JP6077316B2 - Tissue regeneration material and tissue regeneration protein solution - Google Patents

Description

  The present invention relates to a tissue regeneration material and a tissue regeneration protein solution.

  Conventionally, gauze, absorbent cotton, etc. have been used as wound dressings that are temporarily applied to affected areas, such as burns, skin wounds, skin exfoliated wounds, and traumatic skin defect wounds, to protect affected areas due to wounds. ing. Further, in wounds lacking the dermis layer, a carboxymethylcellulose gel such as an intrasite gel or a granu gel that maintains a moist environment on the wound surface in order to promote the formation of granulation tissue as a wound dressing (Patent Documents 1, 2) ) And collagen sponge (Patent Document 3) is used as a scaffold for cells forming granulation tissue. These coating materials do not have high affinity with tissues, particularly cells, and the coating materials themselves do not have therapeutic effects such as an epithelialization promoting effect. Therefore, the treatment depends on the natural healing power until the wound closure, and there is a problem that it takes time until the wound closure (the state in which epithelialization is completed). In particular, there is no covering material (tissue regeneration material) that promotes epithelialization and regenerates tissue, and there is a need for a tissue regeneration material that promotes epithelialization.

Japanese Patent No. 2664539 Japanese Patent No. 3485593 Japanese Patent Laid-Open No. 10-080438

  An object of the present invention is to provide a tissue regeneration material that can shorten the time required for wound closure.

The tissue regeneration material of the present invention is a tissue regeneration material containing protein (Z) and protein (A), wherein (Z) is a polypeptide chain (Y) and / or a polypeptide chain (Y )), The total number of (Y) and (Y ′) in (Z) is 1 to 40 , and the degree of hydrophobicity is 0.2 to 1.2. Is a protein having a structure in which 13 of RGD sequences and 12 of (GAGAGS) ₉ sequences (53) are alternately located , and (Z) and (A) in the tissue regeneration material A tissue regeneration material having a weight ratio ((Z) / (A)) of 400/1 to 10/1; a protein solution for tissue regeneration containing the tissue regeneration material and water.
Polypeptide chain (Y): VPGVG sequence (2) or GVGVP sequence (3) A polypeptide chain having 2 to 160 consecutive amino acid sequences (X).
Polypeptide chain (Y ′): 1-2 amino acids in (Y) Lysine A polypeptide chain that is replaced with

  The tissue regeneration material and the tissue regeneration protein solution of the present invention have an effect that the time required for wound closure can be shortened.

  In the present invention, the protein (Z) and the protein (A) may be extracted from natural products or artificially produced, but are artificially produced from the viewpoint of eliminating animal-derived components. Are preferred. Moreover, as a method used for artificial production, it can be produced by an organic synthesis method (enzyme method, solid phase synthesis method, liquid phase synthesis method, etc.), a gene recombination method, or the like. For organic synthesis methods, see "Biochemistry Experiment Course 1, Protein Chemistry IV (July 1, 1981, edited by the Japanese Biochemical Society, published by Tokyo Chemical Co., Ltd.)" or "Second Biochemistry Experiment Course 2, Protein Chemistry" (Lower) (May 20, 1987, edited by the Japanese Biochemical Society, published by Tokyo Chemical Co., Ltd.), etc. Regarding the gene recombination method, the method described in Japanese Patent No. 3338441 can be applied. Although both the organic synthesis method and the gene recombination method can produce the proteins (Z) and (A), the gene recombination method is preferable from the viewpoint that the amino acid sequence can be easily changed and mass production can be performed at low cost.

  Since the tissue regeneration material of the present invention contains proteins (Z) and (A), it has high affinity with cells, promotes cell proliferation (particularly epithelial cell proliferation), and promotes epithelialization. The time required for wound closure can be shortened. Further, since the proteins (Z) and (A) are proteins, they are highly biodegradable, and the tissue regeneration material in the skin is easily replaced with granulation tissue after wound closure (epithelialization). Therefore, wound healing is accelerated by using the tissue regeneration material of the present invention.

The tissue regeneration material of the present invention is a tissue regeneration material containing protein (Z) and protein (A), wherein (Z) is a polypeptide chain (Y) and / or a polypeptide chain (Y )), The total number of (Y) and (Y ′) in (Z) is 1 to 100, and the hydrophobicity is 0.2 to 1.2. ) Is a protein having at least one of the following minimum amino acid sequences (B) in one molecule, and the weight ratio ((Z) / (A)) of (Z) to (A) in the tissue regeneration material is 400. It is a tissue regeneration material that is / 1 to 10/1.
Polypeptide chain (Y): a polypeptide comprising 2 to 200 consecutive amino acid sequences (X) of any one of VPGVG sequence (2), GVGVP sequence (3), GPP sequence, GAP sequence and GAHGGPGPK sequence (4) chain.
Polypeptide chain (Y ′): A polypeptide chain in which 1 to 100 amino acids in (Y) are each substituted with lysine or arginine.
Minimum amino acid sequence (B): RGD sequence, LDV sequence, REDV sequence (17), YIGSR sequence (18), PDSGR sequence (19), RYVVLPR sequence (20), LGTIPG sequence (21), RNIAEIIKDI sequence (22), IKVAV At least one amino acid sequence selected from the group consisting of sequence (23), LRE sequence, DGEA sequence (24) and HAV sequence;

The protein (Z) has the following polypeptide chain (Y) and / or the following polypeptide chain (Y ′), and the total number of (Y) and (Y ′) in (Z) is 1 to 100 It is a protein having a hydrophobicity of 0.2 to 1.2.
Polypeptide chain (Y): a polypeptide comprising 2 to 200 consecutive amino acid sequences (X) of any one of VPGVG sequence (2), GVGVP sequence (3), GPP sequence, GAP sequence and GAHGGPGPK sequence (4) chain.
Polypeptide chain (Y ′): A polypeptide chain in which 1 to 100 amino acids in (Y) are each substituted with lysine or arginine.
When (Z) has two or more (Y), the types of amino acid sequences (X) constituting each (Y) may be the same or different. That is, the polypeptide chain (Y) may be (Z) having one selected from (VPGVG) _b sequence, (GVGVP) _c sequence, (GPP) _d sequence, (GAP) _e sequence and (GAHGGPGPK) _f sequence. (Z) having two or more kinds selected from (VPGVG) _b sequence, (GVGVP) _c sequence, (GPP) _d sequence, (GAP) _e sequence and (GAHGGPGPK) _f sequence as polypeptide chain (Y) May be. (Here, b to f are the consecutive numbers of the amino acid sequence (X), respectively, and are integers of 2 to 200).

Of the amino acid sequence (X), the VPGVG sequence (2) and the GVGVP sequence (3) are preferable from the viewpoint of cell affinity. That is, when (Z) has a polypeptide chain (Y), (Z) has (VPGVG) _b sequence as the polypeptide chain (Y) and (Z GVGVP) Those having the _c sequence are preferred.
When two or more polypeptide chains (Y) having different amino acid sequences (X) are used, from the viewpoint of cell affinity and shortening of wound closure time, (VPGVG) _b sequence, (GPP) _d sequence, (GVGVP) _c Two or more types selected from a sequence and a (GAHGGPGPK) _f sequence are preferable, and a (GVGVP) _c sequence and a (GAHGGPGPK) _f sequence are particularly preferable.

  When the protein (Z) has a plurality of the same kind of polypeptide chains (Y) in the amino acid sequence (X), the number of consecutive (X) may be the same or different for each (Y). Good. That is, the above b to f may have a plurality of the same polypeptide chains (Y), or may have a plurality of polypeptide chains (Y) having different numbers (b) to b of (X).

  The polypeptide chain (Y) is a sequence in which 2 to 200 amino acid sequences (X) are continuous (b to f are 2 to 200), but from the viewpoint of cell affinity and shortening of wound closure time, the polypeptide chain (Y) is a continuous number. Is preferably 2 to 50 (b to f are 2 to 50), more preferably 2 to 30 (b to f are 2 to 30).

In the polypeptide chain (Y ′), the amino acid sequence (X) constituting the polypeptide chain (Y) is partially substituted with the following amino acid sequence (X ′), and 1 to 100 in the polypeptide (Y) Are polypeptide chains in which the amino acids are substituted with K (lysine) or R (arginine), respectively. Whether it is a polypeptide chain (Y ′) or not is determined when all K and R in the sequence of the protein (Z) are replaced with other amino acids (G, A, V, P or H). Judgment is made depending on whether the chain (Y) is formed.
Amino acid sequence (X ′): An amino acid sequence in which 1 to 2 amino acids in amino acid sequence (X) are substituted with lysine or arginine, respectively.
In (Y ′), the number of amino acids to be substituted among the amino acids in (Y) is preferably 1 to 70, more preferably 1 to 30, from the viewpoint of solubility in water.

  The amino acid sequence (X ′) is preferably GKGVP sequence (13), GKGKP sequence (14), GKGRP sequence (15) and GRGRP sequence (16) from the viewpoint of solubility in water and appropriate hydrophobicity. More preferred are GKGVP sequence (13) and GKGKP sequence (14).

  When the protein (Z) has (Y) having different types and / or consecutive numbers of (X), each is counted as one, and the number of (Y) is the total. The same applies to (Y ′).

