KR100490817B1 - MP-52 Derived Protein Consisting of 119 Amino Acids and Process for Producting the Same - Google Patents

Abstract

The protein consisting of the amino acid sequence of sequence number 1 of human M52, and its dimer. Constructing a plasmid containing DNA added with a codon encoding methionine at the 5 prime end of the DNA sequence encoding the amino acid sequence, solubilizing the inclusion body obtained by transforming E. coli and culturing the E. coli with the plasmid The dimeric protein is obtained by regenerating the protein of the monomer obtained by purification by dimer and purifying it.

This dimer protein is useful for the treatment of cartilage and bone diseases.

Description

MP-52 Derived Protein Consisting of 119 Amino Acids and Process for Producting the Same

The present invention relates to a protein having the amino acid sequence of SEQ ID NO: 1 from M52. The present invention also relates to a therapeutic agent for cartilage and bone diseases comprising the dimer of the protein and the dimer protein. The present invention also relates to a method for producing the protein in bulk and high purity using E. coli transformed with a plasmid containing a DNA sequence capable of expressing the protein. The present invention also relates to a method for treating cartilage and bone disease, which is achieved by administering the dimer protein.

Currently, estrogen, calcitonin, vitamin D3 and its derivatives, and bisphosphinic acid derivatives are known as preventive or therapeutic agents for bone diseases. In recent years, it has been reported that BMP-2, a bone morphogenetic protein (hereinafter referred to as BMP) belonging to the TGF-β gene superfamily, has a bone induction effect on a series of proteins such as BMP-9.

It is also reported that there is a bone inducing action on proteins called MP52 (WO 93/16099 and WO 95/04819). Mature MP52 is thought to be a protein consisting of 120 residues having alanine at the N terminus, and its amino acid sequence is described in these patents.

For a protein derived from a mouse called GDF-5 having an amino acid sequence very similar to that of MP52, Nature, vol. 368, p. 639-643 (1994) and WO 94/15949.

However, it is not easy to manufacture these proteins on an industrial scale and in pure form.

In the genetic production of MP52 it has been attempted to use animal cells such as L-cells. However, it was not easy to manufacture pure MP52 efficiently.

<Start of invention>

The inventors have attempted to produce MP52 in larger quantities using E. coli by genetic engineering techniques. That is, an attempt was made to produce MP52 by Escherichia coli by adding a codon encoding methionine to the 5 prime end of the DNA encoding MP52 starting from alanine. The result is that not only MP52 but also 121 residues of proteins having methionine at the N-terminus and alanine at the N-terminus are eliminated and 119 residues, starting from proline, are very difficult to purely separate MP52 from this mixture. It was.

The present inventors construct a plasmid in which a codon encoding methionine is bound to the 5 prime end of the DNA sequence encoding the amino acid sequence of SEQ ID NO: 1 consisting of 119 residues in which the N-terminal alanine of MP52 is deleted, and the plasmid When introduced using E. coli, it was found that the N-terminus selectively produced the protein described in SEQ ID NO: 1 starting from proline very efficiently.

In addition, it was confirmed that the dimer of the protein described in SEQ ID NO: 1 has cartilage and bone induction activity, and completed the present invention.

The present invention relates to a protein having an amino acid sequence represented by SEQ ID NO: 1. This protein is a protein consisting of 119 residues of amino acids deleted from the N-terminal alanine in human MP52 which is considered to be a mature portion consisting of 120 residues. The protein obtained by the present invention is soluble in aqueous solution. In addition, since the protein of the present invention is characterized by human origin, its own toxicity is low.

The present invention also relates to a therapeutic agent for cartilage and / or bone diseases consisting of a protein dimer having an amino acid sequence represented by SEQ ID NO: 1. More specifically, since the protein dimer of the present invention has cartilage and bone-inducing activity, osteoarthritis such as osteoporosis, congenital cartilage, bone disease, deformed knee arthropathy and deformed femoral arthrosis or cartilage damage such as osteoarthritis and meniscus injury, trauma. Regeneration of bone and cartilage defects due to swelling, prevention of congenital cartilage and bone diseases such as bone and cartilage defects, fractures, cartilage insufficiency, cartilage insufficiency, cartilage aplasia, cleft palate, and bone insufficiency, as well as root and alveolar defects And to therapeutic agents. Moreover, since the protein of this invention has cartilage and bone induction activity, it can be used for the bone transplantation treatment of cosmetic surgery. Such treatments include those in the veterinary surgical area.

