JP3635142B2 - Method for producing anti-TPO antibody - Google Patents

Description

（１）ラット血漿の塩化カルシウム処理・遠心処理・蛋白質分解酵素阻害剤処理 −８０℃で保存した約１００匹分のＸＲＰ（７４２ｍｌ，蛋白濃度５４．８ｍｇ／ｍｌ，総蛋白質量４０６８６ｍｇ）を解凍し、ポリプロピレン製遠心チューブ（ナルゲン社製）に移した。これに各々最終濃度１００ｍＭになるように塩化カルシウム粉末を加え、４℃で一晩静置した。次に８０００ＲＰＭで６０分遠心後、上清を回収した。ＴＰＯ活性を含むこの上清（７４２ｍｌ，蛋白濃度５４．９ｍｇ／ｍｌ，総蛋白質量４０７４０ｍｇ）に、最終濃度１ｍＭの蛋白質分解酵素阻害剤であるｐ−ＡＰＭＳＦ（ｐ−アミノジフェニルメタンスルホニルフルオリド 塩酸塩、和光純薬工業、カタログ番号０１０−１０３９３）を加え、次に述べるＳｅｐｈａｄｅｘ Ｇ−２５カラムによるバッファー交換のステップへ進めた。
このようにして、約１００匹分ごとにひとつのロットにまとめ、塩化カルシウム・ｐ−ＡＰＭＳＦ処理し、合計１１ロット、約１１００匹分のＸＲＰ（総体積８１８４ｍｌ）総蛋白質量４９３００７ｍｇ）の処理を繰り返し、それぞれＳｅｐｈａｄｅｘ Ｇ−２５カラムへ進めた。
（２）Ｓｅｐｈａｄｅｘ Ｇ−２５＜バッファー交換＞
（１）で得られた塩化カルシウム処理後の上清（７４２ｍｌ，蛋白濃度５４．９ｍｇ／ｍｌ、総蛋白質量４０７４０ｍｇ）を、２０ｍＭ Ｔｒｉｓ−ＨＣＩ，ｐＨ８で予め平衡化してあったＳｅｐｈａｄｅｘ Ｇ−２５Ｍカラム（ファルマシア バイオテク社製、カタログ番号１７−００３３−０３；直径１１．３ｃｍ、ベッド高４７ｃｍ）に流速４０〜７０ｍｌ／ｍｉｎで添加し、蛋白質が溶出してくるまでの１３００ｍｌを捨てた。次に、紫外吸収が立ち上がってきた時点から、電気伝導度が５００μＳ／ｃｍに達するまでを集め、２０ｍＭ Ｔｒｉｓ−ＨＣｌ ｐＨ８溶液に置換されたＴＰＯ活性を含む蛋白質画分（１３７７ｍｌ，蛋白濃度２７．５６ｍｇ／ｍｌ）を回収した。ＴＰＯ活性画分の総蛋白質量は、３７８８２ｍｇ、このステップでの蛋白収量は、９３％であった。また、ＴＰＯの相対活性は、２．３であった。
このようにして、全ロットについてそれぞれＳｅｐｈａｄｅｘ Ｇ−２５カラムを実施した結果、合計すると、総体積 ２１１１７ｍｌ、総蛋白質量 ４８０３０６ｍｇ、平均相対活性 １．８、相対活性量 ８６４６００のＳｅｐｈａｄｅｘ Ｇ−２５のＴＰＯ活性画分を得た。
（３）Ｑ−Ｓｅｐｈａｒｏｓｅ ＦＦ＜強陰イオン交換クロマトグラフィー＞
（２）で得られたＳｅｐｈａｄｅｘ Ｇ−２５ＭのＴＰＯ活性画分（１３７５ｍｌ，蛋白濃度２７．５ｍｇ／ｍｌ、総蛋白質量３７８４１ｍｇ、相対活性２．３）を流速４０ｍｌ／ｍｉｎで、Ｑ−Ｓｅｐｈａｒｏｓｅ ＦＦ（ファルマシアバイオテク社製、カタログ番号１７−０５１０−０１；直径 ５ｃｍ、ベッド高 ２７ｃｍ）に添加し、２０ｍＭ Ｔｒｉｓ−ＨＣｌ，ｐＨ８で素通る画分Ｆ１（３９４９ｍｌ，蛋白濃度０．９８ｍｇ／ｍｌ，総蛋白質量３８７０ｍｇ，相対活性０）を溶出した。
次に、１７５ｍＭ ＮａＣｌを含む２０ｍＭ Ｔｒｉｓ−ＨＣｌ，ｐＨ８緩衝液に換えて、ＴＰＯ活性画分Ｆ２（４３７５ｍｌ，蛋白濃度５．３６ｍｇ／ｍｌ）を溶出した。
最後に、１０００ｍＭ ＮａＣｌを含む２０ｍＭ Ｔｒｉｓ−ＨＣｌ ｐＨ８緩衝液で、Ｆ３（１２２１ｍｌ，蛋白濃度３．９ｍｇ／ｍｌ，総蛋白質量４７８３ｍｇ，相対活性３．８）を溶出した。ＴＰＯ活性画分Ｆ２の総蛋白質量は、２３４４０ｍｇ、このステップでのＦ２の蛋白収量は、６１．９％であった。また、ＴＰＯの相対活性は、６．８に上昇した。
このようにして、Ｓｅｐｈａｄｅｘ Ｇ−２５のＴＰＯ活性画分の全ロットをＱ−ＳｅｐｈａｒｏｓｅＦＦにかけた結果、合計すると総体積 ３５８４２ｍｌ、総蛋白質量 ３１４３８４ｍｇ、平均相対活性 ８．６、相対活性量 ２７０４０００のＱ−Ｓｅｐｈａｒｏｓｅ ＦＦのＴＰＯ活性画分Ｆ２を得た。
（４）小麦胚芽アグルチニン（ＷＧＡ）−Ａｇａｒｏｓｅ＜レクチンアフィニティークロマトグラフィー＞
（３）で得られたＱ−Ｓｅｐｈａｒｏｓｅ ＦＦ（７）ＴＰＯ活性画分Ｆ２を３回に分けて、ＷＧＡ−Ａｇａｒｏｓｅ （ホーネン社製、カタログ番号８００２７３；直径 ５ｃｍ、ベッド高 ２２．５ｃｍ）に流速５ｍｌ／ｍｉｎで添加し、ダルベッコ氏リン酸等張緩衝液（ＤＰＢＳ）で素通る画分Ｆ１（９３３６ｍｌ，蛋白濃度２．３０ｍｇ／ｍｌ，総蛋白質量２１４０７ｍｇ，相対活性６．９）を得た。
次に、０．２ＭＮ−アセチル−Ｄ−グルコサミン（ＧｌｃＮＡｃ、ナカライ社製、カタログ番号００５−２０）、１５０ｍＭ ＮａＣｌ）０．０２％アジ化ナトリウムを含む２０ｍＭ Ｎａ Ｐｈｏｓｐｈａｔｅ，ｐＨ７．２緩衝液により溶出されたプールを、限外濾過ユニット（フィルトロン社製、オメガウルトラセット分子量８０００カット）で濃縮し、ＷＧＡ−Ａｇａｒｏｓｅ吸着ＴＰＯ活性画分Ｆ２（２９９３ｍｌ，蛋白濃度０．３７６ｍｇ／ｍｌ）を得た。
このＴＰＯ活性画分Ｆ２の総蛋白質量は、１１２５ｍｇ、このステップでのＦ２の蛋白収量は、４．８％であった。また、ＴＰＯの相対活性は、１０１に上昇した。ここで得られたＦ２は−８０℃で保存した。
このようにして、Ｑ−Ｓｅｐｈａｒｏｓｅ ＦＦのＴＰＯ活性画分Ｆ２の全ロットについて、繰り返しＷＧＡ−Ａｇａｒｏｓｅにかけた結果、総体積 ３３０９４ｍｌ、総蛋白質量 １５０３０ｍｇ、平均相対活性 １３２、相対活性量 １９８７０００のＷＧＡ−ＡｇａｒｏｓｅのＴＰＯ活性画分Ｆ２を得た。
（５）ＴＳＫ−ｇｅｌ ＡＦ−ＢＬＵＥ ６５０ ＭＨ＜色素吸着アフィニティークロマトグラフィー＞
（４）で得られた、合計２１５匹分のＸＲＰから出発したロットＸＷ８のＷＧＡ−Ａｇａｒｏｓｅ吸着ＴＰＯ活性画分とロットＸＷ９のＷＧＡ−Ａｇａｒｏｓｅ吸着ＴＰＯ活性画分Ｆ２をバッチＸＢ６としてまとめた（５９７４ｍｌ，蛋白濃度０．３８８ｍｇ／ｍｌ、総蛋白質量２３１９ｍｇ、相対活性１５０）。
この体積５９７４ｍｌに対し、０．８５ｍｏｌｅｓのＮａＣｌ（２９６．７６ｇ）を加え、最終濃度０．８２２Ｍ ＮａＣｌ，６１３２ｍｌの溶液とした後、ＩＭ ＮａＣｌ，２０ｍＭ Ｎａ Ｐｈｏｓｐｈａｔｅ， ｐＨ７．２で予め平衡化してあったＴＳＫ−ｇｅｌ ＡＦ−ＢＬＵＥ ６５０ ＭＨカラム（トーソー社製、カタログ番号０８７０５ ；直径 ５ｃｍ、ベッド高 ２３ｃｍ）に、流速７ｍｌ／ｍｉｎで添加した。
添加終了後、流速１０ｍｌ／ｍｉｎにて、２０ｍＭ Ｎａ Ｐｈｏｓｐｈａｔｅ， １ＭＮａＣｌ， ｐＨ７．２で溶出される素通り（約８４７０ｍｌ）を集め、これを限外濾過ユニット（フィルトロン社製、オメガウルトラセット分子量８０００カット）で濃縮し、素通り画分Ｆ１（５４３ｍｌ，蛋白濃度２．０５ｍｇ／ｍｌ，総蛋白質量１１１２ｍｇ，相対活性３１）を得た。
