Bone regeneration material

Abstract

Provided is a bone regeneration material that can provide sufficient therapeutic effects when applied to a bone defect site. The bone regeneration material according to the present invention contains a protein (A). The protein (A) has a polypeptide chain (Y) and / or a polypeptide chain (Y'). The total number of the polypeptide chain (Y) and the polypeptide chain (Y') in the protein (A) is 1-100. The polypeptide chain (Y) includes 2-200 contiguous repeats of an amino acid sequence (X), the amino acid sequence (X) being at least one of: the VPGVG sequence (1), which is the amino acid sequence represented by SEQ ID NO: 1; the GVGVP sequence (4), which is the amino acid sequence represented by SEQ ID NO: 4; and the GAHGPAGPK sequence (3), which is the amino acid sequence represented by SEQ ID NO: 3. The polypeptide chain (Y') is derived from the polypeptide chain (Y) by substituting 5% or less of the amino acid residues with lysine and / or arginine residues, and the total number of the lysine and arginine residues is 1-100.

Description

bone regeneration material

[0001] This invention relates to a bone regeneration material.

[0002] Hydroxyapatite is known to be useful as an adsorbent, a material for tooth and bone formation, and a cell scaffold.

[0003] For example, Patent Document 1 discloses a method for producing a hydroxyapatite-containing porous support, comprising the steps of: dispersing a carrier and hydroxyapatite, or a carrier with hydroxyapatite attached to its surface, in a dispersion medium to obtain a dispersion; and drying the dispersion. It also discloses the use of the hydroxyapatite-containing porous support as a bone regeneration material.

[0004] Japanese Patent Publication No. 2020-83715

[0005] Although hydroxyapatite functions to some extent as a material for tooth and bone formation and as a cell scaffold, it has low biocompatibility. When hydroxyapatite is applied to areas with bone loss, it takes a long time for the area to regenerate, resulting in insufficient therapeutic effects.

[0006] This invention was made to solve the above problems, and the object of this invention is to provide a bone regeneration material that can obtain sufficient therapeutic effect when applied to a site of bone loss.

[0007] The present inventors have diligently conducted research to solve the above problems and have arrived at the present invention. That is, the present invention is a bone regeneration material containing protein (A), wherein protein (A) has polypeptide chains (Y) and / or polypeptide chains (Y'), the total number of polypeptide chains (Y) and polypeptide chains (Y') in protein (A) is 1 to 100, the polypeptide chain (Y) is a polypeptide chain in which at least one amino acid sequence (X) from the amino acid sequence VPGVG sequence (1) shown in SEQ ID NO: 1, the amino acid sequence GVGVP sequence (4) shown in SEQ ID NO: 4, and the amino acid sequence GAHGPAGPK sequence (3) shown in SEQ ID NO: 3 is 2 to 200 consecutive units, and the polypeptide chain (Y') is a polypeptide chain in which 5% or less of the amino acids in polypeptide chain (Y) are substituted with lysine and / or arginine, the total number of lysine and arginine is 1 to 100.

[0008] According to the present invention, it is possible to provide a bone regeneration material that can be applied to areas with bone defects and provide sufficient therapeutic effects.

[0009] The present invention will be described in detail below. The bone regeneration material of the present invention contains protein (A).

[0010] The protein (A) in the present invention has a polypeptide chain (Y) and / or a polypeptide chain (Y'), the total number of polypeptide chains (Y) and (Y') in the protein (A) is 1 to 100, the polypeptide chain (Y) is a polypeptide chain in which at least one amino acid sequence (X) from among the amino acid sequences shown in SEQ ID NO: 1 (VPGVG sequence (1), SEQ ID NO: 4 (GVGVP sequence (4)), and SEQ ID NO: 3 (GAHGPAGPK sequence (3)) is 2 to 200 consecutive units, the polypeptide chain (Y') is a polypeptide chain in which 5% or less of the amino acids in the polypeptide chain (Y) are replaced with lysine and / or arginine, the total number of lysine and arginine is 1 to 100.

[0011] The polypeptide chain (Y) may consist of one type of amino acid sequence (X), or two or more types.

[0012] From the viewpoint of bone regeneration ability and biocompatibility, the VPGVG sequence (1) and the GVGVP sequence (4) are preferred as the amino acid sequence (X).

[0013] Specifically, the polypeptide chain (Y) is (VPVGVG) ｂ Array, (GVGVP) ｃ Sequence and (GAHGPAGPK) ｄ Examples include sequences, etc. Note that b to d are integers from 2 to 200, representing the number of consecutive amino acid sequences (X). If a protein (A) molecule contains multiple polypeptide chains (Y), the polypeptide chains (Y) may be identical or different (VPGVG). ｂ Array, (GVGVP) ｃ Sequence and (GAHGPAGPK) ｄ Protein (A) may have one selected from a group of sequences, or it may have two or more. Furthermore, if protein (A) contains multiple polypeptide chains (Y), the number of consecutive amino acid sequences (X) may be the same or different for each polypeptide chain (Y). That is, there may be multiple polypeptide chains (Y) with the same number of consecutive amino acid sequences (X) b-d, or there may be multiple polypeptide chains (Y) with different numbers of b-d. From the viewpoint of bone regeneration ability and biocompatibility, the polypeptide chain (Y) may be (VPGVG). ｂ Sequence and (GVGVP) ｃ The arrangement is preferable.

[0014] The polypeptide chain (Y) is a polypeptide chain having 2 to 200 consecutive amino acid sequences (X) (where b to d are 2 to 200). However, from the viewpoint of bone regeneration ability and biocompatibility, the number of consecutive amino acid sequences (X) is preferably 2 to 100 (where b to d are 2 to 100), more preferably 2 to 50 (where b to d are 2 to 50), and particularly preferably 2 to 40 (where b to d are 2 to 40).

[0015] Polypeptide chain (Y') is a polypeptide chain in which 5% or less of the amino acids in polypeptide chain (Y) are substituted with lysine and / or arginine, and the total number of substituted lysine and arginine atoms is between 1 and 100.

