JP2009079033A - Osteoblast differentiation inducing factor gene and use thereof - Google Patents

Abstract

[Problem] To provide a new factor for inducing differentiation of osteoblasts or promoting maturation, and a medicine using the same.
[MEANS FOR SOLVING PROBLEMS] A medicament comprising the following protein (1) or (2): This osteoblast differentiation inducing factor is useful as an active ingredient such as a therapeutic agent for osteoporosis and fractures.
(1) Protein consisting of the amino acid sequence of SEQ ID NO: 1
(2) A protein consisting of an amino acid sequence in which one or several amino acids are added, deleted, or substituted in SEQ ID NO: 1 and having osteoblast differentiation inducing or maturation promoting activity.

Description

  The present invention relates to a novel osteoblast differentiation inducing factor, a polynucleotide encoding the same, a drug using them, and a method for screening a drug.

Osteoblasts are cells that play an important role in bone formation.
Osteoblasts differentiate from undifferentiated mesenchymal stem cells. First, mesenchymal stem cells differentiate into preosteoblasts that are positive for alkaline phosphatase and have the ability to produce osteopontin and type I collagen.
Furthermore, those that are present on the bone matrix and differentiated until bone matrix synthesis is performed have remarkable alkaline phosphatase activity. While these osteoblasts synthesize bone fibers such as collagen fibers, osteocalcin, and osteopontin, they simultaneously secrete substrate vesicles with strong alkaline phosphatase activity and high concentrations of acidic phospholipids, enriching phosphate and calcium. First formed hydroxyapatite crystals.
When hydroxyapatite crystals break through the membrane of the matrix vesicle and extend out of the vesicle and spread to nearby collagen fibrils to create a more continuous calcified bone matrix, the bones formed by osteoblasts thereafter Substrate formation and calcification continue. This forms a bone.

Mesenchymal stem cells from which osteoblasts are derived differentiate into cells such as adipocytes, muscle cells, and chondrocytes in addition to osteoblasts. Among them, a large number of hormones and cytokines that promote differentiation into osteoblasts are known, and representative examples include bone formation promoting factors (BMP-2: Bone Morphogenetic Protein-2). It is also known that a transcription factor called Runx2 is involved in promoting differentiation from mesenchymal stem cells to osteoblasts by BMP-2.
Many other factors are thought to be involved in the differentiation of mesenchymal stem cells into osteoblasts, but if such factors can be found, the factors themselves and substances that promote their functions Is useful for the treatment of osteoporosis and fractures.

  The main object of the present invention is to provide a new factor that induces osteoblast differentiation or promotes maturation, and a pharmaceutical using the same.

In order to solve the above-mentioned problems, the present inventor repeated research and obtained the following knowledge.
(i) Each protein represented by SEQ ID NOs: 1, 3, 5, 7, 9, and 11 promotes alkaline phosphatase activity, which is a marker for differentiation into osteoblasts in preosteoblasts, and increases the amount of calcification Let
(ii) Since one amino acid sequence exists in the amino acid sequence of these proteins, these proteins are single-transmembrane type transmembrane proteins localized in the cell membrane.
(iii) The protein and a polynucleotide encoding the protein are useful as a pharmaceutical agent for promoting osteogenesis, such as a therapeutic agent for osteoporosis, bone system diseases accompanied by bone loss, and fractures.
(iv) An antibody against this protein and an siRNA against mRNA encoding this protein inhibit bone formation and are useful as pharmaceuticals such as therapeutic agents for ectopic ossification and ectopic calcification.

The present invention has been completed based on the above findings, and provides the following drugs and drug screening methods.
Item 1. A pharmaceutical comprising the following protein (1) or (2):
(1) Protein consisting of the amino acid sequence of SEQ ID NO: 1
(2) A protein having an amino acid sequence in which one or several amino acids are added, deleted, or substituted in SEQ ID NO: 1 and having activity of inducing osteoblast differentiation or promoting maturation. Item 2. The medicine according to Item 1, wherein the protein of (2) consists of the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13.
Item 3. Item 3. The drug according to Item 1 or 2, wherein the drug is an osteogenesis promoter.
Item 4. Item 3. The drug according to Item 1 or 2, wherein the drug is a therapeutic agent for osteoporosis, a bone system disease accompanied by bone loss, or a fracture.

Item 5. A pharmaceutical comprising the following polynucleotide (3) or (4):
(3) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2
(4) A polynucleotide that hybridizes with a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2 under stringent conditions and encodes a protein having osteoblast differentiation-inducing or maturation-promoting activity Item 6. The pharmaceutical according to Item 5, wherein the polynucleotide of (4) comprises the base sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14.
Item 7. Item 7. The drug according to Item 5 or 6, wherein the drug is an osteogenesis promoter.
Item 8. Item 7. The drug according to Item 5 or 6, wherein the drug is a therapeutic agent for osteoporosis, a bone system disease accompanied by bone loss, or a fracture.
Item 9. The pharmaceutical containing the antibody with respect to the following (1) or (2) protein, or the partial protein of (5) or (6).
(1) Protein consisting of the amino acid sequence of SEQ ID NO: 1
(2) A protein comprising an amino acid sequence in which one or several amino acids are added, deleted, or substituted in SEQ ID NO: 1 and having osteoblast differentiation inducing or maturation promoting activity
(5) A partial protein consisting of a partial sequence of SEQ ID NO: 1, comprising at least amino acids 26 to 94 of SEQ ID NO: 1 and having 117 or fewer amino acids
(6) It consists of an amino acid sequence in which one or several amino acids are added, deleted, or substituted in the partial sequence of SEQ ID NO: 1 described in (5) above, and has osteoblast differentiation induction or maturation promoting activity. Partial protein

Item 10. The protein of (2) consists of the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13,
The protein of (6)
(6-1) a partial protein consisting of a partial sequence of SEQ ID NO: 3 comprising at least amino acid numbers 26 to 94 of SEQ ID NO: 3 and having 117 or less amino acids,
(6-2) a partial protein consisting of a partial sequence of SEQ ID NO: 5, comprising at least amino acid numbers 19 to 89 of SEQ ID NO: 5 and having 112 or less amino acids,
(6-3) a partial protein consisting of a partial sequence of SEQ ID NO: 7, comprising at least amino acid numbers 16 to 89 of SEQ ID NO: 7 and having no more than 112 amino acids,
(6-4) a partial protein consisting of a partial sequence of SEQ ID NO: 9, comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 9, and having an amino acid number of 116 or less,
(6-5) a partial protein consisting of a partial sequence of SEQ ID NO: 11, comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 11 and having 116 or less amino acids, or
(6-6) The medicament according to Item 9, which is a partial protein consisting of a partial sequence of SEQ ID NO: 13, comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 13 and having 116 or less amino acids.
Item 11. Item 11. The drug according to Item 9 or 10, wherein the drug is an osteogenesis inhibitor.
Item 12. Item 11. The drug according to Item 9 or 10, wherein the drug is a therapeutic agent for ectopic ossification, ectopic calcification, or a disease caused thereby.
Item 13. A pharmaceutical comprising an siRNA comprising a base sequence complementary to 15 to 30 bases of the following polynucleotide (3) or (4) and having a total length of 30 bases or less.
(3) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2
(4) a polynucleotide that hybridizes with a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2 under stringent conditions and encodes a protein having osteoblast differentiation inducing or maturation promoting activity; Item 14. The pharmaceutical according to Item 13, wherein the polynucleotide of (4) comprises the base acid sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14.

Item 15. Item 15. The drug according to Item 13 or 14, wherein the drug is an osteogenesis inhibitor.
Item 16. Item 15. The drug according to Item 13 or 14, wherein the drug is a therapeutic agent for ectopic ossification, ectopic calcification, or a disease caused thereby.
Item 17. Compare the expression level of the following protein (1) or (2) between the test cell and test substance mixing step and the test cell mixed with the test substance and the test cell not mixed with the test substance. And selecting a test substance that increases the expression level of the protein, a method for screening an osteogenesis promoter, an osteoporosis therapeutic agent, a therapeutic agent for bone system diseases accompanied by a decrease in bone mass, or a therapeutic agent for fracture.
(1) Protein consisting of the amino acid sequence of SEQ ID NO: 1
(2) A protein comprising an amino acid sequence in which one or several amino acids are added, deleted, or substituted in SEQ ID NO: 1 and having activity of inducing osteoblast differentiation or promoting maturation 18. Compare the expression level of the following protein (1) or (2) between the test cell and test substance mixing step and the test cell mixed with the test substance and the test cell not mixed with the test substance. And an osteogenesis inhibitor, an ectopic ossification therapeutic agent, an ectopic calcification therapeutic agent, or an ectopic ossification. Or the screening method of the therapeutic agent of the disease resulting from ectopic calcification.
(1) Protein consisting of the amino acid sequence of SEQ ID NO: 1
(2) A protein comprising an amino acid sequence in which one or several amino acids are added, deleted, or substituted in SEQ ID NO: 1 and having osteoblast differentiation inducing or maturation promoting activity 19. Mixing a test cell and a test substance that differentiates into osteoblasts introduced with a vector containing the following polynucleotide (3) or (4), and mixing the test cell and the test substance mixed with the test substance Comparing the degree of osteoblast differentiation or maturation with a test cell that has not yet been selected, and selecting a test substance that promotes osteoblast differentiation or maturation of the test cell. And a screening method for a therapeutic agent for bone system diseases accompanied by a decrease in bone mass or a therapeutic agent for fracture.
(3) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2
(4) a polynucleotide that hybridizes with a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2 under stringent conditions and encodes a protein having osteoblast differentiation inducing or maturation promoting activity

Item 20. Mixing a test cell and a test substance that differentiates into osteoblasts introduced with a vector containing the polynucleotide of (3) or (4) below, and mixing the test cell and the test substance mixed with the test substance Osteogenesis inhibitor, ectopic, comprising a step of comparing the degree of osteoblast differentiation or maturation with a test cell that has not been tested and selecting a test substance that suppresses osteoblast differentiation or maturation of the test cell A screening method for a therapeutic agent for ossification, a therapeutic agent for ectopic calcification, or a therapeutic agent for diseases caused by ectopic ossification or ectopic calcification.
(3) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2
(4) A polynucleotide that hybridizes with a DNA comprising the nucleotide sequence of SEQ ID NO: 2 under a stringent condition and encodes a protein having osteoblast differentiation-inducing or maturation-promoting activity 21. In the presence or absence of a test substance, mixing a test cell introduced with a vector containing the following polynucleotide (3) or (4) with a cell that differentiates into an osteoblast, and the test substance: Comparing the degree of osteoblast differentiation or maturation in cells that differentiate into osteoblasts in the presence and absence and selecting a test substance that promotes osteoblast differentiation or maturation. A method for screening a formation promoter, an osteoporosis therapeutic agent, a therapeutic agent for bone system diseases accompanied by bone loss, or a therapeutic agent for fracture.
(3) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2
(4) a polynucleotide that hybridizes with a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2 under stringent conditions and encodes a protein having osteoblast differentiation inducing or maturation promoting activity; In the presence or absence of a test substance, mixing a test cell introduced with a vector containing the following polynucleotide (3) or (4) with a cell that differentiates into an osteoblast, and the test substance: Comparing the degree of osteoblast differentiation or maturation in cells that differentiate into osteoblasts in the presence and absence, and selecting a test substance that suppresses osteoblast differentiation or maturation. A screening method for a formation inhibitor, a therapeutic agent for ectopic ossification, a therapeutic agent for ectopic calcification, or a therapeutic agent for a disease caused by ectopic ossification or ectopic calcification.
(3) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2
(4) A polynucleotide that hybridizes with a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2 under stringent conditions and encodes a protein having osteoblast differentiation inducing or maturation promoting activity 23. In the presence or absence of the test substance, in the step of mixing the following (1) or (2) protein and cells that differentiate into osteoblasts, and in the presence and absence of the test substance, Comparing the degree of osteoblast differentiation or maturation in cells that differentiate into osteoblasts, and selecting a test substance that promotes osteoblast differentiation or maturation, an osteogenesis promoter, an osteoporosis therapeutic agent, and bone mass For screening a therapeutic agent for bone system diseases accompanied by a decrease in bone or a therapeutic agent for fractures.
(1) Protein consisting of the amino acid sequence of SEQ ID NO: 1
(2) A protein having an amino acid sequence in which one or several amino acids are added, deleted or substituted in SEQ ID NO: 1 and having osteoblast differentiation inducing or maturation promoting activity 24. In the presence or absence of the test substance, in the step of mixing the following (1) or (2) protein and cells that differentiate into osteoblasts, and in the presence and absence of the test substance, Comparing the degree of osteoblast differentiation or maturation in cells that differentiate into osteoblasts, and selecting a test substance that suppresses osteoblast differentiation or maturation. A screening method for a therapeutic agent, a therapeutic agent for ectopic calcification, or a therapeutic agent for a disease caused by ectopic ossification or ectopic calcification.
(1) Protein consisting of the amino acid sequence of SEQ ID NO: 1
(2) A protein comprising an amino acid sequence in which one or several amino acids are added, deleted, or substituted in SEQ ID NO: 1 and having osteoblast differentiation inducing or maturation promoting activity

Item 25. The pharmaceutical containing the partial protein of the following (5) or (6).
(5) A partial protein consisting of a partial sequence of SEQ ID NO: 1, comprising at least amino acids 26 to 94 of SEQ ID NO: 1 and having 117 or fewer amino acids
(6) It consists of an amino acid sequence in which one or several amino acids are added, deleted, or substituted in the partial sequence of SEQ ID NO: 1 described in (5) above, and has osteoblast differentiation induction or maturation promoting activity. Partial protein term 26. The protein of (6)
(6-1) a partial protein consisting of a partial sequence of SEQ ID NO: 3 comprising at least amino acid numbers 26 to 94 of SEQ ID NO: 3 and having 117 or less amino acids,
(6-2) a partial protein consisting of a partial sequence of SEQ ID NO: 5, comprising at least amino acid numbers 19 to 89 of SEQ ID NO: 5 and having 112 or less amino acids,
(6-3) a partial protein consisting of a partial sequence of SEQ ID NO: 7, comprising at least amino acid numbers 16 to 89 of SEQ ID NO: 7 and having no more than 112 amino acids,
(6-4) a partial protein consisting of a partial sequence of SEQ ID NO: 9, comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 9, and having an amino acid number of 116 or less,
(6-5) a partial protein consisting of a partial sequence of SEQ ID NO: 11, comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 11 and having 116 or less amino acids, or
(6-6) The medicament according to item 25, which is a partial protein consisting of a partial sequence of SEQ ID NO: 13, comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 13, and having 116 or fewer amino acids.
Item 27. Item 27. The drug according to Item 25 or 26, wherein the drug is an osteogenesis promoter.
Item 28. Item 27. The drug according to Item 25 or 26, wherein the drug is a therapeutic agent for osteoporosis, a bone system disease accompanied by bone loss, or a fracture.
Item 29. The pharmaceutical containing the partial protein of the following (5) or (6 ').
(5) A partial protein consisting of a partial sequence of SEQ ID NO: 1, comprising at least amino acids 26 to 94 of SEQ ID NO: 1 and having 117 or fewer amino acids
(6 ') It consists of an amino acid sequence in which one or several amino acids are added, deleted, or substituted in the partial sequence of SEQ ID NO: 1 described in (5) above, and suppresses osteoblast differentiation induction or maturation Partial protein