次に、溶出液を２Ｍ ＮａＳＣＮにかえ、溶出されたＴＳＫ−ｇｅｌ ＡＦ−ＢＬＵＥ ６５０ＭＨ吸着ＴＰＯ活性画分Ｆ２（１４２７ｍｌ，蛋白濃度０．４４７ｍｇ／ｍｌ）を得た。
このＴＰＯ活性画分Ｆ２の総蛋白質量は６３８ｍｇ、このステップでのＦ２の蛋白収量は２７．５％であった。また、ＴＰＯの相対活性は、１５００に上昇した。
このようにして、ＷＧＡ−Ａｇａｒｏｓｅ吸着ＴＰＯ活性画分Ｆ２の全バッチについて、各々ＴＳＫ ＡＦ−ＢＬＵＥ ６５０ＭＨにかけた結果、総体積 １０６５５ｍｌ、総蛋白質量４２３６ｍｇ、平均相対活性 ９０５、相対活性量 ３８３４０００のＴＳＫ−ｇｅｌΛＦ−ＢＬＵＥ ６５０ＭＨのＴＰＯ活性画分Ｆ２を得た。
（６）Ｐｈｅｎｙｌ Ｓｅｐｈａｒｏｓｅ ６ＦＦ／ＬＳ＜疎水相互作用クロマトグラフィー＞
（５）で得られたＴＳＫ−ｇｅｌ ＡＦ−ＢＬＵＥ６５０ＭＨのＴＰＯ活性画分Ｆ２（１４２４ｍｌ，蛋白濃度０．４４７ｍｇ／ｍｌ）総蛋白質量６３８ｍｇ）相対活性１５００）の体積１４２４ｍｌに対し、１．５ｍｏｌｅｓのＡｍｍｏｎｉｕｍ Ｓｕｌｆａｔｅ（２８２．２ｇ）の粉末を加え、最終濃度１．３５ＭＡｍｍｏｎｉｕｍ Ｓｕｌｆａｔｅ， １５８１ｍｌの溶液とした。
これを１．５Ｍ Ａｍｍｏｎｉｕｍ Ｓｕｌｆａｔｅ， ５０ｍＭ Ｎａ Ｐｈｏｓｐｈａｔｅ， ｐＨ７．２で予め平衡化してあったＰｈｅｎｙｌ Ｓｅｐｈａｒｏｓｅ ６ＦＦ（Ｌｏｗ Ｓｕｂ）カラム（ファルマシアバイオテク社製、カタログ番号１７−０９６５−０５；直径 ５ｃｍ、ベッド高 １０ｃｍ）に、流速７ｍｌ／ｍｉｎにて添加し、添加終了後、溶出液を０．８Ｍ Ａｍｍｏｎｉｕｍ Ｓｕｌｆａｔｅ， ３６ｍＭ Ｎａ Ｐｈｏｓｐｈａ｛ｅにかえ、流速１０ｍｌ／ｍｉｎにて溶出される画分（約３１６０ｍｌ）までを集め、これを限外濾過ユニット（フィルトロン社製、オメガウルトラセット分子量８０００カット）で濃縮し、Ｆ１（４８５ｍｌ，蛋白濃度０．１９４ｍｇ／ｍｌ，総蛋白質量９４．２ｍｇ，相対活性０）を得た。
次に、溶出液を２０ｍＭ Ｎａ Ｐｈｏｓｐｈａｔｅ，ｐＨ７．２にかえ、溶出されたＴＰＯ活性画分Ｆ２（約３５００ｍｌ）を得た。これを限外濾過ユニット（フィルトロン社製、オメガウルトラセット分子量８０００カット）で濃縮し、一旦サンプリングした。この段階のＴＰＯ活性画分Ｆ２（２２０ｍｌ）の蛋白濃度は１．４５ｍｇ／ｍｌ，総蛋白質量３１９ｍｇ）このステップでのＦ２の蛋白収量は、５０．０％であった。また、ＴＰＯの相対活性は、１２３０であった。
このようにして、ＴＳＫ−ｇｅｌ ＡＦ−ＢＬＵＥ ６５０ＭＨのＴＰＯ活性画分Ｆ２の全バッチについて、繰り返しＰｈｅｎｙｌ Ｓｅｐｈａｒｏｓｅ ＦＦ／ＬＳにかけた結果、総体積 １９６６ｍｌ、総蛋白質量 ２７６２ｍｇ、平均相対活性 ８４７、相対活性量 ２３３９０００のＰｈｅｎｙｌ Ｓｅｐｈａｒｏｓｅ ＦＦ／ＬＳのＴＰＯ活性画分Ｆ２を得た。
（７）Ｓｅｐｈａｃｒｙｌ Ｓ−２００ＨＲ＜ゲル濾過クロマトグラフィー＞
（６）で得られたＰｈｅｎｙｌ Ｓｅｐｈａｒｏｓｅ ６ＦＦ／ＬＳ（７）ＴＰＯ活性画分Ｆ２（２１７ｍｌ，蛋白濃度１．４５ｍｇ／ｍｌ）総蛋白質量３１５ｍｇ、相対活性１２３０）に、１４４．８ｍｌの５ＭＮａＣｌ溶液を加えて３６２ｍｌの２Ｍ ＮａＣｌとした後、さらに限外濾過ユニット（アミコン社製；ＹＭ３膜、直径７６ｍｍ）で約５０ｍｌまで濃縮した。
これに８Ｍ尿素を等量体積（５０ｍｌ）加え、最終濃度１Ｍ ＮａＣｌ、４Ｍ尿素の溶液約１００ｍｌにした。さらに約８０ｍｌまで濃縮し、最終的に８８．７８ｍｌのサンプルにし、Ｓｅｐｈａｃｒｙｌ Ｓ−２００ＨＲカラム（ファルマシア バイオテク社製、カタログ番号１７−０５８４−０１；直径 ７．５ｃｍ、ベッド高 １００ｃｍ）に、注入した。
その後３ｍｌ／ｍｉｎの流速にてＤＰＢＳで展開し、ボイド体積（１２００ｍｌ）以降４５ｍｌずつ６０本のポリプロピレン製チューブに集めた。この溶出パターンを図１に示した。２本ごとにアッセイにかけ、残りは１１００匹分の全てのＳｅｐｈａｃｒｙｌ Ｓ−２００ＨＲのプロセスを終えるまで、−８５℃にて凍結保存した。アッセイの結果よりＸＢ６では以下のようにフラクションをまとめた（図１）。
（Ｆ１）チューブ番号 １〜１５（ボイド体積付近の分子量９４０００以上の画分）
（Ｆ２）チューブ番号 １６〜２６（分子量９４０００〜３３０００）
（Ｆ３）チューブ番号 ２７〜４４（分子量３３０００〜３０００）
（Ｆ４）チューブ番号 ４５〜５５（分子量３０００以下）
このようにして、Ｐｈｅｎｙｌ Ｓｅｐｈａｒｏｓｅ ６ＦＦ／ＬＳで得たＴＰＯ活性画分Ｆ２の全バッチについて、各々Ｓｅｐｈａｃｒｙｌ Ｓ−２００ＨＲにかけ、それぞれのフラクションについてアッセイを行ない、−８５℃にて凍結保存をした。すべてのバッチについてＳｅｐｈａｃｒｙｌ Ｓ−２００ＨＲ終了後、次の逆相クロマトグラフィー（ＹＭＣ−Ｐａｃｋ ＰＲＯＴＥＩＮ−ＲＰ）を実施する直前に解凍をし、限外濾過ユニット（アミコン社製；ＹＭ３膜、直径７６ｍｍ）で濃縮し、下記の２つの標品を得た。以下、このＳｅｐｈａｃｒｙｌ Ｓ−２００ＨＲのＴＰＯ活性画分Ｆ２の濃縮標品を「高分子ＴＰＯ標品Ｆ２」、Ｓｅｐｈａｃｒｙｌ Ｓ−２００ＨＲのＴＰＯ活性画分Ｆ３の濃縮標品を「低分子ＴＰＯ標品Ｆ３」と言う。ここで述べる高分子ＴＰＯ標品Ｆ２、低分子ＴＰＯ標品Ｆ３とは便宜上、ゲル濾過クロマトグラフィーで溶出位置の異なった画分をまとめたものを称するのであって、必ずしも真の分子量を表現したものではない。
【表２】

以降、低分子ＴＰＯ標品Ｆ３と高分子ＴＰＯ標品Ｆ２とをそれぞれ次の精製ステップに進めた。
以下（８）〜（１１）に低分子ＴＰＯ標品Ｆ３の精製の各ステップについて述べる。
（８）ＹＭＣ−Ｐａｃｋ ＰＲＯＴＥＩＮ−ＲＰ＜逆相クロマトグラフィー＞
（７）で得られた低分子ＴＰＯ標品Ｆ３（総蛋白質量５０．３ｍｇ，蛋白濃度０．１８４ｍｇ／ｍｌ，相対活性２００００，相対活性量１００７０００、総体積２７４ｍｌ）に展開溶媒Ａ（０．０２５％トリフルオロ酢酸（ＴＦＡ））および展開溶媒Ｂ（０．０２５％ ＴＦＡを含む１−プロパノール）を加え、最終体積５０８．６３ｍｌ、最終プロパノール濃度約２０％、ＴＦＡ濃度０．０１２％、蛋白濃度０．０９８９ｍｇ／ｍｌに調製した。ここで不溶物の発生があったので、これを遠心し、上清のみを２５４．３ｍｌ （２５．２ｍｇ）ずつ２回に分けて、予め３０％Ｂで平衡化してあったＹＭＣ−Ｐａｃｋ ＰＲＯＴＥＩＮ−ＲＰ（ＹＭＣ社、カタログ番号Ａ−ＰＲＲＰ−３３−０３−１５ ；直径 ３ｃｍ、ベッド高 ７．５ｃｍ）カラムに流速２ｍｌ／ｍｉｎで添加した。沈殿物は５ｍＭのＣＨＡＰＳ （３−［（３−コラミドプロピル）ジメチルアンモニオ］−１−プロパンスルフォナート；同仁化学研究所製、カタログ番号７５６２１−０３−３）を含む２０ｍＭ酢酸ナトリウム、ｐＨ５．５を２０ｍｌ加えて可溶化し、合わせてカラムに送り込んだ。
