide gel electrophoresis. Electrophoresis was performed using a 10-15% acrylamide gradient gel (Pharmacia) and an electrophoresis apparatus Phast System (Pharmacia). After the electrophoresis, PVD was performed using the blotting device Phast Transfer (Pharmacia).
F membrane (ProBlot, PerkinElmer) with 20
V, proteins were transcribed at 25 mA for 30 minutes. After transfer,
0.2% Coomassie blue / 40% methanol / 10%
Staining was performed with an acetic acid solution, and excess dye was decolorized with a 60% methanol solution. The bands of about 23 kDa and about 28 kDa were cut out, and a protein sequencer (Procise, 492
(Perkin Elmer), and N-terminal amino acid sequence analysis was performed. The results are shown in SEQ ID NO: 1 in the Sequence Listing.

The polycat A fraction No. obtained from the IMR-90 culture solution of about 80 liters by the same method as described above. 10 μl of 20% for each of 37-46
TFA was added, and a reverse phase column (C4, 10%) equilibrated with 30% acetonitrile containing 0.1% TFA was divided into 10 times.
2.1 x 250 mm, Baydac), a linear gradient of 55% acetonitrile in 50 minutes, flow rate 0.2
Elution was performed at ml / min, and peak 5 was collected. After the resulting peak 5 was subjected to reductive pyridylethylation, N-terminal amino acid sequence analysis was performed using a protein sequencer (Procise, Model 492, Perkin Elmer). As a result, the amino acid sequence of the ADIF up to the 40th amino acid from the N-terminal could be determined. The results are shown in SEQ ID NO: 2 in the Sequence Listing.

[0025]

Embodiment 6Cloning of ADIF cDNA fragment i) Isolation of poly (A) ⁺ RNA from IMR-90 cells Fast Track mRNA Isolation Kit (Inv
1) according to the manual.
× 10⁸About 10 μg of poly (A) from one IMR-90 cell ⁺RNA
Isolated.

Ii) Preparation of Mixed Primers Based on the N-terminal amino acid sequence shown in SEQ ID NO: 2 in Sequence Listing obtained in Example 5, the following two types of mixed primers were synthesized. That is, 19 from the 13th (Thr) from the N-terminal
A mixture of oligonucleotides (mix primers, TAE / F) having all nucleotide sequences capable of encoding the amino acid sequence up to the first (Cys) was synthesized. 29th from N-terminal
A mixture (mix primer, CFE / R) of complementary oligonucleotides to all nucleotide sequences capable of encoding the amino acid sequence from (Gly) to 35th (Glu) was synthesized.
Table 1 shows the base sequences of the used mix primers.

[0027]

[Table 1]

Iii) Amplification of ADIF cDNA fragment by PCR Using 1 μg of poly (A) ⁺ RNA obtained in Example 6-i) as a template and a Superscript II cDNA synthesis kit (Gibco BRL), the protocol of the company was used. A single-stranded cDNA is synthesized according to the procedure described above, PCR is performed using this cDNA and the primers described in Example 6-ii), and ADIFc
A DNA fragment was obtained. The composition of the reaction solution is shown below. 10X Ex Taq buffer (Takara Shuzo) 10 μl 2.5 mM dNTP 8 μl cDNA solution 2.5 μl Ex Taq (Takara Shuzo) 1 μl Distilled water 73.5 μl 20 μM Primer TAE / F 2.5 μl 20 μM Primer CFE / R 2.5 μl Trace amount of the above solution After mixing in a centrifuge tube, PCR was performed under the following conditions. After pretreatment at 95 ° C for 3 minutes, 95 ° C for 30 seconds, 50
After repeating the three-stage reaction of 30 ° C. for 30 seconds and 72 ° C. for 1 second 40 times, the mixture was kept at 72 ° C. for 5 minutes. A part of the reaction solution was analyzed by agarose gel electrophoresis, and the
It was confirmed that a cDNA fragment of 68 bp was obtained.

[0029]

Example 7 Cloning of ADIF cDNA Fragment Amplified by PCR
The cDNA fragment obtained in Example 6-iii) was subjected to DNA ligation kit Ver. PT using 2 (Takara Shuzo)
7 Insert into Tvector (Novagen) and use E. coli XL2Bl
ue (Stratagene) was transformed. The obtained transformant is grown and about 68 bp of ADIFcDN.
The plasmid into which the fragment A was inserted was purified according to a conventional method.
This plasmid was named pBSADIF, and the nucleotide sequence of ADIF cDNA inserted into this plasmid was determined using a DNA sequencing kit (Perkin Elmer). The amino acid sequence consisting of 23 amino acids predicted from this nucleotide sequence could be found in the N-terminal amino acid sequence of ADIF used to design the mix primer (SEQ ID NO: 2 in the Sequence Listing). based on the above results,
The cloned 68 bp cDNA is ADIFcD
It was confirmed to be an NA fragment.