The present invention relates to a method for producing a protein consisting of amino acids of 119 residues derived from human MP52 represented by SEQ ID NO: 1 using E. coli.

The present invention also relates to a mechanism for a plasmid containing DNA encoding methionine at the 5 prime end of a DNA sequence encoding an amino acid sequence of 119 residues derived from human MP52 represented by SEQ ID NO: 1. Human MP52cDNA was amplified using the plasmid vector containing the cDNA described in WO 93/16099 as template DNA, and only the mature part using polymerase chain reaction (PCR method). As used herein, the PCR method generally means amplifying a trace fragment of nucleic acid DNA or RNA by a method described in US Pat. No. 4,683,195.

In order to produce the protein of the present invention, it is necessary to construct an appropriate expression vector including the DNA encoding the protein and introduce it into a preferred E. coli host by the technique of genetic engineering. In order to produce a large amount of the protein of the present invention, the following two improvement methods were carried out. 1) How to increase the productivity of the target protein: A method of increasing the translation efficiency reported by M. Nobuhara et al. (Agric. Biol. Chem., 52 (6), 1331-1338. 1988), ie the AT content around the start codon ATG 2) a method of raising the number of copies of the plasmid, that is, a method of replacing the origin of replication from the pBR system to the pUC system. The expression vector (pKOT245) of the present invention was constructed by directly connecting the DNA region encoding the amino acid sequence of the promoter region and SEQ ID NO: 1. This vector is usually assigned to the National Institute of Life Sciences and Human Technology (NIBH) (1-3 Higashi, Ibaraki 껭 Tsukuba-kun, Japan Institute of Technology and Technology) on April 14, 1995 with the accession number FERM P-14895. And as a deposit based on the Butafest Treaty on 10 April 1996 (FERM BP-5499).

The present invention constructs a plasmid containing DNA added with a codon encoding methionine to the 5 prime ends of the DNA sequence encoding the amino acid sequence represented by SEQ ID NO: 1, and transforms E. coli with the plasmid. The present invention relates to a protein of a monomer obtained by solubilizing and purifying an inclusion body obtained by culturing E. coli and a protein dimer of the protein dimer of SEQ ID NO: 1 obtained by refolding and purifying it. That is, the protein of the present invention, after solubilizing the E. coli inclusion body, a single sulfonated MP52 monomer was obtained by SP-Sepharose FF column and Sephacryl S-200 column. Thus, after refolding, isoelectric point precipitation was performed and the purified dimer fraction of the protein was obtained by passing through a RESOURCE RPC column of reverse phase HPLC. Physicochemical properties of the obtained protein were analyzed by analysis by N-terminal amino acid sequence, amino acid composition and electrophoresis.

The present invention also relates to a production method of culturing Escherichia coli incorporating the expression vector of the present invention under conditions of a culture temperature of 28 ° C. to 34 ° C., pH 6 to 8, and dissolved oxygen concentration of 20 to 50%.

The present invention also relates to a method for treating cartilage and bone disease, comprising administering to a human a therapeutic agent comprising an effective amount of a protein dimer as an active ingredient.

The biological activity of the protein dimer of the present invention was evaluated by soft X-ray imaging analysis, histological analysis and time-lapse analysis of ectopic cartilage and bone formation (topic bone formation). In addition, it was confirmed that the protein of the present invention is effective for cartilage and bone reconstruction by the effect on the endovascular ossification, the effect on the regeneration of articular cartilage and the treatment effect on fracture and bone defect.

The protein dimer of the present invention can be administered systemically by intravenous, intramuscular and intraperitoneal administration. In the case of intravenous administration, intravenous injection is possible in addition to the usual intravenous injection.

As an injectable preparation, it can be made into an injectable powder preparation, for example. In this case, one or two or more suitable water-soluble excipients such as mannitol, sucrose, lactose, maltose, glucose and fructose may be added, dissolved in water, dispensed into vials or ampoules, lyophilized and sealed. It can be made into a formulation.

As a topical administration method, the surface of cartilage, bone or tooth of the site is covered with the present protein using collagen paste, fibrin paste or other adhesive. Among these, the bone used for bone transplantation can use the artificial bone which was used conventionally other than natural bone. Artificial bones refer to bones made of natural or artificial inorganic materials such as metals, ceramics and glass. As the artificial inorganic material, hydroxyapatite is preferable. For example, hydroxyapatite is used for the inner material of the artificial bone and for the outer material of the artificial bone. In addition, the protein can be administered to cancerous bone tissue to promote bone reconstruction, and can also be used for cartilage transplantation.