[0016] Whether a protein (A) is a polypeptide chain (Y') is determined by whether replacing all lysine (K) and arginine (R) in the protein (A) sequence with other amino acids [glycine (G), alanine (A), valine (V), proline (P), or histidine (H)] results in a polypeptide chain (Y).

[0017] In the polypeptide chain (Y'), the proportion of substituted lysine and / or arginine is preferably 0.06% to 5%, more preferably 0.5% to 5%, and particularly preferably 1% to 5%, from the viewpoint of bone regeneration ability and biocompatibility.

[0018] Furthermore, the polypeptide chain (Y') may contain an amino acid sequence (X') in which 60% or less of the amino acids in the amino acid sequence (X) are replaced with lysine and / or arginine. Moreover, the types of amino acid sequences (X) and / or amino acid sequences (X') constituting the polypeptide chain (Y') may be one type each, or two or more types.

[0019] Specifically, examples of amino acid sequences (X') include the GKGVP sequence (7), which is the amino acid sequence shown in SEQ ID NO: 7; the GKGKP sequence (8), which is the amino acid sequence shown in SEQ ID NO: 8; the GKGRP sequence (9), which is the amino acid sequence shown in SEQ ID NO: 9; and the GRGRP sequence (10), which is the amino acid sequence shown in SEQ ID NO: 10. From the viewpoint of bone regeneration ability and biocompatibility, at least one sequence selected from the group consisting of the GKGVP sequence (7), the GKGKP sequence (8), and the GRGRP sequence (10) is preferred for the amino acid sequence (X'), and more preferably the GKGVP sequence (7) and the GKGKP sequence (8). The total number of polypeptide chains (Y) and polypeptide chains (Y') in one molecule of protein (A) is 1 to 100. Furthermore, the total number of these is preferably 1 to 80, and more preferably 1 to 60.

[0020] When the total number of polypeptide chains (Y) and polypeptide chains (Y') in one molecule of protein (A) is within the above range, it is preferable from the viewpoints of bone regeneration ability and biocompatibility.

[0021] When protein (A) has polypeptide chains (Y) with different types and / or consecutive numbers of amino acid sequences (X), each is counted as one, and the number of polypeptide chains (Y) is the total thereof. The same applies to polypeptide chain (Y'). Protein (A) in the present invention preferably satisfies the following relational expression (1). 0.50 ≦ [total number of amino acids constituting amino acid sequence (X) contained in protein (A) and amino acids constituting amino acid sequence (X') contained in protein (A)] / [total number of amino acids constituting protein (A)] ≦ 0.80 (1)

[0022] The above ratio of the number of amino acids can be determined by a protein sequencer. Specifically, it can be determined by the following measurement method. <Measurement method> Using two or more cleavage methods that can cleave at specific amino acid residues, protein (A) is decomposed to about 30 residues or less. Then, after separation by high performance liquid chromatography (HPLC), the amino acid sequence is read by a protein sequencer. Peptide mapping is performed from the obtained amino acid sequence to determine the entire sequence of protein (A). Then, "[total number of amino acids constituting amino acid sequence (X) contained in protein (A) and amino acids constituting amino acid sequence (X') contained in protein (A)] / [total number of amino acids constituting protein (A)]" is calculated.

[0023] Protein (A) preferably has a polypeptide chain (S) in which 2 to 50 consecutive GAGAGS sequences (2) shown in SEQ ID NO: 2 are bound from the viewpoints of bone regeneration ability and biocompatibility. In polypeptide chain (S), the number of consecutive GAGAGS sequences (2) is preferably 2 to 40, more preferably 2 to 30, and particularly preferably 2 to 10 from the viewpoints of bone regeneration ability and biocompatibility.

[0024] In protein (A), the ratio of the number of amino acids in all GAGAGS sequences (2) to the total number of amino acids in protein (A) [{number of GAGAGS sequences (2) in protein (A) × 6} / {total number of amino acids in protein (A)} × 100] is preferably 5 to 50%, more preferably 10 to 47.5%, and particularly preferably 20 to 45% from the viewpoint of bone regeneration ability and biocompatibility. The ratio of the number of amino acids in all GAGAGS sequences (2) to the total number of amino acids in protein (A) can be determined by a protein sequencer. Specifically, it is determined by the following measurement method.

[0025] <Ratio of the number of amino acids in all GAGAGS sequences (2) to the total number of amino acids in protein (A)> Protein (A) is degraded to approximately 30 residues or less using two or more cleavage methods that can cleave at specific amino acid residues. After separation by high-performance liquid chromatography (HPLC), the amino acid sequence is read using a protein sequencer. The entire sequence of protein (A) is determined by peptide mapping from the obtained amino acid sequence. Then, the ratio of the number of amino acids in all GAGAGS sequences (2) to the total number of amino acids in protein (A) is calculated using the following formula: Ratio of the number of amino acids in all GAGAGS sequences (2) to the total number of amino acids in protein (A) (%) = [{Number of GAGAGS sequences (2) × 6} / {Total number of amino acids in protein (A)}] × 100

[0026] If protein (A) has a total of two or more polypeptide chains selected from the group consisting of polypeptide chain (Y), polypeptide chain (Y'), and polypeptide chain (S), it may have an intervening amino acid sequence (Z) between them. The intervening amino acid sequence (Z) is a peptide sequence in which one or more amino acids are linked together, and is not a GAGAGS sequence (2), amino acid sequence (X), or amino acid sequence (X'). From the viewpoint of bone regeneration ability and biocompatibility, the number of amino acids constituting the intervening amino acid sequence (Z) is preferably 1 to 30, more preferably 1 to 15, and particularly preferably 1 to 10. Specific examples of the intervening amino acid sequence (Z) include the VAAGY sequence (11), which is the amino acid sequence shown in SEQ ID NO: 11, and the GAAGY sequence (12), which is the amino acid sequence shown in SEQ ID NO: 12. The ratio of the number of amino acids in all intervening amino acid sequences (Z) to the total number of amino acids in protein (A) [Σ{(number of amino acids in intervening amino acid sequences (Z)) × (number of intervening amino acid sequences (Z))} / {total number of amino acids in protein (A)} × 100] is preferably 0 to 25%, more preferably 0 to 22.5%, and particularly preferably 0.01 to 15% from the viewpoint of bone regeneration ability and biocompatibility.

