JP2005002106A - Intercellular adhesive polypeptide - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an intercellular adhesive polypeptide with very high intercellular adhesiveness. <P>SOLUTION: The intercellular adhesive polypeptide has a structure formed by alternately chemically bonding the smallest amino acid sequence (X) exhibiting an intercellular adhesion signal and an auxiliary amino acid sequence (Y). The number of all the amino acids contained in Y is 1-50, and the total content of glycine and alanine contained in Y is 42-100 % based on the total number of amino acids. Preferably, X contains Arg Gly Asp sequences, Leu Asp Val sequences, Leu Arg Glu sequences, His Ala Val sequences, and other specially expressed sequences; and Y contains (Gly Ala)a sequences, (Gly Ala Gly Ala Gly Ser)b sequences, (Gly Ala Gly Ala Gly Tyr)c sequences, (Gly Ala Gly Val Gly Tyr)d sequences, (Gly Ala Gly Tyr Gly Val)e sequences, and äAsp Gly Gly (Ala)fGly Gly Ala}g sequences and/or (Gly Val Pro Gly Val)h sequences. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a cell adhesion polypeptide. More specifically, the present invention relates to a cell adhesion polypeptide that improves the adhesion of cells to a substrate or the like.

Conventionally, human-derived proteins such as collagen, fibronectin, vitronectin, fibrinogen, laminin and cadherin (non-patent document 1) and insect-derived proteins such as fibroin (non-patent document 2) are known as natural cell-adhesive polypeptides. It has been. In addition, as an artificial cell adhesion polypeptide, it has about 13 each of pronectin F {Arg Gly Ap sequence and (Gly Ala Gly Ala Gly Ser) ₉ sequence (12), and is a number average produced by genetically modified E. coli. Polypeptide having a molecular weight of about 100,000, manufactured by Sanyo Chemical Industries, Ltd.) and pronectin L {Ile Lys Val Ala Val sequence (7) and (Gly Ala Gly Ala Gly Ser) ₉ sequence (12) each having about 13 In addition, a polypeptide having a number average molecular weight of about 100,000 produced by genetically modified Escherichia coli, manufactured by Sanyo Chemical Industries, Ltd.} is known (Non-patent Document 3).

Pathophysiology (Vol. 9, No. 7, pp. 527-535, 1990) Biochemical and Biophysical Research Communications (Biochemical and Biophysical Research Communications, Vol.208, No.2, 511-516, 1995) Handbook of Biodegradable Polymers (Abraham, published by Harwood Academic Publishers, Amsterdam 1997 (Abraham J. Domb et al., Handbook of Biodegradable Polymers, published by Harwood Academic Publishers, Amsterdam 1997)

Conventional natural cell-adhesive polypeptides have the problem of low cell-adhesion due to the small number of minimal amino acid sequences that express cell-adhesion signals in one molecule, and further have poor thermal stability against autoclaves and the like. There was a problem.
On the other hand, conventional artificial cell adhesion polypeptides can improve cell adhesion and thermal stability compared to natural cell adhesion polypeptides, but cells can be treated under special conditions such as serum-free medium. Cell adhesion was insufficient.
That is, an object of the present invention is to provide a cell adhesion polypeptide having extremely high cell adhesion.

As a result of intensive studies, the inventor has found that the above object can be achieved by using a specific polypeptide, and has reached the present invention.
That is, the cell adhesion polypeptide of the present invention is characterized by a polypeptide having a structure in which a minimal amino acid sequence (X) and an auxiliary amino acid sequence (Y) showing a cell adhesion signal are alternately chemically bonded. The total number of amino acids contained in the auxiliary amino acid sequence (Y) is 1 to 50, and the total content of glycine (Gly) and alanine (Ala) contained in (Y) is the total number of amino acids in (Y). The point is based on 42 to 100%.

  Since the cell adhesion polypeptide of the present invention has extremely high cell adhesion, cells (NHDF cells, VERO cells, etc.) that have been difficult to adhere to can be efficiently adhered on a substrate. Moreover, since the heat stability with respect to an autoclave etc. is very high, it is easy and safe disinfection. Therefore, the cell adhesion polypeptide (P) of the present invention, the cell adhesion polypeptide-containing substrate, and the production method including the step of culturing cells using the cell adhesion polypeptide-containing substrate are related to cells. It is extremely useful for development, production of useful substances and treatment.

“Cell adhesion” means the property that a specific minimum amino acid sequence is recognized by the integrin receptor of a cell and the cell adheres easily to a substrate. “Cell adhesion signal” means an amino acid sequence having cell adhesion. It means (Osaka Prefectural Maternal and Child Medical Center Magazine, Vol. 8, No. 1, pp. 58-66, 1992). That is, the minimum amino acid sequence showing a cell adhesion signal is the same as the minimum amino acid sequence for cell adhesion.
Examples of the minimal amino acid sequence (X) showing a cell adhesion signal include, for example, “Pathophysiology, Vol. 9, No. 7, pp. 527-535, 1990” and “Osaka Prefectural Maternal and Child Medical Center Magazine, Vol. 8, Vol. No., pages 58-66, 1992 ”, etc. are used.

  Among these minimal amino acid sequences (X), Arg Gly Asp sequence, Leu Asp Val sequence, Arg Glu Asp Val sequence (1), Tyr Ile Gly Ser Arg sequence (2), Pro Asp Ser Gly Arg sequence (3) Arg Tyr Val Val Leu Pro Arg sequence (4), Leu Gly Thr Ile Pro Gly sequence (5), Arg Asn Ile Ala Glu Ile Ile Lys Asp Ile sequence (6), Ile Lys Val Ala Val sequence (7), Leu Arg Glu sequence, Asp Gly Glu Ala sequence (8), Gly Val Lys Gly Asp Lys Gly Asn Pro Gly Trp Pro Gly Ala Pro sequence (9), Gly Glu Phe Tyr Phe Asp Leu Arg Leu Lys Gly Asp Lys sequence (10) His Ala Val sequence, Tyr Lys Leu Asn Val Asn Asp Ser sequence (11) and Tyr Gly Gly Leu Gly Ser Gln Gly Ala Gly Arg sequence (156) are preferable, and Arg Gly Asp is more preferable from the viewpoint of cell adhesion. Sequence, Tyr Ile Gly Ser Arg sequence (2), Ile Lys Val Ala Val sequence (7) and Tyr Gly Gly Leu Gly Ser Gln Gly Ala Gly Arg sequence (156), particularly preferably Arg Gly Asp sequence Is a column.

  The cell adhesion polypeptide (P) may have at least two minimum amino acid sequences (X) in one molecule, but from the viewpoint of cell adhesion, those having 2 to 30 in one molecule are preferable. More preferably, it has 3-20 pieces, and particularly preferably 4-10 pieces. Also, two or more kinds of minimum amino acid sequences (X) may be included in one molecule.

The auxiliary amino acid sequence (Y) is an amino acid sequence existing between the minimum amino acid sequence (X) and the minimum amino acid sequence (X). The auxiliary amino acid sequence (Y) is an amino acid other than the minimum amino acid sequence (X). Any sequence can be used without limitation, but from the viewpoints of crystallinity (higher crystallinity is preferred) of the cell adhesion polypeptide (P) and cell adhesion, the (Gly Ala) a sequence, (Gly Ala Gly Ala Gly Ser) b sequence, (Gly Ala Gly Ala Gly Tyr) c sequence, (Gly Ala Gly Val Gly Tyr) d sequence, (Gly Ala Gly Tyr Gly Val) e sequence, {Asp Gly Gly (Ala) f Gly Gly Ala} g sequence, (Gly Val Pro Gly Val) h sequence, (Gly) i, (Ala) j, and / or (Gly Gly Ala) k sequence, preferably (Gly Ala) a sequence , (Gly Ala Gly Ala Gly Ser) b sequence, (Gly Ala Gly Ala Gly Tyr) c sequence, (Gly Ala Gly Val Gly Tyr) d Columns, (Gly Ala Gly Tyr Gly Val) e, {Asp Gly Gly (Ala) f Gly Gly Ala} g sequence, and / or (Gly Val Pro Gly Val) h sequence, particularly preferably (Gly Ala Gly Ala Gly Ser) b sequence and / or (Gly Val Pro Gly Val) h sequence.
A is an integer of 1 to 25, b, c, d and e are integers of 1 to 8, f is an integer of 1 to 44, f is 1, g is an integer of 1 to 7, and f is 2. g is an integer of 1 to 6, when f is 3 or 4, g is an integer of 1 to 5, when f is 5 or 6, g is an integer of 1 to 4, and when f is an integer of 7 to 10, g is 1 -3, when f is an integer of 11 to 19, g is 1 or 2, when f is an integer of 20 to 44, g is 1, h is an integer of 1 to 10, i and j are integers of 1 to 50, k Is an integer from 1 to 16.