  The protein (Z) has a total of 1 to 100 polypeptide chains (Y) and / or polypeptide chains (Y ′) in one molecule (Z), but 1 to 80 from the viewpoint of shortening the wound closure time. Is preferable, and 1 to 60 is particularly preferable.

In the present invention, the degree of hydrophobicity of (Z) is 0.2 to 1.2, and from the viewpoint of solubility of (Z) in water and gelation, 0.3 to 1.2 is preferable, and more preferable. Is 0.4 to 1.2, particularly preferably 0.5 to 1.0.
The degree of hydrophobicity of (Z) indicates the degree of hydrophobicity of (Z) molecule. (Z) The number of amino acids constituting the molecule (Mα), the degree of hydrophobicity of each amino acid (Nα) And (Z) The total number of amino acids in one molecule can be calculated by applying the following formula. The hydrophobicity of each amino acid is described in the following numerical values in non-patent literature (Leninger's new chemistry, p.346-347).
Hydrophobicity = {Σ (Mα × Nα)} / (M _T )
Mα: (Z) Number of each amino acid in one molecule Nα: Hydrophobicity of each amino acid M _T : (Z) Total number of amino acids in one molecule 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, when (Z) is (GVGVP) ₄ GKGVP (GVGVP) ₃ sequence (8), the degree of hydrophobicity of (Z) = {16 (number of G) × (−0.4) +15 (V Number) × 4.2 + 8 (number of P) × (−1.6) +1 (number of K) × (−3.9)} / 40 (total number of amino acids) = 1.00.

In the present invention, the protein (Z) may further have a GAGAGS sequence (1). When (Z) has a GAGAGS sequence (1), from the viewpoint of gelation of (Z), it has a polypeptide chain (S) in which 2 to 100 GAGAGS sequences (1) are continuously linked. It is preferable.
In the polypeptide chain (S), the number of consecutive sequences (1) is preferably 2 to 100, more preferably 2 to 50, and further preferably 3 to 40, from the viewpoint of gelation. Especially preferably 4 to 30.
In (Z), when having a polypeptide chain (S), (Z) it is sufficient to have one (S) in one molecule. From the viewpoint of gelation, 1 to 20 is preferable, and more preferably 3 to 10 pieces.

  When the protein (Z) has a total of two or more polypeptide chains (Y), polypeptide chains (Y ′) and polypeptide chains (S), an intervening amino acid is interposed between the polypeptide chain and the polypeptide chain. You may have arrangement | sequence (W). (W) is a peptide sequence in which one amino acid, one amino acid sequence (X), one amino acid sequence (X ′), or two or more amino acids are linked. The number of amino acids constituting (W) is preferably 1-30, more preferably 1-15, and particularly preferably 1-10, from the viewpoint of cell affinity and shortening the wound closure time. Specific examples of (W) include VAAGY sequence (5), GAAGY sequence (6), and LGP sequence.

  The protein (Z) may have a terminal amino acid sequence (T) at both ends in addition to (Y), (Y ′), and (S). In the case of having (T), the structure of both ends of (Z) is preferably a structure in which (T) is bonded to (Y) or (Y ′). (T) has one amino acid, an amino acid sequence ( X) One peptide sequence, one amino acid sequence (X ′) or two or more amino acid bonds. From the viewpoint of cell affinity, the number of amino acids constituting (T) is preferably 1 to 100, more preferably 1 to 50, and particularly preferably 1 to 40. Specific examples of (T) include MDPVVLQRRDWENPGVTQLNRLAAHPPFASDPM sequence (7) and the like.

In addition to the above (T), the protein (Z) is a protein or peptide having a special amino acid sequence at the N and / or C terminus of (Z) in order to facilitate purification or detection of the expressed (Z) (Hereinafter referred to as “purification tags”). As the purification tag, an affinity purification tag is used. Examples of such purification tags include polyhistidine 6 × His tag, GTS tag, MBP tag, HQ tag, Myc tag, HA tag, FLAG tag, V5 tag, Xpress tag, AU1 tag, T7 tag, VSV-G. Tag, DDDDK tag, S tag, CruzTag09 ^™ , CruzTag22 ^™ , CruzTag41 ^™ , Glu-Glu tag, Ha. There are 11 tags and KT3 tags.
An example of a combination of each purification tag (i) and a ligand (ii) that recognizes and binds the tag is shown below.
(I-1) glutathione-S-transferase (GTS) (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 XHis tag (ii-7) Nickel or cobalt As a method for introducing the purified tag sequence, a nucleic acid encoding a purified tag is inserted into the 5 'or 3' end of a nucleic acid encoding a protein (Z) in an expression vector. And a method using a commercially available vector for introducing a purification tag.

  The total content (% by weight) of amino acid sequence (X) and amino acid sequence (X ′) in one molecule of protein (Z) is based on the molecular mass of (Z) from the viewpoint of cell affinity and shortening of wound closure time. Is preferably 10 to 80% by weight, and more preferably 15 to 75% by weight.

The total content of the amino acid sequence (X) and the amino acid sequence (X ′) in the protein (Z) can be determined by a protein sequencer. Specifically, it is measured by the following measurement method.
<Method for Measuring Content of Amino Acid Sequence (X) and Amino Acid Sequence (X ′)>
The protein (Z) is decomposed to about 30 residues or less by using two or more kinds of cleavage methods that can cleave at a specific amino acid residue. Then, after separating by high performance liquid chromatography (HPLC), the amino acid sequence is read with a protein sequencer. Peptide mapping is performed from the obtained amino acid sequence to determine the entire protein (Z) sequence. Thereafter, the total content of the amino acid sequence (X) and the amino acid sequence (X ′) is measured by the measurement formula described below.
Total content (%) of amino acid sequence (X) and amino acid sequence (X ′) = [{molecular mass of amino acid sequence (X)} × {number of amino acid sequence (X)} + {of amino acid sequence (X ′) Molecular mass} × {number of amino acid sequences (X ′)}] / {molecular mass of (Z)} × 100

  Ratio of the number of GAGAGS sequence (1), amino acid sequence (X) and amino acid sequence (X ′) in one molecule of protein (Z) (number of GAGAGS sequence (1): amino acid sequence (X) and ( The total number of X ′) is preferably from 1: 2 to 1:20, more preferably from 1: 2 to 1:12, from the viewpoint of cell affinity, shortening of wound closure time and gelation.

The molecular mass of the protein (Z) is preferably 15 to 200 kDa, more preferably 15 to 100 kDa, from the viewpoint of shortening the wound closure time.
In addition, the molecular mass of protein (Z) is calculated | required by the method which isolate | separates a measurement sample by SDS-PAGE (SDS polyacrylamide gel electrophoresis) method, and compares a migration distance with a standard substance.

In the present invention, the protein (Z) has the polypeptide chain (Y) and / or the polypeptide chain (Y ′), so that the cell affinity is high, the epithelialization promoting effect is excellent, and the wound closure is performed. It becomes a material for tissue regeneration with a short time. Further, the protein (Z), which is a material for tissue regeneration, has the polypeptide chain (Y) and / or the polypeptide chain (Y ′) and the hydrophobicity of (Z) is within the above range. Can be dissolved in water and the aqueous solution can be gelled. If the degree of hydrophobicity is too low, the hydrophilicity becomes high and does not gel, and if the degree of hydrophobicity is too high, it cannot be dissolved in water to form a protein solution.
Further, when (Z) has a polypeptide chain (Y) and / or a polypeptide chain (Y ′) and has a hydrophobicity of 0.2 to 1.2, gelation occurs, but (Z) is GAGAGS. When having the arrangement (1), the gelation ability of (Z) (e.g., the time until gelation is appropriate for covering and filling the affected area) is further enhanced. In particular, when the ratio of GAGAGS sequence (1) to amino acid sequences (X) and (X ′) is 1: 2 to 1:20, the ratio is further increased.
If the protein solution gels and the fluidity becomes low, it will be difficult for the protein solution to flow out when administered to the affected area, and the gelled product will replace granulation tissue, promote epithelialization, and shorten the wound closure time. Is preferable because of high.

A part of preferable protein (Z) is illustrated below.
(1) Amino acid sequence (X) is GVGVP sequence (3) (1-1) GVGVP sequence (3) is a protein (Z1) having a continuous polypeptide chain (Y1), more preferably GVGVP A protein (Z11) having a polypeptide chain (Y11) in which two sequences (3) are continuous and a polypeptide chain (S1) in which six GAGAGS sequences (1) are continuous.
Specifically, it has a structure in which 29 amino acid blocks (L-1) {(GVGVP) ₂ (GAGAGS) ₆ } in which a polypeptide chain (Y11) and a polypeptide chain (S1-1) are bonded are repeatedly chemically bonded. Protein (SLP4.1, hydrophobicity 0.47) etc. of sequence (25) having a molecular mass of about 93 kDa.