[0027] Protein (A) may have a terminal amino acid sequence (T) at its end, in addition to the GAGAGS sequence (2), amino acid sequence (X), amino acid sequence (X'), and intervening amino acid sequence (Z), from the viewpoint of biodegradability. The terminal amino acid sequence (T) may be located at one end or at both ends of protein (A). The terminal amino acid sequence (T) does not include the purification tag described later. The terminal structure of protein (A) is preferably a structure in which the terminal amino acid sequence (T) is attached to a polypeptide chain (Y). The terminal amino acid sequence (T) is a peptide sequence in which one or more amino acids are attached, and is a peptide sequence that is not the GAGAGS sequence (2), amino acid sequence (X), or amino acid sequence (X'). From the viewpoint of biodegradability, the number of amino acids constituting the terminal amino acid sequence (T) is preferably 1 to 100, more preferably 1 to 50, and particularly preferably 1 to 40. Examples of terminal amino acid sequences (T) include the MDPVVLQRRDWENPGVTQLNRLAAAHPPFASDPM sequence (13), which is the amino acid sequence shown in Sequence ID No. 13.

[0028] The ratio of the number of amino acids in the terminal amino acid sequence (T) to the total number of amino acids in the protein (A) is preferably 0 to 25%, more preferably 0 to 22.5%, and particularly preferably 0.01 to 15%, from the viewpoint of biodegradability.

[0029] Protein (A) may be produced using bacteria by biotechnology, as described below. In such cases, to facilitate the purification or detection of the expressed protein (A), protein (A) may have a protein or peptide (hereinafter referred to as "purification tag") with a special amino acid sequence at the N or C terminus, in addition to its terminal amino acid sequence (T). Affinity purification tags are used as purification tags. Examples of such purification tags include the 6×His tag, V5 tag, Xpress tag, AU1 tag, T7 tag, VSV-G tag, DDDDK tag, S tag, CruzTag09™, CruzTag22™, CruzTag41™, Glu-Glu tag, Ha.11 tag, and KT3 tag, all of which are made of polyhistidine. Below are examples of combinations of each purification tag (i) and the ligand (ii) that recognizes and binds to that tag. (i-1) Glutathione-S-transferase (GST) (ii-1) Glutathione (i-2) Maltose-binding protein (MBP) (ii-2) Amylose (i-3) HQ tag (ii-3) Nickel (i-4) Myc tag (ii-4) Anti-Myc antibody (i-5) HA tag (ii-5) Anti-HA antibody (i-6) FLAG tag (ii-6) Anti-FLAG antibody (i-7) 6×His tag (ii-7) Nickel or cobalt Methods for introducing the above purified tag sequences include inserting the nucleic acid encoding the purified tag at the 5' or 3' end of the nucleic acid encoding protein (A) in the expression vector, or using a commercially available purified tag introduction vector.

[0030] In protein (A), the ratio of the total number of amino acids in all intervening amino acid sequences (Z) constituting protein (A), the total number of amino acids in all terminal amino acid sequences (T) constituting protein (A), and the total number of amino acids in the purified tag is preferably 0 to 25%, more preferably 0 to 22.5%, and particularly preferably 0.01 to 15%, based on the total number of amino acids in protein (A), from the viewpoint of biodegradability.

[0031] In the case where the protein (A) contains the polypeptide chain (Y) and / or the polypeptide chain (Y') and the polypeptide chain (S), from the viewpoints of bone regeneration ability and biocompatibility, it is preferable that the polypeptide chain (Y) or the polypeptide chain (Y') and the polypeptide chain (S) are alternately chemically bonded.

[0032] In the protein (A), the ratio of the number of GAGAGS sequences (2) to the total number of amino acid sequences (X) and amino acid sequences (X') (GAGAGS sequence (2): total of amino acid sequences (X) and amino acid sequences (X')) is preferably 1:1.5 to 1:20, more preferably 1:1.5 to 1:6, and particularly preferably 1:2 to 1:5 from the viewpoints of bone regeneration ability and biocompatibility. Some examples of the preferred protein (A) are shown below.

[0033] (A1): A protein in which the amino acid sequence (X) is the GVGVP sequence (4) (A11): A protein having a polypeptide chain (Y'1) in which one amino acid in the polypeptide chain (Y1) with 2 to 200 consecutive GVGVP sequences (4) is replaced by lysine (K) (A11-1): A protein having a polypeptide chain (Y'1) and a polypeptide chain (S1) with 2 to 200 consecutive GAGAGS sequences (2) (A11-2): The amino acid sequence shown in SEQ ID NO: 14 with 8 consecutive GVGVP sequences (4) is (GVGVP) ８ The amino acid sequence shown in SEQ ID NO: 6 in which one amino acid of the polypeptide chain (Y11) of the sequence (14) is replaced by lysine (K) is (GVGVP) ４ GKGVP (GVGVP) ３ A protein having the sequence (6) (Y'11) and a polypeptide chain (S1) with 2 to 200 consecutive GAGAGS sequences (2)

[0034] (A11-2-1): The amino acid sequence shown in SEQ ID NO: 5 with 4 consecutive GAGAGS sequences (2) is (GAGAGS) ４ The polypeptide chain (S1-1) of the sequence (5) and (GVGVP) shown in SEQ ID NO: 6 ４ GKGVP (GVGVP) ３The protein (A11-2-1) having sequence (6) specifically includes SELP8K and SELP8K4, which have the following amino acid sequences: SELP8K: (GAGAGS) ４ Array (5) 12 times and (GVGVP) ４ GKGVP (GVGVP) ３ The amino acid sequence shown in Sequence ID No. 15 (GAGAGS) is formed by having 13 sequences (6) that are chemically bonded alternately. ２ The protein SELP8K4:(GAGAGS) has a molecular weight of approximately 80 kDa and possesses the amino acid sequence shown in Sequence ID No. 16, which has a structure in which sequence (15) is chemically bonded. ４ Sequence (5) and (GVGVP) ４ GKGVP (GVGVP) ３ A protein having the amino acid sequence shown in Sequence ID No. 27, with a molecular weight of approximately 30 kDa, has a structure in which each of the sequences (6) is chemically bonded alternately.