For dosage, there are several factors that influence the action of the protein, such as the weight of bone and cartilage where formation is desired, the site and condition of bone and cartilage damage, the age, sex, severity of infection, and time of administration of the patient. And other clinical factors are considered by the attending physician. The dosage may vary depending on the type of carrier used for reconstitution with the present protein. Generally, the dosage is about 10 to 10 ⁶ nanograms of the present protein per target bone and cartilage wet weight when used as a composition with a support, and 0.1 to 10 ⁴ micrograms per patient when applied topically and systemically as an injection. It is advisable to administer the drug at a frequency of once a week to once a day.

A synergistic effect can be expected by simultaneously applying known growth factors, such as insulin-like growth factor (IGF-I), to bone and cartilage regeneration.

As described above, a method for producing the protein of the present invention in an industrial scale and in a pure form has not been reported so far, and is effective as a therapeutic agent for cartilage and bone disease having cartilage and bone inducing activity. In addition, the production method of the present invention can also be applied to the production of the above-described bone induction factor belonging to the TGF-β gene superfamily that has been produced only in animal cells.

1 is a plasmid map of the protein expression vector (pKOT245) of the present invention obtained in Example 1 (2).

2 is a soft X-ray photograph of bone and cartilage calcified tissue induced into the mouse femoral muscle obtained in Example 4 (1).

3 is a tissue staining micrograph of non-lime sections of bone and cartilage calcified tissue induced into the mouse femoral muscle obtained in Example 4 (1).

4 is a tissue staining micrograph showing the changes over time of cartilage and bone induction observed in the mouse femoral muscle obtained in Example 4 (2).

5 is a tissue staining micrograph of the demineralized slice of the rat parietal bone obtained in Example 4 (3).

Fig. 6 is a tissue staining micrograph of the demineralized section of the femoral head including articular cartilage of the rabbit obtained in Example 4 (4).

7 is a femoral soft X-ray photograph of a rat in which a bone defect is created in the femur obtained in Example 4 (5).

<Best Mode for Carrying Out the Invention>

Next, an Example is shown and the effect of this invention is demonstrated concretely. In addition, this invention is not limited by these Examples.

Example 1 Preparation of Vector

(1) Isolation of the variant MP52 mature part

Human MP52cDNA was amplified using the polymerase chain reaction (PCR) using only the plasmid vector (pSK52s) containing the cDNA described in WO 93/16099 as template DNA and the mature part.

Mature MP52 gene according to the method for improving the productivity of the target protein by raising the AT content around the start codon ATG (M, Nobuhara et al. (Agric. Biol. Chem., 52 (6), 1331-1338, 1988). Some DNA was substituted.

Substitution was performed by PCR using the forward PCR primer of SEQ ID NO: 2. The DNA sequence of the PCR primers was the DNA of SEQ ID NO: 2 as the forward primer and the DNA of SEQ ID NO: 3 was used as the reverse primer.

PCR was performed by adding template DNA (10 nanograms), 50 picomol, dNTP (0.2 mmol) and MgCl ₃ (1.5 mmol), respectively, with Taq DNA polymerase (5U) in the same test tube. .

Each cycle was subjected to 30 cycles of PCR consisting of denaturation (94 ° C., 1 minute), primer annealing (55 ° C., 1 minute), and primer extension (72 ° C., 2 minutes) (all of the following PCR were performed under these conditions). ).

The product from the PCR reaction was separated by electrophoresis in 1.5% low melting agarose (FMC) to obtain a fragment consisting of about 360 bp corresponding to the amino acid sequence of SEQ ID NO: 1 (this is referred to as fragment 1).

(2) Construction of E. coli expression vector of this protein

In order to increase the number of copies of the plasmid, the replication origin was replaced by pUC system from pBR system. After cleaving the tac promoter region of the commercially available E. coli expression vector pKK223-3 (purchased from Pharmacia Biotech Co., Ltd.) with restriction enzymes SspI and EcoRI, Mung Bean Nuclease (Sunga Kabushiki Kaisha, Catalog) No. 2420A), and the rrnBT ₁ T ₂ termination sequence region of pKK223-3 was bound to restriction enzymes SalI and SspI by binding to T4DNA ligase (Takara Shuzo Co., Ltd., catalog number 2011A) on the start codon side of fragment 1 Expression vector for production of this protein by binding to the codon side of fragment 1 cleaved and cleaved with SalI and introduced into the SmaI site of pUC18 [pKOT245 (Accession No. POKOTEN-KI No. P-14895)] 1) was constructed The length of the DNA of pKOT245 was 3.7 kb The nucleotide sequence of the protein expression vector of the present invention was determined by Pharmacia ALF DNA sequencer.

