JP2004344033A

JP2004344033A - Antifreeze proteins in fish

Info

Publication number: JP2004344033A
Application number: JP2003142850A
Authority: JP
Inventors: Yoshiyuki Nishinomiya; 佳志西宮; Ai Miura; 愛三浦; Sakae Tsuda; 栄津田
Original assignee: National Institute of Advanced Industrial Science and Technology AIST
Current assignee: National Institute of Advanced Industrial Science and Technology AIST
Priority date: 2003-05-21
Filing date: 2003-05-21
Publication date: 2004-12-09
Also published as: WO2004104201A1

Abstract

【課題】不凍タンパク質を容易に入手可能な魚種から採取し不凍タンパク質の利用促進を図る。
【解決手段】本発明は、日本国内、日本近海水域、あるいは該水域と同等気候域の水域に生息する魚種のうち廃棄魚であるナガガジあるいはシチロウウオから得た１１種の不凍タンパク質、それらのアミノ酸配列及び塩基配列である。さらに本発明は、得られる遺伝子を用いて遺伝子工学的手法により前記不凍タンパク質を多量に生産する方法、並びにこれら不凍タンパク質を有効成分とする凍結濃縮阻害剤或いは凝固点降下剤である。
【選択図】なしAn object of the present invention is to collect antifreeze proteins from readily available fish species to promote the use of antifreeze proteins.
The present invention relates to eleven antifreeze proteins obtained from Nagagaji or Shirakawao which are discarded fish among fish species inhabiting waters in Japan, waters near Japan, or waters having the same climate as the waters. The amino acid sequence and the base sequence. Further, the present invention relates to a method for producing the antifreeze protein in a large amount by a genetic engineering technique using the obtained gene, and a freeze concentration inhibitor or a freezing point depressant containing the antifreeze protein as an active ingredient.
[Selection diagram] None