(1-2) GVGVP sequence (3) is a protein (Z2) having a polypeptide chain (Y′1) in which one amino acid in a continuous polypeptide chain (Y1) is substituted with K (lysine) More preferably, a protein (Z21) having a polypeptide chain (Y′11) that is (GVGVP) ₄ GKGVP (GVGVP) ₃ sequence (8) and a polypeptide chain (S2) that is (GAGAGS) ₄ sequence (9) And (GVGVP) ₄ GKGVP (GVGVP) ₃ a protein (Z22) having a polypeptide chain (Y′11) which is sequence (8) and a polypeptide chain (S3) which is (GAGAGS) ₂ sequence (10), and poly It is a protein (Z23) having a peptide chain (Y′11), a polypeptide chain (S2) and a polypeptide chain (S3).
Specifically, ₄ polypeptide chains (S2) of 4 GAGAGS sequences (1) (GAGAGS) ₄ sequences (9) and 8 polypeptide chains (Y11) of GVGVP sequences (3) (Y11) (GVGVP) ₄ GKGVP (GVGVP) ₃ sequence (8) (Y′11) in which one of V (valine) in the inside is replaced with K (lysine), and these are chemically bonded alternately. The sequence (11) having a molecular mass of about 80 kDa having a structure in which one polypeptide chain (S3) of a sequence (GAGAGS) ₂ sequence (10) having two consecutive GAGAGS sequences (1) is chemically bonded. protein (SELP8K, hydrophobicity 0.618); GAGAGS sequence (1) of two consecutive (GAGAGS) polypeptide chain of ₂ sequences (10) (S3) and (GVGVP) ₄ GKGVP (GV VP) ₃ sequence (8) each have 17 polypeptide chain (Y'11) of the protein sequence with a molecular weight of they have a structure formed by chemically bonding alternately about 82kDa (12) (SELP0K, Hydrophobicity 0.718) etc.

(1-3) GVGVP sequence (3) is a protein (Z3) having a polypeptide chain (Y′2) in which two amino acids in a continuous polypeptide chain (Y1) are replaced by K (lysine) More preferably, a polypeptide chain (Y′21) in which two GKGVP sequences (13) are continuous, a polypeptide chain (S1) in which six GAGAGS sequences (1) are linked, and 10 GAGAGS sequences (1) are bound. This is a protein (Z31) having the polypeptide chain (S4).
Specifically, the amino acid block (L-2) {(GAGAGS) ₆ (GKGVP) _{2 in which the} polypeptide chain (Y′21) is bound to the polypeptide chain (S1) and the polypeptide chain (S4) is further bound. Protein (SLP4.2, hydrophobicity 0.2) having sequence (26) with a molecular mass of about 73 kDa having a structure in which (GAGAGS) ₁₀ } is chemically bonded 10 times repeatedly, etc.

(2) Amino acid sequence (X) is VPGVG sequence (2) (2-1) VPGVG sequence (2) is a protein (Z4) having a continuous polypeptide chain (Y2), more preferably a VPGVG sequence (2) is a protein (Z41) having 160 continuous polypeptide chains (Y21).
Specifically, a protein (ELP1, hydrophobicity 1.2) having a polypeptide mass (Y21) of 160 VPGVG sequences (2) having a molecular mass of about 65 kDa and a sequence (27).
(2-2) a protein (Z5) having a polypeptide chain (Y22) having four consecutive VPGVG sequences (2) and a polypeptide chain (Y23) having eight consecutive VPGVG sequences (2), more preferably A protein chain (Y51) having four consecutive VPGVG sequences (2), a polypeptide chain (Y23) having eight consecutive VPGVG sequences (2), and a GAGAGS sequence (1) (Z51), Specifically, an amino acid block (L-3) {(VPGVG) ₄ (GAGAGS) (VPGVG) in which one GAGAGS sequence (1) is bound to the polypeptide chain (Y22) and further the polypeptide chain (Y23) is bound. protein sequences having a molecular weight of about 220 kDa (28) having a _8} is bound 40 repeating chemical structure (ELP1.1, hydrophobic 1.12), etc.

In the present invention, protein (A) is a protein having at least one of the following minimum amino acid sequences (B) in one molecule.
Minimum amino acid sequence (B): RGD sequence, LDV sequence, REDV sequence (17), YIGSR sequence (18), PDSGR sequence (19), RYVVLPR sequence (20), LGTIPG sequence (21), RNIAEIIKDI sequence (22), IKVAV At least one amino acid sequence selected from the group consisting of sequence (23), LRE sequence, DGEA sequence (24) and HAV sequence.
The number of the minimum amino acid sequence (B) in (A) is preferably from 1 to 50, more preferably from 3 to 40, particularly from the viewpoint of cell adhesion, cell (particularly epithelial cell) proliferation and shortening of the wound closure time. Preferably it is 4-30.
The minimum amino acid sequence (B) is preferably an RGD sequence from the viewpoint of cell adhesion, cell proliferation, and wound closure time.

In the present invention, the protein (A) preferably further has the following amino acid sequence (C) from the viewpoint of degradability of the protein (A).
Amino acid sequence (C): (GA) _g sequence, (GAGAGS) _h sequence, (GAGAGT) _i sequence, (GAGVGT) _j sequence, (GAGTGV) _k sequence, {DGG (A) ₁ GGA} _m sequence, (GVPGV) _n sequence or (GGA) _o sequence.
[G is an integer of 1 to 400, h is an integer of 1 to 200, i is an integer of 1 to 200, j is an integer of 1 to 200, k is an integer of 1 to 200, and l is an integer of 1 to 200. , M is an integer from 1 to 100, n is an integer from 1 to 200, and o is an integer from 1 to 200. ]
Of the amino acid sequence (C), (GAGAGS) _h is preferable from the viewpoint of degradability of the protein (A).

  In the case of having an amino acid sequence (C), the content of (C) is preferably 2 to 50, more preferably 3 to 30 in one molecule of protein (A) from the viewpoint of degradability of protein (A). Particularly preferably 4 to 20, most preferably 5 to 15. The protein (A) may have two or more amino acid sequences (C).

When the protein (A) has (C), the ratio of the number of (B) and (C) in (A) [(B) / (C)] is 0. From the viewpoint of protein degradability. 5 to 2 is preferable, and 0.75 to 1.75 is more preferable. In addition, from the viewpoint of allowing the protein (A) to easily have a β sheet structure, it is preferable that (B) and (C) are alternately positioned, and particularly preferably, the RGD sequence and the (GAGAGS) _h sequence are alternately positioned. That is.

  The protein (A) may contain a branched chain, may be partially crosslinked, and may contain a cyclic structure. However, the protein (A) is preferably not cross-linked from the viewpoint of easy recognition from cells, more preferably a non-cross-linked linear structure, particularly preferably a cyclic structure and not cross-linked. It has a linear structure. The linear structure also includes a β structure (secondary structure in which linear peptides are bent and the portions are arranged in parallel and a hydrogen bond is formed therebetween).

  Protein (A) has a minimum amino acid sequence (B) and an amino acid sequence (C), and, if necessary, another amino acid sequence between (B) and (C) from the viewpoint of cell proliferation and shortening of wound closure time. It is preferable that the structure is formed by alternately chemically bonding via an amino acid sequence, and an amino acid sequence containing an amino acid sequence (D) described later on both ends of (B) and (C) are alternately chemically bonded. More preferably it is. In these cases, the number (unit) of repeating units (BC) of the minimum amino acid sequence (B) and amino acid sequence (C) is preferably 1 to 50 from the viewpoint of cell proliferation and shortening of wound closure time. More preferably, it is 2 to 40, particularly preferably 3 to 30, and most preferably 4 to 20.

  As the amino acid sequence (D), the N-terminal of the minimal amino acid sequence (B) has an RKLPDA sequence (58), a GAAVTG sequence (29), a GLPGPKGD sequence (30), from the viewpoint of cell proliferation and shortening of wound closure time. GGPAVTG sequence (31), AGPKGADGSGPPAVTG sequence (32), GAAVCEPG sequence (33), GAALC VSEPG sequence (34), SPASAALC VSEPG sequence (35), SPASAAVCEPG sequence (36), AGPKGADGSPGPAVCCEPG sequence (37), APGKADPGGPGPAGPGGPGPG (39), which binds at least one amino acid sequence (D) selected from the group consisting of the GPALCVSEPG sequence (40) and the GAAPGAS sequence (41) More preferably, at least one selected from the group consisting of an RKLPDA sequence (58), a GAAVTG sequence (29), a GLPGPKGD sequence (30), a GGPAVTG sequence (31), and an AGPKGADGSPGPAVTG sequence (32), and more preferably an RKLPDA sequence (58 ) And the GAAVTG sequence (29) are particularly preferred.

  As the amino acid sequence (D), the C-terminal of the minimum amino acid sequence (B) has an RKLPDA sequence (58), a SPASAAGY sequence (42), a SPASAALCVS sequence (43), from the viewpoint of cell proliferation and shortening the wound closure time. SPASAAVC sequence (44), AGPKGADGSGPP sequence (45), AGPKGADGSGPPAVC sequence (46), AGPKGADGSPGPALCVS sequence (47), AGPKGADGSP sequence (48), SPASAAGPVGSP sequence (49), CDAGY GSGPS sequence AG51 and CDAGPVGSP sequence Y It is preferable to bind at least one amino acid sequence (D) selected from the group consisting of (52), RKLPDA sequence (58), SPASAAGY sequence (42), SPASAA At least one selected from the group consisting of CVS sequence (43), SPASAAVC sequence (44), AGPKGADGSGPP sequence (45), AGPKGADGSPGPAVC sequence (46), AGPKGADGSPGPALCVS sequence (47), AGPKGADGSP sequence (48) and SPASAAGPVGSP sequence (49) Are more preferred, and RKLPDA sequence (58) and SPASAAGY sequence (42) are particularly preferred.