  In addition to the above sequences, the auxiliary amino acid sequence (Y) includes other amino acids (serine (Ser), threonine (Thr), valine (Val), leucine (Leu), isoleucine (Ile), cysteine (Cys), methionine. (Met), phenylalanine (Phe), tyrosine (Tyr), proline (Pro), tryptophan (Trp), asparagine (Asn), glutamine (Gln), aspartic acid (Asp), glutamic acid (Glu), arginine (Arg), Lysine (Lys), histidine (His), etc.) may be included.

The total content (number%) of glycine (Gly) and alanine (Ala) contained in the auxiliary amino acid sequence (Y) is preferably 42 to 100, more preferably based on the total number of amino acids in the auxiliary amino acid sequence (Y). Is 50 to 95, particularly preferably 55 to 90, more particularly preferably 60 to 85, and most preferably 61 to 80. That is, the lower limit of the total content (number%) of (Gly) and (Ala) is preferably 42, more preferably 50, particularly preferably 55, and more particularly preferably based on the total number of amino acids in (Y). 60, most preferably 61. Similarly, the upper limit is preferably 100, more preferably 95, particularly preferably 90, more particularly preferably 85, and most preferably 80. Within this range, cell adhesion is further improved.
When both glycine (Gly) and alanine (Ala) are included, the content ratio (Gly / Ala) is preferably 0.03 to 40, more preferably 0.08 to 13, particularly preferably 0.2. ~ 4. That is, in this case, the lower limit of the content ratio (Gly / Ala) of glycine (Gly) and alanine (Ala) is preferably 0.03, more preferably 0.08, and particularly preferably 0.2. Similarly, the upper limit is preferably 40, more preferably 13, and particularly preferably 4.

The total number of amino acids (pieces) contained in the auxiliary amino acid sequence (Y) is preferably 1 to 50, more preferably 5 to 47, particularly preferably 10 to 43, and most preferably 15 to 40. Within this range, cell adhesion is further improved.
If the total number of amino acids contained in the auxiliary amino acid sequence (Y) exceeds this range, or if the total content of Gly and Ala is less than this range, the cell adhesion tends to deteriorate.

Examples of the auxiliary amino acid sequence having the (Gly Ala) a sequence include the amino acid sequences of SEQ ID NOs: (13) to (23).
Examples of the auxiliary amino acid sequence having (Gly Ala Gly Ala Gly Ser) b sequence include the amino acid sequences represented by SEQ ID NOs: (24) to (28), (115) and (157).
Examples of the auxiliary amino acid sequence having (Gly Ala Gly Ala Gly Tyr) c sequence include amino acid sequences represented by SEQ ID NOs: (29) to (33).
Examples of the auxiliary amino acid sequence having (Gly Ala Gly Val Gly Tyr) d sequence include the amino acid sequences represented by SEQ ID NOs: (34) to (38).
Examples of the auxiliary amino acid sequence having (Gly Ala Gly Tyr Gly Val) e sequence include amino acid sequences represented by SEQ ID NOs: (39) to (43).
Examples of the auxiliary amino acid sequence having the {Asp Gly Gly (Ala) f Gly Gly Ala} g sequence include amino acid sequences represented by SEQ ID NOs: (44) to (74) and (123).
Examples of the auxiliary amino acid sequence having (Gly Val Pro Gly Val) h sequence include amino acid sequences represented by SEQ ID NOs: (75) to (81) and (119).
Examples of the auxiliary amino acid sequence having the (Gly) i sequence include amino acid sequences represented by SEQ ID NOs: (82) to (94).
Examples of the auxiliary amino acid sequence having the (Ala) j sequence include amino acid sequences represented by SEQ ID NOs: (95) to (107).
Examples of the auxiliary amino acid sequence having (Gly Gly Ala) k sequence include the amino acid sequences represented by SEQ ID NOs: (108) to (114).
Among these SEQ ID NOs: (16) to (28), (30) to (33), (35) to (38), (40) to (43), (47) to (65), (70) -(71), (75)-(78), (93)-(94), (106)-(107), (111)-(115), (119), (123) and (157) are preferred. More preferably, (24) to (28), (47) to (65), (70) to (71), (75) to (78), (115), (119), (123) and (157) ), Particularly preferably (24) to (28), (115), (119), (123) and (157).

  As long as the cell adhesion polypeptide (P) has a structure in which the minimum amino acid sequence (X) and the auxiliary amino acid sequence (Y) are alternately chemically bonded, it may contain a branched chain in part. A part thereof may be cross-linked and may contain a cyclic structure, but is preferably not cross-linked, more preferably a straight-chain structure that is not cross-linked, particularly preferably a cross-linked structure that does not have a cyclic structure. There is no 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).

  If the cell adhesion polypeptide (P) has a structure in which the minimum amino acid sequence (X) and the auxiliary amino acid sequence (Y) are alternately chemically bonded, (X) and (Y) are directly chemically bonded. Or may be chemically bonded via another amino acid. Further, other amino acids may be contained in the terminal part of the cell adhesion polypeptide (P) (from the minimum amino acid sequence (X) or the auxiliary amino acid sequence (Y) to the peptide end). When other amino acids are included, the content thereof is preferably 1 to 1000, more preferably 3 to 300, and particularly preferably 10 to 100 per cell adhesive polypeptide.

  The number of repeating units (XY) (minimum number) of the minimum amino acid sequence (X) and the auxiliary amino acid sequence (Y) in the cell adhesion polypeptide (P) is preferably 2-30, more preferably 3-20. Especially preferably, it is 4-10. Further, the number of the minimum amino acid sequence (X) and the auxiliary amino acid sequence (Y) may be the same or different. When they are different from each other, the content of one is one less than the content of the other {in this case, the auxiliary amino acid sequence (Y) is preferably small}. The content ratio (X / Y) between the minimum amino acid sequence (X) and the auxiliary amino acid sequence (Y) in the cell adhesion polypeptide (P) is preferably 0.66-1.5, more preferably 0.8. 9-1.4, particularly preferably 1.0-1.3.

  The number average molecular weight (Mn) of the cell adhesion polypeptide (P) is preferably 300 to 1,000,000, more preferably 1,000 to 300,000, particularly preferably 3,000 to 100,000. . That is, the upper limit of (Mn) in (P) is preferably 1,000,000, more preferably 300,000, particularly preferably 100,000, and similarly the lower limit is preferably 300, more preferably 1 3,000, particularly preferably 3,000. The number average molecular weight (Mn) is determined by separating a measurement sample {cell adhesive polypeptide etc.} by SDS-PAGE (SDS polyacrylamide gel electrophoresis) and comparing the migration distance with a standard substance. (same as below).

A part of preferable cell adhesion polypeptide (P) is illustrated below.
(1) When the minimum amino acid sequence (X) is Arg Gly Asp sequence (x1) Five of (x1) and Ser Pro Ala Gly Gly (Ala Gly Ala Gly Ser Gly) ₃ Ala Ser Thr Gly sequence (115) (y1 ) And four polypeptides (P1) (116), (x1) and 9 (y1) having a structure in which these are alternately chemically bonded. Polypeptide (P3) having a structure formed by chemical bonding (P2) (117), polypeptide having a structure formed by alternately bonding 15 of (x1) and 14 of (y1) (P3) ) (118), (x1) and Ser Pro Ala (Gly Val Pro Gly Val) ₂ Gly Gly (Gly Ala Gly Ala Gly Ser) ₃ Ala Ser Thr Gly Sequence (119) (y2) Polypeptides (P4) (120), (x1) 8 and (y2) 7 having a structure in which these are alternately chemically bonded Polypeptides (P5) (121), 12 (x1) and 11 (y2) having a structure in which these are alternately chemically bonded, and these are alternately chemically bonded Polypeptides (P6) (122) and (x1) having the following structure: Ser Pro Ala Ala Ser Asp Gly Gly (Ala) ₁₂ Gly Gly Ala Ala Ser Thr Gly Sequence (123) (y3) 10 (polypeptides (P7) (124) and (x1)) and 9 (y3) having a structure in which these are alternately chemically bonded, and these are alternately chemically bonded Polypeptides (P8) (125) having a structure of (15), (x1) and 14 (y3), and having a structure in which these are alternately chemically bonded (P9) (126) these and a four five and (Gly Ala) ₁₃ sequences (x1) (y4) (21 ) Polypeptide (P10) (127) having a structure formed by alternately chemical bonding and polypeptide having a structure formed by alternately bonding 10 of (x1) and 9 of (y4) (P11) (128) and a polypeptide (P12) (129) having a structure in which 15 of (x1) and 14 of (y4) are alternately bonded chemically.