Description

【０００１】
【発明の属する技術分野】
本発明は、日本近海で捕獲される魚種であるナガガジ（ＺｏａｒｃｅｓｅｌｏｎｇａｔｕｓＫｎｅｒ）またはシチロウウオ（Ｂｒａｃｈｙｏｐｓｉｓｒｏｓｔｒａｔｕｓ（Ｔｉｌｅｓｉｕｓ））の体液及びすり身から新たに調製して得られた不凍タンパク質に関するもので、さらに該タンパク質のアミノ酸配列、該タンパク質をコードするＤＮＡ、該ＤＮＡを含むベクター、該ベクターを含む形質転換体等に関する。
【０００２】
【従来の技術】
凍結温度域においては、水を含むあらゆる物質（含水物）の内部では、自然の法則に従い、水分子同士だけが結びついて氷塊を形成する。このとき水分子以外のあらゆる物質は氷塊形成に伴う物理的な圧迫を受け移動を強いられ、含水物中の氷塊以外の位置に追いやられてしまう（凍結濃縮と呼ばれる）。従って、凍結濃縮が起こった含水物を解凍すると、凍結前の内部構造、生理的活性、あるいは風味などが損なわれる。不凍機能を有するタンパク質、即ち不凍タンパク質（Ａｎｔｉｆｒｅｅｚｅｐｒｏｔｅｉｎ）（ＡＦＰ）は、含水物の凍結濃縮を強く抑制する能力を有し、また氷の再結晶阻害や熱ヒステリシスなどの特性を示す。このため、ＡＦＰの添加は冷凍食品などの品質を保持し凍結に伴う細胞や臓器の生理的活性の消失を妨げる効果があると考えられている。また、氷スラリーを使用する冷熱供給システムあるいは冷熱蓄熱等において、氷の再結晶による配管系の閉塞を解消し得る有効な添加剤としての利用も期待されている。これらの他にも食肉、野菜、加工食品、血液、細胞、卵子、精子、移植臓器などの冷凍保存耐性の向上にＡＦＰを応用する試みがなされている。
【０００３】
しかし、現在までＡＦＰが探索・同定された動物は、主として南極海や北極海周辺の極緯度地方あるいは寒帯域にのみ生息する魚種であった。温帯域である日本の近海で大量に漁獲される魚種についてＡＦＰを探索、同定し、そのＡＦＰを新たに利用することが望まれていた。特に、魚類由来のＡＦＰの回収と精製に費やすコストを低減する目的のために、廃棄魚が有するＡＦＰの利用が強く望まれていた。
【０００４】
【非特許文献１】
ＧａｒｔｈＬＦｌｅｔｃｈｅｒ，ＣｈｏｙＬＨｅｗ，ａｎｄＰｅｔｅｒＬＤａｖｉｅｓ（２００１） ”ＡｎｔｉｆｒｅｅｚｅＰｒｏｔｅｉｎｓｏｆＴｅｌｅｏｓｔＦｉｓｈｅｓ” Ａｎｎ．Ｒｅｖ．Ｐｈｙｓｉｏｌ．６３，３５９−３９０
【非特許文献２】
ＣｈｏｙＬ．ＨｅｗａｎｄＤａｎｉｅｌＳ．Ｃ．Ｙａｎｇ（１９９２） ”Ｐｒｏｔｅｉｎｉｎｔｅｒａｃｔｉｏｎｗｉｔｈｉｃｅ”Ｅｕｒ．Ｊ．Ｂｉｏｃｈｅｍ．２０３，３３−４２
【０００５】
【発明が解決しようとする課題】
本発明は、このような従来の問題を解決しようとするものであり、日本近海等で大量に漁獲することができ、さらに廃棄の対象となっている魚種について、ＡＦＰを探索し、得られるＡＦＰを精製し、或いは遺伝子発現、化学合成などの手法を用いて年間を通じて安定的に供給し、不凍能力を有する蛋白質あるいはペプチドを新たに提供するものである。
【０００６】
北海道の野付漁業協同組合においては野付湾内で捕獲される北海シマエビ等を中心にした産業振興に勤めているが、同湾内に生息するナガガジあるいはシチロウウオが北海シマエビを食い荒らしてしまうことが問題になっている。ナガガジあるいはシチロウウオは、底引き網を用いて行う漁の際に、北海シマエビと共に大量に網にかかり、分別されて廃棄魚用のカゴにまとめられ廃棄業者により廃棄される。不凍タンパク質を有するワカサギなど他の魚種が１キログラム当たり３００円〜２，０００円以上の原価で取引きされるのに対して、ナガガジあるいはシチロウウオは原価が０円である。したがって、ナガガジあるいはシチロウウオは不凍タンパク質を回収し精製するための原材料としてこれを低価格に抑えるために最適な魚種であると言える。
【０００７】
【課題を解決するための手段】
かかる状況において、本発明者は、日本、あるいは日本周辺で大量に漁獲されかつ廃棄の対象となっている魚種であるナガガジまたはシチロウウオの組織液および筋肉をすりつぶした液中に存在する不凍タンパク質を見出し、そのアミノ酸配列と遺伝子配列を決定して、本発明を完成させるに至ったものである。
【０００８】
すなわち、本発明は以下のとおりである。
（１）次の（ａ）または（ｂ）で表される不凍タンパク質Ｚ−１
（ａ）配列番号２で表されるアミノ酸配列を有するタンパク質。
（ｂ）配列番号２で表されるアミノ酸配列において、１若しくは複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ、不凍機能を有するタンパク質。
（２）次の（ａ）または（ｂ）で表される不凍タンパク質Ｚ−２
（ａ）配列番号４で表されるアミノ酸配列を有するタンパク質。
（ｂ）配列番号４で表されるアミノ酸配列において、１若しくは複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ、不凍機能を有するタンパク質。
（３）次の（ａ）または（ｂ）で表される不凍タンパク質Ｚ−３
（ａ）配列番号６で表されるアミノ酸配列を有するタンパク質。
（ｂ）配列番号６で表されるアミノ酸配列において、１若しくは複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ、不凍機能を有するタンパク質。
（４）次の（ａ）または（ｂ）で表される不凍タンパク質Ｚ−４
（ａ）配列番号８で表されるアミノ酸配列を有するタンパク質。
（ｂ）配列番号８で表されるアミノ酸配列において、１若しくは複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ、不凍機能を有するタンパク質。
（５）次の（ａ）または（ｂ）で表される不凍タンパク質Ｚ−５
（ａ）配列番号１０で表されるアミノ酸配列を有するタンパク質。
（ｂ）配列番号１０で表されるアミノ酸配列において、１若しくは複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ、不凍機能を有するタンパク質。
（６）次の（ａ）または（ｂ）で表される不凍タンパク質Ｚ−６
（ａ）配列番号１２で表されるアミノ酸配列を有するタンパク質。
（ｂ）配列番号１２で表されるアミノ酸配列において、１若しくは複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ、不凍機能を有するタンパク質。
（７）次の（ａ）または（ｂ）で表される不凍タンパク質Ｚ−７
（ａ）配列番号１４で表されるアミノ酸配列を有するタンパク質。
（ｂ）配列番号１４で表されるアミノ酸配列において、１若しくは複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ、不凍機能を有するタンパク質。
（８）次の（ａ）または（ｂ）で表される不凍タンパク質Ｚ−８
（ａ）配列番号１６で表されるアミノ酸配列を有するタンパク質。
（ｂ）配列番号１６で表されるアミノ酸配列において、１若しくは複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ、不凍機能を有するタンパク質。
（９）次の（ａ）または（ｂ）で表される不凍タンパク質Ｚ−９
（ａ）配列番号１８で表されるアミノ酸配列を有するタンパク質。
（ｂ）配列番号１８で表されるアミノ酸配列において、１若しくは複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ、不凍機能を有するタンパク質。
（１０）次の（ａ）または（ｂ）で表される不凍タンパク質Ｚ−１０
（ａ）配列番号２０で表されるアミノ酸配列を有するタンパク質。
（ｂ）配列番号２０で表されるアミノ酸配列において、１若しくは複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ、不凍機能を有するタンパク質。
（１１）次の（ａ）または（ｂ）で表される不凍タンパク質Ｂ−１
（ａ）配列番号２２で表されるアミノ酸配列を有するタンパク質。
（ｂ）配列番号２２で表されるアミノ酸配列において、１若しくは複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ、不凍機能を有するタンパク質。
（１２）次の（ａ）または（ｂ）で表される不凍タンパク質Ｚ−１をコードするＤＮＡ
（ａ）配列番号２で表されるアミノ酸配列を有するタンパク質。
（ｂ）配列番号２で表されるアミノ酸配列において、１若しくは複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ、不凍機能を有するタンパク質。
（１３）次の（ａ）または（ｂ）で表される不凍タンパク質Ｚ−２をコードするＤＮＡ
（ａ）配列番号４で表されるアミノ酸配列を有するタンパク質。
（ｂ）配列番号４で表されるアミノ酸配列において、１若しくは複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ、不凍機能を有するタンパク質。
（１４）次の（ａ）または（ｂ）で表される不凍タンパク質Ｚ−３をコードするＤＮＡ
（ａ）配列番号６で表されるアミノ酸配列を有するタンパク質。
（ｂ）配列番号６で表されるアミノ酸配列において、１若しくは複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ、不凍機能を有するタンパク質。
（１５）次の（ａ）または（ｂ）で表される不凍タンパク質Ｚ−４をコードするＤＮＡ
（ａ）配列番号８で表されるアミノ酸配列を有するタンパク質。
（ｂ）配列番号８で表されるアミノ酸配列において、１若しくは複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ、不凍機能を有するタンパク質。
（１６）次の（ａ）または（ｂ）で表される不凍タンパク質Ｚ−５をコードするＤＮＡ
（ａ）配列番号１０で表されるアミノ酸配列を有するタンパク質。
（ｂ）配列番号１０で表されるアミノ酸配列において、１若しくは複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ、不凍機能を有するタンパク質。
（１７）次の（ａ）または（ｂ）で表される不凍タンパク質Ｚ−６をコードするＤＮＡ
（ａ）配列番号１２で表されるアミノ酸配列を有するタンパク質。
（ｂ）配列番号１２で表されるアミノ酸配列において、１若しくは複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ、不凍機能を有するタンパク質。
（１８）次の（ａ）または（ｂ）で表される不凍タンパク質Ｚ−７をコードするＤＮＡ
（ａ）配列番号１４で表されるアミノ酸配列を有するタンパク質。
（ｂ）配列番号１４で表されるアミノ酸配列において、１若しくは複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ、不凍機能を有するタンパク質。
（１９）次の（ａ）または（ｂ）で表される不凍タンパク質Ｚ−８をコードするＤＮＡ
（ａ）配列番号１６で表されるアミノ酸配列を有するタンパク質。
（ｂ）配列番号１６で表されるアミノ酸配列において、１若しくは複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ、不凍機能を有するタンパク質。
（２０）次の（ａ）または（ｂ）で表される不凍タンパク質Ｚ−９をコードするＤＮＡ
（ａ）配列番号１８で表されるアミノ酸配列を有するタンパク質。
（ｂ）配列番号１８で表されるアミノ酸配列において、１若しくは複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ、不凍機能を有するタンパク質。
（２１）次の（ａ）または（ｂ）で表される不凍タンパク質Ｚ−１０をコードするＤＮＡ
（ａ）配列番号２０で表されるアミノ酸配列を有するタンパク質。
（ｂ）配列番号２０で表されるアミノ酸配列において、１若しくは複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ、不凍機能を有するタンパク質。
（２２）次の（ａ）または（ｂ）で表される不凍タンパク質Ｂ−１をコードするＤＮＡ
（ａ）配列番号２２で表されるアミノ酸配列を有するタンパク質。
（ｂ）配列番号２２で表されるアミノ酸配列において、１若しくは複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ、不凍機能を有するタンパク質。
（２３）配列番号１，３，５，７，９，１１，１３，１５，１７，１９，又は２１で表される塩基配列であって、それぞれ不凍タンパク質Ｚ−１、Ｚ−２、Ｚ−３、Ｚ−４、Ｚ−５、Ｚ−６、Ｚ−７、Ｚ−８、Ｚ−９、Ｚ−１０又はＢ−１をコードするＤＮＡ。
（２４）上記（１２）乃至（２３）のいずれか１に記載のＤＮＡを含む組換えベクター。
（２５）組換えベクターが発現ベクターである（２４）に記載の組換えベクター。
（２６）宿主生物を（２５）に記載の組換えベクターによって形質転換した形質転換体。
（２７）宿主生物が大腸菌である（２６）に記載の形質転換体。
（２８）（２７）に記載の形質転換体を培地中で培養して不凍機能を有するタンパク質を産生せしめ、該タンパク質を採取することを特徴とする不凍機能を有するタンパク質の製造方法。
（２９）（１）乃至（１２）のいずれか１に記載のタンパク質をペプチド合成機により化学的に合成することを特徴とするタンパク質の製造方法。