[0035] (A11-2-2): This is the amino acid sequence shown in Sequence ID No. 15, which consists of two consecutive GAGAGS sequences (2). ２ The polypeptide chain (S1-2) is sequence (15), and (GVGVP) ４ GKGVP (GVGVP) ３ The protein (A11-2-2) having sequence (6) specifically contains SELP0K, which has the following amino acid sequence: SELP0K: (GAGAGS) ２ Sequence (15) and (GVGVP) ４ GKGVP (GVGVP) ３ A protein having the amino acid sequence shown in Sequence ID No. 17, with a molecular mass of approximately 82 kDa, has a structure in which each of sequence (6) is chemically bonded alternately.

[0036] (A11-3): This is the amino acid sequence shown in Sequence ID No. 18, in which one amino acid of a polypeptide chain consisting of 12 consecutive GVGVP sequences (4) is substituted with lysine (K). ６ GKGVP (GVGVP) ５A protein having sequence (18) (Y'12) and a polypeptide chain (S1) consisting of 2 to 200 consecutive GAGAGS sequences (2) (A11-3-1): This is the amino acid sequence shown in SEQ ID NO: 19, which consists of four consecutive GAGAGS sequences (2) (GAGAGS). ４ Array (19) and (GVGVP) ６ The protein (A11-3-1) containing the GKGVP (GVGVP) 5 sequence (18) specifically includes SELP8K12, which has the following amino acid sequence: SELP8K12: (GAGAGS) ４ Sequence (19) 12 times and (GVGVP) ６ GKGVP (GVGVP) ５ A structure having 13 sequences (18) that are chemically bonded alternately is called (GAGAGS). ２ A protein having the amino acid sequence shown in Sequence ID No. 20, with a molecular mass of approximately 105 kDa, having a structure in which sequence (15) is chemically bonded.

[0037] (A2): A protein whose amino acid sequence (X) is the VPGVG sequence (1). (A21): A protein having a polypeptide chain (Y2) consisting of 2 to 200 consecutive VPGVG sequences (1) and a GAGAGS sequence (2). Specifically, the protein of (A21) includes ELP1.1 having the following amino acid sequence: ELP1.1: GAGAGS sequence (2), the amino acid sequence shown in SEQ ID NO: 24 (VPPGVG). ４ The amino acid sequence is shown in sequence (24) and sequence number 25 (VPVG). ８ Each of the arrays (25) has 40 copies, and these are (VPGVG) ４ Sequence (24), GAGAGS sequence (2), (VPGVG) ８ A protein with a molecular weight of approximately 200 kDa having the amino acid sequence shown in Sequence ID No. 26, which has a structure in which 40 blocks are chemically bonded together in the order of sequence (25).

[0038] (A3): A protein having a polypeptide chain (Y1) consisting of 2 to 200 consecutive GVGVP sequences (4) and a polypeptide chain (S1) consisting of 2 to 200 consecutive GAGAGS sequences (2). Specifically, the protein of (A3) includes SELP6.1 having the following amino acid sequence: SELP6.1: The amino acid sequence shown in SEQ ID NO: 21 (GAGAGS) ８ The amino acid sequence is shown in sequence (21) and sequence number 22 (GVGVP). ４０ A protein having the amino acid sequence shown in Sequence ID No. 23, with a molecular mass of approximately 110 kDa, has a structure in which each of sequence (22) is chemically bonded alternately.

[0039] Among these, it is preferable that SELP8K has the amino acid sequence shown in SEQ ID NO: 16, SELP0K has the amino acid sequence shown in SEQ ID NO: 17, SELP8K12 has the amino acid sequence shown in SEQ ID NO: 20, SELP6.1 has the amino acid sequence shown in SEQ ID NO: 23, ELP1.1 has the amino acid sequence shown in SEQ ID NO: 26, or SELP8K4 has the amino acid sequence shown in SEQ ID NO: 27. Furthermore, protein (A) may be a protein having an amino acid sequence that is 70% or more identical to the amino acid sequence of SELP8K having the amino acid sequence shown in SEQ ID NO: 16, SELP0K having the amino acid sequence shown in SEQ ID NO: 17, SELP8K12 having the amino acid sequence shown in SEQ ID NO: 20, SELP6.1 having the amino acid sequence shown in SEQ ID NO: 23, ELP1.1 having the amino acid sequence shown in SEQ ID NO: 26, or SELP8K4 having the amino acid sequence shown in SEQ ID NO: 27. Moreover, this identity is preferably 80% or more, and more preferably 90% or more.

[0040] The molecular weight of protein (A) determined by SDS-PAGE (SDS polyacrylamide gel electrophoresis) is preferably 15 to 200 kDa, more preferably 30 to 150 kDa, and particularly preferably 70 to 120 kDa, from the viewpoint of biodegradability.

[0041] In the present invention, the hydrophobicity of protein (A) is preferably 0.2 to 1.2, more preferably 0.4 to 1.0, and particularly preferably 0.42 to 0.80, from the viewpoint of bone regeneration ability and biocompatibility. The hydrophobicity of protein (A) indicates the degree of hydrophobicity of the protein (A) molecule and can be calculated by applying the number of amino acid residues constituting the protein (A) molecule (Mα), the hydrophobicity of each amino acid (Nα), and the total number of amino acid residues in one protein (A) molecule (MT) to the following formula. The hydrophobicity of each amino acid is taken from the following values ​​described in non-patent literature (Albert L. Lehninger, David L. Nelson, Lehninger's New Biochemistry, Vol. 1, Hirokawa Shoten, September 2010, pp. 346-347). Hydrophobicity = Σ(Mα × Nα) / (MT) Mα: Number of amino acid residues in one molecule of protein (A) Nα: Hydrophobicity of each amino acid MT: Total number of amino acid residues in one molecule of protein (A) A (Alanine): 1.8 R (Arginine): -4.5 N (Asparagine): -3.5 D (Aspartic acid): -3.5 C (Cysteine): 2.5 Q (Glutamine): -3.5 E (Glutamic acid): -3.5 G (Glycine): -0.4 H (Histidine): -3.2 I (Isoleucine): 4.5 L (Leucine): 3.8 K (Lysine): -3.9 M (Methionine): 1.9 F (Phenylalanine): 2.8 P (Proline): -1.6 S (Serine): -0.8 T (Threonine): -0.7 W (tryptophan): -0.9 Y (tyrosine): -1.3 V (valine): 4.2 For example, protein (A) is (GVGVP) ４ GKGVP (GVGVP) ３ If the sequence is (6), the hydrophobicity of protein (A) = {16 (number of Gs) × (-0.4) + 15 (number of Vs) × 4.2 + 8 (number of Ps) × (-1.6) + 1 (number of Ks) × (-3.9)} / 40 (total number of amino acid residues) = 1.0.

