JP2004229649A - New protein and dna encoding the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a protein based on a physiological activity by analyzing base sequences of cDNA clones contained in a catalogued full-length cDNA library and specifying the physiological activity of the protein encoded with the cDNA clones for those having new sequences and to provide a method for utilizing the DNA encoding the protein. <P>SOLUTION: The protein is (a) a protein comprising a specific amino acid sequence or (b) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted and/or added in the amino acid sequence of the specific sequence and having an apoptosis inhibitory function. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

【０１２８】
表１から明らかな通り、マウス完全長ｃＤＮＡの塩基配列（ｄｎａｆｏｒｍ５４４８２）によりコードされるタンパク質は、次の通り、マウスｃＤＮＡライブラリーＦＡＮＴＯＭ（ ＨＹＰＥＲＬＩＮＫ ｈｔｔｐ：／／ｆａｎｔｏｍ．ｇｓｃ．ｒｉｋｅｎ．ｇｏ．ｊｐ／ ｈｔｔｐ：／／ｆａｎｔｏｍ．ｇｓｃ．ｒｉｋｅｎ．ｇｏ．ｊｐ／）の特定のクローンが有するｃＤＮＡの塩基配列によりコードされるタンパク質と相互作用を有することが明らかとなった。
即ち、ｄｎａｆｏｒｍ５４４８２によりコードされるタンパク質は、ｃｅｌｌ ｄｉｖｉｓｉｏｎ ｃｙｃｌｅ ｐｒｏｔｅｉｎ ２３ （ＣＤＣ２３）と相互作用することが認められた。ＣＤＣ２３は、細胞分裂後期へと導く機能を持つ。本タンパク質がＣＤＣ２３と相互作用することは、細胞分裂の制御に関わり、アポトーシスに影響を与えることが推測された。さらに、ＣＤＣ２３の変異は発癌と関わっており（Ｏｎｃｏｇｅｎｅ ２００３， ２２（１０）： １４８６−９０）、本タンパク質がその過程を制御する可能性が推測された。
【０１２９】
また、本タンパク質は、ｃｙｔｏｋｅｒａｔｉｎ １０、ｋｅｒａｔｉｎ （ｈｏｍｏｌｏｇ ｔｏ ｈｉｇｈ ｓｕｌｆｕｒ ｐｒｏｔｅｉｎ Ｂ２Ｆ）、あるいは、ｐｕｔａｔｉｖｅ ｋｅｒａｔｉｎ−ａｓｓｏｃｉａｔｅｄ ｐｒｏｔｅｉｎとの相互作用をすることが認められた。表皮細胞が形成されるとき、それは基底層で生まれ、外皮に向かって移動し、顆粒細胞層に至ると部分的アポトーシス機構により核や細胞小器官を徐々に失い、ケラチン化していく。本タンパク質は、このような分化過程を制御することが推測された。
【０１３０】
また、本タンパク質は、ｌｅｕｃｉｎｅ−ｒｉｃｈ ｒｅｐｅａｔ−ｃｏｎｔａｉｎｉｎｇ ｐｒｏｔｅｉｎと相互作用することが認められた。Ｌｅｕｃｉｎｅ−ｒｉｃｈ ｒｅｐｅａｔ （ＬＲＲ） は、多くのタンパク質に、直列に並んで見られる２０乃至２９アミノ酸残基のモチーフである。これを持つタンパク質は、ホルモン−レセプター相互作用、酵素阻害、細胞接着、細胞内輸送など多くの機能に関わる。また、最近の知見から、ＬＲＲタンパク質は、哺乳類初期発生、神経系発生、細胞分極、遺伝子発現調節、アポトーシスシグナリングに関わることも知られている。ＬＲＲは、これら多様な機能を発揮させる構造的フレームワークを成すと考えられている。したがって、本タンパク質は、細胞分裂・分化・アポトーシス等の制御に関わっている可能性が推測された。
【０１３１】
また、本タンパク質は、ｈｙｐｏｔｈｅｔｉｃａｌ ｏｕｔｅｒ ａｒｍ ｄｙｎｅｉｎ ｌｉｇｈｔ ｃｈａｉｎ １ ｓｔｒｕｃｔｕｒｅ ｃｏｎｔａｉｎｉｎｇ ｐｒｏｔｅｉｎと相互作用することが認められた。Ｏｕｔｅｒ ａｒｍ ｄｙｎｅｉｎは、モータータンパク質の１つであり、ＡＡＡ＋ファミリーＡＴＰａｓｅに属し、繊毛や鞭毛の運動を担う「軸糸ダイニン」と細胞内において膜小胞などの輸送や細胞分裂などを担う「細胞質ダイニン」の２種類がある。本タンパク質が、ｈｙｐｏｔｈｅｔｉｃａｌ ｏｕｔｅｒ ａｒｍ ｄｙｎｅｉｎ ｌｉｇｈｔ ｃｈａｉｎ １ ｓｔｒｕｃｔｕｒｅ ｃｏｎｔａｉｎｉｎｇ ｐｒｏｔｅｉｎと相互作用したことから、本タンパク質が、繊毛や鞭毛の運動、または、細胞内において膜小胞などの輸送や細胞分裂などに関連することが推測された。また、ｂｅｔａ−ａｄｒｅｎｅｒｇｉｃ ｒｅｃｅｐｔｏｒのアゴニスト刺激が、気道上皮細胞においてｏｕｔｅｒ ａｒｍ ｄｙｎｅｉｎのリン酸化をもたらすことが知られており（Ｊ． Ａｌｌｅｒｇｙ Ｃｌｉｎ． Ｉｍｍｕｎｏｌ． ２００２， １１０（６Ｓｕｐｐｌ）： Ｓ２７５−８１）、気管の繊毛運動等を介して気管支喘息等の呼吸器疾患との関連があることが推測された。
【０１３２】
実施例６ ＰＣＲ法を用いた組織発現解析
本発明のタンパク質をコードするｍＲＮＡの正常マウスおよび疾患マウスでの組織発現変動を検討するために、常法（Ｈｉｇｕｃｈｉ Ｒ， ｅｔ ａｌ．， Ｂｉｏｔｅｃｈｎｏｌｏｇｙ， １１： １０２６−３０ （１９９３））に従い、ＰＣＲ法を用いた組織発現解析を行った。
（１）ｃＤＮＡ合成
以下のマウス（森脇和郎、外１名編、Ｍｏｌｅｃｕｌａｒ Ｍｅｄｉｃｉｎｅ別冊、Ｖｏｌ． ３６「自然発症疾患モデル動物 」、中山書店、１９９９年）の１９組織からトータルＲＮＡを抽出し、オリゴｄＴをプライマーとして逆転写酵素を用いてｃＤＮＡ合成を行った。
（ａ）正常マウスの組織および糖尿病モデルマウスの組織
▲１▼対照マウスＣ５７ＢＬ／ＫｓＪ − ＋ｍ／＋ｍ Ｊｃｌ（メス、８週齢）の全脳、視床、肺、腎臓、骨髄、膵臓、脂肪細胞、肝臓、眼
▲２▼糖尿病モデルマウスＣ５７ＢＬ／ＫｓＪ − ｄｂ／ｄｂ Ｊｃｌ（メス、８週齢）の膵臓、脂肪細胞、肝臓、眼
（ｂ）老化促進マウスの組織
▲１▼正常老化マウス ＳＡＭ Ｒ１／ＴＡ Ｓｌｃ（オス、１３週齢）の海馬、前頭葉皮質
▲２▼老化促進マウス ＳＡＭ Ｐ８／Ｔａ Ｓｌｃ（オス、１５週齢）の海馬、前頭葉皮質
（ｃ） 癌転移モデルマウスの組織
▲１▼対照マウスＢａｌｂ／ｃ（メス、５週齢）の正常結腸
▲２▼癌転移モデルマウスＢａｌｂ／ｃ（メス、６週齢）の結腸癌（マウス腹腔に結腸癌細胞Ｃｏｌｏｎ２６を移植し、２週間後に結腸癌を摘出）
【０１３３】
（２）ＰＣＲ法による定量
下記の５個の、本発明のタンパク質をコードしているｍＲＮＡの発現は、ライトサイクラー定量ＰＣＲ装置（ロシュ・ダイアグノスティクス社製）とＬｉｇｈｔＣｙｃｌｅｒ−ＦａｓｔＳｔａｒｔ ＤＮＡマスターＳＹＢＲ Ｇｒｅｅｎ Ｉ試薬を用いて、製品に添付されているプロトコールに従い定量した。ｄｎａｆｏｒｍ５４４８２の定量ＰＣＲに用いた合成ＤＮＡ配列を以下に示す。
５’側プライマー：（ＧＣＡＧＣＴＴＧＣＡＧＴＴＡＧＡＡＧＣＡ）（配列番号１１）
３’側プライマー：（ＴＡＣＴＣＴＣＡＧＧＣＣＣＣＡＴＧＧＡＡ）（配列番号１２）
定量結果はＧｌｙｃｅｒａｌｄｅｈｙｄｅ ３−ｐｈｏｓｐｈａｔｅ ｄｅｈｙｄｒｏｇｅｎａｓｅ（ＧＡＰＤＨ）を内部標準として、補正した。即ち、各組織での対象遺伝子の発現量（コピー数／μｌ）をＧＡＰＤＨの発現量（コピー数／μｌ）で除し、定数（１×１０^６）（注：１０の６乗）を乗して表示した。その結果を表２に示す。
【０１３４】
【表２】

【０１３５】
表２から明らかな通り、ｄｎａｆｏｒｍ５４４８２は眼で強く発現し、肺での発現が高かった。
【０１３６】
実施例７ ハエホモログを用いたＲＮＡｉ法による機能解析
（１）マウスｃＤＮＡのハエホモログの解析および取得
上記で得られたマウス全長ｃＤＮＡの塩基配列（クローン名：ｄｎａｆｏｒｍ５４４８２）に対するハエ遺伝子のホモログを以下に示す配列解析により予測した。配列解析は、ＯＲＦから翻訳されるタンパク質を考え、アミノ酸配列レベルで行った。マウスクローンｄｎａｆｏｒｍ５４４８２として、配列番号２に示すアミノ酸配列を含むマウス全長ｃＤＮＡクローン（配列番号１）とハエクローン（Ｂｅｒｋｅｌｅｙ Ｄｒｏｓｏｐｈｉｌａ Ｇｅｎｏｍｅ Ｐｒｏｊｅｃｔ（ＢＤＧＰ） Ｄｒｏｓｏｐｈｉｌａ Ｇｅｎｏｍｉｃ Ｓｅｑｕｅｎｃｅ Ｒｅｌｅａｓｅ ３．０）との間においてペアワイズな配列比較をＮＣＢＩ ＢＬＡＳＴＰ ２．２．２ （Ａｌｔｓｃｈｕｌ ｅｔ ａｌ．， Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｒｅｓ． ２５， ３３８９−３４０２ （１９９７））により行い、２種のクローン間において互いに最も良いＥ−ｖａｌｕｅの値を示した関係にあるハエの遺伝子を、予測されたホモログとして同定した（Ｔａｔｕｓｏｖ， Ｋｏｏｎｉｎ ＆ Ｌｉｐｍａｎ， Ｓｃｉｅｎｃｅ ２７８， ６３１ （１９９７））。結果として、マウスｃＤＮＡ（ｄｎａｆｏｒｍ５４４８２）に対するハエホモログを見つけることができた。ハエホモログの遺伝子番号は、マウス：５４４８２（ｄｎａｆｏｒｍ５４４８２）に対してハエ：ＣＧ１２２８４（トランスクリプト番号：ＣＧ１２２８４−ＰＣ）であった。
【０１３７】
（２）ハエ系統の樹立
（ｉ）逆方向反復配列（ｉｎｖｅｒｔｅｄ ｒｅｐｅａｔ）ベクターの作製
上記ハエｃＤＮＡ（ＣＧ１２２８４）のＯＲＦ、５’側約５００ｂｐの断片（以下これを「目的のｃＤＮＡ」と称することがある）を、ハエのｃＤＮＡライブラリー（Ｂｅｒｋｅｌｅｙ Ｄｒｏｓｏｐｈｉｌａ Ｇｅｎｏｍｅ Ｐｒｏｊｅｃｔ（ ｈｔｔｐ：／／ｗｗｗ．ｆｒｕｉｔｆｌｙ．ｏｒｇ）で同定されたｕｎｉｇｅｎｅｓｅｔ； Ｉｎｖｉｔｒｏｇｅｎ社）を鋳型としたＰＣＲによって増幅した。ＰＣＲプライマーは、ＣｐｏＩＡ７（配列番号１３：ＡＡＡＴＴＴＣＧＧＡＣＣＧの３’末端に、目的のｃＤＮＡの５’末端の２１ベースの塩基配列を結合させたもの）とＳｆｉＩＡ３（配列番号１４：ＡＡＡＴＴＴＧＧＣＣＡＴＡＴＡＧＧＣＣの３’末端に、目的のｃＤＮＡの３’末端の２１ベースの塩基配列を結合させたもの）のセット、およびＳｆｉＩＢ７（配列番号１５：ＡＡＡＴＴＴＧＧＣＣＴＡＣＡＴＧＧＣＣに記載の塩基配列の３’末端に、目的のｃＤＮＡの５’末端の２１ベースの塩基配列を結合させたもの）とＣｐｏＩＢ３（配列番号１６：ＡＡＡＴＴＴＣＧＧＴＣＣＧの３’末端に、目的のｃＤＮＡの３’末端の２１ベースの塩基配列を結合させたもの）のセットを用いた。
【０１３８】
ＳｆｉＩＡ３とＳｆｉＩＢ７プライマーを用いてＰＣＲ増幅した約５００ｂｐのＤＮＡ断片をＳｆｉＩで消化し、この消化断片をハエクローニング用ベクター（ｐＵＡＳＴＣＳ１：上田龍他、細胞工学、ｖｏｌ２１， Ｎｏ．８， ９２３−９３２（２００２））をＳｆｉＩで消化したサイト間に挿入しクローニングした。さらに、このベクターをＣｐｏＩで消化したサイト間に、上記ＣｐｏＩＡ７とＣｐｏＩＢ３プライマーを用いてＰＣＲ増幅したＤＮＡ断片をＣｐｏＩ消化したＤＮＡ断片を挿入しクローニングした。この２回のサブクローニングにより、ハエｃＤＮＡのＯＲＦの約５００ｂｐの断片が、ＵＡＳ配列（ＧＡＬ上流活性化配列）の下流に隣接したヒートショックプロテイン７０のベーシックプロモーターの制御下に、逆向きで２カ所挿入されたベクター（ｉｎｖｅｒｔｅｄ ｒｅｐｅａｔ ベクター）が取得された。
【０１３９】
上記で用いたハエトランスフォーメション用ベクターであるｐＵＡＳＴベクターはトランスポゾンＰ因子を利用したベクターで、ＵＡＳ配列とヒートショックプロテイン７０のベーシックプロモーターを利用して、転写促進タンパク質ＧＡＬ４がＵＡＳ配列に結合することによりＵＡＳ配列の下流に挿入した逆方向反復配列の転写を誘導できるベクターである。
【０１４０】
（ｉｉ）ハエの形質転換
通常のＰ因子形質転換法である、上田龍他、細胞工学、ｖｏｌ２１， Ｎｏ．８， ９２３−９３２（２００２）に記載の方法に則って、上記（ｉ）で調製したｉｎｖｅｒｔｅｄ ｒｅｐｅａｔ ベクターＤＮＡをＷ^１１１８系統のハエ（Ｉｎｄｉａｎａ ｓｔｏｃｋ ｃｅｎｔｅｒ： ｈｔｔｐ：／／ｆｌｙｂａｓｅ．ｂｉｏ．ｉｎｄｉａｎａ．ｅｄｕ／ｓｔｏｃｋｓ／ｆｂｓｔｏｃｋ．ｈｆｏｒｍ）の初期胚にマイクロマニュピレーターを用いて微量注入した。これを培養して孵化させ、成虫とした後に、上記文献記載の手順に準じて交配を行った。この交配で、ダブルバランサーとしてＷ^１１１８系統のＳｐ／ＳＭ１、Ｃｙ：Ｐｒ／ＴＭ３、Ｓｂ Ｓｅｒを用いた。この交配により、上記ｉｎｖｅｒｔｅｄ ｒｅｐｅａｔベクターが挿入した染色体を、ホモ接合に持ったハエ（以下、これを「ＩＲ系統」と称する）が作製された。
【０１４１】
（３）ＲＮＡｉ効果の誘導
（ｉ）ＧＡＬ４ドライバーとの交配による変異誘導
上記（２）で得られたＩＲ系統のハエをＡｃｔ５Ｃ−ＧＡＬ４系統のハエ（Ｉｎｄｉａｎａ ｓｔｏｃｋ ｃｅｎｔｅｒ： ｈｔｔｐ：／／ｆｌｙｂａｓｅ．ｂｉｏ．ｉｎｄｉａｎａ．ｅｄｕ／ｓｔｏｃｋｓ／ｆｂｓｔｏｃｋ．ｈｆｏｒｍ）と交配した（図１）。Ａｃｔ５Ｃ−ＧＡＬ４は、全身の細胞で発現する細胞性アクチン遺伝子（Ａｃｔ５Ｃ）のプロモーターに酵母ＧＡＬ４遺伝子をつないだｆｕｓｉｏｎ ｇｅｎｅを遺伝子導入した系統である。従って、このＡｃｔ５Ｃ−ＧＡＬ４導入遺伝子を持ったハエでは全ての細胞で、ＧＡＬ４タンパク質が発現している。
【０１４２】
ＩＲ系統のハエと、Ａｃｔ５Ｃ−ＧＡＬ４系統のハエを交配した子孫（Ｆ１世代、以下これを「ＲＮＡｉ個体」と称する）では、図１にあるように２種類の導入遺伝子が存在する。従って、全ての細胞でＧＡＬ４タンパク質が発現し、ＩＲベクターを強制転写するため、ｄｓＲＮＡが細胞に出現し、ＲＮＡｉ効果を発揮し、ターゲットｍＲＮＡが発現した場合にこれを分解する。従って、その個体の全ての細胞でターゲット遺伝子の機能阻害が起こる。ターゲットｍＲＮＡが発現していない細胞では何の効果ももたらさない。
【０１４３】
（４）表現型の解析および結果
上記（３）で誘導されたＲＮＡｉ効果で、マウスｃＤＮＡ（ｄｎａｆｏｒｍ５４４８２）のハエホモログの機能が阻害されたことによるハエ個体の表現型の変化を観察した。その結果、ＲＮＡｉ個体は半致死、即ち蛹にはなるが脱皮して成虫となる個体は５％以下であることが判明した。
ＲＮＡｉ技術を用いて上記ハエ遺伝子の発現抑制を行い機能阻害することにより、ハエ個体が半致死となったことから、該遺伝子は初期発生あるいは個体の生存・機能維持に重要な役割を果たしていることが分かった。前記の通り、ｄｎａｆｏｒｍ５４４８２（配列番号２５、２６）によりコードされるタンパク質は、アポトーシス抑制作用を有することが推測されており、ＲＮＡｉ個体中において、ハエホモログ遺伝子のアポトーシス抑制作用が阻害されたことにより、個体の発生・分化の過程で、アポトーシスが正常に進行せず、個体は半致死となったものと考えられる。この様に、ｄｎａｆｏｒｍ５４４８２によりコードされるタンパク質は、アポトーシス抑制作用を有しており、発生・分化・生理的機能あるいは病態との関連で重要な役割を果たしていることが推察される。
【０１４４】
上記の通り、ｄｎａｆｏｒｍ５４４８２によりコードされるタンパク質は、実施例４よりアポトーシスの抑制に関与することが示唆され、実施例６より、眼で強く発現し、肺での発現が高いことが示され、実施例５でも、タンパク質−タンパク質相互作用の実験結果から本タンパク質はアポトーシスの抑制や癌と関係すること、呼吸器系疾患と関係することが示唆された。また、実施例７では、ハエのホモログのＲＮＡｉの効果で半致死であったことから、本タンパク質は、発生・分化等において重要な役割を果たし、眼の疾患や肺等の呼吸器系疾患や癌との関連が示唆された。これらのことから、本タンパク質の発現制御物質、機能賦活物質、あるいは機能阻害物質は、眼の疾患、肺や気管等の呼吸器系疾患、神経変性疾患、免疫疾患または癌の治療薬として開発できる可能性がある。
【０１４５】
【発明の効果】
本発明のタンパク質およびそれをコードするＤＮＡは、アポトーシスの抑制に関わる機能を有するものであるので、該タンパク質あるいはそれをコードするＤＮＡを用いて該活性を調節する物質をスクリーニングすることができ、該タンパク質が関連する疾患等に作用し得る医薬の開発に有用である。
【０１４６】
本出願は、２００２年４月３０日付けの日本特許出願（特願２００２−１２８８５９）および２００２年１２月４日付けの日本特許出願（特願２００２−３５２６４１）に基づくものであり、その内容はここに参照として取り込まれる。また、本明細書にて引用した文献の内容もここに参照として取り込まれる。
【０１４７】
【配列表】

【図面の簡単な説明】
【図１】図１は、ＲＮＡｉ個体中の導入遺伝子の誘導方法を示す概念図である。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a novel protein, a DNA encoding the protein, a full-length cDNA encoding the protein, a recombinant vector having the DNA, an oligonucleotide comprising a partial sequence of the DNA, and a transgenic cell into which the DNA has been introduced. And antibodies that specifically bind to the protein.