(2) When the minimum amino acid sequence (X) is Tyr Ile Gly Ser Arg sequence (2) (x2) Five of (x2) and Ser Pro Ala Gly Gly (Ala Gly Ala Gly Ser Gly) ₃ Ala Ser Thr Gly sequence (115) Polypeptides (P13) (130), (x2) and 9 (y1) having a structure comprising four (y1) and alternating chemical bonds thereof A polypeptide (P14) (131) having a structure in which these are alternately chemically bonded (P14), (x2) and 14 (y1), and a structure in which these are alternately chemically bonded Four polypeptides (P15) (132) and (x2) having SerPro Ala (Gly Val Pro Gly Val) ₂ Gly Gly (Gly Ala Gly Ala Gly Ser) ₃ Ala Ser Thr Gly sequence (119) (y2) And (P16) (133), (x2) a polypeptide having a structure in which these are alternately chemically bonded to each other Polypeptides (P17) (134), 12 (x2) and 11 (y2) having 8 and 7 (y2) and having these chemically linked alternately And five polypeptides (P18) (135) and (x2) having a structure in which these are chemically bonded alternately and Ser Pro Ala Ala Ser Asp Gly Gly (Ala) ₁₂ Gly Gly Ala Ala Ser Thr Gly sequence ( 123) Polypeptides (P19) (136), (x2) and 9 (y3) having a structure comprising four of (y3) and alternately chemical bonds thereof Polypeptides (P20) (137) and 15 (x2) and 14 (y3) having a structure in which these are alternately chemically bonded and having a structure in which these are alternately chemically bonded polypeptide (P21) (138), 5 pieces and (Gly Ala) ₁₃ sequences (x2) (y ) Polypeptides (P22) (139), 10 (x2) and 9 (y4) having a structure in which four of (21) and these are alternately chemically bonded Has a structure in which 15 are (x2) and 14 are (y4) having a structure in which these are alternately chemically bonded to each other (P23) (140). Polypeptide (P24) (141) and the like.

(3) When the minimum amino acid sequence (X) is Ile Lys Val Ala Val sequence (7) (x3) 5 of (x3) and Ser Pro Ala Gly Gly (Ala Gly Ala Gly Ser Gly) ₃ Ala Ser Thr Gly sequence (115) Polypeptides (P25) (142), (x3) and 9 (y1) having 4 structures (y1) and alternately chemical bonds thereof And a polypeptide (P26) (143) having a structure in which these are alternately chemically bonded (P3), 15 (x3) and 14 (y1), and a structure in which these are alternately chemically bonded Four polypeptides (P27) (144) and (x3) having SerPro Ala (Gly Val Pro Gly Val) ₂ Gly Gly (Gly Ala Gly Ala Gly Ser) ₃ Ala Ser Thr Gly sequence (119) (y2) And a polypeptide having a structure in which these are alternately chemically bonded (P28) (145), (x3) Polypeptides (P29) (146), (x3) and 11 (y2) having 8 and 7 (y2) and having a structure in which these are alternately chemically bonded And five polypeptides (P30) (147) and (x3) having a structure in which these are alternately chemically bonded to each other and Ser Pro Ala Ala Ser Asp Gly Gly (Ala) ₁₂ Gly Gly Ala Ala Ser Thr Gly sequence ( 123) Polypeptides (P31) (148), 10 (x3) and 9 (y3) having a structure in which four of (y3) and these are alternately chemically bonded Polypeptide (P32) (149) having a structure in which these are alternately chemically bonded (P32) (15) of (x3) and 14 of (y3) and a structure in which these are alternately chemically bonded polypeptide (P33) (150), 5 pieces and (Gly Ala) ₁₃ sequences (x3) (y4 Polypeptide (P34) (151), (x3) and 9 (y4) having a structure in which four of (21) and these are alternately chemically bonded Polypeptide (P35) (152) having a structure formed by alternately chemically bonding, and poly having a structure formed by alternately bonding 15 of (x3) and 14 of (y4) Peptide (P36) (153) etc.

(4) When the minimum amino acid sequence (X) is Tyr Gly Gly Leu Gly Ser Gln Gly Ala Gly Arg sequence (156) (x4) 5 of (x4) and Ser Pro Ala Gly Gly (Ala Gly Ala Gly Ser Gly) ₃ Polypeptides (P40) (159) and (x4) having 4 structures of the Ala Ser Thr Gly sequence (115) (y1) and the structure in which these are alternately bonded (y1) 15 (polypeptides (P41) (160) and (x4)) and 14 (y1) having a structure in which these are alternately chemically bonded to each other, and these are alternately chemical Four polypeptides (P42) (161) and (x4) having a structure formed by binding and Ser Pro Ala (Gly Val Pro Gly Val) ₂ Gly Gly (Gly Ala Gly Ala Gly Ser) ₃ Ala Ser Thr Gly sequence (119) Polypeptide having a structure comprising three of (y2) and alternately having these chemically bonded (P43) 162), (x4) 8 and (y2) 7 and having a structure in which these are alternately chemically bonded (P44) (163), (x4) 12 and (y2) ) And 5 polypeptides of (P45) (164), (x4) and Ser Pro Ala Ala Ser Asp Gly Gly (Ala) ₁₂ Gly Gly Polypeptides (P46) (165), 10 (x4) and (y3) having a structure in which four of Ala Ala Ser Thr Gly sequences (123) (y3) and these are alternately chemically bonded 15 polypeptides of (P47) (166), (x4) and 14 (y3) having a structure in which these are alternately chemically bonded to each other, and these are alternately chemical Five polypeptides (P48) (167) and (x4) having a structure formed by binding (Gly Ala) ₁₃ sequence (y4) and a four (21) polypeptide they have a structure formed by chemically bonding alternately (P49) (168), and 10 (x4) (y4) Polypeptides (P50) (169), 15 (x4) and 14 (y4) having a structure in which these are alternately chemically bonded, and these are alternately chemically It has 5 polypeptides (P51) (170), (x4) and 4 Ala Ser (Ala Gly Ser Gly Ala Gly) ₃ Ala Ser sequences (157) (y5) having a structure formed by binding Polypeptides (P52) (171), 10 (x4) and 9 (y5) having a structure in which these are alternately chemically bonded and having a structure in which these are alternately chemically bonded Polypeptide (P39) (158) and 15 of (x4) and 14 of (y5) Polypeptide (P53) (172) such that the have they have a structure formed by chemically bonding alternately.

  The cell adhesive polypeptide (P) can be usually produced artificially, and can be easily produced by an organic synthesis method (such as an enzyme method, a solid phase synthesis method and a liquid phase synthesis method), a gene recombination method, or the like. Regarding organic synthesis methods, Biochemistry Experiment Course 1, Protein Chemistry IV (July 1, 1981, edited by the Japanese Biochemical Society, published by Tokyo Chemical Co., Ltd.) or Secondary Biochemistry Experiment Course 2, Protein Chemistry (bottom) (May 20, 1987, edited by the Japanese Biochemical Society, published by Tokyo Chemical Co., Ltd.) and the like can be applied. Regarding the gene recombination method, the method described in JP-T-3-502935 can be applied. In the case of the genetic recombination method, since impurities derived from recombinant microorganisms may be contained, it is preferable to purify by affinity purification using an anti-polypeptide antibody or the like, and to make the purity of the polypeptide 60% by weight or more, More preferably, it is 70% by weight or more, and particularly preferably 80% by weight or more. Among these production methods, the gene recombination method is preferable from the viewpoint that the amino acid sequence of the cell adhesion polypeptide can be easily designed and produced.

As cell adhesion polypeptide (P) by the organic synthesis method, polypeptides (P10) to (P12), polypeptides (P22) to (P24), polypeptides (P34) to (P36), and (P49) to (P51).
Examples of the cell adhesion polypeptide (P) obtained by the genetic recombination method include polypeptides (P1) to (P53).

The cell adhesion polypeptide (P) of the present invention can be used as a cell adhesion polypeptide-containing substrate together with the substrate (B).
The material of the base material (B) can be used without limitation as long as it does not adversely affect cells and living organisms. For example, it is dispersed in a culture solution or body fluid when used in cell culture or applied to living organisms of cells. , Materials that are easily dissolved or absorbed {hereinafter, readily biodegradable materials (N1)}, and materials that are difficult to be dispersed, dissolved, or absorbed in a culture solution or body fluid when used in cell culture or when applied to a living body {hereinafter, The hardly biodegradable material (N2)} can be used. In addition, an easily biodegradable material (N1) and a hardly biodegradable material (N2) can be used in combination.

