JP4493327B2 - Cell adhesion polypeptide - Google Patents

Description

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

Cell adhesion of natural cell-adhesive polypeptides {collagen, fibronectin, vitronectin, fibrinogen, laminin, cadherin and other human-derived proteins (non-patent document 1), fibroin and other insect-derived proteins (non-patent document 2)} Artificial cell adhesion polypeptide with improved stability and heat stability {SELPF (Arg Gly Asp sequence and (Gly Ala Gly Ala Gly Ser) ₁₂ sequence (12) each about 12) A polypeptide having a number average molecular weight of about 80,000) and CLP-CB (Arg Gly Asp sequence and (Gly Ala Pro (Gly Pro Pro) ₄ ) ₂ sequence (13)) (A polypeptide having a number average molecular weight of about 40,000 produced by the above), etc.] 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 artificial cell-adhesive polypeptides may act as an immunogen when in contact with living tissue, producing a large amount of antibody in vivo. That is, there is a problem that an artificial cell adhesion polypeptide may have high immunogenicity.
An object of the present invention is to provide a cell adhesion polypeptide with low immunogenicity.

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 adhesive polypeptide of the present invention is characterized by a polypeptide (P) having a minimal amino acid sequence (X) and an auxiliary amino acid sequence (Y) that expresses cell adhesiveness, wherein (X) is Arg Gly is Asp sequence or Ile Lys Val Ala Val sequence (7), (Y) is (Gly Ala Gly Ala Gly Ser) 3 sequences (21) or _{(Gly Val Pro Gly Val) 2} (Gly Ala Gly Ala Gly Ser) 3 The gist is that it is the sequence (104), and the number average molecular weight of the polypeptide is 4,000 to 9,000.

The cell adhesion polypeptide of the present invention has extremely low immunogenicity. In addition, cell adhesion and thermal stability are remarkably excellent.
Therefore, since the cell adhesive polypeptide of the present invention has high safety, it is suitable for research and development related to cells, production of useful substances, treatment, and the like.

“Cell adhesion” means the property that a specific minimum amino acid sequence is recognized by a cell's integrin receptor so that the cell can easily adhere to a substrate (Osaka Prefectural Maternal and Child Medical Center, Vol. 8, No. 1, 58-66, 1992).
Examples of the minimum amino acid sequence (X) for expressing cell adhesion include, for example, “Pathophysiology, Vol. 9, No. 7, pp. 527-535, 1990” and “Osaka Prefectural Maternal and Child Medical Center, 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 and Tyr Lys Leu Asn Val Asn Asp Ser sequence (11) are preferable, and from the viewpoint of cell adhesion, Arg Gly Asp sequence, Tyr Ile Gly Ser Arg sequence (2) and Ile Lys Val are more preferable. Ala Val sequence (7), particularly preferably Arg Gly Asp sequence.

Polypeptide (P) may have at least one minimum amino acid sequence (X) in one molecule, but from the viewpoint of immunogenicity and cell adhesion, etc., one having 1 to 15 in one molecule More preferably, it has 2 to 10, particularly preferably 3 to 7, most preferably 3 to 6.
Two or more kinds of minimum amino acid sequences (X) may be included in one molecule.

The auxiliary amino acid sequence (Y) can be used without limitation as long as it is an amino acid sequence other than the minimum amino acid sequence (X). From the viewpoint of heat resistance and cell adhesiveness of the cell adhesive polypeptide (P), Gly and / or Or those having Ala are preferred, for example, (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, (Gly Gly Ala) k sequence, (Gly Val Gly Val Pro) m sequence, (Gly Pro Pro) n sequence, (Gly Ala Gln Gly Pro Ala Gly Pro Gly) o sequence, (Gly Ala Pro Gly Ala Pro Gly Ser Gln Gly Ala Pro Gly Leu Gln) p sequence and / or (Gly Ala Pro Gly Thr Pro Gly Pro Gln Gly Leu Pro Gly Ser Pro) those having q sequence and the like. Among these, (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, {Asp Gly Gly (Ala) f Gly Gly Ala} g sequence, (Gly Val Pro Gly Val) h sequence, (Gly Val Gly Val Pro) m sequence and / or (Gly Pro Pro) n sequence More preferably (Gly Ala Gly Ala Gly Ser) b sequence, (Gly Val Pro Gly Val) h sequence, (Gly Val Gly Val Pro) m sequence and / or (Gly Pro Pro) n sequence, Particularly preferred are those having the (Gly Ala Gly Ala Gly Ser) b sequence.
A is an integer from 1 to 25, b, c, d and e are integers from 1 to 8, f is an integer from 1 to 44, f is 1 and g is an integer from 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, m is an integer from 1 to 10, n is an integer from 1 to 16, o is an integer from 1 to 5, p is an integer from 1 to 3, and q is an integer from 1 to 3.

  In addition to the above examples, 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.

Examples of those having the (Gly Ala) a sequence include the amino acid sequences of SEQ ID NOs: (14) to (19).
Examples of those having (Gly Ala Gly Ala Gly Ser) b sequence include amino acid sequences of SEQ ID NOs: (20) to (25).
Examples of those having (Gly Ala Gly Ala Gly Tyr) c sequence include the amino acid sequences of SEQ ID NOs: (26) to (31).
Examples of those having (Gly Ala Gly Val Gly Tyr) d sequence include the amino acid sequences of SEQ ID NOs: (32) to (37).
Examples of those having (Gly Ala Gly Tyr Gly Val) e sequence include amino acid sequences of SEQ ID NOs: (38) to (43).
Examples of those having the {Asp Gly Gly (Ala) f Gly Gly Ala} g sequence include the amino acid sequences of SEQ ID NOs: (44) to (49).
Examples of those having (Gly Val Pro Gly Val) h sequence include amino acid sequences of SEQ ID NOs: (50) to (55).
Examples of those having (Gly) i sequence include the amino acid sequences of SEQ ID NOs: (56) to (61).
Examples of those having (Ala) j sequence include the amino acid sequences of SEQ ID NOs: (62) to (67).
Examples of those having (Gly Gly Ala) k sequence include the amino acid sequences of SEQ ID NOs: (68) to (73).
Examples of those having (Gly Val Gly Val Pro) m sequence include the amino acid sequences of SEQ ID NOs: (74) to (79).
Examples of (Gly Pro Pro) having the n sequence include the amino acid sequences of SEQ ID NOs: (80) to (85).
(Gly Ala Gln Gly Pro Ala Gly Pro Gly) Examples of those having o sequence include amino acid sequences of SEQ ID NOs: (86) to (91).
(Gly Ala Pro Gly Ala Pro Gly Ser Gln Gly Ala Pro Gly Leu Gln) As what has p sequence, the amino acid sequence of sequence number (92)-(97), etc. are mentioned.
(Gly Ala Pro Gly Thr Pro Gly Pro Gln Gly Leu Pro Gly Ser Pro) The thing which has q sequence includes the amino acid sequence of sequence number (98)-(103).

  Polypeptide (P) may have at least one auxiliary amino acid sequence (Y) in one molecule, but from the viewpoint of immunogenicity, cell adhesiveness and heat stability, 1 to 15 in one molecule. Those having a number are preferred, more preferably 2 to 10, particularly preferably 3 to 7, most preferably 3 to 6. Two or more auxiliary amino acid sequences (Y) may be included in one molecule.