(3) quality conversion

The transformation was carried out according to the Rubidium chloride method (Genetic Engineering. P. 17, Elsevier (1978)) by Kushner et al. That is, pKOT245 was transferred to the host E. coli W3110M according to the above-described method to make the protein production E. coli of the present invention.

Example 2 Culture

(1) culture

The protein-expressing Escherichia coli of the present invention was modified in SOC medium (Bacto tryptone 20 g / l, Bacto yeast extract 5 g / l, NaCl 0.5 g / l, MgCl ₂ · 6H ₂ O 2.03 g / l, glucose 3.6 g / l), pre-cultured with medium for production (bacto tryptone 5 g / l, citric acid 4.3 g / l, K ₂ HPO ₄ 4.675 g / l, KH ₂ PO ₄ 1.275 g / l) , NaCl 0.865 g / ℓ, FeSO4 · 7H2O 100 mg / ℓ, CuSO 4 · 5H 2 O 1 mg / ℓ, MnSO 4 · nH 2 O 0.5 mg / ℓ, CuCl 2 · 2H 2 O 2 mg / ℓ, Na 2 _{B4O 7 · 10H 2 O 0.225 mg} / ℓ, (NH 4) 5 Mo 7 O 24 · 4H 2 O 0.1 mg / ℓ, ZnSO 4 · 7H 2 O 2.25 mg / ℓ, CoCl 2 · 6H 2 O 6 mg / ℓ 100 ml of a cell suspension was added to 5 L of MgSO ₄ · 7H ₂ O, 5.0 mg / L thiamine HCl, 3 g / L glucose, and cultured with aeration and agitation in a 10 L culture tank. Add isopropyl-β-D-thiogalactopyranoside at a concentration of 1 mM at the end of the propagation period (OD ₅₅₀ = 5.0) and again reach an OD ₅₅₀ of 150. Incubate until. During the culture, the temperature was controlled to 32 ° C., the pH to 7.15 by adding ammonia, and the dissolved oxygen concentration was controlled to 50% of air saturation by increasing the stirring rate to prevent a decrease in dissolved oxygen concentration. In addition, in order to make a high cell concentration, a 50% glucose solution was added at a concentration of 0.2% with a rapid increase in dissolved oxygen concentration as an index.

(2) Preparation of Escherichia Coli Inclusion Body

The cells obtained by the above method were centrifuged to recover the cells, suspended in 25 mM Tris-HCl buffer (pH 7.3) containing 10 mM ethylenediamine tetraacetic acid, and the cell disruption apparatus (manufactured by Gaulin Inc.). The bacteria were disrupted and centrifuged again to recover the precipitate containing the inclusion body.

Example 3 Tablet

(1) Solubilization of Escherichia Coli Inclusion Body

The E. coli inclusion body was washed three times with 1% Triton X-100, then centrifuged at 3000 x g for 30 minutes at 4 ° C, and the resulting precipitate was washed with 20 mM Tris-HCl buffer, 8M urea, 10 mM at pH 3. Solubilization with sonication with DTT, 1 mM EDTA.

(2) monomer purification

The solubilization liquid was centrifuged at 20000xg for 30 minutes at 4 degreeC, and the supernatant was collect | recovered. The resulting supernatant was passed through SP-Sepharose FF (Pharmacia) equilibrated with 20 mM Tris-HCl buffer, 6M urea, 1 mM EDTA, pH 8.3, washed with the same solution and eluted with the same solution containing 0.5M saline. . Na ₂ SO ₃ and Na ₂ S ₄ O ₆ were added to the eluate so that the final concentrations were 111 mM and 13 mM, respectively, and sulfonation was performed at 4 ° C. for 15 hours. The sulfonated solution was a single, sulfonated protein monomer of the present invention by gel filtration with Sephacryl S-200 HR (Pharmacia) equilibrated with 20 mM Tris-HCl buffer, pH 8.3, 6M urea, 0.2 M salt, 1 mM EDTA. Got.