[0002]
[Prior art]
Obtaining cDNA and analyzing its base sequence are indispensable for analyzing the physiological activity of a protein expressed in a living body and developing a method for utilizing the protein based on the activity. Furthermore, creating a library in which full-length cDNAs corresponding to all gene types are cataloged is one of the important issues of the human genome project. The cataloged library means that there is no duplication in the cDNAs contained in the library, and refers to a library containing one type of each cDNA.
[0003]
The full-length cDNA cloning method is described in JP-A-9-248187 and JP-A-10-127291. This method comprises the steps of binding a molecule serving as a tag to a diol structure present at the 5 'cap site of mRNA, using the mRNA bound with the tag molecule as a template, oligo dT as a primer, and reverse transcription to an RNA-DNA complex. And separating the complex having a DNA corresponding to the full length of the mRNA using the function of the tag molecule.
[0004]
As an efficient reverse transcription method, a method for performing the transcription at a high temperature such that the template does not form a higher-order structure has been developed (Japanese Patent Laid-Open No. Hei 10-84661). Furthermore, a cloning vector has been developed which can uniformly clone a DNA fragment contained in a synthesized full-length cDNA library regardless of its chain length (Japanese Patent Application Laid-Open No. H11-9273).
[0005]
A full-length cDNA library produced by such a technique does not necessarily include all the elements that are different evenly as individual elements of the library, and is present only in clones with a high abundance ratio or, conversely, in very small amounts. There are clones. Since a clone existing only in a trace amount is highly likely to be novel, a subtraction method and a normalization method for enriching such a clone have also been developed (Japanese Patent Application Laid-Open No. 2000-325080; Carnini, P. et al. et al., Genomics, 37, 327-336 (1996)).
[0006]
The nucleotide sequence of each clone of the cataloged full-length cDNA library thus obtained can be identified by a known method. However, the physiological activity of the protein encoded by the cDNA remains unknown. It is.
[0007]
[Problems to be solved by the invention]
The present invention analyzes the nucleotide sequence of a cDNA clone contained in a cataloged full-length cDNA library, and among those having a novel sequence, identifies the biological activity of the protein encoded by the cDNA sequence and determines the biological activity. It is an object of the present invention to propose a method of using a protein based thereon and DNA encoding the same.
[0008]
[Means for Solving the Problems]
The present inventors analyzed the nucleotide sequence of the cDNA clone in the mouse full-length cDNA library and searched a database based on the homology of the sequence, and found that the sequence contained BIR (Baculovirus Inhibitor of apoptosis protein Repeat). A sequence specific to the protein was found, and the protein encoded by these cDNAs was identified as a protein having an apoptosis-suppressing function. The expression level of the cDNA in each tissue was analyzed, and the protein encoded by the cDNA was actually expressed, and its interaction activity was analyzed. Furthermore, the physiological activity (function) of the protein was analyzed by inhibiting the expression of the protein encoded by the cDNA. The present invention has been achieved based on these findings.
[0009]
That is, according to the present invention, the following inventions (1) to (15) are provided.
(1) The following protein of (a) or (b):
(A) a protein consisting of the amino acid sequence of SEQ ID NO: 2;
(B) a protein consisting of the amino acid sequence of SEQ ID NO: 2 with one or several amino acids deleted, substituted and / or added, and having an apoptosis-suppressing function.
[0010]
(2) A DNA encoding the protein of (1).
(3) A full-length cDNA encoding the protein according to (1).
(4) Any one of the following DNAs (a), (b) or (c):
(A) DNA having the nucleotide sequence of SEQ ID NO: 1.
(B) a DNA which has a base sequence in which one or several bases are deleted, substituted and / or added in the base sequence of SEQ ID NO: 1, and encodes a protein having an apoptosis-suppressing function.
(C) DNA encoding a protein having a nucleotide sequence capable of hybridizing under stringent conditions with a DNA having the nucleotide sequence of SEQ ID NO: 1 or a sequence complementary thereto, and having an apoptosis-suppressing function.
[0011]
(5) A recombinant vector containing the DNA according to any of (2) to (4).
(6) A transgenic cell into which the DNA according to any one of (2) to (4) or the recombinant vector according to (5) has been introduced, or an individual comprising the cell.
(7) The protein according to (1), which is produced by the cell according to (6).
[0012]
(8) A sense oligonucleotide having the same sequence as 5 to 100 consecutive nucleotides in the base sequence of the DNA according to any of (2) to (4), and an antisense having a sequence complementary to the sense oligonucleotide. An oligonucleotide selected from the group consisting of an oligonucleotide and an oligonucleotide derivative of the sense or antisense oligonucleotide.
[0013]
(9) An antibody or a partial fragment thereof that specifically binds to the protein according to (1) or (7).
(10) The antibody according to (9), wherein the antibody is a monoclonal antibody.
(11) The antibody according to (10), wherein the monoclonal antibody has an action of neutralizing the apoptosis-suppressing function of the protein according to (1) or (7).
[0014]
(12) A method for controlling an activity of a protein, comprising: bringing the protein according to (1) or (7) into contact with a test substance, and measuring a change in activity of the protein caused by the test substance. Screening method.
(13) A screening for a substance regulating the expression of a DNA, comprising bringing the test substance into contact with the gene-transfected cell according to (6) and detecting a change in the expression level of the DNA introduced into the cell. Method.
(14) At least one or more amino acid sequence information selected from the amino acid sequence of the protein according to (1), and / or at least one selected from the nucleotide sequence of the DNA according to any of (2) to (4) A computer-readable recording medium storing one or more base sequence information.
(15) A carrier to which the protein according to (1) and / or the DNA according to any of (2) to (4) are bound.
[0015]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in more detail.
(1) Acquisition of full-length cDNA and analysis of nucleotide sequence
The DNA of the present invention is a protein consisting of the amino acid sequence of SEQ ID NO: 2 or, in the amino acid sequence, one or several (the number is not particularly limited, for example, 20 or less, preferably 15 or less, more preferably 10 or less, still more preferably 5 or less) amino acid residue substitution, deletion, insertion, addition, or inversion, or an apoptosis-suppressing function. Any protein can be used as long as it can encode a protein having the protein. Specifically, it may be only the translation region encoding the amino acid sequence or may include the full length of the cDNA.