  The protein (A) may be further modified with the compound (AM). Compound (AM) includes compounds containing primary, secondary and tertiary amino groups and salts thereof, ammonium salts (AM-1), compounds containing carboxyl groups (AM-2), and sulfo groups Compound (AM-3) as well as a compound (AM-4) containing a hydroxyl group. When it is modified with (AM), the cell proliferation is further improved and the wound closure time can be shortened.

Compounds containing primary, secondary and tertiary amino groups and salts thereof, and compounds containing ammonium salts (AM-1) include polyamines, amino alcohols, halides having amino groups, amino group-containing monomers and Polymers having amino group-containing monomers as constituent monomers, hydrogen halide salts and quaternized compounds thereof can be used.
As the polyamine, a polyamine having at least one primary amino group or secondary amino group (having 2 to 56 carbon atoms) is used, such as an aliphatic polyamine, an alicyclic polyamine, a heterocyclic polyamine, and an aromatic polyamine. Is used.

  Aliphatic polyamines include alkylene diamines (ethylene diamine, propylene diamine, trimethylene diamine, tetramethylene diamine, hexamethylene diamine, etc.), polyalkylene polyamines having 2 to 6 carbon atoms in the alkylene group (diethylene triamine, iminobispropylamine, Triethylenetetramine, tetraethylenepentamine, pentaethylenehexamine, etc.), and alkyl (carbon number 1-18) substitution products thereof (dimethylaminopropylamine, diethylaminopropylamine, dipropylaminopropylamine, methylethylaminopropylamine, Trimethylhexamethylenediamine, N, N-dioctadecylethylenediamine, trioctadecylethylenediamine, methyliminobispropylamine, etc. Etc. The.

  Examples of alicyclic polyamines include 1,3-diaminocyclohexane, 1,3-bis (methylamino) cyclohexane, 1,3-bis (dihydroxyamino) cyclohexane, isophorone diamine, menthane diamine, and 4,4′-methylene dicyclohexane. Examples include diamines.

  Examples of the heterocyclic polyamine include piperazine, N-methylpiperazine, N-aminoethylpiperazine and 1,4-diaminoethylpiperazine.

  Aromatic polyamines include phenylenediamine, N, N′-dimethylphenylenediamine, N, N, N′-trimethylphenylenediamine, diphenylmethanediamine and 2,6-diaminopyridine, tolylenediamine, diethyltolylenediamine, 4, Examples include 4′-bis (methylamino) diphenylmethane and 1-methyl-2-methylamino-4-aminobenzene.

  As the amino alcohol, an amino alcohol having 2 to 58 carbon atoms is used, and an alkanolamine having 2 to 10 carbon atoms [monoethanolamine, diethanolamine, triethanolamine, monoisopropanolamine, monobutanolamine, triethanolamine, triethanolamine, Propanolamine, tributanolamine, N, N-bis (hydroxyethyl) ethylenediamine and N, N, N ′, N′-tetrakis (hydroxyethyl) ethylenediamine, etc.], alkyl (C 1-18) substituted products thereof [ N, N-dimethylethanolamine, N, N-diethylethanolamine, N-ethyldiethanolamine, N-octadecyldiethanolamine, N, N-diethyl-N ′, N′-bis (hydroxyethyl) ethylenediamine, N, - dioctadecyl -N ', N'-bis (hydroxyethyl) ethylenediamine and N, N, N'trioctadecyl -N'- hydroxyethyl ethylenediamine] and the like.

  Examples of the halide having an amino group include halogenated (such as chlorine and bromine) of alkylamines having 2 to 17 carbon atoms, such as aminoethyl chloride, N-methylaminopropyl chloride, N, N-dimethylaminoethyl chloride. N, N-diethylaminoethyl chloride, N, N-dibenzylaminoethyl bromide, N, N-dimethylaminopropyl bromide, N, N-diethylaminopropyl chloride, N, N-dibenzylaminopropyl chloride and the like.

As the amino group-containing monomer, an amino group-containing vinyl compound having 5 to 21 carbon atoms, ethyleneimine, an amino acid having 2 to 20 carbon atoms, or the like is used.
Examples of the amino group-containing vinyl compound include amino group-containing (meth) acrylates, amino group-containing (meth) acrylamides, amino group-containing aromatic vinyl hydrocarbons, amino group-containing allyl ethers, and the like.

  As the amino group-containing (meth) acrylate, aminoethyl (meth) acrylate, N-methylaminoethyl (meth) acrylate, N, N-dimethylaminoethyl (meth) acrylate, N, N-diethylaminopropyl (meth) acrylate, N, N-dipropylaminoethyl (meth) acrylate, N-benzyl-N-methylaminoethyl (meth) acrylate, N, N-dibenzylaminoethyl (meth) acrylate, N, N-dibenzylaminopropyl (meth) ) Acrylate, morpholinoethyl (meth) acrylate, N-methylpipetidinoethyl (meth) acrylate, and the like.

  Examples of amino group-containing (meth) acrylamides include aminoethyl acrylamide, N-methylaminopropyl acrylamide, N, N-dimethylaminoethyl (meth) acrylamide, N, N-diethylaminopropyl (meth) acrylamide, and N, N-dipropyl. Examples include aminoethyl (meth) acrylamide, N-benzyl-N-methylaminoethyl (meth) acrylamide, morpholinoethyl (meth) acrylamide, and N-methylpipetidinoethyl (meth) acrylamide.

  Examples of amino group-containing aromatic vinyl hydrocarbons include aminoethylstyrene, N-methylaminoethylstyrene, N, N-dimethylaminostyrene, N, N-dipropylaminostyrene, and N-benzyl-N-methylaminostyrene. Can be mentioned.

  Examples of the amino group-containing allyl ether include aminoethyl allyl ether, N-methylaminoethyl allyl ether, N, N-dimethylaminoethyl allyl ether, and N, N-diethylaminoethyl allyl ether.

  As amino acids, arginine, histidine, isoleucine, leucine, methionine, phenylalanine, threonine, tryptophan, tyrosine, valine, alanine, asparagine, aspartic acid, glutamine, glutamic acid, proline, cysteine, lysine, serine, glycine, 3-aminopropionic acid , 8-aminoactanoic acid and 20-aminoeicosanoic acid.

Examples of the polymer of the amino group-containing monomer include a vinyl polymer composed of an amino group-containing vinyl compound, polyethyleneimine, and a polypeptide (not including (A)).
The weight average molecular weight of the polymer of amino group-containing monomers is preferably 500 to 1,000,000, more preferably 1,000 to 800,000, and particularly preferably 2,000 to 500,000. The weight average molecular weight can be measured by gel permeation chromatography (GPC) {reference substance: polystyrene standard having a molecular weight of 420 to 20,600,000 (manufactured by Tosoh Corp.)}.

Examples of the hydrogen halide salt include salts of the amino group-containing compound and halides (hydrogen fluoride, hydrogen chloride, hydrogen bromide, hydrogen iodide, etc.).
Specific examples include N, N-dimethylaminoethyl chloride hydrochloride and N, N-diethylaminoethyl chloride hydrochloride.

  Examples of these quaternized products include those obtained by quaternizing these amino groups with a quaternizing agent (such as methyl chloride, ethyl chloride, benzyl chloride, dimethyl carbonate, dimethyl sulfate and ethylene oxide).

  Of these compounds containing primary, secondary and tertiary amino groups and salts thereof and ammonium salts (AM-1), N, N-dimethylamino hydrochloride is preferred from the viewpoint of cell proliferation and shortening of wound closure time. Ethyl chloride and N, N-diethylaminoethyl chloride hydrochloride are preferred, and N, N-dimethylaminoethyl chloride hydrochloride is more preferred.

  The compound (AM-2) containing a carboxyl group is a carboxylic acid having 2 to 30 carbon atoms, specifically, formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, gluconic acid, lactic acid, apple Acid, tartaric acid, citric acid, ascorbic acid, glucuronic acid, maleic acid, fumaric acid, pyruvic acid, aspartic acid, glutamic acid, benzoic acid, anthranilic acid, mesylic acid, salicylic acid, 4-hydroxybenzoic acid and phenylacetic acid . Moreover, the halide etc. which have these carboxyl groups can also be used. Specific examples include chloroacetic acid and chloroformic acid. Of these carboxyl group-containing compounds (AM-2), chloroacetic acid is preferred from the viewpoint of cell proliferation and shortening of wound closure time.

  The compound (AM-3) containing a sulfo group is a sulfonic acid having 2 to 30 carbon atoms, specifically, ethanesulfonic acid, benzenesulfonic acid, pantothenic acid, 2-hydroxyethanesulfonic acid, toluenesulfone. Examples include acid, sulfanilic acid and cyclohexylaminosulfonic acid. Moreover, the halide etc. which have these sulfo groups can also be used. Specific examples include chlorosulfonic acid and chloroethanesulfonic acid. Of these sulfone group-containing compounds (AM-3), chloroethanesulfonic acid is preferred from the viewpoint of cell growth and shortening of wound closure time.