(3) refolding

50 mM Na-glycine buffer, 0.2 M sodium chloride, 16 mM CHAPS, 5 mM EDTA, 2 mM GSH (reduced glutathione), 1 mM GSSG in a sulfonated protein monomer solution of 9 times the pH 9.8 (Oxidized glutathione) was added and then refolded with stirring at 4 ° C for 1 day.

(4) dimer purification

The refolded sample was diluted twice with pure water and isoelectric point precipitation was carried out by adding 6N hydrochloric acid to pH 7.4. The precipitates were collected by centrifugation at 3000 × g 20 minutes and then dissolved in 30% acetonitrile, 0.1% TFA. The solution was diluted 2-fold with pure water and eluted with 0.05% TFA, 25-45% acetonitrile, by passing through a RESOURCE RPC column (Pharmacia) in dull reversed phase HPLC equilibrated with 0.05% TFA, 25% acetonitrile. The eluate was purified using an absorbance photometer and monitored by absorbance at 280 nm to obtain purified protein dimer fractions of the present invention. It was lyophilized with a SpeedVac concentrator (Servant).

(5) Determination of Physicochemical Properties of Purified Protein of the Present Invention

(A) N terminal amino acid sequence analysis

The N-terminal amino acid sequence of the purified protein of the present invention obtained above was analyzed by an amino acid sequencer, model 476A (Applied Biosystems, Inc.), and the amino acid sequence from the N-terminal to the thirtieth amino acid sequence shown in SEQ ID NO: 1 was identified.

(B) amino acid composition analysis

The amino acid composition of the purified protein of the present invention obtained above was examined by an amino acid analyzer [PICO TAG system (Waters)]. The results are shown in Table 1. The numbers shown in the table represent the number of amino acid residues per monomer.

amino acid Measured value Expected value Asx 11.5 12 Glx 10.9 11 Ser 8.4 9 Gly 4.3 4 His 4.0 4 Arg 7.7 7 Thr 5.4 6 Ala 7.3 7 Pro 10.2 10 Tyr 2.9 3 Val 5.7 7 Met 5.1 4 1 / 2Cys 2.6 7 Ile 4.9 6 Leu 10.0 10 Phe 4.0 4 Lys 5.9 6 Trp - 2  The length of the array 119  -: Not detectable

(C) Analysis by electrophoresis

The molecular weight of the purified protein of the present invention obtained above was confirmed by SDS-PAGE under non-reducing conditions, and showed a molecular weight of about 28 KDa.

From the results shown in (a), (b) and (c), it was found that the protein of the present invention is a protein consisting of 119 residues whose N-terminals start singly from Pro.

Example 4 Measurement of Biological Activity

(1) Induction action of ectopic cartilage and bone tissue

About 500 µg of the protein obtained in Example 3 was dissolved in 50 µl of 10 mM hydrochloric acid, diluted with the same solvent, and a solution having a concentration of 1 µg / 10 µl, 10 µg / 10 µl, and 100 µg / 10 µl was prepared. 10 μl was mixed with 150 μl of pig tendon-derived type I collagen solution (high 껜, 0.5%, pH 3, I-AC), neutralized, and freeze-dried to obtain a mixed femur of male 8-week-old male ICR mice. After twenty-one days, the thighs were removed, the skin was peeled off, and the expression rate of the bone and cartilage calcified tissues was examined by soft X-ray imaging. Table 2 shows the results. Its expression was confirmed at a dose of 1 μg / site or more, and its expression was confirmed in all the mice used at doses of 10 μg / site or more.

Dose of MP52 Protein * Expression rate of bone and cartilage calcified tissue Control (Type I Collagen Only) 0/4 1 μg / site 3/4 10 μg / site 4/4 100 μg / site 4/4 * Expression by soft X-ray imaging analysis of bone and cartilage calcified tissues tested in each of the four cases.

2 shows a soft X-ray photograph of typical bone and cartilage calcified tissue at each dose. In FIG. 2, A shows the results of an example in which MP52 protein was inserted into the mouse femoral muscle at doses of 1 μg / site of MP52 protein, B of 10 μg / site of MP52 protein, and C of 100 μg / site of MP protein, respectively. These results confirmed an increase in dose-dependent bone and cartilage calcification tissue. After fixing the femoral sections of these mice, non-limeous fragments were prepared, and von Kossa staining, Alcian blue staining, and hematoxylin-eosin staining were performed.