[0016]
Specifically, as the DNA containing the full-length cDNA, for example, a DNA having the nucleotide sequence of SEQ ID NO: 1 and the like are mentioned. Examples of the translation region include those having a sequence represented by base numbers 125 to 595 of SEQ ID NO: 1. Further, the DNA of the present invention includes not only the full length of the above-mentioned cDNA but also those containing the above-mentioned translation region and a portion adjacent to the 3 'and / or 5' end thereof, which is the minimum necessary for the expression of the translation region. .
[0017]
The DNA of the present invention may be obtained by any method as long as it can be obtained, and specifically, can be obtained, for example, by the following method. First, mRNA is prepared from a suitable animal, preferably a mammalian tissue or the like by a method known per se and generally used. Next, cDNA is synthesized using this mRNA as a template. At this time, in order to synthesize a full-length cDNA, a molecule serving as a tag is chemically bonded to a diol structure specific to a 5 ′ cap (7MeGpppN) site. Using reverse transcription with oligo dT as a primer and using the function of a tag molecule to separate only full-length cDNA (JP-A-9-248187; JP-A-10-127291). Is preferred. In addition, in the case of reverse transcription, in order to prevent the template from forming a higher-order structure and lowering the efficiency of reverse transcription, in the presence of trehalose or the like, use a thermostable reverse transcriptase at a high temperature. It is preferable to use a method of performing reverse transfer (Japanese Patent Laid-Open No. 10-84661). Here, high temperature means 40-80 degreeC.
[0018]
The thus obtained cDNA is cloned by inserting it into an appropriate cloning vector. The vector used herein has a recombination recognition sequence at both ends of a cloning site capable of uniformly cloning DNAs of various chain lengths, and is a linear vector inserted into a host by a method other than infection. (JP-A-11-9273) is preferably used. In the cDNA library thus obtained, not all clones exist uniformly (hereinafter, this may be referred to as "cataloged"), but only a very small amount exists in this library. A clone that does not have a high probability of being new. Therefore, it is preferable to use a subtraction method or a normalization method for enriching such clones (Japanese Patent Laid-Open No. 2000-325080; Carinci, P. et al., Genomics, 37, 327-336 (1996)).
[0019]
The nucleotide sequence of the cataloged cDNA library is analyzed by a commonly used method known per se. In the case of the DNA of the present invention, in the case of full-length cDNA, the base sequence obtained from the sequence based on the terminal 100 is obtained by converting BLAST (http://www.ncbi.nlm.nih.gov/BLAST/; National Center of Biotechnology Information). ), NCBI databases such as Genbank, EMBL, and DDBJ were searched, and even the sequence showing the highest homology was found to have a similarity of 30% or less as a new sequence for the following analysis.
[0020]
Examples of the DNA having the nucleotide sequence of such a full-length cDNA include those having the nucleotide sequence of SEQ ID NO: 1. Examples of the translation region include those having a sequence represented by base numbers 125 to 595 of SEQ ID NO: 1.
[0021]
The thus obtained novel nucleotide sequence is subjected to homology search by BLAST (Basic local alignment search tool; Altschul, SF, et al., J. Mol. Biol., 215, 403-410 (1990)). protein feature search by HMPMAM, which is one of a group of functions such as homology search and HMMER (sequence analysis method using hidden Markov model; Eddy, SR, Bioinformatics 14, 755-763 (1998)). //Pfam.wustl.edu) or the like, the function of the protein encoded by the nucleotide sequence can be estimated.
[0022]
In the homology search by BLAST, the function of the clone to be analyzed can be estimated from various kinds of annotation information associated with hit sequences having sufficiently significant homology obtained as a result of the search. Here, a sufficiently significant hit sequence means that the degree of coincidence between the functional domain portion of the registered amino acid sequence and the corresponding portion of the amino acid sequence encoded by the DNA of the present invention is 10 as an e-value.^-4Show the following or 30% or more.
[0023]
For example, if many of the top-ranked functional domain sequences have been confirmed to have an apoptosis-suppressing function, the clone to be analyzed that is similar in sequence to that will also have the same function, that is, an apoptosis-suppressing function. The prediction holds.
[0024]
In the HMMPFAM, analysis is performed by a method of checking whether or not a sequence to be analyzed has a feature of a sequence included in an entry in a database in which a protein profile called Pfam is accumulated. Profiles are extracted from a series of proteins with the same characteristics, and even if the structure cannot be determined by comparing one-to-one sequences over the entire length, if there is a characteristic region in the sequence, it can be found and its function can be predicted. . A specific example of the protein function prediction performed in this manner will be described below.
[0025]
The amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1 was determined by BLAST search as Baculoviral IAP repeat-containing protein 3, and e-value: 2 × 10^-20And a 35% identity over 156 amino acid residues, and e-value: 1 × 10 with Inhibitor of apoptosis protein.^-18In addition, 53% identity over 67 amino acid residues was further compared with the Apoptosis inhibitor IAP and e-value: 2 × 10^-17With a 30% identity over 154 amino acid residues. From these results, it was presumed that the protein having the amino acid sequence shown in SEQ ID NO: 2 was a protein having an apoptosis-suppressing action.
[0026]
The Baculoviral IAP repeat-containing protein 3 protein is considered to be involved in suppression of apoptosis based on literature information (Nat. Struct. Biol. 6: 648-651 (1999)) in the database, and the Inhibitor of apoptososis protein Is related to the suppression of apoptosis of T cells from the literature information in the database (DNA Cell Biol. 15: 981-988 (1996)). Furthermore, the above-mentioned Apoptosis inhibitor IAP protein is described in the literature information in the database (J. Virol. 67: 2168-2174 (1993)). Was.
When a protein motif was searched by HMMPFAM, Baculovirus Inhibitor of apoptosis protein Repeat (amino acid sequence entered as PIR in Bfam, also described as IAP repeat) was found at amino acids 96 to 156 of SEQ ID NO: 2.
From these facts, it was presumed that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 1 was a protein having a function related to suppression of apoptosis.
[0027]
The DNA of the present invention may be obtained in a state where a base sequence is deleted or inserted in the translated sequence. As a result of performing the homology search or the protein feature search as described above, the base sequence of the DNA is determined. If a deletion or insertion in the DNA is estimated, it is possible to obtain a full-length cDNA having no base deletion or insertion by using a method commonly used in the art such as library screening or PCR cloning. it can. The protein of the present invention is expressed using the full-length cDNA thus obtained, and can be used for functional analysis.
[0028]
Thus obtained, the base sequence is determined, and the DNA of the present invention whose function is estimated is not limited to the base sequence described in SEQ ID NO: 1 or the one having the base sequence shown above as a translation region thereof, In these base sequences, one or several (the number is not particularly limited, but is, for example, 60 or less, preferably 30 or less, more preferably 20 or less, further preferably 10 or less, DNAs encoding proteins having a base sequence in which 5 or less bases are deleted, substituted and / or added, and having an apoptosis-suppressing function, and conditions which are stringent therewith. DNAs that hybridize below and encode proteins having an apoptosis inhibitory function are also included. As described above, these DNAs include a protein consisting of an amino acid sequence in which one or several amino acid sequences are deleted, substituted and / or added in the amino acid sequence of the protein represented by SEQ ID NO: 2, and having an apoptosis-suppressing function. Includes what to code.
[0029]
Here, a DNA that hybridizes under stringent conditions means a homology of 80% or more, preferably 90% or more, more preferably 95% or more by BLAST analysis with the base sequence shown in SEQ ID NO: 1 or its complementary sequence. And DNA containing a base sequence having the property. Further, the hybridization under stringent conditions means that the reaction is carried out in a normal hybridization buffer at a temperature of 40 to 70 ° C, preferably 60 to 65 ° C, and a salt concentration of 15 mM to 300 mM, preferably The washing can be performed according to a method of washing in a washing solution of 15 mM to 60 mM or the like.
[0030]
Further, the DNA of the present invention may be obtained by the above-described method or may be synthesized. The DNA base sequence can be easily replaced with a commercially available kit such as a site-directed mutagenesis kit (Takara Shuzo) or a quick change site-directed mutagenesis kit (Stratagene).
[0031]
Although the nucleotide sequence of SEQ ID NO: 1 is derived from mouse, a human cDNA library was prepared in accordance with the above-described method for preparing a cDNA library. By performing hybridization using a DNA fragment having the described nucleotide sequence as a probe, a DNA encoding a human homolog protein of the protein encoded by the nucleotide sequence represented by SEQ ID NO: 1 can also be obtained. The DNA which hybridizes with the DNA having the nucleotide sequence of SEQ ID NO: 1 or its complementary sequence under stringent conditions also includes such human homolog DNA and human ortholog DNA described below. .
[0032]
Further, it is also possible to predict the base sequence of the human homolog DNA by using informatics, and to obtain the human homolog DNA from the above-mentioned human cDNA library or the like based on the base sequence.
[0033]
In general, as a method for predicting a base sequence encoding a homolog protein of a target protein using informatics, for example, (i) using a base sequence of a target cDNA as a query, A method of performing a homology search on a database (including a cDNA database predicted by informatics) using BLAST or the like; and (ii) BLAST against a human or other EST database using the base sequence of the target cDNA as a query. A method of performing homology search using such a method and linking the sequence of the hit EST with reference to the base sequence of the target cDNA, and (iii) using the base sequence of the target cDNA as a query Homology search for genome database using BLAST etc. In addition, the position on the genome where the gene of the cDNA of interest exists is specified, and Genscan (http://genes.mit.edu/GENSCAN.html) or Sim4 (Genome Res., 8: 977-74 (1998)) and the like, and a method of predicting the nucleotide sequence of a gene portion in the genome.
[0034]
When predicting the nucleotide sequence of the human homolog DNA of mouse-derived cDNA, any of the above methods can be used. However, the cDNA having the nucleotide sequence of SEQ ID NO: 1 of the present invention is novel and has the above (i) Since it is considered that the nucleotide sequence of the human homolog DNA cannot be obtained by the method, it is preferable to use the method described in (ii) or (iii).
[0035]
Based on the predicted nucleotide sequence of the human homolog DNA, a DNA encoding a human homolog protein of the protein encoded by the nucleotide sequence of SEQ ID NO: 1 can be obtained from the above human cDNA library. As a specific acquisition method, for example, PCR is performed using the above human cDNA library as a template, using a primer having a nucleotide sequence complementary to the nucleotide sequence at the 5 ′ end and 3 ′ end of the predicted human homolog DNA. And a method of performing hybridization with the above human cDNA library using a partial sequence of the predicted human homolog DNA as a probe.
[0036]
Generally, a similar gene having a nucleotide sequence having a high homology with the nucleotide sequence of the target gene is referred to as a “homolog”, and the above-described method also aims to obtain a human homolog. It is important to confirm that not only the similarity but also that the gene obtained as a homolog is a family member of the target gene. Genes acquired as "homologs" between two species of organisms are likely to be "orthologs", the same gene evolved from a common ancestral gene, and differ from each other caused by duplication from a common ancestral gene It may be a "paralog" that is a gene.
[0037]
That is, in order for the human-derived DNA obtained as a homologue to have the same function as the protein of the present invention, the function of the protein encoded by the human-derived DNA must be In order to verify the function of the protein of the present invention as a mouse, it is preferable to confirm that the human homolog is an ortholog of a closely related species of the mouse gene of the present invention.
[0038]
For example, the following method is used as a method for confirming the ortholog. (I) First, homology is analyzed between the nucleotide sequence of the cDNA of the present invention and the nucleotide sequence of the obtained human homolog DNA. Next, using the base sequence of the cDNA of the present invention as a query, a homology search was performed on human base sequences contained in international base sequence databases such as DDBJ, EMBL, GenBank, and patent databases, and the base sequence of the obtained human homolog DNA was obtained. And that the base sequence of the query is higher than the base sequence obtained from the database. Further, (ii) homology is analyzed between the nucleotide sequence of the obtained human homolog DNA and the corresponding nucleotide sequence of the cDNA of the present invention. Next, using the base sequence of the obtained human homologous DNA as a query, a homology search was performed on the mouse base sequence contained in the international base sequence database such as DDBJ, EMBL, GenBank, and the patent database, and the base sequence of the cDNA of the present invention was obtained. And that the base sequence of the query is higher than the base sequence obtained from the database and the base sequence of the query. By confirming the above (i) and (ii), the obtained human homolog can be identified as a human ortholog corresponding to the cDNA of the present invention. The homology analysis described in (i) and (ii) above may be performed by comparing amino acid sequences, or by drawing a molecular evolutionary phylogenetic tree and examining it. In addition, it is preferable that the degree of coincidence by the homology analysis described in the above (i) and (ii) be analyzed as the degree of coincidence over the entire length of the query.
[0039]
By performing a homology search by BLAST or a protein feature search by HMMPFAM on the nucleotide sequence of the human homologous DNA or orthologous DNA thus obtained, the function of the protein encoded by the nucleotide sequence can be estimated.
Furthermore, the protein of the present invention is expressed using the obtained full-length cDNA of human homolog DNA or human ortholog DNA, and can be used for confirmation of activity, functional analysis, and the like.
[0040]
The protein of the present invention is expressed using the full-length cDNA thus obtained, and can be used for confirmation of activity, functional analysis, and the like. Further, the protein encoded by the DNA of the present invention has an apoptosis-suppressing function, and is used as a tool for elucidating cell functions and as a therapeutic drug for diseases caused by abnormalities of the protein or a target for the development of therapeutic drugs. Useful as a protein.
[0041]
(2) Protein encoded by the novel cDNA
The translation region of the protein encoded by the DNA of the present invention is, for example, a base sequence of the DNA which is converted into amino acids by three types of reading frames, and the range encoding the longest polypeptide is defined as the translation region of the present invention. The amino acid sequence can be determined. Such an amino acid sequence includes, for example, the one described in SEQ ID NO: 2. Further, the protein of the present invention is not limited to the above amino acid sequence, but comprises an amino acid sequence in which one or several amino acids have been substituted, deleted and / or added, and has an apoptosis-suppressing function. And those having the following.