[0042] In the present invention, protein (A) can be obtained by extraction from natural products, organic synthesis (enzymatic methods, solid-phase synthesis, and liquid-phase synthesis, etc.), and genetic engineering. Regarding organic synthesis, methods described in "Biochemistry Experiment Course 1, Protein Chemistry IV (July 1, 1981, edited by the Japanese Biochemical Society, published by Tokyo Kagaku Dojin Co., Ltd.)" and "Continued Biochemistry Experiment Course 2, Protein Chemistry (Part 2) (May 20, 1987, edited by the Japanese Biochemical Society, published by Tokyo Kagaku Dojin Co., Ltd.)" can be applied. Regarding genetic engineering, methods described in Japanese Patent Publication No. 3338441 can be applied. While protein (A) can be obtained by extraction from natural products, organic synthesis, and genetic engineering, genetic engineering is preferred from the viewpoint that the amino acid sequence can be easily modified and mass production can be carried out at low cost.

[0043] In the bone regeneration material of the present invention, the weight percentage of protein (A) is preferably 90 to 100 wt%, and more preferably 95 to 100 wt%, from the viewpoint of bone regeneration ability and biocompatibility.

[0044] In the bone regeneration material of the present invention, protein (A) may be spongy, gel-like, or cotton-like fibrous, but it is preferably spongy. Spongy protein (A) can be produced by placing an aqueous solution of protein (A) in a container of a predetermined shape and freeze-drying it.

[0045] The bone regeneration material of the present invention may further contain a reinforcing material (B). Examples of reinforcing material (B) include hydroxyapatite, calcium phosphate, bone material, and hydrophobically treated protein. These reinforcing materials (B) function as a cell scaffold for osteoblasts and can improve the bone regeneration ability of the bone regeneration material of the present invention.

[0046] In the bone regeneration material of the present invention, the volume ratio of the reinforcing material (B) to the volume of the protein (A) ([volume of reinforcing material (B)] / [volume of protein (A)]) is preferably 1 to 20, and more preferably 2 to 10, from the viewpoint of bone regeneration ability and biocompatibility.

[0047] For example, it is preferable to use hydroxyapatite in powder or granular form.

[0048] In the bone regeneration material of the present invention, the weight percentage of hydroxyapatite is preferably 1.5 to 6,000 wt%, and more preferably 5 to 2,000 wt%, based on the weight of protein (A), from the viewpoint of bone regeneration ability and biocompatibility.

[0049] It is preferable to use β-tricalcium phosphate (β-TCP) or octacalcium phosphate (OCP) as the calcium phosphate.

[0050] In the bone regeneration material of the present invention, the weight percentage of calcium phosphate is preferably 1.5 to 6,000 wt%, and more preferably 5 to 2,000 wt%, based on the weight of protein (A), from the viewpoint of bone regeneration ability and biocompatibility.

[0051] The bone material may be the same type of bone material as the part in which the bone regeneration material of the present invention is used, or it may be a different type of bone material, but it is preferable that it be the same type of bone material.

[0052] Furthermore, the bone material may be derived from the patient to whom the bone regeneration material of the present invention is applied, or it may be derived from another person.

[0053] The volume-average particle size of the bone material is preferably 50 to 2,000 μm, and more preferably 100 to 1,000 μm.

[0054] In the bone regeneration material of the present invention, the weight ratio of the bone material is preferably 1.5 to 6,000 wt%, and more preferably 5 to 2,000 wt%, based on the weight of protein (A), from the viewpoint of bone regeneration ability and biocompatibility.

[0055] The hydrophobically treated protein is preferably a hydrophobically treated protein (B) which is a hydrophobicized version of protein (A).

[0056] The following is an example of a method for hydrophobicizing protein (A). First, protein (A) is immersed in a solvent such as an alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol, and butanol, etc.). The immersion time is preferably 1 to 168 hours. Next, protein (A) is removed from the solvent and dried. The drying method is not particularly limited and can be air-dried or hot-air-dried. When air-drying is performed, it is preferable to dry at 5 to 30°C for 1 to 168 hours. By this method, hydrophobic protein (B) can be obtained.

[0057] The hydrophobic protein (B) preferably satisfies the following relationship (2): 0.01 ≤ Q / P (2) [In relationship (2), P is the weight of the dried product (DP) obtained by drying the hydrophobic protein (B) at 1 atmosphere and 100°C for 3 hours, and then drying it at 1 atmosphere and 40°C for 15 hours; Q is the weight of the dried product (DQ) obtained by immersing the above dried product (DP) in water 1,000 times the weight of P at 1 atmosphere and 25°C for 72 hours, then removing it, drying it at 1 atmosphere and 100°C for 3 hours, and then drying it at 1 atmosphere and 40°C for 15 hours.]

[0058] Furthermore, it is preferable that the hydrophobic protein (B) satisfies the following relationship (3): 0.3 ≤ R / P (3) [In relationship (3), P is the weight of the dried product (DP) obtained by drying the hydrophobic protein (B) at 1 atmosphere and 100°C for 3 hours, and then drying it at 1 atmosphere and 40°C for 15 hours; R is the weight of the dried product (DR) obtained by immersing the above dried product (DP) in water 1,000 times the weight of P at 1 atmosphere and 25°C for 2 hours, then removing it, drying it at 1 atmosphere and 100°C for 3 hours, and then drying it at 1 atmosphere and 40°C for 15 hours.]