Item 30. The protein of (6 ')
(6′-1) a partial protein consisting of a partial sequence of SEQ ID NO: 3, comprising at least amino acid numbers 26 to 94 of SEQ ID NO: 3 and having 117 or less amino acids,
(6′-2) a partial protein consisting of a partial sequence of SEQ ID NO: 5, comprising at least amino acid numbers 19 to 89 of SEQ ID NO: 5 and having 112 or less amino acids,
(6′-3) a partial protein consisting of a partial sequence of SEQ ID NO: 7, comprising at least amino acid numbers 16 to 89 of SEQ ID NO: 7 and having an amino acid number of 112 or less,
(6′-4) a partial protein consisting of a partial sequence of SEQ ID NO: 9, comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 9 and having 116 or less amino acids,
(6′-5) a partial protein consisting of a partial sequence of SEQ ID NO: 11, comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 11 and having 116 or less amino acids, or
(6′-6) The medicament according to item 29, which is a partial protein consisting of a partial sequence of SEQ ID NO: 13, comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 13, and having 116 or less amino acids.
Item 31. Item 31. The drug according to Item 29 or 30, wherein the drug is an osteogenesis inhibitor.
Item 32. Item 31. The drug according to Item 29 or 30, wherein the drug is a therapeutic agent for ectopic ossification, ectopic calcification, or a disease caused thereby.
Item 33. A pharmaceutical comprising the following polynucleotide (7) or (8):
(7) A polynucleotide consisting of a partial sequence of SEQ ID NO: 2, which comprises at least the base numbers 76 to 282 of SEQ ID NO: 2 and encodes a partial protein having 117 or less amino acids
(8) a polynucleotide that hybridizes with the polynucleotide comprising the partial sequence of SEQ ID NO: 2 according to (7) above under stringent conditions and encodes a partial protein having osteoblast differentiation inducing or maturation promoting activity Item 34. The polynucleotide of (8)
(8-1) a polynucleotide consisting of a partial sequence of SEQ ID NO: 4, which comprises at least the base numbers 76 to 282 of SEQ ID NO: 4 and encodes a partial protein having 117 or less amino acids,
(8-2) a polynucleotide comprising the partial sequence of SEQ ID NO: 6, which comprises at least the base numbers 55 to 267 of SEQ ID NO: 6 and encodes a partial protein having 112 or less amino acids,
(8-3) a polynucleotide comprising the partial sequence of SEQ ID NO: 8, which comprises at least the base numbers 46 to 267 of SEQ ID NO: 8 and encodes a partial protein having 112 or less amino acids,
(8-4) a polynucleotide comprising the partial sequence of SEQ ID NO: 10, comprising at least the base numbers 79 to 279 of SEQ ID NO: 10 and encoding a partial protein having 116 or less amino acids,
(8-5) a polynucleotide comprising the partial sequence of SEQ ID NO: 12, comprising at least the base numbers 79 to 279 of SEQ ID NO: 12 and encoding a partial protein having 116 or less amino acids, or
(8-6) The pharmaceutical according to Item 33, which is a polynucleotide consisting of a partial sequence of SEQ ID NO: 4, which comprises at least the base numbers 79 to 279 of SEQ ID NO: 14 and encodes a partial protein having 116 or less amino acids.

Item 35. Item 35. The drug according to Item 33 or 34, wherein the drug is an osteogenesis promoter.
Item 36. Item 35. The drug according to Item 33 or 34, wherein the drug is a therapeutic agent for osteoporosis, bone system disease accompanied by bone loss, or fracture.
Item 37. A pharmaceutical comprising the following polynucleotide (7) or (8 '):
(7) A polynucleotide consisting of a partial sequence of SEQ ID NO: 2, which comprises at least the base numbers 76 to 282 of SEQ ID NO: 2 and encodes a partial protein having 117 or less amino acids
(8 ') encodes a partial protein that hybridizes with the polynucleotide comprising the partial sequence of SEQ ID NO: 2 described in (7) under stringent conditions and has an activity of suppressing osteoblast differentiation induction or maturation Polynucleotide term 38. (8 ') polynucleotide is
(8'-1) a polynucleotide comprising the partial sequence of SEQ ID NO: 4, which comprises at least the base numbers 76 to 282 of SEQ ID NO: 4 and encodes a partial protein having 117 or less amino acids,
(8′-2) a polynucleotide comprising the partial sequence of SEQ ID NO: 6, which comprises at least the base numbers 55 to 267 of SEQ ID NO: 6 and encodes a partial protein having 112 or less amino acids,
(8′-3) a polynucleotide comprising the partial sequence of SEQ ID NO: 8, which comprises at least the base numbers 46 to 267 of SEQ ID NO: 8 and encodes a partial protein having 112 or less amino acids,
(8'-4) a polynucleotide comprising the partial sequence of SEQ ID NO: 10, comprising at least the base numbers 79 to 279 of SEQ ID NO: 10 and encoding a partial protein having 116 or less amino acids,
(8′-5) a polynucleotide comprising the partial sequence of SEQ ID NO: 12, comprising at least the base numbers 79 to 279 of SEQ ID NO: 12 and encoding a partial protein having 116 or less amino acids, or
(8'-6) The pharmaceutical according to Item 37, which is a polynucleotide comprising a partial sequence of SEQ ID NO: 4, which comprises at least the base numbers 79 to 279 of SEQ ID NO: 14 and encodes a partial protein having 116 or less amino acids.
Item 39. Item 39. The drug according to Item 37 or 38, wherein the drug is an osteogenesis inhibitor.

Item 40. Item 39. The drug according to Item 37 or 38, wherein the drug is a therapeutic agent for ectopic ossification, ectopic calcification, or a disease caused thereby.
Item 41. In the presence or absence of the test substance, the step of mixing the following partial protein (5) or (6) with cells that differentiate into osteoblasts, and in the presence or absence of the test substance Comparing the degree of osteoblast differentiation or maturation in cells that differentiate into osteoblasts, and selecting a test substance that promotes osteoblast differentiation or maturation, an osteogenesis promoter, an osteoporosis therapeutic agent, bone A method for screening for a therapeutic agent for bone system diseases accompanied by a decrease in the amount or a therapeutic agent for fractures.
(5) A partial protein consisting of a partial sequence of SEQ ID NO: 1, comprising at least amino acids 26 to 94 of SEQ ID NO: 1 and having 117 or fewer amino acids
(6) It consists of an amino acid sequence in which one or several amino acids are added, deleted, or substituted in the partial sequence of SEQ ID NO: 1 described in (5) above, and has osteoblast differentiation induction or maturation promoting activity. Partial protein term 42. The protein of (6)
(6-1) a partial protein consisting of a partial sequence of SEQ ID NO: 3 comprising at least amino acid numbers 26 to 94 of SEQ ID NO: 3 and having 117 or less amino acids,
(6-2) a partial protein consisting of a partial sequence of SEQ ID NO: 5, comprising at least amino acid numbers 19 to 89 of SEQ ID NO: 5 and having 112 or less amino acids,
(6-3) a partial protein consisting of a partial sequence of SEQ ID NO: 7, comprising at least amino acid numbers 16 to 89 of SEQ ID NO: 7 and having no more than 112 amino acids,
(6-4) a partial protein consisting of a partial sequence of SEQ ID NO: 9, comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 9, and having an amino acid number of 116 or less,
(6-5) a partial protein consisting of a partial sequence of SEQ ID NO: 11, comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 11 and having 116 or less amino acids, or
(6-6) The method according to Item 41, which is a partial protein consisting of a partial sequence of SEQ ID NO: 13, which comprises at least amino acid numbers 27 to 93 of SEQ ID NO: 13 and has 116 or fewer amino acids.
Item 43. In the presence or absence of the test substance, the step of mixing the following partial protein (5) or (6) with cells that differentiate into osteoblasts, and in the presence or absence of the test substance Comparing osteoblast differentiation or maturation in cells that differentiate into osteoblasts, and selecting a test substance that suppresses osteoblast differentiation or maturation, and an osteogenesis inhibitor, ectopic ossification Screening method for the above, a therapeutic agent for ectopic calcification, or a therapeutic agent for a disease caused by ectopic ossification or ectopic calcification.
(5) A partial protein consisting of a partial sequence of SEQ ID NO: 1, comprising at least amino acids 26 to 94 of SEQ ID NO: 1 and having 117 or fewer amino acids
(6) It consists of an amino acid sequence in which one or several amino acids are added, deleted, or substituted in the partial sequence of SEQ ID NO: 1 described in (5) above, and has osteoblast differentiation induction or maturation promoting activity. Partial protein term 44. The protein of (6)
(6-1) a partial protein consisting of a partial sequence of SEQ ID NO: 3 comprising at least amino acid numbers 26 to 94 of SEQ ID NO: 3 and having 117 or less amino acids,
(6-2) a partial protein consisting of a partial sequence of SEQ ID NO: 5, comprising at least amino acid numbers 19 to 89 of SEQ ID NO: 5 and having 112 or less amino acids,
(6-3) a partial protein consisting of a partial sequence of SEQ ID NO: 7, comprising at least amino acid numbers 16 to 89 of SEQ ID NO: 7 and having no more than 112 amino acids,
(6-4) a partial protein consisting of a partial sequence of SEQ ID NO: 9, comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 9, and having an amino acid number of 116 or less,
(6-5) a partial protein consisting of a partial sequence of SEQ ID NO: 11, comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 11 and having 116 or less amino acids, or
(6-6) The method according to Item 43, which is a partial protein comprising a partial sequence of SEQ ID NO: 13, comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 13 and having 116 or less amino acids.

Item 45. A diagnostic agent comprising an antibody that specifically binds to the following protein (1) or (2), or partial protein (5) or (6).
(1) Protein consisting of the amino acid sequence of SEQ ID NO: 1
(2) A protein comprising an amino acid sequence in which one or several amino acids are added, deleted, or substituted in SEQ ID NO: 1 and having osteoblast differentiation inducing or maturation promoting activity
(5) A partial protein consisting of a partial sequence of SEQ ID NO: 1, comprising at least amino acids 26 to 94 of SEQ ID NO: 1 and having 117 or fewer amino acids
(6) It consists of an amino acid sequence in which one or several amino acids are added, deleted, or substituted in the partial sequence of SEQ ID NO: 1 described in (5) above, and has osteoblast differentiation induction or maturation promoting activity. Partial protein having 46. The protein of (2) consists of the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13,
The protein of (6)
(6-1) a partial protein consisting of a partial sequence of SEQ ID NO: 3 comprising at least amino acid numbers 26 to 94 of SEQ ID NO: 3 and having 117 or less amino acids,
(6-2) a partial protein consisting of a partial sequence of SEQ ID NO: 5, comprising at least amino acid numbers 19 to 89 of SEQ ID NO: 5 and having 112 or less amino acids,
(6-3) a partial protein consisting of a partial sequence of SEQ ID NO: 7, comprising at least amino acid numbers 16 to 89 of SEQ ID NO: 7 and having no more than 112 amino acids,
(6-4) a partial protein consisting of a partial sequence of SEQ ID NO: 9, comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 9, and having an amino acid number of 116 or less,
(6-5) a partial protein consisting of a partial sequence of SEQ ID NO: 11, comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 11 and having 116 or less amino acids, or
(6-6) The diagnostic agent according to item 45, which is a partial protein consisting of a partial sequence of SEQ ID NO: 13, which comprises at least amino acid numbers 27 to 93 of SEQ ID NO: 13 and has 116 or less amino acids.
Item 47. 47. The diagnostic agent according to item 45 or 46, which is used for diagnosis of ectopic ossification, ectopic calcification, or a disease caused thereby.
Item 48. The following (1) or (2) protein or (5) or (6) partial protein and a test substance are contacted, and a test substance that specifically binds to the protein or the partial protein is selected. And a method for screening a diagnostic agent for ectopic ossification, ectopic calcification, or a disease caused by these.
(1) Protein consisting of the amino acid sequence of SEQ ID NO: 1
(2) A protein comprising an amino acid sequence in which one or several amino acids are added, deleted, or substituted in SEQ ID NO: 1 and having osteoblast differentiation inducing or maturation promoting activity
(5) A partial protein consisting of a partial sequence of SEQ ID NO: 1, comprising at least amino acids 26 to 94 of SEQ ID NO: 1 and having 117 or fewer amino acids
(6) It consists of an amino acid sequence in which one or several amino acids are added, deleted, or substituted in the partial sequence of SEQ ID NO: 1 described in (5) above, and has osteoblast differentiation induction or maturation promoting activity. Partial protein term 49. The protein of (2) consists of the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13,
The protein of (6)
(6-1) a partial protein consisting of a partial sequence of SEQ ID NO: 3 comprising at least amino acid numbers 26 to 94 of SEQ ID NO: 3 and having 117 or less amino acids,
(6-2) a partial protein consisting of a partial sequence of SEQ ID NO: 5, comprising at least amino acid numbers 19 to 89 of SEQ ID NO: 5 and having 112 or less amino acids,
(6-3) a partial protein consisting of a partial sequence of SEQ ID NO: 7, comprising at least amino acid numbers 16 to 89 of SEQ ID NO: 7 and having no more than 112 amino acids,
(6-4) a partial protein consisting of a partial sequence of SEQ ID NO: 9, comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 9, and having an amino acid number of 116 or less,
(6-5) a partial protein consisting of a partial sequence of SEQ ID NO: 11, comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 11 and having 116 or less amino acids, or
(6-6) The method according to Item 48, which is a partial protein consisting of the partial sequence of SEQ ID NO: 13, comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 13, and having 116 or fewer amino acids.

According to the present invention, a new human osteoblast differentiation factor was provided. This protein induces or promotes maturation of mesenchymal undifferentiated cells and differentiated preosteoblasts into osteoblasts.
Therefore, this protein is an osteogenesis promoter, specifically osteoporosis such as juvenile osteoporosis (OMIM259750), pseudoglioma syndrome with osteoporosis (OMIM259770); Menkes syndrome (OMIM309400), homocystinuria (OMIM236200) ), Cole-Carpenter dysplasia (OMIM112240), Black dysplasia (OMIM259450), Osteodysplastic dermatosis (OMIM231070), Hyper IgE syndrome with osteopenia (OMIM147060), Jawbone dysplasia It is useful as a therapeutic agent for bone system diseases with bone loss, such as OMIM166260; The numbers in parentheses above are OMIM (Online Mendelian Inheritance in Man) numbers.

Moreover, the polynucleotide which codes this protein can be used as the same pharmaceutical in the form of the vector and liposome which contain it, for example. Furthermore, antibodies against this protein and siRNA against mRNA encoding this protein are useful as osteogenesis inhibitors, specifically, ectopic ossification, ectopic calcification, or therapeutic agents for diseases caused by these. It is.
Further, the human osteoblast inducing factor of the present invention is considered to be a transmembrane protein localized in the cell membrane. Therefore, this protein is a receptor that induces osteoblast differentiation or promotes maturation by binding to any ligand in the living body, or the extracellular region itself or other extracellular region is another factor in the living body. It is considered that osteoblast differentiation is induced or maturation is promoted by acting on or by binding to some receptor in the living body.

  According to the screening method of the present invention, a substance that induces osteoblast differentiation or promotes maturation by increasing the expression level of this protein, or osteoblast differentiation by acting directly or indirectly on this protein. A substance that promotes induction or maturation can be obtained. These substances are useful as osteogenesis promoters.

  Further, according to another screening method of the present invention, a substance that inhibits or suppresses osteoblast differentiation induction or maturation by reducing the expression level of the protein, or acts directly or indirectly on the protein. Substances that inhibit or suppress osteoblast differentiation induction or maturation can be obtained. These substances are useful as osteogenesis inhibitors, specifically, ectopic ossification, ectopic calcification, or therapeutic agents for diseases resulting therefrom.

  The osteoblast-inducing factor of the present invention detects an osteoblast-inducing factor because it promotes alkaline phosphatase activity, which is a marker of osteoblast differentiation in pre-osteoblasts, and increases the amount of calcification. Thus, the presence of ectopic ossification or ectopic calcification can be predicted. Therefore, an antibody that specifically binds to an osteoblast-inducing factor protein or a partial protein thereof is useful as a diagnostic agent for ectopic ossification, ectopic calcification, or a disease caused thereby. In addition, if a substance that specifically binds to an osteoblast-inducing factor protein or a partial protein is screened, a substance useful as a diagnostic agent for ectopic ossification, ectopic calcification, or a disease caused by them can be obtained. Obtainable.