The total content (%) of glycine (Gly) and alanine (Ala) contained in the auxiliary amino acid sequence (Y) is preferably 10 to 100, more preferably based on the total number of amino acids in the auxiliary amino acid sequence (Y). It is 20 to 95, particularly preferably 30 to 90, and most preferably 40 to 85. That is, the lower limit of the total content ratio (%) of (Gly) and (Ala) is preferably 10, more preferably 20, particularly preferably 30, and most preferably 40 based on the total number of amino acids in (Y). Similarly, the upper limit is preferably 100, more preferably 95, particularly preferably 90, and most preferably 85. Within this range, cell adhesion and thermal stability are 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. ~ 5. 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 5. Within this range, the immunogenicity and the like are low and the quality is further improved.

  The polypeptide (P) is formed by chemically bonding a minimum amino acid sequence (X) and an auxiliary amino acid sequence (Y). The polypeptide (P) may partially include a branched chain, may be partially crosslinked, and may include a cyclic structure, but is preferably not crosslinked. Preferred is a linear structure that is not crosslinked, and particularly preferred is a linear structure that has no cyclic structure and is not crosslinked. 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).

  The polypeptide (P) preferably has a structure in which the minimum amino acid sequence (X) and the auxiliary amino acid sequence (Y) are alternately chemically bonded from the viewpoint of cell adhesion and thermal stability. In this case, the number of repeating units (XY) of the minimum amino acid sequence (X) and auxiliary amino acid sequence (Y) in the polypeptide (P) is from the viewpoint of cell adhesion and immunogenicity, etc. 1-15 is preferable, More preferably, it is 2-10, Most preferably, it is 3-7, Most preferably, it is 3-6.

The number average molecular weight (Mn) of the polypeptide (P) is preferably 1,500 to 15,000, more preferably 2,000 to 14,000, particularly preferably 3,000 to 12,000, most preferably 4. , 10,000 to 10,000. That is, the lower limit of (Mn) in (P) is preferably 1,500, more preferably 2,000, particularly preferably 3,000, most preferably 4,000, and similarly the upper limit is 15,000. Is more preferably 14,000, particularly preferably 12,000, and most preferably 10,000.
The number average molecular weight (Mn) can be measured by a known method. For example, a measurement sample {polypeptide etc.} is separated by SDS-PAGE (SDS polyacrylamide gel electrophoresis), and the migration distance is determined from the standard substance. It is obtained by comparing (hereinafter the same).

The cell adhesion polypeptide of the present invention is a polypeptide having a minimum amino acid sequence (X) and an auxiliary amino acid sequence (Y), and the number average molecular weight of the polypeptide is 1,500 to 15,000 without limitation. Preferred examples include polypeptides (P1 to P345).
(1) It has two (x1) containing Arg Gly Asp sequence (x1) as a minimum amino acid sequence (X) and two (Gly Ala Gly Ala Gly Ser) ₃ sequences (21) (y1) Polypeptide (P1) with Mn of about 4,000 having a structure in which these are alternately chemically bonded (P1); three of (x1) and three of (y1), and these are alternately chemically bonded Polypeptide (P2) having an Mn of about 6,000 having a structure; 4 of (x1) and 4 of (y1), and having an Mn of about 8,000 having a structure in which these are alternately chemically bonded Polypeptide (P3); two of (x1) and two of (Gly Val Pro Gly Val) ₂ (Gly Ala Gly Ala Gly Ser) ₃ sequence (104) (y2) are alternately chemically bonded A polypeptide (P4) having an Mn of about 6,000 having the structure formed by: 3 (x1) and 3 (y2) Polypeptide (P5) having an Mn of about 9,000 having a structure in which these are alternately chemically bonded (P5); four (x1) and four (y2), and these are alternately chemically bonded A polypeptide having a structure Mn of about 12,000 (P6); two of (x1) and two of [Gly Ala Pro (Gly Pro Pro) ₄ ] ₂ sequence (13) (y3) Polypeptide (P7) with an Mn of about 7,000 having a structure formed by alternately chemical bonding (P7); a structure having three (x1) and three (y3), which are alternately chemically bonded Polypeptide (P8) having an Mn of about 11,000; a polypeptide having an Mn of about 14,000 having a structure in which four of (x1) and four of (x3) are alternately bonded to each other (P9); (x1) 4 and the (Gly Ala) ₁₀ sequence (15) of four and have these Mn about 8,000 polypeptide having a structure formed by chemically bonding alternately (P10); (x1) 4 and the (Gly Ala Gly Ala Gly Tyr) 3 sequence (27) of four and have these About 9,000 Mn polypeptides having a structure formed by alternately chemical bonding (P11); (x1) and 4 (Gly Ala Gly Val Gly Tyr) ₃ sequences (33) Polypeptide (P12) with Mn of about 9,000 having a structure in which these are alternately chemically bonded (P12); (x1) and 4 (Gly Ala Gly Tyr Gly Val) ₃ sequences (39) In addition, four of Mn about 9,000 polypeptides (P13); (x1) and 4 of {Asp Gly Gly (Ala) ₁₂ Gly Gly Ala} sequence (45) having a structure in which these are chemically bonded alternately. About 8,000 Mn having a structure in which these are alternately and chemically bonded to each other (P14) (X1) 4 and the (Gly Val Pro Gly Val) ₃ sequence (51) of four and a Mn of about 8,000 polypeptide they have a structure formed by chemically bonding alternately (P15); A polypeptide (P16) having an Mn of about 8,000 having 4 structures of (x1) and 4 structures of (Gly) ₂₁ sequence (57) and having these chemically alternated (P16); A polypeptide of about 8,000 Mn (P17) having 4 and (Ala) ₁₉ sequence (63) and having a structure in which these are alternately chemically bonded (P17); Gly Gly Ala) _{7 of the} sequence (69) and having a structure in which these are alternately chemically bonded to each other, an approximately 8,000 Mn polypeptide (P18); (x1) 4 and (Gly Val Gly Val Pro) ₃ of 4 sequences (75), and Mn having a structure in which these are alternately chemically bonded. 00 polypeptide (P19); 4 of (x1) and 4 of (Gly Pro Pro) ₅ sequence (81), and having a structure in which these are alternately chemically bonded, Mn of about 8,000 Polypeptide (P20); Mn approx. 4 having (x1) and 4 (Gly Ala Gln Gly Pro Ala Gly Pro Gly) ₂ sequence (87) having a structure in which these are alternately chemically bonded 9,000 polypeptides (P21); 4 of (x1) and 4 of (Gly Ala Pro Gly Ala Pro Gly Ser Gln Gly Ala Pro Gly Leu Gln) sequence (93), which are alternately chemistry A polypeptide (P22) having an Mn of about 8,000 having a structure formed by binding; (Gly Ala Pro Gly Thr Pro Gly Pro Gln Gly Leu Pro Gly Ser Pro) A polypeptide having a structure obtained by chemically bonding to about 8,000 Mn (P23) and the like.

(2) Polypeptide containing Leu Asp Val sequence (x2) as minimum amino acid sequence (X) Polypeptide obtained by changing Arg Gly Asp sequence (x1) of polypeptides (P1) to (P23) to Leu Asp Val sequence (x2) (P24) to (P46) and the like.