[0059] If the hydrophobic protein (B) satisfies the above relations (2) and (3), it means that the hydrophobic protein (B) is sufficiently hydrophobic.

[0060] In the bone regeneration material of the present invention, the weight percentage of hydrophobic treated protein is preferably 1.5 to 6,000 wt%, and more preferably 5 to 2,000 wt%, based on the weight of protein (A), from the viewpoint of bone regeneration ability and biocompatibility.

[0061] The bone regeneration material of the present invention may contain water, an inorganic salt, and / or phosphoric acid (salt) in addition to the reinforcing material (B).

[0062] Examples of inorganic salts include sodium chloride, potassium chloride, calcium chloride, magnesium chloride, sodium sulfate, potassium sulfate, calcium sulfate, magnesium sulfate, sodium bicarbonate, potassium bicarbonate, calcium bicarbonate, and magnesium bicarbonate. Note that phosphoric acid (salt) is not included in inorganic salts in this specification. From the viewpoint of bone regeneration ability and biocompatibility, the weight percentage of inorganic salts in the bone regeneration material is preferably 0 to 3 wt%, more preferably 0 to 1 wt%, and even more preferably 0.001 to 0.5 wt%, based on the weight of protein (A).

[0063] In this specification, phosphoric acid (salt) means phosphoric acid and / or phosphates other than calcium phosphate. Examples of phosphates include alkali metal salts and alkaline earth metal salts of phosphoric acid, specifically sodium salts, potassium salts, and magnesium salts. From the viewpoint of bone regeneration ability and biocompatibility, the weight percentage of phosphoric acid (salt) in the bone regeneration material is preferably 0.001 to 2 wt%, more preferably 0.001 to 0.5 wt%, and even more preferably 0.001 to 0.05 wt%, based on the weight of protein (A).

[0064] The bone regeneration material of the present invention can promote bone regeneration when applied to areas where bone is missing (i.e., areas of bone loss). Examples of areas to which the bone regeneration material of the present invention can be applied include the skull, nasal bone, cheekbone, maxilla, mandible, palatine bone, clavicle, scapula, humerus, forearm (radius, ulna), pelvic girdle (ilium, ischium, pubis), femur, patella, tibia, fibula, tarsal bones, etc.

[0065] The bone regeneration material of the present invention can suitably regenerate bone in the affected area when applied to wounds and diseases such as fractures, bone defects, and bone depressions resulting from trauma, bone tumors, osteomyelitis, chronic joint diseases, osteoporosis, and surgery. Furthermore, the use of bone regeneration materials to treat fractures, bone defects, and bone depressions resulting from trauma, bone tumors, osteomyelitis, chronic joint diseases, osteoporosis, and surgery constitutes the use of the bone regeneration material of the present invention.

[0066] This specification contains the following information:

[0067] (1) The present disclosure is a bone regeneration material containing a protein (A), wherein the protein (A) has a polypeptide chain (Y) and / or a polypeptide chain (Y'), the total number of polypeptide chains (Y) and (Y') in the protein (A) is 1 to 100, the polypeptide chain (Y) is a polypeptide chain in which at least one amino acid sequence (X) from the amino acid sequence VPGVG sequence (1) shown in SEQ ID NO: 1, the amino acid sequence GVGVP sequence (4) shown in SEQ ID NO: 4, and the amino acid sequence GAHGPAGPK sequence (3) shown in SEQ ID NO: 3 is 2 to 200 consecutive units, and the polypeptide chain (Y') is a polypeptide chain in which 5% or less of the amino acids in the polypeptide chain (Y) are substituted with lysine and / or arginine, the total number of lysine and arginine is 1 to 100.

[0068] Disclosure (2) further comprises a reinforcing material (B), wherein the reinforcing material (B) is at least one selected from the group consisting of hydroxyapatite, calcium phosphate, bone material, and hydrophobically treated protein, as described in Disclosure (1).

[0069] Disclosure (3) is the bone regeneration material according to Disclosure (2), wherein the hydrophobic treated protein is a hydrophobic treated protein (B) which is a hydrophobic version of the protein (A).

[0070] The present disclosure (4) is a bone regeneration material according to any one of the present disclosures (1) to (3), wherein the protein (A) has the amino acid sequence shown in SEQ ID NO: 16, the amino acid sequence shown in SEQ ID NO: 17, the amino acid sequence shown in SEQ ID NO: 20, the amino acid sequence shown in SEQ ID NO: 23, the amino acid sequence shown in SEQ ID NO: 26, the amino acid sequence shown in SEQ ID NO: 27, or an amino acid sequence that is 70% or more identical to these amino acid sequences.

[0071] Disclosure (5) states that the bone regeneration material is a bone regeneration material according to any one of Disclosures (1) to (4) that is applied to a bone defect.

[0072] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0073] <Production Example 1> [Preparation of SELP8K] ○Preparation of SELP8K-producing strain Plasmid pPTS0345 encoding SELP8K was prepared according to the method described in the examples of Japanese Patent Publication No. 4088341. The prepared plasmid was transformed into E. coli to obtain a SELP8K-producing strain. Hereinafter, this SELP8K-producing strain was used to produce (GAGAGS), a type of protein (A). ４ Array (5) 12 times and (GVGVP) ４ GKGVP (GVGVP) ３ This invention provides a method for producing SELP8K (protein (A-1)), which has the amino acid sequence shown in Sequence ID No. 16, having a molecular mass of approximately 80 kDa, and having a structure in which thirteen sequences (6) are chemically bonded alternately.