（３０）（１）乃至（１２）のいずれか１に記載のタンパク質を有効成分として含むことを特徴とする凍結濃縮阻害剤。
（３１）（１）乃至（１２）のいずれか１に記載のタンパク質を有効成分として含むことを特徴とする凝固点降下剤。
（３２）（１）乃至（１２）に記載のタンパク質を含むことを特徴とする氷スラリー。
【０００９】
【発明の実施の形態】
以下、本発明を詳細に説明する。
北極、南極あるいはその近海などの局地や深海に生息する魚類の体液は、温度がマイナス２℃程度まで下がっても凍結しない。これに対して普通の魚の体液はマイナス０．８℃で凍結する。なお、海水はマイナス１．９℃で凍結する。
【００１０】
北極、南極等に生息する魚類の体液の不凍性は、これら魚類が自ら生産する不凍タンパク質あるいは不凍糖蛋白質（ＡＦＧＰ）に起因するものであることが明らかになっている。このうち不凍タンパク質には、４つのタイプのタンパク質があり、それぞれ分子量約３，０００〜４，５００のＡＦＰＩ、分子量約２０，０００のＡＦＰＩＩ、分子量約７，０００のＡＦＰＩＩＩ、および分子量約１１，０００のＡＦＰＩＶに分類されている。ＡＦＧＰの分子量は２，２００から３３，０００の間である。不凍タンパク質は各々、タンパク質のアミノ酸組成およびその高次構造において差異があり、また、同一タイプに分類されているものであっても、魚種によりタンパク質のアミノ酸配列および高次構造はそれぞれ相違する（非特許文献１参照）。
【００１１】
一方、不凍タンパク質の機能について述べると、通常の場合、氷結晶は、水溶液中において氷核が表れると、まず扁平な六角の板状に成長する。板状平面に対し垂直方向への成長は、板状平面方向に対する成長に比べ１００倍程度遅い。これに対して、不凍蛋白質（ＡＦＰ）が存在すると円盤平面方向への氷晶の成長は阻止され、最初に形成された板状体を基底面として、この基底面に対し垂直方向に、順次、より小さい板状体が積み重ねられていき、最終的にはピラミッドを二つ重ねたバイピラミッド型の氷晶にゆっくりと成長していく。したがって、注目する検体中にＡＦＰが存在している場合に限り、検体液を０℃以下にした場合、その検体液中には一般にバイピラミッド型氷結晶、結晶学的には六方偏四角面体（図１の左図）あるいは六方両錘体（図２の左図）とよばれる氷の単結晶が、顕微鏡下に観測される。ＡＦＰが存在する結果、氷結晶上の１２枚の氷層平面に特異的な結合が起こり、このようなバイピラミッド型氷結晶が生成する。このことが、巨視的には、検体の非凍結現象（不凍活性）として観測される。この現象は、浸透圧計（オスモメーター）を用いることにより検体液の凝固点降下あるいは温度ヒステリシスとして定量化することもできる。凝固点降下の測定法を用いて不凍活性を評価する為には、高純度のＡＦＰの水溶液を得る必要がある。これに対してバイピラミダル氷結晶観測による不凍活性評価法は、ＡＦＰさえ存在していれば観測される（非特許文献２参照）。
【００１２】
（１）本発明によるナガガジ又はシチロウウオの血液又はすり身から本発明の不凍タンパク質Ｚ−１〜Ｚ−１２の調製法
ナガガジ魚体１匹の頭部と内臓を取り除き、胴体部分をきり身にして０．１Ｍ重炭酸アンモニウム水溶液（ｐＨ＝７．９）を加え、ジューサーミキサーに入れて粉砕した。このナガガジすり身に対して０．１Ｍの重炭酸アンモニウム水溶液を加えてすり身の懸濁液を作製した。これを６，０００回転で３０分間遠心して上澄み液を得た。この上澄み液を５０ｍＭ酢酸ナトリウム緩衝液（ｐＨ＝３．７）に対して透析した後にこれを１２，０００回転で３０分間遠心して沈殿物を取り除いた。得られた上澄み液に対して陽イオン交換クロマトグラフィーを行った。この操作には５０ｍＭの酢酸ナトリウム緩衝液（ｐＨ＝３．７）を用い溶出には０〜０．５Ｍの塩化ナトリウム勾配を用いた。この操作で得られる２８０ｎｍの吸収が観測される試料液を逆相クロマトグラフィー（ＨＰＬＣ）により精製した。カラムの平衡化と試料液の取り込みには０．１％のトリフルオロ酢酸を用い溶出にはアセトニトリルの直線勾配を用いた。これにより、ナガガジ由来不凍タンパク質を含む複数の溶出液フラクションを得た。得られた不凍タンパク質の活性確認をバイピラミダル氷結晶の観察により行い、純度の確認をＳＤＳ電気泳動により行った。得られた試料を凍結乾燥した後に冷凍保存した。
【００１３】
（２）上記（１）で調製した不凍タンパク質のアミノ酸配列の決定法
プロテインシーケンサーでスタンダードを解析し、ＨＰＬＣによる分離の際の各アミノ酸に相当する溶出時間を決定した。上記（１）で調製した不凍タンパク質の凍結乾燥粉末を酢酸に溶解し、一般的なエドマン分解法を用いて、プロテインシーケンサーによりＮ末端側から順次解析を行った。具体的には、（ｉ）フェニルイソチオシアナート（ＰＩＴＣ）をタンパク質のＮ末端にカップリングしフェニルチオカルバミルタンパク質（ＰＴＣ‐タンパク質）を生成した。次に、（ｉｉ）ＰＴＣ‐タンパク質からトリフルオロ酢酸で２‐アニリノ‐５‐チアゾリノン誘導体（ＡＴＺ‐アミノ酸）としてＮ末端アミノ酸を切り出す。このＡＴＺ‐アミノ酸を酸性下でフェニルチオヒダントイン誘導体（ＰＴＨ‐アミノ酸）に転換した後にＨＰＬＣで分離し、その溶出時間を基にアミノ酸の種類を決定した。Ｎ末端アミノ酸を切り出された残りのタンパク質を原料として（ｉ）と（ｉｉ）のサイクルを繰り返すことで、アミノ酸配列を決定した。
【００１４】
（３）上記で調製した不凍タンパク質の遺伝子のクローニング法及び塩基配列の決定法
ナガガジの肝臓を不凍タンパク質の発現量が増大している厳寒期に生魚から採取した。採取した肝臓を５ｍｍ角に裁断して１０倍量のＲＮＡ安定化剤中に保存した。安定化剤が浸透した肝臓サンプルを素早く低温ですりつぶしＲＮＡを抽出した。このＲＮＡの溶液からオリゴｄＴマトリックスを用いてｍＲＮＡを分離・精製した。精製したｍＲＮＡを鋳型にオリゴｄＴ配列を含むプライマーと逆転写酵素でｃＤＮＡ（１ｓｔｓｔｒａｎｄ）を合成し、続いてＲＮａｓｅＨとＤＮＡｐｏｌｙｍｅｒａｓｅＩでｃＤＮＡ（２ｎｄｓｔｒａｎｄ）を合成した。さらにＰｆｕＤＮＡＰｏｌｙｍｅｒａｓｅでｃＤＮＡの両末端を平滑化しアダプター配列を付加した。こうして構築したｃＤＮＡライブラリーを鋳型に、上記（２）で決定したアミノ酸配列を元に設計したプライマー、オリゴｄＴ配列を含むプライマーを用いてＰｏｌｙｍｅｒａｓｅｃｈａｉｎｒｅａｃｔｉｏｎ（ＰＣＲ）を行い、増幅した遺伝子断片をＴＡクローニングした。クローニングした遺伝子断片の配列はオリゴｄＴ配列を含むプライマーを用い、ＤｙｅＴｅｒｍｉｎａｔｏｒ法により決定した。その際のサイクルシークエンスの条件は９６℃ １０秒、５０℃ ５秒、６０℃ ４分、２５サイクルであった。その結果、ナガガジ由来ｃＤＮＡライブラリーの中でも優位な約５００ｂｐ長の遺伝子断片群に不凍タンパク質をコードする塩基配列が含まれていることが示された。この結果を基に、５００ｂｐのｃＤＮＡを鋳型に、付加したアダプター配列にアニールするプライマーとオリゴｄＴ配列を含むプライマーを用いて約５００ｂｐの遺伝子断片を増幅し、ＴＡクローニングした。クローニングした遺伝子断片の配列は同様にＤｙｅＴｅｒｍｉｎａｔｏｒ法により決定した。
【００１５】
（４）組換えベクターの調製法
増幅した遺伝子断片を０．８％アガロースで電気泳動し、５００ｂｐ長の遺伝子断片を切り出し、精製した。ＴＡクローニング用ベクター５０ｎｇに対し精製した遺伝子断片を２０ｎｇ加え、４℃で一晩Ｔ４ＤＮＡＬｉｇａｓｅによりライゲーション反応を行った。
【００１６】
（５）形質転換体の調製法
氷上で融解したコンピテントセル（ＤＨ５α）に組換えベクター溶液を加え、氷上に３０分間静置した。これを４２℃で１分間熱処理し、すぐに氷上に移して２分間静置した。これにＳＯＣ培地を加え、３７℃で３０分間インキュベートした。１００μｇ／ｍｌアンピシリン含有ＬＢ寒天培地に広げ、３７℃で一晩インキュベートしてコロニーを形成させた。形成された任意のコロニーを１００μｇ／ｍｌアンピシリン含有ＬＢ液体培地に植菌し、３７℃で一晩大腸菌を振とう培養した。増殖した大腸菌をアルカリ‐ＳＤＳ法で破壊し、ＤＮＡ吸着マトリックス及びスピンカラムを用いて組換えベクターを分離・精製した。
【００１７】
（６）形質転換体を使用して不凍タンパク質を製造する方法
プロモーター、ターミネーター配列等を含む組換え体発現用ベクターに不凍タンパク質をコードする塩基配列を組込み、この組換えベクターによる形質転換体を１００μｇ／ｍｌアンピシリン含有ＬＢ液体培地１０ｍｌで前培養した。この培養液１０ｍｌを１００μｇ／ｍｌアンピシリン含有ＬＢ液体培地１０００ｍｌに加え、さらに培養を続けた。培養の温度として２８℃を設定した。培養液の濁度（Ｏ．Ｄ．＝６００）が０．５まで増加した時点でタンパク質の発現誘導物質を添加した。発現誘導後一晩で培養液を回収し、（１）内に記述した陽イオン交換クロマトグラフィーによる方法を用いて不凍蛋白質を精製した。
【００１８】
（７）不凍タンパク質を凍結濃縮阻害剤及び凝固点降下剤として使用する方法含水物の凍結濃縮阻害剤又は凝固点降下剤としてＡＦＰを使用する際には、ＡＦＰを、含水物中のＡＦＰの濃度が、含水物の質量に対して０．０２重量％濃度以上となる量を添加する。特に、０．０３重量％濃度から０．０５重量％濃度の範囲内であるときにＡＦＰは最大の効果を発揮する。このとき、例えば含水物が液体（水溶液、血液など）である場合には、ＡＦＰの粉末又は水溶液をそのまま混合すれば良い。また、含水物が細胞、組織である場合には、注射器などを用いてＡＦＰの水溶液を含水物内部に注入しても良い。また、含水物をＡＦＰの水溶液に浸した後に５０〜２，０００ｋｇ／ｃｍ^２に加圧する等の手段により、含水物内部にＡＦＰを浸透させても良い。
【００１９】
本発明の不凍タンパク質は、前述したように、不凍タンパク質には含水物の凍結濃縮を妨げるはたらきや氷の再結晶化を妨げるはたらきがあるため、上記各魚種から得られた不凍タンパク質がＡＦＰＩ〜ＩＶのどのタイプの不凍タンパク質に分類されるか否かに関わらず、含水物の凍結濃縮阻害剤ないしは氷の再結晶化防止剤として使用でき、例えば冷凍食品あるいは食肉などに混入することによりその品質を持続させるために使用できる。また、この品質維持効果は、細胞（卵子や精子）、組織、臓器、野菜などを長期凍結保存する際にも同様に期待できる。さらに、近年、エネルギー密度が大きい氷スラリーを熱媒体として使用する冷熱供給システムあるいは冷熱蓄熱等が提案されているが、これらにおいては、氷の再結晶による配管系の閉塞の問題があり、本発明の不凍タンパク質は氷の再結晶化を有効に防ぐものであるから、この問題を解決するために有望な手段となりうる。このほか不凍蛋白質をコードする遺伝子を植物体に組み込むことにより、その植物に耐寒性を持たせることも応用技術として期待できる。
【００２０】
【実施例】
以下、実施例を挙げて本発明を具体的に示す。但し、本発明の技術的範囲はこれら実施例に限定されるものではない。
【００２１】
（実施例１）
（１）検体試料
不凍タンパク質の採取源として使用した魚種は以下のとおりである。
ナガガジ（Ｚｏａｒｃｅｓｅｌｏｎｇａｔｕｓｋｎｅｒ、英名Ｎｏｔｃｈｅｄ−Ｆｉｎｅｅｌｐｏｕｔ）：北海道野付湾で漁獲。
シチロウウオ（Ｂｒａｃｈｙｏｐｓｉｓｒｏｓｔｒａｔｕｓ、英名Ｌｏｎｇｓｎｏｕｔｐｏａｃｈｅｒ）：北海道野付湾で漁獲。
【００２２】
（２）不凍活性の観測
採取したナガガジとシチロウウオの血液それぞれ１ｕｌをライカ社製ＤＭＬＢ１００型顕微鏡（ＬｅｉｃａＤＭＬＢ１００ｐｈｏｔｏｍｉｃｒｏｓｃｏｐｅ）の直径１６ｍｍのカバーガラス上に滴下した。これをそのままもう１枚の直径１２．５ｍｍのカバーガラスによりはさみ、これをＤＭＬＢ１００型顕微鏡のステージ部に設置した冷却箱内にセットした。冷却箱の上下には直径１ｍｍの光取り入れ穴をあけ、顕微鏡光源からの光は下側の穴から箱内を通り上側の穴を抜けてレンズに入光させるようにした。この上下の穴により規定される光軸上に検体液をセットすることで、光軸上にある検体液中の物質が顕微鏡観察される。検体液がセットされた冷却箱の中の温度は、リンカム社製ＬＫ６００温度制御装置（ＬＫ６００温度コントローラー）により＋／−０．１℃の誤差で制御される。室温下で検体液をセットした後、温度制御装置により冷却箱内の温度を毎秒０．２℃でマイナス２２℃まで下降させた。およそマイナス１．４℃からマイナス４０℃の間の温度のどこかで検体液の全体が凍結する。凍結の後に毎秒０．１℃で冷却箱内温度を上昇させ零℃で上昇を停止し、そのまま１秒〜１０秒程度の間、零℃を維持していると凍結が溶け、無数のきれつの入った氷結晶状態を経たのちに、数えられる程度の氷の単結晶が水中に浮かんだものが観測された。その瞬間に、冷却箱内の温度をマイナス０．１℃〜マイナス１．０℃程度に下降させて止め、氷結晶の形状を観察した。観察結果を図１に示す。試験に使用した魚種のいずれにおいてもバイピラミダル型の氷晶が観察され、これらの魚種は、いずれも不凍タンパク質を有することを確認した。また、魚肉すり身懸濁液１Ｌ、および乾物懸濁液１Ｌを用いて同様の試験を行ったが、これらの試験においても、すべての魚種について、バイピラミダル型の氷晶が観察された。
【００２３】
（３）ナガガジＡＦＰ− Ｚ−１の調製及びアミノ酸配列決定