サンプルを添加した後、約５０ｍｌの溶媒（展開溶媒Ａ：展開溶媒Ｂ＝３：１）を通液し、まず素通り画分を集めた。次に展開プログラム（１２０分の３０％Ｂ〜４５％Ｂの直線濃度勾配）を開始して、１０ｍｌずつ合計３６本のフラクションをポリプロピレン製チューブに集め取った。これを繰り返して同じチューブに集めたため、最終的に２０ｍｌずつ、合計３６本フラクションとなった。素通り画分はそのまま限外濾過ユニット（アミコン社製：ＹＭ３膜、直径７６ｍｍ）で２０ｍｌまで濃縮した。
素通り画分及びチューブ番号１〜３６の各２０ｍｌのフラクションより０．１ｍｌ取り、２０μｌの５％ＢＳＡを添加後遠心エバポレーションで乾固し、最終的に０．２５ｍｌのＩＭＤＭアッセイ培養液に溶解し、アッセイにかけ、ＴＰＯ活性画分を特定した。この結果、チューブ番号１７〜２７（プロパノール濃度で３６．０〜４３．０％の範囲）にＴＰＯの活性があり、これを低分子ＴＰＯ標品Ｆ３由来のＹＭＣ−Ｐａｃｋ ＰＲＯＴＥＩＮ−ＲＰのＴＰＯ活性画分Ｆ２とした。次のＹＭＣ−Ｐａｃｋ ＣＮ−ＡＰに進める直前まで−８５℃で保存した。低分子ＴＰＯ標品Ｆ３由来のＹＭＣ−Ｐａｃｋ ＰＲＯＴＥＩＮ−ＲＰのＴＰＯ活性画分Ｆ２
総体積 ２２０ｍｌ
蛋白濃度 ０．０１３０ｍｇ／ｍｌ
総蛋白質量 ２．８５ｍｇ
相対活性 １３００００
相対活性量 ３７１０００
（９）ＹＭＣ−Ｐａｃｋ ＣＮ−ＡＰ＜逆相クロマトグラフィー＞
（８）で得られた低分子ＴＰＯ標品Ｆ３由来のＹＭＣ−Ｐａｃｋ ＰＲＯＴＥＩＮ−ＲＰのＴＰＯ活性画分Ｆ２のうち２１４．９ｍｌ（総蛋白質量２．７９ｍｇ，蛋白濃度０．０１３０ｍｇ／ｍｌ，相対活性１３００００，相対活性量３６３００）に、５０％グリセロールを０．６ｍｌ加え、１．８ｍｌまで濃縮した。最終的に体積５ｍｌで、プロパノール濃度は２０％以下、グリセロールは約６％であった。
これを５回（各回の注入蛋白質量０．５５５ｍｇ，体積１ｍｌ）に分けて実施した。毎回ごとに、展開溶媒Ａに０．１％ＴＦＡ）展開溶媒Ｂに０．０５％ＴＦＡを含む１−プロパノールを用い、１５％Ｂで平衡化したＹＭＣ−Ｐａｃｋ ＣＮ−ＡＰ（ＹＭＣ社製、カタログ番号ＡＰ−５１３；直径 ６ｍｍ、ベッド高 ２５０ｍｍ）カラムに、流速０．６ｍｌ／ｍｉｎで注入した。
注入終了後、１５％Ｂから２５％Ｂにプロパノール濃度を上げ、さらに２５％Ｂから５０％Ｂまで６５分の直線濃度勾配で展開した。最後の回に、蛋白を含まない同じ組成の溶液１ｍｌを注入、展開し、カラム内に残存するＴＰＯ活性の回収を行った。合計６回分同じポリプロピレン製チューブに集めたため、各フラクションは、７．２ｍｌずつ４４本となった。
このうち３０μｌ （２４０分の１フラクション）を取り２０μｌの５％ＢＳＡを加え、遠心エバポレーションで乾固し、最終的に０．２４ｍｌのＩＭＤＭアッセイ培養液に溶解し、アッセイにかけ、ＴＰＯ活性画分を特定した。この結果、チューブ番号２８〜３３（プロパノール濃度で３７．０〜４２．０％の範囲）に強いＴＰＯの活性があったため、これを低分子ＴＰＯ標品Ｆ３由来ＹＭＣ−Ｐａｃｋ ＣＮ−ＡＰのメインのＴＰＯ活性画分ＦＡとした。
低分子ＴＰＯ標品Ｆ３由来のＹＭＣ−Ｐａｃｋ ＣＮ−ＡＰのＴＰＯ活性画分ＦＡ
総体積 ４３．２０ｍｌ
蛋白濃度 ０．００８６３ｍｇ／ｍｌ
総蛋白質量 ０．３７３ｍｇ
相対活性 ８０００００
相対活性量 ２９８４００
（１０）Ｃａｐｃｅｌｌ Ｐａｋ Ｃ１ ３００Ａ＜最終の逆相クロマトグラフィー＞ （９）で得られた低分子ＴＰＯ標品Ｆ３由来のＴＰＯ活性画分ＦＡ４３．２０ｍｌのうち４３．１２ｍｌ（総蛋白質量０．３７２ｍｇ，蛋白濃度０．００８６３ｍｇ／ｍｌ，相対活性８０００００，相対活性量２９７６００）に、０．２ｍｌの５０％グリセロールを加え、０．１ｍｌのグリセロール溶液となるまで濃縮した。
これに展開溶媒Ａ（０．１％ＴＦＡ）：展開溶媒Ｂ（０．０５％ＴＦＡを含む１−プロパノール）＝８５；１５ （１５％Ｂ）の溶液２ｍｌを加えて、最終的に、体積２．１ｍｌ）プロパノール濃度が約１４％、グリセロールが約４．８％、蛋白濃度０．１７７ｍｇ／ｍｌのサンプルに調製した。これを１５％Ｂで平衡化したＣａｐｃｅｌｌ Ｐａｋ Ｃ１ ３００Ａ （資生堂製、カタログ番号Ｃ１ ＴＹＰＥ；ＳＧ３００Ａ；直径 ４．６ｍｍ、ベッド高 ２５０ｍｍ）カラムに注入し、２７％Ｂから３８％Ｂまで６５分の直線濃度勾配で流速０．４ｍｌ／ｍｉｎで展開し、ポリプロピレン製チューブ７２本に０．６ｍｌずつ集めた。
各フラクションから、３μｌ （２００分の１フラクション）を取り２０μｌの５％ＢＳＡを加え、最終的に２２５μｌのＩＭＤＭアッセイ培養液に置換し、オリジナル体積から７５倍希釈したものをアッセイにかけた。
各フラクションから、電気泳動のために１μｌ （６００分の１フラクション）を取り、遠心エバポレーションし、還元剤を含まないＳＤＳゲル電気泳動サンプルバッファーを１０μｌ加え、９５℃で５分処理した。これを１５−２５％ＳＤＳ−ポリアクリルアミドプレキャストゲル（第一化学薬品社製）を用いてＳＤＳゲル電気泳動し、２Ｄ−銀染色試薬・「第一」銀染色キット（第一化学薬品社製、カタログ番号１６７９９７、以下「銀染色キット」と言う）で染色した。分子量マーカーには「第一」・ＩＩＩ低分子量マーカー（第一化学薬品社製、カタログ番号１８１０６１、以下「ＤＰＣＩＩＩ」と言う）を用いた。
以上の分析の結果、チューブ番号３５〜４３（プロパノール濃度で３０．０〜３２．５％の範囲）に明らかにＴＰＯの活性があった。このうちチューブ番号３６〜４２（プロパノール濃度で３０．５〜３２．０％の範囲）をメインのＴＰＯ活性画分ＦＡとした。以上の結果を図２に示した。
蛋白質量をクロマトグラムから推定し、アッセイの結果と合わせて評価すると、総蛋白質量３９．６μｇ，蛋白濃度９．４μｇ／ｍｌ，相対活性４８９００００，相対活性量１９３６００となった。ＴＰＯ活性画分チューブ番号３６〜４２のＳＤＳゲル電気泳動像を調べてみると、活性の強さと、染色された濃さが相関するバンドが存在することが明らかとなった。しかもこのバンドの分子量は、見かけ上、即ち還元状態での標準分子量蛋白に対し、１７０００〜１９０００の位置にあり、ＴＰＯの候補となる有力なバンドであることがわかった。
（１１）電気泳動ゲルからのＴＰＯ活性の抽出＜１５％ＳＤＳポリアクリルアミドゲル電気泳動＞
ＴＰＯ活性画分ＦＡの分析例
（１０）で得られた低分子ＴＰＯ標品Ｆ３由来のＴＰＯ活性画分ＦＡ ４２００μｌ（総蛋白質量３９．６μｇ，蛋白濃度９．４μｇ／ｍｌ，相対活性４８９００００，相対活性量１９３６００）の内、５．５μｌ （７６４分の１フラクション）を活性抽出のため、２．５μｌ （１６８０分の１フラクション）を銀染色のためにそれぞれサンプルチューブに取り、遠心エバポレーションし、還元剤不含のＳＤＳゲル電気泳動サンプルバッファー１０μｌを加え、３７℃１時間処理後、室温で１８時間放置することによりＳＤＳ化した。