[0074] ○Culturing of SELP8K-producing strains The overnight culture solution of SELP8K-producing strains grown at 30°C was inoculated into 50 ml of LB medium in a 250 ml flask. Kanamycin was added to the LB medium to a final concentration of 50 μg / ml to form a culture medium, and this culture medium was incubated at 30°C with stirring (200 rpm). When the turbidity of the culture medium reached OD600 = 0.8 (using a UV1700 spectrophotometer manufactured by Shimadzu Corporation), 40 ml of the culture medium was transferred to another flask warmed to 42°C and cultured at 42°C for approximately 2 hours. After that, the cultured culture medium was cooled on ice, the turbidity OD600 of the culture medium was measured, and E. coli was collected by centrifugation.

[0075] Using purified E. coli cells containing SELP8K, the protein was purified from the E. coli biomass by the following steps: 1) cell lysis, 2) removal of insoluble fragments by centrifugation, 3) ammonium sulfate precipitation, 4) ultrafiltration, 5) cation exchange chromatography, 6) ultrafiltration, and 7) freeze-drying. In this way, purified SELP8K (protein (A-1)) with a molecular weight of approximately 80 kDa was obtained.

[0076] Step 1: To 100 g of collected E. coli, 200 g of deionized water was added, and the cells were lysed using a high-pressure homogenizer (55 MPa) to obtain a cell lysate containing the lysed cells. Subsequently, the cell lysate was adjusted to pH 4.0 with glacial acetic acid.

[0077] Step 2: Removal of insoluble fragments by centrifugation. The bacterial lysate was then centrifuged (6300 rpm, 4°C, 30 minutes) and the supernatant was collected.

[0078] Step 3: Ammonium sulfate precipitation. A saturated ammonium sulfate solution was added to the supernatant recovered in Step 2 to achieve an ammonium sulfate concentration of 25 wt%. After standing for 8 to 12 hours, the precipitate was collected by centrifugation. The collected precipitate was dissolved in deionized water. Next, a saturated ammonium sulfate solution was added to the dissolved solution to achieve an ammonium sulfate concentration of 25 wt%. After standing for 8 to 12 hours, the precipitate was collected by centrifugation. The collected precipitate was dissolved in deionized water to obtain a solution.

[0079] Step 4: The solution obtained in Step 3 was subjected to an ultrafiltration apparatus (holofiber: manufactured by GE Healthcare) with a molecular weight cut of 30,000. The solution obtained in Step 3 was subjected to ultrafiltration using 20 times the amount of deionized water to obtain the polypeptide solution after ultrafiltration.

[0080] Step 5: The polypeptide solution, after ultrafiltration, was added to 10 mM sodium acetate buffer to achieve a polypeptide concentration of 20 g / L. This solution was then subjected to an AKTAPrime (Amersham) equipped with a HiPrepSP XL16 / 10 cation exchange column (GE Healthcare). The eluted fraction was collected using 500 mM sodium acetate buffer as the eluate.

[0081] Step 6: The eluted fraction obtained in ultrafiltration step 5 was treated in the same manner as in "4: Ultrafiltration" above to obtain the polypeptide solution after ultrafiltration.

[0082] Step 7: The polypeptide solution obtained in Step 6 was diluted with deionized water so that the freeze-dried polypeptide concentration was 3 g / L, and placed in a stainless steel tray so that the water level was 10 mm or less. Then, it was placed in a freeze-dryer (manufactured by Nippon Techno Service Co., Ltd.) and frozen at -30°C for 24 hours. After freezing, primary drying was performed at -30°C for 110 hours under a vacuum of 5 Pa or less, and secondary drying was performed at 30°C for 48 hours under a vacuum of 5 Pa or less to obtain SELP8K (protein (A-1)). Protein (A-1) was identified using the Western blotting method described below. Furthermore, for protein (A-1), the ratio of "[total number of amino acids constituting amino acid sequence (X) contained in protein (A) and amino acids constituting amino acid sequence (X') contained in protein (A)] / [total number of amino acids constituting protein (A)]" is 0.54. The ratio of the number of GAGAGS sequences (2) to the total number of amino acid sequences (X) and amino acid sequences (X') in one molecule of protein (A-1) (GAGAGS sequence (2): total of amino acid sequences (X) and amino acid sequences (X')) is 1:2.

[0083] Step 8: The protein (A-1) obtained in molding step 7 is diluted with deionized water to obtain SELP8K solution [concentration of protein (A-1): 12.5 g / L], which is then applied to a base area of ​​1.9 mm. ２ One ml was placed in a cylindrical mold with a depth of 1.7 mm, and freeze-dried under the same conditions as in step 7 to form a sponge shape.

[0084] ○Identification of SELP8K (A-1) Analysis was performed by Western blotting using rabbit anti-SELP8K antibody and rabbit anti-6×His antibody (Rockland) against the 6×His tag of the C-terminal sequence. The procedure for Western blotting was as follows. A band showing antibody reactivity was observed at the apparent molecular mass of 80 kDa for each antibody. Furthermore, Table 1 shows the amino acid composition ratio (measured value) of protein (A-1) obtained by amino acid composition analysis using an amino acid analysis system (Prominence, Shimadzu Corporation) and the amino acid composition ratio (theoretical value) of SELP8K inferred from the synthetic gene sequence. From these, it can be seen that protein (A-1) consists of 13 polypeptide chains (Y'11) of the (GVGVP) 4GKGVP (GVGVP) 3 sequence (6), in which one of the valine (V) molecules in the polypeptide chain (Y) consisting of eight consecutive GVGVP sequences (4) is replaced with lysine (K), and 4 consecutive GAGAGS sequences (2) (GAGAGS). ４ The molecule has 12 polypeptide chains (S1-1) of sequence (5), which are chemically bonded alternately, and the amino acid sequence shown in Sequence ID No. 15 is (GAGAGS). ２ It was confirmed that the protein (SELP8K) has the amino acid sequence shown in SEQ ID NO: 16, and that the sequence (15) has a structure formed by chemical bonding.