(3) An Arg Glu Asp Val sequence (1) (x3) as the minimum amino acid sequence (X) Arg Gly Asp sequence (x1) of polypeptides (P1) to (P23) is represented by Arg Glu Asp Val sequence (1) Polypeptides (P47) to (P69) etc. changed to (x3).

(4) Those containing the Tyr Ile Gly Ser Arg sequence (2) (x4) as the minimum amino acid sequence (X) The Arg Gly Asp sequence (x1) of the polypeptides (P1) to (P23) is converted to the Tyr Ile Gly Ser Arg sequence ( 2) Polypeptides (P70) to (P92) etc. changed to (x4).

(5) Containing Pro Asp Ser Gly Arg Sequence (3) (x5) as Minimum Amino Acid Sequence (X) Arg Gly Asp Sequence (x1) of Polypeptides (P1) to (P23) is Pro Asp Ser Gly Arg Sequence ( 3) Polypeptides (P93) to (P115) etc. changed to (x5).

(6) Containing Arg Tyr Val Val Leu Pro Arg Sequence (4) (x6) as Minimum Amino Acid Sequence (X) Arg Gly Asp Sequence (x1) of Polypeptides (P1) to (P23) is Arg Tyr Val Val Leu Polypeptides (P116) to (P138) modified to Pro Arg sequence (4) (x6).

(7) The one containing Leu Gly Thr Ile Pro Gly sequence (5) (x7) as the minimum amino acid sequence (X) The Arg Gly Asp sequence (x1) of polypeptides (P1) to (P23) is obtained as Leu Gly Thr Ile Pro Gly Polypeptide (P139)-(P161) etc. which were changed into sequence (5) (x7).

(8) An Arg Asn Ile Ala Glu Ile Ile Lys Asp Ile Sequence (6) (x8) as the Minimum Amino Acid Sequence (X) Arg Gly Asp Sequence (x1) of Polypeptides (P1) to (P23) Ile Ala Glu Ile Ile Lys Asp Ile Polypeptide (P162)-(P184) etc. changed to sequence (6) (x8).

(9) Those containing the Ile Lys Val Ala Val sequence (7) (x9) as the minimum amino acid sequence (X) The Arg Gly Asp sequence (x1) of the polypeptides (P1) to (P23) is converted into the Ile Lys Val Ala Val sequence ( 7) Polypeptides (P185) to (P207) etc. changed to (x9).

(10) Polypeptide containing Leu Arg Glu sequence (x10) as minimum amino acid sequence (X) Polypeptide obtained by changing Arg Gly Asp sequence (x1) of polypeptides (P1) to (P23) to Leu Arg Glu sequence (x10) (P208) to (P230) and the like.

(11) The one containing Asp Gly Glu Ala sequence (8) (x11) as the minimum amino acid sequence (X) Arg Gly Asp sequence (x1) of polypeptides (P1) to (P23) is Asp Gly Glu Ala sequence (8) Polypeptides (P231) to (P253) changed to (x11).

(12) Those containing the Gly Val Lys Gly Asp Lys Gly Asn Pro Gly Trp Pro Gly Ala Pro sequence (9) (x12) as the minimum amino acid sequence (X) Arg Gly Asp sequence of the polypeptides (P1) to (P23) ( Polypeptides (P254) to (P276) obtained by changing x1) to Gly Val Lys Gly Asp Lys Gly Asn Pro Gly Trp Pro Gly Ala Pro sequence (9) (x12).

(13) Those containing Gly Glu Phe Tyr Phe Asp Leu Arg Leu Lys Gly Asp Lys Sequence (10) (x13) as Minimum Amino Acid Sequence (X) Arg Gly Asp Sequence (x1) of Polypeptides (P1) to (P23) Gly Glu Phe Tyr Phe Asp Leu Arg Leu Lys Polypeptides (P277) to (P299) and the like changed to Gly Asp Lys sequence (10) (x13).

(14) A polypeptide comprising His Ala Val sequence (x14) as minimum amino acid sequence (X) Polypeptide (P1) to Arg Gly Asp sequence (x1) of (P23) changed to His Ala Val sequence (x14) (P300) to (P322) and the like.

(15) Those containing the Tyr Lys Leu Asn Val Asn Asp Ser sequence (11) (x15) as the minimum amino acid sequence (X) The Arg Gly Asp sequence (x1) of the polypeptides (P1) to (P23) is converted to Tyr Lys Leu Asn Polypeptides (P323) to (P345) and the like changed to Val Asn Asp Ser sequence (11) (x15).

Among these examples, polypeptides (P1) to (P23), polypeptides (P70) to (P92) and polypeptides (P185) to (P207) are more preferable, and then polypeptides (P1) to (P P23) and polypeptides (P185) to (P207), particularly preferably polypeptides (P1) to (P23), most preferably (P1) to (P9).

  The polypeptide (P) can be artificially produced, 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 Japanese Patent No. 3338441 can be applied. Although both the organic synthesis method and the gene recombination method can produce the polypeptide (P), the gene recombination method is preferable from the viewpoint that the polypeptide (P) can be mass-produced at low cost.

  The polypeptide (P) is preferably modified with a compound (AM) containing an amino group and / or an ammonio group. When it is modified with (AM), the cell adhesiveness of (P) is further improved.

As the compound (AM) containing an amino group and / or an ammonio group, polyamines, amino alcohols, halides having amino groups, amino group-containing monomers, polymers of amino group-containing monomers, and quaternized products thereof are used. it can.
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 (C1-C18) substitution products thereof (dimethylaminopropylamine, diethylaminopropylamine, dipropylaminopropylamine, methylethylaminopropylamine, Trimethylhexamethylenediamine, N, N-dioctadecylethylenediamine, trioctadecylethylenediamine, methyliminobispropylamine, etc.) And the like.

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.

  As the halide having an amino group, halogenated (such as chlorine and bromine) of alkylamine having 2 to 17 carbon atoms is used, and aminoethyl chloride, N-methylaminopropyl chloride, dimethylaminoethyl chloride, diethylaminoethyl chloride are used. , Dibenzylaminoethyl bromide, dimethylaminopropyl bromide, diethylaminopropyl chloride, 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. In addition, (meth) acryl ... means acryl ... and / or methacryl ...

  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 polypeptide (not including polypeptide (P)). The weight average molecular weight of the amino group-containing monomer polymer is preferably 500 or more, more preferably 1,000 or more, particularly preferably 2,000 or more, and preferably 1,000,000 or less, more preferably 800,000. Hereinafter, it is particularly preferably 500,000 or less. The weight average molecular weight can be measured by gel permeation chromatography (GPC). In addition, as a reference material, for example, a polystyrene standard (manufactured by Tosoh) having a molecular weight of 420 to 20,600,000 can be used.

  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).

  Examples of the method of modifying with a compound (AM) containing an amino group and / or ammonio group include a method of reacting a compound (AM) containing an amino group and / or ammonio group with a polypeptide before modification, and A method of physically adsorbing a compound (AM) containing an amino group and / or an ammonio group to a polypeptide before modification can be applied, and the method can be performed by using a polypeptide (P) and a substrate (B) described later. Among the bonding methods, chemical bonding and / or physical adsorption similar to (1) to (3) can be used, and preferable chemical bonding and / or physical adsorption are also the same.