[0042]
As a method for obtaining the protein of the present invention, the method of transcription / translation of the DNA of the present invention described in the above (1) by an appropriate method is preferably used. Specifically, a recombinant vector inserted into a suitable expression vector or a suitable vector together with a suitable promoter is prepared, and a suitable host microorganism is transformed with the recombinant vector or introduced into a suitable cultured cell. And then can be obtained by purification.
[0043]
When the protein thus obtained is obtained in a free form, it can be converted to a salt by a known method or a method analogous thereto, and conversely, when the protein is obtained in a salt form, it can be converted to a free form or another salt. can do. Such salts of the protein of the present invention are also included in the protein of the present invention. Further, a protein produced by the transformant may be modified before or after purification by applying an appropriate protein modifying enzyme to modify the protein arbitrarily or partially removing the polypeptide. Can be. These modified proteins are also included in the scope of the present invention as long as they have an apoptosis-suppressing function.
[0044]
When producing the protein of the present invention, the vector used for the production of the recombinant vector containing the DNA of the present invention is not particularly limited as long as the DNA is expressed in the transformant. Any of vectors may be used. Of these, usually, a commercially available protein expression vector into which an expression control region DNA such as a promoter suitable for a host into which the DNA is introduced has already been inserted is used. Specific examples of such a protein expression vector include pET3 and pET11 (manufactured by Stratagene) and pGEX (manufactured by Amersham Pharmacia Biotech) when the host is Escherichia coli, and pESP when the host is yeast. -I expression vector (manufactured by Stratagene) and the like, and in the case of insect cells, BacPAK6 (manufactured by Clontech). When the host is an animal cell, examples include ZAP Express (manufactured by Stratagene), pSVK3 (manufactured by Amersham Pharmacia Biotech) and the like.
[0045]
When using a vector into which the expression control region has not been inserted, it is necessary to insert at least a promoter as the expression control region. Here, as the promoter, a promoter contained in a host microorganism or a cultured cell can be used. However, the promoter is not limited thereto. Specifically, for example, when the host is Escherichia coli, T3, T7, tac Lac promoter and the like, and in the case of yeast, for example, nmt1 promoter, Gal1 promoter and the like can be used. When the host is an animal cell, SV40 promoter, CMV promoter and the like are preferably used.
[0046]
When a host capable of functioning with a mammalian-derived promoter is used, a promoter specific to the gene of the present invention can also be used. Insertion of the DNA of the present invention into these vectors may be performed by linking the DNA or a DNA fragment containing the DNA to the amino acid sequence of the protein encoded by the gene DNA downstream of the promoter in the vector.
[0047]
The recombinant vector thus prepared can be used to transform a host described below by a method known per se to prepare a DNA transductant. As a method for introducing the vector into a host, specifically, a heat shock method (J. Mol. Biol., 53, 154 (1970)), a calcium phosphate method (Science, 221, 551, (1983)), DEAE Dextran method (Science, 215, 166, (1982)), in vitro packaging method (Proc. Natl. Acad. Sci. USA, 72, 581, (1975)), virus vector method (Cell, 37, 1053, (1984)). )), And the electric pulse method (Chu. Et al., Nuc. Acids Res., 15, 1331 (1987)).
[0048]
The host for preparing the DNA transfectant is not particularly limited as long as the DNA of the present invention is expressed in the body. For example, Escherichia coli, yeast, baculovirus (arthropod polyhedrosis virus) -insect cells, or Animal cells and the like can be mentioned. Specifically, BL21 and XL-2Blue (manufactured by Stratagene) are used for E. coli, SP-Q01 (manufactured by Stratagene) is used for yeast, and AcNPV is used for baculovirus (J. Biol. Chem., 263, 7406, ( 1988)) and its host, Sf-9 cells (J. Biol. Chem., 263, 7406, (1988)). Examples of animal cells include mouse fibroblast C127 (J. Viol., 26, 291, (1978)) and Chinese hamster ovary cell CHO cells (Proc. Natl. Acad. Sci. USA, 77, 4216, (1980)). Among them, COS-7 cells derived from African green monkey kidney (ATCC CRL1651: American Type Culture Collection preserved cells), HEK293 cells derived from human fetal kidney (ATCC CRL1573) or Human cervical cancer HeLa cells (ATCC CCL-2) are used.
[0049]
In addition to the above-described expression method using a protein expression vector, a homologous recombination technique (AA Vertes et al., Biosci) in which a DNA fragment of the present invention linked to a promoter is directly inserted into a chromosome of a host microorganism. Biotechnol. Biochem., 57, 2036 (1993)) or a transposon or an insertion sequence (AA Vertes et al., Molecular Microbiol., 11, 739, (1994)). It can also be made.
[0050]
The obtained culture is obtained by collecting cells or cells by a method such as centrifugation, suspending the cells or the like in a suitable buffer, and sonicating, lysozyme, and / or freezing and thawing. After the disruption, a crude protein solution is obtained by centrifugation, filtration, or the like, and further purified by a combination of appropriate purification methods. Thus, the protein of the present invention is obtained. In addition to the above-described expression method using the protein expression recombinant vector, protein expression is induced by subjecting the DNA of the present invention obtained in (1) to a cell-free transcription / translation system to obtain the protein of the present invention. be able to. The cell-free transcription / translation system used in the present invention is a system containing all the elements necessary for transcription from DNA to mRNA and translation from mRNA to protein. Refers to any system in which the protein being synthesized is synthesized. Specific examples of the cell-free transcription / translation system include a transcription / translation system prepared based on an eukaryotic cell and a bacterial cell, or an extract from a part thereof. A transcription / translation system prepared based on an extract from Erythrocytes, wheat germ and Escherichia coli (Escherichia coli S30 extract) may be mentioned.
[0051]
Separation and purification of the protein of the present invention from the obtained transcription / translation product of the cell-free transcription / translation system can be carried out by a commonly used method known per se. Specifically, for example, a DNA region encoding an epitope peptide, a polyhistidine peptide, glutathione-S-transferase (GST), a maltose binding protein, or the like is introduced into the DNA to be transcribed and translated, and expressed as described above. The protein can be purified by utilizing the affinity of the protein with a substance having affinity.
[0052]
The expression of the target protein is separated by SDS-polyacrylamide gel electrophoresis or the like, and stained with Coomassie Brilliant Blue (manufactured by Sigma) or detected by an antibody that specifically binds to the protein of the present invention described later. It can be confirmed by the method of performing. In general, it is known that an expressed protein is cleaved (processed) by a proteolytic enzyme present in a living body. The protein of the present invention is, of course, included in the protein of the present invention as long as it has an apoptosis-suppressing function, even if it is a partial fragment of the truncated amino acid sequence.
[0053]
By analyzing the interaction between the thus obtained protein and other proteins and DNA, it is possible to know the multifaceted functions in the living body. As a method for analyzing the interaction, a conventional method known per se can be used. Specifically, for example, yeast two-hybrid method, fluorescence depolarization method, surface plasmon method, phage display method, ribosomal method Display method and the like can be mentioned.
[0054]
(3) Preparation of oligonucleotide
Using the DNA of the present invention or a fragment thereof obtained by the method described in (1) above, an antisense oligonucleotide having a partial sequence of the DNA of the present invention, a sense -An oligonucleotide such as an oligonucleotide can be prepared.
[0055]
Examples of the oligonucleotide include a DNA having the same sequence as 5 to 100 consecutive bases in the base sequence of the DNA or a DNA having a sequence complementary to the DNA. Specific examples include a DNA having the same sequence as 5 to 100 consecutive nucleotides in the base sequence represented by SEQ ID NO: 1 or a DNA having a sequence complementary to the DNA. When used as a sense primer and an antisense primer, the above-mentioned oligonucleotides in which the melting temperature (Tm) and the number of bases of both do not extremely change are preferable. The length of the sequence is generally 5 to 100 bases, preferably 10 to 60 bases, and more preferably 15 to 50 bases.
[0056]
In addition, derivatives of these oligonucleotides can also be used as the oligonucleotide of the present invention. Examples of the oligonucleotide derivative include an oligonucleotide derivative in which a phosphoric diester bond in an oligonucleotide is converted to a phosphorothioate bond, and an oligonucleotide in which a phosphoric diester bond in an oligonucleotide is converted to an N3′-P5 ′ phosphoramidate bond. Nucleotide derivative, oligonucleotide derivative in which ribose and phosphodiester bond in oligonucleotide are converted to peptide nucleic acid bond, oligonucleotide derivative in which uracil in oligonucleotide is substituted with C-5 propynyluracil, uracil in oligonucleotide is Oligonucleotide derivatives substituted with C-5 thiazole uracil, oligonucleotide derivatives substituted with cytosine in the oligonucleotide with C-5 propynylcytosine, oligonucleotides Is an oligonucleotide derivative in which cytosine is substituted by phenoxazine-modified cytosine, an oligonucleotide derivative in which ribose in the oligonucleotide is substituted by 2′-O-propyl ribose, or ribose in the oligonucleotide is 2 Oligonucleotide derivatives substituted with '-methoxyethoxyribose can be mentioned.
[0057]
In addition, the oligonucleotide of the present invention is prepared as a double-stranded RNA, introduced into a transfectant, and inhibits the expression of a target gene by RNA interference (hereinafter, may be abbreviated as “RNAi” in some cases). ) Method. As the RNA interference method, for example, a method described in (Elbashir, S., et al., Nature, 411, 494-498 (2001)) can be used. The double-stranded RNA does not necessarily have to be all RNA, and for example, those described in WO 02/10374 can be used.
[0058]
Here, the target gene may be any DNA as long as it is the DNA of the present invention. A double-stranded RNA consisting of a sequence substantially identical to at least a part of the base sequence of these DNAs (hereinafter sometimes referred to as “double-stranded polynucleotide”) is defined as a part of the base sequence of the target gene. And a sequence substantially the same as a sequence of 15 bp or more, which may be any part. Here, “substantially the same” means that it has 80% or more homology with the sequence of the target gene. The nucleotide length of the nucleotide may be any length from 15 bp to the entire length of the open reading frame (ORF) of the target gene, but a length of about 15 to 500 bp is preferably used. However, it is known that mammalian-derived cells have a signal transduction system activated in response to a long double-stranded RNA of 30 bp or more. This is called an interferon reaction (Mareus, PI, et al., Interferon, 5, 115-180 (1983)), and when the double-stranded RNA enters a cell, PKR (dsRNA-responsive) is obtained. Protein kinase: Non-specifically inhibits the initiation of translation of many genes via Bass, BL, Nature, 411, 428-429 (2001)), and at the same time, 2 ′, 5 ′ oligoadenylate synthetase (Bass, B.L., Nature, 411, 428-429 (2001)), RNaseL is activated, and non-specific degradation of intracellular RNA is caused. These non-specific reactions mask the specific response of the target gene. Therefore, when a mammal, or a cell or tissue derived from the animal is used as a recipient, a double-stranded polynucleotide of 15 to 30 bp, preferably 19 to 24 bp, and most preferably 21 bp is preferably used. The double-stranded polynucleotide does not need to be entirely double-stranded, and includes those in which the 5 'or 3' end is partially protruded, but those in which the 3 'end is protruded by two bases are preferably used.
[0059]
The double-stranded polynucleotide means a double-stranded polynucleotide having complementarity, but may be a self-annealed single-stranded polynucleotide having self-complementarity. The single-stranded polynucleotide having self-complementarity includes, for example, one having an inverted repeat sequence.
[0060]
The method for preparing the double-stranded polynucleotide is not particularly limited, but a known chemical synthesis method is preferably used. In chemical synthesis, a single-stranded polynucleotide having complementarity can be separately synthesized, and can be converted into a double-stranded strand by associating them by an appropriate method. Specific examples of the method of association include a method in which the synthesized single-stranded polynucleotide is mixed, heated to a temperature at which the double-strand is dissociated, and then gradually cooled. The associated double-stranded polynucleotide is confirmed using an agarose gel or the like, and the remaining single-stranded polynucleotide is removed by, for example, decomposing it with an appropriate enzyme.
[0061]
The transfectant into which the double-stranded polynucleotide thus prepared is introduced may be any as long as the target gene can be transcribed into RNA or translated into protein in the cell. Specific examples include those belonging to plant, animal, protozoan, virus, bacterial, or fungal species. The plant may be a monocotyledonous, dicotyledonous or gymnosperm, and the animal may be a vertebrate or invertebrate. Preferred microorganisms are those used in agriculture or industry, and those that are pathogenic for plants or animals. Fungi include organisms in both mold and yeast forms. Examples of vertebrates include mammals, including fish, cows, goats, pigs, sheep, hamsters, mice, rats, and humans, and invertebrates include nematodes and other reptiles, Drosophila melanogaster ( Drosophila), and other insects. Preferably, the cells are vertebrate cells.
[0062]
The transductant means a cell, tissue, or individual. Here, the cell may be from germline or somatic, totipotent, or pluripotent, split or undivided, parenchymal tissue or epithelium, immortalized or transformed, and the like. The cells may be gametes or embryos, in the case of embryos, single-cell or constitutive cells, or cells from multi-cell embryos, including fetal tissue. Furthermore, they may be undifferentiated cells, such as stem cells, or differentiated cells, such as from cells of an organ or tissue, including fetal tissue, or any other cells present in an organism. Differentiating cell types include adipocytes, fibroblasts, muscle cells, cardiomyocytes, endothelial cells, nerve cells, glial, blood cells, megakaryocytes, lymphocytes, macrophages, neutrophils, eosinophils, eosinophils, Includes basophils, mast cells, leukocytes, granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts, hepatocytes and cells of the endocrine or exocrine glands.