As the readily biodegradable material (N1), a natural polymer (N1A), a synthetic polymer (N1B), an inorganic substance (N1C), or the like can be used.
Natural polymers (N1A) include collagen, gelatin, glycosaminoglycan, hyaluronic acid, chondroitin sulfate, keratan sulfate, dermatan sulfate, heparin, elastin, chitin, chitosan, fibrin, alginic acid, starch, dextran, albumin, polyhydroxy Examples include butyric acid, pectin, pectic acid, galactan, pullulan, agarose, cellulose, gluten, and fibroin.
As the synthetic polymer (N1B), a (co) polymer comprising, as an essential monomer, a monomer selected from the group consisting of lactic acid, leucine, glycolic acid, ε-caprolactone, dioxanone, malic acid, lactide, and glycolide (Polyglycolic acid) and synthetic polypeptides that do not contain the cell adhesion polypeptide of the present invention.
As the inorganic substance (N1C), calcium carbonate, calcium phosphate, and the like are used.
Examples of calcium carbonate include light calcium carbonate and heavy calcium carbonate. Examples of calcium phosphate include hydroxyapatite, tricalcium phosphate, and a mixture of these with other calcium phosphates (such as monocalcium hydrogen phosphate).
Among these, natural polymer (N1A) and synthetic polymer (N1B) are preferable, more preferably natural polymer (N1A), particularly preferably collagen, gelatin, hyaluronic acid, chitin, chitosan, fibrin, alginic acid, starch, Dextran, agarose, cellulose and fibroin.

As the hardly biodegradable material (N2), a natural polymer (N2A), a synthetic polymer (N2B), an inorganic substance (N2C), or the like can be used.
Examples of the natural polymer (N2A) include natural fibers (cotton, hair, hemp, silk, etc.) and the like.
Examples of the synthetic polymer (N2B) include polyolefins (polyethylene, polypropylene, and modified products thereof), olefin copolymers {ethylene-vinyl acetate copolymer, ethylene-ethyl (meth) acrylate copolymer, ethylene-methyl ( Meth) acrylate copolymer and ethylene- (meth) acrylic acid copolymer}, polyurethane, polyester, polyacrylic acid, polyamide, polyvinyl chloride, polyvinylidene chloride, polystyrene, fluororesin, silicone resin, cellulose, viscose Rayon, cupra rayon, polynosic, acetate, triacetate, polyacrylonitrile, vinylon, vinylidene and the like are used.
As the inorganic substance (N2C), metal (gold, silver, platinum, titanium, nickel, etc.), ceramics (alumina, zirconia, aluminum nitride, etc.), etc. are used.
Of these, the synthetic polymer (N2B) and the inorganic substance (N2C) are preferable, the synthetic polymer (N2B) is more preferable, and the polyolefin, polyurethane, polyester, polyacrylic acid, polystyrene, and silicone resin are particularly preferable.

  The shape of the substrate (B) is not particularly limited as long as cells can adhere to it, and generally used ones can be used. Plates, petri dishes, T-flasks, roller bottles, microcarrier beads and hollow fibers, Any of a sheet (film, foam (sponge), cloth, etc.) and gel may be used.

  The cell adhesion polypeptide (P) and the substrate (B) are combined by chemical bonding (such as ionic bonding, hydrogen bonding and / or covalent bonding) and / or physical adsorption (adsorption by van der Waals force). A chemical bond is preferable and a covalent bond is more preferable in that the cell adhesion polypeptide (P) and the substrate (B) are firmly bonded.

  As a method for covalently binding the cell adhesion polypeptide (P) and the base material (B), <1> (P) having a primary amino group or secondary amino group and (B) carboxyl <2> a method of reacting a group having a group; <2> a method of reacting (P) a compound having a primary amino group or a secondary amino group and (B) a compound having a hydroxyl group; <3> A method of reacting a hydroxyl group of (P) with a carboxyl group of (B); <4> (P) having a halogen and (B) a hydroxyl group or a mercapto group And a method of reacting <5> (P) with a vinyl group and (B) with an amino group, a hydroxyl group or a carboxyl group. .

These reactions can be carried out by a known method (for example, the method described in “Basics and Experiments of Peptide Synthesis, October 5, 1997, published by Maruzen Co., Ltd.”). Specifically, it is as the following <1> to <5>.
When reacting <1> (P) having a primary amino group or secondary amino group with (B) having a carboxyl group, the carboxyl group of (B) is reacted with a carbodiimide compound in advance. after the {(moiety derived from -OCOR the ^{(B)) R '-N =} C (OCOR) -NH-R'} acylisourea, primary amino group or secondary amino of the this (P) (B) and (P) can be chemically bonded by an amide bond by contacting with a group having a group.
Examples of the carbodiimide compound include N, N′-dicyclohexylcarbodiimide and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride.

When reacting <2> (P) having a primary amino group or secondary amino group with (B) having a hydroxyl group, the hydroxyl group in (B) is After reacting, the imidazole derivative {R-Im, Im is an imidazoline ring, R is derived from (B)}, and this is contacted with (P) one having a primary amino group or a secondary amino group As a result, (B) and (P) can be chemically bonded by an N—C bond.
Examples of the carbonyldiimidazole compound include N, N′-carbonyldiimidazole.

<3> In the case of reacting (P) having a hydroxyl group and (B) having a carboxyl group, the carboxyl group of (B) is reacted with a carbodiimide compound in advance to obtain acylisourea, By bringing this and (P) into contact with one having a hydroxyl group, (B) and (P) can be chemically bonded by an ester bond.

<4> In the case of reacting a halogen group in (P) and a hydroxyl group or mercapto group in (B), by mixing both in the presence or absence of an alkali compound, ( B) and (P) can be chemically bonded by an ether bond or a thioether bond.
Examples of the alkali compound include inorganic alkali compounds (such as sodium hydroxide, potassium hydroxide, and lithium hydroxide) and organic alkali compounds (such as dimethylaminopyridine, ammonia, triethylamine, sodium methoxide, and DBU (registered trademark of San Apro)). Is mentioned.

<5> When reacting (P) having a vinyl group and (B) having an amino group, hydroxyl group or carboxyl group, both are mixed in the presence or absence of an alkali compound. By adding Michael, (B) and (P) can be chemically bonded by a C—N or C—O bond.

As a method for physically adsorbing, ion-bonding and / or hydrogen-bonding the cell adhesion polypeptide (P) to the base material (B), for example, (P) and (B) are introduced into a solvent and mixed. Examples of the method are as follows.
Although there is no restriction | limiting in particular as a solvent, 0.001-50 weight% (preferably 0.01-10 weight%) of inorganic salt, organic acid salt, an amino acid, a vitamin, alcohol, lipid and sugar, an acid, and / or a base Aqueous solutions, water, organic acids, body fluids, and the like can be used.

Inorganic salts include alkali metal halide salts (sodium chloride, potassium chloride, lithium chloride, sodium bromide and lithium bromide), alkaline earth metal halide salts (calcium chloride and magnesium bromide), and alkali metal sulfate salts. (Such as sodium sulfate and potassium hydrogen sulfate), alkaline earth metal sulfates (such as magnesium sulfate and calcium sulfate), heavy metal sulfates (such as copper sulfate and iron sulfate), alkali metal phosphates (sodium phosphate, hydrogen phosphate) Sodium, potassium phosphate and potassium hydrogen phosphate), alkali metal nitrates (such as sodium nitrate and potassium nitrate), heavy metal nitrates (such as iron nitrate and copper nitrate), alkali metal carbonates (such as sodium carbonate and potassium hydrogen carbonate) And alkali metal perhalogenates (sodium perchlorate and lithium perchlorate Umm, etc.), and the like can be used.
As the organic acid salt, an alkali metal salt, alkaline earth metal salt or heavy metal salt of an organic acid having 1 to 6 carbon atoms can be used. Sodium formate, sodium acetate, lithium acetate, sodium tartrate, potassium malate, calcium acetate And copper acetate.

Examples of amino acids include arginine, histidine, isoleucine, leucine, methionine, phenylalanine, threonine, tryptophan, tyrosine, valine, alanine, asparagine, aspartic acid, glutamic acid, proline, serine and glycine.
Examples of vitamins include choline, inositol, nicotinamide, glutamine, vitamin A, vitamin B ₁₂ and vitamin C.
As alcohol, C1-C4 alcohol etc. can be used and methanol, ethanol, isopropyl alcohol, ethylene glycol, butanol, etc. are mentioned.
Examples of lipids / sugars include lipids, monosaccharides, disaccharides, oligosaccharides, amino sugars, and acidic sugars.