The average number (number) of amino groups in the polypeptide (P) modified with the compound (AM) containing an amino group and / or an ammonio group is 0.001 to 100,000 per molecule of the polypeptide (P). Is preferable, more preferably 0.01 to 10,000, and particularly preferably 0.1 to 1,000. That is, the lower limit of the average number (number) of amino groups in (P) is preferably 0.001 per molecule of (P), more preferably 0.01, particularly preferably 0.1, and similarly The upper limit is preferably 100,000, more preferably 10,000, and particularly preferably 1,000.
The average number of amino groups can be quantified by a known method or the like. For example, the trinitrobenzene sulfonic acid (TNBS) method [Protein Chemistry IV (published by Tokyo Chemical Industry Co., Ltd., 1981)] or hydrochloric acid / indicator (bromophenol blue, etc.) ) Total amine value measurement method by titration method [JIS K7237-1986, ASTM D2074-66, etc.] and the like.
Specifically, for example, a compound (AM) containing an amino group and / or an ammonio group having a known number of amino groups is measured by the TNBS method while changing the concentration per unit volume, and a calibration curve (amino group) Number and absorbance graph). Moreover, the polypeptide (P) before modification is measured by the TNBS method, and the obtained absorbance is converted into the number of amino groups using a calibration curve. At the same time, the modified polypeptide is measured by the TNBS method, and the obtained absorbance is converted into the number of amino groups using a calibration curve. By calculating the difference in the number of amino groups before and after the modification, and dividing that value by the number of molecules of the polypeptide (P) used in the measurement, the average number of amino groups modified by the polypeptide (P) And

The cell adhesion polypeptide 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 (nylon), polyvinyl chloride, polyvinylidene chloride, polystyrene, fluororesin, silicone resin, cellulose And chemical fibers (viscose rayon, cupra rayon, polynosic, acetate, triacetate, polyethylene, polypropylene, polyamide, polyester, polyacrylonitrile, vinylon, polyvinyl chloride, vinylidene and polyurea Emissions etc.) and the like can be 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, polyamide (nylon), polystyrene, and silicone are particularly preferable. Resin.

  The shape of the base material (B) is not particularly limited as long as cells can be adhered, and generally used materials can be used. Plates, petri dishes, T-flasks, roller bottles, beads, hollow fibers, sheets ( Any of film, foam (sponge) and cloth) and gel may be used.

  The cell adhesion polypeptide and the base material (B) are combined by chemical bonding (ionic bond, hydrogen bond and / or covalent bond, etc.) 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 and the base material (B) are firmly bonded.

  Examples of the method for covalently binding the cell adhesion polypeptide and the base material (B) include (1) a cell adhesion polypeptide having a primary amino group or a secondary amino group (for example, arginine, histidine , Isoleucine, leucine, methionine, phenylalanine, threonine, tryptophan, tyrosine, valine, alanine, asparagine, aspartic acid, glutamine, glutamic acid, proline, cysteine, lysine, serine, glycine, ornithine, histidine, 3-aminopropionic acid, 8- A cell-adhesive polypeptide comprising aminooctanoic acid and 20-aminoeicosanoic acid as structural units), and (B) having a carboxyl group (for example, polyglycolic acid, minimum amino acid sequence for expressing cell adhesion) (X) not included Polypeptide, modified polyethylene or polypropylene, ethylene- (meth) acrylic acid copolymer, polyacrylic acid, collagen, gelatin, glycosaminoglycan, hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparin, elastin, fibrin, alginic acid , Albumin, pectin, pectic acid, coating materials such as gluten and fibroin); (2) a cell adhesion polypeptide having a primary amino group or a secondary amino group; and (B) Among them, those having a hydroxyl group (for example, polyglycolic acid, ethylene-vinyl acetate copolymer, polyurethane, polyester, cellulose, chemical fiber, collagen, gelatin, glycosaminoglycan, hyaluronic acid, chondroitin sulfate, keratan sulfate, A method of reacting a coating material such as matane sulfate, heparin, elastin, chitin, chitosan, fibrin, alginic acid, starch, dextran, albumin, polyhydroxybutyric acid, pectin, pectic acid, galactan, pullulan, agarose, gluten and fibroin); (3) a cell-adhesive polypeptide having a hydroxyl group (for example, a cell-adhesive polypeptide containing aspartic acid, glutamic acid, serine, threonine, tyrosine, thyronine, hydroxypurine and the like as a structural unit), and (B) (4) a cell-adhesive polypeptide having a halogen atom (for example, a polypeptide modified with a halide having an amino group) and (B) Of which, hydroxyl group A method of reacting with a compound having a mercapto group (for example, a coating material having a structure in which a part or all of the hydroxyl group is replaced with a mercapto group in addition to the above-mentioned one having a hydroxyl group); and (5) a cell An adhesive polypeptide having a vinyl group (for example, a cell adhesive polypeptide modified with an amino group-containing monomer) and (B) having an amino group, a hydroxyl group or a carboxyl group are reacted. Methods and the like.

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).
(1) In the case of reacting a cell-adhesive polypeptide having a primary amino group or a secondary amino group with (B) one having a carboxyl group, the carboxyl group in (B) is converted into a carbodiimide compound in advance. After reacting to give acylisourea {R′—N═C (OCOR) —NH—R ′ (the portion where —OCOR is derived from (B))}, the primary amino group or 2 of the cell adhesive polypeptide By adding a compound having a secondary amino group, a cell adhesion polypeptide can be introduced into (B) by forming an amide bond. Examples of the carbodiimide compound include N, N′-dicyclohexylcarbodiimide and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride.

  (2) In the case of reacting a cell adhesion polypeptide having a primary amino group or a secondary amino group with (B) having a hydroxyl group, the hydroxyl group of (B) is previously carbonyldiimidazole. After reacting with a compound to make an imidazole derivative {R-Im, Im is an imidazoline ring, R is derived from (B)}, a cell adhesive polypeptide having a primary amino group or a secondary amino group is added. Thus, the cell adhesion polypeptide can be introduced into (B) by forming an N—C bond. Examples of the carbonyldiimidazole compound include N, N′-carbonyldiimidazole.

  (3) In the case of reacting a cell adhesive polypeptide 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. Thereafter, by adding a cell-adhesive polypeptide having a hydroxyl group, the cell-adhesive polypeptide can be introduced into (B) by forming an ester bond.

  (4) In the case where a cell adhesive polypeptide having a halogen atom and (B) having a hydroxyl group or a mercapto group are reacted, both are mixed in the presence or absence of an alkali compound. By (B), the cell adhesion polypeptide can be introduced by forming 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 a cell adhesive polypeptide having a vinyl group is reacted with one having an amino group, a hydroxyl group or a carboxyl group in (B), both are reacted in the presence or absence of an alkali compound. The cell adhesion polypeptide can be introduced into (B) by mixing and adding Michael.

  Examples of the method for physically adsorbing, ion-bonding and / or hydrogen-bonding the cell-adhesive polypeptide to the base material (B) include, for example, putting the cell-adhesive polypeptide and (B) in a solvent and mixing them. 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, body fluids and the like can be used.

