JP4361734B2 - 血管形成の調節に有用なトリプトファニル−tRNAシンテターゼ由来ポリペプチド - Google Patents
血管形成の調節に有用なトリプトファニル−tRNAシンテターゼ由来ポリペプチド Download PDFInfo
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Description
「短縮tRNAシンテターゼポリペプチド」とは対応する全長tRNAシンテターゼよりも短いポリペプチドを意味する。
図1に配列番号1のアミノ酸残基94〜471(例えば配列番号12)として示すポリペプチド及び配列番号7のアミノ酸配列をもつポリペプチド又は配列番号6のcDNAによりコードされるポリペプチドは本発明を構成する。本発明は上記ポリペプチドの変異体とポリヌクレオチドの変異体にも関する。ポリヌクレオチド変異体はポリヌクレオチドの天然対立遺伝子変異体でもポリヌクレオチドの非天然変異体でもよい。従って、本発明は配列番号7、配列番号12に示すと同一のポリペプチド及び配列番号6のcDNAによりコードされるポリペプチドをコードするポリヌクレオチドと、配列番号7及び配列番号12のポリペプチドの血管形成阻害フラグメント、誘導体又は類似体をコードする前記ポリヌクレオチドの変異体に関する。このようなヌクレオチド変異体としては欠失変異体、置換変異体及び付加又は挿入変異体が挙げられる。
無内毒素組換えヒトTrpRSを次のように作製した。全長TrpRS(配列番号1のアミノ酸残基1〜471)又は配列番号1の残基94〜471(即ち全長TrpRSの残基94〜471)から主に構成される短縮TrpRS(以下、T2(配列番号12)と言う)と、配列番号1の残基71〜471から主に構成される第2の短縮TrpRS(以下、T1(配列番号13)と言う)を作製した。各プラスミドは6個のヒスチジン残基(例えば配列番号1のアミノ酸残基472〜484)を含むC末端タグと開始メチオニン残基もコードした。His6タグ付きT1は配列番号5のアミノ酸配列をもち、His6タグ付きT2は配列番号7のアミノ酸配列をもつ。
PMNエラスターゼによるヒト全長TrpRSの開裂を試験した。プロテアーゼ:タンパク質比が1:3000となるようにPBS(pH7.4)中PMNエラスターゼでTrpRSを0、15、30又は60分間処理した。開裂後、試料を12.5%SDS−ポリアクリルアミドゲル上で分析した。DNAのヌクレオチド3428〜4738(配列番号2)によりコードされる約53kDaの全長TrpRSをPMNエラスターゼで開裂すると、約46kDaの大きいフラグメント(配列番号5、C末端ヒスチジンタグをもつT1)と約43kDaの小さいフラグメント(配列番号7、C末端ヒスチジンタグをもつT2)が生成された。
Brooksら,Methods Mol.Biol.,129:257−269(1999)及びEliceiriら,Mol.Cell,4:915−924(1999)に記載されている方法に従ってマウスマトリゲル血管形成アッセイを使用してT2(配列番号7)の抗血管形成活性を試験した。文献記載の方法を次のように改変して行った。20nM VEGFを加えた増殖因子無添加マトリゲル(Becton Dickinson,Franklin Lakes,NJ)400μlを無胸腺Wehiマウスに皮下注射した。マトリゲルプラグに2.5μM T2を加えることによりまずT2の抗血管形成活性を試験した。種々の濃度のT2をプラグに加えて力価を測定した。5日目にフルオレセイン標識内皮結合レクチンGriffonia(Bandeiraea) Simplicifolia I、イソレクチンB4(Vector Laboratories,Burlingame,CA)をマウスに静脈注射し、マトリゲルプラグを切除した。RIPA緩衝液(10mMリン酸ナトリウム,pH7.4、150mM塩化ナトリウム、1%Nonidet P−40、0.5%デオキシコール酸ナトリウム、0.1%ドデシル硫酸ナトリウム)中でプラグを粉砕後に分光学的分析により各プラグのフルオレセイン含量を定量した。
網膜に注入したT2の取込みと局在を調べるために、フルオレセイン標識(ALEXA(登録商標)488,Molecular Probes,Inc.,Eugene OR)したT2を生後7日(P7)に硝子体に注入した。P8とP12に眼球を摘出し、4%PFAで15分間固定した。更に網膜から付着性非網膜組織を切除し、4%PFAに4℃で一晩浸した後、ドライアイス上で媒質(TISSUE−TEK(登録商標)O.C.T.,Sakura Fine Technical Co.,日本)で包埋した。クリオスタット切片(10ミクロン)をPBSで再水和し、PBS中5%BSA、2%正常ヤギ血清でブロックした。血管を抗マウスコラーゲンIV抗体で上述のように可視化した。DAPI核染色剤を加えたVECTASHIELD(登録商標)(Vector Laboratories,Burlingame,CA)を使用して組織をカバースリップにマウントした。
Claims (8)
- アミノ酸残基配列:
- 配列番号7のアミノ酸残基配列からなる単離形ポリペプチド。
- 請求項1に記載のポリペプチドを発現する組換え宿主細胞。
- 眼内血管新生を阻害する医薬を製造するための請求項1に記載のポリペプチドの使用。
- アミノ酸残基配列:
- ポリペプチドが配列番号7又は配列番号12のアミノ酸残基配列からなり、かつ水性賦形剤1ml当たり0.1〜0.5mgの範囲の濃度で存在する請求項5に記載の抗血管形成組成物。
- 適当な密閉容器に充填した少なくとも単用量投与に十分な量の請求項1に記載のポリペプチドと、硝子体内注射に適した33ゲージ未満の少なくとも1本のセルフシーリング注射針と、少なくとも1本の精密目盛付き注射器を含む眼内血管新生阻害用キット。
- 組成、その投与方法並びに政府法規に規定される必要な任意安全性及び有効性情報を記載した印刷情報資料を更に含む請求項7に記載のキット。
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US27095101P | 2001-02-23 | 2001-02-23 | |