Hereinafter, the present invention will be described in detail.
Polynucleotide Encoding Osteoblast Differentiation Inducing Factor The polynucleotide encoding the human osteoblast differentiation inducing factor newly found in the present invention is the following polynucleotide (3) or (4).
(3) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2
(4) a polynucleotide that hybridizes with a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2 under stringent conditions and encodes a protein having osteoblast differentiation-inducing or maturation-promoting activity. Includes a polynucleotide having the base sequence and a polynucleotide complementary thereto. Polynucleotides include both DNA and RNA unless otherwise specified. The polynucleotide includes a single-stranded polynucleotide complementary to the single-stranded DNA and a double-stranded polynucleotide in addition to the single-stranded DNA having the base sequence.

The polynucleotide (3) encodes a human osteoblast differentiation factor. The polynucleotide of (3) is obtained by, for example, performing PCR using a primer designed based on SEQ ID NO: 2 and using a human cDNA library library as a template, or by hybridization using a probe designed based on SEQ ID NO: 2. It can be obtained by screening a library library. It can also be obtained by chemical synthesis.
In the present invention, “stringent conditions” refers to general conditions such as those described in Molecular Cloning, A Laboratory Manual, Second Edition, 1989, Vol 2, p11.45, and the like. Specifically, it refers to a case where hybridization occurs at a temperature 5 to 10 ° C. lower than the melting temperature (Tm) of the complete hybrid.

Based on the polynucleotide of (3), the method of obtaining the polynucleotide of (4) will be described. Modifications that do not lose biological function are, for example, polar, charge, The amino acid can be substituted with an amino acid having properties similar to those of the amino acid before substitution in terms of hydrophilicity / hydrophobicity. For example, glycine, alanine, valine, leucine, isoleucine, proline are classified as nonpolar amino acids; serine, threonine, cysteine, methionine, asparagine, glutamine are classified as polar amino acids; phenylalanine, tyrosine, tryptophan Lysine, arginine and histidine are classified as basic amino acids; aspartic acid and glutamic acid are classified as acidic amino acids. Accordingly, substitution can be made by selecting from the same group of amino acids.
When one amino acid has several translation codons, it is of course possible to substitute a base within the translation codon. For example, alanine has four translation codons, GCA, GCC, GCG, and GCT, and the third base of the codon can be substituted between ATGCs.

The polynucleotide of (4) includes, for example, polynucleotides of other biological species corresponding to human polynucleotides. Such polynucleotides can be selected, for example, by NCBI blast search (www.ncbi.nlm.nih.gov/BLAST/). Specifically, for example, the base sequence of SEQ ID NO: 2 is subjected to NCBI blast search to search for sequences having higher homology than base sequence databases and EST databases of other species. By selecting a nucleotide sequence having a nucleotide sequence homology of 90% or more selected by the search, for example, a corresponding gene of another corresponding species can be selected. It can also be identified by screening a gene library of another species using the full length or a part of the polynucleotide of (3), 5′-RACE or the like.
Specific examples of the polynucleotide (4) include a polynucleotide encoding a monkey osteoblast differentiation inducing factor having the base sequence of SEQ ID NO: 4, and a mouse osteoblast differentiation inducing factor having the base sequence of SEQ ID NO: 6. A polynucleotide encoding a rat osteoblast differentiation inducing factor having the base sequence of SEQ ID NO: 8, a polynucleotide encoding a canine osteoblast differentiation inducing factor having the base sequence of SEQ ID NO: 10, Examples thereof include a polynucleotide encoding a horse osteoblast differentiation inducing factor having a base sequence, a polynucleotide encoding a bovine osteoblast differentiation inducing factor having the base sequence of SEQ ID NO: 14, and the like.
That the protein of (4) has osteoblast differentiation induction or maturation promoting activity can be confirmed using the increase in alkaline phosphatase activity and the increase in the amount of calcium deposition described in the Examples as indicators.

Osteoblast differentiation inducing factor The human osteoblast differentiation inducing factor newly found in the present invention is the following protein (1) or (2).
(1) Protein consisting of the amino acid sequence of SEQ ID NO: 1
(2) A protein comprising an amino acid sequence in which one or several amino acids are added, deleted, or substituted in SEQ ID NO: 1 and having osteoblast differentiation inducing or maturation promoting activity
The protein (1) is a human osteoblast differentiation factor. The protein (1) can be obtained from a human cell or tissue by a known protein isolation method. Alternatively, a transformant introduced with DNA consisting of the nucleotide sequence of SEQ ID NO: 2 can be cultured and recovered from the culture. Furthermore, chemical synthesis can also be performed. The method for obtaining the protein (2) from the protein (1) is as described for the polynucleotide.

In the protein of (2), “several” refers to, for example, 5, preferably 3, and more preferably 2.
Specific examples of the protein (2) include a monkey osteoblast differentiation inducing factor having the amino acid sequence of SEQ ID NO: 3, a mouse osteoblast differentiation inducing factor having the amino acid sequence of SEQ ID NO: 5, and the amino acid sequence of SEQ ID NO: 7. Rat osteoblast differentiation inducing factor, canine osteoblast differentiation inducing factor having the amino acid sequence of SEQ ID NO: 9, equine osteoblast differentiation inducing factor having the amino acid sequence of SEQ ID NO: 11, bovine having the amino acid sequence of SEQ ID NO: 14 Examples include osteoblast differentiation inducing factors.
When the amino acid sequence of human osteoblast differentiation inducing factor of SEQ ID NO: 1 was analyzed using PSORT II, a web tool that predicts intracellular localization sites, the region of amino acid numbers 1 to 25 is a signal peptide, The region of numbers 97 to 113 is a transmembrane region, the region of amino acid numbers 26 to 96 is an extramembrane region, and is considered to be a type 1a single-transmembrane protein in which the N-terminus is located outside the cell. From the result of immunostaining, it is considered to be a membrane protein localized in the cell membrane.

Similarly, in the amino acid sequence of the monkey osteoblast differentiation inducing factor of SEQ ID NO: 3, the region of amino acid numbers 1 to 25 is a signal peptide, the region of amino acid numbers 97 to 113 is a transmembrane region, and amino acid numbers 26 to 96 regions are considered to be extramembrane regions.
In the amino acid sequence of the mouse osteoblast differentiation inducing factor of SEQ ID NO: 5, the region of amino acid numbers 1 to 18 is a signal peptide, the region of amino acid numbers 92 to 108 is a transmembrane region, and amino acid numbers 18 to 91 This region is considered to be an extramembranous region.
In the amino acid sequence of the rat osteoblast differentiation inducing factor of SEQ ID NO: 7, the region of amino acid numbers 1 to 15 is a signal peptide, the region of amino acid numbers 92 to 108 is a transmembrane region, and amino acid numbers 16 to 91 This region is considered to be an extramembranous region.

In the amino acid sequence of the canine osteoblast differentiation inducing factor of SEQ ID NO: 9, the region of amino acid numbers 1 to 26 is a signal peptide, the region of amino acid numbers 96 to 112 is a transmembrane region, and amino acid numbers 27 to 95 This region is considered to be an extramembranous region.
Further, in the amino acid sequence of the equine osteoblast differentiation inducing factor of SEQ ID NO: 11, the region of amino acid numbers 1 to 26 is a signal peptide, the region of amino acid numbers 96 to 112 is a transmembrane region, and amino acid numbers 27 to 95 This region is considered to be an extramembranous region.
Further, in the amino acid sequence of the bovine osteoblast differentiation inducing factor of SEQ ID NO: 13, the region of amino acid numbers 1 to 26 is a signal peptide, the region of amino acid numbers 96 to 112 is a transmembrane region, and amino acid numbers 27 to 95 This region is considered to be an extramembranous region.

Medicine
<Osteblast differentiation factor protein>
The osteoblast differentiation inducing factor of (1) or (2) described above is an osteogenesis promoter, specifically juvenile osteoporosis (OMIM259750), pseudoglioglioma syndrome with osteoporosis (OMIM259770), etc. Osteoporosis: Menkes syndrome (OMIM309400), homocystinuria (OMIM236200), Cole-Carpenter osteodysplasia (OMIM112240), black osteodysplasia (OMIM259450), osteodysplastic senile dermatosis (OMIM231070), osteopenia It is useful as an active ingredient for bone system diseases with bone loss such as hyper-IgE syndrome (OMIM147060) and osteophyseal dysplasia (OMIM166260) with bone loss; In the present invention, “treatment” includes alleviation of symptoms, suppression of progression of symptoms, recovery of symptoms, and the like.

When used as an active ingredient of a medicament, the osteoblast differentiation inducing factor protein may be a pharmaceutically acceptable non-toxic salt. Examples of such salts include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts; acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, succinic acid And salts with organic acids such as benzoic acid.
The dosage form of the protein is not particularly limited, and may be either an oral administration agent or a parenteral administration agent. Preferred are dosage forms such as injections, microcapsules and pellets for topical administration. In addition to the protein, injections include conventional additives such as solvents, stabilizers, solubilizers, suspending agents, surfactants, emulsifiers, soothing agents, buffers or preservatives. It may be. The pellet is obtained by mixing the present protein with an excipient, binder, disintegrant, lubricant, stabilizer or wetting agent and molding. Examples of microcapsules include those in which the present protein is filled in biodegradable capsules together with excipients.

  In the case of an injection, it may be administered intravenously or locally to a bone, particularly a damaged part. What is necessary is just to embed in a bone, when it is a microcapsule or a pellet. The dosage of this protein in the case of an injection may be about 1 ng to 1000 mg, preferably about 0.1 μg to 10 mg, more preferably about 10 μg to 10 mg per day for an adult. This dose may be administered until bone formation. In addition, the dose of the protein in the case of microcapsules or pellets to be embedded locally is about 1 ng to 1000 mg, preferably about 0.1 μg to 10 mg, more preferably 10 μg per bone injury site for adults. About 10 mg should be used.

<Polynucleotide encoding osteoblast differentiation inducing factor>
The polynucleotide encoding the osteoblast differentiation inducing factor of (3) or (4) described above is also an active ingredient of an osteogenesis promoter, specifically, a therapeutic agent for osteoporosis, fracture, congenital osteogenesis imperfecta. Useful. The polynucleotide may also be a non-toxic salt. The polynucleotide in this case is usually DNA. This DNA may be a modified DNA.
The polynucleotide may be formulated in the form of an injection, microcapsule, or pellet while being inserted into a known viral vector used for gene therapy such as retrovirus, adenovirus, vaccinia virus and the like. In addition, injections, microcapsules, pellets, etc., in which the above polynucleotides inserted in viral vectors or general expression vectors (for example, expression vectors having SV4 promoter, CMV promoter, elongation promoter, etc.) are encapsulated in liposomes It can also be formulated in the form of
In the case of an injection, it may be administered intravenously or locally to a bone, particularly a damaged part. What is necessary is just to embed in a bone, when it is a microcapsule or a pellet. The dosage of this polynucleotide in the case of an injection may be about 1 ng to 1000 mg, preferably about 0.1 μg to 10 mg, more preferably about 10 μg to 10 mg per day for an adult. This dose may be administered until bone formation. The dosage of this polynucleotide in the case of microcapsules or pellets to be embedded locally is about 1 ng to 1000 mg, preferably about 0.1 μg to 10 mg, more preferably about 1 mg to 10 mg per bone damage site What is necessary is just to be about 10 μg to 10 mg.

<Antibody>
The antibody against the osteoblast differentiation inducing factor protein of (1) or (2) described above is used as an active ingredient of an osteogenesis inhibitor, specifically, a therapeutic agent for ectopic ossification or ectopic calcification. Useful. In addition, therapeutic agents for diseases caused by these, such as arteriosclerosis due to vascular calcification, ischemic heart disease (eg myocardial infarction, angina pectoris) caused by arteriosclerosis, cerebrovascular disease (eg cerebral infarction), etc. Useful as an active ingredient. There are types of arteriosclerosis that involve calcification of the blood vessel wall, and that calcification causes a decrease in arterial function is described in various textbooks (for example, (i) It is described in “Clinical”, Nagai Shoten, published March 10, 2006, page 8, (ii) Eugene Braunwald, “Harrison Internal Medicine” 16th. In addition, an antibody against the partial protein (5) or (6) below which is a partial protein of the osteoblast differentiation inducing factor protein (1) or (2) is also useful as an active ingredient of an osteogenesis inhibitor. The partial protein (6) will be described later.
(5) A partial protein consisting of a partial sequence of SEQ ID NO: 1, comprising at least amino acids 26 to 94 of SEQ ID NO: 1 and having 117 or fewer amino acids
(6) It consists of an amino acid sequence in which one or several amino acids are added, deleted, or substituted in the partial sequence of SEQ ID NO: 1 described in (5) above, and has osteoblast differentiation induction or maturation promoting activity. The partial protein antibody may be a pharmaceutically acceptable non-toxic salt.

  The antibody may be a polyclonal antibody or a monoclonal antibody. Furthermore, derivatives of these antibodies (such as chimeric antibodies) may be used. These antibodies can be produced by a known production method (for example, Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Section 11.12 to 11.13). Specifically, when the antibody of the present invention is a polyclonal antibody, the protein can be used to immunize non-human animals such as rabbits and obtained from the sera of the immunized animals according to a conventional method. In the case of a monoclonal antibody, for example, a non-human animal such as a mouse is immunized with a polypeptide having the present protein or a partial sequence thereof, and spleen cells and myeloma cells of the immunized animal are fused. It can be obtained from the resulting hybridoma (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Section 11.4-11.11).

  The dosage form and administration route of the antibody are as described for the osteoblast differentiation factor protein of the present invention. The dose of the antibody in the case of an injection may be about 1 ng to 1000 mg, preferably about 0.1 μg to 10 mg, more preferably about 10 μg to 10 mg per day for an adult. This dose may be administered until bone formation. In addition, the dose of the antibody in the case of a microcapsule or pellet embedded locally is about 1 ng to 1000 mg, preferably about 0.1 μg to 10 mg, more preferably about 10 μg It should be about 10mg.

<SiRNA>
The siRNA having a base sequence complementary to 15 to 30 bases, preferably 18 to 25 bases of the polynucleotide of (3) or (4) described above and having a total length of 30 bases or less, preferably 25 bases or less It is useful as an active ingredient of an agent, specifically, a therapeutic agent for ectopic ossification or ectopic calcification. In addition, effective therapeutic agents for diseases caused by these, such as arteriosclerosis due to vascular calcification, ischemic heart disease (myocardial infarction, angina pectoris) caused by arteriosclerosis, and cerebrovascular disease (cerebral infarction, etc.) Useful as an ingredient. siRNA design methods are well known, e.g., Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference.Ui-Tei K, Naito Y, Takahashi F, Haraguchi T, Ohki-Hamazaki H, Juni A, Ueda R, Saigo K. Nucleic Acids Res. 2004 Feb 9; 32 (3): 936-48. Print 2004. PMID: 14769950.

  The siRNA can be formulated as an injection, microcapsule, or pellet as it is or encapsulated in liposomes. The route of administration is the same as for polynucleotides. The dosage of siRNA in the case of an injection may be about 1 ng to 1000 mg, preferably about 0.1 μg to 10 mg, more preferably about 10 μg to 10 mg per day for an adult. This dose may be administered until bone formation. Moreover, the dosage of siRNA in the case of microcapsules or pellets to be embedded locally is about 1 ng to 1000 mg, preferably about 0.1 μg to 10 mg, more preferably 10 μg It should be about 10mg.