As inorganic salts, metal halide salts, metal sulfate salts, metal phosphate salts, metal nitrate salts, metal carbonate salts and metal perhalogenates can be used, for example, sodium chloride, sodium sulfate, sodium phosphate, calcium chloride. , Iron nitrate, potassium chloride, magnesium sulfate, sodium carbonate, sodium hydrogen phosphate, potassium phosphate, potassium hydrogen phosphate, copper sulfate, iron sulfate, lithium chloride, sodium bromide, lithium bromide, sodium perchlorate Examples include lithium chlorate.
Examples of the organic acid salt include sodium formate, sodium acetate, lithium acetate, and sodium tartrate.

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 B12, and vitamin C.
As alcohol, C1-C4 alcohol etc. can be used, For example, methanol, ethanol, isopropyl alcohol, butanol, etc. are mentioned.
Examples of lipids and sugars include lipids, monosaccharides, disaccharides, oligosaccharides, amino sugars, and acidic sugars.

Examples of the acid include inorganic acids and organic acids having 1 to 6 carbon atoms, and examples thereof include hydrochloric acid, phosphoric acid, acetic acid, formic acid, phenol and sulfuric acid.
As the base, inorganic bases and organic bases having 2 to 6 carbon atoms can be used, and examples thereof include sodium hydroxide, potassium hydroxide, ammonia and triethylamine.
Examples of water include distilled water, ion exchange water, tap water, and ion exchange distilled water.
Examples of the body fluid include blood, plasma, serum and urine.
Among these solvents, an aqueous solution containing an inorganic salt, an acid and / or a base, and water are preferable, and an aqueous solution containing an inorganic salt, an acid and / or a base is more preferable.

The content of the cell adhesion polypeptide in the substrate of the present invention is preferably 0.1 ng / cm ² or more, more preferably 1 ng / cm ² or more per unit area of the substrate from the viewpoint of cell adhesion. In particular, it is preferably 10 ng / cm ² or more, most preferably 100 ng / cm ² or more, preferably 100 mg / cm ² or less, more preferably 10 mg / cm ² or less, particularly preferably 1 mg / cm ² or less, most preferably Preferably, it is 100 μg / cm ² or less. In the present invention, the unit area means the surface area of the surface of the base material (B) to which cells to be cultured can adhere, and minute irregularities (for example, 1 μm or less) such that cells do not enter. Is handled as a flat surface, but for the purpose of increasing the unit area, the surface area of the rib is included in the unit area for ribs or the like.
Although the measuring method of content of the cell-adhesive polypeptide per unit area is not specifically limited, For example, an immunoassay can be utilized. Specifically, a part of the surface of the substrate (for example, a 1 cm × 1 cm square shape) is cut out, and an enzyme labeled with an antibody that binds to a cell adhesive polypeptide (hereinafter referred to as enzyme-labeled antibody 1) is reacted. Then, by measuring the enzyme amount of the reacted enzyme-labeled antibody 1, the content of the cell adhesive polypeptide per unit area can be measured.
The enzyme-labeled antibody 1 is usually obtained by chemically bonding an enzyme and a specific antibody and can be chemically bonded by a known method. For example, an enzyme (for example, peroxidase, β-D-galactosidase, alkaline phosphatase, glucose-6-phosphate dehydrogenase, etc.) and a specific antibody are mixed by glutaraldehyde method, periodic acid method, maleimide method, pyridyl disulfide method, etc. Methods for chemical coupling, etc. (Ultrasensitive enzyme immunoassay, written by Ishikawa Shinji, Japan Society of Publishing Press, 1993; Enzyme Immunoassay, translated by Ishikawa Yuji, Tokyo Chemical Co., Ltd., 1989; and Enzyme Antibody Method, Keiichi Watanabe) Et al., Interdisciplinary Planning Co., Ltd., 1992). A specific antibody is an antibody that specifically binds to a cell adhesion polypeptide and can be prepared by a known method. For example, a polyclonal antibody production method and a monoclonal antibody production method (enzyme immunoassay, translated by Junji Ishikawa, Tokyo Chemical Co., Ltd., 1989; and enzyme antibody method, edited by Keiichi Watanabe, et al. . The affinity constant for the cross-reactive antigen of the specific antibody is preferably as small as possible. For example, when the affinity constant of the specific antibody for the cell adhesion polypeptide is 1, the affinity constant for the cross-reactive antigen is preferably 1 or less. More preferably, it is 0.1 or less, and particularly preferably 0.01 or less. These affinity constants can be obtained by the method described in Enzyme Immunoassay (translated by Yuji Ishikawa, Tokyo Kagaku Dojin, 1989).

The cell-adhesive polypeptide-containing substrate of the present invention may be sterilized as necessary.
Although it does not specifically limit as a sterilization method, Radiation sterilization, ethylene oxide gas sterilization, plasma sterilization, gamma ray sterilization, alcohol sterilization, autoclave sterilization, dry heat sterilization, etc. are mentioned.

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 polypeptide-containing substrate of the present invention is not limited as long as it is a cell, but even if the cell-adhesive polypeptide comes into contact with a living tissue, the cell-adhesive polypeptide immunogen Due to their extremely low properties, normal cells derived from mammals, cell lines derived from mammals, and insect cells, which are mainly used for production of useful substances such as pharmaceuticals and treatment, 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 cells, skeletal muscle stem cells, hematopoietic stem cells, neural stem cells, hepatic stem cells (oval cells, small hepatocytes, etc.), adipose tissue stem cells, embryonic stem (ES) cells, epidermal stem cells, intestinal stem cells, sperm stem cells 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, Cells, human striped swallowtail cells, swallowtail butterfly cells, American cockroach cells, and nettle cottonweed cells (Tn-5 cells, HIGH FIVE cells, MG1 cells, 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. 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.
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 included, the content (% by weight) varies depending on the type of the antibacterial agent, but is preferably 10 ₋₄ to 10, more preferably 10 ₋₅ to 1, particularly preferably based on the weight of the medium. 10 _{−6 to} 0.1. That is, in this case, the lower limit of the content (% by weight) of the antibacterial agent is preferably 10 ₋₄ , more preferably 10 ₋₅ , particularly preferably 10 ₋₆ 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 type of cells used and the type of cell-adhesive polypeptide-containing substrate, but is preferably 10 or more, more preferably 100, per 1 cm ₂ of the surface area of the cell-adhesive polypeptide-containing substrate. One or more, particularly preferably 1,000 or more, and preferably 10 million or less, more preferably 1 million or less, and particularly preferably 100,000 or less.
The concentration of cells dispersed in the medium (cells / mL) is not particularly limited, but is preferably 100 or more, more preferably 1000 or more, particularly preferably 10,000 or more per 1 mL of the medium. The number is preferably 100 million or less, more preferably 10 million or less, and particularly preferably 1 million or less.

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 and collected after the culture, (1) a method of recovering by treating with a chelating agent such as EDTA and / or a proteolytic enzyme such as trypsin or collagenase, 2) A method of recovering by scraping with a scraper or the like, (3) a method of recovering by lowering the temperature of the cell-adhesive polypeptide-containing substrate (B) and / or the medium from 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).

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to this.

<Example 1>
(1) Preparation of Cell Adhesive Polypeptide (P1) According to the method described in Examples in Japanese Patent No. 3338441, two Arg Gly Asp sequences (x1) and (Gly Ala Gly Ala Gly Ser) ₃ A polypeptide having an Mn of about 4,000 having a structure comprising two of the sequences (21) and (y1) and alternately chemical bonds thereof is produced by genetically modified Escherichia coli and purified by column chromatography. A cell adhesion polypeptide was obtained. Further, this polypeptide was autoclaved (120 ° C., 20 minutes) to obtain a cell adhesion polypeptide (P1).