[0063]
As a method for introducing a double-stranded polynucleotide into a transfectant, when the transfectant is a cell or tissue, calcium phosphate method, electroporation method, lipofection method, virus infection, double-stranded polynucleotide solution Immersion, transformation, or the like. Examples of a method for introducing the gene into an embryo include microinjection, electroporation, and virus infection. When the recipient is a plant, a method of injecting or perfusing the plant into a body cavity or stromal cells, or spraying is used. In the case of an individual animal, it is introduced systemically by oral, topical, parenteral (including subcutaneous, intramuscular and intravenous administration), vaginal, rectal, nasal, ocular and intraperitoneal administration. A method, an electroporation method, a virus infection, or the like is used. For methods for oral introduction, the double-stranded polynucleotide can be mixed directly with the food of the organism. Furthermore, when introduced into an individual, it can be administered, for example, by administration as an implanted long-term release preparation or the like, or by ingesting an introduced body into which a double-stranded polynucleotide has been introduced.
[0064]
The amount of the double-stranded polynucleotide to be introduced can be appropriately selected depending on the introduced substance and the target gene, but it is preferable to introduce an amount sufficient to introduce at least one copy per cell. Specifically, for example, when the transfectant is a human cultured cell and the double-stranded polynucleotide is introduced by the calcium phosphate method, 0.1 to 1000 nM is preferable.
By suppressing the expression of the gene of the present invention in the transfectant by RNA interference, the function of the protein encoded by the gene of the present invention can be confirmed, or a new function can be analyzed.
[0065]
(4) Antibodies that specifically bind to the protein of the present invention
As a method for preparing an antibody that specifically binds to the protein of the present invention, a commonly used known method can be used. For a polypeptide used as an antigen, an epitope (antigen determination An appropriate sequence can be selected and used as the group. As a method for selecting an epitope, commercially available software such as Epitope Adviser (manufactured by Fujitsu Kyushu System Engineering Co., Ltd.) can be used.
[0066]
As the polypeptide used as the above antigen, a synthetic peptide synthesized according to a known method or the protein of the present invention itself can be used. The polypeptide serving as an antigen may be prepared in an appropriate solution or the like according to a known method and immunized to a mammal, for example, a rabbit, a mouse, a rat, or the like. It is preferable to use an antigen peptide as a conjugate with a suitable carrier protein or to add an adjuvant or the like for immunization.
[0067]
The route of administration of the antigen upon immunization is not particularly limited, and any route such as subcutaneous, intraperitoneal, intravenous, or intramuscular may be used. Specifically, for example, a method of inoculating BALB / c mice several times every several days to several weeks with an antigen polypeptide is used. The amount of the antigen to be taken is preferably about 0.3 to 0.5 mg / time when the antigen is a polypeptide, but is appropriately adjusted depending on the type of the polypeptide and the animal species to be immunized.
[0068]
After immunization, blood is appropriately collected as a test, and an increase in the antibody titer is confirmed by a method such as enzyme-linked immunosorbent assay (hereinafter sometimes referred to as “ELISA”) or Western blotting. Blood is collected from animals with increased titers. A polyclonal antibody can be obtained by subjecting this to an appropriate treatment used for antibody preparation. Specifically, for example, there is a method of obtaining a purified antibody obtained by purifying an antibody component from serum according to a known method. For purification of the antibody component, methods such as centrifugation, ion exchange chromatography, and affinity chromatography can be used.
[0069]
In addition, a monoclonal antibody can be prepared by using a hybridoma fused with spleen cells and myeloma cells of the animal according to a known method (Milstein, et al., Nature, 256, 495 (1975)). . The monoclonal antibody can be obtained, for example, by the following method.
[0070]
First, antibody-producing cells are obtained from an animal whose antibody titer has increased due to immunization with the above-described antigen. The antibody-producing cells are plasma cells and lymphocytes which are precursor cells thereof, which may be obtained from any of the individuals, but is preferably obtained from the spleen, lymph nodes, peripheral blood and the like. As a myeloma to be fused with these cells, generally, a cell line obtained from a mouse, for example, a P3X63-Ag8.653 (ATCC: CRL) which is an 8-azaguanine-resistant mouse (derived from BALB / c) myeloma cell line is used. -1580), P3-NS1 / 1Ag4.1 (RIKEN cell bank: RCB0095) and the like are preferably used. For cell fusion, antibody-producing cells and myeloma cells are mixed at an appropriate ratio, and 50% polyethylene is added to an appropriate cell fusion medium such as RPMI1640 or Iskov's modified Dulbecco's medium (IMDM) or Dulbecco's modified Eagle's medium (DMEM). It can be carried out by using a solution in which glycol (PEG) is dissolved. It can also be carried out by the electrofusion method (U. Zimmer-mann. Et al., Naturewissenschaften, 68, 577 (1981)).
[0071]
Hybridomas were prepared using a myeloma cell line that was resistant to 8-azaguanine, using 5% CO2 in a normal medium (HAT medium) containing an appropriate amount of hypoxanthine / aminopterin / thymidine (HAT) solution.₂At 37 ° C. for an appropriate time. This selection method can be appropriately selected and used depending on the myeloma cell line to be used. The antibody titer of the antibody produced by the selected hybridoma is analyzed by the above-described method, the hybridoma producing the antibody having a high antibody titer is separated by a limiting dilution method or the like, and the separated fused cells are cultured in an appropriate medium. The monoclonal supernatant can be obtained by purifying the resulting culture supernatant by an appropriate method such as ammonium sulfate fractionation and affinity chromatography. For purification, a commercially available monoclonal antibody purification kit can also be used. Furthermore, ascites containing a large amount of the monoclonal antibody of the present invention can be obtained by growing the antibody-producing hybridoma obtained above in the abdominal cavity of an animal of the same strain as the immunized animal or a nude mouse.
[0072]
When a human-derived protein is obtained as the protein of the present invention, the above-described method is applied to a Severe combined immunodeficiency (SCID) mouse transplanted with human peripheral blood lymphocytes using the polypeptide or a partial peptide thereof as an antigen. A humanized antibody can also be prepared by immunization in the same manner as described above and preparing a hybridoma of the antibody-producing cells of the immunized animal and human myeloma cells (Mosier, DE, et al. Nature, 335, 256-259 (1988); Duchosal, MA, et al., Nature, 355, 258-262 (1992)).
[0073]
Further, RNA is extracted from the obtained hybridoma producing the human antibody, a gene encoding the target human antibody is cloned, this gene is inserted into an appropriate vector, and this is introduced into an appropriate host. By expression, human antibodies can be produced in larger quantities. Here, an antibody with low binding to an antigen can be obtained as an antibody with even higher binding by using an evolutionary engineering technique known per se. A partial fragment such as a monovalent antibody can be prepared by cleaving the Fab and Fc portions using, for example, papain or the like, and collecting the Fab portion using an affinity column or the like.
[0074]
The thus-obtained antibody that specifically binds to the protein of the present invention can also be used as a neutralizing antibody that specifically binds to the protein of the present invention and thereby inhibits the apoptosis-suppressing function of the protein. There is no particular limitation on the method of selecting a substance that inhibits the activity of the protein. For example, it is possible to contact an antibody with the DNA transfectant prepared in (2) above and determine whether the function of the target protein in the transfectant is inhibited. And a method of analyzing the above.
[0075]
Such a neutralizing antibody may be used alone when the clinical application is performed, or may be used as a pharmaceutical composition by mixing with a pharmaceutically acceptable carrier. At this time, the ratio of the active ingredient to the carrier can be varied between 1 and 90% by weight. Such drugs can be administered in various forms, such as tablets, capsules, granules, powders, orally administered by syrup or the like, or injections, drops, liposomes, Parenteral administration with suppositories and the like can be mentioned. In addition, the dose can be appropriately selected depending on symptoms, age, body weight, and the like.
[0076]
(5) Confirmation of the activity of the protein of the present invention
The protein of the present invention is prepared as a recombinant protein as described in the above (2), and by analyzing this, it can be confirmed that it has the activity estimated in the above (1). Furthermore, analysis can also be performed by combining the antibody and the like prepared as described in (4) above.
Since the protein of the present invention is structurally similar to Baculoviral IAP repeat-containing protein and Inhibitor of apoptosis protein, it was speculated that it is a molecule having an apoptosis inhibitory action.
[0077]
The apoptosis-suppressing function of the protein of the present invention can be verified, for example, by the following method (Nature 379 (1996) 349-353). A vector for expressing the gene encoding the protein of the present invention and a vector not containing an insert for negative control were transferred to CHO cells, HeLa cells, Rat-1 cells, etc. according to a conventional method such as an adenovirus method or a lipofectamine method. One day after transient gene transfer, induction of apoptosis by serum removal, menadione treatment (1 μM to 50 μM), or TNF-α treatment (TNF-α 20 units / ml and cycloheximide 30 μg / ml) After 3 days of culture, the apoptosis-suppressing activity of the protein of the present invention can be examined by calculating the survival rate by measuring trypan blue staining, MTT staining, or eluted LDL activity.
The above-described functional assay system can be used for screening for an expression controlling substance of the protein of the present invention, and for screening a substance for activating or inhibiting a function of the protein of the present invention. Note that confirmation of the activity of the protein of the present invention is not limited to the method described above.
[0078]
Examples of the method for analyzing the function of the protein of the present invention include, for example, (i) a method for comparative analysis of the expression state in each tissue, disease, or developmental stage, and (ii) an interaction with another protein or DNA. A method of analyzing, (iii) a method of introducing into a suitable cell or individual, and analyzing a phenotypic change, and (iv) a method of analyzing the phenotypic change by inhibiting the expression of the protein in a suitable cell or individual. And the like. Moreover, according to such a method, the activity specific to the target protein can be analyzed from many aspects.
[0079]
In the method (i), the expression of the protein of the present invention can be analyzed at the mRNA level or the protein level. When the expression level is analyzed at the mRNA level, for example, an in situ hybridization method (In situ hybridization: Application to Developmental Biology & Medicine., Ed. by Harry, N. ed. 1990)), a hybridization method using a DNA chip, a quantitative PCR method, and the like. In the case of analysis at the protein level, ELISA using an antibody that specifically binds to the protein of the present invention described later, Western blotting, tissue staining, and the like can be mentioned. Here, when a known variant is present in the protein to be analyzed, it is preferable to use a probe that is present only in the cDNA encoding the protein to be analyzed and does not hybridize with the cDNA encoding the known variant. In the case of the quantitative PCR method, a method of selecting primers capable of producing amplified fragments having different lengths between the target cDNA and the known variant (Wong, Y., Neuroscience Let., 320: 141-145 (2002)) and the like Is mentioned. Also, when analyzing at the protein level, it is preferable to use an antibody that reacts only with the target protein and does not react with a known variant.
[0080]
In the method (ii), the function of the protein of the present invention can be analyzed by examining the presence or absence of interaction between the protein of the present invention and a known protein. As a method for analyzing the interaction, a conventional method known per se can be used, and specifically, for example, yeast two-hybrid method, fluorescence depolarization method, surface plasmon method, phage display method, ribosomal display And the like. Also in this method, when a known variant exists in the protein to be analyzed, it is preferable to analyze the interacting substance of the known variant in the same manner to identify a substance that specifically interacts with the target protein.
[0081]
In the above method (iii), the cells into which the cDNA of the present invention is introduced are not particularly limited, but human cultured cells are particularly preferably used. Methods for introducing DNA into cells include those described in (2) above. In addition, the phenotype of the introduced cells can be observed with a microscope, such as cell viability, cell growth rate, cell differentiation, neurite outgrowth when cells are neurons, localization and migration of intracellular proteins, etc. And those that can be analyzed by biochemical experiments, such as changes in the expression of specific proteins in cells. These phenotypes are associated with the target protein by introducing the DNA of the present invention or DNA encoding the known variant into cells in the same manner when a known variant exists in the target protein, and performing comparative analysis. The phenotype can be identified. Further, since it is known that the protein of the present invention has an apoptosis-suppressing function, it is also preferable to analyze the protein by focusing on the phenotype and the like found in diseases associated with apoptosis suppression.
[0082]
The method (iv) can be efficiently performed by a method using an oligonucleotide described below or an RNA interference method. In this method, when a known variant is present in the target protein to be analyzed, a similar analysis is performed for the known variant and other variants, and a target protein-specific function is identified by comparative analysis. be able to.
[0083]
(6) Screening for a molecule that regulates the activity of the protein of the present invention
By screening for a substance that specifically binds to the protein of the present invention and has an action of inhibiting, antagonizing, or enhancing the function (activity) of the protein of the present invention, a function modulator of the protein of the present invention (hereinafter, referred to as (Sometimes referred to as "modulators").
[0084]
This method of screening for a regulatory substance may be any method as long as it can obtain a substance that specifically binds to the protein of the present invention and has an activity of inhibiting, antagonizing or enhancing the activity of the protein. For example, a method of first contacting a protein of the present invention with a test substance and selecting the test substance based on the binding property to the protein as an index, and then selecting a test substance using a change in the activity of the protein of the present invention as an index is used. be able to.
[0085]
The test substance may be any substance that interacts with the protein of the present invention and may affect the activity of the protein, and specifically, for example, a peptide, Examples include proteins, non-peptidic compounds, low molecular compounds, synthetic compounds, fermentation products, cell extracts, animal tissue extracts, and the like. These substances may be novel substances or known substances. As a method for analyzing the interaction between the test substance and the protein of the present invention, a conventional method known per se can be used. Specifically, for example, yeast two-hybrid method, fluorescence depolarization method, surface plasmon method Phage display method, ribosomal display method, or the competition analysis method with the antibody described in the above (4). A substance found to bind to the protein of the present invention by such a method is then analyzed by analyzing how the activity of the protein of the present invention is affected in the presence of the substance. Whether it is used as a modulator is identified.
