JP2003210166A - Cell-adhered substrate for poikilothermic animal- originated cell - Google Patents

Cell-adhered substrate for poikilothermic animal- originated cell

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Publication number
JP2003210166A
JP2003210166A JP2002328721A JP2002328721A JP2003210166A JP 2003210166 A JP2003210166 A JP 2003210166A JP 2002328721 A JP2002328721 A JP 2002328721A JP 2002328721 A JP2002328721 A JP 2002328721A JP 2003210166 A JP2003210166 A JP 2003210166A
Authority
JP
Japan
Prior art keywords
gly
sequence
cell
cells
pro
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
JP2002328721A
Other languages
Japanese (ja)
Inventor
Sukehito Kurokawa
祐人 黒川
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sanyo Chemical Industries Ltd
Original Assignee
Sanyo Chemical Industries Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sanyo Chemical Industries Ltd filed Critical Sanyo Chemical Industries Ltd
Priority to JP2002328721A priority Critical patent/JP2003210166A/en
Publication of JP2003210166A publication Critical patent/JP2003210166A/en
Pending legal-status Critical Current

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Abstract

<P>PROBLEM TO BE SOLVED: To provide a cell-adhered substrate which can efficiently proliferate a poikilothermic animal-originated cell in a state free from a risk that an infectious substance such as a prion or a human being-infecting virus is contained. <P>SOLUTION: This cell-adhered substrate is characterized by adhering (A) the poikilothermic animal-originated cell to (B) the substrate with (X) a cell- adhesive artificial peptide and/or (Y) a cell-adhesive auxiliary artificial peptide. (X) is a peptide which contains the minimum amino acid sequence (sh) exhibiting a cell-adhesive signal and has an amino acid sequence (jh) obtained by polymerizing a repeating unit comprising at least three amino acids in a polymerization degree of at least 3. (Y) is a peptide which does not contain the minimum amino acid sequence (sh) and has an amino acid sequence (jh) obtained by polymerizing a repeating unit comprising at least three amino acids in a polymerization degree of at least 3. (A) is preferably an insect cell. <P>COPYRIGHT: (C)2003,JPO

Description

【発明の詳細な説明】Detailed Description of the Invention

【0001】[0001]

【発明の属する技術分野】本発明は、細胞接着基材に関
する。さらに詳しくは、変温動物由来細胞と基材とを接
着させてなる細胞接着基材に関する。
TECHNICAL FIELD The present invention relates to a cell adhesion substrate. More specifically, it relates to a cell-adhesive substrate obtained by adhering cells derived from a thermophilic animal and a substrate.

【0002】[0002]

【従来の技術】従来、変温動物由来細胞と基材とを接着
させてなる細胞接着基材として、天然由来のコラーゲン
や絹蛋白質を基材にコーティングしたものに変温動物由
来細胞を接着させてなる細胞接着基材が知られている
(特許文献1)。
2. Description of the Related Art Conventionally, as a cell-adhesive substrate obtained by adhering cells derived from a temperature-altering animal to a substrate, a cell derived from a temperature-altering animal is adhered to a substrate coated with naturally-derived collagen or silk protein. A cell-adhesive substrate composed of the following is known (Patent Document 1).

【0003】[0003]

【特許文献1】特開平11−243948号公報。[Patent Document 1] Japanese Patent Application Laid-Open No. 11-243948.

【0004】[0004]

【発明が解決しようとする課題】天然由来のコラーゲン
や絹蛋白質を基材にコーティングしたものに変温動物由
来細胞を接着させてなる細胞接着基材は、天然由来の蛋
白質を用いるため蛋白質中にプリオンやヒト感染性のウ
イルス等の感染物質が含有される危険性があるという問
題点及び細胞接着性が低いという問題点があった。すな
わち、本発明の目的は、天然由来の蛋白質を使用せず、
細胞接着性の高い細胞接着基材を提供することである。
A cell-adhesive base material obtained by adhering cells derived from a temperature-altering animal to a base material coated with naturally-occurring collagen or silk protein is used in the protein because the naturally-occurring protein is used. There are problems that there is a risk of containing infectious substances such as prions and human infectious viruses, and that cell adhesion is low. That is, the object of the present invention is to use no naturally derived protein,
It is intended to provide a cell adhesive substrate having high cell adhesiveness.

【0005】[0005]

【課題を解決するための手段】本発明者は、鋭意研究を
重ねてきた結果、特定のペプチドを使用することにより
上記目的を達成することを見いだし本発明に到達した。
すなわち、本発明の細胞接着基材の特徴は、変温動物由
来細胞(A)及び基材(B)と、細胞接着性人工ペプチ
ド(X)及び/又は細胞接着補助人工ペプチド(Y)を
用いて、変温動物由来細胞(A)及び基材(B)を接着
させてなる点にある。
As a result of intensive studies, the present inventors have found that the above object can be achieved by using a specific peptide, and have reached the present invention.
That is, the feature of the cell adhesion substrate of the present invention is that the cells (A) and the substrate (B) derived from a thermophilic animal and the cell-adhesive artificial peptide (X) and / or the cell-adhesion assisting artificial peptide (Y) are used. Then, the cells (A) derived from the different temperature animal and the base material (B) are adhered to each other.

【0006】[0006]

【発明の実施の形態】細胞接着性人工ペプチド(X)
は、細胞接着シグナルを現わす最小アミノ酸配列(s
h)を有している。なお、「細胞接着シグナルを現わ
す」とは、特定の最小アミノ酸配列(sh)が細胞のイ
ンテグリンレセプターに認識されることを意味する(大
阪府立母子医療センター雑誌、第8巻 第1号、58〜
66頁、1992年)。
BEST MODE FOR CARRYING OUT THE INVENTION Cell-adhesive artificial peptide (X)
Is the minimum amino acid sequence (s
h). The expression "cell adhesion signal" means that a specific minimal amino acid sequence (sh) is recognized by the cell integrin receptor (Osaka Prefectural Medical Center for Maternal and Childhood, Vol. 8, No. 1, 58). ~
66, 1992).

【0007】細胞接着シグナルを現わす最小アミノ酸配
列(sh)としては、例えば、「病態生理、第9巻 第
7号、527〜535頁、1990年」や「大阪府立母
子医療センター雑誌、第8巻 第1号、58〜66頁、
1992年」に記載されているもの等が用いられる。こ
れらの最小アミノ酸配列(sh)の中で好ましいもの
は、Arg Gly Asp配列、Leu Asp Val配列、Arg Glu Asp
Val配列(1)、Tyr IleGly Ser Arg配列(2)、Pro A
sp Ser Gly Arg配列(3)、Arg Tyr Val Val Leu Pro
Arg配列(4)、Leu Gly Thr Ile Pro Gly配列(5)、
Arg Asn Ile AlaGlu Ile Ile Lys Asp Ile配列(6)、
Ile Lys Val Ala Val配列(7)、Leu Arg Glu配列、As
p Gly Glu Ala 配列(8)、Gly Val Lys Gly Asp Lys
Gly AsnPro Gly Trp Pro Gly Ala Pro配列(9)、Gly
Glu Phe Tyr Phe Asp Leu Arg Leu Lys Gly Asp Lys
(10)、His Ala Val配列及びTyr Lys Leu Asn Val A
sn Asp Ser配列(11)である。さらに好ましいもの
は、変温動物由来細胞(A)への接着性が高い点で、Ar
g Gly Asp配列、Tyr Ile Gly Ser Arg配列(2)、及び
Ile Lys Val Ala Val配列(7)である。なお、アミノ
酸配列は3文字表記で表し、( )内にアミノ酸配列表
中の対応する配列番号を記載する(以下、同じであ
る。)。
The minimum amino acid sequence (sh) that expresses a cell adhesion signal is, for example, "Pathophysiology, Vol. 9, No. 7, pp. 527-535, 1990" and "Osaka Prefectural Maternal and Child Medical Center Magazine, 8". Volume 1, pages 58-66,
Those described in "1992" are used. Preferred among these minimal amino acid sequences (sh) are Arg Gly Asp sequence, Leu Asp Val sequence, Arg Glu Asp sequence.
Val sequence (1), Tyr IleGly Ser Arg sequence (2), Pro A
sp 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 AlaGlu Ile Ile Lys Asp Ile sequence (6),
Ile Lys Val Ala Val sequence (7), Leu Arg Glu sequence, As
p Gly Glu Ala sequence (8), Gly Val Lys Gly Asp Lys
Gly AsnPro Gly Trp Pro Gly Ala Pro Sequence (9), Gly
Glu Phe Tyr Phe Asp Leu Arg Leu Lys Gly Asp Lys
(10), His Ala Val sequence and Tyr Lys Leu Asn Val A
This is the sn Asp Ser sequence (11). More preferable is Ar because it has high adhesiveness to cells (A) derived from a thermophilic animal.
g Gly Asp sequence, Tyr Ile Gly Ser Arg sequence (2), and
Ile Lys Val Ala Val sequence (7). The amino acid sequence is represented by a three-letter code, and the corresponding sequence number in the amino acid sequence list is shown in parentheses (hereinafter the same).

【0008】細胞接着性人工ペプチド(X)は、前記最
小アミノ酸配列(sh)を1分子中に少なくとも1個有
すればよいが、変温動物由来細胞(A)への接着性が高
い点で、1分子中に2〜50個有するものが好ましく、
さらに好ましくは1分子中に3〜30個、特に好ましく
は1分子中に5〜20個有するものである。細胞接着性
人工ペプチド(X)の数平均分子量は、300〜3,0
00,000が好ましく、さらに好ましくは1,000
〜1,000,000、特に好ましくは3,000〜3
00,000である。すなわち、(X)の数平均分子量
の上限は3,000,000が好ましく、さらに好まし
くは1,000,000、特に好ましくは300,00
0であり、同様に下限は300が好ましく、さらに好ま
しくは1,000、特に好ましくは3,000である。
なお、数平均分子量は、SDS−PAGE(SDSポリ
アクリルアミドゲル電気泳動)法で、測定サンプルを水
中で分離し、泳動距離を標準物質と比較することによっ
て求めるものである(以下、同じである。)。
The artificial cell-adhesive peptide (X) may have at least one minimum amino acid sequence (sh) in one molecule, but it has high adhesiveness to cells (A) derived from a thermophilic animal. It is preferable to have 2 to 50 in one molecule,
It is more preferably 3 to 30 in one molecule, and most preferably 5 to 20 in one molecule. The number average molecular weight of the cell-adhesive artificial peptide (X) is 300 to 3,0.
0,000 is preferable, more preferably 1,000
~ 1,000,000, particularly preferably 3,000-3
It is 0,000. That is, the upper limit of the number average molecular weight of (X) is preferably 3,000,000, more preferably 1,000,000, and particularly preferably 300,000.
Similarly, the lower limit is preferably 300, more preferably 1,000, and particularly preferably 3,000.
The number average molecular weight is determined by SDS-PAGE (SDS polyacrylamide gel electrophoresis) method by separating the measurement sample in water and comparing the migration distance with a standard substance (hereinafter the same. ).

【0009】細胞接着性人工ペプチド(X)としては、
例えば、Arg Gly Asp Ser配列(14)からなるペプチ
ド、Gly Arg Gly Asp Ser配列(15)からなるペプチ
ド、Gly Arg Gly Asp Ser Pro配列(16)からなるペ
プチド、Arg Gly Asp Ser ProAla Ser Ser Lys Pro配列
(17)からなるペプチド、Ala Val Thr Gly Arg GlyA
sp Ser Pro Ala Ser Ala配列(18)からなるペプチ
ド、Pro Gly Ala Ser Ile Lys Val Ala Val Ser Ala Gl
y Pro Ser配列(19)からなるペプチド、Cys Ser Arg
Ala Arg Lys Gln Ala Ala Ser Ile Lys Val Ala Val S
er Ala Asp Arg配列(20)からなるペプチド、Val Cy
s Glu Pro Gly Tyr Ile Gly Ser Arg CysAsp配列(2
1)からなるペプチド及びこれらの少なくとも一種のペ
プチドからなる重合体等が例示できる。重合体として
は、例えば、(Arg Gly Asp Ser)4配列(22)、(Ar
g Gly Asp Ser)8配列(23)、(Arg Gly Asp Ser)
16配列(24)、(Gly Arg Gly Asp Ser)8配列(2
5)、(Gly Arg Gly Asp Ser Pro)8配列(26)、
(Arg Gly Asp Ser Pro Ala Ser Ser Lys Pro)4配列
(27)、(Ala Val Thr Gly ArgGly Asp Ser Pro Ala
Ser Ala)4配列(28)、(Pro Gly Ala Ser Ile Lys
Val Ala Val Ser Ala Gly Pro Ser)4配列(29)、
(Cys Ser Arg Ala Arg LysGln Ala Ala Ser Ile Lys V
al Ala Val Ser Ala Asp Arg)4配列(30)、又は(V
al Cys Glu Pro Gly Tyr Ile Gly Ser Arg Cys Asp)4
(31)配列からなるペプチド等が挙げられる。該重合
体の重合度は、2〜50が好ましく、さらに好ましくは
3〜30、特に好ましくは4〜20、最も好ましくは4
〜16である。
As the cell-adhesive artificial peptide (X),
For example, a peptide consisting of Arg Gly Asp Ser sequence (14), a peptide consisting of Gly Arg Gly Asp Ser sequence (15), a peptide consisting of Gly Arg Gly Asp Ser Pro sequence (16), Arg Gly Asp Ser ProAla Ser Ser Lys Pro Peptide consisting of sequence (17), Ala Val Thr Gly Arg GlyA
Peptide consisting of sp Ser Pro Ala Ser Ala sequence (18), Pro Gly Ala Ser Ile Lys Val Ala Val Ser Ala Gl
Cys Ser Arg, a peptide consisting of y Pro Ser sequence (19)
Ala Arg Lys Gln Ala Ala Ser Ile Lys Val Ala Val S
Peptide consisting of er Ala Asp Arg sequence (20), Val Cy
s Glu Pro Gly Tyr Ile Gly Ser Arg CysAsp sequence (2
Examples include the peptide consisting of 1) and a polymer containing at least one of these peptides. Examples of the polymer include (Arg Gly Asp Ser) 4 sequence (22) and (Ar
g Gly Asp Ser) 8 sequences (23), (Arg Gly Asp Ser)
16 sequences (24), (Gly Arg Gly Asp Ser) 8 sequences (2
5), (Gly Arg Gly Asp Ser Pro) 8 sequences (26),
(Arg Gly Asp Ser Pro Ala Ser Ser Lys Pro) 4 sequences (27), (Ala Val Thr Gly ArgGly Asp Ser Pro Ala
Ser Ala) 4 sequences (28), (Pro Gly Ala Ser Ile Lys
Val Ala Val Ser Ala Gly Pro Ser) 4 sequences (29),
(Cys Ser Arg Ala Arg LysGln Ala Ala Ser Ile Lys V
al Ala Val Ser Ala Asp Arg) 4 sequence (30), or (V
al Cys Glu Pro Gly Tyr Ile Gly Ser Arg Cys Asp) 4
(31) Peptides consisting of the sequence and the like are mentioned. The degree of polymerization of the polymer is preferably 2 to 50, more preferably 3 to 30, particularly preferably 4 to 20, and most preferably 4.
~ 16.

【00010】細胞接着補助人工ペプチド(Y)は、細
胞接着シグナルを現わす最小アミノ酸配列(sh)を含
まないペプチドであって、少なくとも3個のアミノ酸か
らなる反復単位が、少なくとも重合度3で重合した構造
(アミノ酸配列)(jh)を有するペプチドである。少
なくとも3個のアミノ酸からなる反復単位としては、例
えば、「特表平3−502935号公報、反復単位」や
「Abraham J. Dombら著、Handbook of Biodegradable P
olymers, Harwood Academic Publishers発行, Amsterda
m、1997年、ブロック配列」に記載されているもの等が
用いられる。これらの反復単位のうち、Gly Ala Gly Al
a Gly Ser配列(12)、Gly Val Gly Val Pro配列(1
3)、Gly Pro Pro配列、Gly Ala Gln Gly Pro Ala Gly
ProGly 配列(32)、Gly Ala Pro Gly Ala Pro Gly
Ser Gln Gly Ala Pro Gly Leu Gln配列(33)及びGly
Ala Pro Gly Thr Pro Gly Pro Gln Gly Leu Pro Gly S
er Pro配列(34)が好ましく、変温動物由来細胞
(A)への接着性が高いという観点から、さらに好まし
くはGly Ala Gly Ala Gly Ser配列(12)、GlyVal Gl
y Val Pro配列(13)及びGly Pro Pro配列、特に好ま
しくはGly Ala Gly Ala Gly Ser配列(12)である。
The cell adhesion-assisting artificial peptide (Y) is a peptide that does not contain the minimum amino acid sequence (sh) that expresses a cell adhesion signal, and a repeating unit consisting of at least 3 amino acids is polymerized at a polymerization degree of at least 3. It is a peptide having the structure (amino acid sequence) (jh). Examples of the repeating unit consisting of at least three amino acids include, for example, "Table 3 of Japanese Patent Publication No. 3-502935, Repeating unit" and "Abraham J. Domb et al., Handbook of Biodegradable P
Published by olymers, Harwood Academic Publishers, Amsterda
m, 1997, block sequence ”and the like are used. Of these repeating units, Gly Ala Gly Al
a Gly Ser sequence (12), Gly Val Gly Val Pro sequence (1
3), Gly Pro Pro array, Gly Ala Gln Gly Pro Ala Gly
ProGly array (32), Gly Ala Pro Gly Ala Pro Gly
Ser Gln Gly Ala Pro Gly Leu Gln sequence (33) and Gly
Ala Pro Gly Thr Pro Gly Pro Gln Gly Leu Pro Gly S
er Pro sequence (34) is preferable, and more preferably Gly Ala Gly Ala Gly Ser sequence (12) and GlyVal Gl from the viewpoint of high adhesiveness to cells (A) derived from a thermophilic animal.
y Val Pro sequence (13) and Gly Pro Pro sequence, particularly preferably Gly Ala Gly Ala Gly Ser sequence (12).