  Examples of the compound (AM-4) containing a hydroxyl group include methanol, ethanol, propanol, butanol and the like. Moreover, the halide etc. which have these hydroxyl groups can also be used. Specific examples include chloroethanol and chloropropanol. Of these hydroxyl group-containing compounds (AM-4), chloroethanol is preferred from the viewpoint of cell proliferation and shortening of wound closure time.

As a method of modifying with compound (AM), (1) a method of chemically bonding {covalent bond, ionic bond and / or hydrogen bond, etc.} between compound (AM) and protein (A) before modification, and ( 2) A method in which the compound (AM) is physically adsorbed (adsorbed by van der Waals force) to the unmodified polypeptide can be applied.
Among these, from the viewpoint of cell proliferation and shortening the wound closure time, the chemical bonding method (1) is preferable, and covalent bonding is more preferable.

  (1) In the method of chemically bonding {covalent bond, ionic bond and / or hydrogen bond, etc.} between compound (AM) and protein (A) before modification, (A) is an amino acid residue. It preferably has an amino acid having a reactive group {hydroxyl group, carboxyl group, mercapto group, primary or secondary amino group, etc.}. Of the reactive groups, from the viewpoint of cell proliferation, a hydroxyl group, a carboxyl group and a primary amino group are preferable, a hydroxyl group and a carboxyl group are more preferable, and a hydroxyl group is particularly preferable. Further, it is sufficient that at least one reactive group is contained in one molecule of (A), but from the viewpoint of cell proliferation and shortening of wound closure time, those having 2 to 50 in one molecule are preferable, and more preferable. Have from 3 to 30, more preferably from 5 to 20 per molecule.

  As a method for chemical bonding, a known method can be applied, and examples thereof include a method described in JP-A-2007-51127. A reaction solvent may be used for the chemical bond formation reaction, and a known solvent can be used, for example, water, lithium bromide aqueous solution, lithium perchlorate aqueous solution, methanol, ethanol, isopropanol, acetone, dimethyl Examples include sulfoxide, dimethylacetamide, and tetrahydrofuran.

  Specific examples of covalently bonding the compound (AM) and the protein (A) before modification include amino acids (eg, Ser and Tyr) containing hydroxyl groups in the side chains in (A) as primary, secondary and Method of alkyl etherification with tertiary amino group and / or alkyl halide having ammonium salt, carboxyl group, sulfone group, hydroxyl group, and amino acid containing carboxyl group in side chain (for example, Asp and Glu) The method of esterifying with the alcohol which has an amino group and / or ammonium salt is mentioned.

Suitable examples of protein (A) are shown below.
A polypeptide {“pronectin F”, pronectin (with a structure (54) of about 110,000 Mn having 13 RGD sequences and 12 of (GAGAGS) ₉ sequences (53) and having an alternating structure thereof PRONECTIN is a registered trademark (Japan and USA) of Sanyo Chemical Industries, Ltd. Manufactured by Sanyo Chemical Industries Co., Ltd.
A protein obtained by modifying pronectin F with compound (AM), specifically, a polypeptide ("pronectin F plus") modified with N, N-dimethylaminoethyl chloride hydrochloride as compound (AM);
A polypeptide of sequence (56) of about 20,000 Mn (“pronectin F2”) having 5 structures of RGD sequence and 5 of (GAGAGS) ₃ sequence (55), which are alternately located;
A polypeptide of sequence (57) of about 10,000 Mn (“pronectin F3”) having three RGD sequences and three (GAGAGS) ₃ sequences (55) and having an alternating structure thereof;
A six and RKLPDA sequence RGD sequences and six (58) and 12 (GAGAGS) ₉ sequences (53), these RGD sequence, (GAGAGS) ₉ sequences (53), RKLPDA sequence (58) , (GAGAGS) ₉ polypeptide ("pronectin FT") having a sequence (59) of about 110,000 Mn having a structure alternately located in the order of sequence (53);
A polypeptide having a sequence of about 120,000 Mn (“pronectin FT2”) obtained by chemically binding the amino acid sequence RKLPDA sequence (58) to the Ser residue of “pronectin F”.

  The molecular mass of (A) is preferably 300 to 3,000,000, more preferably 1,000 to 1,000,000, and particularly preferably 3,000 to 3,000, from the viewpoint of cell proliferation and shortening of the wound closure time. 300,000. The molecular mass of (A) is obtained by separating (A) by SDS-PAGE (SDS polyacrylamide gel electrophoresis) and comparing the migration distance with a standard substance (hereinafter the same). is there).

  The tissue regeneration material of the present invention contains the protein (Z) and protein (A), and the weight ratio ((Z) / (A) of (Z) and (A) in the tissue regeneration material. )) Is 400/1 to 10/1, but from the viewpoint of shortening the wound closure time, the weight ratio is preferably 400/1 to 50/1.

In the tissue regeneration material of the present invention, the cell adhesion rate of the protein (A) is preferably 80 to 100%, more preferably 85 to 100%, particularly preferably from the viewpoint of promoting epithelialization and shortening the wound closure time. Is 90 to 100%.
The protein (A) has high cell adhesion because it has the minimum amino acid sequence (B). When the ratio of the minimum amino acid sequence (B) in (A) increases, the cell adhesion rate of (A) increases. Get higher. Due to the high cell adhesion rate of (A), when a tissue regeneration material containing protein (Z) and protein (A) is used, appropriate cell adhesion is obtained, epithelial cells are easy to expand, and wound closure is shortened. Increases effectiveness.
Further, the cell adhesion rate of the protein (Z) is preferably 0 to 20%, more preferably 0 to 18%, particularly preferably 3 to 15% from the viewpoint of promoting epithelialization and shortening the wound closure time. .
The protein (Z) has an amino acid sequence (X) and thus has low cell adhesion, and when the proportion of the amino acid sequence (X) in (Z) increases, the cell adhesion rate of (Z) becomes lower. . In addition, since the cell adhesion rate of (Z) is low, when it is used as a tissue regeneration material containing protein (Z) and protein (A), it becomes appropriate cell adhesion, and epithelial cells are easy to expand, Increases closure shortening effect.
Cell adhesion rate: When a substrate having 0.1 μg of protein (A) or protein (Z) per 1 cm ² is prepared and mouse gingival epithelial cells are seeded on the substrate, the amount of mouse gingival epithelial cells that adhere to the substrate Percentage.

Specifically, the cell adhesion rate is measured by the following measuring method.
<Measurement method of cell adhesion rate>
Dissolve 1 mg of the protein of the measurement sample in 1 mL of deionized water, and further dilute with 0.02 M phosphate buffer (PBS containing pH 7.2, purity 99.5 wt% sodium chloride 0.85 wt%). 10 μg / mL protein aqueous solution is prepared. This aqueous solution is added to a 96-well polystyrene plate at 50 μL / hole and allowed to stand at 25 ° C. for 2 hours.
After standing for 2 hours, the solvent was removed using an aspirator, washed twice with 100 μL / hole of physiological saline (0.9 wt / volume% sodium chloride aqueous solution), and further washed with 100 μL / hole of deionized water. Create a substrate with 0.1 μg protein per cm ² surface of the substrate.
The amount of protein per surface area of the prepared substrate is confirmed by performing the following measurement. A mixed solution (C) of Reagent A solution: Reagent B solution: Reagent C solution = 25: 24: 1 attached to Micro BCA ^™ protein assay kit (manufactured by THERMO Fisher Scientific) is prepared. The mixed solution (C) is added to the substrate at 100 μL / hole and allowed to stand at 37 ° C. for 2 hours.
After 2 hours of standing, the chelate production amount of bicinchoninic acid (BCA) and Cu ⁺ is measured at room temperature using a plate reader (MTP-32 manufactured by Corona Electric Co., Ltd.) with an absorbance of 450 nm (control wavelength 630 nm). Further, the coating amount is obtained from a calibration curve prepared beforehand with bovine serum albumin.
To the prepared substrate, DMEM medium without serum was added at 50 μL / well and stored in a 37 ° C. incubator for 1 hour. After 1 hour, mouse gingival epithelial cells are added at 10,000 cells / 50 μL / well and left to stand for 2 hours in an incubator at 37 ° C. and a carbon dioxide concentration of 5% by volume.
After completion of the culture, the medium is removed using an aspirator, and physiological saline is added at 100 μL / well, taking care not to directly contact the cells, and the physiological saline is removed using an aspirator. Next, PBS is added at 50 μL / well, Tetracolor One (Seikagaku Corporation) is added at 10 μL / hole, and left in an incubator at 37 ° C. and a carbon dioxide concentration of 5 vol% for 4 hours.
After 4 hours, the amount of formazan produced is measured with a plate reader at an absorbance of 450 nm (reference wavelength 630 nm). Since the number of adhered cells is proportional to the height of absorbance, the number of adhered cells is calculated from a calibration curve prepared in advance. The cell adhesion rate is calculated by the following formula from the number of adhered cells and the number of seeded cells.
Cell adhesion rate (%) = number of adherent cells / number of seeded cells × 100

Examples of the method for applying the tissue regeneration material of the present invention to the affected area include the following methods.
(1) A protein solution for tissue regeneration described later containing a predetermined amount of protein (Z), protein (A) and water is prepared at 4 to 25 ° C. The protein solution for tissue regeneration may contain an inorganic salt and / or phosphoric acid (salt) as necessary. Moreover, the protein solution for tissue regeneration may be warmed immediately before administration to the affected part, if necessary.
(2) The tissue regeneration protein solution is administered to the affected area (wound, etc.).
(3) After administration, coat with an appropriate dressing so that the protein solution for tissue regeneration does not flow out of the affected area.