分子量マーカーには、プレステインド・ローレンジマーカー（Ｂｉｏ−Ｒａｄ社１６１−０３０５）、及びＤＰＣＩＩＩマーカーを用いた。これらのサンプルを常法（Ｌａｅｍｍｌｉ）Ｎａｔｕｒｅ、２２７巻、６８０−６８５頁、（１９７０））に従って、マイクロスラブゲルを用いた１５％ＳＤＳポリアクリルアミドゲル電気泳動を４℃にて実施した。泳動終了後、直ちに銀染色に付す部分をナイフで切断し固定液に入れ、銀染色キットを用いて銀染色した。
一方、活性を切り出すべき部分を、分子量の全域に渡って、ナイフを用いて幅１．５〜２．５ｍｍの３４本のゲルにスライスし、小林の方法（小林幹彦、生化学、第５９巻、第９号（１９８７））を改良した方法でゲルの破砕を行った。微細な断片に破砕されたゲルに各々０．３ｍｌの抽出バッファー（２０ｍＭ Ｔｒｉｓ−ＨＣｌ，ｐＨ８，５００ｍＭ ＮａＣｌ，０．０５％ＢＳＡ）を加え、４℃で６時間振とうし、抽出を行った。
次に最終濃度２０ｍＭの５００ｍＭリン酸カリウム、ｐＨ６．８を加え、４℃で１時間振とうし、沈殿したＳＤＳを除くためウルトラフリーＣ３ＧＶ ０．２２μｍフィルター付濾過ユニット（ミリポア社製、型番ＵＦＣ３ ＯＧＶ ０Ｓ）に移し、１０００ｘｇ（４０００ＲＰＭ）で１５分間遠心し、濾液を回収した。これをウルトラフリーＣ３−ＬＧＣ分子量１００００カット限外濾過ユニット（ミリポア社製、型番ＵＦＣ３ ＬＧＣ ００）に移し３０００ｘｇ（７０００ＲＰＭ）で遠心した。濃縮液が約５０μｌに達した時点で、３００μｌの２０ｍＭ Ｎａ Ｐｈｏｓｐｈａｔｅ，ｐＨ７．２のバッファーを加え、再び限外濾過を行った。
これを２回繰り返し、残存するＳＤＳを除去した。さらにアッセイ培養液に対し同様な操作を繰り返し、最終的に３００μｌに調製した。これを滅菌し、ＴＰＯ活性を測定した。
このような実験の結果、銀染色で明瞭に検出できた蛋白質は、ＤＰＣＩＩＩマーカーに対し、見かけ上の分子量約１７０００〜１９０００、 １４０００、１１０００の３種であった。
Ｃａｐｃｅｌｌ Ｐａｋ Ｃ１カラムのＴＰＯ活性画分の電気泳動で、活性の強さと、染色されたバンドの濃さが相関する見かけ上の分子量が約１７０００〜１９０００のバンドが観察できたが、本実験においてもＴＰＯ活性が検出された見かけ上の分子量は約１７０００〜１９０００であった。以上の結果を図３に示した。
以上の結果、ＴＰＯ活性を示す蛋白質は、最終的に電気泳動ゲル上で確認可能なまでにＣａｐｃｅｌｌ Ｐａｋ Ｃ１ ３００Ａカラムの活性画分中に精製されたと確認できた。
このサンプルを１５％ＳＤＳポリアクリルアミドゲル電気泳動（非還元下）して銀染色されたバンドの濃さから、全ＴＰＯ活性画分中の見かけ上の分子量約１７０００〜１９０００のＴＰＯ候補蛋白質の量は、約１．７μｇであった。
以下（１２）〜（１５）に高分子ＴＰＯ標品Ｆ２の精製の各ステップについて述べる。
（１２）ＹＭＣ−Ｐａｃｋ ＰＲＯＴＥＩＮ−ＲＰ＜逆相クロマトグラフィー＞
（７）で得られた高分子ＴＰＯ標品Ｆ２（総蛋白質量２５７ｍｇ，蛋白濃度０．８９４ｍｇ／ｍｌ，相対活性７８４０，相対活性量２０１５０００、総体積２８７ｍｌ）に展開溶媒Ａ（０．０２５％ ＴＦＡ）およびサンプルの３分の１容の９５．８ｍｌの展開溶媒Ｂ（０．０２５％ ＴＦＡを含む１−プロパノール）を加え、最終体積３８３ｍｌ、最終プロパノール濃度約２５％、ＴＦＡ濃度０．００６％、蛋白濃度０．６７１ｍｇ／ｍｌとした。ここで不溶物の発生があったので、遠心後の上清のみを６２．３ｍｌ（４２．８ｍｇ）ずつ６回に分けて、予め３０％Ｂで平衡化してあったＹＭＣ−Ｐａｃｋ ＰＲＯＴＥＩＮ−ＲＰ（ＹＭＣ社製、カタログ番号Ａ−ＰＲＲＰ−３３−０３−１５；直径 ３ｃｍ、ベッド高 ７．５ｃｍ）カラムに流速２ｍｌ／ｍｉｎで注入した。沈殿物は５ｍＭのＣＨＡＰＳを含む２０ｍＭ酢酸ナトリウム、ｐＨ５．５を１０ｍｌ加えて可溶化できたので、合わせてカラムに送り込んだ。
それぞれサンプルを注入した後、約５０ｍｌの溶媒（展開溶媒Ａ：展開溶媒Ｂ＝３：１）を通液し、素通り画分を集めた後、展開プログラム（１２０分の３０％Ｂから４５％Ｂの直線濃度勾配）を開始し、１５ｍｌずつ合計２４本のフラクションをポリプロピレン製チューブに集め取った。１回目から６回目まで同じ様に繰り返し、最終的に９０ｍｌずつのフラクションが２４本となった。素通り画分とチューブ番号１はそのまま限外濾過ユニット（アミコン社製；ＹＭ３膜、直径７６ｍｍ）で９０ｍｌまで濃縮した。
素通りを含むチューブ番号１から２４までのフラクションより０．３ｍｌ取り、１０μｌの５％ＢＳＡを添加後遠心エバポレーションで乾固し、最終的に０．３ｍｌのＩＭＤＭアッセイ培養液に溶解し、アッセイにかけ、ＴＰＯ活性画分を特定した。この結果、チューブ番号１０〜１５（プロパノール濃度で３４．０〜３９．５％の範囲）にＴＰＯの活性があり、これを高分子ＴＰＯ標品Ｆ２由来のＹＭＣ−Ｐａｃｋ ＰＲＯＴＥＩＮ−ＲＰのＴＰＯ活性画分Ｆ２とした。次のＹＭＣ−Ｐａｃｋ ＣＮ−ＡＰに進める直前まで、−８５℃で保存した。
高分子ＴＰＯ標品Ｆ２由来のＹＭＣ−Ｐａｃｋ ＰＲＯＴＥＩＮ−ＲＰのＴＰＯ活性画分Ｆ２
総体積 ５４０ｍｌ
蛋白濃度 ０．０２１ｍｇ／ｍｌ
総蛋白質量 １１．４ｍｇ
相対活性 ２２７０００
相対活性量 ２５８８０００
（１３）Ｓｕｐｅｒｄｅｘ ７５ｐｇ＜ＣＨＡＰＳ存在下でのゲル濾過クロマトグラフィー＞
（１２）で得られた高分子ＴＰＯ標品Ｆ２由来のＹＭＣ−Ｐａｃｋ ＰＲＯＴＥＩＮ−ＲＰのＴＰＯ活性画分Ｆ２のうち、５３８．２ｍｌ（総蛋白質量１１．３ｍｇ，蛋白濃度０．０２１ｍｇ／ｍｌ，相対活性２２７０００，相対活性量２５６５０００）に５０％グリセロールを０．６ｍｌ添加後、遠心エバポレーション濃縮した。次に、６ｍｌの２０ｍＭ ＣＨＡＰＳを加えた。さらに、１８ｍｌの２０ｍＭ ＣＨＡＰＳを加え攪拌し、４℃に移し、４１時間後に最初のサンプルをＨｉＬｏａｄ２６／６０ Ｓｕｐｅｒｄｅｘ ７５ｐｇ（ファルマシア バイオテク社製、カタログ番号１７−１０７０−０１；直径 ２．６ｃｍ、ベッド高 ６０ｃｍ）カラムに注入し、流速１ｍｌ／ｍｉｎで、５ｍＭ ＣＨＡＰＳを含むＤＰＢＳで展開した。一回に４ｍｌ（蛋白濃度０．４６６ｍｇ／ｍｌ，蛋白質量１．８６ｍｇ）のサンプルをカラムに注入した。
６回目に分けてカラムで展開し、全てのＹＭＣ−Ｐａｃｋ ＰＲＯＴＥＩＮ−ＲＰのＴＰＯ活性画分をＳｕｐｅｒｄｅｘ ７５ｐｇカラムで分取した。フラクションは５ｍｌずつ６回分、即ち合計３０ｍｌのフラクションが４５本となった。
それぞれのフラクションより０．１ｍｌ取り、１０μｌの５％ＢＳＡを添加後、遠心エバポレーションで乾固し、最終的に０．２５ｍｌのＩＭＤＭアッセイ培養液に溶解し、アッセイにかけ、ＴＰＯ活性画分を特定した。この結果、チューブ番号１３〜３１（分子量で７８０００〜３０００の範囲）にＴＰＯの活性があったため、これを高分子ＴＰＯ標品Ｆ２由来のＳｕｐｅｒｄｅｘ ７５ｐｇのＴＰＯ活性画分Ｆ２とした。
高分子ＴＰＯ標品Ｆ２由来のＳｕｐｅｒｄｅｘ ７５ｐｇのＴＰＯ活性画分Ｆ２
総体積 ５４０ｍｌ
蛋白濃度 ０．００２１６ｍｇ／ｍｌ
総蛋白質量 １．１７ｍｇ
相対活性 １７５００００
相対活性量 ２０４１０００