Fig. 3 shows a micrograph of the tissue staining of a sample encased with type I collagen at a dose of 10 μg / site of the present protein. In FIG. 3, A represents von Cossa stain, B represents Alcian blue stain, and C represents hematoxylin-eosin stain, respectively.

In FIG. 3 (A), the arrow ct portion represents calcified tissue, and the arrow cc portion represents calcified chondrocytes. In FIG. 3 (B), the arrow rc portion represents the remaining cartilage tissue. In FIG. 3 (C), the arrow ad portion represents fat cells, the arrow bm portion represents bone marrow cells, the arrow Ib portion represents lamellar bone, the arrow ob portion represents osteoblasts, and the arrow wb portion represents fibroblasts, respectively. In Fig. 3, it was clear that osteoblasts, bone marrow cells, and calcified chondrocytes were formed by the administration of the MP52 protein, and bone and cartilage calcified tissues were formed.

From the results of Example 4, it became clear that the protein dimer of the present invention has cartilage and bone inducing action.

(2) Over time analysis of ectopic ossification

A freeze-dried product prepared in the same manner as in Example 4 (1) containing about 3 μg of the protein obtained in Example 3 was placed in the femoral muscles of the ICR mice, and after 3,7,10,14,21 and 28 days Tissues were extracted, fixed with 10% formalin, and then the tissue sections were subjected to hematoxylin-eosin staining (HE) and von Kossa staining. 4 shows an optical micrograph of the stained sections.

On day 3 (FIG. 4A, HE), the appearance of undifferentiated mesenchymal cells (mc) comprising morphologically connective fibrous cells was found between the inserted collagen fibers (co) and the surrounding muscle tissue (m). It became. From day 7 (FIG. 4B, HE) to day 10 (FIG. 4C, HE), undifferentiated mesenchymal cells (mc) accumulate and proliferate in this region, and the enlargement of undifferentiated mesenchymal cells and changes to total chondrogenic cells Found. On day 14 (FIG. 4D, HE; FIG. 4E, von Kossa), formation of calcified cartilage tissue (arrow cc) and bone tissue (arrow b) was confirmed. On day 21 (FIG. 4F, HE; FIG. 4G, von Kossa), the formation of bone marrow cells (arrow bm) was observed, while almost no calcified cartilage tissue observed on day 14 was found so that the calcified cartilage tissue was bone tissue (arrow b). Was thought of as On day 28 (FIG. 4H, HE), extensive bone marrow cells (bm) were found and bone tissue (b) formed appeared to be in the process of absorption.

Thus, it was clear that rMP52 is a protein capable of inducing cartilage tissue in ectopicity and subsequently inducing cartilage bone formation, as is known for conventional BMPs.

(3) effect on intraosseous ossification

The protein obtained in Example 3 was dissolved in physiological phosphate buffer (pH 3.4) containing 0.01% human serum albumin, and 0.01 µg / 20 µl, 0.1 µg / 20 µl and 1 µg / 20 µl solution were prepared, 20 μl was injected into the periosteum of the left and right parietal bones of Sprague-Doley rats at the same site once daily for 12 days using a microsyringe from day 1 after birth. An equal amount of the solution was injected into the other parietal periosteum. In addition, the solvent was injected into both osteoblasts to compare and contrast. One day after the final administration, demineralized hematoxylin-eosin staining specimens were taken across the administration site after extraction and fixation of the left and right parietal bone, and the thickness of the left and right parietal bone administration site was measured on a micrograph to determine the thickness of the solvent administration site in the same individual. The ratio of the thickness of the protein administration site of the present invention to was calculated. Table 3 shows the result. Moreover, when 0.1 microgram / 20 microliters of the protein of this invention were continuously injected in FIG. 5, the example (FIG. 5B) on the optical micrograph of a tissue section was shown with that of the opposite solvent administration group (FIG. 5A).

Activation and proliferation of periosteal cells (p) is indicated by the administration of the protein of the present invention, and simultaneously activated osteoblasts (arrow ob) appear in the parietal bone and the periphery of the periosteum (p). It was clear that the thickness of b) increased at the site of administration. This has been shown to be useful for the treatment of osteoporosis, fractures and alveolar, and root defects, at least when topically infused with the protein of the present invention, thereby enhancing membrane ossification.