[0086]
As a specific analysis method, specifically, the method described in the above (5) can be used. If the activity of inhibiting apoptosis is increased as compared to the absence of the substance, the substance may function as an activator of the inhibition of apoptosis. The substance can be identified as possibly functioning as an inhibitor of apoptosis.
Here, for the purpose of screening for a pharmaceutically active ingredient, it is preferable to use the above-mentioned human homolog protein or ortholog protein for the DNA or recombinant protein of the present invention to be used. Furthermore, the substances screened by the above method may be selected as drug candidates by screening in vivo.
[0087]
Since the protein of the present invention seems to have an apoptosis-suppressing function, the expression controlling substance, function-activating substance, or function-inhibiting substance of the protein of the present invention includes Alzheimer's disease, Parkinson's disease, and other neurodegenerative diseases such as ischemic encephalopathy. Prevention or prevention of cancer, muscular atrophy, acquired immunodeficiency syndrome, immune diseases, diseases involving tumor necrosis factor (TNF) such as endotoxin shock and other cytokines (IL-1, IL-6, etc.) It can be used as a therapeutic pharmaceutical composition.
[0088]
Such modulators can be used alone for the clinical application, but can also be used as pharmaceutical compositions by mixing with a pharmaceutically acceptable carrier. At this time, the ratio of the active ingredient to the carrier can be varied between 1 and 90% by weight. Such drugs can be administered in various forms, such as tablets, capsules, granules, powders, orally administered by syrup or the like, or injections, drops, liposomes, Parenteral administration with suppositories and the like can be mentioned. In addition, the dose can be appropriately selected depending on symptoms, age, body weight, and the like.
[0089]
(7) Screening of the DNA expression regulator of the present invention
Examples of the screening method include a method of analyzing the expression level of the protein of the present invention or the mRNA encoding the same in the presence of a test substance. Specifically, for example, cells expressing the protein of the present invention described in (2) above are cultured in an appropriate medium containing a test substance, and the amount of the protein of the present invention expressed in the cells is determined by ELISA. And the like, or by analyzing the amount of mRNA encoding the protein of the present invention in the cells by quantitative reverse transcription PCR, Northern blotting, or the like.
[0090]
As the test substance, those described in the above (6) can be used. According to this analysis, if the amount of the protein or mRNA expressed in the cells cultured in the absence of the test substance increases as compared with the amount of the test substance, the substance functions as a substance for promoting the expression of the DNA of the present invention. If it is possible and, on the contrary, decreases, it can be determined that the substance can be used as a substance inhibiting the expression of the DNA of the present invention.
[0091]
The above-mentioned active ingredient can be used alone for clinical application, but can also be used as a pharmaceutical composition by blending it with a pharmaceutically acceptable carrier. At this time, the ratio of the active ingredient to the carrier can be varied between 1 and 90% by weight. Such drugs can be administered in various forms, such as tablets, capsules, granules, powders, orally administered by syrup or the like, or injections, drops, liposomes, Parenteral administration with suppositories and the like can be mentioned. In addition, the dose can be appropriately selected depending on symptoms, age, body weight, and the like.
[0092]
(8) The DNA-introduced animal of the present invention
The transfected DNA containing the DNA of the present invention described in the above (1) is constructed, introduced into a fertilized egg of a mammal other than a human, and this is transplanted into a female individual uterus to generate the DNA. A non-human mammal into which DNA has been introduced can be produced. More specifically, for example, after superovulation of a female individual by hormone administration, it is mated with a male, a fertilized egg is extracted from an oviduct on the first day after mating, and the introduced DNA is microinjected into the fertilized egg. It is introduced by the method described above. Thereafter, after culturing by an appropriate method, the surviving fertilized eggs are transplanted into the uterus of a pseudopregnant female individual (foster parent) to give birth. Whether or not the target DNA has been introduced into the newborn can be identified by performing Southern blot analysis on DNA extracted from cells of the individual. Examples of mammals other than humans include mice, rats, guinea pigs, hamsters, rabbits, goats, pigs, dogs, cats, and the like.
[0093]
The thus-obtained DNA-introduced animal of the present invention obtains its offspring by crossing this individual and subculturing it in a normal breeding environment while confirming that the introduced DNA is stably retained. be able to. In addition, the offspring can be obtained by repeating in vitro fertilization, and the strain can be maintained.
The non-human mammal into which the DNA of the present invention has been introduced can be used as an analysis of the function of the DNA of the present invention in a living body, or as a screening system for a substance regulating the function.
[0094]
(9) Other uses of the protein of the present invention and DNA containing a nucleotide sequence encoding the same
The protein of the present invention can be used as a carrier having it bound on a substrate. In addition, a nucleotide sequence encoding the protein of the present invention, for example, a DNA having the nucleotide sequence of SEQ ID NO: 1 and a partial fragment thereof can be used as a carrier obtained by binding them on a substrate. These may be hereinafter referred to as “protein chips”, “DNA chips” or “DNA arrays” (DNA microarrays and DNA macroarrays). These protein chips or DNA chips or arrays may contain other proteins and DNAs in addition to the proteins and DNAs of the present invention.
[0095]
Here, a resin substrate such as a nylon film or a polypropylene film, a nitrocellulose film, a glass plate, a silicon plate, or the like is used as a substrate for binding proteins and DNA. When using a fluorescent substance or the like, a glass plate or a silicon plate containing no fluorescent substance is preferably used. The binding of the protein or DNA to the base can be easily carried out by a commonly used method known per se. These protein chips, DNA chips, or DNA arrays are also included in the scope of the present invention.
[0096]
In addition, the amino acid sequence of the protein of the present invention and the nucleotide sequence of DNA can also be used as sequence information. Here, the nucleotide sequence of the DNA includes the nucleotide sequence of the corresponding RNA. That is, by storing the obtained amino acid sequence or base sequence in an appropriate recording medium in a predetermined format readable by a computer, a database of the amino acid sequence or base sequence can be constructed. This database may contain the base sequences of other types of proteins and DNAs encoding them. Further, in the present invention, the database also means a computer system that writes the above-mentioned sequence on an appropriate recording medium and performs a search according to a predetermined program. Examples of suitable recording media include magnetic media such as flexible disks, hard disks, and magnetic tapes; optical disks such as CD-ROM, MO, CD-R, CD-RW, DVD-R, and DVD-RW; and semiconductor memories. And the like.
[0097]
【Example】
Hereinafter, the present invention will be described in detail with reference to examples, but the scope of the present invention is not limited to these examples.
Example 1  Preparation of cDNA library
(1) Preparation of mRNA
mRNA-prepared mouse (C57BL / 6) 0.5 to 1 g of each organ or tissue is homogenized with 10 ml of a suspension, and 1 ml of 2M sodium acetate at pH 4.0 and the same amount of phenol / chloroform (5: 1 by volume). The mixture was added and extracted. When the same amount of isopropanol was added to the aqueous layer after the extraction, RNA separated and precipitated from the aqueous phase. After incubating the sample on ice for 1 hour, the precipitate was collected in a refrigerated centrifuge at 4,000 rpm for 15 minutes. The sample was washed with 70% ethanol, dissolved in 8 ml of water, and added with 2 ml of 5 M NaCl, 1% of CTAB (cetyltrimethy-lammonium bromide), 4 ml of urea, and 16 ml of an aqueous solution of pH 7.0 containing 50 mM Tris to remove RNA. The precipitate was removed to remove the polysaccharide (CTAB precipitation).
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Subsequently, the RNA was centrifuged at 4,000 rpm for 15 minutes at room temperature to dissolve the RNA in 4 ml of 7M guanidine-C1. After adding twice the volume of ethanol, the mixture was incubated on ice for 1 hour, centrifuged at 4,000 rpm for 15 minutes, and the resulting precipitate was washed with 70% ethanol to collect RNA, which was dissolved again in water. RNA purity was measured by reading the OD ratios 260/280 (> 1.8) and 230/260 (<0.45).
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(2) Preparation of first strand cDNA
Using 15 μg of the mRNA prepared in (1) above, 5-methyl-dCTP, dATP, dTTP, and dGTP were converted to 0.54 mM and 0.6 M trehalose in a reaction volume of 165 μl using reverse transcriptase 3,000 units. , 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂A reverse transcription reaction was performed under the conditions of 10 mM DTT, 52 ng / μl BSA, and RNase inhibitor 5 units. Oligonucleotide (SEQ ID NO: 3) containing a recognition sequence of restriction enzyme XhoI (wherein V represents A, G, or C, and N represents A, G, C, or T), using 12.6 μl as a primer Was.
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At the start of the reaction, 1/4 of the reaction solution is collected, and 1.5 μl of [α-32P] -dGTP (3000 Ci / mmol, 10 μCi / μl; manufactured by Amersham) is added to the first strand cDNA. Was measured for synthesis efficiency. 0.5 μl of the RI-labeled reaction solution was spotted on DE-81 paper, and the RI activity before and after washing three times with 0.5 M sodium phosphate buffer (pH 7.0) was measured and calculated. Thereafter, the RI-labeled reaction solution and the non-labeled reaction solution were mixed, 8 μl of 0.5 M EDTA, 2 μl of 10% SDS, and 20 μg of proteinase K were added, and the mixture was heated at 45 ° C. for 15 minutes. After extraction with phenol / chloroform and ethanol precipitation, the precipitate was dissolved in 47 μl of RNase-free water (hereinafter referred to as RNase-free water).
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(3) 5 'cap structure and addition of biotin to 3' end
Biotinylated RNA Diol In order to bind biotin to the diol site (present at both the 5 'end of the Cap structure and the ribose at the 3' end of the poly A chain), a two-step reaction was performed. They are the oxidation of a diol group followed by the coupling reaction of biotin hydrazide with an oxidized RNA. First, 15 μg of the RNA-first strand cDNA complex obtained by the reverse transcription reaction was placed in a 50 μl reaction mixture using a 6.6 mM sodium acetate buffer (pH 4.5) and sodium periodate as an oxidizing agent. Processed. This oxidation reaction was performed on ice for 45 minutes under light-shielded conditions.
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Subsequently, 11 μl of 5 M sodium chloride, 0.5 μl of 10% SDS, and the same amount of isopropanol were added, left on ice for 60 minutes, and centrifuged at 4 ° C. for 15 minutes at 15,000 rpm to obtain a precipitate. The precipitate was washed with 70% ethanol and redissolved in 50 μl of RNase-free water. 5 μl of 1 M sodium acetate (pH 6.1), 5 μl of 10% SDS, and 150 μl of 10 mM biotin hydrazide (manufactured by Sigma) were added to the sample, and the mixture was reacted at room temperature (22 to 26 ° C.) overnight. Finally, 5 μl of 5 M NaCl, 75 μl of 1 M sodium acetate (pH 6.1) and 2.5 volumes of ethanol were added, and the mixture was cooled on ice for 1 hour, centrifuged at 4 ° C. for 15 minutes, and biotinylated. After the reaction, the reaction solution was centrifuged for 15 minutes to precipitate the RNA-DNA complex again. The precipitate was washed once with 70% ethanol and once with 80% ethanol, and dissolved in 70 μl of RNase-free water.
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(4) Selection of full-length cDNA by RNase I
By treating the biotinylated RNA-DNA complex obtained in the above (3) with RNase I that digests single-stranded RNA, mRNA whose mRNA was not completely elongated during the reverse transcription reaction, and mRNA of mRNA The biotin residue labeled at the 3 'end was removed. Specifically, 10 μl of 10 × RNase I buffer (100 mM Tris-HCl (pH 7.5), 50 mM EDTA, 2 M NaOAc) was added to 70 μl of the sample obtained in (3), and RNase I (RNase One).^TM200 units (Promega), and the single-stranded RNA was digested at 37 ° C. for 15 minutes.
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(5) Collection of full-length cDNA
In order to prevent non-specific adsorption of cDNA to magnetic beads coated with streptavidin, 5 μg (500 μl) of 100 μg of yeast tRNA (treated with DNase I) was used as magnetic beads (MPG) particles coated with streptomyvit. (CPG, NJ)) and left on ice for 1 hour, followed by washing with a solution of 50 mM EDTA and 2 M NaCl.
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The beads were suspended in 500 μl of a solution of 50 mM EDTA and 2 M NaCl, and the RNase I-treated cDNA obtained in (4) above was added. By stirring for 30 minutes at room temperature, the magnetic beads and the full-length cDNA were bound. The beads capturing the full-length cDNA were washed 4 times with a solution of 50 mM EDTA and 2 M NaCl, and once with 0.4% SDS, 50 μg / μl yeast tRNA, 10 mM NaCl, 0.2 mM EDTA, and 10 mM Tris-HCl (pH 7.5). RNase H buffer (20 mM Tris-HCl (pH 7.5), 10 mM MgCl 2, once with 20% glycerol, once with a 50 μg / μl aqueous solution of yeast tRNA.₂After washing once with 20 mM KCl, 0.1 mM EDTA, and 0.1 mM dithiothreitol (DTT), the cells were suspended in RNase H buffer (100 μl), RNase H (3 units) was added, and the mixture was heated at 37 ° C. for 30 minutes. Thereafter, 1 μl of 10% SDS and 2 μl of 0.5 M EDTA were added, and the mixture was exposed to 65 ° C. for 10 minutes, and the supernatant was collected.
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The thus recovered single-stranded full-length cDNA was extracted with phenol / chloroform, and the volume of the solution was reduced to 100 μl or less using a speed bag, and then subjected to G25 / G100 Sephadex chromatography. The fraction having RI activity was collected in a silicon-treated microtube, and 2 μg of glycogen was added, and the precipitate obtained by ethanol precipitation was dissolved in 30 μl of ultrapure water.
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(6) Addition of oligo dG to single-stranded cDNA
30 μl of the single-stranded cDNA recovered in the above (5) was mixed with 200 mM sodium cacodylate (pH 6.9), 1 mM MgCl 2 in a final volume of 50 μl of the reaction solution.₂, 1 mM CoCl₂Under the conditions of 1 mM 2-mercaptoethanol and 100 μM dGTP, oligo dG addition reaction was carried out at 37 ° C. for 30 minutes using 32 units of terminal deoxynucleotidyl transferase (TaKaRa). At the end of the reaction, EDTA was added to 50 mM and dissolved in 31 μl of ultrapure water through a series of extractions with phenol / chloroform and ethanol precipitation.