（１４）ＹＭＣ−Ｐａｃｋ ＣＮ−ＡＰ＜逆相クロマトグラフィー＞
（１３）で得られた高分子ＴＰＯ標品Ｆ２由来のＳｕｐｅｒｄｅｘ ７５ｐｇのＴＰＯ活性画分Ｆ２（分子量７８０００〜３０００）５４０ｍｌのうち、５１３．２ｍｌ（総蛋白質量１．１１ｍｇ，蛋白濃度０．００２１６ｍｇ／ｍｌ，相対活性１７５００００，相対活性量１９４３０００）に１０分の１容の展開液Ｂ（０．０５％ＴＦＡを含む１−プロパノール）を加えた後、展開溶媒Ａ（０．１％ＴＦＡ）と展開液Ｂを用いて１５％Ｂで平衡化したＹＭＣ−Ｐａｃｋ ＣＮ−ＡＰ （ＹＭＣ社製、カタログ番号ＡＰ−５１３；直径 ６ｍｍ、ベッド高 ２５０ｍｍ）カラムに、流速０．６ｍｌ／ｍｉｎで注入した。注入終了後、１５％Ｂから２５％Ｂにプロパノール濃度を上げ、さらに２５％Ｂから５０％Ｂまでの６５分の直線濃度勾配で展開した。
ＹＭＣ−Ｐａｃｋ ＣＮ−ＡＰカラムに進めるにあたり、全インプットサンプルの２０分の１をまずパイロット的に進め、活性が良好に回収できることを確認できた。そこで、残りの２０分の１９を２回に分けて分取した。つまり合計３回の展開をおこなった。計３回分で分取された各フラクションを同じポリプロピレン製チューブ４４本に集めたので、合計３．６ｍｌずつとなった。
このうち５μｌ （７２０分の１フラクション）を取り、最終的に０．２５ｍｌのＩＭＤＭアッセイ培養液に置換した。アッセイの結果、チューブ番号２４〜３０（プロパノール濃度で３６．０〜４２．０％の範囲）に極めて強いＴＰＯの活性があったため、これを高分子ＴＰＯ標品Ｆ２由来のＹＭＣ−Ｐａｃｋ ＣＮ−ＡＰのメインのＴＰＯ活性画分ＦＡとした。
高分子ＴＰＯ標品Ｆ２由来のＳｕｐｅｒｄｅｘ ７５ｐｇのＴＰＯ活性画分ＦＡ
総体積 ２５．２０ｍｌ
蛋白濃度 ０．０２４６ｍｇ／ｍｌ
総蛋白質量 ０．６２０ｍｇ
相対活性 ７０００００
相対活性量 ４３４０００
（１５）Ｃａｐｃｅｌｌ Ｐａｋ Ｃ１ ３００Ａ＜最終の逆相クロマトグラフィー＞
（１４）で得られた高分子ＴＰＯ標品Ｆ２由来のＹＭＣ−Ｐａｃｋ ＣＮ−ＡＰのＴＰＯ活性画分ＦＡ２５．２０ｍｌのうち、２４．６６ｍｌ（総蛋白質量０．６０６ｍｇ，蛋白濃度０．０２４６ｍｇ／ｍｌ，相対活性７０００００，相対活性量４２４０００）に、０．４ｍｌの５０％グリセロールを加え、遠心エバポレーションで濃縮した。
最終的に、プロパノール濃度は数％、グリセロールは１０％、蛋白濃度０．３０３ｍｇ／ｍｌの２ｍｌのサンプルとなった。展開溶媒Ａ（０．１％ＴＦＡ）、展開溶媒Ｂ（０．０５％ＴＦＡを含む１−プロパノール）を用いて、１５％Ｂで平衡化したＣａｐｃｅｌｌ Ｐａｋ Ｃ１３００Ａ（資生堂 カタログ番号Ｃ１ ＴＹＰＥ：ＳＧ３００Ａ；直径 ４．６ｍｍ、ベッド高 ２５０ｍｍ）カラムに注入し、２７％Ｂから３８％Ｂまで６５分の直線濃度勾配で流速０．４ｍｌ／ｍｉｎで展開し、ポリプロピレン製チューブ７２本に０．６ｍｌずつ集めた。
各フラクションから、０．７５μｌ （８００分の１フラクション）を取り、２０μｌの５％ＢＳＡを加え、最終的に２２５μｌのＩＭＤＭアッセイ培養液に置換し、オリジナル体積から３００倍希釈したものをアッセイにかけた。
各フラクションから、電気泳動のために２μｌ （３００分の１フラクション）を取り、遠心エバポレーションし、１０μｌの還元剤を含まないＳＤＳ電気泳動サンプルバッファーを加え、９５℃で５分処理した。これを１５〜２５％ＳＤＳ−ポリアクリルアミドプレキャストゲル（第一化学薬品製）を用いてＳＤＳゲル電気泳動し、銀染色キットで染色した。分子量マーカーにはＤＰＣＩＩＩマーカーを用いた。
以上の分析の結果、チューブ番号３３〜３９（プロパノール濃度で２９．５〜３１．５％の範囲）に明らかにＴＰＯの活性があった。このうち、チューブ番号３４〜３９（プロパノール濃度で３０．０〜３１．５％の範囲）をメインのＴＰＯ活性画分ＦＡとした。メインのＴＰＯ活性画分のＳＤＳゲル電気泳動像を調べてみると、（１０）に述べた低分子ＴＰＯ標品Ｆ３から出発したものと同じく見かけ上１７０００〜２２０００の分子量範囲に、活性の強さと、染色された濃さが相関するバンドが存在することが明らかとなった。
＜実施例２＞
ラット精製ＴＰＯの部分アミノ酸配列の分析
岩松の方法（岩松ら、新基礎生化学実験法、第４巻、３３〜８４頁（丸善刊）；岩松明彦、生化学、第６３巻、第２号、１３９−１４３頁、（１９９１） ；Ａｋｉｈｉｒｏ Ｉｗａｍａｓｔｕ、Ｅｌｅｃｔｏｒｏｐｈｏｒｅｓｉｓ、第１３巻、１４２−１４７頁、（１９９２））により、実施例１の（１０）で得られたＣａｐｃｅｌｌ Ｐａｋ Ｃ１ ３００ＡカラムのＴＰＯ画分ＦＡ中のラットＴＰＯ候補タンパク質のアミノ酸配列の分析を行った。即ち、サンプルをＳＤＳゲル電気泳動し、電気的にポリビニリデンジフルオリド（ＰＶＤＦ）膜に転写した。次いで、ＰＶＤＦ膜上の蛋白質を還元Ｓ−アルキル化した後、系統的、段階的に３種のプロテアーゼで限定酵素分解し、ペプチドフラグメント化し、これを逆相クロマトグラフィーで分離精製し、得られたペプチドを高感度アミノ酸配列決定法により分析した。以下にその詳細を述べる。
低分子ＴＰＯ標品Ｆ３由来のＣａｐｃｅｌｌ ＰａｋＣ１ ３００ＡのＴＰＯ画分ＦＡのＴＰＯ候補蛋白質の分析例
（１）Ｃａｐｃｅｌｌ Ｐａｋ Ｃ１ ３００ＡカラムのＴＰＯ画分ＦＡ（チューブ番号３６〜４２）の濃縮
実施例１の（１０）で得られた低分子ＴＰＯ標品Ｆ３由来の、ＣａｐｃｅｌｌＰａｋ Ｃ１３００ＡカラムのＴＰＯ活性画分ＦＡ（チューブ番号３６〜４２）４２００μｌ（総蛋白質量３９．６μｇ，蛋白濃度９．４μｇ／ｍｌ，相対活性４８９００００，相対活性量１９３６００）のうち、４１５１μｌ（全フラクションの９８．８％）をアミノ酸配列のための分析に進めた。クロマトグラムから推定される蛋白質量は３９．１μｇであるが、このうちＳＤＳゲル電気泳動で銀染色された見かけ上の分子量約１７０００〜１９０００のＴＰＯ候補蛋白質の量は、約１．６μｇであった。
このサンプルにグリセロールを添加し、遠心エバポレーションで濃縮し、５μｌのグリセロール溶液とした。これに還元剤を含まないＳＤＳ電気泳動サンプルバッファー、及びｐＨを調整するために１Ｍ Ｔｒｉｓ−ＨＣｌ，ｐＨ８を加え、最終的に、２００ｍＭ Ｔｒｉｓ−ＨＣｌ，ｐＨ８．０，５０ｍＭ Ｔｒｉｓ−ＨＣｌ，ｐＨ６．８，１．１％ＳＤＳ，２ｍＭ ＥＤＴＡ，０．０２％ＢＰＢ，３０％グリセロールを含む約２５μｌのサンプルにした。
このサンプルを過度に加熱することなく十分にＳＤＳ化するために、まず室温に１４時間置き、次に６０℃で５分処理した。
（２）電気泳動
常法に従って、マイクロスラブゲル（４．０％アクリルアミド濃縮ゲル、１５％アクリルアミド分離ゲル）を調製し、ＳＤＳゲル電気泳動を室温下で１２．５ｍＡ）次いで１７．５ｍＡの一定電流にて２時間かけて実施した。分子量マーカーには、プレステインド・ローレンジマーカー（Ｂｉｏ−Ｒａｄ社１６１−０３０５）、及びＤＰＣＩＩＩマーカーを用いた。泳動終了後直ちＰＶＤＦ膜に転写した（次項）。