The amount of each component in the tissue regeneration protein solution is the same as that of the protein solution for tissue regeneration of the present invention described later, and the preferred range is also the same.
The protein solution for tissue regeneration is in the form of a solution immediately after mixing the protein (A), protein (Z), and water. However, the fluidity becomes low and gels by applying stimuli such as passage of time and heat. It is preferable.
From the viewpoint of gelling the tissue regeneration protein solution in a short time, the temperature of the tissue regeneration protein solution is preferably 25 to 80 ° C. When it is 80 ° C. or lower, gelation can be achieved without deteriorating the function of the tissue regeneration material, and the time until gelation is appropriate.
Moreover, when using the protein solution for tissue regeneration, the temperature of the protein solution for tissue regeneration at the time of application is preferably 4 to 80 ° C., more preferably 4 from the viewpoint of thermal stability and handling property of the tissue regeneration material. -60 ° C, then more preferably 25-50 ° C, particularly preferably 30-40 ° C.

  The method of administration to the affected area is not particularly limited as long as the affected area can be covered, but from the viewpoint of burden on the patient, it is preferable to inject so as to fill the defective part of the affected area.

The dressing material used in the above (3) is not particularly limited, and examples thereof include polyurethane, silicone, polyvinyl alcohol, polypropylene, polyester, polystyrene, polyethylene, ethylene vinyl acetate copolymer, and nylon.
The dressing can be used without limitation as long as it can be coated so that the tissue regeneration protein solution does not flow out of the affected area after administration of the tissue regeneration protein solution, but a film shape is preferred.

  The tissue regeneration material of the present invention is presumed to have low antigenicity because it does not contain animal-derived serum or the like. Moreover, since protein (Z) and protein (A) have a biological origin arrangement | sequence, it is guessed that biocompatibility is high. Furthermore, since protein (Z) and protein (A) can be mass-produced at low cost by bacteria such as Escherichia coli, tissue regeneration materials can be easily obtained.

The protein solution for tissue regeneration of the present invention is a protein solution for tissue regeneration containing the tissue regeneration material and water.
The content (% by weight) of the tissue regeneration material in the tissue regeneration protein solution is preferably 5.03 to 30.3, more preferably, based on the total weight of the tissue regeneration material and water, from the viewpoint of wound healing. Is 10.05-30.1.
The content (% by weight) of protein (Z) in the tissue regeneration protein solution is 5 based on the total weight of the tissue regeneration material and water from the viewpoint of gelation of the tissue regeneration protein solution and wound healing. -30 is preferable, More preferably, it is 10-30.
The content (% by weight) of the protein (A) in the tissue regeneration protein solution is preferably 0.03 to 0.3 based on the total weight of the tissue regeneration material and water from the viewpoint of shortening the wound closure time. More preferably, it is 0.05-0.1.
From the viewpoint of wound healing, the content (% by weight) of the tissue regeneration material in the tissue regeneration protein solution is preferably 5.03 to 30.3, more preferably 10 based on the weight of the tissue regeneration protein solution. 0.05 to 30.1.
The content (% by weight) of protein (Z) in the tissue regeneration protein solution is preferably 5 to 30, based on the weight of the tissue regeneration protein solution, from the viewpoint of gelation of the protein solution and wound healing, More preferably, it is 10-30.
The content (% by weight) of protein (A) in the protein solution for tissue regeneration is:
From the viewpoint of shortening the wound closure time, 0.03-0.3 is preferable and 0.05-0.1 is more preferable based on the weight of the protein solution for tissue regeneration.

The water in the protein solution for tissue regeneration is not particularly limited as long as it is sterilized. Examples of the sterilization method include water passing through a microfiltration membrane having a pore size of 0.2 μm or less, and an ultrafiltration membrane. And water passed through a reverse osmosis membrane, deionized water heated at 121 ° C. for 20 minutes in an autoclave and overheat sterilized, and the like.
The content (% by weight) of water in the protein solution for tissue regeneration is 69.7 to 94.94 based on the total weight of the material for tissue regeneration and water from the viewpoint of solubility of the proteins (Z) and (A). 97 is preferable, More preferably, it is 69.9-89.95.
The content (% by weight) of water in the protein solution for tissue regeneration is 69.7 to 94.97 based on the weight of the protein solution for tissue regeneration from the viewpoint of solubility of the proteins (Z) and (A). It is preferably 69.9 to 89.95, more preferably 69.9 to 89.65, and particularly preferably 88.35.

The protein solution for tissue regeneration may contain an inorganic salt and phosphoric acid (salt) in addition to the material for tissue regeneration and water.
Examples of the inorganic salt include sodium chloride, potassium chloride, calcium chloride, magnesium chloride, sodium sulfate, potassium sulfate, calcium sulfate, magnesium sulfate, sodium hydrogen carbonate, potassium hydrogen carbonate, calcium hydrogen carbonate, and magnesium hydrogen carbonate. Phosphate is not included in inorganic salts.
The salt content (wt%) in the tissue regeneration protein solution is preferably 0 to 1.3, more preferably 0, based on the weight of the tissue regeneration protein solution, from the viewpoint of making it equivalent to a human body fluid. 0.5 to 1.3, then more preferably 0.7 to 1.1, and particularly preferably 0.85 to 0.95.

Phosphoric acid (salt) means phosphoric acid and / or phosphate.
Examples of phosphoric acid (salt) in the protein solution for tissue regeneration include phosphoric acid and phosphate.
Examples of the salt include alkali metal salts and alkaline earth metal salts, and specific examples include sodium salts, potassium salts, calcium salts, and magnesium salts.
The content (% by weight) of phosphoric acid (salt) in the tissue regeneration protein solution is preferably 0 to 0.30, more preferably, based on the weight of the tissue regeneration protein aqueous solution (B), from the viewpoint of wound healing. Is 0.10 to 0.30, then more preferably 0.12 to 0.28, and particularly preferably 0.14 to 0.26.

  The pH of the protein solution for tissue regeneration is preferably 5 to 9, more preferably 6 to 8, from the viewpoint of tissue affinity.

  Since the tissue regeneration protein solution of the present invention contains the tissue regeneration material, it is a material that promotes the healing of wounds caused by diseases or wounds such as burns, skin wounds, skin exfoliation wounds, and traumatic skin defect wounds. It is effective as (especially a material that promotes epithelialization). In particular, a protein solution containing a tissue regeneration material and water in the above-mentioned preferred range and gelling is useful as a wound covering material or filler.

  Hereinafter, although an example and a comparative example explain the present invention further, the present invention is not limited to these.

<Production Example 1>
Production of SELP8K A plasmid pPT0345 encoding SELP8K was prepared according to the method described in Examples of Japanese Patent No. 4088341.
The prepared plasmid was transformed into E. coli to obtain a SELP8K production strain. Hereinafter, a method for producing SELP8K using this SELP8K production strain will be described.
An overnight culture of SELP8K producing strain grown at 30 ° C. was used to inoculate 50 ml of LB medium in a 250 ml flask. Kanamycin was added to a final concentration of 50 μg / ml, and the culture was incubated at 30 ° C. with stirring (200 rpm). When the culture solution reached OD600 = 0.8 (absorbance meter UV1700, manufactured by Shimadzu Corporation), 40 ml was transferred to a flask pre-warmed to 42 ° C. and incubated at the same temperature for about 2 hours. The culture was cooled on ice and the OD600 of the culture was measured. E. coli was collected by centrifugation. In order to remove the protein from the collected E. coli, lysis was performed by ultrasonic disruption (4 ° C., 30 seconds × 10 times).
The protein produced by this E. coli was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene fluoride membrane. Thereafter, Western blot analysis was performed using an anti-rabbit SELP8K antibody as a primary antibody and an anti-rabbit IgG HRP-labeled antibody (manufactured by GE Healthcare) as a secondary antibody. The apparent molecular mass of the product was about 80 kDa. Therefore, it was found that the SELP8K producing strain produced SELP8K having an anti-rabbit SELP8K antibody reactivity with an apparent molecular mass of 80,000.

SELP8K purification The SELP8K obtained above was purified from E. coli biomass by lysis of cells, removal of insoluble debris by centrifugation, and affinity chromatography. In this way, SELP8K protein (Z-1), which is a protein (Z) having a molecular mass of about 80 kDa, was obtained.

○ Identification of SELP8K The obtained SELP8K (Z-1) was identified by the following procedure.
The analysis was performed by Western blotting using an anti-rabbit SELP8K antibody and an anti-rabbit 6 × His antibody (Roland) against a 6 × His tag of the C-terminal sequence. A protein band with an apparent molecular mass of 80,000 showed antibody reactivity to each antibody. As a result of subjecting the obtained protein to amino analysis, the product was found to be glycine (43.7%), alanine (12.3%), serine (5.3%), proline (11.7%) and valine. (21.2%). The product also contained 1.5% lysine. Table 1 below shows the correlation between the composition of the purified product and the predicted theoretical composition deduced from the synthetic gene sequence.
Therefore, SELP8K protein (Z-1) is a polypeptide chain (Y) in which 8 GVGVP sequences (3) are continuous, and one of V in (Y) is substituted with K (GVGVP) ₄ GKGVP (GVGVP) ₃ has twelve polypeptide chain (Y) of the sequence (8) 13 and GAGAGS sequences (1) are consecutive four (GAGAGS) ₄ sequence (9), it is chemically bonded alternately It was confirmed that the protein has the sequence (11).