ナガガジの身を包丁で切断しミキサーを用いてすり身にした。このすり身２０ｍｌに対して２０ｍｌの０．１Ｍの重炭酸アンモニウム水溶液を加えることで、すり身の懸濁液４０ｍｌを調製した。これをプラスチック試験管に入れ、６，０００回転で３０分間遠心して、約２０ｍｌの上澄み液を得た。この上澄み液を、５０ｍＭ酢酸ナトリウム緩衝液（ｐＨ＝３．７）に対して透析し、共雑タンパク質を凝集させた。これを１２，０００回転で３０分間遠心して取り除き、上澄み液を得た。この上澄み液に対して陽イオン交換クロマトグラフィーを行い、１ｍｌずつの溶出液を２８０ｎｍの吸収を検出しながらフラクションコレクターにより回収した。陽イオン交換クロマトグラフィーにはＡｍｅｒｓｈａｍＰｈａｒｍａｃｉａＢｉｏｔｅｃｈのＦＰＬＣシステムとＢＩＯ−ＲＡＤのＨｉｇｈ−Ｓカラムを用いた。５０ｍＭの酢酸ナトリウム緩衝液（ｐＨ＝３．７）を用いてカラムの平衡化と上澄み液の取り込みをおこない溶出は流速１ｍｌ／分で０〜０．５Ｍの塩化ナトリウムの直線勾配をかけることで行った。ここまでの操作は全て４℃で行った。次に、２８０ｎｍの吸収の観測された試料液をＴＯＳＯのＨＰＬＣシステムとＯＤＳカラムを用いた逆相クロマトグラフィーにより精製した。０．１％のトリフルオロ酢酸を用いてカラムの平衡化と試料液の取り込みを行い、溶出にはアセトニトリルの直線勾配を用いた。溶出試料の吸光度は２１４ｎｍと２８０ｎｍで検出し、単一のタンパク質を含む溶出液フラクションを得た後にこれを凍結乾燥した。この凍結乾燥粉末を０．１Ｍの重炭酸アンモニウム水溶液に溶解し、そのバイピラミダル氷結晶の観測をおこなうことで不凍タンパク質であることを確認し、不凍タンパク質Ｚ−１を得た。こうしてナガガジＡＦＰ−Ｚ−１のサンプルを精製した後、これを凍結乾燥したサンプルを用いたアミノ酸配列の決定を行った。乾燥状態のサンプルを酢酸に溶解し、ポリブレン処理したカートリッジフィルターに添加した。ＡｐｐｌｉｅｄＢｉｏｓｙｓｔｅｍｓ社製４９１プロテインシークエンサーでエドマン分解し、Ｎ末端側から順次アミノ酸配列を決定した。この結果、ＡＦＰ−Ｚ−１のアミノ酸配列は配列表の配列番号２に示すとおりである。
【００２４】
（４）全ＲＮＡの単離
全ＲＮＡの単離に関する基本的な操作はプロトコール集（”Ｒｎｅａｓｙ^ＲＰｒｏｔｅｃｔａｎｄＲＮＡｌａｔｅｒ^ＴＭＨａｎｄｂｏｏｋ” 及び ”ＲＮｅａｓｙ^ＲＭｉｎｉＨａｎｄｂｏｏｋ” （ＱＩＡＧＥＮ））に従った。また、サンプルとするナガガジの肝臓は不凍タンパク質の発現量が増大している厳寒期に生魚から採取した。採取した１〜２ｇの肝臓を５ｍｍ角に裁断して１０倍量のＲＮＡｌａｔｅｒ（ＱＩＡＧＥＮ）中に保存した。この肝臓サンプル６０ｍｇを液体窒素中で粉断し３０ｍｇずつＢｕｆｆｅｒＲＬＴ６００μｌに懸濁した後、ＱＩＡｓｈｒｅｄｄｅｒ（ＱＩＡＧＥＮ）でさらに粉砕した。遠心により回収した上清に等量の７０％エタノールを加え、スピンカラムを通すことでＲＮＡをカラムに吸着させた。ＢｕｆｆｅｒＲＷ１、ＢｕｆｆｅｒＲＰＥでカラムを洗浄後、ＲＮａｓｅフリー水３０μｌでそれぞれＲＮＡを溶出した。
【００２５】
（５）ｍＲＮＡの単離
ｍＲＮＡの単離はＯｌｉｇｏｔｅｘＴＭ−ｄＴ３０＜Ｓｕｐｅｒ＞ｍＲＮＡＰｕｒｉｆｉｃａｔｉｏｎｋｉｔ（ＴａＫａＲａ）を用いて行い、操作は添付されているプロトコールに従った。１．１μｇ／μｌのｔｏｔａｌＲＮＡ溶液１５０μｌに対し２ｘＢｉｎｄｉｎｇＢｕｆｆｅｒ１５０μｌ、ＯｌｉｇｏｔｅｘＴＭ−ｄＴ３０＜Ｓｕｐｅｒ＞１５μｌを加え７０℃で３分間加熱した後、室温で１０分間放置した。遠心操作にてＯｌｉｇｏｔｅｘＴＭ−ｄＴ３０＜Ｓｕｐｅｒ＞を回収後、ＷａｓｈＢｕｆｆｅｒに懸濁しスピンカラムでＯｌｉｇｏｔｅｘ−ｄＴ３０＜Ｓｕｐｅｒ＞を洗浄した。ＯｌｉｇｏｔｅｘＴＭ−ｄＴ３０＜Ｓｕｐｅｒ＞に吸着したｍＲＮＡを７０℃で加熱しておいたＲＮａｓｅフリー水４０μｌで溶出した。
【００２６】
（６）ｃＤＮＡライブラリーの構築
ｃＤＮＡライブラリーの構築はＺＡＰ−ｃＤＮＡＳｙｎｔｈｅｓｉｓＫｉｔ（ＳＴＲＡＴＡＧＥＮＥ^Ｒ）で行った。基本的操作は ”ｃＤＮＡＳｙｎｔｈｅｓｉｓＫｉｔ，ＺＡＰ−ｃＤＮＡ^ＲＳｙｎｔｈｅｓｉｓＫｉｔ，ａｎｄＺＡＰ−ｃＤＮＡ^ＲＧｉｇａｐａｃｋ^ＲＩＩＩＧｏｌｄＣｌｏｎｉｎｇＫｉｔＩＮＳＴＲＵＣＴＩＯＮＭＡＮＵＡＬ” （ＳＴＲＡＴＡＧＥＮＥ^Ｒ）に従った。上述のように回収したｍＲＮＡを鋳型としメチル化されたｄＣＴＰを含むｄＮＴＰ（ｄＡＴＰ、ｄＣＴＰ、ｄＧＴＰ、ｄＴＴＰ）基質として用いＳｔｒａｔａＳｃｒｉｐｔＲＴａｓｅを加え、４２℃ １時間反応を行うことでｆｉｒｓｔ−ｓｔｒａｎｄｃＤＮＡを逆転写合成した。次にＲＮａｓｅＨ、ＤＮＡｐｏｌｙｍｅｒａｓｅ、ｄＮＴＰを加え、１６℃ ２．５時間反応を行い、ｓｅｃｏｎｄ−ｓｔｒａｎｄｃＤＮＡを合成した。ＰｆｕＤＮＡｐｏｌｙｍｅｒａｓｅでｃＤＮＡの末端を平滑化した後、Ｔ４ＤＮＡｌｉｇａｓｅ
でアダプター配列を結合した。
【００２７】
（７）塩基配列の決定
構築したｃＤＮＡライブラリーの一部を０．８％アガロースで電気泳動したところ、５００ｂｐの遺伝子断片が主として確認された。そこで、プロテインシークエンサーにより決定されたアミノ酸配列を元に縮重プライマーを設計し、このプライマーとｐｏｌｙＡ配列に相補的なプライマーを用いて５００ｂｐのｃＤＮＡを鋳型にＥｘＴａｑ^ＴＭ（ＴａＫａＲａ）でＰＣＲ（ｐｏｌｙｍｅｒａｓｅｃｈａｉｎｒｅａｃｔｉｏｎ）を行った。ＰＣＲ産物をｐＧＥＭ^Ｒ−ＴＥａｓｙ（Ｐｒｏｍｅｇａ）にクローニングし、このプラスミドで大腸菌を形質転換した。形質転換体を１００μｇ／ｍｌアンピシリン含有ＬＢ寒天培地上に広げ、形成された任意のコロニーを１００μｇ／ｍｌアンピシリン含有ＬＢ液体培地に植菌し、３７℃で一晩大腸菌を振とう培養した。増殖した大腸菌からアルカリ−ＳＤＳ法でプラスミドを分離精製し、Ｔ７ｐｒｏｍｏｔｅｒプライマーとＢｉｇＤｙｅ^ＲＴｅｒｍｉｎａｔｏｒｖ３．１ＣｙｃｌｅＳｅｑｕｅｎｃｉｎｇＫｉｔ（ＡｐｐｌｉｅｄＢｉｏｓｙｓｔｅｍｓ）を用いてシークエンス反応を行いＡＢＩＰＲＩＳＭ^ＴＭ３１０ＧｅｎｅｔｉｃＡｎａｌｙｚｅｒ及び３１００ＧｅｎｅｔｉｃＡｎａｌｙｚｅｒ（ＡｐｐｌｉｅｄＢｉｏｓｙｓｔｅｍｓ）で塩基配列を解析した。この解析結果から分離精製した不凍タンパク質Ｚ−１と高い相同性を有するアミノ酸配列をコードする塩基配列郡が５００ｂｐのｃＤＮＡに含まれていることが確認された。そこで、この５００ｂｐのｃＤＮＡを鋳型にリンカー配列及びｐｏｌｙＡ配列にアニールするプライマーを用いてＥｘＴａｑ^ＴＭ（ＴａＫａＲａ）でＰＣＲ（ｐｏｌｙｍｅｒａｓｅｃｈａｉｎｒｅａｃｔｉｏｎ）を行い、不凍タンパク質をコードする塩基配列全長を増幅した。これらＰＣＲ産物をｐＧＥＭ^Ｒ−ＴＥａｓｙ（Ｐｒｏｍｅｇａ）にクローニングし、遺伝子断片の配列を前述の方法で解析した。得られる不凍タンパク質Ｚ−１の塩基配列は配列表の配列番号１に示すとおりである。
【００２８】
ナガガジ由来の残り９種類の新規なＡＦＰ−Ｚ−２〜Ｚ−１０のアミノ酸配列を上記と同様な方法により調製し、それらの遺伝子のＣＤＳ領域の塩基配列を上記と同様の方法により決定した。新規なＡＦＰ−Ｚ−２〜Ｚ−１０のアミノ酸配列は配列表の配列番号４，６，８，１０，１２，１４，１６，１８及び２０に、塩基配列は配列番号３，５，７，９，１１，１３，１５，１７及び１９に示す。これらの塩基配列は、既に塩基配列が明らかにされているマクロゾアルケルスアメリカヌス由来のタイプＩＩＩ型不凍タンパク質と約７５〜９０％の相同性を示した。このようにして、ナガガジ由来のタイプＩＩＩ型の新規新規なＡＦＰ−Ｚ−２〜Ｚ−１０が得られた。
【００２９】
（実施例２）
（１）シチロウウオＡＦＰ−Ｂ−１のアミノ酸配列決定
実施例１のナガガジＡＦＰの精製と同様の精製法により、シチロウウオのすり身４０ｍｌからＴＯＳＯＨＨＰＬＣシステムとＴＳＫ−ＧＥＬＯＤＳ−８０ＴＳ（４．６ｍｍ（ＩＤ）ｘ１５．０ｃｍ（Ｌ）、ＴＯＳＯＨ）を用いてＡＦＰを高純度に精製した後、不凍活性を確認し（図２参照）、その凍結乾燥したサンプルを用いてアミノ酸配列の決定を行った。乾燥状態のサンプルを酢酸に溶解し、ポリブレン処理したカートリッジフィルターに添加した。ＡｐｐｌｉｅｄＢｉｏｓｙｓｔｅｍｓ社製４９１プロテインシークエンサーでエドマン分解し、Ｎ末端側から順次アミノ酸配列を決定した。この結果、ＡＦＰ−Ｂ−１のアミノ酸配列は配列表の配列番号２２に示すとおりである。
【００３０】
（２）全ＲＮＡの単離
全ＲＮＡの単離の基本的な操作はプロトコール集（”ＲＮｅａｓｙ^ＲＰｒｏｔｅｃｔａｎｄＲＮＡｌａｔｅｒＴＭＨａｎｄｂｏｏｋ” 及び ”ＲＮｅａｓｙ^ＲＭｉｎｉＨａｎｄｂｏｏｋ” （ＱＩＡＧＥＮ））に従った。また、サンプルとする各種魚類の肝臓は不凍タンパク質の発現量が増大している厳寒期に生魚から採取した。採取した１〜２ｇの肝臓を５ｍｍ角に裁断して１０倍量のＲＮＡｌａｔｅｒ（ＱＩＡＧＥＮ）中に保存した。この肝臓サンプル６０ｍｇを液体窒素中で粉断し３０ｍｇずつＢｕｆｆｅｒＲＬＴ６００μｌにけん濁した後、ＱＩＡｓｈｒｅｄｄｅｒ（ＱＩＡＧＥＮ）でさらに粉砕した。遠心により回収した上清に等量の７０％エタノールを加え、スピンカラムを通すことでＲＮＡをカラムに吸着させた。ＢｕｆｆｅｒＲＷ１、ＢｕｆｆｅｒＲＰＥでカラムを洗浄後、ＲＮａｓｅフリー水３０μｌでそれぞれＲＮＡを溶出した。
【００３１】
（３）ｍＲＮＡの単離
ｍＲＮＡの単離はＯｌｉｇｏｔｅｘＴＭ−ｄＴ３０＜Ｓｕｐｅｒ＞ｍＲＮＡＰｕｒｉｆｉｃａｔｉｏｎｋｉｔ（ＴａＫａＲａ）を用いて行い、操作は添付されているプロトコールに従った。１．１μｇ／μｌのｔｏｔａｌＲＮＡ溶液１５０μｌに対し２ｘＢｉｎｄｉｎｇＢｕｆｆｅｒ１５０μｌ、ＯｌｉｇｏｔｅｘＴＭ−ｄＴ３０＜Ｓｕｐｅｒ＞１５μｌを加え７０℃で３分間加熱した後、室温で１０分間放置した。遠心操作にてＯｌｉｇｏｔｅｘＴＭ−ｄＴ３０＜Ｓｕｐｅｒ＞を回収後、ＷａｓｈＢｕｆｆｅｒにけん濁しスピンカラムでＯｌｉｇｏｔｅｘ−ｄＴ３０＜Ｓｕｐｅｒ＞を洗浄した。ＯｌｉｇｏｔｅｘＴＭ−ｄＴ３０＜Ｓｕｐｅｒ＞に吸着したｍＲＮＡを７０℃で加熱しておいたＲＮａｓｅフリー水４０μｌで溶出した。
【００３２】
（４）ｃＤＮＡライブラリーの構築
ｃＤＮＡライブラリーの構築はＺＡＰ−ｃＤＮＡＳｙｎｔｈｅｓｉｓＫｉｔ（ＳＴＲＡＴＡＧＥＮＥ^Ｒ）で行った。基本的操作は ”ｃＤＮＡＳｙｎｔｈｅｓｉｓＫｉｔ，ＺＡＰ−ｃＤＮＡ^ＲＳｙｎｔｈｅｓｉｓＫｉｔ，ａｎｄＺＡＰ−ｃＤＮＡ^ＲＧｉｇａｐａｃｋ^ＲＩＩＩＧｏｌｄＣｌｏｎｉｎｇＫｉｔＩＮＳＴＲＵＣＴＩＯＮＭＡＮＵＡＬ” （ＳＴＲＡＴＡＧＥＮＥ^Ｒ）に従った。上述のように回収したｍＲＮＡを鋳型としメチル化されたｄＣＴＰを含むｄＮＴＰ（ｄＡＴＰ、ｄＣＴＰ、ｄＧＴＰ、ｄＴＴＰ）基質として用いＳｔｒａｔａＳｃｒｉｐｔＲＴａｓｅを加え、４２℃ １時間反応を行うことでｆｉｒｓｔ−ｓｔｒａｎｄｃＤＮＡを逆転写合成した。次にＲＮａｓｅＨ、ＤＮＡｐｏｌｙｍｅｒａｓｅ、ｄＮＴＰを加え、１６℃ ２．５時間反応を行い、ｓｅｃｏｎｄ−ｓｔｒａｎｄｃＤＮＡを合成した。ＰｆｕＤＮＡｐｏｌｙｍｅｒａｓｅでｃＤＮＡの末端を平滑化した後、Ｔ４ＤＮＡｌｉｇａｓｅ
でアダプター配列を結合した。
【００３３】
（５）塩基配列の決定
プロテインシークエンサーにより決定されたアミノ酸配列を元に縮重プライマーを設計し、このプライマーとｐｏｌｙＡ配列に相補的なプライマーを用いてｃＤＮＡを鋳型にＥｘＴａｑＴＭ（ＴａＫａＲａ）でＰＣＲ（ｐｏｌｙｍｅｒａｓｅｃｈａｉｎｒｅａｃｔｉｏｎ）を行った。ＰＣＲ産物をｐＧＥＭ^Ｒ−ＴＥａｓｙ（Ｐｒｏｍｅｇａ）にクローニングし、このプラスミドで大腸菌を形質転換した。形質転換体を１００μｇ／ｍｌアンピシリン含有ＬＢ寒天培地上に広げ、形成された任意のコロニーを１００μｇ／ｍｌアンピシリン含有ＬＢ液体培地に植菌し、３７℃で一晩大腸菌を振とう培養した。増殖した大腸菌からアルカリ−ＳＤＳ法でプラスミドを分離精製し、Ｔ７ｐｒｏｍｏｔｅｒプライマーとＢｉｇＤｙｅ^ＲＴｅｒｍｉｎａｔｏｒｖ３．１ＣｙｃｌｅＳｅｑｕｅｎｃｉｎｇＫｉｔ（ＡｐｐｌｉｅｄＢｉｏｓｙｓｔｅｍｓ）を用いてシークエンス反応を行いＡＢＩＰＲＩＳＭＴＭ３１０ＧｅｎｅｔｉｃＡｎａｌｙｚｅｒ及び３１００ＧｅｎｅｔｉｃＡｎａｌｙｚｅｒ（ＡｐｐｌｉｅｄＢｉｏｓｙｓｔｅｍｓ）で塩基配列を解析した。この解析結果から不凍タンパク質のＣ末端をコードする塩基配列を決定し、この配列に相補的なプライマーとリンカー配列にアニールするプライマーを用いてｃＤＮＡを鋳型にＥｘＴａｑＴＭ（ＴａＫａＲａ）でＰＣＲ（ｐｏｌｙｍｅｒａｓｅｃｈａｉｎｒｅａｃｔｉｏｎ）を行い、不凍タンパク質をコードする塩基配列全長を増幅した。このＰＣＲ産物をｐＧＥＭ^Ｒ−ＴＥａｓｙ（Ｐｒｏｍｅｇａ）にクローニングし、遺伝子断片の配列を前述の方法で解析した。この結果、シチロウウオ由来ののＡＦＰ−Ｂ−１の塩基配列は配列番号２１に示すとおりである。シチロウウオ由来ののＡＦＰ−Ｂ−１の塩基配列は配列が明らかにされているリプテルスアメリカヌス（ｓｅａｒａｖｅｎ（Ｈｅｍｉｔｒｉｐｔｅｒｕｓａｍｅｒｉｃａｎｕｓ））由来のタイプＩＩ型不凍タンパク質と約６３％の相同性を示した。このようにして、タイプＩＩ型の不凍タンパク質がシチロウウオのすり身から分離、精製された。
【００３４】
【発明の効果】
本発明により日本近海で漁獲される魚であって、通常廃棄されているナガガシ或いはシチロウウオから１１種類の新規な不凍タンパク質を採取し、これらのアミノ酸配列及び塩基配列を決定した。また、得られる遺伝子を用いて遺伝子工学的手法により前記不凍タンパク質を多量に生産することを可能とした。
【００３５】
これら不凍タンパク質は、前述のとおり、例えばアイスクリーム、冷凍食品等における氷晶の成長による食味劣化、組織破壊を防止し、また、氷スラリーを使用する冷熱供給システムあるいは冷熱蓄熱等において、氷の再結晶による配管系の閉塞を解消し得る有効な添加剤として期待されているものである。さらに卵子や精子、移植臓器等の低温長期保存冷凍保存においても有望な物質である。
【００３６】
【配列表】