また、分析に付したサンプルの一部を、非還元のまま、及びジチオスレイトール（ＤＴＴ）で還元化し、１５〜２５％ポリアクリルアミドプレキャストゲル（第一化学薬品社製；マルチゲル１５／２５、カタログ番号２１１０７２）で電気泳動した。これを銀染色キットを用いて銀染色したところ、ＴＰＯと期待されたバンドは、還元下において分子量約１９０００であり、Ｃａｐｃｅｌｌ Ｐａｋ Ｃ１ ３００ＡカラムでのＴＰＯ活性画分中のＴＰＯ候補蛋白質の純度が、数％程度であることが確認できた。また非還元・還元それぞれの移動度が異なるため、分子内部に少なくとも一つ以上のＳ−Ｓ結合を持つことが示唆された。
（３）ＰＶＤＦ膜へのエレクトロブロット法による転写・バンドの検出
セミドライ転写装置（マリソル社製、ウエットフォー転写装置モデルＫＳ−８４６０）を用いて、常法に従い、１６０ｍＡ（１１〜１７Ｖ）の一定電流で１時間かけてＰＶＤＦ膜（アプライドバイオシステムズ社製ＰｒｏＢｌｏｔｔ、カタログ番号４００９９４）に転写を行った。陽極液に、０．３Ｍ Ｔｒｉｓ，２０％メタノール，ｐＨ１０．４、転写膜液に、２５ｍＭ Ｔｒｉｓ，２０％メタノール，ｐＨ１０．４、陰極液に、２５ｍＭ Ｔｒｉｓ， ４０ｍＭアミノカプロン酸，２０％メタノール，ｐＨ１０．４を用いた。
転写された膜をポンソーＳ染色液（１００ｍ１中０．１ｇのポンソーＳと１ｍ１の酢酸を含む）で染色したところ、複数のバンドが染色され、この中にＴＰＯと期待された分子量約１９０００のバンドを確認することができた。これを切り出し、ペプチドの断片化に進めた。（次項）
（４）ペプチドフラグメント化とペプチドマッピング・アミノ酸配列分析
ＰＶＤＦ膜上に転写・還元Ｓ−アルキル化されたＴＰＯ候補蛋白質の断片化を系統的に行うために、次の三つのプロテアーゼにより、段階的に限定的酵素分解を行った。
一次消化 リシルエンドペプチダーゼ（Ａｃｈｒｏｍｏｂａｃｔｅｒ ｌｙｔｉｃｕｓ ｍ４９７−１、和光純薬工業製、カタログ番号１２９−０２５４１）二次消化 エンドプロテイナーゼＡｓｐ−Ｎ（ベーリンガー・マンハイム社製、カタログ番号１０５４５８９）
三次消化 トリプシン−ＴＰＣＫ （Ｗｏｒｔｈｉｎｇｔｏｎ Ｂｉｏｃｈｅｍｉｃａｌ社製、カタログ番号３７４０）
各々の酵素消化で得られたペプチド断片を回収し、展開溶媒Ａに０．０５％ＴＦＡ）展開溶媒Ｂに０．０２％ＴＦＡを含むイソプロパノール：アセトニトリル＝７：３の混液を用いて、Ｗａｋｏｓｉｌ−ＩＩ ５Ｃ１８ Ｃ１８逆相カラム（和光純薬工業製；直径２，０ｍｍ）長さ１５０ｍｍ）で、カラム温度３０℃、流速０．２５ｍｌ／分、１％Ｂから５０％Ｂを３０分の直線濃度勾配にて展開することによりマッピング（図４）し、得られたペプチドフラグメントを回収した。それぞれのペプチドフラグメントを気相アミノ酸シークエンサー（島津製作所製、ＰＰＳＱ−２）にてエドマン分解後、順次回収されたＮ末端のＰＴＨアミノ酸を、アイソクラティック溶出法によるＣ１８逆相カラムクロマトグラフィーにて同定を行った。この結果を次にまとめた。
一次消化 リシルエンドペプチダーゼによるペプチド断片のアミノ酸配列
フラグメント名 アミノ酸配列
【表３】

【表４】

【表５】

以上の配列のうち、（）付きで示したものは、系統的酵素消化から演鐸推定しうるアミノ酸残基である。
（５）得られたアミノ酸配列の類似性分析＜ホモロジーサーチ＞
得られたアミノ酸配列が、すでに報告されている既知の蛋白質に含まれているかどうか、あるいは、類似配列をもつ蛋白質があるかどうかについて、配列解析ソフトウエアであるマックベクター（Ｋｏｄａｋ ＩｎｔｅｒｎａｔｉｏｎａｌＢｉｏｔｅｃｈｎｏｌｏｇｉｅｓ， Ｉｎｃ．）を用いて分析した。既知蛋白質あるいは既知遺伝子の情報は、Ｅｎｔｒｅｚ Ｒｅｌｅａｓｅ ６データベース（米国Ｎａｔｉｏｎａｌ Ｃｅｎｔｅｒ ｆｏｒ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ Ｉｎｆｏｒｍａｔｉｏｎ，Ｎａｔｉｏｎａｌ Ｌｉｂｒａｒｙ ｏｆ Ｍｅｄｉｃｉｎｅ，Ｎａｔｉｏｎａｌ Ｉｎｓｔｉｔｕｔｅｓ ｏｆ Ｈｅａｌｔｈ）１９９３年８月１５日発行）を利用した。これに含まれる各種データベースは以下の通りである。
Ｅｎｔｒｅｚ Ｒｅｌｅａｓｅ ６データベース
ＮＣＢＩ−ＧｅｎＢａｎｋ，Ａｕｇｕｓｔ １５， １９９３（Ｒｅｌｅａｓｅ７８．０）ＥＭＢＬ，Ｊｕｌｙ １５，１９９３（Ｒｅｌｅａｓｅ３５．０ ｐｌｕｓ ｕｐｄａｔｅｓ）ＤＤＢＪ，Ｊｕｌｙ １５，１９９３
ＳＷＩＳＳ−ＰＲＯＴ，Ａｐｒｉｌ，１９９３（Ｒｅｌｅａｓｅ ２５．０）
ＰＩＲ，Ｊｕｎｅ ３０，１９９３（Ｒｅｌｅａｓｅ ３７，０）
ＰＤＢ，Ａｐｒｉｌ，１９９３
ＰＲＦ，Ｍａｙ，１９９３
ｄｂＥＳＴ，Ｊｕｌｙ １５，１９９３（Ｒｅｌｅａｓｅ １．１０）
Ｕ．Ｓ．ａｎｄ Ｅｕｒｏｐｉａｎ Ｐａｔｅｎｔｓ（一部）
この結果、ＡＰ１２の配列（Ｋ）ＤＳＦＬＡＤＶＫは、ラットのＣｏｒｔｉｃｏｓｔｅｒｏｉｄ−ｂｉｎｄｉｎｇ ｇｌｏｂｕｌｉｎ （ＣＢＧ）ｐｒｅｃｕｒｓｏｒ［ＰＩＲデータベース登録番号 Ａ４００６６；Ｓｍｉｔｈ ａｎｄ Ｈａｍｍｏｎｄ：“Ｒａｔ ｃｏｒｔｉｃｏｓｔｅｒｏｉｄ−ｂｉｎｄｉｎｇ ｇｌｏｂｕｌｉｎ；ｐｒｉｍａｒｙ ｓｔｒｕｃｔｕｒｅ ａｎｄ ｍｅｓｓｅｎｇｅｒ ｒｉｂｏｎｕｃｌｅｉｃ ａｃｉｄ ｌｅｖｅｌｓ ｉｎ ｔｈｅ ｌｉｖｅｒ ｕｎｄｅｒ ｄｉｆｆｅｒｅｎｔｐｈｙｓｉｏｌｏｇｉｃａｌ ｃｏｎｄｉｔｉｏｎｓ．” Ｍｏｌ．Ｅｎｄｏｃｒｉｎｏｌ．． （１９８９），３，４２０−４２６，］の内部配列ＫＤＳＦＬＡＤＶＫと完全一致した。
さらによく調べてみると、ＡＰ３の配列（Ｋ）ＸＹＹＥＳＺ（（ＸはＡ、Ｓ、Ｇ、Ｍ、Ｑのどれか）、（ＺはＥまたはＫ））と類似性の高いＫＱＹＹＥＳＥという配列が、ラットＣＢＧのアミノ酸配列に含まれていることが判明した。これらのＡＰ１２、ＡＰ３に相当する配列はラットＣＢＧでは連続しており、ＫＤＳＦＬＡＤＶＫＱＹＹＥＳＥという内部アミノ酸配列に相当する。
しかしながら、ＡＰ１２、ＡＰ３以外のフラグメントのアミノ酸配列に関しては、類似性を考慮すべき既知の蛋白質や遺伝子は見つからなかった。
＜実施例３＞
血小板減少症ラット血漿由来ＴＰＯの生物学的特性分析
（１） ラットＣＦＵ−ＭＫアッセイ系（液体培養系）において
代表例として、血小板減少症ラット血漿からのＴＰＯ部分精製標品（実施例１の（８）に記載したＹＭＣ Ｐａｃｋ Ｐｒｏｔｅｉｎ−ＲＰカラムＴＰＯ活性画分Ｆ２）を用いた場合の用量反応曲線を図５に示した。培養を経時的に顕微鏡下で観察したところ、日を追って巨核球の分化、成熟の進行、即ち細胞サイズの増大が認められ、おそらく、細胞の増加も起こっていることと思われた。特に顕著な変化として、培養最終日の４日目に数多くの巨核球による突起形成が認められた（培養３日目ではほとんど認められない）。この突起形成は、ｃｙｔｏｐｌａｓｍｉｃ ｐｒｏｃｅｓｓ ｆｏｒｍａｔｉｏｎ（ＬｅｖｅｎとＹｅｅ、 Ｂｌｏｏｄ、６９巻、１０４６−１０５２頁、（１９８７））、あるいは、ｐｒｏｐｌａｔｅｌｅｔ ｐｒｏｃｅｓｓ ｆｏｒｍａｔｉｏｎ （Ｔｏｐｐら、Ｂｌｏｏｄ７、７６巻、９１２−９２４頁、（１９９０））などと呼ばれ、巨核球からさらに分化の進んだ血小板の前駆構造体であり、現在までのところｉｎｖｉｔｒｏで観察できる巨核球分化の最終形態と考えられている。ＴＰＯ標品単独でこのような形態変化が高い頻度で認められたことから、本因子は単独でＣＦＵ−ＭＫの増殖・分化を促進し、成熟巨核球を生成させ、さらに最終的に血小板産生まで進行させる可能性が考えられる。