<Partial protein of osteoblast differentiation factor>
When the osteoblast differentiation inducing factor of the present invention is localized in the cell membrane and functions as a ligand, the following (5) or (6) partial protein having osteoblast differentiation induction or maturation promoting activity is: It is useful as an active ingredient of a bone formation promoter, specifically a bone system disease accompanied by bone loss or a fracture treatment agent.
(5) A partial protein consisting of a partial sequence of SEQ ID NO: 1, comprising at least amino acids 26 to 94 of SEQ ID NO: 1 and having 117 or fewer amino acids
(6) It consists of an amino acid sequence in which one or several amino acids are added, deleted, or substituted in the partial sequence of SEQ ID NO: 1 described in (5) above, and has osteoblast differentiation induction or maturation promoting activity. Partial protein
As the protein of (6),
(6-1) a partial protein (monkey) consisting of a partial sequence of SEQ ID NO: 3, comprising at least amino acid numbers 26 to 94 of SEQ ID NO: 3 and having 117 or less amino acids,
(6-2) a partial protein (mouse) comprising at least the amino acid numbers 19 to 89 of SEQ ID NO: 5 and comprising the partial sequence of SEQ ID NO: 5 having an amino acid number of 112 or less,
(6-3) a partial protein (rat) comprising the partial sequence of SEQ ID NO: 7, comprising at least amino acid numbers 16 to 89 of SEQ ID NO: 7 and having 112 or less amino acids,
(6-4) a partial protein (dog) comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 9 and comprising a partial sequence of SEQ ID NO: 9, wherein the number of amino acids is 116 or less,
(6-5) a partial protein (horse) comprising at least the partial sequence of SEQ ID NO: 11, comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 11, and having 116 or less amino acids,
(6-6) A partial protein (bovine) comprising the partial sequence of SEQ ID NO: 13, comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 13 and having 116 or fewer amino acids
Is mentioned.

When the osteoblast differentiation inducing factor of the present invention is localized in the cell membrane and functions as a receptor, it has the following activity to suppress osteoblast differentiation induction or maturation of (5) or (6 ′) Since the partial protein competes with the full-length protein, it is useful as an active ingredient of an osteogenesis inhibitor, specifically, a therapeutic agent for ectopic ossification or ectopic calcification. In addition, effective therapeutic agents for diseases caused by these, such as arteriosclerosis due to vascular calcification, ischemic heart disease (myocardial infarction, angina pectoris) caused by arteriosclerosis, and cerebrovascular disease (cerebral infarction, etc.) Useful as an ingredient.
(5) A partial protein consisting of a partial sequence of SEQ ID NO: 1, comprising at least amino acids 26 to 94 of SEQ ID NO: 1 and having 117 or fewer amino acids
(6 ') It consists of an amino acid sequence in which one or several amino acids are added, deleted, or substituted in the partial sequence of SEQ ID NO: 1 described in (5) above, and suppresses osteoblast differentiation induction or maturation Partial protein
As a protein of (6 '),
(6'-1) a partial protein (monkey) comprising the partial sequence of SEQ ID NO: 3, comprising at least amino acid numbers 26 to 94 of SEQ ID NO: 3 and having 117 or less amino acids,
(6′-2) a partial protein (mouse) comprising at least the amino acid numbers 19 to 89 of SEQ ID NO: 5 and comprising the partial sequence of SEQ ID NO: 5 having an amino acid number of 112 or less,
(6'-3) a partial protein comprising the partial sequence of SEQ ID NO: 7 (rat), comprising at least amino acid numbers 16 to 89 of SEQ ID NO: 7 and having an amino acid number of 112 or less,
(6′-4) a partial protein (dog) comprising at least the partial sequence of SEQ ID NO: 9, comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 9 and having an amino acid number of 116 or less,
(6′-5) a partial protein (horse) consisting of a partial sequence of SEQ ID NO: 11, comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 11 and having an amino acid number of 116 or less,
(6'-6) Partial protein (bovine) comprising the partial sequence of SEQ ID NO: 13, comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 13 and having 116 or fewer amino acids
Is mentioned.
The partial protein can be chemically synthesized based on each amino acid sequence. It can also be obtained by culturing a transformant introduced with DNA encoding the partial protein.
The partial protein dosage form, administration route, dosage, etc. are the same as for the full-length protein.

<Polynucleotide encoding partial protein of osteoblast differentiation inducing factor>
When the osteoblast differentiation inducing factor of the present invention is localized in the cell membrane and functions as a ligand, the following partial protein having osteoblast differentiation induction or maturation promoting activity of (7) or (8): The encoding polynucleotide is useful as an active ingredient of a bone formation promoter, specifically a bone system disease accompanied by bone loss or a fracture treatment agent.
(7) A polynucleotide consisting of a partial sequence of SEQ ID NO: 2, which comprises at least the base numbers 76 to 282 of SEQ ID NO: 2 and encodes a partial protein having 117 or less amino acids
(8) a polynucleotide that hybridizes with the polynucleotide comprising the partial sequence of SEQ ID NO: 2 according to (7) above under stringent conditions and encodes a partial protein having osteoblast differentiation inducing or maturation promoting activity
As the polynucleotide of (8),
(8-1) a polynucleotide (monkey) comprising the partial sequence of SEQ ID NO: 4, comprising at least the base numbers 76 to 282 of SEQ ID NO: 4 and encoding a partial protein having 117 or less amino acids;
(8-2) a polynucleotide (mouse) comprising a partial sequence of SEQ ID NO: 6 which comprises at least the base numbers 55 to 267 of SEQ ID NO: 6 and encodes a partial protein having 112 or less amino acids;
(8-3) a polynucleotide (rat) comprising a partial sequence of SEQ ID NO: 8, which comprises at least the base numbers 46 to 267 of SEQ ID NO: 8 and encodes a partial protein having 112 or less amino acids,
(8-4) a polynucleotide (dog) comprising the partial sequence of SEQ ID NO: 10, which comprises at least the base numbers 79 to 279 of SEQ ID NO: 10 and encodes a partial protein having 116 or less amino acids,
(8-5) a polynucleotide (horse) comprising a partial sequence of SEQ ID NO: 12, which comprises at least the base numbers 79 to 279 of SEQ ID NO: 12 and encodes a partial protein having 116 or less amino acids,
(8-6) a polynucleotide comprising the partial sequence of SEQ ID NO: 4 (bovine) encoding at least a partial protein having 116 or less amino acids, comprising at least base numbers 79 to 279 of SEQ ID NO: 14
Is mentioned.

When the osteoblast differentiation inducing factor of the present invention is localized in the cell membrane and functions as a receptor, it has the following activity to suppress osteoblast differentiation induction or maturation of (7) or (8 ′) The polynucleotide encoding the partial protein is useful as an active ingredient of an osteogenesis inhibitor, specifically, a therapeutic agent for ectopic ossification or ectopic calcification. In addition, effective therapeutic agents for diseases caused by these, such as arteriosclerosis due to vascular calcification, ischemic heart disease (myocardial infarction, angina pectoris) caused by arteriosclerosis, and cerebrovascular disease (cerebral infarction, etc.) Useful as an ingredient.
(7) A polynucleotide consisting of a partial sequence of SEQ ID NO: 2, which comprises at least the base numbers 76 to 282 of SEQ ID NO: 2 and encodes a partial protein having 117 or less amino acids
(8 ') a polynucleotide encoding a partial protein that hybridizes with a polynucleotide comprising the partial sequence of SEQ ID NO: 2 described in (7) above under stringent conditions and has osteoblast differentiation inducing or maturation promoting activity nucleotide
As the polynucleotide (8 ′),
(8'-1) a polynucleotide (monkey) comprising the partial sequence of SEQ ID NO: 4, comprising at least the base numbers 76 to 282 of SEQ ID NO: 4 and encoding a partial protein having 117 or less amino acids,
(8'-2) a polynucleotide (mouse) comprising a partial sequence of SEQ ID NO: 6, which comprises at least the base numbers 55 to 267 of SEQ ID NO: 6 and encodes a partial protein having 112 or less amino acids,
(8′-3) a polynucleotide (rat) comprising a partial sequence of SEQ ID NO: 8, which comprises at least the base numbers 46 to 267 of SEQ ID NO: 8 and encodes a partial protein having 112 or less amino acids,
(8'-4) a polynucleotide (dog) comprising a partial sequence of SEQ ID NO: 10, which comprises at least the base numbers 79 to 279 of SEQ ID NO: 10 and encodes a partial protein having 116 or less amino acids,
(8'-5) a polynucleotide (horse) comprising a partial sequence of SEQ ID NO: 12, comprising at least the base numbers 79 to 279 of SEQ ID NO: 12 and encoding a partial protein having 116 or less amino acids,
(8′-6) a polynucleotide comprising the partial sequence of SEQ ID NO: 4 (bovine) encoding at least a partial protein having 116 or less amino acids, comprising at least base numbers 79 to 279 of SEQ ID NO: 14
Is mentioned.
A polynucleotide encoding a partial protein can be obtained, for example, by screening cDNA libraries of various organisms using primers designed based on the base sequence.
The dosage form, administration route, and dosage of the polynucleotide encoding the partial protein are the same as those of the full-length polynucleotide.

Medicinal screening method
<First screening method>
A first screening method for a medicament of the present invention comprises the above (1) between a step of mixing a test cell and a test substance, and a test cell mixed with the test substance and a test cell not mixed with the test substance. And (2) comparing the expression levels of the protein and selecting a test substance that increases or decreases the expression level of the protein.
When selecting a test substance that increases the protein expression level, it is possible to screen for an osteogenesis promoter, specifically, osteoporosis, a bone system disease associated with bone loss, and a fracture treatment agent. When selecting a test substance that reduces the protein expression level, screening for a bone formation inhibitor, specifically, a therapeutic agent for ectopic ossification, ectopic calcification, or a disease caused by them it can. The first screening method can be used regardless of whether the osteoblast differentiation inducing factor of the present invention is a ligand or a receptor.

The test cells may be derived from any organism, but human therapeutics can be obtained efficiently by using human cells. The test cell may be any cell that differentiates into osteoblasts, such as human or mouse primary cultured osteoblasts; human osteoblastic cells such as MG-63, HS-Os-1, and Saos-2; Examples include mouse preosteoblasts such as MC3T3-E1.
The type of test substance is not particularly limited. Examples include proteins, peptides, DNAs encoding these, low molecular weight compounds, cell extracts, cell nuclear extracts, plant extracts, and microorganism culture supernatants. A low molecular weight compound is preferable in consideration of absorption and tissue transfer when used as a medicine.
Test cells are subcultured using a medium suitable for the cells and cultured so as to be in a logarithmic growth phase. When contacting the test substance, the cells are seeded so as to be about 50 to 70% confluent and cultured for about 12 hours to 14 days. Next, the test substance is added to the cell culture so as to have a concentration of about 1 nM to 10 μM, for example, depending on the type. Then, it is incubated at about 20 to 37 ° C. for about 1 hour to 2 months.

Then, compare the expression level of the protein (1) or (2) in the test cells mixed with the test substance and the expression level of the protein in the test cells not mixed with the test substance. That's fine. The expression level may be detected by RT-PCR or Western blotting. The comparison may be performed after measuring the expression level in each case, but may be performed directly by visual observation or the like.
By using the test substance, when the expression level of the osteoblast-inducing factor protein is, for example, 80% or less, preferably 50% or less of the control in which the test substance is not contacted, it may be determined that the expression is suppressed. Further, when the expression level of the osteoblast-inducing factor protein is, for example, 120% or more, preferably 150% or more of the control, it may be determined that the expression is promoted.

<Second screening method>
When the osteoblast-inducing factor of the present invention is a receptor, the polynucleotide of the present invention can be used for the second screening method for pharmaceuticals shown below.
That is, the second screening method for the medicament of the present invention comprises a step of mixing a test cell and a test substance that differentiates into an osteoblast into which a vector containing the polynucleotide of (3) or (4) above is introduced. Between the test cells mixed with the test substance and the test cells not mixed with the test substance, the degree of differentiation or maturation into osteoblasts is compared. Selecting a test substance to be promoted or suppressed.

When selecting a test substance that promotes differentiation or maturation of test cells into osteoblasts, an osteogenesis promoter can be screened, and a test substance that suppresses differentiation or maturation of test cells into osteoblasts can be selected. When selecting, an osteogenesis inhibitor can be screened.
The test cells may be cells that differentiate into osteoblasts, and for example, those described in the item of the first screening method can be used. The types of test substances are as described above.
Test cells are subcultured using a medium suitable for the cells and cultured so as to be in a logarithmic growth phase. When contacting the test substance, the cells are seeded so as to be about 50 to 70% confluent and cultured for about 24 hours to 2 months. Next, the test substance is added to the cell culture so as to have a concentration of about 1 nM to 10 μM, for example, depending on the type. Then, it is incubated at about 20 to 37 ° C. for about 1 hour to 2 months.

Thereafter, the degree of differentiation or maturation into osteoblasts may be compared between test cells mixed with the test substance and test cells not mixed with the test substance. That is, the maturity of the test cells as osteoblasts may be compared. Maturity is an indicator of the expression level (transcription amount, protein amount) of differentiation markers such as bone sialoprotein (BSP) and osteocalcin (OCN), alkaline phosphatase activity, and calcium deposition in the test cells. Can be compared. This comparison may be performed after measuring values in both cases where the test substance is mixed and not mixed, but may be directly compared by visual observation or the like.
The expression level of the differentiation marker can be detected by, for example, RT-PCT or Western blotting. The alkaline phosphatase activity can be detected, for example, by measuring the absorbance of the produced paranitrophenol at around 420 nm using paranitrophenyl phosphate as a substrate. It is also possible to visually detect the intensity of blue-violet coloration due to staining. The amount of calcium deposition can be detected by, for example, a chelate coloring method (ortho-fresolphthalein complexone (OCPC) method). In addition, the intensity of reddish brown color developed by alizarin red staining can be detected visually.
When the test substance is used, if the osteoblast differentiation inducing activity is, for example, 80% or less, preferably 50% or less of the control that does not contact the test substance, it is determined that osteoblast differentiation induction or maturation is suppressed. That's fine. Further, when the osteoblast differentiation inducing activity is, for example, 120% or more, preferably 150% or more of the control, it may be determined that osteoblast differentiation induction or maturation is promoted.

<Third screening method>
When the osteoblast-inducing factor of the present invention is a ligand, the polynucleotide of the present invention can be used in the third method for screening pharmaceuticals shown below.
That is, the third screening method for the medicament of the present invention comprises a test cell and an osteoblast into which a vector containing the polynucleotide (3) or (4) is introduced in the presence or absence of a test substance. Comparing the degree of differentiation or maturation of osteoblasts into cells that differentiate into osteoblasts in the presence and absence of the test substance Selecting a test substance that promotes or suppresses the differentiation or maturation of.
When a test substance that promotes differentiation or maturation of cells that differentiate into osteoblasts is selected, an osteogenesis promoter can be screened, and osteoblasts that differentiate into osteoblasts can be screened. When selecting a test substance that suppresses differentiation or maturation of bone, an osteogenesis inhibitor can be screened.

The type of test cell is not particularly limited. Cells that differentiate into osteoblasts can also be used as test cells. Moreover, the kind of cell which differentiates into an osteoblast is not limited, For example, what was illustrated by the 1st screening method can be used. The biological species of these cells is not limited, but human cells are preferred.
Test cells and cells that differentiate into osteoblasts are subcultured using a medium suitable for each cell and cultured so as to be in a logarithmic growth phase. The mixing ratio of the two is usually about the same number. When contacting the test substance, both cells are seeded so as to be about 50 to 70% confluent and cultured for about 24 hours to 2 months. Next, the test substance is added to the cell culture so as to have a concentration of about 1 nM to 10 μM, for example, depending on the type. Then, it is incubated at about 20 to 37 ° C. for about 1 hour to 2 months.
Thereafter, the degree of differentiation or maturation of cells that differentiate into osteoblasts between when mixed with the test substance and when not mixed with the test substance, that is, the degree of maturation as osteoblasts Should be compared. The maturity comparison method is as described above.
When the test substance is used, if the osteoblast differentiation inducing activity is, for example, 80% or less, preferably 50% or less of the control that does not contact the test substance, it is determined that osteoblast differentiation induction or maturation is suppressed. That's fine. Further, when the osteoblast differentiation inducing activity is, for example, 120% or more, preferably 150% or more of the control, it may be determined that osteoblast differentiation induction or maturation is promoted.