【00011】細胞接着補助人工ペプチド(Y)は、反
復単位を少なくとも重合度3で重合した構造(jh)を
有するペプチドであればよいが、変温動物由来細胞
(A)への接着性が高い点で、反復単位を、1分子中に
3〜10,000個有するものが好ましく、さらに好ま
しくは1分子中に10〜3,000個、特に好ましくは
1分子中に30〜1,000個有するものである。細胞
接着補助人工ペプチド(Y)の数平均分子量は、500
〜5,000,000が好ましく、さらに好ましくは
1,500〜1,500,000、特に好ましくは5,
000〜500,000である。すなわち、(Y)の数
平均分子量の上限は5,000,000が好ましく、さ
らに好ましくは1,500,000、特に好ましくは5
00,000であり、同様に下限は500が好ましく、
さらに好ましくは1,500、特に好ましくは5,00
0である。
The artificial cell adhesion promoting peptide (Y) may be any peptide having a structure (jh) obtained by polymerizing repeating units at a degree of polymerization of at least 3, but has high adhesiveness to cells (A) derived from a thermophilic animal. In this respect, those having 3 to 10,000 repeating units in one molecule are preferable, 10 to 3,000 repeating units are more preferable in one molecule, and 30 to 1,000 repeating units are particularly preferable in one molecule. It is a thing. The number average molecular weight of the cell adhesion-assisting artificial peptide (Y) is 500.
To 5,000,000 are preferable, more preferably 1,500 to 1,500,000, and particularly preferably 5,
It is 000 to 500,000. That is, the upper limit of the number average molecular weight of (Y) is preferably 5,000,000, more preferably 1,500,000, and particularly preferably 5.
And the lower limit is preferably 500,
More preferably 1,500, particularly preferably 5,000
It is 0.

【0012】細胞接着補助人工ペプチド(Y)として
は、例えば、「特表平3−502935号公報」や「Ab
raham J. Dombら著、Handbook of Biodegradable Polym
ers, Harwood Academic Publishers発行, Amsterdam、1
997年」に記載されているもの等が用いられる。これら
の細胞接着補助人工ペプチド(Y)のうち、1分子中に
Gly Ala Gly Ala Gly Ser配列(12)を約170個有
する数平均分子量約10万のペプチド(a)、1分子中
に(Gly Ala Gly Ala Gly Ser)9配列(35)とGly Al
a Ala Gly Tyr配列(36)とをそれぞれ約18個づつ
有する数平均分子量約10万のペプチド(b)、(Gly
Val Gly Val Pro)8配列(37)と(GlyAla Gly Ala G
ly Ser)8配列(38)とをそれぞれ約11個づつ有す
る数平均分子量約10万のペプチド(c)、(Gly Val
Gly Val Pro)12配列(39)と(Gly Ala Gly Ala Gly
Ser)8配列(38)とをそれぞれ約8個づつ有する数
平均分子量約10万のペプチド(d)、(Gly Val Gly
Val Pro)16配列(40)と(Gly Ala Gly Ala Gly Se
r)8配列(38)とをそれぞれ約7個づつ有する数平均
分子量約10万のペプチド(e)、(Gly Val Gly Val
Pro)8配列(37)と(Gly Ala Gly Ala Gly Ser)6
列(41)とをそれぞれ約12個づつ有する数平均分子
量約10万のペプチド(f)、及び(Gly Val Gly Val
Pro)8配列(37)と(Gly Ala Gly Ala Gly Ser)4
列(42)とをそれぞれ約12個づつ有する数平均分子
量約10万のペプチド(g)が好ましく、さらに好まし
くはペプチド(a)及びペプチド(b)である。
Examples of the cell adhesion-assisting artificial peptide (Y) include, for example, "Japanese Patent Publication No. 3-502935" and "Ab".
Raham J. Domb et al., Handbook of Biodegradable Polym
Published by ers, Harwood Academic Publishers, Amsterdam, 1
Those described in "997" are used. Among these cell adhesion-assisting artificial peptides (Y),
A peptide having about 170 Gly Ala Gly Ala Gly Ser sequences (12) and a number average molecular weight of about 100,000 (a), (Gly Ala Gly Ala Gly Ser) 9 sequence (35) and Gly Al in one molecule.
a peptide having a number average molecular weight of about 100,000, each having about 18 Ala Gly Tyr sequences (36) (b), (Gly
Val Gly Val Pro) 8 sequences (37) and (GlyAla Gly Ala G
ly Ser) 8 sequences (38) and 11 peptides each having a number average molecular weight of about 100,000 (Gly Val).
Gly Val Pro) 12 sequences (39) and (Gly Ala Gly Ala Gly
Ser) 8 sequence (38) each having about 8 peptides (d) with a number average molecular weight of about 100,000 (Gly Val Gly
Val Pro) 16 sequences (40) and (Gly Ala Gly Ala Gly Se
r) 8 sequences (38) and 7 peptides each (7) each having a number average molecular weight of about 100,000, (Gly Val Gly Val
Pro) 8 sequence (37) and (Gly Ala Gly Ala Gly Ser) 6 sequence (41) each having about 12 peptides (f) having a number average molecular weight of about 100,000, and (Gly Val Gly Val
Pro) 8 sequence (37) and (Gly Ala Gly Ala Gly Ser) 4 sequence (42) each having about 12 each, and preferably a peptide (g) having a number average molecular weight of about 100,000, more preferably the peptide (a). And peptide (b).

【0013】細胞接着性人工ペプチド(X)及び細胞接
着補助人工ペプチド(Y)は、人工的に製造されるもの
であり、例えば、有機合成法(酵素法、固相合成法及び
液相合成法等)、及び遺伝子組み換え法によって容易に
製造できる。有機合成法に関しては、例えば、「生化学
実験講座1、タンパク質の化学IV(1981年7月1
日、日本生化学会編、株式会社東京化学同人発行)」や
「続生化学実験講座2、タンパク質の化学(下)(昭和
62年5月20日、日本生化学会編、株式会社東京化学
同人発行)」に記載されている方法等が適用できる。
The cell-adhesive artificial peptide (X) and the cell-adhesion assisting artificial peptide (Y) are artificially produced, and include, for example, organic synthetic methods (enzymatic method, solid phase synthetic method and liquid phase synthetic method). Etc.) and a gene recombination method. Regarding the organic synthesis method, for example, “Biochemistry Experiment Course 1, Protein Chemistry IV (July 1, 1981)
"Japan, Japan Biochemical Society, edited by Tokyo Kagaku Doujin") and "Sequel Biochemistry Experiment Course 2, Protein Chemistry (below)" (May 20, 1987, edited by Biochemical Society of Japan, Published by Tokyo Kagaku Doujin) ) ”Is applicable.

【0014】遺伝子組み換え法に関しては、例えば、
「特表平3−502935号公報」に記載されている方
法等が適用できる。なお、遺伝子組み換え法による場
合、組み換え微生物由来の不純物を含むことがあるた
め、抗ペプチド抗体等を用いたアフィニティ精製等によ
って精製し、ペプチドの純度を80重量%以上にするこ
とが好ましく、さらに好ましくは90重量%以上、特に
好ましくは95重量%以上である。これらのうち、細胞
接着性人工ペプチド(X)及び細胞接着補助人工ペプチ
ド(Y)のアミノ酸配列を容易に設計・製造できるとい
う観点から、遺伝子組み換え法が好ましい。
Regarding the gene recombination method, for example,
The method and the like described in “Tokuhyo Hyo 3-502935” can be applied. In the case of the gene recombination method, impurities derived from a recombinant microorganism may be contained. Therefore, it is preferable to purify the peptide by affinity purification using an anti-peptide antibody or the like so that the purity of the peptide is 80% by weight or more, and further preferable. Is 90% by weight or more, particularly preferably 95% by weight or more. Of these, the gene recombination method is preferable from the viewpoint that the amino acid sequences of the cell-adhesive artificial peptide (X) and the cell-adhesion assisting artificial peptide (Y) can be easily designed and manufactured.

【0015】有機合成法による細胞接着性人工ペプチド
(X1)としては、例えば、Arg Gly Asp Ser配列(1
4)、Gly Arg Gly Asp Ser配列(15)、Gly Arg Gly
AspSer Pro配列(16)及びArg Gly Asp Ser Pro Ala
Ser Ser Lys Pro配列(17)からなる群より選ばれる
少なくとも1種のアミノ酸配列を有するペプチド等が挙
げられる。有機合成法による細胞接着補助人工ペプチド
(Y1)としては、例えば、GlyAla Gly Ala Gly Ser配
列(12)及び/又はGly Val Gly Val Pro配列(1
3)を有するペプチド等が挙げられる。
Examples of the cell-adhesive artificial peptide (X1) produced by the organic synthesis method include, for example, Arg Gly Asp Ser sequence (1
4), Gly Arg Gly Asp Ser sequence (15), Gly Arg Gly
AspSer Pro Sequence (16) and Arg Gly Asp Ser Pro Ala
Examples include peptides having at least one amino acid sequence selected from the group consisting of Ser Ser Lys Pro sequence (17). Examples of the cell adhesion-assisting artificial peptide (Y1) obtained by the organic synthesis method include, for example, GlyAla Gly Ala Gly Ser sequence (12) and / or Gly Val Gly Val Pro sequence (1
3) and the like.

【0016】遺伝子組み換え法による細胞接着性人工ペ
プチド(X2)としては、例えば、Arg Gly Asp配列、T
yr Ile Gly Ser Arg配列(2)及びIle Lys Val Ala Va
l配列(7)からなる群より選ばれる少なくとも1種の
アミノ酸配列を有するペプチド等が挙げられる。遺伝子
組み換え法による細胞接着補助人工ペプチド(Y2)と
しては、例えば、Gly Ala Gly Ala Gly Ser配列(1
2)、Gly Val Gly Val Pro配列(13)及びGly Ala P
ro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro
配列(43)からなる群より選ばれる少なくとも1種の
アミノ酸配列を有するペプチド等が挙げられる。
Examples of the cell-adhesive artificial peptide (X2) obtained by the gene recombination method include Arg Gly Asp sequence, T
yr Ile Gly Ser Arg sequence (2) and Ile Lys Val Ala Va
Examples thereof include peptides having at least one kind of amino acid sequence selected from the group consisting of l sequence (7). Examples of the artificial cell adhesion-assisting peptide (Y2) produced by the gene recombination method include Gly Ala Gly Ala Gly Ser sequence (1
2), Gly Val Gly Val Pro sequence (13) and Gly Ala P
ro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro
Examples thereof include a peptide having at least one kind of amino acid sequence selected from the group consisting of sequence (43).

【0017】細胞接着性人工ペプチド(X)及び細胞接
着補助人工ペプチド(Y)は、同一分子内にあってもよ
く、すなわち、(X)及び(Y)に換えて、細胞接着シ
グナルを現わす最小アミノ酸配列(sh)と、少なくと
も3個のアミノ酸からなる反復単位を少なくとも重合度
3で重合してなるアミノ酸配列(jh)(細胞接着シグ
ナルを現わす最小アミノ酸配列(sh)を含まない)と
を有するペプチド(Z)を用いることができる。このよ
うな細胞接着性人工ペプチド(X)と細胞接着補助人工
ペプチド(Y)とが同一分子内にあるペプチド(Z)
は、変温動物由来細胞(A)へのペプチドの接着性が高
いという観点から好ましい。
The cell-adhesive artificial peptide (X) and the cell-adhesion assisting artificial peptide (Y) may be in the same molecule, that is, in place of (X) and (Y), a cell-adhesion signal is expressed. A minimum amino acid sequence (sh) and an amino acid sequence (jh) formed by polymerizing repeating units consisting of at least 3 amino acids at a degree of polymerization of at least 3 (excluding the minimum amino acid sequence (sh) expressing a cell adhesion signal) The peptide (Z) having can be used. Peptide (Z) in which the cell-adhesive artificial peptide (X) and the cell-adhesion assisting artificial peptide (Y) are in the same molecule
Is preferable from the viewpoint that the peptide has high adhesiveness to the cells (A) derived from a thermophilic animal.

【0018】ペプチド(Z)は、最小アミノ酸配列(s
h)を1分子中に少なくとも1個有すればよいが、変温
動物由来細胞(A)への接着性が高い点で、1分子中に
2個以上有するものが好ましく、さらに好ましくは3個
以上、特に好ましくは5個以上有するものであり、ま
た、50個以下有するものが好ましく、さらに好ましく
は1分子中に30個以下、特に好ましくは1分子中に2
0個以下有するものである。また、ペプチド(Z)は、
アミノ酸配列(jh)を1分子中に少なくとも1個有す
ればよいが、熱に対する安定性の観点から、1分子中に
2個以上有するものが好ましく、さらに好ましくは3個
以上、特に好ましくは5個以上有するものであり、ま
た、50個以下有するものが好ましく、さらに好ましく
は1分子中に30個以下、特に好ましくは1分子中に2
0個以下有するものである。
The peptide (Z) has a minimum amino acid sequence (s
It suffices to have at least one h) in one molecule, but it is preferable to have two or more in one molecule, and more preferably three in terms of high adhesiveness to cells (A) derived from a thermophilic animal. As described above, particularly preferably 5 or more, and preferably 50 or less, more preferably 30 or less in one molecule, particularly preferably 2 in 1 molecule.
It has 0 or less. Further, the peptide (Z) is
It is sufficient that at least one amino acid sequence (jh) is contained in one molecule, but from the viewpoint of stability to heat, one having two or more amino acids in one molecule is preferable, more preferably three or more, and particularly preferably 5 It is preferable that it has not less than 50 and not more than 50, more preferably not more than 30 in one molecule, particularly preferably not more than 2 in one molecule.
It has 0 or less.

【0019】ペプチド(Z)において、最小アミノ酸配
列(sh)とアミノ酸配列(jh)との個数の比率
{(jh)/(sh)は、0.2以上が好ましく、さら
に好ましくは0.5以上、特に好ましくは0.8以上で
あり、また、5以下が好ましく、さらに好ましくは2以
下、特に好ましくは1.3以下である。最小アミノ酸配
列(sh)とアミノ酸配列(jh)とは、ペプチド
(Z)のβターン構造の取りやすさ等の観点から、最小
アミノ酸配列(sh)とアミノ酸配列(jh)とが交互
に位置することが好ましい。
In the peptide (Z), the ratio of the number of the minimum amino acid sequence (sh) to the amino acid sequence (jh) {(jh) / (sh) is preferably 0.2 or more, more preferably 0.5 or more. , Particularly preferably 0.8 or more, 5 or less, more preferably 2 or less, particularly preferably 1.3 or less. The minimum amino acid sequence (sh) and the amino acid sequence (jh) are located alternately with the minimum amino acid sequence (sh) and the amino acid sequence (jh) from the viewpoint of ease of taking the β-turn structure of the peptide (Z). It is preferable.

【0020】ペプチド(Z)としては、例えば、「特表
平3−502935号公報」や「Abraham J. Dombら
著、Handbook of Biodegradable Polymers, Harwood Ac
ademicPublishers発行, Amsterdam、1997年」等に記載
の(Gly Ala Gly Ala Gly Ser) 9配列(35)とArg Gl
y Asp配列とをそれぞれ約12個づつ有する数平均分子
量約10万のペプチド(h)、(Gly Ala Gly Ala Gly
Ser)9配列(35)とTyrIle Gly Ser Arg配列(2)と
をそれぞれ約13個づつ有する数平均分子量約10万の
ペプチド(i)、(Gly Ala Gly Ala Gly Ser)9配列
(35)とIle LysVal Ala Val配列(7)とをそれぞれ
約12個づつ有する数平均分子量約10万るペプチド
(j)、(Gly Val Gly Val Pro)8配列(37)と(Gl
y Ala Gly Ala Gly Ser)12配列(44)とArg Gly Asp
配列とをそれぞれ約12個づつ有する数平均分子量約1
0万のペプチド(k)、及び(Gly Ala Pro Gly Pro Pr
o GlyPro Pro Gly Pro Pro Gly Pro Pro)2(45)配
列とArg Gly Asp配列とをそれぞれ約6個づつ有する数
平均分子量約5万のペプチド(l)等が挙げられる。
As the peptide (Z), for example, "special table
No. 3-502935, "Abraham J. Domb et al.
By: Handbook of Biodegradable Polymers, Harwood Ac
Published by ademic Publishers, Amsterdam, 1997 "etc.
Of (Gly Ala Gly Ala Gly Ser) 9Sequence (35) and Arg Gl
Number average molecule having about 12 y Asp sequences each
About 100,000 peptides (h), (Gly Ala Gly Ala Gly
Ser)9Sequence (35) and TyrIle Gly Ser Arg sequence (2)
Of about 100,000 having a number average molecular weight of about 13 each
Peptide (i), (Gly Ala Gly Ala Gly Ser)9Array
(35) and the Ile LysVal Ala Val sequence (7), respectively
Peptides with a number average molecular weight of about 100,000 having about 12 each
(J), (Gly Val Gly Val Pro)8Sequences (37) and (Gl
y Ala Gly Ala Gly Ser) 12 sequences (44) and Arg Gly Asp
Number average molecular weight of about 1 each having about 12 sequences and about 1
0,000 peptides (k), and (Gly Ala Pro Gly Pro Pr
o GlyPro Pro Gly Pro Pro Gly Pro Pro)2(45) distribution
Number with about 6 columns and 6 Arg Gly Asp sequences
Peptide (l) having an average molecular weight of about 50,000 can be used.