[0030]

Example 8 Preparation of DNA Probe Using the plasmid inserted with the 68 bp ADIF cDNA fragment prepared in Example 7 as a template and performing PCR under the conditions of Example 6-iii), this ADIF cDNA
The fragment was amplified. About 68 bp by agarose electrophoresis
After isolation of the ADIF cDNA fragment of QIAEX II DN
Purification was performed using an A isolation kit (Qiagen). This cDNA was labeled with [α ³² P] dCTP using Megaprime DNA Labeling Kit (Amersham) and used as a probe for screening for full-length ADIF cDNA.

[0031]

Example 9 Preparation of cDNA Library Using poly (A) ⁺ RNA obtained in Example 6-i) and 2.5 μg of the template as a template, using a Great-Strength cDNA Synthesis Kit (Clontech) according to the company protocol, oligo
cDNA synthesis using (dT) primer, EcoRI-SalI-Not-
After addition of I adapter and cDNA size fractionation, ethanol precipitation was performed, followed by dissolution in 10 μl of TE buffer. 0.1 μg of the obtained adapter-added cDNA
Pre-cut with EcoRI using T4 DNA ligase1
μg of λZAP express vector (Stratagene) was inserted. The cDNA recombinant phage DNA solution thus obtained was subjected to an in vitro packaging reaction using Gigapack Gold II (Stratagene) to prepare a λZAP express recombinant phage.

[0032]

Example 10 Screening of Recombinant Phage The recombinant phage obtained in Example 9 was infected with Escherichia coli XL1-Blue MRF '(Stratagene) at 37 ° C. for 15 minutes, and then heated at 50 ° C. to a concentration of 0.7%. It was added to NZY medium containing agar and poured onto NZY agar plate. After overnight culture at 37 ° C., Hybond N (Amersham) was adhered to the plate on which the plaque was formed for about 30 seconds. The filter was alkali-denatured according to a conventional method, neutralized, immersed in 2XSSC solution, and immobilized on the filter by UV crosslink (Stratagene). The obtained filter was immersed in Rapid Hybridization Buffer (Amersham) containing 100 μg / ml salmon sperm DNA,
And heat-denatured DNA probe (2
X10 ⁵ cpm / ml) and transfer to the above buffer at 65 ° C
For one night. After the reaction, the filter was washed twice with 2 × SSC and twice with 0.1 × SSC and 0.1% SDS solution at 65 ° C. for 10 minutes. Some of the obtained positive clones were purified by one more screening. Those having an insert of about 2.0 kb were used below. The purified phage is infected with Escherichia coli XL1-Blue MRF 'according to the protocol of λZAP Express Cloning Kit (Stratagene) and helper phage ExAssist (Stratagene)
After infecting the culture supernatant with E. coli XLOLR (Stratagene) and picking up a kanamycin-resistant strain, pBKCMV (Stratagene) as described in 2.
A transformant having the plasmid pBKADIF into which the 0 kb insert was inserted was obtained. This transformant is XLOLR / pBKADIF
As the accession number FERMBP-64 on August 11, 1998 by the Ministry of International Trade and Industry
Deposited as 59. A transformant having this plasmid was grown, and the plasmid was purified by a conventional method.

[0033]

Example 11 cDNA encoding the entire amino acid sequence of ADIF
Determination of the base sequence of ADIF cDNA obtained in Example 10
The determination was performed using an NA sequencing kit (PerkinElmer). The primers used were T3 and T7 primers (Stratagene) and synthetic primers designed based on the nucleotide sequence of ADIF cDNA, and the sequences are shown in SEQ ID NOs: 4 to 11 in the Sequence Listing. The nucleotide sequence of ADIF thus determined is shown in SEQ ID NO: 12 in the Sequence Listing, and the amino acid sequence deduced from the sequence is shown in SEQ ID NO: 13 in the Sequence Listing.
Are shown below.

[0034]

Example 12 Recombinant type using 293 / EBNA cells
Production of ADIF i) Preparation of ADIF cDNA Expression Plasmid The plasmid pBKADIF into which the approximately 2.0 kb ADIF cDNA obtained in Example 9 was inserted was digested with the restriction enzyme SalI.
DIF cDNA was excised, separated by agarose electrophoresis, and purified using QIAEXII DNA isolation kit (Qiagen). This ADIFcDN
A was digested with the restriction enzyme XhoI in advance, and ligated with Ver. Ligation kit Ver. 2
(Takara Shuzo) to transform Escherichia coli DH5α (Gibco BRL). The resulting transformant was grown and the expression plasmid pCEPADIF into which ADIF cDNA was inserted was purified using a Qiagen column (Qiagen). The ADIF expression plasmid pCEPADIF was precipitated with ethanol, dissolved in sterile distilled water, and used for the following procedure.