（２） コロニーアッセイ系において
血小板減少症ラット血漿からのＴＰＯ部分精製標品について、ラットの非分離骨髄細胞、分離・濃縮各段階の細胞、あるいはＧｐＩＩｂ／ＩＩＩａ^＋ＣＦＵ−ＭＫ画分を用いたコロニーアッセイ系で検定したところ、ラット血漿由来のＴＰＯは、巨核球コロニーを形成させた。ＴＰＯによって形成される巨核球コロニーと他の既知サイトカイン、即ち、ラットＩＬ−３、マウスＧＭ−ＣＳＦ、あるいはヒトＥＰＯによって形成される巨核球コロニーを比較すると、ＴＰＯによって形成される巨核球コロニーには、個々のコロニーを構成する巨核球数は少ないが、各々の巨核球のサィズが大きい、即ち成熟度が進んでいるという特徴がある。さらに、他の細胞系統のコロニーはほとんど形成されず、ＴＰＯが示すＭｅｇ−ＣＳＦ活性は巨核球特異的な活性と考えられる。これらのことから、ＴＰＯは、本コロニーアッセイ系においてＭｅｇ−ＣＳＦ活性を発揮する他の既知サイトカイン、即ち、ラットＩＬ−３、マウスＧＭ−ＣＳＦ、あるいはヒトＥＰＯとは、生物学的特性を異にし、ユニークなＭｅｇ−ＣＳＦ活性を発揮することが明白となった。
ヒト骨髄細胞、あるいはヒト瞬帯血細胞由来のＣＤ３４^＋ＤＲ^＋細胞画分に対しても血小板減少症ラット血漿からのＴＰＯ部分精製標品はＭｅｇ−ＣＳＦ活性を示し、有意な数のヒト巨核球コロニーを形成させた。このことは本因子に種特異性のないことを示している。
＜実施例４＞
ラットＴＰＯ産生細胞の特定化
（１） ラットＴＰＯ産生臓器の探索
まず、ラットＴＰＯの部分アミノ酸配列に基づくラットＴＰＯ遺伝子のクローニング、あるいは発現クローニングのためのｍＲＮＡ供給源を確保する目的で、ラットＴＰＯ産生臓器の探索、特定化を行った。当初、Ｐ５５抗体投与により血小板減少症にしたラットから経時的に骨髄、肺、肝臓、脾臓を摘出し、その細胞（肺、肝臓の場合は臓器切片）の培養上清を採取し、ラットＣＦＵ−ＭＫアッセイ系にて上清中の活性を評価したが、明確な結果は得られなかった。続いて、ラットにおける肝臓とＴＰＯ産生との関連性を示唆する報告（Ｓｉｅｍｅｎｓｍａら、Ｊ．Ｌａｂ．Ｃｌｉｎ．Ｍｅｄ．、８６巻、８１７−８３３頁、（１９７５））を考慮して、Ｐ５５抗体投与により血小板減少症にしたラットの肝臓からコラゲナーゼかん流法にて調製した肝細胞を培養し、その上清からＷＧＡ−Ａｇａｒｏｓｅカラムに吸着した画分をＶｙｄａｃ ｐｈｅｎｙｌ逆相カラムに展開したところ、ラットＣＦＵ−ＭＫアッセイ系にてラット血漿由来ＴＰＯ活性と同じ位置に極めて類似した活性が認められた。正常ラット肝細胞の培養上清からも弱いながらも活性が認められた。これらの結果から、肝臓がＴＰＯ産生臓器の１つである可能性が強く示唆された。
（２） ラットＴＰＯ産生細胞株のスクリーニング
上記の結果を基に、ラットＴＰＯ産生細胞株のスクリーニングを行った。まず、２０種類のラット肝臓由来細胞株をそれぞれの継代培養用の培養液中でほぼコンフルエントになるまで培養した後、培養液に含まれる血清を５％ＦＣＳに統一したそれぞれの培養液で置換して、さらに３日間培養を継続し、それぞれの培養上清を採取した。その上清を（１）に記した方法で部分精製して、ＴＰＯ産生の有無を調べたところ、３種類のラット肝実質細胞由来細胞株、即ち、ＭｃＡ−ＲＨ８９９４細胞（ＡＴＣＣ寄託番号ＣＲＬ１６０２、Ｂｅｃｋｅｒら、“Ｏｎｃｏｄｅｖｅｌｏｐｍｅｎｔａｌ Ｇｅｎｅ Ｅｘｐｒｅｓｓｉｏｎ″ ｅｄ．ｂｙ Ｆｉｓｈｍａｎ ａｎｄ Ｓｅｌｌ、Ａｃａｄｅｍｉｃ Ｐｒｅｓｓ、 ＮＹ、２５９−２７０頁、（１９７６）、大日本製薬より購入）、Ｈ４−ＩＩ−Ｅ細胞（ＡＴＣＣ寄託番号ＣＲＬ１５４８、Ｐｉｔｏｔら、Ｎａｔ． Ｃａｎｃｅｒ Ｉｎｓｔ．Ｍｏｎｏｇｒ．、１３巻、２２９−２４５頁、（１９６４）、大日本製薬より購入）、およびＨＴＣ細胞（Ｔｈｏｍｐｓｏｎら、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ、５６巻、２９６−３０３頁、（１９６６）、大日本製薬より購入）から明らかにＴＰＯ活性の産生が確認された。
（３） ＭｃＡ−ＲＨ８９９４細胞、Ｈ４−ＩＩ−Ｅ細胞、およびＨＴＣ細胞が産生するＴＰＯ活性の詳細な分析
これらの３種類のラット細胞株から分泌されるＴＰＯ活性と平行して精製を進めていたラット血漿由来のＴＰＯ活性を、生化学的性質と生物学的性質の両面からさらに詳細に比較検討した。
ＭｃＡ−ＲＨ８９９４細胞を１０％ＦＣＳを含むａｌｐｈａ−ＭＥＭ（−）培養液に浮遊させて、底面積１７５ｃｍ^２の組織培養用培養プラスティックフラスコに１×１０^６個／フラスコになるように入れ、５％炭酸ガス培養器中にて３７℃で３日間培養した後、５％ＦＣＳを含むＩＭＤＭ培養液に置き換え、さらに３日間培養し、上清を回収した。Ｈ４−ＩＩ−Ｅ細胞を１０％ＦＣＳを含むＤｕｌｂｅｃｃｏ改変Ｅａｇｌｅ培養液（グルコース４．５ｇ／ｌ含有）（以下、ＤＭＥＭ培養液）に浮遊させて、底面積１７５ｃｍ^２の組織培養用プラスティックフラスコに５×１０^５個／フラスコになるように入れ、５％炭酸ガス培養器中にて３７℃で３日間培養した後、５％ＦＣＳを含むＩＭＤＭ培養液に置き換え、さらに３日間培養し、上清を回収した。また、ＨＴＣ細胞を５％ＦＣＳを含むＤＭＥＭ培養液に浮遊させて、底面積１７５ｃｍ^２の組織培養用プラスティックフラスコに２．５×１０^５個／フラスコになるように入れ、５％炭酸ガス培養器中にて３７℃で３日間培養した後、５％ＦＣＳを含むＩＭＤＭ培養液に置き換え、さらに３日間培養し、上清を回収した。
このようにして得た３種類の細胞株の培養上清それぞれ２リットルから、実施例１−２に記載したＸＲＰからのＴＰＯの精製法に従って、細胞株由来ＴＰＯの部分精製を行った。以下に概略を述べる。