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(7) Second strand cDNA synthesis
The synthesis of the second-strand cDNA using the first-strand cDNA as a template was performed as follows. In a reaction system having a final volume of 60 μl, a second strand low buffer (200 mM Tris-HCl (pH 8.75), 100 mM KCl, 100 mM (NH₄)₂SO₄, 20 mM MgSO₄3% of 1% Triton X-100, 1 mg / μl BSA, second strand high buffer (200 mM Tris-HCl (pH 9.2), 600 mM KCl, 20 mM MgCl₂) 3 μl, 0.25 mM each of dCTP, dATP, dTTP, and dGTP, 6 μl of β-NADH, 31 μl of oligo dG-added first-strand cDNA, 600 ng of second-strand primer-adapter (SEQ ID NO: 4), and Ex Taq DNA polymerase ( Second-strand cDNA was synthesized using 15 units of TaKaRa Ex Taq (TaKaRa), 150 units of thermostable DNA ligase (Ampligase; Epicentre), and 3 units of heat-resistant RNase H (Hybridase; Epicentre).
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The reaction was stopped by adding 1 μl of 0.5 M EDTA, and further heated at 45 ° C. for 15 minutes in the presence of 1 μl of 10% SDS and 10 μg of proteinase K to finally dissolve the protein components. A double-stranded full-length cDNA purified by extraction with ethanol / chloroform and ethanol precipitation was obtained.
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(8) Preparation of library
The double-stranded full-length cDNA obtained by the above method was inserted into a λZAPIII vector and recovered as a library. The λZAPIII vector is obtained by modifying SEQ ID NO: 5, which is a partial sequence of the multiple cloning site of λZAPII (manufactured by STRATAGENE) vector, to SEQ ID NO: 6 and newly introducing two SfiI sites.
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Further, a λPS (RIKEN) vector was prepared, and cDNA was inserted. λPS (RIKEN) (named λ-FLC-1 (FLC means FULL-LENGTH cDNA)) is a λPS vector of MoBiTec (Germany) modified for cDNA. That is, BamHI and SalI convenient for cDNA insertion are respectively introduced into cloning sites existing on both sides of a 10 kbp stuffer, and a 6 kb DNA fragment is inserted into an XbaI site so that a cDNA of about 0.5 kb to about 13 kb can be cloned. (JP-A-2000-325080). Using this λ-FLC-1, for example, in the case of a lung cDNA library, the average chain length of the insert was 2.57 kb, and it was possible to actually clone an insert of 0.5 kb to 12 kb. In the case of the conventional method λZAP, the average chain length of the insert was 0.97 kb, indicating that the use of λ-FLC-1 enables the cloning of a large-sized cDNA more efficiently than λZAP.
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Example 2  Normalization / subtraction of full-length cDNA library
(1) Preparation of driver
The mRNA prepared in Example 1 (1) (hereinafter sometimes referred to as “(a) RNA driver”) and the RNA prepared by in vitro transcription reaction were used as drivers. The latter RNA is further divided into two types (hereinafter referred to as “(b) RNA driver” and “(c) RNA driver”). One is obtained by recovering cDNA from RNA-cDNA removed by normalization and cloning into a phage vector. After infection with Escherichia coli, 1000 to 2000 plaques per starting material are mixed to form one library (mini-library), which is converted into plasmid DNA by a conventional method (the phage is infected again with Escherichia coli together with helper phage to form a phagemid). , And another infection to obtain plasmid DNA).
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The obtained DNA was subjected to an in vitro transcription reaction (using T3 RNA polymerase or T7 RNA polymerase), treated with DNase I (RQ1-RNase free; manufactured by Promega) and Proteinase K, and then extracted with phenol / chloroform. (B) RNA driver Got. At this time, as a starting material, a mini-library is prepared from each of nine types of tissues (pancreas, liver, lung, kidney, brain, spleen, testes, small intestine, stomach), and the nine types of mini-libraries are mixed. To obtain RNA. As another RNA, a library (about 20,000 clones) already stored as a non-overlapping clone is cultured, and the obtained DNA is subjected to in vitro transcription reaction in the same manner as (b) RNA driver (c). ) RNA driver.
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These three kinds of RNAs were labeled with biotin using Label-IT Biotin Labeling Kit (manufactured by Mirus Corporation), added to tester cDNA at a ratio of 1: 1: 1, and reacted with Rot10 (42 ° C.). The second strand was synthesized with respect to the supernatant collected after the treatment with streptavidin beads (CPG).
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Example 3  Nucleotide sequencing of full-length cDNA clones
(1) clone rearray
One representative clone was selected from each cluster. Representative clones were selected with Q-bot (GENETIX LIMITED) and arrayed on a 384-well plate. At that time, E. coli was cultured in 50 μl of LB medium at 30 ° C. for 18 to 24 hours. At this time, when the cDNA library was introduced into the PS vector and transformed Escherichia coli DH10B, 100 mg / ml ampicillin and 50 mg / ml kanamycin were added, introduced into the Zap vector, and introduced into the SOLR system. If so, 100 mg / ml ampicillin and 25 mg / ml streptavidin were added.
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(2) Extraction of plasmid and InsSizing
Each of the clones cultured in the above (1) is further cultured in 1.3 ml of an HT solution containing 100 mg / ml of ampicillin, and the cells are collected by centrifugation. Then, QIAprep 96 Turbo (manufactured by QIAGEN) is used. To recover and purify the plasmid DNA. In order to examine the chain length of the cDNA inserted in the obtained plasmid, 1/30 of the plasmid DNA obtained above was digested with the restriction enzyme PvuII and subjected to 1% agarose gel electrophoresis.
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(3) Sequencing
Three types of sequencers were used to analyze the full-length nucleotide sequence of the full-length cDNA inserted into the thus obtained plasmid. In addition, plasmids were divided into two categories: those having insertion sequences shorter than 2.5 kb and those having longer insertion sequences. Among these, the nucleotide sequence of the clone having an insertion sequence shorter than 2.5 kb was analyzed from both ends. At this time, the plasmid was prepared using the primers described in SEQ ID NO: 7 (sense strand) and 8 (antisense strand) when the vector was PS, and SEQ ID NO: 9 (sense strand) when the vector was Zap. , And 10 (antisense strand) using the Thermosequenase Primer Cycle Sequencing Kit (manufactured by Amersham Pharmacia Biotech), and analyzed using Licor DNA4200 (long read sequencer).
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Gaps that could not be analyzed by the above nucleotide sequence analysis were determined by the primer walking method. At this time, ABI Prism 377 and / or ABI Prism 3700 (manufactured by Applied Biosystems Inc.), BigDye terminator kit and Cycle Sequencing FS Ready Reaction Kit (Applied Systems, Inc.) were used.
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In addition, the sequence of a clone in which the inserted cDNA was longer than 2.5 kb was determined by the shotgun method. At that time, Shimadzu RISA 384 and DYEnamic ET terminator cycle sequencing kit (manufactured by Amersham Pharmacia Biotech) were used. To generate a shotgun library, 48 DNA fragments grown by PCR from 48 independent representative clones were used. The ends of the amplified DNA fragment were blunt-ended with T4 DNA polymerase.
This DNA fragment was inserted into a pUC18 vector, and Escherichia coli DH10B was transformed with the recombinant vector. A plasmid DNA was prepared from this Escherichia coli in the same manner as in the above (2).
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About those representative clones, the base sequence was determined by base sequence analysis from both ends, and the base sequences were ligated on a computer, and then subjected to sharing using Double Stroke Sharing Device (manufactured by Fire Inc.). Nucleotide sequence determination by the shotgun method was performed with duplication of 12 to 15 clones. The gaps whose sequence could not be determined by this nucleotide sequence determination were determined by primer walking in the same manner as described above.
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Example 4  Analysis of nucleotide sequence
As shown in SEQ ID NO: 1, dnform 54482 was composed of 1531 bases, of which base numbers 125 to 595 were open reading frames (including a stop codon). The amino acid sequence predicted from the open reading frame consists of 156 amino acid residues (SEQ ID NO: 2). When a homology search was performed on the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 1 using BLAST, the SPTR protein database (integrating the SWISS-PROT protein sequence database and the TrEMBL nucleic acid translation database) contained (i ) Database registration symbol Q13490, Baculoviral IAP repeat-containing protein 3 is e-value: 2 × 10^-20And (ii) the database registration code Q90660, Inhibitor of apoptosis protein, e-value: 1 × 10^-18And (iii) the database registration code P41436, an Apoptosis inhibitor IAP, with e-value: 2 × 10^-17In 154 amino acid residues with a 30% match. From these results, it was inferred that the protein having the amino acid sequence shown in SEQ ID NO: 2 was a protein having an apoptosis-suppressing action.
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The protein (i) is related to the inhibition of apoptosis based on literature information (Nat. Struct. Biol. 6: 648-651 (1999)) in the database, and the protein (ii) is related to the database From the literature information (DNA Cell Biol. 15: 981-988 (1996)), it is suggested that the protein is related to the suppression of apoptosis of T cells, and the protein (iii) is described in the literature information (J. Virol. 67) in the database. : 2168-2174 (1993)).
In addition, a protein motif search by HMMPFAM was performed on the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 1. When amino acid numbers 96 to 156 in SEQ ID NO: 2 were found, Baculovirus Inhibitor of apoptosis protein Repeat (amino acid that is entered as a BIR in Pfam) Sequence, also described as IAP repeat).
From these facts, it was presumed that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 1 was a protein having a function related to suppression of apoptosis.
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Example 5  Protein-protein interaction analysis
Using the two-hybrid method in mammalian cells (Suzuki, H., et al., Genome Research, 11, 1758-1765 (2001)), the nucleotide sequence of one kind of mouse full-length cDNA (dnaform 54482) is encoded. The protein-protein interaction of the protein was comprehensively analyzed.
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(1) Rapid sample preparation using PCR method
The CheckMate mammarian two-hybrid system (Promega) was used for the two-hybrid experiments on mammalian cells. Samples for protein-protein interaction analysis were a plasmid vector pBIND having a Gal4 gene DNA binding region inserted downstream of a CMV promoter, a plasmid vector pACT having a VP16 gene transcription activation region inserted downstream of a CMV promoter, and 5 Was prepared using a plasmid vector pG5luc in which a reporter luciferase gene was inserted downstream of the Gal4 binding region and the TATA box. A fusion gene of the Gal4 gene and a protein coding sequence of one kind of mouse full-length cDNA (dnaform 39540), and the VP16 gene and a mouse cDNA library FANTOM (http://fantom.gsc.liken.go.jp/) A fusion gene with the protein coding sequence of the full-length cDNA of each clone was prepared basically by combining ligation using a common sequence and a two-step PCR method according to the protocol of Promega (Suzuki, H., et al.). al., Genome Research, 11, 1758-1765 (2001)).
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The protein coding sequence of the mouse cDNA was PCR-amplified using a forward primer having a common sequence on the 5 ′ side and a gene-specific sequence on the 3 ′ side and an M13 universal primer, and then the above amplification product and pBIND or pACT were amplified. A PCR amplification product (with a common sequence added to the 3 ′ side) is mixed, and a second-stage PCR amplification is performed using nested primers to express a fusion protein of Gal4 and mouse protein (BIND sample) Alternatively, a vector (ACT sample) for expressing a fusion protein of VP16 and mouse protein was constructed.
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(2) Two-hybrid experiments on high-throughput mammalian cells
BIND and ACT samples prepared by the PCR method were used directly without further purification. 0.25 μl each of the BIND sample and the ACT sample, 30 ng pG5luc, and 9.5 μl Opti-MEM medium (Lifetech) were dispensed into a 384-well plate. 10 μl of LF2000 transfection reagent (Lifetech) diluted 32 times in Opti-MEM medium was added to the wells, mixed, incubated for 20 minutes, and then suspended in F12 medium at 1,300 cells / μl in CHO-K1 Chinese hamster. 20 μl of the cell solution was added and well suspended. The assay sample is₂After culturing for 20 hours in an incubator, luciferase activity was measured using Steady-Glo Luciferase Assay System (Promega) to confirm the interaction. Table 1 shows the results.
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[Table 1]

[0128]
As is clear from Table 1, the protein encoded by the nucleotide sequence (dnaform54482) of the mouse full-length cDNA is as follows, using the mouse cDNA library FANTOM (HYPERLINK http://fantom.gsc.liken.go.jp/http). ///Fantom.gsc.riken.go.jp/) has been found to interact with the protein encoded by the nucleotide sequence of the cDNA of the specific clone.
That is, it was confirmed that the protein encoded by dnaform54482 interacts with cell division cycle protein 23 (CDC23). CDC23 has a function of leading to late cell division. It was speculated that the interaction of this protein with CDC23 is involved in the control of cell division and affects apoptosis. Furthermore, the mutation of CDC23 is associated with carcinogenesis (Oncogene 2003, 22 (10): 1486-90), and it was speculated that the present protein may regulate the process.
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In addition, it was confirmed that the present protein interacts with cytokeratin 10, keratin (homology to high sulfur protein B2F), or putative keratin-associated protein. When epidermal cells are formed, they are born in the basal layer, migrate toward the outer skin, and, at the granular cell layer, gradually lose nuclei and organelles by a partial apoptotic mechanism and become keratinized. It was speculated that this protein controls such a differentiation process.