[0085]

[0086] <Western Blotting Method> 20 μL of Western blot sample was mixed with 10 μL of 3×SDS buffer [containing 150 mM Tris HCl (pH 6.8), 300 mM dithiothreitol, 6 wt% sodium dodecyl sulfate (SDS), 0.3 wt% bromophenol blue, and 30 wt% glycerol] and heated at 95°C for 5 minutes to prepare the electrophoresis sample. 15 μL of this electrophoresis sample was used for SDS-PAGE. The gel after electrophoresis was transferred to a polyvinylidene fluoride membrane (hereinafter abbreviated as "membrane"), and the membrane was blocked by immersion in blocking buffer [containing 20 mM Tris (pH 7.6), 137 mM NaCl, 0.1 wt% Tween 20, and 5 wt% skim milk] and shaking at room temperature for 1 hour. After blocking treatment, the membrane was washed for 2 minutes with TBS-T [containing 20 mM Tris (pH 7.6), 137 mM NaCl, and 0.1 wt% Tween 20]. Next, the membrane was immersed in the primary antibody solution (primary antibody: anti-SELP8K antibody and anti-His-tag antibody (manufactured by Rockland) diluted 1 / 500 with TBS-T) and left to stand overnight at 4°C to allow the antibody reaction to occur. After the reaction, the membrane was washed four times with TBS-T for 5 minutes each. Then, the membrane was immersed in a solution of a secondary antibody that could bind to the primary antibody and was conjugated with horseradish peroxidase as a labeling enzyme (secondary antibody: ECL anti-rabbit IgG HRP linked F(ab')2 fragment (GE Healthcare) diluted 1 / 2000 with TBS-T), and allowed to stand at room temperature for 30 minutes to allow the antibody reaction to occur. After the reaction, the membrane was washed four times with TBS-T for 5 minutes each, and then the enzymatic reaction was performed using the ECL-Advance Western Blotting Detection Kit (GE Healthcare). The membrane was exposed to light using a luminometer ForECL (GE Healthcare), and the bands were observed using a chemiluminescence fluorescence imaging system.

[0087] <Production Example 2> [Preparation of hydrophobic SELP8K (B-1)] SELP8K (A-1) after "Step 8: Molding" in <Production Example 1> above was immersed in methanol and left to stand at 4°C for 16 hours. After 16 hours, SELP8K (A-1) was removed and air-dried at 25°C for 16 hours. Through these steps, hydrophobic SELP8K (B-1) was obtained.

[0088] <Example 1> A bone regeneration material was obtained using "SELP8K," a protein (A-1) produced in Production Example 1, as the structure. The evaluation described below was performed using this bone regeneration material.

[0089] <Comparative Example 1> Instead of the protein (A-1) produced in Production Example 1, a hydrophobized SELP8K (B-1) produced in Production Example 2 was used as the structure to obtain a bone regeneration material. The evaluation described below was performed using this bone regeneration material.

[0090] <Comparative Example 2> A bone regeneration material was obtained in the same manner as in Comparative Example 1, except that "hydrophobized SELP8K (B-1)" was replaced with "hydroxyapatite". The evaluation described below was performed using this bone regeneration material.

[0091] <Comparative Example 3> A bone regeneration material was obtained in the same manner as in Comparative Example 1, except that "hydrophobized SELP8K (B-1)" was replaced with "β-TCP". The evaluation described below was performed using this bone regeneration material. Evaluation Method

[0092] (1) Evaluation of bone regeneration ability (deformation angle measurement) SD rats (female, 8 weeks old) were subjected to inhalation anesthesia, and the tibia was exposed to create a cylindrical defect with a diameter of 5 mm and a depth of 2 mm. 4 mg of bone regeneration material was administered to the defect, and after wound closure, the rats were kept for a predetermined period. After 8 weeks, the tibia was harvested and micro-CT images were taken. The angle between the proximal tibial axis and the distal bone axis was measured from the micro-CT images. The results are shown in Table 2.

[0093] (2) Evaluation of remodeling The area of ​​bone defects was measured from micro-CT images taken to evaluate bone regeneration capacity (deformation angle measurement). The results are shown in Table 2.

[0094]

[0095] As shown in Table 2, when the bone regeneration material according to Example 1 was used, deformation was less likely to occur during bone regeneration, and the bone defect area was small, indicating high bone regeneration capacity. From these results, it can be said that the bone regeneration material of the present invention can improve bone regeneration capacity when applied to areas with bone defects, and also exhibits excellent biocompatibility.

[0096] The bone regeneration material of the present invention exhibits high bone regeneration ability when applied to areas with bone loss, and also possesses excellent biocompatibility, making it suitable for various applications, including medical use.

Claims

1. A bone regeneration material containing protein (A), wherein protein (A) has polypeptide chains (Y) and / or polypeptide chains (Y'), the total number of polypeptide chains (Y) and polypeptide chains (Y') in protein (A) is 1 to 100, the polypeptide chain (Y) is a polypeptide chain in which at least one amino acid sequence (X) from the amino acid sequence VPGVG sequence (1) shown in SEQ ID NO: 1, the amino acid sequence GVGVP sequence (4) shown in SEQ ID NO: 4, and the amino acid sequence GAHGPAGPK sequence (3) shown in SEQ ID NO: 3 is 2 to 200 consecutive units, and the polypeptide chain (Y') is a polypeptide chain in which 5% or less of the amino acids in polypeptide chain (Y) are substituted with lysine and / or arginine, the total number of lysine and arginine units is 1 to 100.

2. The bone regeneration material according to claim 1, further comprising a reinforcing material (B), wherein the reinforcing material (B) is at least one selected from the group consisting of hydroxyapatite, calcium phosphate, bone material, and hydrophobically treated protein.

3. The bone regeneration material according to claim 2, wherein the hydrophobic treated protein is a hydrophobic treated protein (B) which is a hydrophobicized version of protein (A).

4. The bone regeneration material according to any one of claims 1 to 3, wherein the protein (A) has the amino acid sequence shown in SEQ ID NO: 16, the amino acid sequence shown in SEQ ID NO: 17, the amino acid sequence shown in SEQ ID NO: 20, the amino acid sequence shown in SEQ ID NO: 23, the amino acid sequence shown in SEQ ID NO: 26, the amino acid sequence shown in SEQ ID NO: 27, or an amino acid sequence that is 70% or more identical to these amino acid sequences.

5. The bone regeneration material according to any one of claims 1 to 3, wherein the bone regeneration material is applied to a bone defect.

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