Dose of Protein of the Invention (μl / site / day) Parietal bone thickness at the site of administration (µm) Thickness ratio (B / A) Solvent Administration (A) Protein Administration of the Invention (B) 0 (solvent) 128 ± 7 141 ± 20 1.10 ± 0.16 0.01 134 ± 9 167 ± 30 1.27 ± 0.33 0.1 119 ± 19 190 ± 29 1.60 ± 0.10 * One 132 ± 19 225 ± 25 1.70 ± 0.14 ** The thickness of the parietal bone and its ratio represent the mean ± standard deviation of four cases. * P <0.05, ** P <0.01 vs left and right parietal solvent administration group (Williams method)

(4) effect on regeneration of articular cartilage

After cutting the skin and joints of the right knee of six 12-week-old male rabbits (New Zealand White), exposing the patella of the femur to avoid damaging the tendon, and using a medical drill to the area 5 mm in diameter The articular cartilage and bone defects which penetrated to the bone marrow cavity of the bone were prepared, and a freeze-dried product containing about 10 µg of the protein obtained in Example 3 or free of type I collagen in the defect site was prepared in the same manner as in Example 4 (1). Three cases were prepared, the joints and skin were sutured, and after 3 weeks, the femoral head was removed and fixed, and all demineralized tissue sections were prepared and subjected to Alcian blue staining. 6 shows optical micrographs of each typical example. When only the Type I collagen lyophilized material was added (FIGS. 6A and 6B), only the fibrous tissue (f) appeared in the defect site (arrow d), but the type I collagen containing about 10 µg of the protein of the present invention obtained in Example 3 In the case of inserting (Figs. 6C and 6D), the formation of chondrocytes (ch) with the deposition of an alkian blue staining positive substrate extracellularly in the defect site was confirmed.

In addition, the formed tissues constructed a cell-like structure similar to the layered structures such as the stationary cell layer, the proliferating cell layer, and the hypertrophy cell layer observed in normal articular cartilage tissue from the outside of the defect site to the inside. These findings were found in all the rabbits used, indicating that the protein of the present invention is effective for the restoration of articular cartilage degeneration tissue caused by cartilage regeneration, in particular deformable arthrosis or osteoarthritis and the like.

(5) Therapeutic effect against cartilage and bone defect

Thirty Sprague Dawley male rats (approximately 15 weeks old) were used to incise the skin tissue of the femur, exposing the femur in the surrounding muscle tissue and a 5 mm circumferential bone with a dental drill in the middle of the femoral stem. A defect was created and both ends of the remaining femur were fixed with a polyethylene plate using a screw made of stainless steel. The protein 0, about 1, 10 or 100 µg-containing collagen freeze-dried product obtained in Example 3 was placed in the bone defect site and the skin tissue was closed. The results of their soft X-ray photographing immediately after the addition and 12 weeks are shown in FIG. 7. 7A is a photograph at the time of bone defect creation. As shown in Figs. 7B to E, with type I collagen alone (Fig. 7B) and 1 μg of protein of the present study type I collagen (Fig. 7C), at the 12th week, there was a slight spine at both ends of the defect. , Only the formation of arrow cs) was shown, and bone union was not reached. On the other hand, in the case where 10 or 100 μg-containing type I collagen (FIGS. D and E) were added, the formation of the ribs (arrow cs) was confirmed throughout the bone defect site, and bone union was confirmed on the X-ray. At 12 weeks, the femur was removed, the polyethylene plate was removed, and the femoral major axis direction 15 mm including the bone defect creation part was continuously operated in the scanning mode at 1 mm intervals by the double X-ray absorption method (Aloka, DCS-600). Injected perpendicularly to each other, the bone salt at the center of 3mm equivalent bone defect was accumulated, and both ends were fixed with resin and mounted on a bone strength measuring device (Malto Works, MZ-500), 180 ° / min. The maximum torsional force required to rotate the lower resin at break speed and break the femur was measured (Table 4). Incorporation of the protein of the present invention into the mouse femoral defect site showed dose-dependent increase in bone inflammation at the same time as well as an increase in bone strength (torsional force) at the site. It was shown to be effective for aggregate reconstruction at the defect site.