<Fourth screening method>
The osteoblast-inducing factor of the present invention can be used in the following fourth method for screening pharmaceuticals, regardless of whether it is a ligand or a receptor.
That is, the fourth screening method for the medicament of the present invention comprises a step of mixing the protein of (1) or (2) and a cell that differentiates into an osteoblast in the presence or absence of a test substance. , Comparing the degree of osteoblast differentiation or maturation in cells that differentiate into osteoblasts in the presence and absence of the test substance, and selecting the test substance that promotes or suppresses osteoblast differentiation or maturation The method includes a process.
Further, the partial protein of (5) or (6) can be used instead of the osteoblast differentiation inducing factor of (1) or (2). Examples of the partial protein (6) include the aforementioned partial proteins of monkey, mouse, rat, dog, horse, and cow.
When a test substance that promotes osteoblast differentiation or maturation is selected, an osteogenesis promoter can be screened. When a test substance that suppresses osteoblast differentiation or maturation is selected, an osteogenesis inhibitor is selected. Can be screened.

Cells that differentiate into osteoblasts are subcultured using a medium suitable for the cells and cultured so as to be in a logarithmic growth phase. When contacting the protein of (1) or (2) and the test substance, the cells are seeded so as to be about 50 to 70% confluent and cultured for about 12 hours to 14 days. Next, the protein (1) or (2) is suspended or dissolved in the cell culture so as to have a concentration of about 1 × 10 ^{−6 to} 10 mg / ml. At the same time or thereafter, the test substance is added to the cell culture so as to have a concentration of, for example, about 1 nM to 10 μM, depending on the type. Then, it is incubated at about 20 to 37 ° C. for about 1 hour to 2 months.
Thereafter, the degree of differentiation or maturation of cells that differentiate into osteoblasts, that is, the degree of maturity as osteoblasts, may be compared between the case where the test substance is added and the case where the test substance is not added. . The maturity comparison method is as described above.
When the test substance is used, the maturity of osteoblasts is, for example, 80% or less, preferably 50% or less of the control not using the test substance, and it is determined that osteoblast differentiation or maturation is suppressed. That's fine. Further, when osteoblast differentiation or maturation is, for example, 120% or more, preferably 150% or more of the control, it may be determined that osteoblast differentiation or maturation is promoted.

Diagnostic Agent The osteoblast-inducing factor of the present invention promotes alkaline phosphatase activity, which is a marker of osteoblast differentiation in pre-osteoblasts, and increases the amount of calcification. If a blast inducer is detected, the presence of ectopic ossification or ectopic calcification can be predicted. Therefore, a substance capable of specifically detecting an osteoblast-inducing factor is useful as a diagnostic agent for ectopic ossification, ectopic calcification, or a disease caused thereby.

<Antibody>
An antibody that specifically binds to the osteoblast differentiation inducing factor protein of (1) or (2) above, and the partial protein of the osteoblast differentiation inducing factor protein of (1) or (2) above (5) or The antibody that specifically binds to the partial protein of (6) is useful as a diagnostic agent for ectopic ossification, ectopic calcification, or a disease caused thereby. Known methods for detecting proteins using antibodies include, for example, radioimmunoassay (RIA), ELISA (solid phase enzyme immunoassay), Ustan blotting, immunoprecipitation, immunohistochemistry and the like. In addition, it is possible to identify an osteoblast differentiation inducing factor expression site by administering a diagnostic agent containing an antibody to a living body and detecting the location of the diagnostic agent.

<Diagnostic drug screening method>
The method for screening a diagnostic agent of the present invention comprises the steps of bringing the protein of (1) or (2) above or the partial protein of (5) or (6) into contact with a test substance, and the specific protein and partial protein. And selecting a test substance that binds automatically. In the contacting step, for example, a sample containing a cell expressing the protein (1) or (2) or the partial protein (5) or (6) (culture solution, body fluid, tissue, cell lysate, tissue It is possible to use a method in which a test substance is added to a disrupted solution and the like and incubated for an appropriate time. Whether or not it is a test substance that specifically binds can be determined, for example, by comparison with a case where it is brought into contact with an antibody that specifically binds.
The obtained substance is useful as a diagnostic agent for ectopic ossification, ectopic calcification, or diseases resulting therefrom. When the obtained substance is used as a diagnostic agent, a known label (radiolabel, enzyme label, fluorescent label, etc.) can be used.

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.
Example 1 (determination of amino acid sequence and base sequence)
The amino acid sequences and base sequences of osteoblast differentiation inducing factors for each species were obtained from the NCBI (National Center for Biotechnology Information) database. Accession numbers are: Human: AAH96825.1 / BC096825, Monkey: XP_001164009 / XM_001164009, Mouse: NP_666274 / NM_146162, Rat: XP_222278 / XM_222278, Dog: XP_854479 / XM_849386, Horse: XP_001497070 / XM_001497020, Cattle: NP_0010 / NP_0010 .
The amino acid sequences of human, monkey, mouse, rat, dog, horse, and bovine osteoblast differentiation factor proteins are shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, and 13, respectively. In addition, the base sequences of DNAs encoding them are shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 14, respectively.

  FIG. 1 is an alignment diagram of amino acid sequences of human, mouse, rat, dog, horse, cow and monkey osteoblast differentiation inducing factor proteins. Preparation of alignment diagrams and identification of consensus sequences were performed using gene sequence information analysis software MacVector (MacVector). The amino acid sequence of the osteoblast differentiation factor is highly conserved among higher vertebrates, suggesting the importance of this protein.

Example 2 (Localization of human osteoblast differentiation factor protein in mouse preosteoblast cell line MC3T3-E1)
(1) Culture of preosteoblast cell line MC3T3-E1 A cell line (RCB1126) obtained from the RIKEN Cell Bank was maintained in αMEM medium (sigma-aldrich, USA, M8042) containing 10% fetal bovine serum. % of were cultured in a humidified atmosphere containing CO _2.
(2) Preparation of human osteoblast differentiation inducing factor protein expression plasmid A DNA fragment encoding human osteoblast differentiation inducing factor protein was obtained by PCR. As a template for PCR, cDNA prepared from human fetal brain total RNA (Clontech Laboratories, USA, 636526) using SuperScript II reverse transcriptase (Invitrogen, USA, 18064-014) was used. Based on the human osteoblast differentiation factor base sequence (NCBI accession number: BC096825), two oligonucleotides (5'GGTACCATGGTTTCGGCGGCAGCCCCCAGCCTC3 '(SEQ ID NO: 15) and 5'GGATCCGACACTGGGGTGGACACTGCTGC3' (SEQ ID NO: 16)) were synthesized (Gene Design, Japan), used as a primer. The resulting DNA fragment is cloned into the pGEM-T Easy vector (Promega, USA, A1360) and confirmed by ABI PRISM 310 Genetic Analyzer using DYEnamic ET Terminator Cycle Sequencing Kit (Amersham Biosciences, USA, US81050) did.

  Subsequently, the pBluescript II KS vector (Stratagene, USA) was subcloned into a modified vector for the purpose of adding a FLAG tag to the C-terminus. This modified vector is gGATCC (= BamHI) -GATTACAAGGATGACGACGATAAG (SEQ ID NO: 17) (= DYKDDDDK (SEQ ID NO: 18): FLAG tag) -TAA-GCggccgc (= TAA stop codon-NotI) between BamHI and NotI of the multicloning site. Is inserted. A DNA fragment encoding a protein with a FLAG tag added to the C-terminus of human osteoblast differentiation inducing factor protein with restriction enzymes KpnI and NotI was excised and subcloned into a pcDNA3 vector (invitrogen, USA, A-150228). Insert a DNA fragment excised from these plasmids with the restriction enzymes HindIII and NotI into the BamHI-NotI site of the Nolan-GFP virus vector (distributed from Dr. Gary Nolan laboratory (Stanford University)) using the BamHI-HindIII linker. To obtain the target expression plasmid.

(3) Production of human osteoblast differentiation factor protein expression virus
The expression plasmid obtained in (2) and the helper plasmid pGag-Pol were temporarily introduced into HEK293 cell-derived packaging cells. Specifically, 20 μg of the expression plasmid and 10 μg of the helper plasmid were obtained by seeding 3 × 10 ⁶ cells in a 10 cm plate the day before using the calcium phosphate method. After incubating with 10 ml of medium for 24 hours at 37 ° C, confirm that gene transfer is sufficient by GFP expression, replace the medium with 6 ml, and then collect the virus-containing medium every 24 hours and fresh The addition of the medium was repeated, and a total of 18 ml of virus solution was obtained in 3 days. The virus solution was filtered through a PVDF membrane filter (0.45 μm pore), snap-frozen with liquid nitrogen, and stored at −80 ° C. until use.

(4) Establishment of human osteoblast differentiation factor protein stable expression cell line (infection)
1 × 10 ⁵ cells of MC3T3-E1 cells are seeded on a 6 cm plate. The next day, 2 ml of medium and 2 ml of thawed virus solution were mixed with 32 μg of polybrene and added at the time of medium exchange. After 24 hours, the culture medium was replaced with the maintenance medium, and it was confirmed that almost all cells expressed GFP 3 days after the infection. These were used in experiments as human osteoblast differentiation inducing factor protein stable expression cell lines.

(5) Confirmation of protein localization by immunostaining method Cover glass (MATSUNAMI, Japan, 24 × 24mm Thickness NO.1) is placed in a 35mm plate and coated with gelatin. The stably expressing cells 1 × 10 ⁵ cells obtained in (3) were seeded, cultured for 2 days, and then immunostained. All experimental operations were performed at room temperature.
(5-1) Fixed / Transparent / Blocking
After washing twice with 1 × PBS, fixed with 4% paraformaldehyde / PBS for 15 minutes, washed twice with 1 × PBS, then 15% with 0.1% TritonX-100 (Wako, Japan, 168-11805) / PBS After permeabilization for 1 minute, washing twice with 1 × PBS, blocking was performed with 2% bovine serum albumin (Sigma-Aldrich, USA, A2153) / PBS.
(5-2) Antibody antigen reaction and visualization Monoclonal ANTI-FLAG M2 Antibody (SIGMA, USA, F1804) is added as a primary antibody to 2% bovine serum albumin / PBS to a dilution of 1: 1000. did. After incubation for 60 minutes, the cells were washed twice with 0.1% bovine serum albumin / PBS. Adjust and use 1: 400 dilution in bovine serum albumin / PBS. At the same time, 4 ′, 6′-diamidino-2-phenylindole (DAPI) (SIGMA, USA, D8417) was added at a dilution of 1: 1000 for the purpose of nuclear staining. After the reaction of the secondary antibody, it was washed twice with 0.1% bovine serum albumin / PBS and sealed in a slide glass using a water-soluble mounting agent.
(5-3) Observation and image processing The immunostained slice tissue was observed and photographed with a confocal microscope LSM510 (Carl Zeiss, Oberkochen, Germany), and image processing was performed with the image processing software Zeuss LSM Image Browser. It was.

(6) Reagents / solutions The reagents and solutions used in Example 2 are as follows.
20 × PBS: NaCl 160 g, KCl 4 g, Na ₂ HPO4 · 7H ₂ O 43.4 g, KH ₂ PO ₄ 4 g / Adjusted to 1 L with purified water.
4% paraformaldehyde / PBS: 4 g paraformaldehyde (MERCK, Germany 104005) / 94 ml purified water, 5 ml 20 × PBS / 100 ml total
(7) Resulting stained image is shown in FIG. The amorphous part stained with the red fluorescent dye is the osteoblast differentiation inducing factor protein, and the circular part (blue fluorescent dye part) present inside and outside the amorphous part indicates the cell nucleus. The amorphous part (red fluorescent dye part) shows a pattern that expresses the endoplasmic reticulum and the cell membrane, and this is a membrane protein predicted from the sequence of the osteoblast differentiation inducing factor protein and is localized in the cell membrane This suggests the possibility of having functions related to cell-cell interactions.

Example 3 (Expression pattern of osteoblast differentiation inducing factor in mouse developmental stage)
The animal experiment was conducted according to the protocol approved by the Osaka Bioscience Institute Animal Experiment Committee.
(1) Preparation of DIG probe
DIG probes were prepared using DIG RNA Labeling Mix (Roche, Switzerland, 1 277 073) and T3 / T7 RNA polymerase (Roche, Switzerland, 1 031 171/0 881 775). The DNA regions used as templates were mouse osteoblast differentiation inducing factor: AK078473 (1-2093), mouse collagen 1: AK086730 (1-1712), osteopontin: NM_009263 (310-1296), mouse osteocalcin: NM_009263 (1 -470). 50 μl of RNA probe prepared from 11 μg of template DNA was added to the hybridization solution to a concentration of 5% and used.

(2) Whole mount in situ hybridization
(2-1) Preparation of samples Mouse fetuses were collected from pregnant mice deeply anesthetized with ether, fixed with 4% paraformaldehyde / PBS at 4 ° C overnight, returned to room temperature, and washed with 1 x PBT. 70% methanol (Nacalai Tesque 21915-35), 80% methanol, 90% methanol, 100% methanol and so on in order to increase the concentration. Subsequently, the methanol concentration was lowered from 100% to 70%, and the concentration was returned to 1 × PBT. The mouse embryo after rehydration was used for the experimental operation.
(2-2) Pretreatment / Hybridization The sample was immersed in 6% hydrogen peroxide (Wako, Japan, 081-04215) / PBT and bleached. Wash with 1 × PBT, treat with 10 μg / ml proteinase K (Sigma-Aldrich, USA, P2308) / PBT for 15 minutes, and then immerse in 2 mg / ml glycine (Sigma-Aldrich, USA, G7403) for 10 minutes. I stopped. After washing with 1 × PBT, the cells were fixed with 4% paraformaldehyde / PBS for 20 minutes. Wash with 1xPBT (operate at room temperature so far), soak in prehybridization solution at 70 ° C for 1 hour, replace with hybridization solution with DIG probe added, and perform hybridization at 70 ° C overnight .
(2-3) Washing / antibody hybridization Washing solution 1 (70 ° C) for 30 min x 3 times, Washing solution 3 (65 ° C) for 30 min x 3 times, 1 x TBST (room temperature) 3 minutes × 5 washes were sequentially performed. After blocking with 10% sheep serum / 1 × TBST for 2.5 hours (room temperature), the cells were immersed in an Ab mix solution and antibody hybridization (4 ° C.) was performed overnight.
(2-4) Cleaning
Washing was performed in order of 1 × TBST for 3 minutes × 5 times and 1.5 hours × 5 times in order (room temperature), and then at 4 ° C. overnight.
(2-5) Coloring and shooting (both at room temperature)
After washing with 1 × NTMT for 10 minutes × 3 times, a reaction mix solution was added, and the color reaction was carried out in the dark. After the signal was observed, the plate was washed with 1 × NTMT for 10 minutes × 2 times and immersed in 1 × PBT (pH 5.5) for 10 minutes. After fixing with 4% paraformaldehyde / PBS for 1 hour, it was transferred to 1 × PBT, and photographed with a stereomicroscope StemiSV11 (Carl Zeiss, Oberkochen, Germany).