Ii) Expression of ADIF The ADIF expression plasmid pCEPAD obtained in Example 12-i)
Using the IF, the recombinant ADI
F was expressed. 1x10 ⁶ 293-EBNA cells (Invitroge
Was seeded in a 75 cm ² T-flask in IMDM (Life Technologies) containing 10% fetal bovine serum, and cultured at 37 ° C. overnight in a CO ₂ incubator (5% CO ₂ ). pCEP
ADIF was performed using FuGENE6 (Boehringer Mannheim) 29
3-EBNA cells were transfected. 7 μg of DNA,
20 ml of FuGENE6 was used. Transfected 293-
EBNA cells were incubated in a CO ₂ incubator (5% C
After culturing at O ₂₎ in, and replaced with serum-free medium, after two more days of culture, the culture solution recovery and added, after three more days incubation, culture medium was harvested from five 75 cm ² T-flasks ADI
200 ml of a culture solution containing F was obtained.

Iii) Purification of recombinant ADIF 200 ml of culture solution containing ADIF obtained in Example 12-ii)
Was adjusted to pH 6 with acetic acid, and then loaded onto a cation column (HiTrapSP, 1 ml, Pharmacia) equilibrated with 50 mM BisTris-HCl, pH 6.0 containing 0.1% CHAPS. After washing with 50 mM BisTris-HCl containing 0.1% CHAPS, pH 6.0, elution was performed at a linear gradient of 1 M in NaCl concentration for 60 minutes, at a flow rate of 1 ml / min, and fractionation was performed at 1 ml / fraction. went. Using 10 μl of each fraction,
ADIF activity was measured according to the method described in Example 2, and 7 ml of an ADIF active fraction eluted with about 0.3 to 0.4 M NaCl was obtained. The resulting ADIF active fraction (7 ml) was concentrated to about 200 μl using a centrifugal concentrator (Centricon 10, Amicon), and 0.1% CHAPS and O.C. 5M
50 mM Tris-HCl containing NaCl, pH 7.5
Gel filtration column (Superdex200) equilibrated with
HR, 1 × 30 cm, Pharmacia).
1% CHAPS and O.D. 50 mM with 5 M NaCl
The solution was developed with Tris-HCl, pH 7.5 at a flow rate of 0.5 ml / min, and fractionated at 0.5 ml / fraction. Using 5 μl of each fraction, ADIF activity was measured according to the method described in Example 2, and 2 ml of an ADIF active fraction eluted at an elution time of about 24 to 27 minutes was obtained.

[0037]

Example 13 Osteoblast-based cell proliferation activity of ADIF The osteoblast-based cell proliferation activity was measured by the following method. That is, MC3T3-E1 cells (RIKEN CELL BANK RCB1126), which are mouse osteoblast-like cells, and HOS cells (ATCC CRL-1543), which are human osteosarcoma-derived cells, were each treated with α-MEM (GIBCO) containing 10% fetal bovine serum. 2 × 10 ³ pcs in BRL)
The cells were seeded at / 100 μl / well in a 96-well plate and cultured at 37 ° C. overnight. After washing each well with serum-free α-MEM, 100 μl of serum-free α-MEM was added to the well.
Was added and the cells were further cultured for 2 days. Two days later, the medium in each well was replaced with α-MEM containing 0.2% bovine serum albumin, and the recombinant ADIF obtained in Example 12 diluted with α-MEM containing 0.2% bovine serum albumin was added thereto. The culture was continued for another 22 hours. After 22 hours, ³ H-thymidine (manufactured by Amersham) was added at 1 μCi / well, and the cells were further cultured for 2 hours. Two hours later, the cells were washed with PBS, peeled off with 0.5% trypsin, and collected on a glass filter using a cell harvester (Packard). The radioactivity incorporated into the cells in each well was measured using Matrix 96 (Packard), and the value was defined as the amount of synthesized DNA. The results are shown in FIGS.
As a result, by adding ADIF, the uptake of ³ H-thymidine into each cell increases in a concentration-dependent manner,
It was confirmed that the synthesis increased. Therefore, ADI
By using F, osteoblasts can be proliferated.