<Production Example 2>
○ Synthesis of protein (A1-1) (pronectin F plus) 13 RGD sequences and 12 (GAGAGS) ₉ sequences (53) according to the method described in the examples in JP-T-3-502935 A polypeptide (pronectin F) having a sequence (54) of about 110,000 Mn having a structure in which these are alternately located was produced by genetically modified Escherichia coli to obtain a protein (A1).
After dissolving 50 mg of protein (A1) and 150 mg of N, N-dimethylaminoethyl chloride (special grade reagent) in 1.5 mL of 4.5 M lithium perchlorate aqueous solution at 20 to 40 ° C., the solution is 20 to 40 ° C. Then, 1.325 mL of 4.5 M aqueous lithium perchlorate solution in which 100 mg of sodium hydroxide (special grade reagent) was dissolved was added dropwise at a constant rate over 45 to 50 seconds. After stirring at room temperature (25 ° C.) for 1 hour, the reaction solution was dialyzed against 10 L of deionized water for 48 hours using a dialysis membrane having a molecular weight cut off of 12,000 to 14,000. In the first 12 hours, deionized water was changed every 4 hours. The obtained aqueous solution was freeze-dried under the conditions of −20 ° C. and 0.1 kPa or less for 24 hours to obtain a water-soluble protein (A1-1). The number of introduced N, N-dimethylaminoethyl chloride hydrochloride was measured in accordance with the method described in the examples in JP-T-10-500701, and was found to be 12 per molecule.

<Production Example 3>
In the same manner as in Production Example 1, except that “plasmid pPT0364 encoding SELP0K” is used instead of “plasmid pPT0345 encoding SELP8K”, the SELP0K protein (Z-2 of the sequence (12) having a molecular mass of about 82 kDa is used. )

<Production Example 4>
In the same manner as in Production Example 1, except that "plasmid pPT0102-1 encoding ELP1.1" was used instead of "plasmid pPT0345 encoding SELP8K", the ELP1.EL of sequence (28) having a molecular mass of about 220 kDa was used. 1 protein (Z-3) was obtained.

<Production Example 5>
In the same manner as in Production Example 1, except that “pSY139-1 encoding SLP4.1” is used instead of “plasmid pPT0345 encoding SELP8K”, SLP4.1 of the sequence (25) having a molecular mass of about 93 kDa is used. A protein (Z-4) was obtained.

<Production Example 6>
In the same manner as in Production Example 1, except that “plasmid pPT0345 encoding ELP1” is used instead of “plasmid pPT0345 encoding SELP8K”, the ELP1 protein (Z-5) having a molecular mass of about 65 kDa (27) is used. )

<Production Example 7>
In the same manner as in Production Example 1, except that “pSY1398-2 encoding SLP4.2” is used instead of “plasmid pPT0345 encoding SELP8K”, SLP4.2 of the sequence (26) having a molecular mass of about 73 kDa is used. A protein (Z-6) was obtained.

<Production Example 8>
In the same manner as in Production Example 1, except that “pSY1398 encoding SLP4” is used instead of “plasmid pPT0345 encoding SELP8K”, the SLP4 protein (Z′-1) having a molecular mass of about 70 kDa (60 ′) )

<Production Example 9>
In the same manner as in Production Example 1, except that “pPT0312 encoding CLP3.7” is used instead of “plasmid pPT0345 encoding SELP8K”, a CLP3.7 protein of sequence (61) having a molecular mass of about 66 kDa ( Z′-2) was obtained.

<Production Example 10>
In the same manner as in Production Example 1, except that “plasmid pPT0345 encoding DCP2” is used instead of “plasmid pPT0345 encoding SELP8K”, a DCP2 protein (Z′−) having a molecular mass of about 80 kDa in sequence (62) is used. 3) was obtained.

<Production Example 11>
In the same manner as in Production Example 1, except that "plasmid pPT0102-2 encoding ELP1.2" is used instead of "plasmid pPT0345 encoding SELP8K", ELP1. Two proteins (Z′-4) were obtained.

<Measurement of cell adhesion rate>
1 mg of each of the proteins (Z-1) to (Z-6) and (A1-1) is dissolved in 1 mL of deionized water, and 0.02 M phosphate buffer (pH 7.2, purity 99.5% by weight). A 10 μg / mL protein aqueous solution was prepared by diluting with PBS containing 0.85 wt% sodium chloride. This aqueous solution was added to a 96-well polystyrene plate (Nippon Becton Dickinson Co., Ltd.) at 50 μL / hole and allowed to stand at 25 ° C. for 2 hours.
After leaving for 2 hours, the solvent was removed using an aspirator, washed twice with 100 μL / hole of physiological saline (0.9 wt / volume% sodium chloride aqueous solution), and further washed with 100 μL / hole of deionized water, Substrates (K1) to (K7) were prepared.
A mixed solution (C) of Reagent A solution: Reagent B solution: Reagent C solution = 25: 24: 1 attached to Micro BCA ^™ protein assay kit (manufactured by THERMO Fisher Scientific) was prepared. 100 μL / hole of the mixed solution (C) was added to the substrates (K1) to (K7), and the mixture was allowed to stand at 37 ° C. for 2 hours.
Two hours after standing, the amount of chelate produced between bicinchoninic acid (BCA) and Cu ⁺ was measured at room temperature using a plate reader (MTP-32 manufactured by Corona Electric Co., Ltd.) with an absorbance of 450 nm (control wavelength 630 nm). Furthermore, when the coating amount was obtained from a calibration curve prepared beforehand with bovine serum albumin, the amount of protein per 1 cm ² of the surface of the substrate was all 0.1 μg.
To the prepared substrates (K1) to (K7), 50 μL / well of serum-free DMEM medium (ICN Biomedicals) was added and stored in a 37 ° C. incubator for 1 hour. One hour later, mouse gingival epithelial cells were added at 10,000 cells / 50 μL / well and left to stand for 2 hours in an incubator at 37 ° C. and a carbon dioxide concentration of 5% by volume.
After completion of the culture, the medium is removed using an aspirator, and physiological saline is added at 100 μL / well, taking care not to directly contact the cells, and the physiological saline is removed using an aspirator. Next, PBS was added at 50 μL / well, and Tetra Color One (Seikagaku Corporation) was added at 10 μL / hole, and left in an incubator with a carbon dioxide concentration of 5% by volume at 37 ° C. for 4 hours.
After 4 hours, the amount of formazan produced was measured using a plate reader (MTP-32 manufactured by Corona Electric Co., Ltd.) at an absorbance of 450 nm (reference wavelength 630 nm). Since the number of adhered cells is proportional to the height of absorbance, the number of adhered cells was calculated from a calibration curve prepared in advance. The cell adhesion rate was calculated from the number of adhered cells and the number of seeded cells by the following formula. The results are shown in Table 2.
Cell adhesion rate (%) = number of adherent cells / number of seeded cells × 100

<Examples 1-6>
In a plastic tube container, the proteins (Z-1) to (Z-6) obtained in Production Examples 1 and 3 to 7 were dissolved in 20 mg and 80 μL of 4 ° C. phosphate buffer (PBS), respectively. The protein (A1-1) 0.2mg was melt | dissolved in this, and protein solution (B1)-(B6) was produced.

<Comparative Examples 1-4>
In a plastic tube container, the proteins (Z′-1) to (Z′-4) obtained in Production Examples 8 to 11 were dissolved in 20 mg and 80 μL of 4 ° C. phosphate buffer (PBS), respectively. The protein (A1-1) 0.2 mg was melt | dissolved in this, and protein solution (B7)-(B10) was produced.

<Evaluation of gelation ability of protein solution>
The protein solutions (B1) to (B10) were allowed to stand at 37 ° C., and it was confirmed whether gelation occurred after 2 hours and 4 hours. To check if the gel has been formed, the plastic tube container (Eppendorf tube, 1.5 mL) containing the protein solution (100 μL) is turned over. If the solution does not drip, it is gelled. It was judged. The results are shown in Table 3. In the table, those in which the protein solution was gelled were marked with ◯, and those that were not gelled were marked with ×.

From the results in Table 3, proteins (Z′-1) to (Z ′) having no polypeptide chain (Y) or (Y ′) and having a hydrophobicity outside the range of 0.2 to 1.2. -3) and protein solutions (B7) to (B10) containing a protein (Z′-4) having a polypeptide chain (Y) but having a hydrophobicity outside the above range may not gel. I understand.
On the other hand, a protein having a polypeptide chain (Y) and / or a polypeptide chain (Y ′) and having a hydrophobicity of 0.2 to 1.2 and including proteins (Z-1) to (Z-6) It turns out that solution (B1)-(B6) gelatinizes. Therefore, it turns out that a wound part can be filled and a wound part can be coat | covered and sealed. In particular, the protein solutions (B1) and (B2) containing the protein (Z-1) or (Z-2) are gelled after 2 hours and are effective in covering and sealing the wound part more quickly. I understand that.