(2) Preparation of cell-adhesive polypeptide-containing substrate (PB1) 1 mg of cell-adhesive polypeptide (P1) was dissolved in 1 mL of 4.5 M lithium perchlorate aqueous solution, and further 99.5 wt% sodium chloride. Was diluted 20-fold with 0.02M phosphate buffer (pH 7.2, PBS) containing 0.85% by weight to prepare a P1 solution (P1 concentration: 50 μg / mL). This P1 solution 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 / well of physiological saline and further washed with 100 μL / hole of deionized water to produce a cell-adhesive polypeptide-containing substrate (PB1) (P1 attachment) Amount: about 1 μg / cm ₂ ).

The adhesion amount of the cell adhesive polypeptide (P1) on the cell adhesive polypeptide-containing substrate (PB1) was measured by the following procedure.
(1) Contains 1% by weight of bovine serum albumin in a standard plate (H1) with a known amount of cell adhesion polypeptide (P1) and a cell adhesion polypeptide-containing substrate (PB1) with an unknown amount of adhesion PBS was added at 50 μL / well and allowed to stand at room temperature (25 ° C.) for 2 hours. Thereafter, the solution was removed using an aspirator.
The standard plate is prepared in substantially the same manner as the preparation of the cell-adhesive polypeptide-containing substrate (PB1) described above, but the amount of adhesion is dialyzed and freeze-dried from the P1 aqueous solution after the cell-adhesive polypeptide is attached. Then, the weight of the unattached cell adhesive polypeptide was determined, and the weight of the unattached cell adhesive polypeptide was subtracted from the weight of the unattached cell adhesive polypeptide.

(2) PBS containing 0.001% by weight of peroxidase-labeled anti-P1 antibody, 1% by weight of bovine serum albumin and 0.2% by weight of Tween 20 was added at 50 μL / well and reacted at 37 ° C. for 2 hours. . After the reaction, PBS containing 0.2% by weight of Tween 20 was added at 100 μL / well and washed 3 times to remove the solution.
The peroxidase-labeled anti-P1 antibody was prepared by immunizing the rabbit with the cell-adhesive polypeptide (P1) according to the polyclonal antibody production method (Enzyme Immunoassay, translated by Junji Ishikawa, Tokyo Chemical Co., Ltd., 1989). By obtaining an antibody and binding the anti-P1 antibody and peroxidase (manufactured by Toyobo Co., Ltd.) by the maleimide method (Enzyme immunoassay, translated by Junji Ishikawa, Tokyo Chemical Co., Ltd., 1989), a peroxidase-labeled anti-P1 antibody Got.
(3) A mixed solution containing 20% by weight of OLYDAS exclusive color developer set (manufactured by Sanyo Kasei Kogyo Co., Ltd.) and 80% by weight of ion-exchanged water was added at 50 μL / hole and reacted at 37 ° C. for 1 hour. After the reaction, the absorbance was measured at a wavelength of 380 nm.
(4) A calibration curve was prepared using the absorbance obtained from the standard plate (H1), and the adhesion amount of the cell-adhesive polypeptide-containing substrate (PB1) was obtained from the calibration curve.
Thereafter, the adhesion amount of the cell adhesive polypeptide was measured in the same manner.

<Example 2>
(1) Preparation of Cell Adhesive Polypeptide (P2) According to the method described in Examples in Japanese Patent No. 3338441, three Arg Gly Asp sequences (x1) and (Gly Ala Gly Ala Gly Ser) ₃ A polypeptide with a Mn of about 6,000 having a structure comprising three of the sequences (21) and (y1) and alternating chemical bonds between them is produced by genetically modified Escherichia coli and purified by column chromatography. A cell adhesion polypeptide was obtained. Further, the polypeptide was autoclaved (120 ° C., 20 minutes) to obtain a cell adhesion polypeptide (P2).

(2) Preparation of cell-adhesive polypeptide-containing substrate (PB2) Cell-adhesive polypeptide-containing substrate (PB1) of Example 1 except that (P2) is used instead of cell-adhesive polypeptide (P1) In the same manner as in the preparation of (1), a cell-adhesive polypeptide-containing substrate (PB2) was prepared (P2 adhesion amount: about 1 μg / cm ₂ ).

<Example 3>
(1) Preparation of Cell Adhesive Polypeptide (P3) According to the method described in Examples in Japanese Patent No. 3338441, four Arg Gly Asp sequences (x1) and (Gly Ala Gly Ala Gly Ser) ₃ A polypeptide having an Mn of about 8,000 having a structure in which four of the sequences (21) and (y1) are alternately bonded to each other is produced by genetically modified Escherichia coli and purified by column chromatography. A cell adhesion polypeptide was obtained. Further, the polypeptide was autoclaved (120 ° C., 20 minutes) to obtain a cell adhesion polypeptide (P3).

(2) Preparation of cell adhesion polypeptide-containing substrate (PB3) Cell adhesion polypeptide-containing substrate (PB1) of Example 1 except that (P3) is used instead of cell adhesion polypeptide (P1) In the same manner as in the preparation of (1), a cell-adhesive polypeptide-containing substrate (PB3) was prepared (P3 adhesion amount: about 1 μg / cm ₂ ).

<Example 4>
(1) Preparation of Cell Adhesive Polypeptide (P5) According to the method described in Examples in Japanese Patent No. 3338441, three Arg Gly Asp sequences (x1) and (Gly Val Pro Gly Val) ₂ ( Gly Ala Gly Ala Gly Ser) _{3 of the} sequence (104) (y2) and a polypeptide having an Mn of about 9,000 having a structure in which these are alternately chemically bonded, is produced by genetically modified E. coli, Purification by column chromatography gave a cell adhesion polypeptide. Further, this polypeptide was autoclaved (120 ° C., 20 minutes) to obtain a cell adhesion polypeptide (P5).

(2) Preparation of cell-adhesive polypeptide-containing substrate (PB4) Cell-adhesive polypeptide-containing substrate (PB1) of Example 1 except that (P5) is used instead of cell-adhesive polypeptide (P1) In the same manner as in the preparation of (1), a cell-adhesive polypeptide-containing substrate (PB4) was produced (P5 adhesion amount: about 1 μg / cm ₂ ).

< Reference Example 1 >
(1) Preparation of Cell Adhesive Polypeptide (P8) According to the method described in Examples in Japanese Patent No. 3338441, three Arg Gly Asp sequences (x1) and [Gly Ala Pro (Gly Pro Pro) ₄ ] A polypeptide having an Mn of about 11,000 having a structure in which _{two of the two} sequences (13) and (y3) are alternately and chemically bonded is produced by genetically modified Escherichia coli and subjected to column chromatography. Purification gave a cell adhesion polypeptide. Further, the polypeptide was autoclaved (120 ° C., 20 minutes) to obtain a cell adhesion polypeptide (P8).

(2) Preparation of cell-adhesive polypeptide-containing substrate (PB5) Cell-adhesive polypeptide-containing substrate (PB1) of Example 1 except that (P8) is used instead of cell-adhesive polypeptide (P1) In the same manner as in the preparation of (1), a cell-adhesive polypeptide-containing substrate (PB5) was prepared (P8 adhesion amount: about 1 μg / cm ₂ ).