【0021】市場から入手できるペプチド(X)として
は、商品名を記載すると例えば、RGDS[Arg Gly As
p Ser配列(14)を1個含有してなるペプチド、数平
均分子量約400、(株)ペプチド研究所製]、GRG
DS[Gly Arg Gly Asp Ser配列(15)を1個含有し
てなるペプチド、数平均分子量約500、(株)ペプチ
ド研究所製]、RetroNectin(リコンビナン
トヒトフィブロネクチンCH−296)[ヒトフィブロ
ネクチン細胞接着シグナルであるCS1シグナルと細胞
接着ドメインTypeIII及びヘパリン結合ドメイン
IIを1つずつ有する数平均分子量約6万のペプチド、
宝酒造(株)製(Arg Gly Asp Ser配列(14)を約1
個含有)]、及びRGDS−Protein A「Arg
Gly Asp配列をProtein A(IgG結合ドメイ
ン)に挿入した数平均分子量約3万のペプチド、宝酒造
(株)製(Arg Gly Asp Ser配列(14)を約1個含
有)]等が挙げられる。
[0021] Peptide (X) available on the market can be described in the trade name as, for example, RGDS [Arg Gly As
Peptide containing one p Ser sequence (14), number average molecular weight of about 400, manufactured by Peptide Institute Co., Ltd., GRG
DS [peptide containing one Gly Arg Gly Asp Ser sequence (15), number average molecular weight of about 500, manufactured by Peptide Laboratories], RetroNectin (recombinant human fibronectin CH-296) [human fibronectin cell adhesion signal A peptide having a number average molecular weight of about 60,000, each having one CS1 signal and one cell adhesion domain Type III and one heparin-binding domain II,
Takara Shuzo Co., Ltd. (Arg Gly Asp Ser sequence (14)
Individual)), and RGDS-Protein A "Arg
Examples include a peptide having a number average molecular weight of about 30,000 in which a Gly Asp sequence is inserted into Protein A (IgG binding domain), manufactured by Takara Shuzo Co., Ltd. (containing about one Arg Gly Asp Ser sequence (14)), and the like.

【0022】市場から入手できるペプチド(Z)として
は、商品名を記載すると例えば、プロネクチンF[1分
子中にArg Gly Asp配列と(Gly Ala Gly Ala Gly Ser)
9配列(35)とを各々約12個有し、遺伝子組み換え
大腸菌により製造される数平均分子量約10万のペプチ
ド、三洋化成工業(株)製]、プロネクチンFプラス
[プロネクチンFをジメチルアミノエチルクロライドと
反応させて水溶性にしたもの、三洋化成工業(株)
製]、プロネクチンL[1分子中にIle Lys Val AlaVal
配列(7)と(Gly Ala Gly Ala Gly Ser)9配列(3
5)とを各々約12個有し、遺伝子組み換え大腸菌によ
り製造される数平均分子量約10万のペプチド、三洋化
成工業(株)製]等が挙げられる。なお、市場から入手
できるペプチド(Y)は知られていないが、上記の製造
方法により容易に得ることができる。
As the commercially available peptide (Z), when the trade name is described, for example, Pronectin F [Arg Gly Asp sequence and (Gly Ala Gly Ala Gly Ser) in one molecule]
Peptide having about 12 each of 9 sequences (35) and having a number average molecular weight of about 100,000 produced by genetically engineered Escherichia coli, manufactured by Sanyo Kasei Co., Ltd., Pronectin F plus [Pronectin F dimethylaminoethyl chloride] Made water-soluble by reacting with Sanyo Chemical Industry Co., Ltd.
], Pronectin L [Ile Lys Val AlaVal in one molecule]
Sequence (7) and (Gly Ala Gly Ala Gly Ser) 9 sequence (3
5) and 12 each of which have a number average molecular weight of about 100,000 produced by genetically engineered Escherichia coli, Sanyo Chemical Industry Co., Ltd.] and the like. Although the peptide (Y) available on the market is not known, it can be easily obtained by the above production method.

【0023】変温動物由来細胞(A)としては、魚類
(例えば、コイ、ナマズ、サケ、マス、サバ及びスズキ
等)、両生類(例えば、カエル、サンショウウオ及びイ
モリ等)、は虫類(例えば、カメ、トカゲ、ヘビ及びワ
ニ等)、原索動物(例えば、ヒゲムシ、ホヤ及びタリア
等)、棘皮動物(例えば、ヒトデ、ナマコ及びウニ
等)、節足動物(例えば、昆虫、甲殻類、クモ、ヤス
デ、ムカデ及びカブトガニ等)、環形動物(例えば、ゴ
カイ、ミミズ、ヒル及びイムシ等)、軟体動物(例え
ば、カイ、イカ及びタコ等)、扁形動物(例えば、渦
虫、吸虫、二生類及び条虫等)、刺胞動物(例えば、ヒ
ドロ虫、鉢虫及び花虫等)、海綿動物(例えば、石灰海
綿類、六放海綿類、四軸海綿類及び珪角海綿類等)及び
原生動物(例えば、鞭毛虫、肉質虫及び胞子虫等)等の
岩波生物学辞典(山田常雄ら編集、株式会社岩波書店発
行)に記載の変温動物からなる群より選ばれる少なくと
も1種の動物に由来する細胞等が挙げられる。
Examples of cells (A) derived from a thermophilic animal include fishes (eg, carp, catfish, salmon, trout, mackerel and sea bass), amphibians (eg, frog, salamander and newt), and reptiles (eg, turtle, Lizards, snakes and crocodiles), protozoa (eg, bedbugs, ascidians and taryas), echinoderms (eg, starfish, sea cucumbers and sea urchins), arthropods (eg, insects, crustaceans, spiders, millipedes, etc.) Centipedes and horseshoe crabs, etc., annelids (eg, mosquitoes, earthworms, leeches and worms), molluscs (eg, kai, squid, octopus, etc.), flatworms (eg, whirlworms, trematodes, amphibians, tapeworms, etc.) ), Cnidarians (for example, hydrozoa, beetles and flower worms), sponges (for example, lime sponges, six-spray sponges, tetraaxial sponges and horned sponges) and protozoa (for example, Flagellate, meat Insects and Iwanami Biological Dictionary spores insects, etc.), etc. (Tsuneo Yamada et al., Eds., Cells, and the like derived from at least one animal selected from the group consisting of heterothermic according to Iwanami Shoten published) LTD.

【0024】これらのうち、魚類の細胞、両生類の細
胞、は虫類の細胞及び節足動物の細胞が好ましく、さら
に好ましくは両生類の細胞及び節足動物の細胞、特に好
ましくは節足動物の細胞、さらに特に好ましくは昆虫の
細胞、甲殻類の細胞、クモ類の細胞及びヤスデ類の細
胞、より特に好ましくは昆虫の細胞及び甲殻類の細胞、
最も好ましくは昆虫の細胞である。
Of these, fish cells, amphibian cells, reptile cells and arthropod cells are preferred, amphibian cells and arthropod cells are more preferred, and arthropod cells are particularly preferred. Particularly preferably insect cells, crustacean cells, arachnid cells and millipedes cells, more particularly preferably insect cells and crustacean cells,
Most preferred are insect cells.

【0025】昆虫の細胞としては、カイコ細胞、クワコ
細胞、サクサン細胞、シンジュサン細胞、ヨトウガ細
胞、クワゴマダラヒトリ細胞、ハマキムシ細胞、ショウ
ジョウバエ細胞、センチニクバエ細胞、ヒトスジシマカ
細胞、アゲハチョウ細胞、ワモンゴキブリ細胞及びイラ
クサキンウワバ細胞等が挙げられ、これらのうち、カイ
コ細胞、サクサン細胞、ヨトウガ細胞、イラクサキンウ
ワバ細胞、クワゴマダラヒトリ細胞、ショウジョウバエ
細胞、センチニクバエ細胞、アゲハチョウ細胞及びワモ
ンゴキブリ細胞が好ましく、さらに好ましくはカイコ細
胞、ヨトウガ細胞、イラクサキンウワバ細胞、センチニ
クバエ細胞及びワモンゴキブリ細胞、特に好ましくはカ
イコ細胞、ヨトウガ細胞及びイラクサキンウワバ細胞で
ある。
Examples of insect cells include silkworm cells, mulberry cells, saxan cells, synjusan cells, syringa cells, stilt cells, Dermatophagoid cells, Drosophila melanogaster cells, Aedes albopictus cells, Aedes albopictus cells, swallowtail cells, and nettle cockroaches cells and nettles. Examples thereof include mosquito cells, and among these, silkworm cells, saxan cells, armyworm cells, nettle buckwheat cells, locust-dwelling cells, Drosophila melanogaster cells, sentinel fly cells, swallowtail cells and American cockroaches cells are preferable, and silkworms are more preferable. Cells, armyworm cells, nettle fruit cells, sentinel fly cells and American cockroaches cells, particularly preferably silkworm cells, armyworm cells and nettleworm cells.

【0026】カイコ細胞としてはBmN細胞及びBoM
o細胞等が挙げられ、ヨトウガ細胞としてはSf9細胞
及びSf21細胞等が挙げられ、イラクサキンウワバ細
胞としてはTn−5細胞、HIGH FIVE細胞及び
MG1細胞等が挙げられる。変温動物由来細胞(A)の
由来は、細胞が採取できる部位であれば何れでもよい
が、細胞採取し易いという観点から、胚、卵巣、脂肪体
および血球が好ましい。
BmN cells and BoM as silkworm cells
o cells and the like, Syogoga cells include Sf9 cells and Sf21 cells and the like, and nettle buckwheat cells include Tn-5 cells, HIGH FIVE cells, MG1 cells and the like. The cells of the different temperature animal (A) may be derived from any site where cells can be collected, but from the viewpoint of easy cell collection, embryos, ovaries, fat pads and blood cells are preferable.

【0027】変温動物由来細胞(A)は、遺伝子を導入
(組み換え)した変温動物由来細胞であってもよい。細
胞に遺伝子を導入する方法としては、例えば、マイクロ
インジェクション法、リン酸カルシウム法、エレクトロ
ポレーション法、プロトプラスト融合法、DEAEデキ
ストラン法、リポフェクション法、カチオン性物質利用
法、ウイルス法及びパーティクルガン法等の遺伝子工学
キーワードブック(緒方宣邦ら著、株式会社羊土社発
行、2000年)や生物化学実験法45組換えタンパク
質生産法(塚越規弘編著、株式会社学会出版センター、
2001年)に記載の方法等が適用できる。これらのう
ちカチオン性物質利用法及びウイルス法が好ましい。
The cell (A) derived from a thermophilic animal may be a cell derived from a thermophilic animal into which a gene has been introduced (recombined). Examples of the method for introducing a gene into cells include, for example, microinjection method, calcium phosphate method, electroporation method, protoplast fusion method, DEAE dextran method, lipofection method, cationic substance utilization method, virus method and particle gun method. Engineering Keyword Book (Nobunoku Ogata et al., Published by Yodosha Co., Ltd., 2000) and Biochemical Experimental Method 45 Recombinant protein production method (edited by Norihiro Tsukakoshi, Academic Publishing Center, Inc.)
The method described in 2001) can be applied. Of these, the cationic substance utilization method and the virus method are preferable.

【0028】基材(B)としては特に制限はなく、細胞
が接着でき、細胞培養ができるものであれば何れも使用
できる。基材(B)の形状としては、細胞培養に一般に
用いられる形状[例えば、細胞培養の技術(日本組織培
養学会編集、株式会社朝倉書店発行、1999年)に記
載の形状]等が使用でき、例えば、板状、穴付きプレー
ト(6穴、24穴、96穴プレート等)、シャーレ、フ
ラスコ、ボトル、ビーズ及びファイバー等が挙げられ
る。基材(B)の材質としては、細胞培養に一般に用い
られる材質[例えば、細胞培養の技術(日本組織培養学
会編集、株式会社朝倉書店発行、1999年)に記載の
材質]等が使用でき、例えば、ガラス、シリコーン、ポ
リスチレン、ポリエチレン、ポリプロピレン、ナイロ
ン、アクリル樹脂及びポリウレタン等が挙げられる。
The base material (B) is not particularly limited, and any material can be used as long as it can adhere cells and culture cells. As the shape of the base material (B), a shape generally used for cell culture [for example, a shape described in the technology of cell culture (edited by The Japanese Tissue Culture Society, published by Asakura Shoten Co., Ltd., 1999)] can be used, For example, a plate shape, a plate with holes (6 hole, 24 hole, 96 hole plate, etc.), a petri dish, a flask, a bottle, beads, fibers and the like can be mentioned. As the material of the base material (B), materials generally used for cell culture [for example, materials described in cell culture technology (edited by Japan Tissue Culture Society, published by Asakura Shoten Co., Ltd., 1999)] can be used, Examples thereof include glass, silicone, polystyrene, polyethylene, polypropylene, nylon, acrylic resin and polyurethane.

【0029】本発明の細胞接着基材は、ペプチド(X)
及び/又はペプチド(Y)を、基材(B)の表面に付着
した状態及び/又は基材(B)に練り込まれた状態とす
ることにより生産することができる。ペプチド(X)及
び/又はペプチド(Y)が、基材(B)の表面に付着し
た状態及び/又は基材(B)に練り込んだ状態のうち、
ペプチド(X)及び/又はペプチド(Y)の使用量が少
なくて済むという観点から、基材(B)の表面に付着し
た状態が好ましい。
The cell adhesion substrate of the present invention comprises the peptide (X)
And / or the peptide (Y) can be produced by making it adhere to the surface of the base material (B) and / or kneaded into the base material (B). Of the state where the peptide (X) and / or the peptide (Y) is attached to the surface of the base material (B) and / or the state of being kneaded into the base material (B),
From the viewpoint that the amount of the peptide (X) and / or the peptide (Y) used is small, the state of being attached to the surface of the base material (B) is preferable.

【0030】ペプチド(X)及び/又はペプチド(Y)
を基材(B)の表面に付着した状態にさせる方法として
は、ペプチド(X)及び/又はペプチド(Y)を物理的
に接触させる方法及び化学的に結合させる方法等が挙げ
られる。
Peptide (X) and / or peptide (Y)
Examples of the method for bringing the substrate into the state of being attached to the surface of the base material (B) include a method of physically contacting the peptide (X) and / or the peptide (Y) and a method of chemically bonding them.

【0031】ペプチド(X)及び/又はペプチド(Y)
を物理的に接触させる方法としては、例えば、ペプチド
(X)及び/又はペプチド(Y)を溶媒(S1)等に溶
解又は分散させた溶液又は分散液を、基材(B)に塗
工、含浸又は混合させる方法等が適用できる。そしてそ
の後、乾燥させることによって、(X)及び/又は
(Y)が付着した細胞接着基材を得ることができる(例
えば、「Esty,A.Biomedical Pro
ducts 1991年」に記載の方法等)。
Peptide (X) and / or peptide (Y)
As a method of physically contacting with, for example, a solution or dispersion obtained by dissolving or dispersing the peptide (X) and / or the peptide (Y) in the solvent (S1) or the like is applied to the base material (B), A method of impregnation or mixing can be applied. Then, after that, the cell-adhesive substrate having (X) and / or (Y) attached thereto can be obtained by drying (for example, “Esty, A. Biomedical Pro.
Ducts 1991 ”, etc.).

【0032】溶媒(S1)としては特に制限はないが、
無機塩、有機酸塩、アミノ酸、ビタミン、アルコール、
脂質・糖、酸及び/又は塩基を含有する水溶液、水及び
体液等が使用できる。水溶液中の無機塩、有機酸塩、ア
ミノ酸、ビタミン、アルコール、脂質・糖、酸及び/又
は塩基の含有量は、水溶液の重量に基づいて、0.00
1〜50重量%が好ましく、さらに好ましくは0.01
〜10重量%である。すなわち、この含有量の上限は5
0重量%が好ましく、さらに好ましくは10重量であ
り、またこの含有量の下限は0.001重量%が好まし
く、さらに好ましくは0.01重量である。
The solvent (S1) is not particularly limited,
Inorganic salts, organic acid salts, amino acids, vitamins, alcohols,
Aqueous solutions containing lipids / sugars, acids and / or bases, water and body fluids can be used. The content of the inorganic salt, organic acid salt, amino acid, vitamin, alcohol, lipid / sugar, acid and / or base in the aqueous solution is 0.00 based on the weight of the aqueous solution.
1 to 50% by weight is preferable, and 0.01 is more preferable.
10 to 10% by weight. That is, the upper limit of this content is 5
The content is preferably 0% by weight, more preferably 10% by weight, and the lower limit of this content is preferably 0.001% by weight, more preferably 0.01% by weight.