【図面の簡単な説明】
【図１】バイピラミッド型氷結晶の形状を示す模式図および写真である。この図において左は六方両錘体の模式図を、右はナガガジの血液に観測された六方両錘体の氷結晶の写真を示す。
【図２】バイピラミッド型氷結晶の形状を示す模式図および写真である。この図において、左は六方偏四角面体の模式図を右はシチロウウオの血液に観測された六方偏四角面体の氷結晶の写真を示す。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to an antifreeze protein newly prepared from body fluids and surimi of fishes of the species Nagasaki (Zoarces elongatus Kner) or Blackfish (Brachyopsis rostratus (Tilesius)) captured in the waters around Japan. The present invention relates to an amino acid sequence of the protein, a DNA encoding the protein, a vector containing the DNA, a transformant containing the vector, and the like.
[0002]
[Prior art]
In the freezing temperature range, inside water-containing substances (water-containing substances), only water molecules are linked to each other to form ice blocks according to the law of nature. At this time, all substances other than water molecules are forced to move due to physical pressure accompanying the formation of ice blocks, and are driven to a position other than the ice blocks in the hydrated substance (called freeze concentration). Therefore, when the hydrated substance that has undergone freeze concentration is thawed, the internal structure before freezing, physiological activity, flavor, and the like are impaired. A protein having an antifreeze function, that is, an antifreeze protein (AFP) has an ability to strongly suppress freeze-concentration of a hydrate, and exhibits properties such as inhibition of ice recrystallization and thermal hysteresis. Therefore, it is considered that the addition of AFP has the effect of maintaining the quality of frozen foods and the like and preventing the loss of the physiological activity of cells and organs due to freezing. Further, in a cold heat supply system or a cold heat storage system using an ice slurry, it is also expected to be used as an effective additive capable of eliminating clogging of a piping system due to recrystallization of ice. In addition to these, attempts have been made to apply AFP to improving the cryopreservation resistance of meat, vegetables, processed foods, blood, cells, ova, sperm, transplanted organs and the like.
[0003]
However, the animals for which AFP has been searched and identified to date have been mainly fish species that live only in polar latitudes around the Antarctic and Arctic Oceans or in the cold regions. It has been desired to search and identify AFPs for fish species caught in large quantities in the temperate zone near Japan, and to newly use the AFPs. In particular, for the purpose of reducing the cost of collecting and purifying AFP derived from fish, the use of AFP of waste fish has been strongly desired.
[0004]
[Non-patent document 1]
Garth L Fletcher, Choy L Hew, and Peter L Davies (2001) "Antifreeze Proteins of Teleost Fishes" Ann. Rev .. Physiol. 63, 359-390
[Non-patent document 2]
Choy L. Hew and Daniel S.A. C. Yang (1992) "Protein interaction with ice" Eur. J. Biochem. 203, 33-42
[0005]
[Problems to be solved by the invention]
The present invention is intended to solve such a conventional problem. A large amount of fish can be caught in the waters around Japan and the like, and AFPs can be searched for fish species to be discarded and obtained. AFP is purified or supplied stably throughout the year using techniques such as gene expression and chemical synthesis to newly provide a protein or peptide having antifreeze ability.
[0006]
The Notsuke Fisheries Cooperative in Hokkaido is engaged in industrial promotion centered on the North Sea prawns captured in Notsuke Bay, but the problem is that Nagahaji or Shichirouo, which inhabit the Bay, eat the North Sea prawns. I have. When fishing using a bottom seine, a large amount of Nagagaji or beetle fish are caught in a net together with North Sea prawns, separated, collected into a basket for discarded fish, and disposed of by a waste disposal company. Other fish species, such as smelt with antifreeze protein, are traded at a cost of 300-2,000 yen or more per kilogram, while Nagagaji or Shirouuo cost 0 yen. Therefore, it can be said that Nagaji or Beetlefish is the most suitable fish species as a raw material for recovering and refining antifreeze proteins and to keep them low in price.
[0007]
[Means for Solving the Problems]
In such a situation, the present inventor has determined that the antifreeze protein present in the tissue fluid and muscle-mashed fluid of Nagagashi or Beechfish, a fish species that has been caught in large quantities in or around Japan and is subject to disposal. The present inventors have found and determined the amino acid sequence and gene sequence thereof and have completed the present invention.
[0008]
That is, the present invention is as follows.
(1) Antifreeze protein Z-1 represented by the following (a) or (b)
(A) a protein having the amino acid sequence represented by SEQ ID NO: 2;
(B) a protein comprising an amino acid sequence represented by SEQ ID NO: 2 in which one or more amino acids have been deleted, substituted or added, and having an antifreeze function;
(2) Antifreeze protein Z-2 represented by the following (a) or (b):
(A) a protein having the amino acid sequence represented by SEQ ID NO: 4;
(B) a protein comprising an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 4, and having an antifreeze function;
(3) Antifreeze protein Z-3 represented by the following (a) or (b):
(A) a protein having the amino acid sequence represented by SEQ ID NO: 6;
(B) a protein consisting of the amino acid sequence represented by SEQ ID NO: 6 in which one or more amino acids have been deleted, substituted or added, and having an antifreeze function;
(4) Antifreeze protein Z-4 represented by the following (a) or (b):
(A) a protein having the amino acid sequence represented by SEQ ID NO: 8;
(B) a protein consisting of the amino acid sequence represented by SEQ ID NO: 8 with one or more amino acids deleted, substituted or added, and having an antifreeze function;
(5) Antifreeze protein Z-5 represented by the following (a) or (b):
(A) a protein having the amino acid sequence represented by SEQ ID NO: 10;
(B) a protein comprising an amino acid sequence represented by SEQ ID NO: 10 in which one or more amino acids have been deleted, substituted or added, and having an antifreeze function;
(6) Antifreeze protein Z-6 represented by the following (a) or (b):
(A) a protein having the amino acid sequence represented by SEQ ID NO: 12;
(B) a protein consisting of the amino acid sequence represented by SEQ ID NO: 12 with one or more amino acids deleted, substituted or added, and having an antifreeze function;
(7) Antifreeze protein Z-7 represented by the following (a) or (b)
(A) a protein having the amino acid sequence represented by SEQ ID NO: 14;
(B) a protein comprising an amino acid sequence represented by SEQ ID NO: 14 in which one or more amino acids have been deleted, substituted or added, and having an antifreeze function;
(8) Antifreeze protein Z-8 represented by the following (a) or (b)
(A) a protein having the amino acid sequence represented by SEQ ID NO: 16;
(B) a protein consisting of the amino acid sequence represented by SEQ ID NO: 16 with one or more amino acids deleted, substituted or added, and having an antifreeze function;
(9) Antifreeze protein Z-9 represented by the following (a) or (b)
(A) a protein having the amino acid sequence represented by SEQ ID NO: 18;
(B) a protein comprising an amino acid sequence represented by SEQ ID NO: 18 in which one or more amino acids have been deleted, substituted or added, and having an antifreeze function;
(10) Antifreeze protein Z-10 represented by the following (a) or (b):
(A) a protein having the amino acid sequence represented by SEQ ID NO: 20;
(B) a protein consisting of the amino acid sequence represented by SEQ ID NO: 20 with one or more amino acids deleted, substituted or added, and having an antifreeze function;
(11) Antifreeze protein B-1 represented by the following (a) or (b):
(A) a protein having the amino acid sequence represented by SEQ ID NO: 22;
(B) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 22, and which has an antifreeze function;
(12) DNA encoding antifreeze protein Z-1 represented by the following (a) or (b):
(A) a protein having the amino acid sequence represented by SEQ ID NO: 2;
(B) a protein comprising an amino acid sequence represented by SEQ ID NO: 2 in which one or more amino acids have been deleted, substituted or added, and having an antifreeze function;
(13) DNA encoding antifreeze protein Z-2 represented by the following (a) or (b):
(A) a protein having the amino acid sequence represented by SEQ ID NO: 4;
(B) a protein comprising an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 4, and having an antifreeze function;
(14) DNA encoding antifreeze protein Z-3 represented by the following (a) or (b):
(A) a protein having the amino acid sequence represented by SEQ ID NO: 6;
(B) a protein consisting of the amino acid sequence represented by SEQ ID NO: 6 in which one or more amino acids have been deleted, substituted or added, and having an antifreeze function;
(15) DNA encoding antifreeze protein Z-4 represented by the following (a) or (b):
(A) a protein having the amino acid sequence represented by SEQ ID NO: 8;
(B) a protein consisting of the amino acid sequence represented by SEQ ID NO: 8 with one or more amino acids deleted, substituted or added, and having an antifreeze function;
(16) DNA encoding antifreeze protein Z-5 represented by the following (a) or (b):
(A) a protein having the amino acid sequence represented by SEQ ID NO: 10;
(B) a protein comprising an amino acid sequence represented by SEQ ID NO: 10 in which one or more amino acids have been deleted, substituted or added, and having an antifreeze function;
(17) DNA encoding antifreeze protein Z-6 represented by the following (a) or (b):
(A) a protein having the amino acid sequence represented by SEQ ID NO: 12;
(B) a protein consisting of the amino acid sequence represented by SEQ ID NO: 12 with one or more amino acids deleted, substituted or added, and having an antifreeze function;
(18) DNA encoding antifreeze protein Z-7 represented by the following (a) or (b):
(A) a protein having the amino acid sequence represented by SEQ ID NO: 14;
(B) a protein comprising an amino acid sequence represented by SEQ ID NO: 14 in which one or more amino acids have been deleted, substituted or added, and having an antifreeze function;
(19) DNA encoding antifreeze protein Z-8 represented by the following (a) or (b):
(A) a protein having the amino acid sequence represented by SEQ ID NO: 16;
(B) a protein consisting of the amino acid sequence represented by SEQ ID NO: 16 with one or more amino acids deleted, substituted or added, and having an antifreeze function;
(20) DNA encoding antifreeze protein Z-9 represented by the following (a) or (b):
(A) a protein having the amino acid sequence represented by SEQ ID NO: 18;
(B) a protein comprising an amino acid sequence represented by SEQ ID NO: 18 in which one or more amino acids have been deleted, substituted or added, and having an antifreeze function;
(21) DNA encoding antifreeze protein Z-10 represented by the following (a) or (b):
(A) a protein having the amino acid sequence represented by SEQ ID NO: 20;
(B) a protein consisting of the amino acid sequence represented by SEQ ID NO: 20 with one or more amino acids deleted, substituted or added, and having an antifreeze function;
(22) DNA encoding antifreeze protein B-1 represented by the following (a) or (b):
(A) a protein having the amino acid sequence represented by SEQ ID NO: 22;
(B) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 22, and which has an antifreeze function;
(23) a base sequence represented by SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or 21, and the antifreeze proteins Z-1, Z-2, and Z DNA encoding -3, Z-4, Z-5, Z-6, Z-7, Z-8, Z-9, Z-10 or B-1.
(24) A recombinant vector containing the DNA of any one of (12) to (23).
(25) The recombinant vector according to (24), wherein the recombinant vector is an expression vector.
(26) A transformant obtained by transforming a host organism with the recombinant vector according to (25).
(27) The transformant according to (26), wherein the host organism is Escherichia coli.
(28) A method for producing a protein having an antifreeze function, comprising culturing the transformant according to (27) in a medium to produce a protein having an antifreeze function, and collecting the protein.
(29) A method for producing a protein, which comprises chemically synthesizing the protein according to any one of (1) to (12) using a peptide synthesizer.
(30) A freeze concentration inhibitor comprising the protein according to any one of (1) to (12) as an active ingredient.
(31) A freezing-point depressant comprising the protein according to any one of (1) to (12) as an active ingredient.
(32) An ice slurry comprising the protein according to (1) to (12).
[0009]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail.
The body fluids of fish inhabiting the Arctic, Antarctic or near the sea or in the deep sea do not freeze even when the temperature drops to about minus 2 ° C. In contrast, normal fish body fluids freeze at -0.8 ° C. Seawater freezes at minus 1.9 ° C.
[0010]