Examples of the acid include inorganic acids (mineral acids) and organic acids having 1 to 6 carbon atoms, and examples include hydrochloric acid, phosphoric acid, sulfuric acid, perchloric acid, acetic acid, formic acid, malic acid, phenol and catechol.
Examples of the base include inorganic bases and organic bases having 2 to 6 carbon atoms, such as sodium hydroxide, potassium hydroxide, lithium hydroxide, dimethylaminopyridine, sodium methoxide and DBU (registered trademark of San Apro), ammonia, Examples include triethanolamine and triethylamine.
Examples of water include distilled water, ion exchange water, tap water, and ion exchange distilled water.
As an organic acid, a C1-C6 carboxylic acid etc. can be used, Formic acid, an acetic acid, propionic acid, hexanoic acid, etc. are mentioned.
Examples of the body fluid include blood, plasma, serum and urine.
Among these solvents, aqueous solutions and water containing inorganic salts, acids and / or bases are preferred, and aqueous solutions containing inorganic salts, acids and / or bases are more preferred.

The content of the cell adhesive polypeptide (P) in the cell adhesive polypeptide-containing substrate of the present invention is preferably 0.1 ng / cm ^{2 to} 100 mg / cm ² per unit area of the substrate (B), It is more preferably 1 ng / cm ^{2 to} 10 mg / cm ² , particularly preferably 10 ng / cm ^{2 to 1} mg / cm ² , most preferably 100 ng / cm ^{2 to} 100 μg / cm ² . That is, the lower limit of the content of (P) is preferably 0.1 ng / cm ² , more preferably 1 ng / cm ² , particularly preferably 10 ng / cm ² , and most preferably 100 ng / cm ² per unit area of (B). cm ^2, and also similarly limit is preferably 100 mg / cm ^2, more preferably 10 mg / cm ^2, particularly preferably 1 mg / cm ^2, and most preferably 100 [mu] g / cm ^2. In addition, a unit area means the surface area of the surface where the cell to culture | cultivate can adhere | attach among the surfaces of a base material (B). Here, minute unevenness (for example, 1 μm or less) that does not allow cells to enter is handled as a flat surface, but for ribs or the like provided for the purpose of increasing the unit area, the surface area of the rib is a unit. Included in the area.
The method for measuring the content of the cell adhesion polypeptide (P) per unit area is not particularly limited, but an immunological measurement method can be used. For example, a part of the surface of the cell adhesion polypeptide-containing substrate ( For example, a 1 cm × 1 cm square shape) is cut out, an antibody labeled with an enzyme (hereinafter referred to as an enzyme-labeled antibody) is reacted with an antibody that binds to the cell adhesion polypeptide (P), and the enzyme amount of the reacted enzyme-labeled antibody By measuring, the content of the cell adhesive polypeptide (P) per unit area can be measured. In addition, by labeling an antibody with an isotope, dye, fluorescent substance or luminescent substance instead of an enzyme, and measuring the radiation dose, dye quantity, fluorescence intensity or luminescence intensity instead of the enzyme quantity, The content of the cell adhesion polypeptide (P) can also be measured.

The cell-adhesive polypeptide-containing substrate of the present invention may be sterilized as necessary.
Examples of the sterilization method include radiation sterilization, ethylene oxide gas sterilization, plasma sterilization, γ-ray sterilization, alcohol sterilization, autoclave sterilization, and dry heat sterilization. Of these, autoclave sterilization and dry heat sterilization are preferred because they are easy to sterilize.
The heating temperature (° C.) for autoclave sterilization and dry heat sterilization is preferably 40 to 180, more preferably 60 to 160, and particularly preferably 80 to 140. That is, the lower limit of the heating temperature (° C.) in this case is preferably 40, more preferably 60, particularly preferably 80, and similarly, the upper limit is preferably 180, more preferably 160, particularly preferably 140. is there.
The heating time in autoclave sterilization and dry heat sterilization is preferably 1 second to 5000 minutes, more preferably 10 seconds to 500 minutes, and particularly preferably 1 to 100 minutes. That is, the lower limit of the heating time in this case is preferably 1 second, more preferably 10 seconds, particularly preferably 1 minute, and similarly the upper limit is preferably 5000 minutes, more preferably 500 minutes, particularly preferably. 100 minutes.
The pressure in the tank (MPa) when autoclaving is preferably 0.002 to 5, more preferably 0.01 to 1, and particularly preferably 0.05 to 0.2. That is, the lower limit of the pressure in the tank (MPa) in this case is preferably 0.002, more preferably 0.01, particularly preferably 0.05, and similarly the upper limit is preferably 5, more preferably 1, particularly preferably 0.2.

As a method for producing cells including the step of culturing cells using a cell-adhesive polypeptide-containing substrate, a known method except that the cell-adhesive polypeptide-containing substrate of the present invention is used instead of the known culture substrate. The method is applicable.
As the cell culture process, the cell-adhesive polypeptide-containing substrate of the present invention or an appropriate container (a petri dish, plate, flask, beaker, fermenter, etc.) containing the cell-adhesive polypeptide-containing substrate of the present invention is used. And a method of culturing under appropriate conditions by adding a medium (ME) in which cells (CE) are suspended.

The cell (CE) that can adhere to the cell-adhesive peptide-containing substrate of the present invention is not limited as long as it is a cell, but normal cells derived from mammals, established cell lines derived from mammals, and insect cells are suitable. .
Normal cells derived from mammals include cells involved in the skin (epithelial cells, fibroblasts, vascular endothelial cells, smooth muscle cells, etc.), cells involved in blood vessels (vascular endothelial cells, smooth muscle cells, fibroblasts, etc.) ), Cells involved in muscle (muscle cells, etc.), cells involved in fat (adipocytes, etc.), cells involved in nerves (nerve cells, etc.), cells involved in liver (liver parenchymal cells, etc.), involved in pancreas Cells (such as pancreatic islet cells), cells involved in the kidney (renal epithelial cells, proximal tubular epithelial cells and mesangial cells, etc.), cells involved in the lung and bronchi (epithelial cells, fibroblasts, vascular endothelial cells) And smooth muscle cells), cells involved in the eyes (photocells, corneal epithelial cells and corneal endothelial cells, etc.), cells involved in prostate (epithelial cells, stromal cells, smooth muscle cells, etc.), cells involved in bone (Osteoblasts, bone cells and osteoclasts ), Cells involved in cartilage (such as chondroblasts and chondrocytes), cells involved in teeth (such as periodontal ligament cells and osteoblasts), cells involved in blood (such as leukocytes and erythrocytes), and stem cells {eg, Bone marrow undifferentiated mesenchymal stem cell, skeletal muscle stem cell, hematopoietic stem cell, neural stem cell, hepatic stem cell (oval cell, small hepatocyte, etc.), adipose tissue stem cell, embryonic stem (ES) cell, epidermal stem cell, intestinal stem cell, sperm stem cell Embryonic reproductive stem (EG) cells, pancreatic stem cells (pancreatic ductal epithelial stem cells, etc.), leukocyte stem cells, lymphoid stem cells, corneal stem cells, progenitor cells (adipose precursor cells, vascular endothelial precursor cells, cartilage precursor cells, lymphoid cells) Progenitor cells, NK progenitor cells, etc.)} and the like.

  Examples of cell lines derived from mammals include 3T3 cells, A549 cells, AH130 cells, B95-8 cells, BHK cells, BOSC23 cells, BS-C-1 cells, C3H10T1 / 2 cells, C-6 cells, and CHO. Cells, COS cells, CV-1 cells, F9 cells, FL cells, FL5-1 cells, FM3A cells, G-361 cells, GP + E-86 cells, GP + envAm12 cells, H4-II-E cells, HEK293 cells, HeLa cells, HEp-2 cells, HL-60 cells, HTC cells, HUVEC cells, IMR-32 cells, IMR-90 cells, K562 cells, KB cells, L cells, L5178Y cells, L-929 cells, MA104 cells, MDBK cells, MDCK Cells, MIA PaCa-2 cells, N18 cells, Namalwa cells, NG108-15 cells, RK cell, OC10 cell, OTT6050 cell, P388 cell, PA12 cell, PA317 cell, PC-12 cell, PG13 cell, QGH cell, Raji cell, RPMI-1788 cell, SGE1 cell, Sp2 / O-Ag14 cell, ST2 cell, Examples include THP-1 cells, U-937 cells, V79 cells, VERO cells, WI-38 cells, ψ2 cells, and ψCRE cells. {Cell culture technology (edited by the Japanese Society for Tissue Culture, published by Asakura Shoten Co., Ltd., 1999) Year)}.

  Insect cells include silkworm cells (BmN cells, BoMo cells, etc.), mulberry cells, sakusan cells, synjusan cells, mushroom cells (Sf9 cells, Sf21 cells, etc.), stag beetle hitori cells, caterpillar cells, Drosophila cells, sentinic fly, Cell, human striped cell, swallowtail butterfly cell, American cockroach cell, and nettle cottonweed cell (Tn-5 cell, HIGH FIVE cell, MG1 cell, etc.) etc. {Insect Bio Factory (edited by Shigeru Kimura, published by Industrial Research Institute, Inc., 2000) Year).