(3) Paraffin section in situ hybridization
(3-1) Preparation of sample Mouse fetus was collected from pregnant mice deeply anesthetized with ether, fixed with 4% paraformaldehyde / PBS at 4 ° C overnight, washed as it was with 1x PBS at 4 ° C, Dehydration and secondary fixation were performed with ethanol (Nacalai Tesque, Japan, 14713-95). (Soaked sequentially in 70 → 80 → 90 → 100% ethanol). The sample returned to room temperature was clarified with 100% xylene (Wako, Japan, 244-00081), and then immersed in paraffin (Sakura Finetech Japan, Japan, No. 7810) at 60 ° C. The dehydration with ethanol took 4 days, and the xylene / paraffin process took 9 hours. Samples were embedded in paraffin in plastic molds and the completed blocks were stored at 4 ° C.
(3-2) Preparation of slices The block was sliced with a rotary microtome HM340E (Carl Zeiss, Oberkochen, Germany), placed on a slide glass, stretched for 30 minutes on a 48 ° C stretcher plate, and then placed in a 70 ° C dryer. And dried for 10 minutes to ensure complete contact with the slide glass. Store the finished sample at 4 ° C until use.
(3-3) Deparaffinization / Pretreatment / Hybridization Samples were immersed in xylene, ethanol (100 → 90 → 80 → 70 → 50%), and 1 × PBS in this order for deparaffinization and rehydration. Heat treatment in citrate buffer (100 ° C) for 20 minutes, treatment with 0.2N HCl solution for 10 minutes, treatment with 4μg / ml proteinase K / PBS for 2 minutes, and then immersion in 2mg / ml glycine for 10 minutes I stopped. After washing 3 times with 1 × PBS, the cells were fixed with 4% paraformaldehyde / PBS for 15 minutes. The plate was washed with 1 × PBS (up to room temperature operation so far), immersed in a prehybridization solution at 60 ° C. for 1 hour, then replaced with a hybridization solution to which a DIG probe was added, and hybridization was carried out at 60 ° C. overnight.

(3-4) Washing / antibody hybridization Washing solution 1 (60 ° C) for 15 min x 3 times, Washing solution 3 (55 ° C) for 15 min x 3 times, 1 x TBST (room temperature) Washing for 10 minutes × 3 times was sequentially performed. After blocking with 5% sheep serum / 1 × TBST for 1 hour (room temperature), it was immersed in an Ab mix solution and antibody hybridization (4 ° C.) was performed for 2 hours.
(3-5) Coloring and shooting (both at room temperature)
After washing with 1 × TBST for 15 minutes × 4 times, and with 1 × NTMT for 10 minutes × 3 times, a reaction mix solution was added to carry out a color reaction with light shielding. After the signal was observed, the plate was washed with 1 × NTMT for 5 minutes × 2 times and immersed in 1 × PBT (pH 5.5) for 1 minute. After fixing with 4% paraformaldehyde / PBS for 10 minutes, it was transferred to 1 × PBT and sealed with a cover glass using a water-soluble mounting agent. Images were taken with an optical microscope Axioskop2 plus (Carl Zeiss, Oberkochen, Germany).

(4) Reagents / solutions The reagents and solutions used in Example 3 are as follows.
1 x PBT: 0.1% Tween-20 / polyoxyethylene sorbitan monolaurate (Nacalai Tesque, Japan, 356-24) / PBS
Prehybridization / hybridization solution: 50% Formamide, 5 × SCC (pH4.5), 50μg / ml yeast RNA (Roche, Switzerland, 109233)
Cleaning solution 1: 50% formamide, 5 × SCC (pH 4.5), 1% SDS
Cleaning solution 3: 50% formamide, 2 x SCC (pH 4.5)
10 x TBS: NaCl 8g, KCl 0.2g, 1M Tris-HCl (pH7.5) 25ml / purified water 75ml
1 x TBST: 0.1% Tween-20 / TBS
Ab mix solution: Anti-Digoxigenin-AP Fab fragments (Roche, Switzerland, 1 093 274) were diluted 1 × 2000 in 1 × TBST and used.
1 × NTMT: 100 mM NaCl, 100 mM Tris-HCl (pH 9.5), 50 mM MgCl ₂ , 0.1% Tween-20, 2 mM Levamisole (Sigma-Aldrich, USA, L9756)
Reaction mix solution: 75 mg / ml Nitrotetrazolium blue (Sigma-Aldrich, USA, N6876) 6.75 μl, 50 mg / ml 5-Bromo-4-chloro-3-indolylphosphate disodium salt (Sigma-Aldrich, USA, B6149) 5.25 μl in 1 × Prepared in addition to NTMT.
Citrate buffer: 0.1M citric acid aqueous solution 9.5ml + 0.1M sodium citrate aqueous solution 41.5ml / purified water 449ml

(5) The result image is shown in FIG. As shown in FIG. 3 (A), the osteoblast differentiation inducing factor is first expressed in limb buds (embryo 9.5 and 10.5 embryos) in the developmental process of mice, and then the mesenchyme surrounding the finger cartilage primordia. The cells showed expression (embryonic day 12.5 embryos). At the late embryonic stage, osteoblast-specific expression was observed in the extremities.
FIG. 3 (B) is a comparison between osteoblast differentiation inducing factors in osteoblasts and other various markers. Collagen 1 is a protein secreted from relatively early osteoblasts, and osteocalcin is a protein secreted from mature osteoblasts. Osteopontin was used as a marker for calcified tissue (including calcified cartilage).
From the results of FIGS. 3 (A) and 3 (B), it can be seen that the osteoblast differentiation inducing factor is expressed from the immature stage to the mature stage of the osteoblast. This suggests that the osteoblast differentiation inducing factor has a strong differentiation inducing or maturation promoting activity.

Example 4 (Promoting differentiation and maturation of a preosteoblast cell line by an osteoblast differentiation inducing factor)
The purpose of this study was to examine whether the osteoblast differentiation inducing factor has a function for differentiation or maturation of a pre-osteoblast cell line. The cells used were the human osteoblast differentiation inducing factor protein stable expression cell line prepared in (2) of Example 2 and the control cell line prepared in the same manner (in (3) and (4) of Example 2). Two types, one prepared using an empty Nolan-GFP virus vector instead of the human osteoblast differentiation factor protein expression plasmid).

(1) Induction of differentiation of the preosteoblast cell line MC3T3-E1
In order to induce differentiation of MC3T3-E1, 10 mM β-glycerol phosphate (Sigma-Aldrich, USA, 50020) and 50 g / ml. L-ascorbic acid (Sigma-Aldrich, USA, A9659) plus differentiation medium was used. Cells were seeded at 3 × 10 ⁴ / well in a 12-well plate, cultured with maintenance medium for 2 days, and culture with differentiation medium was started from a confluent state.
(2) Alkaline phosphatase activity Alkaline phosphatase (ALP) is used as an early differentiation marker for osteoblasts. In this study, cells were evaluated for alkaline phosphatase staining and LabAssay ^™ ALP kit (Wako, Japan, 291-58601).
(2-1) Alkaline phosphatase staining (reaction at room temperature)
Cells (human osteoblast differentiation inducing factor protein stable expression cell line and control strain) on the 2nd day of culture in maintenance medium (0 days of differentiation start) and 2nd day of culture in differentiation medium (2 days of differentiation start) After washing with 1 × PBS, fix with 4% paraformaldehyde / PBS for 10 minutes, wash with 1 × PBS, replace with ALP buffer, then replace with ALP coloring solution, react for 15 minutes with light shielding, Photographed with a microscope SteREO Lumar. V12 (Carl Zeiss, Oberkochen, Germany).
(2-2) Measurement of ALP activity using LabAssay ^TM ALP Kit Cells stably cultured on differentiation medium (Day 2 of differentiation initiation) and Day 5 (Day 5 of differentiation initiation) After washing the cell line and control line with 1x PBS, replace with ALP lysate and collect the cells with a cell scraper. This cell lysate was used for sonication / centrifugation, and the supernatant was used for measurement of ALP activity using LabAssay ^™ ALP kit. The ALP activity was calculated as the activity per protein weight according to the manual attached to the kit. That is, the enzyme activity was determined by dividing the number of moles of paranitrophenol produced per minute under the conditions of pH 9.8 and 37 ° C. by the amount of protein.

(3) Calcium deposition (calcification)
As an indicator of late differentiation of osteoblasts, calcium deposition is used together with various markers (BSP, OCN, etc.). This time, the deposited calcium was quantified by alizarin red staining and calcium C-test _Wako (Wako, Japan, 272-21801).
(3-1) Alizarin Red Staining Cells on the 18th day of culture with differentiation medium (18 days after differentiation start) were washed with 1 × PBS, fixed with 70% ethanol for 10 minutes, and replaced with alizarin red staining solution. Time standing. Photographed with a stereo microscope SteREO Lumar. V12 (Carl Zeiss, Oberkochen, Germany) after washing with purified water.
(3-2) Quantification of Calcium Deposited with Calcium C-Test _Wako (Wako, Japan, 272-21801) Cells on differentiation medium 18 days (18 days of differentiation) and 24 days (24 days of differentiation) After washing with 1 × PBS, replace with 0.6N HCl solution and collect cells with a cell scraper. The collected cells were rocked in 0.6N HCl solution for 24 hours at room temperature. After centrifugation, the supernatant was used for calcium quantification with Calcium C-Test _Wako . The amount of calcium deposition was calculated as the weight per well according to the manual attached to the kit.

(4) Reagents / solutions The reagents and solutions used in Example 4 are as follows.
ALP buffer: 100 mM Tris (pH 9.5), 100 mM NaCl, 50 mM MgCl
Alizarin Red staining solution: 1% Arizarin Red S (Sigma-Aldrich, USA, 01-2180-2) / purified water (adjusted to pH 4.2 with HCl solution)

(5) Results In this example, the differentiation ability was compared between the human osteoblast differentiation factor protein stable expression cell line and the control cell line. Alkaline phosphatase was used as an early differentiation marker for osteoblasts, and the amount of deposited calcium was used as an indicator of late differentiation.
FIG. 4 shows the results of alkaline phosphatase staining and alizarin red staining, and FIG. 5 is a graph showing alkaline phosphatase activity and calcium deposition.
Alkaline phosphatase staining (blue purple) shows no difference before differentiation induction, but on the second day of differentiation induction, the human osteoblast differentiation inducer protein stable expression cell line shows stronger staining than the control cell line. Indicated. Alkaline phosphatase activity was higher in both the 2nd and 5th days of induction in the human osteoblast differentiation factor protein stable expression cell line than in the control cell line.
In addition, staining with alizarin red, a pigment that forms a colored (reddish brown) calcium rack, shows that the human osteoblast differentiation inducing factor protein stable expression cell line is stronger than the control cell line on the 18th day of differentiation induction. showed that. Moreover, in the calcium quantification by the kit, the human osteoblast differentiation inducing factor protein stable expression cell line showed a higher value than the control cell line on the 18th and 24th day of induction. These data indicate that osteoblast differentiation inducing factors have an effect of promoting osteoblast differentiation and maturation.

Example 5 (differentiation induction by partial protein of human and mouse osteoblast differentiation inducing factor in mouse preosteoblast cell line MC3T3-E1)
Mouse pre-osteoblast cell line that stably expresses human and mouse osteoblast differentiation-inducing factor and two types of partial proteins (extracellular region only, extracellular region + transmembrane region + part of intracellular region) MC3T3-E1 was used to examine whether the partial protein of the osteoblast differentiation inducing factor has a function for differentiation or maturation of the preosteoblast cell line.
FIG. 6 is a schematic diagram showing the entire coding length of the mouse (human) osteoblast differentiation inducing factor, only the extracellular region, and part of the extracellular region + transmembrane region + intracellular region. The expressed proteins are human osteoblast differentiation inducing factor full length: amino acid numbers 1-283 of SEQ ID NO: 1, human osteoblast differentiation inducing factor extracellular region: amino acid numbers 1-94 of SEQ ID NO: 1, human osteoblast, respectively. Cell differentiation inducing factor extracellular region + transmembrane region + part of intracellular region: amino acid number 1-117 of SEQ ID NO: 1, mouse osteoblast differentiation inducing factor full length: amino acid number 1-280 of SEQ ID NO: 5, mouse bone Blast differentiation inducing factor extracellular region: amino acid numbers 1 to 89 of SEQ ID NO: 5, mouse osteoblast differentiation inducing factor extracellular region + transmembrane region + part of intracellular region: amino acid numbers 1 to 112 of SEQ ID NO: 5 It hits. Here, the part described as a part of the intracellular region is 4 amino acid residues in both human and mouse, and is included in the transmembrane region on the database UniProtKB / Swiss-Prot.
The MC3T3-E1 cells that express the full length human osteoblast differentiation factor were those established in Example 2 and used in this example.

(1) Cultivation of preosteoblast cell line MC3T3-E1 Culture was performed as described in Example 2 (1).
(2) Establishment of a human osteoblast differentiation inducing factor partial protein expression cell line DNA fragment encoding a partial protein using the human osteoblast differentiation inducing factor protein (full length) expression plasmid prepared in Example 2 (2) as a template Was amplified by PCR. The primers used were the extracellular region of human osteoblast differentiation inducing factor: 5'GGATCCATGGTTTCGGCGGCAGCCCCCAGCCTC3 '(sequence 19), 5'GGATCCGTACTGGCGGAAGAAGTCCACTATC3' (sequence 20), extracellular region of human osteoblast differentiation inducing factor + transmembrane region + Part of the intracellular region: 5'GGATCCATGGTTTCGGCGGCAGCCCCCAGCCTC3 '(sequence 19), 5'GGATCCGATGACCGCGGCACAGACGATG3' (sequence 21). Using the obtained DNA fragment, according to the method of Example 2 (2) to (4), a cell line expressing the extracellular region of human osteoblast differentiation inducing factor, and human osteoblast differentiation inducing factor A cell line expressing a part of the extracellular region + transmembrane region + intracellular region was established.

(3) Establishment of full length and partial protein expression cell line of mouse osteoblast differentiation inducer According to the method of Example 2 (2), cDNA obtained by reverse transcription of total RNA prepared from mouse 12.5 day fetus was used as a template. PCR was performed to amplify a DNA fragment encoding the mouse osteoblast differentiation factor full-length protein and two types of partial proteins (extracellular region only, extracellular region + transmembrane region + part of intracellular region). . Total RNA was prepared according to the protocol using TRIzol reagent (Invitrogen, USA, 15596-018). The primers used were mouse osteoblast differentiation factor full length: 5'GGTACCATGGTCCCCTGGTTCCTCCTGTCTC3 '(sequence 22), 5'GGATCCGACACTGGGGGAGACTCTGTTGCAG3' (sequence 23), mouse osteoblast differentiation inducer extracellular region: 5'GGATCCATGGTCCCCTGGTTCCTCCTGTCTC3 ' ), 5′GGATCCGTACTGCCGGAAGAAATCCATGATC3 ′ (sequence 25), mouse osteoblast differentiation inducer extracellular region + transmembrane region + part of intracellular region: 5′GGATCCATGGTCCCCTGGTTCCTCCTGTCTC3 ′ (sequence 24), 5′GGATCCGATGAGGGCGGCGCGCAGACTATGAAC3 ′ (sequence 26) It is. Using the obtained DNA fragment, according to the method of Example 2 (2) to (4), a cell line expressing the full length of mouse osteoblast differentiation inducing factor and the extracellular region of mouse osteoblast differentiation inducing factor A cell line that expresses a part of an extracellular region + transmembrane region + intracellular region of a mouse osteoblast differentiation inducing factor was established.