It has been found that the antifreeze properties of the body fluids of fish that inhabit the Arctic, Antarctic, etc. are due to antifreeze proteins or antifreeze glycoproteins (AFGP) that these fish produce themselves. Among them, antifreeze proteins include four types of proteins, AFPI having a molecular weight of about 3,000 to 4,500, AFPII having a molecular weight of about 20,000, AFPIII having a molecular weight of about 7,000, and a molecular weight of about 11,000. 000 AFPIV. The molecular weight of AFGP is between 2,200 and 33,000. Each antifreeze protein has a difference in the amino acid composition of the protein and its higher-order structure, and even if they are classified into the same type, the amino acid sequence and the higher-order structure of the protein differ depending on the fish species. (See Non-Patent Document 1).
[0011]
On the other hand, regarding the function of antifreeze proteins, ice crystals usually grow into flat hexagonal plates when ice nuclei appear in an aqueous solution. The growth in the direction perpendicular to the plate-like plane is about 100 times slower than the growth in the direction of the plate-like plane. On the other hand, when the antifreeze protein (AFP) is present, the growth of ice crystals in the direction of the disk plane is prevented, and the plate-like body formed first is used as a base surface, and the plate-like body is sequentially formed in a direction perpendicular to the base surface. Then, smaller plate-like bodies are stacked, and eventually grow slowly into a bipyramid ice crystal in which two pyramids are stacked. Therefore, only when AFP is present in the sample of interest, when the sample solution is cooled to 0 ° C. or lower, the sample solution generally contains a bipyramid ice crystal, and a crystallographically hexagonal tetrahedron ( A single crystal of ice called a hexagonal pyramid (left figure in FIG. 2) or a hexagonal bipyramid (left figure in FIG. 2) is observed under a microscope. As a result of the presence of AFP, specific binding occurs to the twelve ice layer planes on the ice crystal, and such a bipyramid ice crystal is generated. This is macroscopically observed as a non-freezing phenomenon (antifreeze activity) of the specimen. This phenomenon can be quantified as a freezing point drop or temperature hysteresis of the sample liquid by using an osmometer (osmometer). In order to evaluate the antifreeze activity using the method of measuring the freezing point depression, it is necessary to obtain a high-purity aqueous solution of AFP. On the other hand, the antifreeze activity evaluation method by bipyramidal ice crystal observation is observed as long as AFP is present (see Non-Patent Document 2).
[0012]
(1) A method for preparing the antifreeze proteins Z-1 to Z-12 of the present invention from the blood or surimi of Nagaji or Beetlefish according to the present invention
The head and internal organs of one Nagafish fish body were removed, and the body was cut off, 0.1M ammonium bicarbonate aqueous solution (pH = 7.9) was added, and the mixture was ground in a juicer mixer. A 0.1 M aqueous solution of ammonium bicarbonate was added to the Nagami surimi to prepare a surimi suspension. This was centrifuged at 6,000 rpm for 30 minutes to obtain a supernatant. The supernatant was dialyzed against a 50 mM sodium acetate buffer (pH = 3.7) and centrifuged at 12,000 rpm for 30 minutes to remove a precipitate. Cation exchange chromatography was performed on the obtained supernatant. For this operation, a 50 mM sodium acetate buffer (pH = 3.7) was used, and a 0-0.5 M sodium chloride gradient was used for elution. The sample solution obtained by this operation, at which absorption at 280 nm was observed, was purified by reverse phase chromatography (HPLC). 0.1% trifluoroacetic acid was used for column equilibration and sample uptake, and a linear gradient of acetonitrile was used for elution. As a result, a plurality of eluate fractions containing Nagaji-derived antifreeze protein were obtained. The activity of the obtained antifreeze protein was confirmed by observing bipyramidal ice crystals, and the purity was confirmed by SDS electrophoresis. The obtained sample was lyophilized and then stored frozen.
[0013]
(2) Method for determining amino acid sequence of antifreeze protein prepared in (1) above
The standards were analyzed using a protein sequencer, and the elution time corresponding to each amino acid during separation by HPLC was determined. The freeze-dried powder of the antifreeze protein prepared in the above (1) was dissolved in acetic acid, and analyzed sequentially from the N-terminal side by a protein sequencer using a general Edman degradation method. Specifically, (i) phenylisothiocyanate (PITC) was coupled to the N-terminus of the protein to generate a phenylthiocarbamyl protein (PTC-protein). Next, (ii) the N-terminal amino acid is cut out from the PTC-protein as a 2-anilino-5-thiazolinone derivative (ATZ-amino acid) with trifluoroacetic acid. The ATZ-amino acid was converted to a phenylthiohydantoin derivative (PTH-amino acid) under acidic conditions, separated by HPLC, and the type of amino acid was determined based on the elution time. The amino acid sequence was determined by repeating the cycles (i) and (ii) using the remaining protein from which the N-terminal amino acid was cut out as a raw material.
[0014]
(3) Cloning method of antifreeze protein gene prepared above and determination of base sequence
Nagaji liver was collected from raw fish during the severe cold season when the expression level of antifreeze protein was increased. The collected liver was cut into 5 mm squares and stored in a 10-fold amount of RNA stabilizer. Liver samples impregnated with the stabilizer were quickly ground at low temperature to extract RNA. MRNA was separated and purified from this RNA solution using an oligo dT matrix. Using purified mRNA as a template, cDNA (1st strand) was synthesized with a primer containing an oligo dT sequence and reverse transcriptase, and then cDNA (2nd strand) was synthesized with RNase H and DNA polymerase I. Further, both ends of the cDNA were blunted with Pfu DNA Polymerase, and an adapter sequence was added. Using the thus constructed cDNA library as a template, Polymerase chain reaction (PCR) was performed using a primer designed based on the amino acid sequence determined in (2) above and a primer containing an oligo dT sequence, and the amplified gene fragment was subjected to TA. Cloned. The sequence of the cloned gene fragment was determined by the DyeTerminator method using a primer containing an oligo dT sequence. The conditions of the cycle sequence at that time were 96 ° C. for 10 seconds, 50 ° C. for 5 seconds, 60 ° C. for 4 minutes, and 25 cycles. As a result, it was shown that the base sequence encoding the antifreeze protein was contained in a group of gene fragments having a length of about 500 bp, which was superior in the Nagaji-derived cDNA library. Based on the results, a gene fragment of about 500 bp was amplified using a 500 bp cDNA as a template, a primer that anneals to the added adapter sequence and a primer containing an oligo dT sequence, and TA cloned. The sequence of the cloned gene fragment was similarly determined by the Dye Terminator method.
[0015]
(4) Preparation of recombinant vector
The amplified gene fragment was electrophoresed on 0.8% agarose, and a 500 bp gene fragment was cut out and purified. 20 ng of the purified gene fragment was added to 50 ng of the TA cloning vector, and a ligation reaction was performed at 4 ° C. overnight using T4 DNA Ligase.
[0016]
(5) Preparation of transformant
The recombinant vector solution was added to competent cells (DH5α) melted on ice, and allowed to stand on ice for 30 minutes. This was heat-treated at 42 ° C. for 1 minute, immediately transferred to ice and allowed to stand for 2 minutes. To this was added an SOC medium and incubated at 37 ° C. for 30 minutes. The cells were spread on an LB agar medium containing 100 μg / ml ampicillin, and incubated at 37 ° C. overnight to form colonies. An arbitrary colony thus formed was inoculated into an LB liquid medium containing 100 μg / ml ampicillin, and cultured with shaking E. coli at 37 ° C. overnight. The grown Escherichia coli was destroyed by the alkali-SDS method, and the recombinant vector was separated and purified using a DNA adsorption matrix and a spin column.
[0017]
(6) Method for producing antifreeze protein using transformant
A base sequence encoding an antifreeze protein was incorporated into a recombinant expression vector containing a promoter, a terminator sequence, and the like, and a transformant with this recombinant vector was precultured in 10 ml of an LB liquid medium containing 100 μg / ml ampicillin. 10 ml of this culture was added to 1000 ml of LB liquid medium containing 100 μg / ml ampicillin, and the culture was further continued. 28 ° C. was set as the culture temperature. When the turbidity of the culture (OD = 600) increased to 0.5, a protein expression inducer was added. The culture solution was collected overnight after the induction of expression, and the antifreeze protein was purified using the method by cation exchange chromatography described in (1).
[0018]
(7) Method of using antifreeze protein as freeze-concentration inhibitor and freezing-point depressant When using AFP as a freeze-concentration inhibitor or freezing-point depressant for hydrated products, the concentration of AFP in the hydrated product is reduced. Is added in an amount of 0.02% by weight or more based on the weight of the hydrated substance. In particular, when the concentration is in the range of 0.03% by weight to 0.05% by weight, AFP exerts its maximum effect. At this time, for example, when the hydrate is a liquid (aqueous solution, blood, etc.), the powder or the aqueous solution of AFP may be mixed as it is. When the hydrate is a cell or tissue, an aqueous solution of AFP may be injected into the hydrate using a syringe or the like. Further, after immersing the hydrated product in the aqueous solution of AFP, 50 to 2,000 kg / cm ² AFP may be permeated into the hydrated material by, for example, pressurizing the AFP.
[0019]
As described above, the antifreeze protein of the present invention has a function of preventing freeze-concentration of water-containing substances and a function of preventing recrystallization of ice. Can be used as an inhibitor of freeze-concentration of hydrates or an agent for preventing recrystallization of ice, regardless of which type of antifreeze protein of AFPI to IV is used. It can be used to maintain its quality. This quality maintaining effect can also be expected when cells (eggs and sperm), tissues, organs, vegetables and the like are stored for a long period of time in a frozen state. Further, in recent years, a cold heat supply system or a cold heat storage system using an ice slurry having a large energy density as a heat medium has been proposed. However, in these systems, there is a problem of clogging of a piping system due to recrystallization of ice. Since antifreeze proteins effectively prevent recrystallization of ice, they can be a promising means to solve this problem. In addition, application of a gene encoding an antifreeze protein into a plant body to provide the plant with cold resistance can also be expected as an applied technique.
[0020]
【Example】
Hereinafter, the present invention will be specifically described with reference to examples. However, the technical scope of the present invention is not limited to these examples.
[0021]
(Example 1)
(1) Sample sample
The fish species used as sources for collecting antifreeze proteins are as follows.
Nagagaji (Zoarces elongatus kner, English name Notched-Fineelport): Caught in Notsuke Bay, Hokkaido.
Whitefish (Brachyopsis rostratus, English name Longsout poacher): Caught in Notsuke Bay, Hokkaido.
[0022]
(2) Observation of antifreeze activity
One ul of each of the collected Nagagaji and beetfish blood was dropped on a 16 mm diameter cover glass of a Leica DMLB100 photomicroscope manufactured by Leica. This was directly sandwiched between another cover glass having a diameter of 12.5 mm, and this was set in a cooling box provided on the stage of a DMLB100 type microscope. Light intake holes having a diameter of 1 mm were formed on the upper and lower sides of the cooling box, and light from a microscope light source passed through the inside of the box from the lower hole, passed through the upper hole, and entered the lens. By setting the sample liquid on the optical axis defined by the upper and lower holes, the substance in the sample liquid on the optical axis is observed with a microscope. The temperature in the cooling box in which the sample liquid is set is controlled with an error of +/− 0.1 ° C. by an LK600 temperature controller (LK600 temperature controller) manufactured by Linkham Inc. After the sample solution was set at room temperature, the temperature in the cooling box was decreased by 0.2 ° C./second to −22 ° C. by the temperature controller. At some point between about minus 1.4 ° C and minus 40 ° C, the entire sample liquid freezes. After freezing, the temperature in the cooling box is raised at 0.1 ° C per second and stopped at 0 ° C. If the temperature is kept at 0 ° C for about 1 second to 10 seconds, the freezing will melt and countless cracks will occur. After passing through the entered ice crystal state, countable single crystals of ice were observed floating in water. At that moment, the temperature in the cooling box was lowered to about −0.1 ° C. to −1.0 ° C. and stopped, and the shape of the ice crystal was observed. The observation results are shown in FIG. Bipyramidal ice crystals were observed in any of the fish species used in the test, and it was confirmed that each of these fish species had antifreeze proteins. Similar tests were performed using 1 L of the fish meat surimi suspension and 1 L of the dry matter suspension. In these tests, bipyramidal ice crystals were observed for all fish species.