The medium (ME) used in the cell culture method using the cell-adhesive polypeptide-containing substrate of the present invention can be any medium used for general cell culture without particular limitation, and RPMI medium, MEM medium, BME medium, DME medium. , ΑMEM medium, IMEM medium, ES medium, DM-160 medium, Fisher medium, F12 medium, WE medium, ASF103 medium, ASF104 medium, ASF301 medium, CHO-S-SFMII medium, CD-CHO medium, VP-SFM medium, OPTIPRO ^™ SFM medium, 293SFMII medium, CD293 medium, Grace medium, IPL-41 medium, Schneider medium, TC-100 medium, Sf-900II medium, Ex-cell405 medium, Express-Five medium, Drosophila medium, and mixed media thereof Is mentioned The
Moreover, what added serum etc. to these culture media can also be utilized. However, since the cell-adhesive polypeptide-containing substrate of the present invention has high cell adhesion activity without using serum, it is preferable not to use serum when there is a concern about serum-derived biohazards such as virus infection. .
Serum includes human serum and animal serum (bovine serum, horse serum, goat serum, sheep serum, pig serum, rabbit serum, chicken serum, rat serum, mouse serum, etc.), of which human serum Bovine serum and horse serum are preferred. Moreover, the origin of animal serum includes serum derived from adults, serum derived from pups, serum derived from newborns, serum derived from fetuses, etc. Among these, serum derived from pups, serum derived from newborns, and fetuses derived from fetuses These are preferable, more preferably neonatal-derived serum, and fetal-derived serum, and particularly preferably fetal-derived serum. Further, the serum may be subjected to inactivation treatment, antibody removal treatment, or the like.
When using serum, the amount of serum used (% by weight) is preferably 0.1 to 50, more preferably 0.3 to 30, and particularly preferably 1 to 20, based on the weight of the medium. That is, the lower limit of the amount of serum used (% by weight) is preferably 0.1, more preferably 0.3, particularly preferably 1 based on the weight of the medium, and similarly the upper limit is preferably 50, More preferably, it is 30, particularly preferably 20.

If necessary, the medium can contain a cell growth factor as an additive to increase the cell growth rate or the cell activity.
Cell growth factors include fibroblast growth factor, transforming growth factor, epithelial growth factor, hepatocyte growth factor, platelet-derived growth factor, insulin-like growth factor, vascular endothelial growth factor, nerve growth factor, stem cell factor, leukemia Inhibitory factors, osteogenic factors, heparin-binding epithelial cell growth factor, neurotrophic factor, connective tissue growth factor, angiopoietin, cytokines, interleukins, adrenamodulin and natriuretic peptides are included. Of these, fibroblast growth factor, transforming growth factor, insulin-like growth factor, and osteogenic factor are preferable, and more preferably fibroblast, from the viewpoint that the range of applicable cells is wide and the healing period can be further shortened. Growth factors, transforming growth factors and insulin-like growth factors.
When a cell growth factor is used, the content (% by weight) of the cell growth factor varies depending on the type of cell growth factor, but is preferably 10 ⁻¹⁶ to 10 ⁻³ , more preferably 10 ⁻ based on the weight of the medium. ¹⁴ to 10 ⁻⁵ , particularly preferably 10 ⁻¹² to 10 ⁻⁷ . That is, in this case, the lower limit of the content (% by weight) of the cell growth factor is preferably 10 ⁻¹⁶ , more preferably 10 ⁻¹⁴ , particularly preferably 10 ⁻¹² , based on the weight of the medium. The upper limit is preferably 10 ⁻³ , more preferably 10 ⁻⁵ , and particularly preferably 10 ⁻⁷ .

These media can further contain an antibacterial agent (amphotericin B, gentamicin, penicillin, streptomycin, etc.). When the antibacterial agent is contained, the content (% by weight) varies depending on the type of the antibacterial agent, but is preferably 10 ⁻⁶ to 10, more preferably 10 ⁻⁵ to 1, particularly preferably based on the weight of the medium. 10 ^{−4 to} 0.1. That is, in this case, the lower limit of the content (% by weight) of the antibacterial agent is preferably 10 ⁻⁶ , more preferably 10 ⁻⁵ , particularly preferably 10 ⁻⁴ based on the weight of the medium, and similarly. The upper limit is preferably 10, more preferably 1, and particularly preferably 0.1.

The amount of cells seeded varies depending on the cells used and the type of the cell-adhesive polypeptide-containing substrate (B), but preferably 10 to 10 million cells per 1 cm ² surface area of (B), more preferably 100 ˜1,000,000, particularly preferably 1,000 to 100,000.
Moreover, there is no restriction | limiting in particular as the density | concentration (cell / mL) of the cell disperse | distributed to a culture medium, However, 100-100 million per 1mL of culture media are preferable, More preferably, it is 1000-10 million, Most preferably, it is 10,000- One million.

There are no particular limitations on the culture conditions, and carbon dioxide (CO ₂ ) concentration of 1 to 20% by volume, 5 to 45 ° C. for 1 hour to 100 days, and if necessary, culture conditions are changed every 1 to 10 days. Etc. are applicable. Preferable conditions are conditions in which the culture is performed if the medium is changed every 2 to 3 days at a CO ₂ concentration of 3 to 10% by volume, 30 to 40 ° C., 1 to 20 days, and 2 to 3 days.

  When the cells are detached from the cell-adhesive polypeptide-containing substrate after collection and collected, <1> a method of collecting with a chelating agent such as EDTA and / or a proteolytic enzyme such as trypsin or collagenase, < 2> A method of collecting by scraping with a scraper or the like, <3> A method of collecting by lowering the temperature of the cell-adhesive polypeptide-containing substrate (B) and / or the medium below the temperature of the culture conditions, and the like.

  Examples of the cell (CE2) obtained by cell culture using the cell-adhesive polypeptide-containing substrate include the same cells (CE) that can adhere to the cell-adhesive peptide-containing substrate. Note that the cell (CE) may be differentiated to obtain a cell (CE2) different from the original cell (CE). For example, when the original cell (CE) is an undifferentiated bone marrow mesenchymal stem cell and the cell (CE2) is an osteoblast, a chondrocyte, an adipocyte, or the like (Tissue Engineering, Minoru Ueda, Foundation) Nagoya University Press, October 10, 1999).

A cell adhesion polypeptide (P) of the present invention, a cell adhesion polypeptide-containing substrate, and a production method comprising a cell culture step using the cell adhesion polypeptide-containing substrate, Suitable for production and treatment of useful substances.
For research and development, substitutes for animal experiments (toxicity tests, irritancy tests, metabolic function tests, etc.), gene transfer, screening of target substances (cells, bioactive substances, genes, harmful chemical substances, medicinal ingredients, etc.) and cells It can be used for purification and functional analysis.
For the production of useful substances, it can be used for the production of cytokines, thrombolytic agents, blood coagulation factor preparations, vaccines, hormones, antibiotics, antibodies and growth factors.
For treatment, skin, skull, muscle, skin tissue, bone, cartilage, blood vessel, nerve, tendon, ligament, follicular tissue, mucosal tissue, periodontal tissue, dentin, bone marrow, retina, serosa, gastrointestinal tract and fat Can be used for treatment of tissues such as lung, liver, pancreas and kidney.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.
<Example 1>
(1) Preparation of Cell Adhesive Polypeptide [P1] According to the method described in Examples in JP-T-3-502935, five Arg Gly Asp sequences (x1) and Ser Pro Ala Gly Gly ( Ala Gly Ala Gly Ser Gly) ₃ Ala Ser Thr Gly [Thr Ser Thr Gly] having a structure in which four of the Ala Ser Thr Gly sequences (115) and (y1) are alternately bonded to each other and having a number average molecular weight of about 20,000 Arg Gly Asp Ser Pro Ala Gly Gly (Ala Gly Ala Gly Ser Gly) ₃ Ala Ser Thr Gly] ₄ Arg Gly Asp Ser Pro Ala Gly Gly (Ala Gly Ala Gly Ser Gly) ₃ Ala Ser sequence (116) Manufactured by genetically modified E. coli. Further, this polypeptide was autoclaved (120 ° C., 20 minutes) to obtain a cell adhesion polypeptide (116) [P1].

(2) Preparation of Cell Adhesive Polypeptide-Containing Substrate [PB1A] 1 mg of cell adhesion polypeptide (P1) was dissolved in 1 mL of 4.5 M lithium perchlorate aqueous solution, and further 99.5 wt% sodium chloride was added in an amount of 0.001. P1 solution A (P1 concentration: 50 μg / mL) was prepared by diluting 20-fold with 0.02M phosphate buffer (pH 7.2, PBS) containing 85% by weight. This P1 solution A was put into a 96-well polystyrene plate (Nippon Becton Dickinson Co., Ltd.) at 50 μL / hole and left at 27 ± 3 ° C. for 2 hours. After removing the solution using an aspirator, it was washed twice with 100 μL / hole of physiological saline and further washed with 100 μL / hole of deionized water to prepare a cell-adhesive polypeptide-containing substrate [PB1A] (P1 attachment) Amount: about 1 μg / cm ² ).