[0038]

Industrial Applicability According to the present invention, there is provided an osteogenesis promoter containing an adipocyte formation inhibitory factor (ADIF) as an active ingredient. According to the present invention, a preparation having excellent osteoblast proliferation promoting activity is provided, and is useful as an agent for preventing and / or treating a bone loss disease such as osteoporosis.

[0039]

[Sequence List] SEQUENCE LISTING <110> Snow Brand Milk Products Co., Ltd. <120> New protein and its production method <130> YTP98025 <160> 13 <210> 1 <211> 20 <212> PRT <213> Homo Sapiens <400> 1 Xaa Xaa Gln Gln Xaa Gly Arg Leu Xaa Leu Gln Asn Thr Ala Glu Ile 1 5 10 15 Gln His Xaa Leu 20 <210> 2 <211> 40 <212> PRT <213> Homo Sapiens <400> 2 Xaa Ser Gln Gln Lys Gly Arg Leu Ser Leu Gln Asn Thr Ala Glu Ile 1 5 10 15 Gln His Cys Leu Val Asn Ala Gly Asp Val Gly Cys Gly Val Phe Glu 20 25 30 Cys Phe Glu Asn Asn Xaa Xaa Glu 35 40 <210> 3 <211> 40 <212> PRT <213> Homo Sapiens <400> 3 Ser Ser Gln Gln Lys Gly Arg Leu Ser Leu Gln Asn Thr Ala Glu Ile 1 5 10 15 Gln His Cys Leu Val Asn Ala Gly Asp Val Gly Cys Gly Val Phe Glu 20 25 30 Cys Phe Glu Asn Asn Ser Cys Glu 35 40 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthesized DNA <400> 4 AATTAACCCT CACTAAAGGG 20 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <22 0> <223> Description of Artificial Sequence: Synthesized DNA <400> 5 GTAATACGAC TCACTATAGG GC <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthesized DNA < 400> 6 AAGAGGGGAG CACAAAGGAT <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthesized DNA <400> 7 GTCCCTGCAG AATACAGCGG <210> 8 <211> 20 < 212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthesized DNA <400> 8 CAAGGACTTG CTGCTGCACG <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthesized DNA <400> 9 TGGACGGCGT GGAGGAAAGA <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthesized DNA <400> 10 GCTCAAGATG GAGCACAGGC < 210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthesized DNA <400> 11 GTTGTGCAGA AAAGTCATGC <210> 12 <211> 882 < 212> DNA <213> Homo Sapiens <400> 12 ATGACCCTGG CTTTGGTGTT GGCCACCTTT GACCCGGCGC GGGGGACCGA CGCCACCAAC 60 CCACCCGAGG GTCCCCAAGA CAGGAGCTCC CAGCAGAAAG GCCGCCTGTC CCTGCAGAAT 120 ACAGCGGAGA TCCAGCACTG TTTGGTCAAC GCTGGCGATG TGGGGTGTGG CGTGTTTGAA 180 TGTTTCGAGA ACAACTCTTG TGAGATTCGG GGCTTACATG GGATTTGCAT GACTTTTCTG 240 CACAACGCTG GAAAATTTGA TGCCCAGGGC AAGTCATTCA TCAAAGACGC CTTGAAATGT 300 AAGGCCCACG CTCTGCGGCA CAGGTTCGGC TGCATAAGCC GGAAGTGCCC GGCCATCAGG 360 GAAATGGTGT CCCAGTTGCA GCGGGAATGC TACCTCAAGC ACGACCTGTG CGCGGCTGCC 420 CAGGAGAACA CCCGGGTGAT AGTGGAGATG ATCCATTTCA AGGACTTGCT GCTGCACGAA 480 CCCTACGTGG ACCTCGTGAA CTTGCTGCTG ACCTGTGGGG AGGAGGTGAA GGAGGCCATC 540 ACCCACAGCG TGCAGGTTCA GTGTGAGCAG AACTGGGGAA GCCTGTGCTC CATCTTGAGC 600 TTCTGCACCT CGGCCATCCA GAAGCCTCCC ACGGCGCCCC CCGAGCGCCA GCCCCAGGTG 660 GACAGAACCA AGCTCTCCAG GGCCCACCAC GGGGAAGCAG GACATCACCT CCCAGAGCCC 720 AGCAGTAGGG AGACTGGCCG AGGTGCCAAG GGTGAGCGAG GTAGCAAGAG CCACCCAAAC 780 GCCCATGCCC GAGGCAGAGT CGGGGGCCTT GGGGCTCAGG GACCTTCCGG AAGCAGCGAG 840 TGGGAAGACG AACAGTCTGA GTATTCTGAT ATCCGGAGGT GA 882 <210> 13 <211> 293 <212> PRT <213> Homo Sapiens <400> 13 Met Thr Leu Ala Leu Val Leu Ala Thr Phe Asp Pro Ala Arg Gly Thr -28 -25- 20 -15 Asp Ala Thr Asn Pro Pro Glu Gly Pro Gln Asp Arg Ser Ser Gln Gln -10 -5 -1 1 Lys Gly Arg Leu Ser Leu Gln Asn Thr Ala Glu Ile Gln His Cys Leu 5 10 15 20 Val Asn Ala Gly Asp Val Gly Cys Gly Val Phe Glu Cys Phe Glu Asn 25 30 35 Asn Ser Cys Glu Ile Arg Gly Leu His Gly Ile Cys Met Thr Phe Leu 40 45 50 His Asn Ala Gly Lys Phe Asp Ala Gln Gly Lys Ser Phe Ile Lys Asp 55 60 65 Ala Leu Lys Cys Lys Ala His Ala Leu Arg His Arg Phe Gly Cys Ile 70 75 80 Ser Arg Lys Cys Pro Ala Ile Arg Glu Met Val Ser Gln Leu Gln Arg 85 90 95 100 Glu Cys Tyr Leu Lys His Asp Leu Cys Ala Ala Ala Gln Glu Asn Thr 105 110 115 Arg Val Ile Val Glu Met Ile His Phe Lys Asp Leu Leu Leu His Glu 120 125 130 Pro Tyr Val Asp Leu Val Asn Leu Leu Leu Thr Cys Gly Glu Glu Val 135 140 145 Lys Glu Ala Ile Thr His Ser Val Gln Val Gln Cys Glu Gln Asn Trp 150 155 160 Gly Ser Leu Cys Ser Ile Leu Ser Phe Cys Thr Ser Ala Ile Gln Lys 165 170 175 180 Pro Pro Thr Ala Pro Pro Glu Arg Gln Pro Gln Val Asp Arg Thr Lys 185 190 195 Leu Ser Arg Ala His His Gly Glu Ala Gly His His Leu Pro Glu Pro 200 205 210 Ser Ser Arg Glu Thr Gly Arg Gly Ala Lys Gly Glu Arg Gly Ser Lys 215 220 225 Ser His Pro Asn Ala His Ala Arg Gly Arg Val Gly Gly Leu Gly Ala 230 235 240 Gln Gly Pro Ser Gly Ser Ser Glu Trp Glu Asp Glu Gln Ser Glu Tyr 245 250 255 260 Ser Asp Ile Arg Arg 265