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In addition, the present protein was found to interact with leucine-rich repeat-containing protein. Leucine-rich repeat (LRR) is a 20-29 amino acid residue motif found in many proteins in tandem. Proteins having this are involved in many functions such as hormone-receptor interaction, enzyme inhibition, cell adhesion, and intracellular transport. It is also known from recent findings that the LRR protein is involved in early mammalian development, nervous system development, cell polarization, regulation of gene expression, and apoptosis signaling. LRR is thought to form a structural framework that performs these various functions. Therefore, it was speculated that this protein may be involved in the control of cell division, differentiation, apoptosis and the like.
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Further, it was confirmed that the present protein interacts with the hypothetical outer arm dynein light chain 1 structure containing protein. Outer arm dynein is one of the motor proteins and belongs to the AAA + family ATPase, and “axonal dynein” is responsible for ciliary and flagella movement and “cytoplasmic dynein is responsible for intracellular transport and cell division of membrane vesicles and the like. There are two types. Since the present protein interacts with the hypothetical outer arm dynein light chain 1 structure containing protein, the present protein is involved in cilia or flagella movement, or transport or cell division of membrane vesicles or the like in cells. Was speculated. Also, it is known that agonist stimulation of beta-adrenergic receptor causes phosphorylation of outer arm dynin in airway epithelial cells (J. Allergy Clin. Immunol. 2002, 110 (6Suppl): S275-81), It is presumed that there is a connection with respiratory diseases such as bronchial asthma through ciliary movement and the like.
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Example 6  Tissue expression analysis using PCR method
In order to examine changes in tissue expression of mRNA encoding the protein of the present invention in normal and diseased mice, PCR is performed according to a conventional method (Higuchi R, et al., Biotechnology, 11: 1026-30 (1993)). Was used to perform tissue expression analysis.
(1) cDNA synthesis
Total RNA was extracted from 19 tissues of the following mice (Kazuo Moriwaki, one other edition, Molecular Medicine, separate volume, Vol. 36, “Spontaneous Disease Model Animals”, Nakayama Shoten, 1999), and reverse transcription was performed using oligo dT as a primer. CDNA synthesis was performed using the enzyme.
(A) Tissue of normal mouse and tissue of diabetic model mouse
{Circle around (1)} Control mouse C57BL / KsJ − + m / + m Jcl (female, 8 weeks old) whole brain, thalamus, lung, kidney, bone marrow, pancreas, fat cells, liver, eyes
(2) Diabetes model mouse C57BL / KsJ-db / db Jcl (female, 8 weeks old) pancreas, adipocyte, liver, eye
(B) Tissue of senescence accelerating mouse
(1) Normally aged mouse SAM R1 / TA Slc (male, 13 weeks old) hippocampus, frontal cortex
(2) Senescence-accelerated mouse SAM P8 / Ta Slc (male, 15 weeks old) hippocampus, frontal cortex
(C) Tissue of cancer metastasis model mouse
(1) Normal colon of control mouse Balb / c (female, 5 weeks old)
(2) Colon metastasis model mouse Balb / c (female, 6 weeks old) colon cancer (colon cancer cells Colon 26 are transplanted into the mouse abdominal cavity, and colon cancer is removed 2 weeks later)
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(2) Quantification by PCR method
The following five mRNAs encoding the protein of the present invention were expressed in a product using a light cycler quantitative PCR device (manufactured by Roche Diagnostics) and a LightCycler-FastStart DNA master SYBR Green I reagent. Quantification was performed according to the attached protocol. The synthetic DNA sequence used for the quantitative PCR of dnaform 54482 is shown below.
5 'side primer: (GCAGCTTGCAGTTAGAAGCA) (SEQ ID NO: 11)
3'-side primer: (TACTCTCAGGCCCCCATGGAA) (SEQ ID NO: 12)
The quantitative results were corrected using Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal standard. That is, the expression amount (copy number / μl) of the target gene in each tissue was divided by the expression amount of GAPDH (copy number / μl) to obtain a constant (1 × 10⁶) (Note: 10 to the sixth power). Table 2 shows the results.
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[Table 2]

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As is clear from Table 2, dnaform54482 was strongly expressed in the eyes and highly expressed in the lungs.
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Example 7  Functional analysis by RNAi method using fly homolog
(1) Analysis and acquisition of mouse cDNA fly homologs
The homologue of the fly gene with respect to the nucleotide sequence (clone name: dnaform54482) of the mouse full-length cDNA obtained above was predicted by the following sequence analysis. The sequence analysis was performed at the amino acid sequence level considering the protein translated from the ORF. As a mouse clone dnaform54482, a mouse full-length cDNA clone containing the amino acid sequence shown in SEQ ID NO: 2 (SEQ ID NO: 1) and a fly clone (Berkeley Drosophila Genome Project (BDGP) Drosophila Genomic Sequence). Was performed by NCBI BLASTP 2.2.2 (Altschul et al., Nucleic Acids Res. 25, 3389-3402 (1997)), and the two types of clones showed the best E-value value to each other. The fly gene was identified as a predicted homolog (Tatusov, Koonin & Lipman, Scie) 278, 631 (1997)). As a result, a fly homologue against mouse cDNA (dnaform 54482) could be found. The gene number of the fly homolog was mouse: 54482 (dnaform 54482) and fly: CG12284 (transcript number: CG12284-PC).
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(2) Establishment of fly strain
(I) Construction of inverted repeat vector
An ORF of the above fly cDNA (CG12284), a fragment of about 500 bp on the 5 ′ side (hereinafter, this may be referred to as “target cDNA”) was converted into a fly cDNA library (Berkeley Drosophila Genome Project (http: // www. (fruitfly.org) (invitrogen) as a template. The PCR primers were CpoIA7 (SEQ ID NO: 13: the 3 ′ end of AAAATTTCGGACCG bound to the 21 base sequence of the 5 ′ end of the cDNA of interest) and SfiIA3 (SEQ ID NO: 14: 3 ′ end of AAAATTTGGCCATATAGGCC, A set of 3'-terminal 21 'base sequence of the target cDNA and SfiIB7 (SEQ ID NO: 15: 3'-end of the base sequence described in AAAATTTGCCTCATAGGCC, 5'-terminal 21' of the target cDNA) A set of base nucleotide sequences joined together and CpoIB3 (SEQ ID NO: 16: AAATTTCGGTCCG linked to the 3 'end 21 base sequence of the target cDNA at the 3' end) were used.
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A DNA fragment of about 500 bp which was PCR-amplified using SfiIA3 and SfiIB7 primers was digested with SfiI, and this digested fragment was used for a fly cloning vector (pUAASTCS1: Ryu Ueda et al., Cell Engineering, vol 21, No. 8, 923-932 (2002). )) Was inserted between the sites digested with SfiI and cloned. Furthermore, a CpoI-digested DNA fragment obtained by PCR-amplifying a DNA fragment using the above-mentioned CpoIA7 and CpoIB3 primers was inserted between sites where this vector was digested with CpoI, and cloned. By this two subclonings, an approximately 500 bp fragment of the fly cDNA ORF was inserted at two locations in the reverse direction under the control of the basic promoter of heat shock protein 70 adjacent to the downstream of the UAS sequence (GAL upstream activation sequence). The obtained vector (inverted repeat vector) was obtained.
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The fly transformation vector pUAST vector used above is a vector using a transposon P factor, and the transcription promoting protein GAL4 is bound to the UAS sequence using the UAS sequence and the basic promoter of heat shock protein 70. Is a vector capable of inducing transcription of an inverted repeat sequence inserted downstream of a UAS sequence.
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(Ii) Transformation of fly
Ryu Ueda et al., Cell Engineering, vol. 8, 923-932 (2002), and the inverted repeat vector DNA prepared in (i) above is converted to W¹¹¹⁸A micromanipulator was used to microinject the early embryos of the strain fly (Indiana stock center: http://flybase.bio.indiana.edu/stocks/fbstock.hform). This was cultured and hatched to obtain an adult, and then crossed according to the procedure described in the above-mentioned literature. In this cross, W as a double balancer¹¹¹⁸The strains Sp / SM1, Cy: Pr / TM3, and Sb Ser were used. By this crossing, a fly having a homozygous chromosome into which the inverted repeat vector was inserted (hereinafter referred to as “IR strain”) was produced.
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(3) Induction of RNAi effect
(I) Mutation induction by crossing with GAL4 driver
The fly of the IR strain obtained in the above (2) was crossed with a fly of the Act5C-GAL4 strain (Indiana stock center: http://flybase.bio.indiana.edu/stocks/fbstock.hform) (FIG. 1). Act5C-GAL4 is a strain in which fusion gene in which the yeast GAL4 gene is linked to the promoter of the cellular actin gene (Act5C) expressed in cells of the whole body is introduced. Therefore, the GAL4 protein is expressed in all cells in the fly having the Act5C-GAL4 transgene.
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As shown in FIG. 1, two types of transgenes are present in progeny (F1 generation, hereinafter referred to as “RNAi individuals”) in which an IR fly and an Act5C-GAL4 fly are crossed. Therefore, since the GAL4 protein is expressed in all cells and the IR vector is forcibly transcribed, dsRNA appears in the cells, exerts an RNAi effect, and degrades when the target mRNA is expressed. Therefore, the function of the target gene is inhibited in all cells of the individual. It has no effect on cells in which the target mRNA is not expressed.
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(4) Phenotype analysis and results
With the RNAi effect induced in the above (3), a change in the phenotype of the fly individual due to the inhibition of the function of the fly homolog of the mouse cDNA (dnaform 54482) was observed. As a result, it was found that 5% or less of the RNAi individuals were semi-lethal, that is, they became pupae but molted and became adults.
By inhibiting the expression of the fly gene using RNAi technology and inhibiting its function, the fly individual became semi-lethal, and thus the gene plays an important role in early development or in maintaining the survival and function of the individual. I understood. As described above, it is estimated that the protein encoded by dnaform54482 (SEQ ID NOS: 25 and 26) has an apoptosis-suppressing action. Apoptosis did not progress normally during the development and differentiation of, and it is considered that the individual became semi-lethal. As described above, the protein encoded by dnaform 54482 has an apoptosis-suppressing effect, and is presumed to play an important role in relation to development, differentiation, physiological functions, or pathological conditions.
[0144]
As described above, the protein encoded by dnaform54482 is suggested from Example 4 to be involved in suppression of apoptosis, and Example 6 shows that it is strongly expressed in eyes and highly expressed in lungs. Also in Example 5, the experimental results of the protein-protein interaction suggested that the present protein is associated with suppression of apoptosis, cancer, and respiratory disease. Further, in Example 7, the protein was semi-lethal due to the effect of RNAi of a fly homolog, and thus this protein plays an important role in development / differentiation and the like. The association with cancer was suggested. From these facts, the expression control substance, function activator, or function inhibitor of the present protein can be developed as a therapeutic drug for eye diseases, respiratory diseases such as lungs and trachea, neurodegenerative diseases, immune diseases or cancer. there is a possibility.
[0145]
【The invention's effect】
Since the protein of the present invention and the DNA encoding the same have a function related to the suppression of apoptosis, a substance that regulates the activity can be screened using the protein or the DNA encoding the protein, It is useful for the development of medicines that can act on diseases associated with proteins.
[0146]
This application is based on a Japanese patent application filed on April 30, 2002 (Japanese Patent Application No. 2002-128859) and a Japanese patent application filed on December 4, 2002 (Japanese Patent Application No. 2002-352641). Here incorporated by reference. The contents of the documents cited in the present specification are also incorporated herein by reference.
[0147]
[Sequence list]

[Brief description of the drawings]
FIG. 1 is a conceptual diagram showing a method for inducing a transgene in an RNAi individual.

Claims (15)

The following protein (a) or (b):
(A) a protein consisting of the amino acid sequence of SEQ ID NO: 2;
(B) a protein consisting of the amino acid sequence of SEQ ID NO: 2 with one or several amino acids deleted, substituted and / or added, and having an apoptosis-suppressing function. A DNA encoding the protein according to claim 1. A full-length cDNA encoding the protein according to claim 1. DNA of any of the following (a), (b) or (c):
(A) DNA having the nucleotide sequence of SEQ ID NO: 1.
(B) DNA encoding a protein having a base sequence in which one or several bases have been deleted, substituted and / or added in the base sequence of SEQ ID NO: 1, and having an apoptosis-suppressing function.
(C) a DNA encoding a protein having a nucleotide sequence capable of hybridizing under stringent conditions with a DNA having the nucleotide sequence of SEQ ID NO: 1 or a sequence complementary thereto, and having an apoptosis-suppressing function. A recombinant vector comprising the DNA according to claim 2. A gene-introduced cell into which the DNA according to any one of claims 2 to 4 or the recombinant vector according to claim 5 has been introduced, or an individual comprising the cell. A protein according to claim 1, which is produced by the cell according to claim 6. A sense oligonucleotide having the same sequence as 5 to 100 consecutive bases in the base sequence of the DNA according to any one of claims 2 to 4, an antisense oligonucleotide having a sequence complementary to the sense oligonucleotide, and An oligonucleotide selected from the group consisting of oligonucleotide derivatives of the sense or antisense oligonucleotide. An antibody or a partial fragment thereof that specifically binds to the protein according to claim 1. The antibody according to claim 9, wherein the antibody is a monoclonal antibody. The antibody according to claim 10, wherein the monoclonal antibody has an action of neutralizing the apoptosis-suppressing function of the protein according to claim 1 or 7. A method for screening for an activity-regulating substance of a protein, comprising bringing the protein according to claim 1 or 7 into contact with a test substance, and measuring a change in the activity of the protein due to the test substance. A method for screening a substance regulating the expression of a DNA, comprising bringing the test substance into contact with the gene-transfected cell according to claim 6, and detecting a change in the expression level of the DNA introduced into the cell. At least one or more amino acid sequence information selected from the amino acid sequence of the protein according to claim 1 and / or at least one or more nucleotide sequence selected from the nucleotide sequence of the DNA according to any one of claims 2 to 4. A computer-readable recording medium that stores information. A carrier to which the protein according to claim 1 and / or the DNA according to any one of claims 2 to 4 are bound.

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