<Example 6>
(1) Preparation of Cell Adhesive Polypeptide (P1E) In a glass test tube, 0.2 g of N, N′-carbonyldiimidazole was dissolved in 5 mL of dimethyl sulfoxide. 10 mg of P1) was added and reacted for 1 hour at 37 ° C. and 90 times / minute in a shaking thermostat. After 1 hour, 0.1 g of ethylenediamine was added and reacted for 24 hours with shaking at 37 ° C. and 90 times / minute. Next, the reaction solution was transferred into a dialysis tube and dialyzed with 2 L of distilled water for 2 hours 5 times. After dialysis, freeze-drying (reduced pressure of 10 Pa, −20 ° C., 48 hours) gave a cell adhesion polypeptide (P1E) modified with ethylenediamine (Mn about 4,000).

(2) Preparation of cell-adhesive polypeptide-containing substrate (PB6) Cell-adhesive polypeptide-containing substrate (PB1) of Example 1 except that (P1E) is used instead of cell-adhesive polypeptide (P1) In the same manner as in the preparation of (1), a cell-adhesive polypeptide-containing substrate (PB6) was prepared (P1E adhesion amount: about 0.5 μg / cm ₂ ).

<Example 7>
(1) Preparation of Cell Adhesive Polypeptide (P185) According to the method described in Examples in Japanese Patent No. 3338441, two Ile Lys Val Ala Val sequences (7) (x9) and (Gly Ala Gly Ala Gly Ser) A polypeptide having an Mn of about 4,000 having a structure in which two of the _three sequences (21) and (y1) are alternately bonded to each other is produced by genetically modified Escherichia coli and subjected to column chromatography. To obtain a cell adhesion polypeptide. Further, the polypeptide was autoclaved (120 ° C., 20 minutes) to obtain a cell adhesion polypeptide (P185).

(2) Preparation of cell-adhesive polypeptide-containing substrate (PB7) Cell-adhesive polypeptide-containing substrate (PB1) of Example 1 except that (P185) is used instead of cell-adhesive polypeptide (P1) In the same manner as in the preparation of (1), a cell-adhesive polypeptide-containing substrate (PB7) was prepared (P185 adhesion amount: about 1 μg / cm ₂ ).

<Example 8>
(1) Preparation of Cell Adhesive Polypeptide (P186) According to the method described in Examples in Japanese Patent No. 3338441, three Ile Lys Val Ala Val sequences (7) (x9) and (Gly Ala Gly Ala Gly Ser) A polypeptide having an Mn of about 6,000 having a structure in which _{three of the three} sequences (21) and (y1) are alternately bonded to each other is produced by recombinant E. coli, and column chromatography To obtain a cell adhesion polypeptide. Further, the polypeptide was autoclaved (120 ° C., 20 minutes) to obtain a cell adhesion polypeptide (P186).

(2) Preparation of cell-adhesive polypeptide-containing substrate (PB8) Cell-adhesive polypeptide-containing substrate (PB1) of Example 1 except that (P186) is used instead of cell-adhesive polypeptide (P1) In the same manner as in the preparation of (1), a cell-adhesive polypeptide-containing substrate (PB8) was prepared (P186 adhesion amount: about 1 μg / cm ₂ ).

<Example 9>
(1) Preparation of Cell Adhesive Polypeptide (P187) According to the method described in Examples in Japanese Patent No. 3338441, four Ile Lys Val Ala Val sequences (7) (x9) and (Gly Ala Gly Ala Gly Ser) A polypeptide having Mn of about 8,000 having a structure in which four of the _three sequences (21) and (y1) are alternately bonded to each other is produced by recombinant E. coli, and column chromatography To obtain a cell adhesion polypeptide. Further, this polypeptide was autoclaved (120 ° C., 20 minutes) to obtain a cell adhesion polypeptide (P187).

(2) Preparation of cell adhesive polypeptide-containing substrate (PB9) Cell adhesive polypeptide-containing substrate (PB1) of Example 1 except that (P187) is used instead of cell adhesive polypeptide (P1) In the same manner as in the preparation of (), a cell-adhesive polypeptide-containing substrate (PB9) was prepared (P187 adhesion amount: about 1 μg / cm ₂ ).

<Example 10>
(1) Preparation of Cell Adhesive Polypeptide (P189) According to the method described in Examples in Japanese Patent No. 3338441, three Ile Lys Val Ala Val sequences (7) (x9) and (Gly Val Pro Gly Val) ₂ (Gly Ala Gly Ala Gly Ser) ₃ Recombinant polypeptide of about 9,000 Mn having a structure in which _{three of the} sequences (104) (y2) are alternately bonded to each other Manufactured with E. coli and purified by column chromatography to obtain a cell adhesion polypeptide. Further, this polypeptide was autoclaved (120 ° C., 20 minutes) to obtain a cell adhesion polypeptide (P189).

(2) Preparation of cell adhesive polypeptide-containing substrate (PB10) Cell adhesive polypeptide-containing substrate (PB1) of Example 1 except that (P189) is used instead of cell adhesive polypeptide (P1) In the same manner as in (1), a cell-adhesive polypeptide-containing substrate (PB10) was prepared (P189 adhesion amount: about 1 μg / cm ₂ ).

< Reference Example 2 >
(1) Preparation of Cell Adhesive Polypeptide (P192) According to the method described in Examples in Japanese Patent No. 3338441, three Ile Lys Val Ala Val sequences (7) (x9) and [Gly Ala Pro (Gly Pro Pro) ₄ ] _{2 of the} sequence (13) (y3), and a polypeptide having an Mn of about 11,000 having a structure in which these are alternately chemically bonded, is produced by genetically modified E. coli, Purification by column chromatography gave a cell adhesion polypeptide. Further, the polypeptide was autoclaved (120 ° C., 20 minutes) to obtain a cell adhesion polypeptide (P192).

(2) Preparation of cell-adhesive polypeptide-containing substrate (PB11) Cell-adhesive polypeptide-containing substrate (PB1) of Example 1 except that (P192) is used instead of cell-adhesive polypeptide (P1) In the same manner as in the preparation of (1), a cell-adhesive polypeptide-containing substrate (PB11) was prepared (P192 adhesion amount: about 1 μg / cm ₂ ).

<Example 12>
(1) Preparation of Cell Adhesive Polypeptide (P185E) The same procedure as in the preparation of Cell Adhesive Polypeptide (P1E) of Example 6 was used except that (P185) was used instead of cell adhesive polypeptide (P1). Thus, a cell adhesion polypeptide [P185E] was prepared (Mn about 4,000).

(2) Preparation of cell-adhesive polypeptide-containing substrate (PB12) Cell-adhesive polypeptide-containing substrate (PB1) of Example 1 except that (P185E) is used instead of cell-adhesive polypeptide (P1). In the same manner as in the preparation of (1), a cell-adhesive polypeptide-containing substrate (PB12) was prepared (P185E adhesion amount: about 0.5 μg / cm ₂ ).

<Comparative Example 1>
(1) Preparation of Cell Adhesive Polypeptide (H1) According to the method described in Examples in Japanese Patent No. 3338441, about 12 Arg Gly Asp sequences (x1) and (Gly Ala Gly Ala Gly Ser) A polypeptide (P346) having an Mn of about 80,000 having about 12 of ₁₂ sequences (12) and a structure in which these are alternately chemically bonded was designated as a cell adhesion polypeptide (H1).