(3) Preparation of Cell Adhesive Polypeptide-Containing Substrate [PB1B] 1 mg of cell adhesive polypeptide (P1) is dissolved in 1 mL of formic acid and further diluted 10-fold with formic acid to obtain P1 solution B (P1 concentration: 100 μg). / ML). This P1 solution B was poured into a 96-well polystyrene plate (Nippon Becton Dickinson Co., Ltd.) at 16 μL / hole and allowed to stand at 27 ± 3 ° C. for 12 hours. After formic acid was completely dried, reduced pressure (10 mmHg for 2 hours) ) Drying was performed. After drying, it was washed twice with 100 μL / well of physiological saline and further washed with 100 μL / hole of deionized water to prepare a cell-adhesive polypeptide-containing substrate [PB1B] (P1 adhesion amount: about 5 μg / cm ^2). ).

<Example 2>
(1) Preparation of Cell Adhesive Polypeptide [P5] According to the method described in the Examples of JP-T-3-502935, eight Arg Gly Asp sequences (x1) and Ser Pro Ala (Gly Val Pro Gly Val) ₂ Gly Gly (Gly Ala Gly Ala Gly Ser) ₃ Ala Ser Thr Gly 7 (7) of sequence (119) (y2) and number average molecular weight having a structure in which these are alternately chemically bonded 30,000 Thr Ser Thr Gly [Arg Gly Asp Ser Pro Ala (Gly Val Pro Gly Val) ₂ Gly Gly (Gly Ala Gly Ala Gly Ser) ₃ Ala Ser Thr Gly] ₇ Arg Gly Asp Ser Pro Ala (Gly Val Pro Gly Val) ₂ Gly Gly (Gly Ala Gly Ala Gly Ser) ₃ Ala Ser sequence (121) polypeptide was produced in E. coli. Further, this polypeptide was autoclaved (120 ° C., 20 minutes) to obtain a cell adhesion polypeptide (121) [P5].

(2) Preparation of cell-adhesive polypeptide-containing substrate [PB2A] The cell-adhesive polypeptide-containing substrate [PB1A] of Example 1 was used except that P5 was used instead of cell-adhesive polypeptide (P1). In the same manner as in the preparation, a cell-adhesive polypeptide-containing substrate [PB2A] was produced (P5 adhesion amount: about 1 μg / cm ² ).

(3) Preparation of cell-adhesive polypeptide-containing substrate [PB2B] The cell-adhesive polypeptide-containing substrate [PB1B] of Example 1 was used except that P5 was used instead of cell-adhesive polypeptide (P1). In the same manner as in the preparation, a cell-adhesive polypeptide-containing substrate [PB2B] was produced (P5 adhesion amount: about 5 μg / cm ² ).

<Example 3>
(1) Preparation of Cell Adhesive Polypeptide [P7] According to the method described in the examples in JP-T-3-502935, five Arg Gly Asp sequences (x1) and Ser Pro Ala Ala Ser Asp Gly Gly (Ala) ₁₂ Gly Gly Ala Ala Ser Thr Gly Thr Ser Thr Gly having a structure in which four of the sequences (123) (y3) and these are alternately chemically bonded have a number average molecular weight of about 20,000 [Arg Gly Asp Ser Pro Ala Ala Ser Asp Gly Gly (Ala) ₁₂ Gly Gly Ala Ala Ser Thr Gly] ₄ Arg Gly Asp Ser Pro Ala Ala Ser Asp Gly Gly (Ala) ₁₂ Gly Gly Ala Ala Ser of sequence (124) The polypeptide was produced by genetically modified E. coli. Further, this polypeptide was autoclaved (120 ° C., 20 minutes) to obtain a cell adhesion polypeptide (124) [P7].

(2) Preparation of cell-adhesive polypeptide-containing substrate [PB3A] The cell-adhesive polypeptide-containing substrate [PB1A] of Example 1 was used except that P7 was used instead of cell-adhesive polypeptide (P1). In the same manner as in the preparation, a cell-adhesive polypeptide-containing substrate [PB3A] was prepared (P7 adhesion amount: about 1 μg / cm ² ).

(3) Preparation of cell-adhesive polypeptide-containing substrate [PB3B] The cell-adhesive polypeptide-containing substrate [PB1B] of Example 1 was used except that P7 was used instead of cell-adhesive polypeptide (P1). In the same manner as in the preparation, a cell-adhesive polypeptide-containing substrate [PB3B] was produced (P7 adhesion amount: about 5 μg / cm ² ).

<Example 4>
(1) Preparation of Cell Adhesive Polypeptide [P39] Tyr Gly Gly Leu Gly Ser Gln Gly Ala Gly Arg Sequence (156) (x4) According to the method described in Examples in Japanese Patent Publication No. Hei 3-502935 ) And 9 of Ala Ser (Ala Gly Ser Gly Ala Gly) ₃ Ala Ser sequence (157) (y5), and a number average molecular weight of about 50,000 having a structure in which these are alternately chemically bonded Met His His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met Ala Asp Ile Gly Ser Ser Arg Met Thr Ser Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Ser (Tyr Gly Gly Leu Gly Ser Gln Gly Ala Gly Arg (Ala Ser (Ala Gly Ser Gly Ala Gly) ₃ Ala Ser)) ₉ Tyr Gly Gly Leu Gly Ser Gln Gly Ala Gly Arg Ala Ser Met Ser Arg Val Asp Leu Lys Leu Ala Ala Ala Leu Glu His His His His His His Sequence (158) The peptide was produced by genetically modified E. coli. Further, this polypeptide was autoclaved (120 ° C., 20 minutes) to obtain a cell adhesion polypeptide (158) [P39].

(2) Preparation of cell-adhesive polypeptide-containing substrate [PB4A] The cell-adhesive polypeptide-containing substrate [PB1A] of Example 1 was used except that P39 was used instead of cell-adhesive polypeptide (P1). In the same manner as in the preparation, a cell-adhesive polypeptide-containing substrate [PB4A] was produced (P39 adhesion amount: about 1 μg / cm ² ).

<Comparative Example 1>
(1) Preparation of Cell Adhesive Polypeptide [P4] According to the method described in the examples in JP-T-3-502935, 14 Arg Gly Asp sequences (x1) and Ser Pro Ala Ser Ala Ala Gly Tyr (Gly Ala Gly Ala Gly Ser) ₉ Gly Ala Ala Val Thr Gly 13 (13) of the sequence (154) (y5) and a number average molecular weight of about 100,000 Met Asp Pro Val Val Leu Gln Arg Arg Asp Trp Glu Asn Pro Gly Val Thr Gln Leu Asn Arg Leu Ala Ala His Pro Pro Phe Ala Ser Asp Pro Met Gly Ala Gly Ser (Gly Ala Gly Ala Gly Ser) ₆ Gly Ala Ala Val Thr Gly [Arg Gly Asp Ser Pro Ala Ser Ala Ala Gly Tyr (Gly Ala Gly Ala Gly Ser) ₉ Gly Ala Ala Val Thr Gly] ₁₃ Arg Gly Asp Ser Pro Ala Ser Ala Ala Gly Tyr (Gly Ala Gly Ala Gly Ser) ₂ Gly Ala Gly Ala Met Asp Pro Gly Arg Tyr Gln Leu Ser Ala Gly Arg Tyr His Tyr Gln Leu Val Trp Cys Gln Lys polypeptide (155) produced by genetically modified E. coli did. Further, this polypeptide was autoclaved (120 ° C., 20 minutes) to obtain a cell adhesion polypeptide (155) [P37].

(2) Preparation of cell adhesive polypeptide-containing substrate [PB5A] The cell adhesive polypeptide-containing substrate [PB1A] of Example 1 was used except that P37 was used instead of the cell adhesive polypeptide (P1). In the same manner as in the preparation, a cell adhesive polypeptide-containing substrate [PB5] was produced (P37 adhesion amount: about 1 μg / cm ² ).

(3) Preparation of cell-adhesive polypeptide-containing substrate [PB4B] The cell-adhesive polypeptide-containing substrate [PB1B] of Example 1 was used except that P37 was used instead of cell-adhesive polypeptide (P1). In the same manner as in the preparation, a cell-adhesive polypeptide-containing substrate [PB4B] was produced (P37 adhesion amount: about 5 μg / cm ² ).