【0033】無機塩としては、ハロゲン化金属塩、硫酸
金属塩、リン酸金属塩、硝酸金属塩、炭酸金属塩及び過
ハロゲン酸金属等が使用でき、塩化ナトリウム、硫酸ナ
トリウム、リン酸ナトリウム、塩化カルシウム、硝酸
鉄、塩化カリウム、硫酸マグネシウム、炭酸ナトリウ
ム、リン酸水素ナトリウム、リン酸カリウム、リン酸水
素カリウム、硫酸銅、硫酸鉄、塩化リチウム、臭化ナト
リウム、臭化リチウム過塩素酸ナトリウム及び過塩素酸
リチウム等が挙げられる。有機酸塩としては、蟻酸ナト
リウム、酢酸ナトリウム、酢酸リチウム及び酒石酸ナト
リウム等が挙げられる。
As the inorganic salt, metal halides, metal sulfates, metal phosphates, metal nitrates, metal carbonates, metal perhalogenates and the like can be used. Sodium chloride, sodium sulfate, sodium phosphate, chloride Calcium, 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 and sodium perchlorate Examples thereof include lithium chlorate. Examples of the organic acid salt include sodium formate, sodium acetate, lithium acetate and sodium tartrate.

【0034】アミノ酸としては、アルギニン、ヒスチジ
ン、イソロイシン、ロイシン、メチオニン、フェニルア
ラニン、スレオニン、トリプトファン、チロシン、バリ
ン、アラニン、アスパラギン、アスパラギン酸、グルタ
ミン酸、プロリン、セリン及びグリシン等が挙げられ
る。ビタミンとしては、コリン、イノシトール、ニコチ
ンアミド、グルタミン、ビタミンA、ビタミンB12及び
ビタミンC等が挙げられる。アルコールとしては、炭素
数1〜4のアルコール等が使用でき、メタノール、エタ
ノール、イソプロピルアルコール及びブタノール等が挙
げられる。脂質・糖としては、脂質、単糖、2糖、オリ
ゴ糖、アミノ糖及び酸性糖等が挙げられる。
Examples of the 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 the alcohol, an alcohol having 1 to 4 carbon atoms can be used, and examples thereof include methanol, ethanol, isopropyl alcohol and butanol. Examples of lipids / sugars include lipids, monosaccharides, disaccharides, oligosaccharides, amino sugars and acidic sugars.

【0035】酸としては、無機酸及び炭素数1〜6の有
機酸等が使用でき、塩酸、燐酸、酢酸、蟻酸、フェノー
ル及び硫酸等が挙げられる。塩基としては、無機塩基及
び炭素数2〜6の有機塩基等が使用でき、水酸化ナトリ
ウム、水酸化カリウム、アンモニア及びトリエチルアミ
ン等が挙げられる。水としては、蒸留水、イオン交換
水、水道水及びイオン交換蒸留水等が挙げられる。体液
としては、血液、血漿、血清及び尿等が挙げられる。こ
れらの溶媒(S1)の中で、無機塩、酸及び/又は塩基
を含有する水溶液及び水が好ましく、さらに好ましくは
無機塩、酸及び/又は塩基を含有する水溶液である。
As the acid, inorganic acids and organic acids having 1 to 6 carbon atoms can be used, 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-exchanged water, tap water, and ion-exchanged distilled water. The body fluid includes blood, plasma, serum, urine and the like. Among these solvents (S1), 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.

【0036】さらに、ペプチド(X)及び/又はペプチ
ド(Y)を培地に溶解又は分散させることにより、その
培地溶液(分散液)を変温動物由来細胞(A)の培地と
して使用し、変温動物由来細胞(A)の培養するととも
に、ペプチド(X)及び/又はペプチド(Y)を基材
(B)の表面に付着させることもできる。この場合、通
常、変温動物由来細胞(A)は基材上で培養されるが、
培地中で培養された変温動物由来細胞(A)が基材に接
着される場合もありうる。
Furthermore, by dissolving or dispersing the peptide (X) and / or the peptide (Y) in a medium, the medium solution (dispersion) is used as a medium for the animal (A) having a different temperature, and the temperature is changed. It is also possible to attach the peptide (X) and / or peptide (Y) to the surface of the base material (B) while culturing the animal-derived cell (A). In this case, the cells (A) derived from a thermophilic animal are usually cultured on the substrate,
In some cases, cells (A) derived from a thermophilic animal cultured in a medium may adhere to the substrate.

【0037】ペプチド(X)及び/又はペプチド(Y)
を化学的に結合させる方法としては、例えば、N−ヒド
ロキシコハク酸イミド又はカルボジイミド等の存在下
に、基材(B)にペプチド(X)及び/又はペプチド
(Y)をエステル化又はアミド化により固定させ、洗浄
乾燥させる方法等が適用できる。なお、この場合、反応
溶媒を使用してもよく、反応溶媒としては公知のものが
使用でき、例えば、溶媒(S1)及び有機溶剤等が用い
られる。有機溶媒としては、アセトン、エタノール、ジ
メチルスルホキシド、ジメチルホルムアミド、ジメチル
アセトアミド及びテトラヒドロフラン等が挙げられる。
これらのうち好ましくは、水及び有機溶媒である。ペプ
チド(X)及び/又はペプチド(Y)を化学的に結合さ
せる場合、化学反応に関与する官能基としては、カルボ
キシル基、アミノ基及び水酸基等が挙げられる。
Peptide (X) and / or peptide (Y)
Examples of the method for chemically coupling the peptide include, for example, esterification or amidation of the peptide (X) and / or peptide (Y) on the base material (B) in the presence of N-hydroxysuccinimide or carbodiimide. A method of fixing, washing and drying can be applied. In this case, a reaction solvent may be used, and a known reaction solvent can be used. For example, the solvent (S1) and an organic solvent are used. Examples of the organic solvent include acetone, ethanol, dimethylsulfoxide, dimethylformamide, dimethylacetamide and tetrahydrofuran.
Of these, water and organic solvents are preferable. When the peptide (X) and / or the peptide (Y) are chemically bonded, examples of the functional group involved in the chemical reaction include a carboxyl group, an amino group and a hydroxyl group.

【0038】ペプチド(X)及び/又はペプチド(Y)
を基材(B)に練り込まれた状態にさせる方法として
は、成形前の基材(B)の溶液にペプチド(X)及び/
又はペプチド(Y)を投入した混合物を、所定の形状に
成形する方法等が挙げられ、例えば、熱可塑性樹脂(ポ
リエチレン、ポリスチレン及びポリアミド等)を融点温
度以上にして流動可能状態とした後、ペプチド(X)及
び/又はペプチド(Y)を投入し、その混合物を押出成
形や射出成形などで加工する方法や、熱硬化性樹脂(尿
素樹脂、メラミン樹脂、フェノール樹脂、エポキシ樹
脂、不飽和ポリエステル、アルキド樹脂、ウレタン樹
脂、エボナイト及びシリコーン樹脂等)に、ペプチド
(X)及び/又はペプチド(Y)を投入し、その混合物
を硬化温度以上にして加工する方法等が適用できる。
Peptide (X) and / or peptide (Y)
As a method for bringing the base material (B) into a kneaded state, the peptide (X) and //
Alternatively, a method of molding a mixture into which the peptide (Y) is put into a predetermined shape may be used. For example, a thermoplastic resin (polyethylene, polystyrene, polyamide, etc.) may be melted at a temperature above its melting point to make it flowable, and then the peptide (X) and / or peptide (Y) are added and the mixture is processed by extrusion molding or injection molding, or a thermosetting resin (urea resin, melamine resin, phenol resin, epoxy resin, unsaturated polyester, For example, a method may be applied in which the peptide (X) and / or the peptide (Y) is added to an alkyd resin, a urethane resin, an ebonite, a silicone resin, etc., and the mixture is processed at a curing temperature or higher.

【0039】ペプチド(X)及び/又はペプチド(Y)
を基材(B)の表面に付着させる場合、ペプチド(X)
及び/又はペプチド(Y)の含有溶液(分散液)中の
(X)及び/又は(Y)の濃度は、(X)及び/又は
(Y)の含有溶液(分散液)の重量に基づいて、0.0
01μg/g〜999mg/gが好ましく、さらに好ま
しくは0.01μg/g〜100mg/g、特に好まし
くは0.1μg/g〜10mg/g、最も好ましくは1
μg/g〜1mg/gである。すなわち、この場合、濃
度の上限は999mg/gが好ましく、さらに好ましく
は100mg/g、特に好ましくは10mg/g、最も
好ましくは1mg/gであり、同様に下限は0.001
μg/gが好ましく、さらに好ましくは0.01μg/
g、特に好ましくは0.1μg/g、最も好ましくは1
μg/gである。
Peptide (X) and / or peptide (Y)
When the peptide is attached to the surface of the substrate (B), the peptide (X)
And / or the concentration of (X) and / or (Y) in the solution (dispersion) containing the peptide (Y) is based on the weight of the solution (dispersion) containing (X) and / or (Y). , 0.0
01 μg / g to 999 mg / g is preferable, more preferably 0.01 μg / g to 100 mg / g, particularly preferably 0.1 μg / g to 10 mg / g, most preferably 1
It is μg / g to 1 mg / g. That is, in this case, the upper limit of the concentration is preferably 999 mg / g, more preferably 100 mg / g, particularly preferably 10 mg / g, most preferably 1 mg / g, and similarly the lower limit is 0.001.
μg / g is preferable, and 0.01 μg / g is more preferable.
g, particularly preferably 0.1 μg / g, most preferably 1
μg / g.

【0040】ペプチド(X)及び/又はペプチド(Y)
を基材(B)の表面に付着させた場合、ペプチド(X)
及び/又はペプチド(Y)の含有量としては、基材
(B)の培養単位面積あたり、0.1ng/cm2〜1
00mg/cm2が好ましく、さらに好ましくは1ng
/cm2〜10mg/cm2、特に好ましくは10ng/
cm 2〜1mg/cm2、最も好ましくは100ng/c
2〜100μg/cm2である。なお、培養単位面積
は、基材(B)の表面のうち、培養される細胞が接着し
得る表面の表面積を意味する。ここで、細胞が入り込ま
ないような微小な凹凸(例えば、1μm以下)は平坦な
表面として取扱うが、培養単位面積を高める目的でリブ
(畝)等が設けてあるものについてはそのリブの表面積
を培養単位面積に含まれる。また、基材(B)の培養単
位面積は、基材メーカーがカタログに記載している基材
の培養単位面積をそのまま適用できる。培養単位面積あ
たりのペプチド(X)及び/又はペプチド(Y)の含有
量の測定方法は特に限定されないが、例えば、免疫学的
測定法等が利用できる。免疫学的測定法としては、例え
ば、ペプチド(X)及び/又はペプチド(Y)と結合す
る抗体に酵素を標識したもの(以下、酵素標識抗体)を
基材(B)の培養単位面積に反応させ、基材(B)の培
養単位面積に結合した酵素標識抗体の酵素量を測定する
方法等が挙げられる。
Peptide (X) and / or peptide (Y)
When the peptide is attached to the surface of the substrate (B), the peptide (X)
And / or the content of the peptide (Y) is a base material.
(B) per culture unit area 0.1 ng / cm2~ 1
00 mg / cm2Is preferred, and more preferably 1 ng
/ Cm2-10mg / cm2, Particularly preferably 10 ng /
cm 2~ 1 mg / cm2, Most preferably 100 ng / c
m2~ 100 μg / cm2Is. In addition, culture unit area
Cells of the surface of the substrate (B) to which the cultured cells adhere
It means the surface area of the surface to be obtained. Where the cells get in
Fine irregularities that do not exist (for example, 1 μm or less) are flat
Handled as the surface, but ribs for the purpose of increasing the culture unit area
For those with (ridges) etc., the surface area of the rib
Is included in the culture unit area. In addition, the culture unit of the substrate (B)
The base area is the base material that the base material manufacturer lists in the catalog.
The culture unit area of can be applied as it is. Culture unit area
Of peptide (X) and / or peptide (Y)
The method for measuring the amount is not particularly limited.
Measurement methods can be used. As an immunological measurement method, for example,
A peptide (X) and / or a peptide (Y)
Labeled with an enzyme (hereinafter, enzyme-labeled antibody)
Culture the base material (B) by reacting it with the culture unit area of the base material (B).
Measure the amount of enzyme-labeled antibody bound to the nutrient area
Methods and the like.

【0041】ペプチド(X)及び/又はペプチド(Y)
を基材(B)に練り込ませる場合、ペプチド(X)及び
/又はペプチド(Y)の含有混合物中の(X)及び/又
は(Y)の濃度は、含有混合物の重量に基づいて、0.
01μg/g〜999mg/gが好ましく、さらに好ま
しくは0.1μg/g〜100mg/g、特に好ましく
は1μg/g〜10mg/g、最も好ましくは10μg
/g〜10mg/gである。すなわち、この場合、濃度
の上限は999mg/gが好ましく、さらに好ましくは
100mg/g、特に好ましくは10mg/g、最も好
ましくは1mg/gであり、同様に下限は0.01μg
/gが好ましく、さらに好ましくは0.1μg/g、特
に好ましくは1μg/gmg/g、最も好ましくは10
μg/gである。
Peptide (X) and / or peptide (Y)
When kneading the base material (B), the concentration of (X) and / or (Y) in the mixture containing peptide (X) and / or peptide (Y) is 0 based on the weight of the mixture. .
01 μg / g to 999 mg / g is preferable, more preferably 0.1 μg / g to 100 mg / g, particularly preferably 1 μg / g to 10 mg / g, most preferably 10 μg.
/ G-10 mg / g. That is, in this case, the upper limit of the concentration is preferably 999 mg / g, more preferably 100 mg / g, particularly preferably 10 mg / g, most preferably 1 mg / g, and similarly the lower limit is 0.01 μg.
/ G is preferred, 0.1 μg / g is more preferred, 1 μg / g mg / g is particularly preferred, and 10 is most preferred.
μg / g.

【0042】ペプチド(X)及び/又はペプチド(Y)
を、基材(B)の表面に付着又は基材(B)に練り込ん
だ後、必要に応じて滅菌処理を施してもよい。滅菌方法
としては特に制限はなく、例えば、放射線滅菌、エチレ
ンオキサイドガス滅菌、γ線滅菌、アルコール滅菌、オ
ートクレーブ滅菌及び乾熱滅菌等が挙げられる。
Peptide (X) and / or peptide (Y)
May be attached to the surface of the base material (B) or kneaded into the base material (B), and then sterilized as necessary. The sterilization method is not particularly limited, and examples thereof include radiation sterilization, ethylene oxide gas sterilization, γ-ray sterilization, alcohol sterilization, autoclave sterilization, and dry heat sterilization.

【0043】変温動物由来細胞(A)と基材(B)とを
接着させる方法としては、細胞を基材に接着させる一般
的な方法[例えば、細胞培養の技術(日本組織培養学会
編集、株式会社朝倉書店発行、1999年)、生物化学
実験法45組換えタンパク質生産法(塚越規弘編著、株
式会社学会出版センター、2001年)及び昆虫バイオ
工場(木村滋編著、株式会社工業調査会発行、2000
年)等に記載の方法]等が使用できる。例えば、0.1
〜10,000万cellsの変温動物由来細胞(A)
及び0.001〜100mLの培地を、0.01〜1,
000平方センチメートルの培養単位面積を有する基材
(B)に投入し、0〜60℃の空気や水等の雰囲気下、
1分〜1週間培養して細胞を接着させる方法等が挙げら
れる。なお、ペプチド(X)及び/又はペプチド(Y)
はあらかじめ基材(B)の表面に付着及び/又は基材
(B)に練り込ませておくことが好ましい。
As a method for adhering the cells (A) derived from the temperature-altering animal and the base material (B), a general method for adhering cells to the base material [eg, cell culture technology (edited by the Japan Tissue Culture Society, Published by Asakura Shoten Co., Ltd. (1999), Biochemical Experimental Method 45 Recombinant protein production method (edited by Norihiro Tsukakoshi, Academic Society Publishing Center, 2001) and insect bio plant (edited by Shigeru Kimura, published by Industrial Research Board) , 2000
Year)) etc.] etc. can be used. For example, 0.1
~ 10 million cells of thermothermic animal-derived cells (A)
And 0.001 to 100 mL of the medium, 0.01 to 1,
It is put into a base material (B) having a culture unit area of 000 square centimeters, and the atmosphere such as air or water at 0 to 60 ° C.
Examples include a method of culturing for 1 minute to 1 week to adhere cells. The peptide (X) and / or the peptide (Y)
Is preferably adhered to the surface of the base material (B) and / or kneaded into the base material (B) in advance.