(4) Induction of differentiation of pre-osteoblast cell line by partial protein of mouse osteoblast differentiation factor 3 types of cell lines expressing full length or partial protein of mouse osteoblast differentiation factor established in (3) above, The control cell line (see Example 4) was cultured in a differentiation medium, and alkaline phosphatase activity and calcium deposition were measured. Specifically, cells were seeded at 3 × 10 ⁴ / well in a 12-well plate and cultured for 2 days with the maintenance medium of Example 2 (1). From confluent state, the differentiation medium (10 mM β- Culture with glycerol phosphate (Sigma-Aldrich, USA, 50020) and 50 g / ml L-ascorbic acid (Sigma-Aldrich, USA, A9659)) was started.

LabAssay ^™ ALP kit (Wako, Japan, 291-58601) was used for the measurement of alkaline phosphatase activity. After washing each cell on differentiation medium 1.5 days (1.5 days of differentiation start), 3 days (3 days of differentiation start) and 6.5 days (6.5 days of differentiation start) with 1x PBS, Replace and collect cells with a cell scraper. This cell lysate was used for sonication / centrifugation, and the supernatant was used for measurement of ALP activity using LabAssay ^™ ALP kit. The ALP activity was calculated as the activity per protein weight according to the manual attached to the kit. That is, the enzyme activity was determined by dividing the number of moles of paranitrophenol produced per minute under the conditions of pH 9.8 and 37 ° C. by the amount of protein.

Calcium C-Test _Wako (Wako, Japan, 272-21801) was used for quantification of deposited calcium. Cells on the differentiation medium 28th day (28th day of differentiation) and 42th day (42th day of differentiation) were washed with 1xPBS, then replaced with 0.6N HCl solution, and the cells were collected using a cell scraper. . The collected cells were rocked in 0.6N HCl solution for 24 hours at room temperature. After centrifugation, the supernatant was used for calcium quantification with Calcium C-Test _Wako . The amount of calcium deposition was calculated as the weight per well according to the manual attached to the kit.

(5) Induction of differentiation of pre-osteoblast cell line by partial protein of human osteoblast differentiation factor Two types of cell lines expressing partial protein of human osteoblast differentiation factor established in (2) above, Examples The cell line expressing the full-length human osteoblast differentiation inducing factor established in 2 and a control cell line (see Example 4) were cultured in a differentiation medium, and the amount of calcium deposition was measured. Specifically, cells were seeded at 3 × 10 ⁴ / well in a 12-well plate and cultured for 2 days with the maintenance medium of Example 2 (1). From confluent state, the differentiation medium (10 mM β- Culture with glycerol phosphate (Sigma-Aldrich, USA, 50020) and 50 g / ml L-ascorbic acid (Sigma-Aldrich, USA, A9659)) was started.

Calcium C-Test _Wako (Wako, Japan, 272-21801) was used for quantification of deposited calcium. Cells on the 33rd day of culture in differentiation medium (33 days after differentiation) were washed with 1X PBS, then replaced with 0.6N HCl solution, and the cells were collected with a cell scraper. The collected cells were rocked in 0.6N HCl solution for 24 hours at room temperature. After centrifugation, the supernatant was used for calcium quantification with Calcium C-Test _Wako . The amount of calcium deposition was calculated as the weight per well according to the manual attached to the kit.

(5) Results The results are shown in FIGS. 7 to 9, the full length expression cell line is “full length”, the control cell line is “control”, the cell line expressing a partial protein consisting only of the extracellular region is “extracellular region”, the extracellular region + A cell line expressing a partial protein consisting of a transmembrane region + a part of an intracellular region was designated as “extracellular + transmembrane region”.

  FIG. 7 is a graph showing alkaline phosphatase activity in a cell line expressing the full-length or partial protein of a mouse osteoblast differentiation inducing factor. As is clear from FIG. 7, the cell line expressing the partial protein consisting of the extracellular region + transmembrane region + part of the intracellular region is higher than the control cell line in any differentiation induction time, and is a full-length expressing cell line. Showed alkaline phosphatase activity equivalent to. In addition, the cell line expressing a partial protein consisting only of the extracellular region showed significantly higher alkaline phosphatase activity than the full-length expressing cell line.

  FIG. 8 is a graph showing the amount of calcium deposition in a cell line expressing the full length or partial protein of a mouse osteoblast differentiation inducing factor. As is clear from FIG. 8, the cell line expressing the partial protein consisting of the extracellular region + transmembrane region + part of the intracellular region is higher than the control cell line in any differentiation induction time, and is a full-length expressing cell line. Calcium deposition amount equal to or greater than A cell line expressing a partial protein consisting only of the extracellular region showed a significantly higher amount of calcium deposition than the full-length expressing cell line.

  FIG. 9 is a graph showing the amount of calcification in a cell line expressing the full length or partial protein of human osteoblast differentiation inducing factor. As is clear from FIG. 8, the cell line expressing a partial protein consisting of extracellular region + transmembrane region + part of intracellular region is higher in calcium than control cell line and full-length expressing cell line on the 33rd day of differentiation induction. Deposition amount was shown. A cell line expressing a partial protein consisting only of the extracellular region showed a significantly higher amount of calcium deposition than the full-length expressing cell line.

  From the above results, it was shown that the partial protein consisting of extracellular region + transmembrane region + part of intracellular region has the effect of promoting osteoblast differentiation, and the partial protein consisting only of the extracellular region is very It was shown to have a strong osteoblast differentiation promoting effect. That is, it was revealed that osteoblast differentiation promoting effects (alkali phosphatase activity induction, enhanced calcium deposition, etc.), which are physiological activities of osteoblast differentiation inducing factors, can also be obtained by the partial protein. As a partial protein, the promoting effect was obtained both in the case of only the extracellular region or in the case of including the extracellular region and the transmembrane region, and the effect was more remarkable than that due to the full-length protein. Furthermore, the promotion effect was seen most strongly in the case of only the extracellular region. These results are considered to suggest that the osteoblast-inducing factor has activity as a ligand from the viewpoint of cell-cell interaction.

It is the figure which compared the amino acid sequence of the osteoblast differentiation inducer protein between vertebrates. It is a photograph which shows the localization in the mouse | mouth preosteoblast of a human osteoblast differentiation inducer. (A) is a photograph showing the expression pattern of a mouse osteoblast differentiation inducing factor in the mouse developmental stage. (B) is a photograph comparing the expression of mouse osteoblast differentiation inducing factor with the expression of other mouse differentiation markers. It is a photograph showing the results of alkaline phosphatase staining and alizarin red staining of mouse preosteoblasts stably expressing human osteoblast differentiation inducing factors. It is a graph which shows the alkaline phosphatase activity and the amount of calcium deposition of the mouse | mouth preosteoblast which stably expressed the human osteoblast differentiation inducer. FIG. 4 is a schematic diagram showing a coding length of a mouse (human) osteoblast differentiation inducing factor, only an extracellular region, and part of the extracellular region + transmembrane region + intracellular region. It is a graph which shows the alkaline phosphatase activity in the cell strain which expresses the full length or partial protein of a mouse | mouth osteoblast differentiation inducer. It is a graph which shows the calcium deposition amount in the cell strain which expresses the full length or partial protein of a mouse | mouth osteoblast differentiation inducing factor. It is a graph which shows the calcium deposition amount in the cell strain which expresses the full length or partial protein of a human osteoblast differentiation inducer.

Claims (49)