<Comparative example 2>
(1) Preparation of cell adhesion polypeptide [P5] Cell adhesion polypeptide [P38] is obtained by autoclaving (120 ° C, 20 minutes) of bovine-derived collagen type I (Nippon Becton Dickinson Co., Ltd.). It was.

(2) Preparation of Cell Adhesive Polypeptide-Containing Substrate [PB6A] 3. P15 solution A (concentration of P38) was obtained by diluting 3.15 mg / mL cell adhesion polypeptide (P38) 63-fold with 0.012N hydrochloric acid aqueous solution. : 50 μg / mL). This P38 solution A was put into a 96-well polystyrene plate (Nippon Becton Dickinson Co., Ltd.) at 50 μL / hole and left at 27 ± 3 ° C. for 2 hours. After removing the solution using an aspirator, it was washed twice with 100 μL / hole of physiological saline and further washed with 100 μL / hole of deionized water to produce a cell-adhesive polypeptide-containing substrate [PB6A] (P38 attachment) Amount: about 1 μg / cm ² ).

(3) Preparation of Cell Adhesive Polypeptide-Containing Substrate [PB5B] 3.15 mg / mL cell adhesive polypeptide (P38) was diluted 31.5 times with formic acid to obtain P38 solution B (P38 concentration: 100 μg). / ML). This P38 solution B was poured into a 96-well polystyrene plate (Nippon Becton Dickinson Co., Ltd.) at 16 μL / hole and allowed to stand at 27 ± 3 ° C. for 12 hours. After formic acid was completely dried, reduced pressure (10 mmHg) for 2 hours. ) Drying was performed. After drying, the cells were washed twice with 100 μL / well of physiological saline and further washed with 100 μL / well of deionized water to prepare a cell-adhesive polypeptide-containing substrate [PB5B] (P38 adhesion amount: about 5 μg / cm ^2). ).

<Comparative Example 3>
(1) Preparation of Substrate [H7A] Substrate [H7A] was prepared in the same manner as in Cell Adhesive Polypeptide-Containing Substrate [PB1A] of Example 1, except that the cell adhesive polypeptide (P1) was not used. Produced.
(2) Preparation of Substrate [H6B] Substrate [H6B] was prepared in the same manner as Cell Adhesive Polypeptide-Containing Substrate [PB1B] in Example 1 except that cell adhesive polypeptide (P1) was not used. Produced.

<Evaluation 1: Cell adhesion activity of NHDF cells (normal human skin fibroblasts)>
(1) An aqueous solution containing 0.2% by weight of bovine serum albumin in PBS was put into an evaluation substrate (PB1A to PB6A and H7A) at 50 μL / hole and left at 27 ± 3 ° C. for 2 hours. After removing the solution using an aspirator, it was washed twice with 100 μL / hole of physiological saline and further washed with 100 μL / hole of deionized water. Next, DMEM (manufactured by ICN Biomedicals) was added at 100 μL / well and stored in a 37 ° C. incubator for 1 hour. After 1 hour, NHDF cells were added at 10,000 cells / well and left to stand for 1 hour in an incubator at 37 ° C. and a CO ₂ concentration of 5% by volume.

(2) After completion of the culture, the medium is removed using an aspirator, and 100 μL / well is taken so that ion-exchanged water containing 0.85% by weight of sodium chloride (hereinafter referred to as physiological saline) is not directly applied to the cells. And the physiological saline was removed using an aspirator. Next, PBS was added at 50 μL / hole, and Tetra Color One (Seikagaku Corporation) was added at 10 μL / hole, and left in an incubator at 37 ° C. and 5% by volume of CO ₂ for 4 hours.

(3) 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 (control wavelength 630 nm), and this value was defined as cell adhesion activity. The cell adhesion activity is proportional to the absorbance. These results are shown in Table 1 (these results are average data for 8 holes each). The tetrazolium salt of Tetracolor One is reduced by intracellular mitochondrial dehydrogenase to produce formazan and develop color.

<Evaluation 2: Cell adhesion activity of VERO cells (African green monkey kidney-derived cells)>
(1) An aqueous solution containing 0.2% by weight of bovine serum albumin in PBS was put into an evaluation substrate (PB1B to PB5B and H6B) at 50 μL / hole and left at 27 ± 3 ° C. for 2 hours. After removing the solution using an aspirator, it was washed twice with 100 μL / hole of physiological saline and further washed with 100 μL / hole of deionized water. Next, DMEM (manufactured by ICN Biomedicals) was added at 100 μL / well and stored in a 37 ° C. incubator for 1 hour. After 1 hour, VERO cells were added at 10,000 cells / well and left to stand for 1 hour in an incubator at 37 ° C. and 5% by volume of CO ₂ .

(2) After completion of the culture, the medium was removed using an aspirator, and physiological saline was added at 100 μL / well, taking care not to directly hit the cells, and the physiological saline was removed using an aspirator. Next, PBS was added at 50 μL / hole, and Tetra Color One (Seikagaku Corporation) was added at 10 μL / hole, and left in an incubator at 37 ° C. and 5% by volume of CO ₂ for 4 hours.

(3) 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 (control wavelength 630 nm), and this value was defined as cell adhesion activity. The cell adhesion activity is proportional to the absorbance. These results are shown in Table 2 (these results are average data for 8 holes each).

  From the results of Tables 1 and 2, it can be seen that the cell adhesive polypeptide of the present invention has extremely high cell adhesiveness compared to the case where the cell adhesive polypeptide or cell adhesive polypeptide of the comparative example is not used. .

Claims (8)

A polypeptide having a structure in which a minimum amino acid sequence (X) and an auxiliary amino acid sequence (Y) that express a cell adhesion signal are alternately chemically bonded, and the total number of amino acids contained in (Y) is 1 to 50 And the total content of glycine (Gly) and alanine (Ala) contained in (Y) is 42 to 100% by number based on the total number of amino acids in (Y). Polypeptide. The minimum amino acid sequence (X) showing a cell adhesion signal is Arg Gly Asp sequence, Leu Asp Val sequence, Arg Glu Asp Val sequence (1), Tyr Ile Gly Ser Arg sequence (2), Pro Asp Ser Gly Arg sequence ( 3), Arg Tyr Val Val Leu Pro Arg sequence (4), Leu Gly Thr Ile Pro Gly sequence (5), Arg Asn Ile Ala Glu Ile Ile Lys Asp Ile sequence (6), Ile Lys Val Ala Val sequence (7) , Leu Arg Glu sequence, Asp Gly Glu Ala sequence (8), Gly Val Lys Gly Asp Lys Gly Asn Pro Gly Trp Pro Gly Ala Pro sequence (9), Gly Glu Phe Tyr Phe Asp Leu Arg Leu Lys Gly Asp Lys sequence ( 10) at least one selected from the group consisting of His Ala Val sequence, Tyr Lys Leu Asn Val Asn Asp Ser sequence (11), and Tyr Gly Gly Leu Gly Ser Gln Gly Ala Gly Arg sequence (156) The cell adhesion polypeptide described. The auxiliary amino acid sequence (Y) is (Gly Ala) a sequence, (Gly Ala Gly Ala Gly Ser) b sequence, (Gly Ala Gly Ala Gly Tyr) c sequence, (Gly Ala Gly Val Gly Tyr) d sequence, (Gly Ala Gly Tyr Gly Val) e sequence, {Asp Gly Gly (Ala) f The cell adhesion polypeptide according to claim 1 or 2, which has at least one selected from the group consisting of a Gly Gly Ala} g sequence and a (Gly Val Pro Gly Val) h sequence.
A is an integer of 1 to 25, b, c, d and e are integers of 1 to 8, f is an integer of 1 to 44, f is 1, g is an integer of 1 to 7, and f is 2. g is an integer of 1 to 6, when f is 3 or 4, g is an integer of 1 to 5, when f is 5 or 6, g is an integer of 1 to 4, and when f is an integer of 7 to 10, g is 1 When g is an integer of 11 to 19, g is 1 or 2, and when f is an integer of 20 to 44, g is 1 and h is an integer of 1 to 10. The cell adhesion polypeptide according to any one of claims 1 to 3, wherein the number of repeating units (XY) of the minimum amino acid sequence (X) and the auxiliary amino acid sequence (Y) is 2 to 30. The cell adhesion polypeptide according to any one of claims 1 to 4, wherein the content ratio (X / Y) of the minimum amino acid sequence (X) and the auxiliary amino acid sequence (Y) is 0.66-1.5. (116) to (118), (120) to (122), (124) to (153) and (158) to (172) any one of SEQ ID NOs selected from the group consisting of (172) peptide. A cell-adhesive polypeptide-containing substrate comprising the cell-adhesive polypeptide (P) according to any one of claims 1 to 6 and the substrate (B). A method for producing cells, comprising a step of culturing cells using the cell-adhesive polypeptide-containing substrate according to claim 7.