<Examples 7 to 12>
○ Preparation of protein solution SELP8K (Z-1) and protein (A1-1) were dissolved in a phosphate buffer solution (PBS) at 4 ° C in the ratios shown in Table 4 to prepare protein solutions (1) to (6). did.

<Comparative Examples 5-8>
Each component was mixed in the ratio of Table 4, and protein solution (7)-(10) was produced.

<Comparative Example 9>
200 mg of intrasite gel (manufactured by Smith and Nephew) and 2 mg of protein (A1-1) were dissolved in 798 μL of 4 ° C. PBS to prepare a gel solution (11).

<Comparative Example 10>
200 mg of granugel (manufactured by Convatec) and 2 mg of protein (A1-1) were dissolved in 798 μL of 4 ° C. PBS to obtain a gel solution (12).

<Examples 13 to 18>
SELP0K (Z-5) and protein (A1-1) were dissolved in PBS at 4 ° C. in the ratios shown in Table 5 to prepare protein solutions (13) to (18).

<Comparative Examples 11-13>
SELP0K (Z-5) and protein (A1-1) were dissolved in PBS at 4 ° C. at the ratio shown in Table 5 to prepare protein solutions (19) to (21).

<Examples 19 to 22>
ELP1 (Z-5) and protein (A1-1) were dissolved in PBS at 4 ° C. in the ratios shown in Table 6 to prepare protein solutions (22) to (25).

<Comparative Examples 14-16>
ELP1 (Z-5) and protein (A1-1) were dissolved in PBS at 4 ° C. in the ratios shown in Table 6 to prepare protein solutions (26) to (28).

<Evaluation>
(Treatment test with a full-thickness layer model using healthy guinea pigs)
Healthy guinea pig std Hartley (manufactured by SLC Japan) 7 weeks old was depilated under anesthesia, and a 10 x 10 mm full-thickness skin defect wound was created with the fat layer completely exposed on the sterilized guinea pig dorsal skin, After hemostasis and drying, the protein solutions (1) to (10), (13) to (28) and the gel solutions (11) to (12) obtained in Examples 7 to 12 and Comparative Examples 5 to 10 were respectively obtained. 80 μL was injected and a polyurethane film was attached. Then, gauze was put on each wound part, and the wound part periphery was fixed with the nylon thread | yarn. The protein solutions (1) to (9) and (13) to (28) were all gelled 4 hours after being injected into the wound. On the 10th day of the treatment period, the specimen was pseudo-dead, the skin including the wound was collected, and a pathological specimen (HE staining) was prepared.
Using the pathological specimen on the 10th day of the treatment period, the epithelialization length extended from the normal tissue was measured using a micro ruler (trade name: Micro Ruler for microscope, manufactured by Techjam Co., Ltd.). The results are shown in Tables 4-6.

From the evaluation results in Table 4, Comparative Example 8 using only the protein (A1-1) had a short epithelialization length of 1.1 mm, and only (A1-1) had a low effect on shortening the wound closure time. Moreover, also in Comparative Examples 9 and 10 in which the protein (A1-1) and granugel or intrasite gel were mixed, the epithelialization length was as short as 0.7 to 0.8 mm, and the wound closure time shortening effect was low. Therefore, it can be seen that the effect of shortening the wound closure time is low only by mixing the gelled product having no polypeptide chain (Y) or (Y ′) with (A1-1).
In addition, the tissue regeneration material of Comparative Example 7 using only protein (Z-1) was shorter than Examples 7 to 12 of the present invention, although it had a certain effect on epithelialization. When the tissue regeneration materials of Comparative Examples 5 and 6 in which the weight ratio of (Z-1) to (A1-1) is outside the range of 400/1 to 10/1 are used, Comparative Example 7 and In comparison, the epithelialization length was comparable or less.
On the other hand, when the tissue regeneration material of Examples 7 to 12 in which the weight ratio of (Z-1) to (A1-1) is within the range of 400/1 to 10/1 is used, the epithelialization length is It is 4.3 mm or more, and it can be seen that the effect of promoting epithelialization is higher than when only (Z-1) is used, and the wound closure time can be shortened. In particular, Examples 7 to 10 in which the weight ratio of (Z-1) to (A1-1) is 400/1 to 50/1 have a long epithelialization length of 5.0 mm or more, and have an epithelialization promoting effect. Since it is extremely high, it can be seen that the wound closure time can be greatly shortened.
Similarly, from the evaluation results of Table 5 and Table 6, when (Z-2) or (Z-5) is used, (Z-2) or (Z-5) is the same as in (Z-1). ) And (A1-1) in the case where the tissue regeneration material of Comparative Examples 11 to 14 having a weight ratio outside the range of 400/1 to 10/1 was used, compared with Comparative Example 7, epithelialization The length was comparable or less.
On the other hand, when the tissue regeneration material of Examples 13 to 22 in which the weight ratio of (Z-2) or (Z-5) to (A1-1) is in the range of 400/1 to 10/1 is used. It is found that the epithelialization length is 5.1 mm or more, the epithelialization promoting effect is higher than when only (Z-2) or (Z-5) is used, and the wound closure time can be shortened.

  The tissue regeneration material of the present invention is excellent in epithelialization promoting action, has a short wound closure time, and has high biodegradability. Therefore, it is effective not only as a wound dressing material that protects diseases such as burns, skin-cutting wounds, skin exfoliation wounds, and traumatic skin defect wounds, but also as a tissue regeneration material that promotes epithelialization.

Claims (8)

A tissue regeneration material containing protein (Z) and protein (A),
(Z) has the following polypeptide chain (Y) and / or the following polypeptide chain (Y ′), and the total number of (Y) and (Y ′) in (Z) is 1 to 40 A protein having a hydrophobicity of 0.2 to 1.2,
(A) is a protein having 13 RGD sequences and 12 (GAGAGS) ₉ sequences (53), and having a structure in which these are alternately located ;
A tissue regeneration material in which the weight ratio ((Z) / (A)) of (Z) to (A) in the tissue regeneration material is 400/1 to 10/1.
Polypeptide chain (Y): VPGVG sequence (2) or GVGVP sequence (3) A polypeptide chain having 2 to 160 consecutive amino acid sequences (X).
Polypeptide chain (Y ′): 1-2 amino acids in (Y) Lysine A polypeptide chain that is replaced with Protein (Z) as polypeptide chain (Y) (VPGVG) ₁₂ (Array), (GVGVP) ₂ Sequence or (VPGVG) ₁₆₀ (GVGVP) as a protein or polypeptide chain (Y ') having a sequence _Four GKGVP (GVGVP) _Three Sequence or (GKGVP) ₂ The tissue regeneration material according to claim 1, which is a protein having a sequence. Protein (Z) is (GVGVP) _Four GKGVP (GVGVP) _Three (GAGAGS) _Four A protein having a sequence, (GVGVP) _Four GKGVP (GVGVP) _Three (GAGAGS) ₂ A protein having a sequence, (VPGVG) _Four GAGAGS (VPGVG) ₈ A protein having a sequence, (GAGAGS) ₆ (GVGVP) ₂ A protein having a sequence, (VPGVG) ₁₆₀ Protein with sequence or (GAGAGS) ₆ (GKGVP) ₂ (GAGAGS) _Ten The tissue regeneration material according to claim 1 or 2, which is a protein having a sequence. Protein (Z) is (GVGVP) _Four GKGVP (GVGVP) _Three 13 sequences and (GAGAGS) _Four A protein having a structure having 12 sequences and alternately chemically bonding them (GVGVP) _Four GKGVP (GVGVP) _Three 17 sequences and (GAGAGS) ₂ A protein having a structure having 17 sequences and alternately chemically bonding them, (VPGVG) _Four GAGAGS (VPGVG) ₈ A protein having a structure with 40 consecutive sequences, (GAGAGS) ₆ (GVGVP) ₂ A protein having a structure having 29 consecutive sequences, (VPGVG) ₁₆₀ Protein with sequence or (GAGAGS) ₆ (GKGVP) ₂ (GAGAGS) _Ten The tissue regeneration material according to any one of claims 1 to 3, which is a protein having a structure in which 10 sequences are continuous. Protein (Z) is (GVGVP) _Four GKGVP (GVGVP) _Three 13 sequences and (GAGAGS) _Four A protein having a structure having 12 sequences and alternately chemically bonding them (GVGVP) _Four GKGVP (GVGVP) _Three 17 sequences and (GAGAGS) ₂ The tissue regeneration material according to any one of claims 1 to 4, which is a protein having a structure having 17 sequences and alternately having these chemically bonded, or a protein having a structure having 160 consecutive (VPGVG) sequences. The tissue regeneration material according to any one of claims 1 to 5, wherein the protein (A) is modified with N, N-dimethylaminoethyl chloride hydrochloride. The tissue regeneration material according to any one of claims 1 to 6 , wherein the protein (Z) has a molecular mass of 15 to 200 kDa as determined by SDS-PAGE (SDS polyacrylamide gel electrophoresis). A tissue regeneration protein solution comprising the tissue regeneration material according to any one of claims 1 to 7 and water.