[0023]
(3) Preparation of Nagafuji AFP-Z-1 and amino acid sequence determination
The Nagagaji body was cut with a kitchen knife and ground using a mixer. By adding 20 ml of a 0.1 M aqueous solution of ammonium bicarbonate to 20 ml of the surimi, 40 ml of a surimi suspension was prepared. This was placed in a plastic test tube and centrifuged at 6,000 rpm for 30 minutes to obtain about 20 ml of a supernatant. The supernatant was dialyzed against a 50 mM sodium acetate buffer (pH = 3.7) to aggregate contaminating proteins. This was removed by centrifugation at 12,000 rpm for 30 minutes to obtain a supernatant. The supernatant was subjected to cation exchange chromatography, and 1 ml of each eluate was collected by a fraction collector while detecting absorption at 280 nm. Amersham Pharmacia Biotech FPLC system and BIO-RAD High-S column were used for cation exchange chromatography. The column was equilibrated with 50 mM sodium acetate buffer (pH = 3.7) and the supernatant was taken up. Elution was performed by applying a linear gradient of 0 to 0.5 M sodium chloride at a flow rate of 1 ml / min. Was. All the operations so far were performed at 4 ° C. Next, the sample solution in which the absorption at 280 nm was observed was purified by reverse phase chromatography using a TOSO HPLC system and an ODS column. The column was equilibrated and the sample solution was taken up using 0.1% trifluoroacetic acid, and a linear gradient of acetonitrile was used for elution. The absorbance of the eluted sample was detected at 214 nm and 280 nm, and an eluate fraction containing a single protein was obtained and lyophilized. This freeze-dried powder was dissolved in a 0.1 M aqueous solution of ammonium bicarbonate, and its bipyramidal ice crystals were observed to confirm that it was an antifreeze protein. Thus, antifreeze protein Z-1 was obtained. After purifying a sample of Nagaagaji AFP-Z-1, the amino acid sequence was determined using a freeze-dried sample. The dried sample was dissolved in acetic acid and added to a polybrene-treated cartridge filter. Edman degradation was performed using a 491 protein sequencer manufactured by Applied Biosystems, and the amino acid sequence was determined sequentially from the N-terminal side. As a result, the amino acid sequence of AFP-Z-1 is as shown in SEQ ID NO: 2 in the sequence listing.
[0024]
(4) Isolation of total RNA
The basic procedure for isolation of total RNA is described in the protocol ("Rneasy"). ^R Protect and RNAlater ^TM Handbook ”and“ RNeasy ” ^R Mini Handbook "(QIAGEN) was used. Liver of Nagagaji as a sample was collected from a raw fish in the severe cold season when the expression level of antifreeze protein was increased. The liver sample was cut in a 10-fold amount of RNAlater (QIAGEN), and 60 mg of the liver sample was pulverized in liquid nitrogen, and 30 mg of the liver sample was suspended in 600 μl of Buffer RLT, followed by further pulverization with QIAshredder (QIAGEN). An equal volume of 70% ethanol was added to the collected supernatant, and the RNA was adsorbed onto the column by passing through a spin column.After washing the column with Buffer RW1 and Buffer RPE, the RNA was eluted with 30 μl of RNase-free water.
[0025]
(5) mRNA isolation
The mRNA was isolated using OligotexTM-dT30 <Super> mRNA Purification kit (TaKaRa), and the operation was performed according to the attached protocol. To 150 μl of 1.1 μg / μl total RNA solution, 150 μl of 2 × Binding Buffer and 15 μl of OligotexTM-dT30 <Super> were added, heated at 70 ° C. for 3 minutes, and left at room temperature for 10 minutes. After recovering Oligotex ™ -dT30 <Super> by centrifugation, the suspension was suspended in Wash Buffer, and Oligotex-dT30 <Super> was washed with a spin column. The mRNA adsorbed on OligotexTM-dT30 <Super> was eluted with 40 µl of RNase-free water heated at 70 ° C.
[0026]
(6) Construction of cDNA library
The construction of the cDNA library is performed using ZAP-cDNA Synthesis Kit (STRATAGENE). ^R ). The basic operation is "cDNA Synthesis Kit, ZAP-cDNA" ^R Synthesis Kit, and ZAP-cDNA ^R Gigapack ^R III Gold Cloning Kit INSTRUCTION MANUAL ”(STRATAGENE ^R ). Using the mRNA collected as described above as a template, dNTPs containing methylated dCTP (dATP, dCTP, dGTP, dTTP), using StrataScript RTase, and reacting at 42 ° C. for 1 hour to reverse first-strand cDNA. Photo-synthesized. Next, RNase H, DNA polymerase, and dNTP were added, and the mixture was reacted at 16 ° C. for 2.5 hours to synthesize second-strand cDNA. After blunting the ends of the cDNA with Pfu DNA polymerase, T4 DNA ligase was used.
Ligated the adapter sequence.
[0027]
(7) Determination of base sequence
When a part of the constructed cDNA library was electrophoresed on 0.8% agarose, a 500 bp gene fragment was mainly confirmed. Therefore, a degenerate primer was designed based on the amino acid sequence determined by the protein sequencer, and using this primer and a primer complementary to the poly A sequence, a 500 bp cDNA as a template and Ex Taq ^TM PCR (polymerase chain reaction) was performed using (TaKaRa). PCR product to pGEM ^R E. coli was transformed with this plasmid by cloning into -T Easy (Promega). The transformant was spread on an LB agar medium containing 100 μg / ml ampicillin, and any formed colony was inoculated into an LB liquid medium containing 100 μg / ml ampicillin, and cultured at 37 ° C. overnight with shaking E. coli. The plasmid was separated and purified from the grown E. coli by the alkali-SDS method, and T7 promoter primer and BigDye were used. ^R Perform a sequence reaction using Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and perform ABI PRISM. ^TM The nucleotide sequence was analyzed using 310 Genetic Analyzer and 3100 Genetic Analyzer (Applied Biosystems). From the results of this analysis, it was confirmed that the base sequence encoding an amino acid sequence having high homology to the antifreeze protein Z-1 separated and purified was contained in the 500 bp cDNA. Therefore, using this 500 bp cDNA as a template and a primer that anneals to a linker sequence and a poly A sequence, Ex Taq ^TM PCR (polymerase chain reaction) was performed using (TaKaRa) to amplify the entire base sequence encoding the antifreeze protein. These PCR products were pGEM ^R It was cloned into -T Easy (Promega), and the sequence of the gene fragment was analyzed by the method described above. The nucleotide sequence of the obtained antifreeze protein Z-1 is as shown in SEQ ID NO: 1 in the sequence listing.
[0028]
The amino acid sequences of the remaining nine new AFP-Z-2 to Z-10 derived from Nagaji were prepared by the same method as described above, and the nucleotide sequences of the CDS regions of those genes were determined by the same method as described above. The amino acid sequences of the new AFP-Z-2 to Z-10 are shown in SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18 and 20 in the sequence listing, and the nucleotide sequences are shown in SEQ ID NOs: 3, 5, 7, and 9, 11, 13, 15, 17 and 19. These nucleotide sequences showed about 75 to 90% homology with a type III antifreeze protein derived from Macrozoarques americanus whose nucleotide sequence had already been determined. In this way, new type AFP-Z-2 to Z-10 of type III derived from Nagaagaji were obtained.
[0029]
(Example 2)
(1) Determination of amino acid sequence of AFP-B-1
According to the same purification method as that of Nagagaji AFP of Example 1, a TOSOH HPLC system and TSK-GEL ODS-80TS (4.6 mm (ID) x 15.0 cm (L), TOSOH) were used from 40 ml of a surimi from a stickfish. After AFP was purified to high purity, the antifreeze activity was confirmed (see FIG. 2), and the amino acid sequence was determined using the lyophilized sample. The dried sample was dissolved in acetic acid and added to a polybrene-treated cartridge filter. Edman degradation was performed using a 491 protein sequencer manufactured by Applied Biosystems, and the amino acid sequence was determined sequentially from the N-terminal side. As a result, the amino acid sequence of AFP-B-1 is as shown in SEQ ID NO: 22 in the sequence listing.
[0030]
(2) Isolation of total RNA
The basic procedure for the isolation of total RNA is the protocol collection ("RNeasy"). ^R Protect and RNAlater ™ Handbook ”and“ RNeasy ” ^R According to Mini Handbook "(QIAGEN), the livers of various fishes as samples were collected from raw fish during the severe cold season when the expression level of antifreeze proteins was increased. The liver sample (60 mg) was shredded in liquid nitrogen, suspended in 600 μl of Buffer RLT (30 μl each), and further crushed by QIAshredder (QIAGEN). An equal volume of 70% ethanol was added to the supernatant collected by the above step, and the RNA was adsorbed on the column by passing through a spin column.After washing the column with Buffer RW1 and Buffer RPE, the RNA was eluted with 30 μl of RNase-free water. .
[0031]
(3) mRNA isolation
The mRNA was isolated using OligotexTM-dT30 <Super> mRNA Purification kit (TaKaRa), and the operation was performed according to the attached protocol. To 150 μl of 1.1 μg / μl total RNA solution, 150 μl of 2 × Binding Buffer and 15 μl of OligotexTM-dT30 <Super> were added, heated at 70 ° C. for 3 minutes, and left at room temperature for 10 minutes. After recovering OligotexTM-dT30 <Super> by centrifugation, the suspension was suspended in Wash Buffer, and Oligotex-dT30 <Super> was washed with a spin column. The mRNA adsorbed on OligotexTM-dT30 <Super> was eluted with 40 µl of RNase-free water heated at 70 ° C.
[0032]
(4) Construction of cDNA library
The construction of the cDNA library is performed using ZAP-cDNA Synthesis Kit (STRATAGENE). ^R ). The basic operation is "cDNA Synthesis Kit, ZAP-cDNA" ^R Synthesis Kit, and ZAP-cDNA ^R Gigapack ^R III Gold Cloning Kit INSTRUCTION MANUAL ”(STRATAGENE ^R ). Using the mRNA collected as described above as a template, dNTPs containing methylated dCTP (dATP, dCTP, dGTP, dTTP), using StrataScript RTase, and reacting at 42 ° C. for 1 hour to reverse first-strand cDNA. Photo-synthesized. Next, RNase H, DNA polymerase, and dNTP were added, and the mixture was reacted at 16 ° C. for 2.5 hours to synthesize second-strand cDNA. After blunting the ends of the cDNA with Pfu DNA polymerase, T4 DNA ligase was used.
Ligated the adapter sequence.
[0033]
(5) Determination of base sequence
A degenerate primer is designed on the basis of the amino acid sequence determined by the protein sequencer, and PCR (polymerase chain reaction) is performed using Ex Taq ™ (TaKaRa) using this primer and a primer complementary to the polyA sequence and cDNA as a template. Was. PCR products to pGEM ^R E. coli was transformed with this plasmid by cloning into -T Easy (Promega). The transformant was spread on an LB agar medium containing 100 μg / ml ampicillin, and any formed colony was inoculated into an LB liquid medium containing 100 μg / ml ampicillin, and cultured at 37 ° C. overnight with shaking E. coli. The plasmid was separated and purified from the grown E. coli by the alkali-SDS method, and T7 promoter primer and BigDye were used. ^R The sequence reaction was carried out using Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems), and ABI PRISMTM 310 Genetic Analyzer and 3100 Genetic Analyzer were analyzed by Apisizer. The nucleotide sequence encoding the C-terminus of the antifreeze protein was determined from the results of this analysis, and PCR (polymerase chain) was performed using Ex Taq ™ (TaKaRa) with cDNA as a template, using a primer complementary to this sequence and a primer that anneals to the linker sequence. reaction) to amplify the entire nucleotide sequence encoding the antifreeze protein. This PCR product is called pGEM ^R It was cloned into -T Easy (Promega), and the sequence of the gene fragment was analyzed by the method described above. As a result, the nucleotide sequence of AFP-B-1 derived from rosewood is as shown in SEQ ID NO: 21. The nucleotide sequence of AFP-B-1 derived from rosehipfish showed about 63% homology with the type II antifreeze protein derived from Lipterus americanus (sea raven (Hemitripterus americanus)) whose sequence was revealed. In this way, the type II antifreeze protein was separated and purified from the brown fish surimi.
[0034]
【The invention's effect】
According to the present invention, 11 types of novel antifreeze proteins were collected from a long-tailed Nagashi or beetle fish, which are fish caught in the waters around Japan, and their amino acid sequences and nucleotide sequences were determined. Further, it has become possible to produce the antifreeze protein in a large amount by a genetic engineering technique using the obtained gene.
[0035]
As described above, these antifreeze proteins prevent the deterioration of taste and tissue destruction due to the growth of ice crystals in ice cream, frozen foods, and the like.In addition, in a cold heat supply system using a cold slurry or cold heat storage, etc. It is expected as an effective additive capable of resolving clogging of a piping system due to recrystallization. Furthermore, it is a promising substance for long-term storage at low temperatures and frozen storage of eggs, sperm, transplanted organs and the like.
[0036]
[Sequence list]