【0044】変温動物由来細胞(A)が基材(B)に接
着したことの確認方法としては、一般的な確認方法[例
えば、細胞培養の技術(日本組織培養学会編集、株式会
社朝倉書店発行、1999年)、生物化学実験法45組
換えタンパク質生産法(塚越規弘編著、株式会社学会出
版センター、2001年)及び昆虫バイオ工場(木村滋
編著、株式会社工業調査会発行、2000年)等に記載
の方法]が使用できる。例えば、血球計数法(コールタ
ーカウンターの使用など)、顕微鏡観察法(計算盤の使
用など)及び染色法(トリパンブルー、エリスロシン−
B、ニグロシン、クリスタルバイオレット、テトラゾリ
ウム塩の使用など)等が挙げられる。本発明の細胞接着
基材において、基材(B)に接着した変温動物由来細胞
(A)の数は、基材(B)の培養単位面積当り、50〜
5,000万cells/cm2が好ましく、さらに好
ましくは500〜500万cells/cm2、特に好
ましくは5,000〜50万cells/cm2であ
る。すなわち、変温動物由来細胞(A)の数の上限は、
5,000万cells/cm2が好ましく、さらに好
ましくは500万cells/cm2、特に好ましくは
50万cells/cm2、また、この下限は50ce
lls/cm2が好ましく、さらに好ましくは500c
ells/cm2、特に好ましくは5,000cell
s/cm2である。
As a method for confirming that the cells (A) derived from a temperature-altering animal have adhered to the base material (B), a general confirmation method [for example, cell culture technique (edited by the Japan Tissue Culture Society, Asakura Shoten Co., Ltd. (Published, 1999), Biochemical Experimental Method 45 Recombinant protein production method (edited by Norihiro Tsukakoshi, Academic Society Publishing Center, 2001) and insect bio plant (edited by Shigeru Kimura, published by Industrial Research Board, 2000) Etc.] can be used. For example, hemocytometry (using a Coulter counter, etc.), microscopy (using a calculator, etc.) and staining (trypan blue, erythrosine-
B, nigrosine, crystal violet, use of tetrazolium salt, etc.) and the like. In the cell-adhesive substrate of the present invention, the number of cells (A) derived from a thermophilic animal adhered to the substrate (B) is 50 to 50 per unit culture area of the substrate (B).
It is preferably 50 million cells / cm 2 , more preferably 5 to 5 million cells / cm 2 , and particularly preferably 50 to 500,000 cells / cm 2 . That is, the upper limit of the number of cells (A) derived from a thermophilic animal is
It is preferably 50 million cells / cm 2 , more preferably 5 million cells / cm 2 , particularly preferably 500,000 cells / cm 2 , and the lower limit is 50 ce.
lls / cm 2 is preferable, more preferably 500 c
ells / cm 2 , particularly preferably 5,000 cells
s / cm 2 .

【0045】本発明の細胞接着基材を用いて細胞培養し
細胞を生産する方法としては、例えば、本発明の細胞接
着基材及び培地等を用いて、培地を適時交換しながら細
胞を培養する方法等が挙げられる。なお、基材(B)に
培地と変温動物由来細胞(A)とを接触させて、基材
(B)の表面に変温動物由来細胞(A)を接着させた
後、培地を適時交換して細胞を培養する方法等でもよ
い。本発明の細胞接着基材を用いて細胞培養し細胞を生
産する方法において、細胞培養の条件としては特に制限
はないが例えば、0〜60℃の空気や水等の雰囲気下、
1時間〜1カ月培養する方法などが挙げられる。なお、
培地交換しながら細胞培養することが好ましく、その交
換頻度としては、1〜7日毎が好ましく、さらに好まし
くは1〜3日毎である。本発明の細胞接着基材を用いて
細胞培養し細胞を生産する方法によって得られる変温動
物由来細胞(A)の数は、基材(B)の培養単位面積当
り、100〜1億cells/cm2が好ましく、さら
に好ましくは1,000〜1,000万cells/c
2、特に好ましくは1万〜100万cellsであ
る。すなわち、変温動物由来細胞(A)の数の上限は、
1億cells/cm2が好ましく、さらに好ましくは
1,000万cells/cm2、特に好ましくは10
0万cells/cm2、また、この下限は100ce
lls/cm2が好ましく、さらに好ましくは1,00
0cells/cm2、特に好ましくは1万cells
/cm2である。
As a method for producing cells by culturing cells using the cell-adhesive substrate of the present invention, for example, cells are cultured using the cell-adhesive substrate of the present invention, a medium and the like while appropriately changing the medium. Methods and the like. It should be noted that the medium and the cells of different temperature of animal origin (A) are brought into contact with the base material (B) to adhere the cells of origin of the different temperature animal (A) to the surface of the base material (B), and then the medium is replaced at appropriate times. Alternatively, a method of culturing cells may be used. In the method of culturing cells using the cell-adhesive substrate of the present invention to produce cells, the conditions for cell culture are not particularly limited, but for example, in an atmosphere of 0 to 60 ° C., such as air or water,
Examples include a method of culturing for 1 hour to 1 month. In addition,
The cells are preferably cultured while the medium is exchanged, and the frequency of exchange is preferably 1 to 7 days, more preferably 1 to 3 days. The number of cells (A) derived from a thermophilic animal obtained by the method of culturing cells using the cell-adhesive substrate of the present invention to produce cells is 100 to 100 million cells / unit of culture unit area of the substrate (B). cm 2 is preferable, and more preferably 10 to 10 million cells / c.
m 2 , particularly preferably 10,000 to 1,000,000 cells. That is, the upper limit of the number of cells (A) derived from a thermophilic animal is
100 million cells / cm 2 is preferable, 10 million cells / cm 2 is more preferable, and 10 is particularly preferable.
0,000 cells / cm 2 , and this lower limit is 100ce
lls / cm 2 is preferable, and more preferably 100
0 cells / cm 2 , particularly preferably 10,000 cells
/ Cm 2 .

【0046】変温動物由来細胞(A)と基材(B)とを
接着させる方法および本発明の細胞接着基材を用いて細
胞培養し細胞を生産する方法において、培地の使用量と
しては、使用する基材(B)の種類などによって異なる
が、基材(B)の培養単位面積当り、0.0003〜3
00mL/cm2が好ましく、さらに好ましくは0.0
03〜30mL/cm2、特に好ましくは0.03〜3
mL/cm2である。すなわち、培地の使用量の上限
は、300mL/cm2が好ましく、さらに好ましくは
30mL/cm2、特に好ましくは3mL/cm2、ま
た、この下限は0.0003mL/cm2が好ましく、
さらに好ましくは0.003mL/cm2、特に好まし
くは0.03mL/cm2である。変温動物由来細胞
(A)と基材(B)とを接着させる方法および本発明の
細胞接着基材を用いて細胞培養し細胞を生産する方法に
おいて、培養に用いられる培地としては、細胞培養に使
用される公知のものが使用でき、例えば、Grace培
地、IPL−41培地、Schneider培地、TC
−100培地、Sf−900II培地、Ex−cell
405培地、Express−Five培地、Dros
ophila培地、MEM培地、DMEM培地、ハムF
−12培地、RPMI培地、及びこれらの培地の混合物
等が挙げられる。
In the method for adhering the cells (A) derived from a temperature-altering animal and the substrate (B) and the method for producing cells by culturing cells using the cell-adhesive substrate of the present invention, the amount of medium used is Although it depends on the type of the substrate (B) used, etc., 0.0003 to 3 per unit culture area of the substrate (B)
00 mL / cm 2 is preferable, and 0.0 is more preferable.
03 to 30 mL / cm 2 , particularly preferably 0.03 to 3
It is mL / cm 2 . That is, the upper limit of the amount of the medium is preferably 300 mL / cm 2, more preferably 30 mL / cm 2, particularly preferably 3 mL / cm 2, also the lower limit is preferably 0.0003mL / cm 2,
It is more preferably 0.003 mL / cm 2 , and particularly preferably 0.03 mL / cm 2 . In the method for adhering the cells (A) and the substrate (B) derived from the thermophilic animal and the method for producing cells by culturing cells using the cell-adhesive substrate of the present invention, the medium used for culturing is cell culture. The well-known thing used for can be used, for example, Grace medium, IPL-41 medium, Schneider medium, TC.
-100 medium, Sf-900II medium, Ex-cell
405 medium, Express-Five medium, Dros
optila medium, MEM medium, DMEM medium, Ham F
-12 medium, RPMI medium, and a mixture of these mediums.

【0047】これらの培地には、抗菌剤(アンホテリシ
ンB、ゲンタマイシン、ペニシリン及びストレプトマイ
シン等)、血清(ヒト血清、ウシ血清、ウマ血清及びヒ
ツジ血清等)等公知の添加剤を含有させることができ
る。抗菌剤を含有する場合、培地中の抗菌剤の濃度は、
培地の容量に基づいて、1ng/L〜100g/Lが好
ましく、さらに好ましくは10ng/L〜10g/Lで
ある。すなわち、この場合、抗菌剤濃度の上限は100
g/Lが好ましく、さらに好ましくは10g/Lであ
り、同様に下限は1ng/Lが好ましく、さらに好まし
くは10ng/Lである。
These media may contain known additives such as antibacterial agents (amphotericin B, gentamicin, penicillin, streptomycin, etc.), serum (human serum, bovine serum, horse serum, sheep serum, etc.). When the antibacterial agent is contained, the concentration of the antibacterial agent in the medium is
Based on the volume of the medium, 1 ng / L to 100 g / L is preferable, and 10 ng / L to 10 g / L is more preferable. That is, in this case, the upper limit of the antibacterial agent concentration is 100.
g / L is preferable, more preferably 10 g / L, and similarly, the lower limit is preferably 1 ng / L, more preferably 10 ng / L.

【0048】血清は含有しない方が好ましいが、血清を
含有する場合、培地中の血清の濃度は、血清由来のウイ
ルス等による感染の危険性を下げるために、培地の全容
量に対して、1×10-6〜50%(v/v)が好まし
く、さらに好ましくは1×10 -6〜10%(v/v)で
あり、特に好ましくは1×10-6〜2%(v/v)であ
る。すなわち、この場合、血清濃度の上限は50%(v
/v)が好ましく、さらに好ましくは10%(v/v)
であり、特に好ましくは2%(v/v)であり、同様に
下限は1×10-6%(v/v)が好ましく、さらに好ま
しくは1×10-6%(v/v)であり、特に好ましくは
1×10-6%(v/v)である。
Serum is preferably not contained, but serum is not included.
If included, the concentration of serum in the medium is
In order to reduce the risk of infection by loose
1 x 10 for quantity-6~ 50% (v / v) is preferred
More preferably 1 × 10 -6-10% (v / v)
Yes, particularly preferably 1 × 10-6~ 2% (v / v)
It That is, in this case, the upper limit of the serum concentration is 50% (v
/ V) is preferred, more preferably 10% (v / v)
And particularly preferably 2% (v / v),
The lower limit is 1 × 10-6% (V / v) is preferred and more preferred
It is 1 × 10-6% (V / v), particularly preferably
1 x 10-6% (V / v).

【0049】細胞接着性人工ペプチド(X)及び/又は
細胞接着補助人工ペプチド(Y)を用いることにより、
変温動物由来細胞(A)、特に昆虫細胞を効率良く基材
(B)に接着させることができるため、有用蛋白質を生
産できるウイルス感染の昆虫細胞を効率良く作製するこ
とができる。このウイルスとしては、昆虫細胞に感染で
きるウイルスで有れば制限なく、バキュロウイルス等が
挙げられる{「生物化学実験法45組換えタンパク質生
産法、塚越規弘編著、株式会社学会出版センター発行、
2001年」や「昆虫バイオ工場、木村滋編著、株式会
社工業調査会発行、2000年」}。ウイルス感染の昆
虫細胞の作製方法としては、例えば、トランスファーベ
クターに発現させたい遺伝子を組込み、昆虫感染性のウ
イルスDNAとともに昆虫細胞にトランスフェクトした
後、プラークアッセイによってウイルス感染昆虫細胞を
選別する方法などが挙げられる{「生物化学実験法45
組換えタンパク質生産法、塚越規弘編著、株式会社学会
出版センター発行、2001年」や「昆虫バイオ工場、
木村滋編著、株式会社工業調査会発行、2000
年」}。
By using the cell-adhesive artificial peptide (X) and / or the cell-adhesion assisting artificial peptide (Y),
Since cells (A) derived from a thermophilic animal, particularly insect cells, can be efficiently attached to the substrate (B), virus-infected insect cells capable of producing useful proteins can be efficiently produced. This virus is not limited as long as it is a virus that can infect insect cells, and includes baculovirus, etc. {"Biochemical Experimental Method 45 Recombinant Protein Production Method, edited by Norihiro Tsukakoshi, published by Academic Society Publishing Center, Inc.,
2001 ”and“ Insect Bio Factory, edited by Shigeru Kimura, published by Industrial Research Institute Co., Ltd., 2000 ”}. As a method for producing virus-infected insect cells, for example, a method of incorporating a gene to be expressed in a transfer vector, transfecting insect cells with insect-infectious viral DNA, and then selecting virus-infected insect cells by plaque assay, etc. {{"Biochemistry Experimental Method 45"
Recombinant protein production method, edited by Norihiro Tsukakoshi, published by Japan Society for Publishing, 2001 "and" insect bio plant, "
Edited by Shigeru Kimura, Published by Industrial Research Institute, 2000
Year"}.

【0050】ウイルス感染の昆虫細胞等の変温動物由来
細胞(A)が生産する有用蛋白質としては、例えば、ワ
クチン、検査・診断薬用の原料、獣医薬、畜産薬、医薬
及び殺虫剤等が挙げられる{「生物化学実験法45組換
えタンパク質生産法、塚越規弘編著、株式会社学会出版
センター発行、2001年」や「昆虫バイオ工場、木村
滋編著、株式会社工業調査会発行、2000年」}。
Examples of useful proteins produced by cells (A) derived from thermophilic animals such as virus-infected insect cells include vaccines, raw materials for test / diagnostic agents, veterinary medicines, livestock medicines, medicines and insecticides. Responded {"Biochemical Experimental Method 45 Recombinant Protein Production Method, edited by Norihiro Tsukakoshi, published by Academic Society Publishing Center, 2001" and "Insect Bio Factory, edited by Shigeru Kimura, published by Industrial Research Institute, 2000"} .

【0051】[0051]

【実施例】以下に実施例を掲げて本発明を更に詳しく説
明するが、本発明はこれら実施例のみに限定されるもの
ではない。
The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

【0052】<実施例1>(Sf9細胞と基材との接着
1) (1)ペプチド(Z)の準備 特表平3−502935号公報中の実施例記載の方法に
準じて、Arg Gly Asp配列と(Gly Ala Gly Ala Gly Se
r)9配列(35)とを各々約12個有し、数平均分子量
約10万のペプチド"SLPF"を遺伝子組み換え大腸菌
により製造した。
<Example 1> (Adhesion 1 of Sf9 cells and substrate) (1) Preparation of peptide (Z) Arg Gly Asp was prepared according to the method described in Example in JP-A-3-502935. Sequence and (Gly Ala Gly Ala Gly Se
r) A peptide "SLPF" having about 12 each of 9 sequences (35) and having a number average molecular weight of about 100,000 was produced by genetically modified Escherichia coli.

【0053】(2)SLPFの基材への付着 SLPFの1mgを4.5M過塩素酸リチウム水溶液の
1mLに溶解し、さらに、99.5%の塩化ナトリウム
を0.85重量%で含有する0.02M,pH7.2の
リン酸緩衝液(以下、PBS)で100倍希釈して、S
LPF水溶液(10μg/mL)を作製した。このSL
PF水溶液を24穴のポリスチレンプレート(日本ベク
トン・ディッキンソン株式会社製)に1mL/穴で投入
し25℃で2時間静置させた。その後残存するSLPF
水溶液をアスピレーターで吸引除去し、さらに3mLの
蒸留水で穴を3回洗浄して、SLPF付着量が約1μg
/cm2のSLPF付着基材を作製した。
(2) Adhesion of SLPF to substrate 1 mg of SLPF was dissolved in 1 mL of 4.5 M aqueous lithium perchlorate solution, and 99.5% sodium chloride was added at 0.85% by weight. Dilute 100 times with a phosphate buffer solution (hereinafter, PBS) of 0.02M, pH 7.2, and add S
An LPF aqueous solution (10 μg / mL) was prepared. This SL
The PF aqueous solution was put into a 24-well polystyrene plate (manufactured by Nippon Becton, Dickinson and Company) at 1 mL / well and allowed to stand at 25 ° C. for 2 hours. SLPF remaining after that
The aqueous solution is suctioned off with an aspirator, and the holes are washed three times with 3 mL of distilled water to give an SLPF adhesion amount of about 1 μg.
/ Cm 2 of SLPF adhered substrate was prepared.

【0054】(3)Sf9細胞(ウイルス未感染細胞)
の接着 このSLPF付着基材の1穴当たりに1mLのSf−9
00II培地(インビトロジェン株式会社製)及び20
万cells分のSf9細胞(インビトロジェン株式会
社製)液とを投入し28℃で1時間静置させて、Sf9
を基材に接着させて、本発明の細胞接着基材1を得た
(Sf9の接着量:約5万cells/cm2)。
(3) Sf9 cells (virus-uninfected cells)
Adhesion of 1 mL of Sf-9 per hole of this SLPF-attached substrate
00II medium (Invitrogen Corporation) and 20
Sf9 cells (manufactured by Invitrogen Co., Ltd.) solution for 10,000 cells was added and allowed to stand at 28 ° C. for 1 hour to give Sf9.
Was adhered to a base material to obtain a cell-adhesive base material 1 of the present invention (adhesion amount of Sf9: about 50,000 cells / cm 2 ).

【0055】<実施例2> (Sf9細胞と基材との接着2) (1)ペプチド(X)の準備 Arg Gly Asp Ser配列(14)を有するFibrone
ctin Active Fragment(以下、F
AF、株式会社ペプチド研究所製)をそのまま使用し
た。
<Example 2> (Adhesion 2 between Sf9 cells and substrate) (1) Preparation of peptide (X) Fibrone having Arg Gly Asp Ser sequence (14)
ctin Active Fragment (hereinafter, F
AF, manufactured by Peptide Institute Inc.) was used as it was.