[Brief description of the drawings]

FIG. 1 shows Superose in Example 3-vii).
The 12-fraction roughly purified product (Sample 7) was
4 shows an elution profile when loaded on a W column.

FIG. 2 Heparin in Example 3-viii)
5 shows an elution profile when a 5PW roughly purified product (Sample 8) was loaded on a Polycat A column.

FIG. 3 shows the polycat A elution fraction No. in Example 3-ix). 43 shows an elution profile obtained by fractionation of No. 43 by a reversed-phase column.

FIG. 4 ADI of peak 5 eluted from a reversed-phase column
Shows F activity.

FIG. 5 shows the results of SDS-PAGE of a final purified sample under reducing and non-reducing conditions.

[Explanation of symbols]

Lane 1: Molecular weight marker (SDS-PAGE: under non-reducing conditions) Lane 2: ADIF (SDS-PAGE: under non-reducing conditions) Lane 3: Molecular weight marker (SDS-PAGE: under reducing conditions) Lane 4: ADIF (SDS- PAGE: under reducing conditions)

FIG. 6 shows the amount of DNA synthesized by MC3T3-E1 cells when ADIF was added in Example 13.

FIG. 7 shows the amount of DNA synthesized by HOS cells when ADIF was added in Example 13.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) (C12P 21/02 C12R 1:91) (72) Inventor Masataku Tomohoshi Omatsuyama, Ishibashi-cho, Shimotsuga-gun, Tochigi 1-3-3 SK Mansion 3-EF Term (Reference) 4B024 AA01 BA80 CA01 CA04 CA09 CA11 DA03 DA06 EA03 EA04 GA11 HA08 HA09 HA19 4B064 AG01 BA18 CA02 CA10 CA19 CC24 CE04 CE06 CE07 CE11 CE12 CE14 CE17 DA01 4C084 AA17 BA02 BA05 CA25 CA56 DB54 NA14 ZA971 ZB211 4H045 AA10 AA20 AA30 BA10 CA40 DA00 EA28 FA72

Claims (1)

[Claims] An osteogenesis promoter comprising an adipocyte formation inhibitory factor (ADIF) as an active ingredient.