A pharmaceutical comprising the following protein (1) or (2):
(1) Protein consisting of the amino acid sequence of SEQ ID NO: 1
(2) A protein comprising an amino acid sequence in which one or several amino acids are added, deleted, or substituted in SEQ ID NO: 1 and having osteoblast differentiation inducing or maturation promoting activity   The pharmaceutical according to claim 1, wherein the protein of (2) consists of the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13.   The medicament according to claim 1 or 2, wherein the medicament is an osteogenesis promoter.   The medicament according to claim 1 or 2, which is a therapeutic agent for osteoporosis, a bone system disease accompanied by bone loss, or a fracture. A pharmaceutical comprising the following polynucleotide (3) or (4):
(3) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2
(4) a polynucleotide that hybridizes with a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2 under stringent conditions and encodes a protein having osteoblast differentiation inducing or maturation promoting activity   The pharmaceutical according to claim 5, wherein the polynucleotide of (4) comprises the base sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14.   The medicament according to claim 5 or 6, wherein the medicament is an osteogenesis promoter.   The medicament according to claim 5 or 6, which is a therapeutic agent for osteoporosis, a bone system disease accompanied by bone loss, or a fracture. The pharmaceutical containing the antibody with respect to the following (1) or (2) protein, or the partial protein of (5) or (6).
(1) Protein consisting of the amino acid sequence of SEQ ID NO: 1
(2) A protein comprising an amino acid sequence in which one or several amino acids are added, deleted, or substituted in SEQ ID NO: 1 and having osteoblast differentiation inducing or maturation promoting activity
(5) A partial protein consisting of a partial sequence of SEQ ID NO: 1, comprising at least amino acids 26 to 94 of SEQ ID NO: 1 and having 117 or fewer amino acids
(6) It consists of an amino acid sequence in which one or several amino acids are added, deleted, or substituted in the partial sequence of SEQ ID NO: 1 described in (5) above, and has osteoblast differentiation induction or maturation promoting activity. Partial protein The protein of (2) consists of the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13,
The partial protein of (6)
(6-1) a partial protein consisting of a partial sequence of SEQ ID NO: 3 comprising at least amino acid numbers 26 to 94 of SEQ ID NO: 3 and having 117 or less amino acids,
(6-2) a partial protein consisting of a partial sequence of SEQ ID NO: 5, comprising at least amino acid numbers 19 to 89 of SEQ ID NO: 5 and having 112 or less amino acids,
(6-3) a partial protein consisting of a partial sequence of SEQ ID NO: 7, comprising at least amino acid numbers 16 to 89 of SEQ ID NO: 7 and having no more than 112 amino acids,
(6-4) a partial protein consisting of a partial sequence of SEQ ID NO: 9, comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 9, and having an amino acid number of 116 or less,
(6-5) a partial protein consisting of a partial sequence of SEQ ID NO: 11, comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 11 and having 116 or less amino acids, or
(6-6) The medicament according to claim 9, which is a partial protein consisting of a partial sequence of SEQ ID NO: 13, comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 13, and having 116 or less amino acids.   The medicament according to claim 9 or 10, wherein the medicament is an osteogenesis inhibitor.   The medicament according to claim 9 or 10, which is a therapeutic agent for ectopic ossification, ectopic calcification, or a disease caused thereby. A pharmaceutical comprising an siRNA comprising a base sequence complementary to 15 to 30 bases of the following polynucleotide (3) or (4) and having a total length of 30 bases or less.
(3) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2
(4) a polynucleotide that hybridizes with a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2 under stringent conditions and encodes a protein having osteoblast differentiation inducing or maturation promoting activity   14. The pharmaceutical according to claim 13, wherein the polynucleotide of (4) consists of the base acid sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14.   The medicine according to claim 13 or 14, wherein the medicine is an osteogenesis inhibitor.   The medicament according to claim 13 or 14, which is a therapeutic agent for ectopic ossification, ectopic calcification, or a disease caused thereby. Compare the expression level of the following protein (1) or (2) between the test cell and test substance mixing step and the test cell mixed with the test substance and the test cell not mixed with the test substance. And selecting a test substance that increases the expression level of the protein, a method for screening an osteogenesis promoter, an osteoporosis therapeutic agent, a therapeutic agent for bone system diseases accompanied by a decrease in bone mass, or a therapeutic agent for fracture.
(1) Protein consisting of the amino acid sequence of SEQ ID NO: 1
(2) A protein comprising an amino acid sequence in which one or several amino acids are added, deleted, or substituted in SEQ ID NO: 1 and having osteoblast differentiation inducing or maturation promoting activity Compare the expression level of the following protein (1) or (2) between the test cell and test substance mixing step and the test cell mixed with the test substance and the test cell not mixed with the test substance. And an osteogenesis inhibitor, an ectopic ossification therapeutic agent, an ectopic calcification therapeutic agent, or an ectopic ossification. Or the screening method of the therapeutic agent of the disease resulting from ectopic calcification.
(1) Protein consisting of the amino acid sequence of SEQ ID NO: 1
(2) A protein comprising an amino acid sequence in which one or several amino acids are added, deleted, or substituted in SEQ ID NO: 1 and having osteoblast differentiation inducing or maturation promoting activity Mixing a test cell and a test substance that differentiates into osteoblasts introduced with a vector containing the following polynucleotide (3) or (4), and mixing the test cell and the test substance mixed with the test substance Comparing the degree of osteoblast differentiation or maturation with a test cell that has not yet been selected, and selecting a test substance that promotes osteoblast differentiation or maturation of the test cell. And a screening method for a therapeutic agent for bone system diseases accompanied by a decrease in bone mass or a therapeutic agent for fracture.
(3) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2
(4) a polynucleotide that hybridizes with a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2 under stringent conditions and encodes a protein having osteoblast differentiation inducing or maturation promoting activity Mixing a test cell and a test substance that differentiates into osteoblasts introduced with a vector containing the polynucleotide of (3) or (4) below, and mixing the test cell and the test substance mixed with the test substance Osteogenesis inhibitor, ectopic, comprising a step of comparing the degree of osteoblast differentiation or maturation with a test cell that has not been tested and selecting a test substance that suppresses osteoblast differentiation or maturation of the test cell A screening method for a therapeutic agent for ossification, a therapeutic agent for ectopic calcification, or a therapeutic agent for diseases caused by ectopic ossification or ectopic calcification.
(3) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2
(4) a polynucleotide that hybridizes with a DNA comprising the nucleotide sequence of SEQ ID NO: 2 under a stringent condition and encodes a protein having osteoblast differentiation induction or maturation promoting activity In the presence or absence of a test substance, mixing a test cell introduced with a vector containing the following polynucleotide (3) or (4) with a cell that differentiates into an osteoblast, and the test substance: Comparing the degree of osteoblast differentiation or maturation in cells that differentiate into osteoblasts in the presence and absence and selecting a test substance that promotes osteoblast differentiation or maturation. A method for screening a formation promoter, an osteoporosis therapeutic agent, a therapeutic agent for bone system diseases accompanied by bone loss, or a therapeutic agent for fracture.
(3) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2
(4) a polynucleotide that hybridizes with a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2 under stringent conditions and encodes a protein having osteoblast differentiation inducing or maturation promoting activity In the presence or absence of a test substance, mixing a test cell introduced with a vector containing the following polynucleotide (3) or (4) with a cell that differentiates into an osteoblast, and the test substance: Comparing the degree of osteoblast differentiation or maturation in cells that differentiate into osteoblasts in the presence and absence, and selecting a test substance that suppresses osteoblast differentiation or maturation. A screening method for a formation inhibitor, a therapeutic agent for ectopic ossification, a therapeutic agent for ectopic calcification, or a therapeutic agent for a disease caused by ectopic ossification or ectopic calcification.
(3) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2
(4) a polynucleotide that hybridizes with a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2 under stringent conditions and encodes a protein having osteoblast differentiation inducing or maturation promoting activity In the presence or absence of the test substance, in the step of mixing the following (1) or (2) protein and cells that differentiate into osteoblasts, and in the presence and absence of the test substance, Comparing the degree of osteoblast differentiation or maturation in cells that differentiate into osteoblasts, and selecting a test substance that promotes osteoblast differentiation or maturation, an osteogenesis promoter, an osteoporosis therapeutic agent, and bone mass For screening a therapeutic agent for bone system diseases accompanied by a decrease in bone or a therapeutic agent for fractures.
(1) Protein consisting of the amino acid sequence of SEQ ID NO: 1
(2) A protein comprising an amino acid sequence in which one or several amino acids are added, deleted, or substituted in SEQ ID NO: 1 and having osteoblast differentiation inducing or maturation promoting activity In the presence or absence of the test substance, in the step of mixing the following (1) or (2) protein and cells that differentiate into osteoblasts, and in the presence and absence of the test substance, Comparing the degree of osteoblast differentiation or maturation in cells that differentiate into osteoblasts, and selecting a test substance that suppresses osteoblast differentiation or maturation. A screening method for a therapeutic agent, a therapeutic agent for ectopic calcification, or a therapeutic agent for a disease caused by ectopic ossification or ectopic calcification.
(1) Protein consisting of the amino acid sequence of SEQ ID NO: 1
(2) A protein comprising an amino acid sequence in which one or several amino acids are added, deleted, or substituted in SEQ ID NO: 1 and having osteoblast differentiation inducing or maturation promoting activity The pharmaceutical containing the partial protein of the following (5) or (6).
(5) A partial protein consisting of a partial sequence of SEQ ID NO: 1, comprising at least amino acids 26 to 94 of SEQ ID NO: 1 and having 117 or fewer amino acids
(6) It consists of an amino acid sequence in which one or several amino acids are added, deleted, or substituted in the partial sequence of SEQ ID NO: 1 described in (5) above, and has osteoblast differentiation induction or maturation promoting activity. Partial protein The protein of (6)
(6-1) a partial protein consisting of a partial sequence of SEQ ID NO: 3 comprising at least amino acid numbers 26 to 94 of SEQ ID NO: 3 and having 117 or less amino acids,
(6-2) a partial protein consisting of a partial sequence of SEQ ID NO: 5, comprising at least amino acid numbers 19 to 89 of SEQ ID NO: 5 and having 112 or less amino acids,
(6-3) a partial protein consisting of a partial sequence of SEQ ID NO: 7, comprising at least amino acid numbers 16 to 89 of SEQ ID NO: 7 and having no more than 112 amino acids,
(6-4) a partial protein consisting of a partial sequence of SEQ ID NO: 9, comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 9, and having an amino acid number of 116 or less,
(6-5) a partial protein consisting of a partial sequence of SEQ ID NO: 11, comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 11 and having 116 or less amino acids, or
(6-6) The medicament according to claim 25, which is a partial protein consisting of a partial sequence of SEQ ID NO: 13, comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 13, and having 116 or fewer amino acids.   The medicament according to claim 25 or 26, wherein the medicament is an osteogenesis promoter.   27. The medicament according to claim 25 or 26, which is a therapeutic agent for osteoporosis, a bone system disease accompanied by bone loss, or a fracture. The pharmaceutical containing the partial protein of the following (5) or (6 ').
(5) A partial protein consisting of a partial sequence of SEQ ID NO: 1, comprising at least amino acids 26 to 94 of SEQ ID NO: 1 and having 117 or fewer amino acids
(6 ') It consists of an amino acid sequence in which one or several amino acids are added, deleted, or substituted in the partial sequence of SEQ ID NO: 1 described in (5) above, and suppresses osteoblast differentiation induction or maturation Partial protein The protein of (6 ')
(6′-1) a partial protein consisting of a partial sequence of SEQ ID NO: 3, comprising at least amino acid numbers 26 to 94 of SEQ ID NO: 3 and having 117 or less amino acids,
(6′-2) a partial protein consisting of a partial sequence of SEQ ID NO: 5, comprising at least amino acid numbers 19 to 89 of SEQ ID NO: 5 and having 112 or less amino acids,
(6′-3) a partial protein consisting of a partial sequence of SEQ ID NO: 7, comprising at least amino acid numbers 16 to 89 of SEQ ID NO: 7 and having an amino acid number of 112 or less,
(6′-4) a partial protein consisting of a partial sequence of SEQ ID NO: 9, comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 9 and having 116 or less amino acids,
(6′-5) a partial protein consisting of a partial sequence of SEQ ID NO: 11, comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 11 and having 116 or less amino acids, or
(6'-6) The medicament according to claim 29, which is a partial protein consisting of a partial sequence of SEQ ID NO: 13, comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 13 and having 116 or less amino acids.   The medicament according to claim 29 or 30, wherein the medicament is an osteogenesis inhibitor.   The medicament according to claim 29 or 30, wherein the medicament is a therapeutic agent for ectopic ossification, ectopic calcification, or a disease caused thereby. A pharmaceutical comprising the following polynucleotide (7) or (8):
(7) A polynucleotide consisting of a partial sequence of SEQ ID NO: 2, which comprises at least the base numbers 76 to 282 of SEQ ID NO: 2 and encodes a partial protein having 117 or less amino acids
(8) a polynucleotide that hybridizes with the polynucleotide comprising the partial sequence of SEQ ID NO: 2 according to (7) above under stringent conditions and encodes a partial protein having osteoblast differentiation inducing or maturation promoting activity The polynucleotide of (8)
(8-1) a polynucleotide consisting of a partial sequence of SEQ ID NO: 4, which comprises at least the base numbers 76 to 282 of SEQ ID NO: 4 and encodes a partial protein having 117 or less amino acids,
(8-2) a polynucleotide comprising the partial sequence of SEQ ID NO: 6, which comprises at least the base numbers 55 to 267 of SEQ ID NO: 6 and encodes a partial protein having 112 or less amino acids,
(8-3) a polynucleotide comprising the partial sequence of SEQ ID NO: 8, which comprises at least the base numbers 46 to 267 of SEQ ID NO: 8 and encodes a partial protein having 112 or less amino acids,
(8-4) a polynucleotide comprising the partial sequence of SEQ ID NO: 10, comprising at least the base numbers 79 to 279 of SEQ ID NO: 10 and encoding a partial protein having 116 or less amino acids,
(8-5) a polynucleotide comprising the partial sequence of SEQ ID NO: 12, comprising at least the base numbers 79 to 279 of SEQ ID NO: 12 and encoding a partial protein having 116 or less amino acids, or
(8-6) The medicament according to claim 33, which is a polynucleotide comprising a partial sequence of SEQ ID NO: 4, which comprises at least the base numbers 79 to 279 of SEQ ID NO: 14 and encodes a partial protein having 116 or less amino acids.   The medicament according to claim 33 or 34, wherein the medicament is an osteogenesis promoter.   The medicament according to claim 33 or 34, which is a therapeutic agent for osteoporosis, a bone system disease accompanied by bone loss, or a fracture. A pharmaceutical comprising the following polynucleotide (7) or (8 '):
(7) A polynucleotide consisting of a partial sequence of SEQ ID NO: 2, which comprises at least the base numbers 76 to 282 of SEQ ID NO: 2 and encodes a partial protein having 117 or less amino acids
(8 ') encodes a partial protein that hybridizes with the polynucleotide comprising the partial sequence of SEQ ID NO: 2 described in (7) under stringent conditions and has an activity of suppressing osteoblast differentiation induction or maturation Polynucleotide (8 ') polynucleotide is
(8'-1) a polynucleotide comprising the partial sequence of SEQ ID NO: 4, which comprises at least the base numbers 76 to 282 of SEQ ID NO: 4 and encodes a partial protein having 117 or less amino acids,
(8′-2) a polynucleotide comprising the partial sequence of SEQ ID NO: 6, which comprises at least the base numbers 55 to 267 of SEQ ID NO: 6 and encodes a partial protein having 112 or less amino acids,
(8′-3) a polynucleotide comprising the partial sequence of SEQ ID NO: 8, which comprises at least the base numbers 46 to 267 of SEQ ID NO: 8 and encodes a partial protein having 112 or less amino acids,
(8'-4) a polynucleotide comprising the partial sequence of SEQ ID NO: 10, comprising at least the base numbers 79 to 279 of SEQ ID NO: 10 and encoding a partial protein having 116 or less amino acids,
(8′-5) a polynucleotide comprising the partial sequence of SEQ ID NO: 12, comprising at least the base numbers 79 to 279 of SEQ ID NO: 12 and encoding a partial protein having 116 or less amino acids, or
(8'-6) The pharmaceutical according to claim 37, which is a polynucleotide comprising a partial sequence of SEQ ID NO: 4, which comprises at least the base numbers 79 to 279 of SEQ ID NO: 14 and encodes a partial protein having 116 or less amino acids. .   The medicament according to claim 37 or 38, wherein the medicament is an osteogenesis inhibitor.   The medicament according to claim 37 or 38, which is a therapeutic agent for ectopic ossification, ectopic calcification, or a disease caused thereby. In the presence or absence of the test substance, the step of mixing the following partial protein (5) or (6) with cells that differentiate into osteoblasts, and in the presence or absence of the test substance Comparing the degree of osteoblast differentiation or maturation in cells that differentiate into osteoblasts, and selecting a test substance that promotes osteoblast differentiation or maturation, an osteogenesis promoter, an osteoporosis therapeutic agent, bone A method for screening for a therapeutic agent for bone system diseases accompanied by a decrease in the amount or a therapeutic agent for fractures.
(5) A partial protein consisting of a partial sequence of SEQ ID NO: 1, comprising at least amino acids 26 to 94 of SEQ ID NO: 1 and having 117 or fewer amino acids
(6) It consists of an amino acid sequence in which one or several amino acids are added, deleted, or substituted in the partial sequence of SEQ ID NO: 1 described in (5) above, and has osteoblast differentiation induction or maturation promoting activity. Partial protein The protein of (6)
(6-1) a partial protein consisting of a partial sequence of SEQ ID NO: 3 comprising at least amino acid numbers 26 to 94 of SEQ ID NO: 3 and having 117 or less amino acids,
(6-2) a partial protein consisting of a partial sequence of SEQ ID NO: 5, comprising at least amino acid numbers 19 to 89 of SEQ ID NO: 5 and having 112 or less amino acids,
(6-3) a partial protein consisting of a partial sequence of SEQ ID NO: 7, comprising at least amino acid numbers 16 to 89 of SEQ ID NO: 7 and having no more than 112 amino acids,
(6-4) a partial protein consisting of a partial sequence of SEQ ID NO: 9, comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 9, and having an amino acid number of 116 or less,
(6-5) a partial protein consisting of a partial sequence of SEQ ID NO: 11, comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 11 and having 116 or less amino acids, or
(6-6) The method according to claim 41, which is a partial protein consisting of a partial sequence of SEQ ID NO: 13, comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 13, and having 116 or fewer amino acids. In the presence or absence of the test substance, the step of mixing the following partial protein (5) or (6) with cells that differentiate into osteoblasts, and in the presence or absence of the test substance Comparing osteoblast differentiation or maturation in cells that differentiate into osteoblasts, and selecting a test substance that suppresses osteoblast differentiation or maturation, and an osteogenesis inhibitor, ectopic ossification Screening method for the above, a therapeutic agent for ectopic calcification, or a therapeutic agent for a disease caused by ectopic ossification or ectopic calcification.
(5) A partial protein consisting of a partial sequence of SEQ ID NO: 1, comprising at least amino acids 26 to 94 of SEQ ID NO: 1 and having 117 or fewer amino acids
(6) It consists of an amino acid sequence in which one or several amino acids are added, deleted, or substituted in the partial sequence of SEQ ID NO: 1 described in (5) above, and has osteoblast differentiation induction or maturation promoting activity. Partial protein The protein of (6)
(6-1) a partial protein consisting of a partial sequence of SEQ ID NO: 3 comprising at least amino acid numbers 26 to 94 of SEQ ID NO: 3 and having 117 or less amino acids,
(6-2) a partial protein consisting of a partial sequence of SEQ ID NO: 5, comprising at least amino acid numbers 19 to 89 of SEQ ID NO: 5 and having 112 or less amino acids,
(6-3) a partial protein consisting of a partial sequence of SEQ ID NO: 7, comprising at least amino acid numbers 16 to 89 of SEQ ID NO: 7 and having no more than 112 amino acids,
(6-4) a partial protein consisting of a partial sequence of SEQ ID NO: 9, comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 9, and having an amino acid number of 116 or less,
(6-5) a partial protein consisting of a partial sequence of SEQ ID NO: 11, comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 11 and having 116 or less amino acids, or
(6-6) The method according to claim 43, which is a partial protein consisting of a partial sequence of SEQ ID NO: 13, comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 13, and having 116 or fewer amino acids. A diagnostic agent comprising an antibody that specifically binds to the following protein (1) or (2), or partial protein (5) or (6).
(1) Protein consisting of the amino acid sequence of SEQ ID NO: 1
(2) A protein comprising an amino acid sequence in which one or several amino acids are added, deleted, or substituted in SEQ ID NO: 1 and having osteoblast differentiation inducing or maturation promoting activity
(5) A partial protein consisting of a partial sequence of SEQ ID NO: 1, comprising at least amino acids 26 to 94 of SEQ ID NO: 1 and having 117 or fewer amino acids
(6) It consists of an amino acid sequence in which one or several amino acids are added, deleted, or substituted in the partial sequence of SEQ ID NO: 1 described in (5) above, and has osteoblast differentiation induction or maturation promoting activity. Partial protein The protein of (2) consists of the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13,
The protein of (6)
(6-1) a partial protein consisting of a partial sequence of SEQ ID NO: 3 comprising at least amino acid numbers 26 to 94 of SEQ ID NO: 3 and having 117 or less amino acids,
(6-2) a partial protein consisting of a partial sequence of SEQ ID NO: 5, comprising at least amino acid numbers 19 to 89 of SEQ ID NO: 5 and having 112 or less amino acids,
(6-3) a partial protein consisting of a partial sequence of SEQ ID NO: 7, comprising at least amino acid numbers 16 to 89 of SEQ ID NO: 7 and having no more than 112 amino acids,
(6-4) a partial protein consisting of a partial sequence of SEQ ID NO: 9, comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 9, and having an amino acid number of 116 or less,
(6-5) a partial protein consisting of a partial sequence of SEQ ID NO: 11, comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 11 and having 116 or less amino acids, or
(6-6) The diagnostic agent according to claim 45, which is a partial protein consisting of a partial sequence of SEQ ID NO: 13, comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 13 and having 116 or less amino acids.   The diagnostic agent according to claim 45 or 46, which is used for diagnosis of ectopic ossification, ectopic calcification, or a disease caused thereby. The following (1) or (2) protein or (5) or (6) partial protein and a test substance are contacted, and a test substance that specifically binds to the protein or the partial protein is selected. And a method for screening a diagnostic agent for ectopic ossification, ectopic calcification, or a disease caused by these.
(1) Protein consisting of the amino acid sequence of SEQ ID NO: 1
(2) A protein comprising an amino acid sequence in which one or several amino acids are added, deleted, or substituted in SEQ ID NO: 1 and having osteoblast differentiation inducing or maturation promoting activity
(5) A partial protein consisting of a partial sequence of SEQ ID NO: 1, comprising at least amino acids 26 to 94 of SEQ ID NO: 1 and having 117 or fewer amino acids
(6) It consists of an amino acid sequence in which one or several amino acids are added, deleted, or substituted in the partial sequence of SEQ ID NO: 1 described in (5) above, and has osteoblast differentiation induction or maturation promoting activity. Partial protein The protein of (2) consists of the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13,
The protein of (6)
(6-1) a partial protein consisting of a partial sequence of SEQ ID NO: 3 comprising at least amino acid numbers 26 to 94 of SEQ ID NO: 3 and having 117 or less amino acids,
(6-2) a partial protein consisting of a partial sequence of SEQ ID NO: 5, comprising at least amino acid numbers 19 to 89 of SEQ ID NO: 5 and having 112 or less amino acids,
(6-3) a partial protein consisting of a partial sequence of SEQ ID NO: 7, comprising at least amino acid numbers 16 to 89 of SEQ ID NO: 7 and having no more than 112 amino acids,
(6-4) a partial protein consisting of a partial sequence of SEQ ID NO: 9, comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 9, and having an amino acid number of 116 or less,
(6-5) a partial protein consisting of a partial sequence of SEQ ID NO: 11, comprising at least amino acid numbers 27 to 93 of SEQ ID NO: 11 and having 116 or less amino acids, or
(6-6) The method according to claim 48, which is a partial protein consisting of a partial sequence of SEQ ID NO: 13, which comprises at least amino acid numbers 27 to 93 of SEQ ID NO: 13 and has 116 or fewer amino acids.