【0056】(2)FAFの基材への付着 FAFの1mgをPBSの100mLに溶解し、FAF
水溶液(10μg/mL)を作製した。そのFAF水溶
液を24穴のポリスチレンプレート(日本ベクトン・デ
ィッキンソン株式会社製)に1mL/穴で投入し25℃
で2時間静置させた。その後、残存するFAF水溶液を
アスピレーターで吸引除去し、さらに3mLの蒸留水で
穴を3回洗浄して、FAF付着量が約1μg/cm2
FAF付着基材を作製した。
(2) Adhesion of FAF to substrate Dissolve 1 mg of FAF in 100 mL of PBS,
An aqueous solution (10 μg / mL) was prepared. The FAF aqueous solution was put into a 24-well polystyrene plate (manufactured by Nippon Becton Dickinson Co., Ltd.) at 1 mL / well and 25 ° C.
And allowed to stand for 2 hours. Then, the remaining FAF aqueous solution was removed by suction with an aspirator, and the hole was washed three times with 3 mL of distilled water to prepare a FAF-adhered substrate having an FAF adhesion amount of about 1 μg / cm 2 .

【0057】(3)Sf9細胞の接着 このFAF付着基材の1穴当たりに1mLのSf−90
0II培地(インビトロジェン株式会社製)及び20万
cells分のSf9細胞(インビトロジェン株式会社
製)液とを投入し28℃で1時間静置させて、Sf9を
基材に接着させて、本発明の細胞接着基材2を得た(S
f9の接着量:約4万cells/cm 2)。
(3) Adhesion of Sf9 cells 1 mL of Sf-90 per hole of this FAF-attached substrate
0II medium (manufactured by Invitrogen Corporation) and 200,000
Sf9 cells for cells (Invitrogen Corporation)
Liquid) and allowed to stand at 28 ° C for 1 hour to remove Sf9.
The cell-adhesive substrate 2 of the present invention was obtained by adhering to the substrate (S
Adhesion amount of f9: About 40,000 cells / cm 2).

【0058】<実施例3> (Sf9細胞と基材との接着3) (1)ペプチド(Y)の準備 特表平3−502935号公報中の実施例記載の方法に
準じて、(Gly Ala Gly Ala Gly Ser)9配列(35)を
約170個有し、数平均分子量約10万のペプチド"S
LP4"を遺伝子組み換え大腸菌により製造した。
<Example 3> (Adhesion 3 between Sf9 cells and substrate) (1) Preparation of peptide (Y) According to the method described in Example in JP-A-3-502935 (Gly Ala). Gly Ala Gly Ser) Peptide “S” having about 170 sequences 9 (35) and a number average molecular weight of about 100,000
LP4 "was produced by recombinant E. coli.

【0059】(2)SLP4の基材への付着 SLP4の1mgを4.5M過塩素酸リチウム水溶液の
1mLに溶解し、さらに、PBSで100倍希釈し、S
LP4水溶液(10μg/mL)を作製した。そのSL
P4水溶液を24穴のポリスチレンプレート(日本ベク
トン・ディッキンソン株式会社製)に1mL/穴で投入
し25℃で2時間静置させた。 その後残存するSLP
4水溶液をアスピレーターで吸引除去し、さらに3mL
の蒸留水で穴を3回洗浄して、SLP4付着量が約1μ
g/cm2のSLP4付着基材を作製した。
(2) Adhesion of SLP4 to the base material 1 mg of SLP4 was dissolved in 1 mL of a 4.5 M aqueous solution of lithium perchlorate, and further diluted 100-fold with PBS to give S.
An LP4 aqueous solution (10 μg / mL) was prepared. That SL
The P4 aqueous solution was put into a 24-well polystyrene plate (manufactured by Nippon Becton, Dickinson and Company) at 1 mL / well and allowed to stand at 25 ° C. for 2 hours. SLP remaining after that
4 Aspirator removes 4 aqueous solution by suction, and further 3mL
Wash the hole 3 times with the distilled water of the
A g / cm 2 SLP4 adhered substrate was prepared.

【0060】(3)Sf9細胞の接着 このSLP4付着基材の1穴当たりに1mLのSf−9
00II培地(インビトロジェン株式会社製)及び20
万cells分のSf9細胞(インビトロジェン株式会
社製)液とを投入し28℃で1時間静置させて、Sf9
を基材に接着させて、本発明の細胞接着基材3を得た
(Sf9の接着量:約4万cells/cm2)。
(3) Adhesion of Sf9 cells 1 mL of Sf-9 per well of this SLP4 adherent substrate
00II medium (Invitrogen Corporation) and 20
Sf9 cells (manufactured by Invitrogen Co., Ltd.) solution for 10,000 cells was added and allowed to stand at 28 ° C. for 1 hour to give Sf9.
Was adhered to a base material to obtain a cell-adhesive base material 3 of the present invention (adhesion amount of Sf9: about 40,000 cells / cm 2 ).

【0061】<実施例4> (HIGH FIVEと基材との接着1B)実施例1の
Sf9細胞の代わりにHIGH FIVE(ウイルス未
感染細胞)を使用し、Sf−900II培地の代わりに
Express−Five培地(インビトロジェン株式
会社製)を使用した以外は、実施例1と同様にして、本
発明の細胞接着基材1Bを得た(HIGH FIVEの
接着量:約7万cells/cm2)。
Example 4 (Adhesion 1B of HIGH FIVE and Substrate) HIGH FIVE (virus-uninfected cells) was used in place of the Sf9 cells of Example 1, and Express-Five was used in place of the Sf-900II medium. A cell adhesion substrate 1B of the present invention was obtained in the same manner as in Example 1 except that a medium (manufactured by Invitrogen Corporation) was used (adhesion amount of HIGH FIVE: about 70,000 cells / cm 2 ).

【0062】<実施例5> (HIGH FIVEと基材との接着2B)実施例2の
Sf9細胞の代わりにHIGH FIVE(ウイルス未
感染細胞)を使用し、Sf−900II培地の代わりに
Express−Five培地(インビトロジェン株式
会社製)を使用した以外は、実施例2と同様にして、本
発明の細胞接着基材2Bを得た(HIGH FIVEの
接着量:約5万cells/cm2)。
<Example 5> (Adhesion 2B of HIGH FIVE to the substrate) HIGH FIVE (virus-uninfected cells) was used in place of the Sf9 cells of Example 2, and Express-Five was used in place of the Sf-900II medium. A cell adhesion substrate 2B of the present invention was obtained in the same manner as in Example 2 except that a medium (manufactured by Invitrogen Corporation) was used (adhesion amount of HIGH FIVE: about 50,000 cells / cm 2 ).

【0063】<実施例6> (HIGH FIVEと基材との接着3B)実施例3の
Sf9細胞の代わりにHIGH FIVE(ウイルス未
感染細胞)を使用し、Sf−900II培地の代わりに
Express−Five培地(インビトロジェン株式
会社製)を使用した以外は、実施例3と同様にして、本
発明の細胞接着基材3Bを得た(HIGH FIVEの
接着量:約5万cells/cm2)。
<Example 6> (Adhesion 3B of HIGH FIVE to a substrate) HIGH FIVE (virus-uninfected cells) was used in place of the Sf9 cells of Example 3, and Express-Five was used in place of the Sf-900II medium. A cell adhesion substrate 3B of the present invention was obtained in the same manner as in Example 3 except that a medium (manufactured by Invitrogen Corporation) was used (adhesion amount of HIGH FIVE: about 50,000 cells / cm 2 ).

【0064】<比較例1> (Sf9細胞と基材との接着4) (1)コラーゲンの準備 ウシ由来のコラーゲンタイプ1(以下、COL、日本ベ
クトン・ディッキンソン株式会社製、Arg Gly Asp Ser
配列(14)を含む天然蛋白質)をそのまま使用した。
<Comparative Example 1> (Adhesion 4 between Sf9 cells and substrate) (1) Preparation of collagen Collagen type 1 derived from bovine (hereinafter, COL, manufactured by Nippon Becton Dickinson Co., Ltd., Arg Gly Asp Ser)
The native protein containing sequence (14) was used as is.

【0065】(2)COLの基材への付着 COLの1mgを0.05N塩酸水溶液の100mLに
溶解し、COL水溶液(10μg/mL)を作製した。
そのCOL水溶液を24穴のポリスチレンプレート(日
本ベクトン・ディッキンソン株式会社製)に1mL/穴
で投入し25℃で2時間静置させた。その後、残存する
COL水溶液をアスピレーターで吸引除去し、さらに3
mLの蒸留水で穴を3回洗浄して、COL付着量が約1
μg/cm2のCOL付着基材を作製した。
(2) Adhesion of COL to base material 1 mg of COL was dissolved in 100 mL of 0.05N hydrochloric acid aqueous solution to prepare an aqueous COL solution (10 μg / mL).
The COL aqueous solution was put into a 24-well polystyrene plate (manufactured by Nippon Becton Dickinson Co., Ltd.) at 1 mL / well and allowed to stand at 25 ° C. for 2 hours. After that, the remaining COL aqueous solution is removed by suction with an aspirator, and further 3
Wash the hole 3 times with mL of distilled water, and the COL adhesion amount is about 1
A μg / cm 2 COL-adhered substrate was prepared.

【0066】(3)Sf9細胞の接着 このCOL付着基材の1穴当たりに1mLのSf−90
0II培地(インビトロジェン株式会社製)及び20万
cells分のSf9細胞(インビトロジェン株式会社
製)液とを投入し28℃で1時間静置させて、Sf9を
基材に接着させて、比較用の細胞接着基材4を得た(S
f9の接着量:約3万cells/cm 2)。
(3) Adhesion of Sf9 cells 1 mL of Sf-90 per hole of this COL-attached substrate
0II medium (manufactured by Invitrogen Corporation) and 200,000
Sf9 cells for cells (Invitrogen Corporation)
Liquid) and allowed to stand at 28 ° C for 1 hour to remove Sf9.
It was adhered to a base material to obtain a cell adhesion base material 4 for comparison (S
Adhesion amount of f9: about 30,000 cells / cm 2).

【0067】<比較例2> (Sf9細胞と基材との接着5) (1)フィブロインの準備 フィブロイン(以下、FIB、和光純薬工業株式会社
製、Gly Ala Gly Ala Gly Ser配列(12)を含む天然
蛋白質)をそのまま使用した。
Comparative Example 2 (Adhesion 5 between Sf9 cells and substrate) (1) Preparation of fibroin Fibroin (hereinafter, FIB, manufactured by Wako Pure Chemical Industries, Ltd., Gly Ala Gly Ala Gly Ser Sequence (12)) The natural protein containing) was used as it was.

【0068】(2)FIBの基材への付着 FIBの1mgを4.5M過塩素酸リチウム水溶液の1
mLに溶解し、さらに、PBSで100倍希釈し、FI
B水溶液(10μg/mL)を作製した。 そのFIB
水溶液を24穴のポリスチレンプレート(日本ベクトン
・ディッキンソン株式会社製)に1mL/穴で投入し2
5℃で2時間静置させた。その後、残存するFIB水溶
液をアスピレーターで吸引除去し、さらに3mLの蒸留
水で穴を3回洗浄して、FIB付着量が約1μg/cm
2のFIB付着基材を作製した。
(2) Attachment of FIB to Substrate 1 mg of FIB was added to 1 M of 4.5 M lithium perchlorate aqueous solution.
Dissolve it in mL and dilute it 100 times with PBS.
A B aqueous solution (10 μg / mL) was prepared. That FIB
The aqueous solution was added to a 24-well polystyrene plate (manufactured by Nippon Becton Dickinson Co., Ltd.) at 1 mL / well and 2
It was allowed to stand at 5 ° C for 2 hours. After that, the remaining FIB aqueous solution is removed by suction with an aspirator, and the hole is washed three times with 3 mL of distilled water to give an FIB adhesion amount of about 1 μg / cm 2.
Two FIB-attached substrates were prepared.

【0069】(3)Sf9細胞の接着 このFIB付着基材の1穴当たりに1mLのSf−90
0II培地(インビトロジェン株式会社製)及び20万
cells分のSf9細胞(インビトロジェン株式会社
製)液とを投入し28℃で1時間静置させて、Sf9を
基材に接着させて、比較用の細胞接着基材5を得た(S
f9の接着量:約3万cells/cm 2)。
(3) Adhesion of Sf9 cells 1 mL of Sf-90 per hole of this FIB-attached substrate
0II medium (manufactured by Invitrogen Corporation) and 200,000
Sf9 cells for cells (Invitrogen Corporation)
Liquid) and allowed to stand at 28 ° C for 1 hour to remove Sf9.
It was adhered to a base material to obtain a cell adhesion base material 5 for comparison (S
Adhesion amount of f9: about 30,000 cells / cm 2).

【0070】<比較例3> (HIGH FIVEと基材との接着4B)比較例1の
Sf9細胞の代わりにHIGH FIVE(ウイルス未
感染細胞)を使用し、Sf−900II培地の代わりに
Express−Five培地(インビトロジェン株式
会社製)を使用した以外は、比較例1と同様にして、本
発明の細胞接着基材4Bを得た(HIGH FIVEの
接着量:約5万cells/cm2)。
Comparative Example 3 (Adhesion 4B of HIGH FIVE and Substrate) HIGH FIVE (virus-uninfected cells) was used instead of the Sf9 cells of Comparative Example 1, and Express-Five was used instead of the Sf-900II medium. A cell adhesion substrate 4B of the present invention was obtained in the same manner as in Comparative Example 1 except that a medium (manufactured by Invitrogen Corporation) was used (adhesion amount of HIGH FIVE: about 50,000 cells / cm 2 ).

【0071】<比較例4> (HIGH FIVEと基材との接着5B)比較例2の
Sf9細胞の代わりにHIGH FIVE(ウイルス未
感染細胞)を使用し、Sf−900II培地の代わりに
Express−Five培地(インビトロジェン株式
会社製)を使用した以外は、比較例2と同様にして、本
発明の細胞接着基材5Bを得た(HIGH FIVEの
接着量:約4万cells/cm2)。
Comparative Example 4 (Adhesion 5B of HIGH FIVE to Substrate) HIGH FIVE (virus-uninfected cells) was used instead of the Sf9 cells of Comparative Example 2, and Express-Five was used instead of the Sf-900II medium. A cell adhesion substrate 5B of the present invention was obtained in the same manner as in Comparative Example 2 except that a medium (manufactured by Invitrogen Corporation) was used (adhesion amount of HIGH FIVE: about 40,000 cells / cm 2 ).

【0072】<評価1A>実施例1〜3及び比較例1〜
2のSf9接着基材について、Sf9の接着性を評価す
るために、これらの基材中の培地をアスピレーターで吸
引除去し、基材の1穴当たりに0.5mLのPBS及び
0.1mLのテトラカラーワン(商品名:生化学工業株
式会社製テトラゾリウム塩)を投入し25℃で4時間静
置させた。なお、テトラカラーワンのテトラゾリウム塩
が細胞内ミトコンドリアのデヒドロゲナーゼにより還元
されて、ホルマザンを生成することにより発色する。そ
の後、分光光度計を用いて450nm(対照波長630
nm)の吸光度を測定し、この値を接着活性とした。こ
れらの結果を表1に示す。
<Evaluation 1A> Examples 1 to 3 and Comparative Examples 1 to 1
In order to evaluate the adhesiveness of Sf9 with respect to the Sf9 adhesive substrate of No. 2, the medium in these substrates was removed by suction with an aspirator, and 0.5 mL of PBS and 0.1 mL of tetra per well of the substrate were used. Color One (trade name: tetrazolium salt manufactured by Seikagaku Corporation) was added and the mixture was allowed to stand at 25 ° C. for 4 hours. The tetrazolium salt of tetracolorone is reduced by intracellular mitochondrial dehydrogenase to form formazan, which causes color development. Then, using a spectrophotometer, 450 nm (control wavelength 630
nm) absorbance was measured and this value was taken as the adhesive activity. The results are shown in Table 1.

【0073】[0073]

【表1】 [Table 1]

【0074】<評価1B>実施例4〜6及び比較例3〜
4のHIGH FIVE接着基材について、HIGH
FIVEの接着性を評価するために、これらの基材中の
培地をアスピレーターで吸引除去し、基材の1穴当たり
に0.5mLのPBS及び0.1mLのテトラカラーワ
ンを投入し25℃で4時間静置させた。その後、分光光
度計を用いて450nm(対照波長630nm)の吸光
度を測定し、この値を接着活性とした。これらの結果を
表2に示す。
<Evaluation 1B> Examples 4 to 6 and Comparative Examples 3 to
For the HIGH FIVE adhesive base material of No. 4, HIGH
In order to evaluate the adhesiveness of FIVE, the medium in these substrates was removed by suction with an aspirator, 0.5 mL of PBS and 0.1 mL of Tetracolor One were added to each hole of the substrate, and at 25 ° C. Let stand for 4 hours. Then, the absorbance at 450 nm (control wavelength 630 nm) was measured using a spectrophotometer, and this value was taken as the adhesive activity. The results are shown in Table 2.

【0075】[0075]

【表2】 表1及び2の結果から、本発明の細胞接着基材は、比較
例に比べて接着活性が極めて高いことが判る。
[Table 2] From the results of Tables 1 and 2, it can be seen that the cell adhesion substrate of the present invention has extremely high adhesive activity as compared with the comparative example.