(2) Preparation of cell adhesive polypeptide-containing substrate (HB1) Cell adhesive polypeptide-containing substrate (PB1) of Example 1 except that (H1) is used instead of cell adhesive polypeptide (P1) In the same manner as in (1), a cell-adhesive polypeptide-containing substrate (HB1) was prepared (H1 adhesion amount: about 1 μg / cm ₂ ).

<Comparative example 2>
(1) Preparation of cell adhesion polypeptide (H2) According to the method described in Examples in Japanese Patent No. 3338441, about 12 Ile Lys Val Ala Val sequences (7) (x9) and (Gly Ala Gly Ala Gly Ser) ₁₂ sequence (12) of about 12 and a Mn of about 80,000 they have a structure formed by chemically bonding alternately polypeptide (P347) a cell adhesion polypeptide (H2) It was.

(2) Preparation of cell adhesive polypeptide-containing substrate (HB2) Cell adhesive polypeptide-containing substrate (PB1) of Example 1 except that (H2) is used instead of cell adhesive polypeptide (P1) In the same manner as in the preparation of (2), a cell-adhesive polypeptide-containing substrate (HB2) was prepared (H2 adhesion amount: about 1 μg / cm ₂ ).

<Comparative Example 3>
A 96-hole polystyrene plate (Nippon Becton Dickinson Co., Ltd.) was used as it was to form a base material (B1).

<Evaluation 1: Immunogenicity of cell adhesion polypeptide>
(1) Antiserum preparation of cell adhesion polypeptide 0.2 g of complete Freund's adjuvant (manufactured by Difco) containing 0.5% by weight of cell adhesion polypeptide (P1) was added to a mouse (Balb / c ) Injection into the abdominal cavity of each of three animals. Thereafter, the same injection was performed three times at one month intervals (total of four injections). About 5 mL of blood was collected from the tail vein of the mice 5 months after the first injection. Three bloods were mixed to make about 0.3 mL, and then left overnight in a refrigerator (6 ± 4 ° C.) to obtain about 0.1 mL of antiserum (PA1).
Cell adhesion polypeptides (P2), (P3), (P5), (P8), (P1E), (P185), (P186), (P187), (P189), (P192), (P185E), (P185) The same operation was performed for H1) and (H2) to obtain antisera (PA2) to (PA12), (HA1) and (HA2).

(2) Evaluation of antiserum PBS containing 1% by weight of bovine serum albumin was introduced into a cell adhesion polypeptide-containing substrate (PB1) at 50 μL / well and allowed to stand at room temperature (25 ° C.) for 2 hours. . Thereafter, the solution was removed using an aspirator.
PBS containing 0.2% by weight of antiserum (PA1), 1% by weight of bovine serum albumin and 0.2% by weight of Tween 20 was added at 50 μL / well and reacted at 37 ° C. for 1 hour. After the reaction, PBS containing 0.2% by weight of Tween 20 was added at 100 μL / well and washed 3 times to remove the solution.
Next, PBS containing 0.05% by weight of peroxidase-labeled anti-mouse Ig antibody (manufactured by DAKO), 1% by weight of bovine serum albumin and 0.2% by weight of Tween 20 was added at 50 μL / well, and 37 The reaction was carried out at 0 ° C for 1 hour. After the reaction, PBS containing 0.2% by weight of Tween 20 was added at 100 μL / well and washed 3 times to remove the solution.
Finally, a mixed solution containing 20% by weight of an OLYDAS color developing solution set (manufactured by Sanyo Chemical Industries Co., Ltd.) and 80% by weight of ion-exchanged water was added at 50 μL / hole and reacted at 37 ° C. for 1 hour. After the reaction, absorbance was measured at a wavelength of 380 nm, and this value was regarded as immunogenicity. Immunogenicity is proportional to the height of the absorbance.
The antiserum (PA2) to (PA12), (HA1) and (HA2) were similarly evaluated using the cell adhesion polypeptide-containing substrates (PB2) to (PB12), (HB1) and (HB2). The immunogenicity was determined for each. These results are shown in Table 1 (these results are average data for 8 holes each).

  From the results in Table 1, it can be seen that the cell adhesive polypeptide of the present invention has extremely low immunogenicity as compared with the cell adhesive polypeptide of the comparative example.

<Evaluation 2: Cell adhesion of VERO cells (African green monkey kidney-derived cells)>
Each of the cell adhesion polypeptide-containing substrates (PB1) to (PB6), (HB1) and the substrate (B1) to which the cell adhesion polypeptide was not attached was measured by the following procedure.
PBS containing 1% by weight of bovine serum albumin was added to each substrate at 50 μL / well and left at room temperature (25 ° 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 20,000 cells / well and left to stand for 1 hour in an incubator at 37 ° C. and 5% by volume of CO 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.
Four hours later, 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. Cell adhesion is proportional to the absorbance. These results are shown in Table 2 (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 3: Cell adhesion of HT1080 cells (fibrosarcoma-derived cells)>
The cell adhesion polypeptide-containing substrate (PB7) to (PB12), (HB2) and the substrate (B1) to which the cell adhesion polypeptide was not attached were measured by the following procedure.
PBS containing 1% by weight of bovine serum albumin was added to each substrate at 50 μL / well and left at room temperature (25 ° 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, HT1080 cells were added at 20,000 cells / well and left to stand for 1 hour in an incubator at 37 ° C. and a CO ₂ concentration of 5% by volume.
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.
Four hours later, 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. Cell adhesion is proportional to the absorbance. These results are shown in Table 3 (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.

  From the results of Table 2 and Table 3, the cell adhesive polypeptide of the present invention has extremely high cell adhesiveness compared to the case where the cell adhesive polypeptide is not used (Comparative Example 3), and the conventional artificial cell adhesion. It can be seen that the cell adhesion is equal to or greater than that of the sexual polypeptide (Comparative Example 1 and Comparative Example 2).

The cell adhesion polypeptide 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, research and development, and production of useful substances. And suitable for treatment and the like.
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.

Claims (5)

A polypeptide (P) having a minimum amino acid sequence (X) and an auxiliary amino acid sequence (Y) that express cell adhesion,
(X) is Arg Gly Asp sequence or Ile Lys Val Ala Val sequence (7),
(Y) is (Gly Ala Gly Ala Gly Ser) ₃ sequence (21) or (Gly Val Pro Gly Val) ₂ (Gly Ala Gly Ala Gly Ser) ₃ sequence (104),
A cell adhesion polypeptide, wherein the polypeptide has a number average molecular weight of 4,000 to 9,000. 2. A structure in which a minimal amino acid sequence (X) and an auxiliary amino acid sequence (Y) are alternately chemically bonded. A cell adhesion polypeptide according to 1. The cell adhesion polypeptide according to claim 1 or 2 , wherein the cell adhesion polypeptide is modified with a compound (AM) containing an amino group and / or an ammonio group. A cell-adhesive polypeptide-containing substrate comprising the cell-adhesive polypeptide according to any one of claims 1 to 3 and a substrate (B). A method for producing cells, comprising a step of culturing cells using the cell-adhesive polypeptide-containing substrate according to claim 4 .