[Brief description of the drawings]
FIG. 1 is a schematic diagram and a photograph showing the shape of a bipyramid ice crystal. In this figure, the left shows a schematic diagram of a hexagonal bipyramid, and the right shows a photograph of ice crystals of the hexagonal bipyramid observed in Nagaji blood.
FIG. 2 is a schematic diagram and a photograph showing the shape of a bipyramid ice crystal. In this figure, the left side is a schematic diagram of a hexagonal tetrahedron, and the right side is a photograph of ice crystals of the hexagonal tetrahedron observed in blood of a beetle fish.

Claims

Antifreeze protein Z-1 represented by the following (a) or (b):
(A) a protein having the amino acid sequence represented by SEQ ID NO: 2;
(B) a protein comprising an amino acid sequence represented by SEQ ID NO: 2 in which one or more amino acids have been deleted, substituted or added, and having an antifreeze function;

Antifreeze protein Z-2 represented by the following (a) or (b):
(A) a protein having the amino acid sequence represented by SEQ ID NO: 4;
(B) a protein comprising an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 4, and having an antifreeze function;

Antifreeze protein Z-3 represented by the following (a) or (b):
(A) a protein having the amino acid sequence represented by SEQ ID NO: 6;
(B) a protein consisting of the amino acid sequence represented by SEQ ID NO: 6 in which one or more amino acids have been deleted, substituted or added, and having an antifreeze function;

Antifreeze protein Z-4 represented by the following (a) or (b):
(A) a protein having the amino acid sequence represented by SEQ ID NO: 8;
(B) a protein consisting of the amino acid sequence represented by SEQ ID NO: 8 with one or more amino acids deleted, substituted or added, and having an antifreeze function;

Antifreeze protein Z-5 represented by the following (a) or (b):
(A) a protein having the amino acid sequence represented by SEQ ID NO: 10;
(B) a protein comprising an amino acid sequence represented by SEQ ID NO: 10 in which one or more amino acids have been deleted, substituted or added, and having an antifreeze function;

Antifreeze protein Z-6 represented by the following (a) or (b):
(A) a protein having the amino acid sequence represented by SEQ ID NO: 12;
(B) a protein consisting of the amino acid sequence represented by SEQ ID NO: 12 with one or more amino acids deleted, substituted or added, and having an antifreeze function;

Antifreeze protein Z-7 represented by the following (a) or (b):
(A) a protein having the amino acid sequence represented by SEQ ID NO: 14;
(B) a protein comprising an amino acid sequence represented by SEQ ID NO: 14 in which one or more amino acids have been deleted, substituted or added, and having an antifreeze function;

Antifreeze protein Z-8 represented by the following (a) or (b):
(A) a protein having the amino acid sequence represented by SEQ ID NO: 16;
(B) a protein consisting of the amino acid sequence represented by SEQ ID NO: 16 with one or more amino acids deleted, substituted or added, and having an antifreeze function;

Antifreeze protein Z-9 represented by the following (a) or (b):
(A) a protein having the amino acid sequence represented by SEQ ID NO: 18;
(B) a protein comprising an amino acid sequence represented by SEQ ID NO: 18 in which one or more amino acids have been deleted, substituted or added, and having an antifreeze function;

Antifreeze protein Z-10 represented by the following (a) or (b):
(A) a protein having the amino acid sequence represented by SEQ ID NO: 20;
(B) a protein consisting of the amino acid sequence represented by SEQ ID NO: 20 with one or more amino acids deleted, substituted or added, and having an antifreeze function;

Antifreeze protein B-1 represented by the following (a) or (b):
(A) a protein having the amino acid sequence represented by SEQ ID NO: 22;
(B) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 22, and which has an antifreeze function;

DNA encoding antifreeze protein Z-1 represented by the following (a) or (b):
(A) a protein having the amino acid sequence represented by SEQ ID NO: 2;
(B) a protein comprising an amino acid sequence represented by SEQ ID NO: 2 in which one or more amino acids have been deleted, substituted or added, and having an antifreeze function;

DNA encoding antifreeze protein Z-2 represented by the following (a) or (b):
(A) a protein having the amino acid sequence represented by SEQ ID NO: 4;
(B) a protein comprising an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 4, and having an antifreeze function;

DNA encoding antifreeze protein Z-3 represented by the following (a) or (b):
(A) a protein having the amino acid sequence represented by SEQ ID NO: 6;
(B) a protein consisting of the amino acid sequence represented by SEQ ID NO: 6 in which one or more amino acids have been deleted, substituted or added, and having an antifreeze function;

DNA encoding antifreeze protein Z-4 represented by the following (a) or (b):
(A) a protein having the amino acid sequence represented by SEQ ID NO: 8;
(B) a protein consisting of the amino acid sequence represented by SEQ ID NO: 8 with one or more amino acids deleted, substituted or added, and having an antifreeze function;

DNA encoding antifreeze protein Z-5 represented by the following (a) or (b):
(A) a protein having the amino acid sequence represented by SEQ ID NO: 10;
(B) a protein comprising an amino acid sequence represented by SEQ ID NO: 10 in which one or more amino acids have been deleted, substituted or added, and having an antifreeze function;

DNA encoding antifreeze protein Z-6 represented by the following (a) or (b):
(A) a protein having the amino acid sequence represented by SEQ ID NO: 12;
(B) a protein consisting of the amino acid sequence represented by SEQ ID NO: 12 with one or more amino acids deleted, substituted or added, and having an antifreeze function;

DNA encoding antifreeze protein Z-7 represented by the following (a) or (b):
(A) a protein having the amino acid sequence represented by SEQ ID NO: 14;
(B) a protein comprising an amino acid sequence represented by SEQ ID NO: 14 in which one or more amino acids have been deleted, substituted or added, and having an antifreeze function;

DNA encoding antifreeze protein Z-8 represented by the following (a) or (b):
(A) a protein having the amino acid sequence represented by SEQ ID NO: 16;
(B) a protein consisting of the amino acid sequence represented by SEQ ID NO: 16 with one or more amino acids deleted, substituted or added, and having an antifreeze function;

DNA encoding antifreeze protein Z-9 represented by the following (a) or (b):
(A) a protein having the amino acid sequence represented by SEQ ID NO: 18;
(B) a protein comprising an amino acid sequence represented by SEQ ID NO: 18 in which one or more amino acids have been deleted, substituted or added, and having an antifreeze function;

DNA encoding antifreeze protein Z-10 represented by the following (a) or (b):
(A) a protein having the amino acid sequence represented by SEQ ID NO: 20;
(B) a protein consisting of the amino acid sequence represented by SEQ ID NO: 20 with one or more amino acids deleted, substituted or added, and having an antifreeze function;

DNA encoding antifreeze protein B-1 represented by the following (a) or (b):
(A) a protein having the amino acid sequence represented by SEQ ID NO: 22;
(B) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 22, and which has an antifreeze function;

A nucleotide sequence represented by SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or 21, each comprising an antifreeze protein Z-1, Z-2, Z-3; DNA encoding Z-4, Z-5, Z-6, Z-7, Z-8, Z-9, Z-10 or B-1.

A recombinant vector comprising the DNA according to any one of claims 12 to 23.

The recombinant vector according to claim 24, wherein the recombinant vector is an expression vector.

A transformant obtained by transforming a host organism with the recombinant vector according to claim 25.

The transformant according to claim 26, wherein the host organism is Escherichia coli.

A method for producing an antifreeze protein, comprising culturing the transformant according to claim 27 in a medium to produce an antifreeze protein, and collecting the protein.

A method for producing a protein, comprising chemically synthesizing the protein according to any one of claims 1 to 12 using a peptide synthesizer.

A freeze concentration inhibitor comprising the protein according to any one of claims 1 to 12 as an active ingredient.

A freezing point depressant comprising the protein according to any one of claims 1 to 12 as an active ingredient.

An ice slurry comprising the protein according to any one of claims 1 to 12.