【0076】<評価2A>実施例1〜3及び比較例1〜
2のSf9接着基材の細胞増殖活性を評価するために、
Sf9接着基材の1穴当たりに1mLのSf−900I
I培地(インビトロジェン株式会社製)を投入し28℃
で3日間静置させてSf9を増殖させた後、培地をアス
ピレーターで吸引除去し、基材の1穴当たりに0.5m
LのPBS及び0.1mLのテトラカラーワン(生化学
工業株式会社製)を投入し25℃で4時間静置させた。
その後、分光光度計を用いて450nm(対照波長63
0nm)の吸光度を測定し、この値を増殖活性とした。
これらの結果を表3に示す。
<Evaluation 2A> Examples 1 to 3 and Comparative Examples 1 to 1
In order to evaluate the cell proliferating activity of the Sf9 adhesive substrate of No. 2,
1 mL of Sf-900I per hole of Sf9 adhesive substrate
I medium (manufactured by Invitrogen Co., Ltd.) is added and 28 ° C.
After allowing it to stand for 3 days to grow Sf9, the medium is suctioned off with an aspirator and 0.5 m per hole of the substrate.
L PBS and 0.1 mL of Tetra Color One (manufactured by Seikagaku Corporation) were added and the mixture was allowed to stand at 25 ° C. for 4 hours.
Then use a spectrophotometer to measure 450 nm (control wavelength 63
The absorbance at 0 nm) was measured, and this value was defined as the growth activity.
The results are shown in Table 3.

【0077】[0077]

【表3】 [Table 3]

【0078】<評価2B>実施例4〜6及び比較例3〜
4のHIGH FIVE接着基材の細胞増殖活性を評価
するために、HIGH FIVE接着基材の1穴当たり
に1mLのExpress−Five培地(インビトロ
ジェン株式会社製)を投入し28℃で3日間静置させて
HIGH FIVEを増殖させた後、培地をアスピレー
ターで吸引除去し、基材の1穴当たりに0.5mLのP
BS及び0.1mLのテトラカラーワンを投入し25℃
で4時間静置させた。その後、分光光度計を用いて45
0nm(対照波長630nm)の吸光度を測定し、この
値を増殖活性とした。これらの結果を表4に示す。
<Evaluation 2B> Examples 4 to 6 and Comparative Examples 3 to
In order to evaluate the cell proliferation activity of the HIGH FIVE adhesive base material of No. 4, 1 mL of Express-Five medium (manufactured by Invitrogen Corporation) was added to each hole of the HIGH FIVE adhesive base material and allowed to stand at 28 ° C. for 3 days. After growing HIGH FIVE by aspiration, the medium was removed by suction with an aspirator, and 0.5 mL of P was added per well of the substrate.
Add BS and 0.1 mL of Tetra Color One, 25 ℃
And allowed to stand for 4 hours. Then, using a spectrophotometer,
The absorbance at 0 nm (control wavelength 630 nm) was measured, and this value was defined as the growth activity. The results are shown in Table 4.

【0079】[0079]

【表4】 表3及び4の結果から、本発明の細胞接着基材は、比較
例に比べて細胞増殖性が極めて高いことが判る。
[Table 4] From the results of Tables 3 and 4, it can be seen that the cell-adhesive substrate of the present invention has extremely high cell proliferation property as compared with the comparative example.

【0080】[0080]

【発明の効果】本発明の細胞接着基材は、変温動物由来
細胞(A)を極めて効率良く基材に接着でき、さらに変
温動物由来細胞(A)を効率良く増殖できる。すなわ
ち、本発明の細胞接着基材は、天然由来のコラーゲンや
絹蛋白質等を使用しなくても、細胞接着性が極めて高
い。従って、本発明の細胞接着基材は、プリオンやヒト
感染性のウイルス等の感染物質が含有される危険性があ
る天然由来の蛋白質等を含まないため、極めて安全性が
高い。また、極めて効率よく変温動物由来細胞を増殖さ
せることができる。
INDUSTRIAL APPLICABILITY The cell-adhesive substrate of the present invention is capable of adhering cells (A) derived from a temperature-altered animal to the substrate very efficiently and further efficiently growing cells (A) derived from a temperature-altered animal. That is, the cell-adhesive substrate of the present invention has extremely high cell-adhesiveness without using naturally-derived collagen, silk protein, or the like. Therefore, the cell-adhesive substrate of the present invention does not contain naturally-occurring proteins or the like that may contain infectious substances such as prions and human infectious viruses, and is therefore extremely safe. In addition, cells derived from a thermophilic animal can be proliferated extremely efficiently.

【0081】[0081]

【配列表】 <110>三洋化成工業株式会社;SANYO CHEMICAL INDUSTRIES,LTD. <120>変温動物由来細胞の細胞接着基材 <130>P5796 <150>特願2001-351232 <151>2001-11-16 <160>45 <210>1 <211>4 <212>PRT <213>Homo sapiens <400>1 Arg Glu Asp Val 1 <210>2 <211>5 <212>PRT <213>Homo sapiens <400>2 Tyr Ile Gly Ser Arg 1 5 <210>3 <211>5 <212>PRT <213>Homo sapiens <400>3 Pro Asp Ser Gly Arg 1 5 <210>4 <211>7 <212>PRT <213>Homo sapiens <400>4 Arg Tyr Val Val Leu Pro Arg 1 5 <210>5 <211>6 <212>PRT <213>Homo sapiens <400>5 Leu Gly Thr Ile Pro Gly 1 5 <210>6 <211>10 <212>PRT <213>Homo sapiens <400>6 Arg Asn Ile Ala Glu Ile Ile Lys Asp Ile 1 5 10 <210>7 <211>5 <212>PRT <213>Homo sapiens <400>7 Ile Lys Val Ala Val 1 5 <210>8 <211>4 <212>PRT <213>Homo sapiens <400>8 Asp Gly Glu Ala 1 <210>9 <211>15 <212>PRT <213>Homo sapiens <400>9 Gly Val Lys Gly Asp Lys Gly Asn Pro Gly Trp Pro Gly Ala Pro 1 5 10 15 <210>10 <211>13 <212>PRT <213>Homo sapiens <400>10 Gly Glu Phe Tyr Phe Asp Leu Arg Leu Lys Gly Asp Lys 1 5 10 <210>11 <211>8 <212>PRT <213>Homo sapiens <400>11 Tyr Lys Leu Asn Val Asn Asp Ser 1 5 <210>12 <211>6 <212>PRT <213>Bombyx mori <400>12 Gly Ala Gly Ala Gly Ser 1 5 <210>13 <211>5 <212>PRT <213>Homo sapiens <400>13 Gly Val Gly Val Pro 1 5 <210>14 <211>4 <212>PRT <213>Homo sapiens <400>14 Arg Gly Asp Ser 1 <210>15 <211>5 <212>PRT <213>Homo sapiens <400>15 Gly Arg Gly Asp Ser 1 5 <210>16 <211>6 <212>PRT <213>Homo sapiens <400>16 Gly Arg Gly Asp Ser Pro 1 5 <210>17 <211>10 <212>PRT <213>Homo sapiens <400>17 Arg Gly Asp Ser Pro Ala Ser Ser Lys Pro 1 5 10 <210>18 <211>12 <212>PRT <213>Artificial Sequence <400>18 Ala Val Thr Gly Arg Gly Asp Ser Pro Ala Ser Ala 1 5 10 <210>19 <211>14 <212>PRT <213>Artificial Sequence <400>19 Pro Gly Ala Ser Ile Lys Val Ala Val Ser Ala Gly Pro Ser 1 5 10 <210>20 <211>19 <212>PRT <213>Artificial Sequence <400>20 Cys Ser Arg Ala Arg Lys Gln Ala Ala Ser Ile Lys Val Ala Val Ser 1 5 10 15 Ala Asp Arg <210>21 <211>12 <212>PRT <213>Artificial Sequence <400>21 Val Cys Glu Pro Gly Tyr Ile Gly Ser Arg Cys Asp 1 5 10 <210>22 <211>16 <212>PRT <213>Artificial Sequence <400>22 Arg Gly Asp Ser Arg Gly Asp Ser Arg Gly Asp Ser Arg Gly Asp Ser 1 5 10 15 <210>23 <211>32 <212>PRT <213>Artificial Sequence <400>23 Arg Gly Asp Ser Arg Gly Asp Ser Arg Gly Asp Ser Arg Gly Asp Ser 1 5 10 15 Arg Gly Asp Ser Arg Gly Asp Ser Arg Gly Asp Ser Arg Gly Asp Ser 20 25 30 <210>24 <211>64 <212>PRT <213>Artificial Sequence <400>24 Arg Gly Asp Ser Arg Gly Asp Ser Arg Gly Asp Ser Arg Gly Asp Ser 1 5 10 15 Arg Gly Asp Ser Arg Gly Asp Ser Arg Gly Asp Ser Arg Gly Asp Ser 20 25 30 Arg Gly Asp Ser Arg Gly Asp Ser Arg Gly Asp Ser Arg Gly Asp Ser 35 40 45 Arg Gly Asp Ser Arg Gly Asp Ser Arg Gly Asp Ser Arg Gly Asp Ser 50 55 60 <210>25 <211>40 <212>PRT <213>Artificial Sequence <400>25 Gly Arg Gly Asp Ser Gly Arg Gly Asp Ser Gly Arg Gly Asp Ser Gly 1 5 10 15 Arg Gly Asp Ser Gly Arg Gly Asp Ser Gly Arg Gly Asp Ser Gly Arg 20 25 30 Gly Asp Ser Gly Arg Gly Asp Ser 35 40 <210>26 <211>48 <212>PRT <213>Artificial Sequence <400>26 Gly Arg Gly Asp Ser Pro Gly Arg Gly Asp Ser Pro Gly Arg Gly Asp 1 5 10 15 Ser Pro Gly Arg Gly Asp Ser Pro Gly Arg Gly Asp Ser Pro Gly Arg 20 25 30 Gly Asp Ser Pro Gly Arg Gly Asp Ser Pro Gly Arg Gly Asp Ser Pro 35 40 45 <210>27 <211>40 <212>PRT <213>Artificial Sequence <400>27 Arg Gly Asp Ser Pro Ala Ser Ser Lys Pro Arg Gly Asp Ser Pro Ala 1 5 10 15 Ser Ser Lys Pro Arg Gly Asp Ser Pro Ala Ser Ser Lys Pro Arg Gly 20 25 30 Asp Ser Pro Ala Ser Ser Lys Pro 35 40 <210>28 <211>48 <212>PRT <213>Artificial Sequence <400>28 Ala Val Thr Gly Arg Gly Asp Ser Pro Ala Ser Ala Ala Val Thr Gly 1 5 10 15 Arg Gly Asp Ser Pro Ala Ser Ala Ala Val Thr Gly Arg Gly Asp Ser 20 25 30 Pro Ala Ser Ala Ala Val Thr Gly Arg Gly Asp Ser Pro Ala Ser Ala 35 40 45 <210>29 <211>56 <212>PRT <213>Artificial Sequence <400>29 Pro Gly Ala Ser Ile Lys Val Ala Val Ser Ala Gly Pro Ser Pro Gly 1 5 10 15 Ala Ser Ile Lys Val Ala Val Ser Ala Gly Pro Ser Pro Gly Ala Ser 20 25 30 Ile Lys Val Ala Val Ser Ala Gly Pro Ser Pro Gly Ala Ser Ile Lys 35 40 45 Val Ala Val Ser Ala Gly Pro Ser 50 55 <210>30 <211>76 <212>PRT <213>Artificial Sequence <400>30 Cys Ser Arg Ala Arg Lys Gln Ala Ala Ser Ile Lys Val Ala Val Ser 1 5 10 15 Ala Asp Arg Cys Ser Arg Ala Arg Lys Gln Ala Ala Ser Ile Lys Val 20 25 30 Ala Val Ser Ala Asp Arg Cys Ser Arg Ala Arg Lys Gln Ala Ala Ser 35 40 45 Ile Lys Val Ala Val Ser Ala Asp Arg Cys Ser Arg Ala Arg Lys Gln 50 55 60 Ala Ala Ser Ile Lys Val Ala Val Ser Ala Asp Arg 65 70 75 <210>31 <211>48 <212>PRT <213>Artificial Sequence <400>31 Val Cys Glu Pro Gly Tyr Ile Gly Ser Arg Cys Asp Val Cys Glu Pro 1 5 10 15 Gly Tyr Ile Gly Ser Arg Cys Asp Val Cys Glu Pro Gly Tyr Ile Gly 20 25 30 Ser Arg Cys Asp Val Cys Glu Pro Gly Tyr Ile Gly Ser Arg Cys Asp 35 40 45 <210>32 <211>9 <212>PRT <213>Artificial Sequence <400>32 Gly Ala Gln Gly Pro Ala Gly Pro Gly 1 5 <210>33 <211>15 <212>PRT <213>Artificial Sequence <400>33 Gly Ala Pro Gly Ala Pro Gly Ser Gln Gly Ala Pro Gly Leu Gln 1 5 10 15 <210>34 <211>15 <212>PRT <213>Artificial Sequence <400>34 Gly Ala Pro Gly Thr Pro Gly Pro Gln Gly Leu Pro Gly Ser Pro 1 5 10 15 <210>35 <211>54 <212>PRT <213>Artificial Sequence <400>35 Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala 1 5 10 15 Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala 20 25 30 Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser 35 40 45 Gly Ala Gly Ala Gly Ser 50 <210>36 <211>5 <212>PRT <213>Artificial Sequence <400>36 Gly Ala Ala Gly Tyr 1 5 <210>37 <211>40 <212>PRT <213>Artificial Sequence <400>37 Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 1 5 10 15 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 20 25 30 Gly Val Pro Gly Val Gly Val Pro 35 40 <210>38 <211>48 <212>PRT <213>Artificial Sequence <400>38 Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala 1 5 10 15 Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala 20 25 30 Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser 35 40 45 <210>39 <211>60 <212>PRT <213>Artificial Sequence <400>39 Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 1 5 10 15 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 20 25 30 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 35 40 45 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 50 55 60 <210>40 <211>80 <212>PRT <213>Artificial Sequence <400>40 Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 1 5 10 15 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 20 25 30 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 35 40 45 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 50 55 60 Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 65 70 75 80 <210>41 <211>36 <212>PRT <213>Artificial Sequence <400>41 Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala 1 5 10 15 Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala 20 25 30 Gly Ala Gly Ser 35 <210>42 <211>24 <212>PRT <213>Artificial Sequence <400>42 Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala 1 5 10 15 Gly Ser Gly Ala Gly Ala Gly Ser 20 <210>43 <211>15 <212>PRT <213>Artificial Sequence <400>43 Gly Ala Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro 1 5 10 15 <210>44 <211>72 <212>PRT <213>Artificial Sequence <400>44 Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala 1 5 10 15 Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala 20 25 30 Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser 35 40 45 Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala 50 55 60 Gly Ser Gly Ala Gly Ala Gly Ser 65 70 <210>45 <211>30 <212>PRT <213>Artificial Sequence <400>45 Gly Ala Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly 1 5 10 15 Ala Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro 20 25 30 [Sequence list] <110> SANYO CHEMICAL INDUSTRIES, LTD. <120> Cell-adhesive base material for cells derived from temperature-altering animals <130> P5796 <150> Japanese Patent Application 2001-351232 <151> 2001-11-16 <160> 45 <210> 1 <211> 4 <212> PRT <213> Homo sapiens <400> 1 Arg Glu Asp Val   1 <210> 2 <211> 5 <212> PRT <213> Homo sapiens <400> 2 Tyr Ile Gly Ser Arg   1 5 <210> 3 <211> 5 <212> PRT <213> Homo sapiens <400> 3 Pro Asp Ser Gly Arg   1 5 <210> 4 <211> 7 <212> PRT <213> Homo sapiens <400> 4 Arg Tyr Val Val Leu Pro Arg   1 5 <210> 5 <211> 6 <212> PRT <213> Homo sapiens <400> 5 Leu Gly Thr Ile Pro Gly   1 5 <210> 6 <211> 10 <212> PRT <213> Homo sapiens <400> 6 Arg Asn Ile Ala Glu Ile Ile Lys Asp Ile   1 5 10 <210> 7 <211> 5 <212> PRT <213> Homo sapiens <400> 7 Ile Lys Val Ala Val   1 5 <210> 8 <211> 4 <212> PRT <213> Homo sapiens <400> 8 Asp Gly Glu Ala   1 <210> 9 <211> 15 <212> PRT <213> Homo sapiens <400> 9 Gly Val Lys Gly Asp Lys Gly Asn Pro Gly Trp Pro Gly Ala Pro   1 5 10 15 <210> 10 <211> 13 <212> PRT <213> Homo sapiens <400> 10 Gly Glu Phe Tyr Phe Asp Leu Arg Leu Lys Gly Asp Lys   1 5 10 <210> 11 <211> 8 <212> PRT <213> Homo sapiens <400> 11 Tyr Lys Leu Asn Val Asn Asp Ser   1 5 <210> 12 <211> 6 <212> PRT <213> Bombyx mori <400> 12 Gly Ala Gly Ala Gly Ser   1 5 <210> 13 <211> 5 <212> PRT <213> Homo sapiens <400> 13 Gly Val Gly Val Pro   1 5 <210> 14 <211> 4 <212> PRT <213> Homo sapiens <400> 14 Arg Gly Asp Ser   1 <210> 15 <211> 5 <212> PRT <213> Homo sapiens <400> 15 Gly Arg Gly Asp Ser   1 5 <210> 16 <211> 6 <212> PRT <213> Homo sapiens <400> 16 Gly Arg Gly Asp Ser Pro   1 5 <210> 17 <211> 10 <212> PRT <213> Homo sapiens <400> 17 Arg Gly Asp Ser Pro Ala Ser Ser Lys Pro   1 5 10 <210> 18 <211> 12 <212> PRT <213> Artificial Sequence <400> 18 Ala Val Thr Gly Arg Gly Asp Ser Pro Ala Ser Ala   1 5 10 <210> 19 <211> 14 <212> PRT <213> Artificial Sequence <400> 19 Pro Gly Ala Ser Ile Lys Val Ala Val Ser Ala Gly Pro Ser   1 5 10 <210> 20 <211> 19 <212> PRT <213> Artificial Sequence <400> 20 Cys Ser Arg Ala Arg Lys Gln Ala Ala Ser Ile Lys Val Ala Val Ser   1 5 10 15 Ala Asp Arg <210> 21 <211> 12 <212> PRT <213> Artificial Sequence <400> 21 Val Cys Glu Pro Gly Tyr Ile Gly Ser Arg Cys Asp   1 5 10 <210> 22 <211> 16 <212> PRT <213> Artificial Sequence <400> 22 Arg Gly Asp Ser Arg Gly Asp Ser Arg Gly Asp Ser Arg Gly Asp Ser   1 5 10 15 <210> 23 <211> 32 <212> PRT <213> Artificial Sequence <400> 23 Arg Gly Asp Ser Arg Gly Asp Ser Arg Gly Asp Ser Arg Gly Asp Ser   1 5 10 15 Arg Gly Asp Ser Arg Gly Asp Ser Arg Gly Asp Ser Arg Gly Asp Ser              20 25 30 <210> 24 <211> 64 <212> PRT <213> Artificial Sequence <400> 24 Arg Gly Asp Ser Arg Gly Asp Ser Arg Gly Asp Ser Arg Gly Asp Ser   1 5 10 15 Arg Gly Asp Ser Arg Gly Asp Ser Arg Gly Asp Ser Arg Gly Asp Ser              20 25 30 Arg Gly Asp Ser Arg Gly Asp Ser Arg Gly Asp Ser Arg Gly Asp Ser          35 40 45 Arg Gly Asp Ser Arg Gly Asp Ser Arg Gly Asp Ser Arg Gly Asp Ser      50 55 60 <210> 25 <211> 40 <212> PRT <213> Artificial Sequence <400> 25 Gly Arg Gly Asp Ser Gly Arg Gly Asp Ser Gly Arg Gly Asp Ser Gly   1 5 10 15 Arg Gly Asp Ser Gly Arg Gly Asp Ser Gly Arg Gly Asp Ser Gly Arg              20 25 30 Gly Asp Ser Gly Arg Gly Asp Ser          35 40 <210> 26 <211> 48 <212> PRT <213> Artificial Sequence <400> 26 Gly Arg Gly Asp Ser Pro Gly Arg Gly Asp Ser Pro Gly Arg Gly Asp   1 5 10 15 Ser Pro Gly Arg Gly Asp Ser Pro Gly Arg Gly Asp Ser Pro Gly Arg              20 25 30 Gly Asp Ser Pro Gly Arg Gly Asp Ser Pro Gly Arg Gly Asp Ser Pro          35 40 45 <210> 27 <211> 40 <212> PRT <213> Artificial Sequence <400> 27 Arg Gly Asp Ser Pro Ala Ser Ser Lys Pro Arg Gly Asp Ser Pro Ala   1 5 10 15 Ser Ser Lys Pro Arg Gly Asp Ser Pro Ala Ser Ser Lys Pro Arg Gly              20 25 30 Asp Ser Pro Ala Ser Ser Lys Pro          35 40 <210> 28 <211> 48 <212> PRT <213> Artificial Sequence <400> 28 Ala Val Thr Gly Arg Gly Asp Ser Pro Ala Ser Ala Ala Val Thr Gly   1 5 10 15 Arg Gly Asp Ser Pro Ala Ser Ala Ala Val Thr Gly Arg Gly Asp Ser              20 25 30 Pro Ala Ser Ala Ala Val Thr Gly Arg Gly Asp Ser Pro Ala Ser Ala          35 40 45 <210> 29 <211> 56 <212> PRT <213> Artificial Sequence <400> 29 Pro Gly Ala Ser Ile Lys Val Ala Val Ser Ala Gly Pro Ser Pro Gly   1 5 10 15 Ala Ser Ile Lys Val Ala Val Ser Ala Gly Pro Ser Pro Gly Ala Ser              20 25 30 Ile Lys Val Ala Val Ser Ala Gly Pro Ser Pro Gly Ala Ser Ile Lys          35 40 45 Val Ala Val Ser Ala Gly Pro Ser      50 55 <210> 30 <211> 76 <212> PRT <213> Artificial Sequence <400> 30 Cys Ser Arg Ala Arg Lys Gln Ala Ala Ser Ile Lys Val Ala Val Ser   1 5 10 15 Ala Asp Arg Cys Ser Arg Ala Arg Lys Gln Ala Ala Ser Ile Lys Val              20 25 30 Ala Val Ser Ala Asp Arg Cys Ser Arg Ala Arg Lys Gln Ala Ala Ser          35 40 45 Ile Lys Val Ala Val Ser Ala Asp Arg Cys Ser Arg Ala Arg Lys Gln      50 55 60 Ala Ala Ser Ile Lys Val Ala Val Ser Ala Asp Arg  65 70 75 <210> 31 <211> 48 <212> PRT <213> Artificial Sequence <400> 31 Val Cys Glu Pro Gly Tyr Ile Gly Ser Arg Cys Asp Val Cys Glu Pro   1 5 10 15 Gly Tyr Ile Gly Ser Arg Cys Asp Val Cys Glu Pro Gly Tyr Ile Gly              20 25 30 Ser Arg Cys Asp Val Cys Glu Pro Gly Tyr Ile Gly Ser Arg Cys Asp          35 40 45 <210> 32 <211> 9 <212> PRT <213> Artificial Sequence <400> 32 Gly Ala Gln Gly Pro Ala Gly Pro Gly   1 5 <210> 33 <211> 15 <212> PRT <213> Artificial Sequence <400> 33 Gly Ala Pro Gly Ala Pro Gly Ser Gln Gly Ala Pro Gly Leu Gln   1 5 10 15 <210> 34 <211> 15 <212> PRT <213> Artificial Sequence <400> 34 Gly Ala Pro Gly Thr Pro Gly Pro Gln Gly Leu Pro Gly Ser Pro   1 5 10 15 <210> 35 <211> 54 <212> PRT <213> Artificial Sequence <400> 35 Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala   1 5 10 15 Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala              20 25 30 Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser          35 40 45 Gly Ala Gly Ala Gly Ser      50 <210> 36 <211> 5 <212> PRT <213> Artificial Sequence <400> 36 Gly Ala Ala Gly Tyr   1 5 <210> 37 <211> 40 <212> PRT <213> Artificial Sequence <400> 37 Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly   1 5 10 15 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val              20 25 30 Gly Val Pro Gly Val Gly Val Pro          35 40 <210> 38 <211> 48 <212> PRT <213> Artificial Sequence <400> 38 Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala   1 5 10 15 Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala              20 25 30 Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser          35 40 45 <210> 39 <211> 60 <212> PRT <213> Artificial Sequence <400> 39 Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly   1 5 10 15 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val              20 25 30 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly          35 40 45 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro      50 55 60 <210> 40 <211> 80 <212> PRT <213> Artificial Sequence <400> 40 Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly   1 5 10 15 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val              20 25 30 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly          35 40 45 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val      50 55 60 Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro  65 70 75 80 <210> 41 <211> 36 <212> PRT <213> Artificial Sequence <400> 41 Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala   1 5 10 15 Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala              20 25 30 Gly Ala Gly Ser          35 <210> 42 <211> 24 <212> PRT <213> Artificial Sequence <400> 42 Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala   1 5 10 15 Gly Ser Gly Ala Gly Ala Gly Ser              20 <210> 43 <211> 15 <212> PRT <213> Artificial Sequence <400> 43 Gly Ala Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro   1 5 10 15 <210> 44 <211> 72 <212> PRT <213> Artificial Sequence <400> 44 Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala   1 5 10 15 Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala              20 25 30 Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser          35 40 45 Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala      50 55 60 Gly Ser Gly Ala Gly Ala Gly Ser  65 70 <210> 45 <211> 30 <212> PRT <213> Artificial Sequence <400> 45 Gly Ala Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly   1 5 10 15 Ala Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro              20 25 30