Dose (μg / site) of the protein of the invention Osteitis in Mice Femur Bone Defect (mg) Torsional force (kgfcm) The number of courtesy Collagen alone 120.2 ± 24.5 2.92 ± 0.09 6 One 176.9 ± 36.4 6.24 ± 1.00 8 10 277.4 ± 63.9 9.35 ± 3.14 8 100 374.8 ± 67.1 * 4.034 ± 7.64 * 8 The mean value ± quasi error is shown. * p <0.05 versus Student's t-test

A protein consisting of a dimer of a protein having an amino acid sequence set forth in SEQ ID NO: 1 has cartilage and bone inducing activity and is useful as a therapeutic agent for cartilage and bone patients. In addition, by modifying the expression vector of the protein of the present invention, it is possible to produce the protein on an industrial scale and in pure form by genetic engineering techniques using E. coli.

Sequence table

SEQ ID NO: 1

Sequence length: 119

Sequence form: amino acid

Topology: straight

Sequence Type: Peptide

Fragment type: N-terminal fragment

origin:

Biometric: Person (homo sapiens)

Type of Tissue: Human Fetus

Characteristic of the sequence:

Presence location:

Other information: 383rd-501th amino acid sequence of the MP52 amino acid sequence.

<SEQ ID NO 1>

SEQ ID NO: 2

Sequence length: 27

Sequence form: Nucleic acid

Strands: One strand

Topology: straight

Sequence Type: Other Nucleic Acids

Origin: none

Creature: None

State Name: None

Sequence Features: Forward PCR Primer for MP52 Mature Isolation

<SEQ ID NO 2>

SEQ ID NO: 3

Sequence length: 26

Sequence form: Nucleic acid

Strands: One strand

Topology: straight

Sequence Type: Other Nucleic Acids

Origin: none

Creature: None

State name: None

Sequence Characteristics: Reverse PCR Primer for MP52 Mature Isolation

<SEQ ID NO 3>

Claims (23)

A protein having the amino acid sequence set forth in SEQ ID NO: 1. A protein consisting of a homodimer of the protein according to claim 1. A pharmaceutical composition for treating cartilage and bone disease containing a therapeutically effective amount of a protein according to claim 2 and a pharmaceutical carrier. The pharmaceutical composition of claim 3, wherein the cartilage and bone diseases are osteoporosis. 4. The pharmaceutical composition of claim 3, wherein the cartilage and bone diseases are deformable arthrosis or osteoarthritis. The pharmaceutical composition of claim 3, wherein the cartilage and bone diseases are fractures and bone defects. The pharmaceutical composition of claim 3, wherein the cartilage and bone diseases are defects of the root and alveolar. A method for producing a protein of claim 1 using E. coli transformed with a plasmid containing DNA capable of expressing the protein of claim 1. The manufacturing method of Claim 8 which builds the plasmid containing DNA which added the codon which codes a methionine to the 5 prime terminal of the DNA sequence which codes the amino acid sequence of sequence number SEQ ID NO: 1. (i) constructing a plasmid containing a DNA sequence encoding the amino acid sequence set forth in SEQ ID NO: 1 having methionine at the N-terminus, (ii) transforming E. coli with the plasmid, (iii) solubilizing the inclusion body obtained by culturing E. coli, (iv) purifying monomeric protein from the solubilized solution (v) refolding the monomer into a dimeric protein and purifying it Method for producing a dimeric protein according to claim 2 comprising a. A method for the treatment of cartilage and bone diseases, comprising administering to a non-human animal a pharmaceutical composition comprising an effective amount of the homodimer protein according to claim 2 as an active ingredient. The method of claim 11, wherein the cartilage and bone disease is osteoporosis. The method of claim 11, wherein the cartilage and bone diseases are deformable arthrosis or osteoarthritis. The method of claim 11, wherein the cartilage and bone diseases are fractures, bone defects. The method of claim 11, wherein the cartilage and bone diseases are defects of the root, alveolar. 4. The pharmaceutical composition of claim 3, wherein the cartilage and bone disease are cartilage damage or defect. The pharmaceutical composition of claim 16, wherein the cartilage disease is a joint semicircle injury. The pharmaceutical composition of claim 3, wherein the cartilage and bone diseases are congenital. 19. The pharmaceutical composition of claim 18, wherein the cartilage and bone diseases are cartilage insufficiency, cartilage dysplasia, cartilage aplasia, cleft palate or osteopenia. 4. A pharmaceutical composition according to claim 3 for bone transplantation or cartilage transplantation or induction of new cartilage or bone. The pharmaceutical composition according to any one of claims 3 to 7 and 16 to 20, for systemic or topical administration. The pharmaceutical composition according to any one of claims 3 to 7 and 16 to 20 for administration of an injection formulation. The pharmaceutical composition of claim 20, wherein natural or artificial bone is utilized.

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