PCT/US2002/005185 WO2002067970A1 (en) | 2001-02-23 | 2002-02-22 | TRYPTOPHANYL-tRNA SYNTHETASE DERIVED POLYPEPTIDES USEFUL FOR THE REGULATION OF ANGIOGENESIS |
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JP2009157410A Division JP4448925B2 (ja) | 2001-02-23 | 2009-07-02 | 血管形成の調節に有用なトリプトファニル−tRNAシンテターゼ由来ポリペプチド |
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JP4361734B2 true JP4361734B2 (ja) | 2009-11-11 |
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JP2009157410A Expired - Fee Related JP4448925B2 (ja) | 2001-02-23 | 2009-07-02 | 血管形成の調節に有用なトリプトファニル−tRNAシンテターゼ由来ポリペプチド |
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US (1) | US20030017564A1 (ja) |
EP (2) | EP2080520A1 (ja) |
JP (2) | JP4361734B2 (ja) |
KR (1) | KR100869914B1 (ja) |
CN (1) | CN100486992C (ja) |
AT (1) | ATE420653T1 (ja) |
AU (2) | AU2002306558B2 (ja) |
BR (1) | BR0207449A (ja) |
CA (1) | CA2438273C (ja) |
DE (1) | DE60230860D1 (ja) |
ES (1) | ES2318063T3 (ja) |
HK (1) | HK1069322A1 (ja) |
IL (3) | IL157558A0 (ja) |
MX (1) | MXPA03007586A (ja) |
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PL (1) | PL208008B1 (ja) |
RU (1) | RU2297425C2 (ja) |
WO (1) | WO2002067970A1 (ja) |
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US5116868A (en) | 1989-05-03 | 1992-05-26 | The Johns Hopkins University | Effective ophthalmic irrigation solution |
US5292362A (en) | 1990-07-27 | 1994-03-08 | The Trustees Of Columbia University In The City Of New York | Tissue bonding and sealing composition and method of using the same |
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ZA200307383B (en) | 2005-03-30 |
US20030017564A1 (en) | 2003-01-23 |
RU2003128420A (ru) | 2005-02-20 |
CA2438273A1 (en) | 2002-09-06 |
NZ528193A (en) | 2006-02-24 |
WO2002067970A1 (en) | 2002-09-06 |
EP1377305B1 (en) | 2009-01-14 |
KR100869914B1 (ko) | 2008-11-21 |
CN100486992C (zh) | 2009-05-13 |
RU2297425C2 (ru) | 2007-04-20 |
EP1377305A4 (en) | 2005-02-09 |
PL364596A1 (en) | 2004-12-13 |
IL201755A (en) | 2015-01-29 |
JP2009261409A (ja) | 2009-11-12 |
DE60230860D1 (de) | 2009-03-05 |
ATE420653T1 (de) | 2009-01-15 |
CA2438273C (en) | 2013-06-18 |
ES2318063T3 (es) | 2009-05-01 |
CN1527719A (zh) | 2004-09-08 |
BR0207449A (pt) | 2004-04-06 |
MXPA03007586A (es) | 2003-12-11 |
AU2007219318A1 (en) | 2007-10-18 |
IL157558A (en) | 2010-06-16 |
AU2007219318B2 (en) | 2009-12-17 |
IL157558A0 (en) | 2004-03-28 |
EP2080520A1 (en) | 2009-07-22 |
JP4448925B2 (ja) | 2010-04-14 |
JP2004532010A (ja) | 2004-10-21 |
EP1377305A1 (en) | 2004-01-07 |
HK1069322A1 (en) | 2005-05-20 |
KR20030078935A (ko) | 2003-10-08 |
AU2002306558B2 (en) | 2007-06-28 |
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