───────────────────────────────────────────────────── フロントページの続き Fターム(参考) 4B029 AA08 AA21 BB11 CC02 CC08 GA03 GB04 GB05 4B033 NA01 NA02 NA16 NB01 NB12 NB13 NB57 NC04 ND02 NE02 NF06 4B064 AG01 CA10 CA19 CC24 DA20 4B065 AA90X AB01 AC14 BA01 CA24    ─────────────────────────────────────────────────── ─── Continued front page    F-term (reference) 4B029 AA08 AA21 BB11 CC02 CC08                       GA03 GB04 GB05                 4B033 NA01 NA02 NA16 NB01 NB12                       NB13 NB57 NC04 ND02 NE02                       NF06                 4B064 AG01 CA10 CA19 CC24 DA20                 4B065 AA90X AB01 AC14 BA01                       CA24

Claims (7)

【特許請求の範囲】[Claims] 【請求項1】細胞接着性人工ペプチド(X)及び/又は
細胞接着補助人工ペプチド(Y)を用いて、変温動物由
来細胞(A)及び基材(B)を接着させてなることを特
徴とする細胞接着基材。
1. A cell-adhesive artificial peptide (X) and / or a cell adhesion-assisting artificial peptide (Y) are used to adhere cells (A) and a substrate (B) derived from a thermophilic animal. A cell adhesion substrate.
【請求項2】 細胞接着性人工ペプチド(X)を必須構
成成分として用いてなり、(X)の細胞接着シグナルを
現わす最小アミノ酸配列(sh)が、アミノ酸3文字表
記で表わされる、Arg Gly Asp配列、Leu Asp Val配列、
Arg Glu AspVal配列(1)、Tyr Ile Gly Ser Arg配列
(2)、Pro Asp Ser Gly Arg配列(3)、Arg Tyr Val
Val Leu Pro Arg配列(4)、Leu Gly Thr Ile Pro Gl
y配列(5)、Arg Asn Ile Ala Glu Ile Ile Lys Asp I
le配列(6)、Ile Lys Val Ala Val配列(7)、Leu A
rg Glu配列、Asp Gly Glu Ala 配列(8)、Gly Val Ly
s Gly Asp Lys Gly Asn Pro Gly Trp Pro Gly Ala Pro
配列(9)、Gly Glu Phe Tyr Phe Asp Leu Arg Leu Ly
s Gly Asp Lys(10)、His Ala Val配列及びTyr Lys
Leu Asn Val Asn Asp Ser配列(11)からなる群より
選ばれる少なくとも1種の配列である請求項1記載の細
胞接着基材。
2. Arg Gly, which comprises a cell-adhesive artificial peptide (X) as an essential component, and the minimum amino acid sequence (sh) that expresses the cell-adhesion signal of (X) is represented by the three-letter amino acid code. Asp sequence, Leu Asp Val sequence,
Arg Glu AspVal 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 Gl
y sequence (5), Arg Asn Ile Ala Glu Ile Ile Lys Asp I
le sequence (6), Ile Lys Val Ala Val sequence (7), Leu A
rg Glu sequence, Asp Gly Glu Ala sequence (8), Gly Val Ly
s Gly Asp Lys Gly Asn Pro Gly Trp Pro Gly Ala Pro
Sequence (9), Gly Glu Phe Tyr Phe Asp Leu Arg Leu Ly
s Gly Asp Lys (10), His Ala Val sequence and Tyr Lys
The cell adhesion substrate according to claim 1, which is at least one kind of sequence selected from the group consisting of Leu Asn Val Asn Asp Ser sequence (11).
【請求項3】 細胞接着補助人工ペプチド(Y)を必須
構成成分として用いてなり、(Y)のアミノ酸配列が、
アミノ酸3文字表記で表わされる、Gly AlaGly Ala Gly
Ser(12)及び/又はGly Val Gly Val Pro(13)
である請求項1又は2記載の細胞接着基材。
3. An artificial peptide (Y) for assisting cell adhesion is used as an essential component, wherein the amino acid sequence of (Y) is
Gly AlaGly Ala Gly represented by the three letter amino acid code
Ser (12) and / or Gly Val Gly Val Pro (13)
The cell adhesion substrate according to claim 1 or 2.
【請求項4】 変温動物由来細胞(A)が昆虫細胞であ
る請求項1〜3のいずれかに記載の細胞接着基材。
4. The cell-adhesive substrate according to claim 1, wherein the cell (A) derived from a thermophilic animal is an insect cell.
【請求項5】 細胞接着性人工ペプチド(X)及び/又
は細胞接着補助人工ペプチド(Y)を用いて、変温動物
由来細胞(A)と基材(B)とを接着させることを特徴
とする細胞接着基材の生産方法。
5. A cell-adhesive artificial peptide (X) and / or a cell adhesion-assisting artificial peptide (Y) are used to adhere cells (A) derived from a thermophilic animal to a substrate (B). A method for producing a cell adhesion substrate.
【請求項6】 請求項4に記載の細胞接着基材を用いて
細胞培養することを特徴とするウイルス感染昆虫細胞の
生産方法。
6. A method for producing virus-infected insect cells, which comprises culturing cells using the cell adhesion substrate according to claim 4.
【請求項7】 請求項6記載の生産方法で生産されるウ
イルス感染昆虫細胞を用いることを特徴とする有用蛋白
質の生産方法。
7. A method for producing a useful protein, which comprises using a virus-infected insect cell produced by the production method according to claim 6.
JP2002328721A 2001-11-16 2002-11-12 Cell-adhered substrate for poikilothermic animal- originated cell Pending JP2003210166A (en)

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JP2001-351232 2001-11-16
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005002106A (en) * 2003-05-21 2005-01-06 Sanyo Chem Ind Ltd Intercellular adhesive polypeptide
JP2005170810A (en) * 2003-12-09 2005-06-30 Sanyo Chem Ind Ltd Intercellular adhesive polypeptide
US7989160B2 (en) 2006-02-13 2011-08-02 Alethia Biotherapeutics Inc. Polynucleotides and polypeptide sequences involved in the process of bone remodeling
US8168181B2 (en) 2006-02-13 2012-05-01 Alethia Biotherapeutics, Inc. Methods of impairing osteoclast differentiation using antibodies that bind siglec-15
US8221969B2 (en) 2004-04-19 2012-07-17 Sanyo Chemical Industries, Ltd. Method of producing virus
JP5161581B2 (en) * 2005-10-28 2013-03-13 株式会社クラレ Cell culture container and cell culture method
JP2013135740A (en) * 2011-12-28 2013-07-11 Kyocera Medical Corp Medical material
US9493562B2 (en) 2012-07-19 2016-11-15 Alethia Biotherapeutics Inc. Anti-Siglec-15 antibodies

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JP4510512B2 (en) * 2003-05-21 2010-07-28 三洋化成工業株式会社 Cell adhesion polypeptide
JP2005002106A (en) * 2003-05-21 2005-01-06 Sanyo Chem Ind Ltd Intercellular adhesive polypeptide
JP2005170810A (en) * 2003-12-09 2005-06-30 Sanyo Chem Ind Ltd Intercellular adhesive polypeptide
JP4493327B2 (en) * 2003-12-09 2010-06-30 三洋化成工業株式会社 Cell adhesion polypeptide
US8221969B2 (en) 2004-04-19 2012-07-17 Sanyo Chemical Industries, Ltd. Method of producing virus
JP5161581B2 (en) * 2005-10-28 2013-03-13 株式会社クラレ Cell culture container and cell culture method
US8540988B2 (en) 2006-02-13 2013-09-24 Alethia Biotherapeutics Inc. Antibodies that bind polypeptides involved in the process of bone remodeling
US9040246B2 (en) 2006-02-13 2015-05-26 Alethia Biotherapeutics Inc. Methods of making antibodies that bind polypeptides involved in the process of bone remodeling
US8431126B2 (en) 2006-02-13 2013-04-30 Alethia Biotherapeutics Inc. Antibodies that bind polypeptides involved in the process of bone remodeling
US9695419B2 (en) 2006-02-13 2017-07-04 Daiichi Sankyo Company, Limited Polynucleotides and polypeptide sequences involved in the process of bone remodeling
US7989160B2 (en) 2006-02-13 2011-08-02 Alethia Biotherapeutics Inc. Polynucleotides and polypeptide sequences involved in the process of bone remodeling
US9067984B2 (en) 2006-02-13 2015-06-30 Alethia Biotherapeutics Inc. Methods of impairing osteoclast differentiation using antibodies that bind Siglec-15
US8168181B2 (en) 2006-02-13 2012-05-01 Alethia Biotherapeutics, Inc. Methods of impairing osteoclast differentiation using antibodies that bind siglec-15
US8900579B2 (en) 2009-10-06 2014-12-02 Alethia Biotherapuetics Inc. Siglec-15 antibodies in treating bone loss-related disease
US8741289B2 (en) 2009-10-06 2014-06-03 Alethia Biotherapeutics Inc. Siglec 15 antibodies in treating bone loss-related disease
US9388242B2 (en) 2009-10-06 2016-07-12 Alethia Biotherapeutics Inc. Nucleic acids encoding anti-Siglec-15 antibodies
US9617337B2 (en) 2009-10-06 2017-04-11 Daiichi Sankyo Company, Limited Siglec-15 antibodies in treating bone loss-related disease
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US9493562B2 (en) 2012-07-19 2016-11-15 Alethia Biotherapeutics Inc. Anti-Siglec-15 antibodies

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