まず、限外濾過器により培養上清を約６倍に濃縮した後、ＳｅｐｈａｄｅｘＧ−２５カラムで２０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．０）にバッファー交換した。溶出液をＱ−Ｓｅｐｈａｒｏｓｅ ＦＦカラムに添加し、２０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．０）で洗滌後、吸着画分を１７５ｍＭ ＮａＣｌを含む２０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．０）で溶出した。この画分をＷＧＡ−Ａｇａｒｏｓｅカラムに添加し、ＰＢＳで洗った後、吸着画分を０．２Ｍ ＧｌｃＮＡｃと０．１５Ｍ ＮａＣｌを含む２０ｍＭＮａ Ｐｈｏｓｐｈａｔｅ（ｐＨ７．２）により溶出した。この溶出液をＴＳＫ−ｇｅｌ ＡＦ−ＢＬＵＥ ６５０ＭＨカラムに添加し、ＩＭ ＮａＣｌを含む２０ｍＭ Ｎａ Ｐｈｏｓｐｈａｔｅ（ｐＨ７．２）で洗った後、２ＭＮａＳＣＮにより溶出した。これをＰｈｅｎｙｌ−Ｓｅｐｈａｒｏｓｅ ６ ＦＦ／ＬＳカラムに添加し、１．５Ｍ硫酸アンモニウム（Ａｍｍｏｎｌｕｍ Ｓｕｌｆａｔｅ）を含む５０ｍＭ Ｎａ Ｐｈｏｓｐｈａｔｅ （ｐＨ７．２）、次いで０．８Ｍ硫酸アンモニウムを含む３６ｍＭＮａ Ｐｈｏｓｐｈａｔｅで洗った後、２０ｍＭＮａ Ｐｈｏｓｐｈａｔｅ（ｐＨ７．２）により溶出した。この吸着画分を、濃縮後、逆相ＶｙｄａｃＰｒｏｔｅｉｎＣ４カラム（Ｔｈｅ Ｓｅｐａｒａｔｉｏｎｓ Ｇｒｏｕｐ社製 カタログ番号２１４ＴＰ５１０１５；直径１ｃｍ、ベッド高１５ｃｍ）で分画した。展開溶媒Ａに０．１％ＴＦＡ、展開溶媒Ｂに０，０５％ＴＦＡを含む１−プロパノールを用い、予めカラムを２０％Ｂで平衡化し、サンプルを注入後、流速１ｍｌ／ｍｉｎで２０％Ｂから４０％Ｂまで９０分の直線濃度勾配により溶出した。この結果、どの細胞株由来のＴＰＯ活性も、３０％から４３％濃度の１−プロパノールで溶出された。
各精製段階の標品をラットＣＦＵ−ＭＫアッセイ系にて活性測定した結果、３種類の細胞株由来のＴＰＯ活性はいずれもＸＲＰ由来のＴＰＯ活性と極めて類似した挙動を示した（実施例１−２を参照）（表６に、ラットＣＦＵ−ＭＫアッセイ系での各段階における相対比活性、活性収率などを記載）。さらに、最終段階の逆相カラムからの溶出画分をラットＣＦＵ−ＭＫアッセイ系にて活性測定した結果、３種類の細胞株由来のＴＰＯ活性とＸＲＰ由来のＴＰＯ活性は同じピーク位置に溶出されていた。この逆相カラムの活性画分を中心に、ラットＧｐＩＩｂ／ＩＩＩａ^＋ＣＦＵ−ＭＫの分離・濃縮過程の付着細胞除去段階で得られる非付着性細胞を用いてコロニーアッセイを行ったところ、いずれもラットＣＦＵ−ＭＫアッセイ系での活性の溶出パターンにほぼ一致して巨核球コロニーの形成が認められ（表７）、また、ＸＲＰ由来のＴＰＯ活性と同様に、３種類の細胞株由来の活性はいずれも専ら巨核球コロニーを形成させ、他の系統のコロニーはほとんど形成させなかった。
実施例１−２に記載した方法に従って、プールした逆相カラムの活性画分をＳＤＳポリアクリルアミドゲル電気泳動に供し、泳動後にゲルから蛋白質を抽出して、ラットＣＦＵ−ＭＫアッセイ系にて活性測定したところ、ＸＲＰ由来のＴＰＯは見かけの分子量１７０００〜２２０００、ＭｃＡ−ＲＨ８９９４細胞由来のＴＰＯは見かけの分子量３３０００〜３９０００、Ｈ４−ＩＩ−Ｅ細胞由来のＴＰＯは見かけの分子量３１０００〜３８０００、ＨＴＣ細胞由来のＴＰＯは見かけの分子量１７０００〜２２０００、および分子量２８０００〜３５０００を有していた。
以上のように、ＭｃＡ−ＲＨ８９９４細胞、Ｈ４−ＩＩ−Ｅ細胞、およびＨＴＣ細胞が産生するＴＰＯ活性は、ＸＲＰ由来のＴＰＯと比べて、見かけ上の分子量の点で生化学的性質を若干異にするものの、生物学的性質において同等であることが明らかとなった。本発明において、ここで特筆すべきことは、血液中に存在するＴＰＯ活性を持つ分子が、産生細胞において、あるいは産生細胞から分泌後に、分子内の特定な、あるいは不特定な位置で切断されたものである可能性を示唆する。また、ＴＰＯ遺伝子（ｍＲＮＡやｃＤＮＡ）においても様々な長さのものが存在する可能性をも示したものである。
【表６】

【表７】

＜実施例５＞
ｃＤＮＡライブラリー作製用発現ベクター（ｐＥＦ１８Ｓ）の構築
ベクターへのｃＤＮＡ断片の組み込みが容易で、クローニングしたｃＤＮＡの発現効率が高いベクターを作製し、ＴＰＯｃＤＮＡのクロニングと発現に備えた。すなわち、発現効率が高いプロモーターとして知られるエロンゲーションファクター１α（ＥＦ１α）のプロモーターを、扱いやすい発現ベクターｐＭＥ１８ＳのＳＲαプロモーターと入れ替え、発現ベクターｐＥＦ１８Ｓを構築した（図６参照）。エロンゲーションファクター１αのプロモーターは発現ベクターｐＥＦ−ＢＯＳ（Ｍｉｚｕｓｈｉｍａら、Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｒｅｓ．、１８、５３２２、１９９０）１μｇを制限酵素ＨｉｎｄＩＩＩとＥｃｏＲＩで部分的に消化した後、２％アガロースゲル（ＦＭＣ Ｂｉｏ Ｐｒｏｄｕｃｔｓ社製）を用いた電気泳動にかけ、約１２００ｂｐのＤＮＡ断片を分離し、プレップ−Ａ−ジーンＤＮＡ精製キット（バイオラッド社製；Ｗｉｌｌｉｓら、Ｂｉｏ Ｔｅｃｈｎｉｑｕｅｓ、 ９、９２−９９、１９９０の方法にもとづいたもので、多孔性シリカベースのマトリックスを利用した吸着により選択的にＤＮＡを精製するキット）を用いて精製した。このＤＮＡ断片１００ｎｇを同様にＨｉｎｄＩＩＩとＥｃｏＲＩで消化した５０ｎｇの発現ベクターｐＭＥ１８Ｓ（Ｌｉｕら、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ、９０、８９５７−８９６１、１９９３）につなぎこんだ。宿主菌にはコンピテント・ハイＥ．ｃｏｌｉＤＨ５（東洋紡績社製；Ｈａｎａｈａｎら、Ｊ．Ｍｏｌ．Ｂｉｏｌ．、１６６、５５７−５８０、１９８３の方法の変法により作製したコンピテントな宿主菌）を用い、得られたコロニー１２個をランダムに選びプラスミドＤＮＡを精製し、それらＤＮＡの制限酵素での消化パターンにより目的のプラスミド（ｐＥＦ１８Ｓ）を含む１０クローンの中から１個を選択し、大量にプラスミドＤＮＡを調製した。
プラスミドＤＮＡの精製は本質的にＭｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ［Ｓａｍｂｒｏｏｋら、Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ Ｐｒｅｓｓ（１９８９）］に記載されているようにして実施した。
すなわち、上記のようにして得られたクローンｐＭＥ１８Ｓを５０μｇ／ｍｌのＡｍｐｉｃｉｌｉｎを含む５０ｍｌのＬＢ培地（１％Ｂａｃｔｏ−ｔｒｙｐｔｏｎｅ、 ０．５％Ｂａｃｌｏ−ｙｅａｓｔ ｅｘｔｒａｃｔ、０．５％ＮａＣｌ）で一夜培養した後、遠心分離により得た菌体を４ｍｌのＴＥＧ−ｌｙｓｏｚｙｍｅ（２５ｍＭ Ｔｒｉｓ−Ｃｌ（ｐＨ８）、１０ｍＭ ＥＤＴＡ、５０ｍＭ Ｇｌｕｃｏｓｅ、０．５％ｌｙｓｏｚｙｍｅ）溶液に懸濁し、８ｍ１の０．２ＮＮａＯＨ／１％ＳＤＳ溶液を加えてよく懸濁する。さらに３Ｍ ｐｏｔａｓｓｉｕｍ／５Ｍ ａｃｅｔａｔｅ溶液を６ｍｌ加えてよく懸濁した後遠心し、上清を得る。上清はフェノールークロロホルム（１：１）処理後、等量のイソプロパノールを加えて遠心し、ペレットを得た。ペレットはＴＥ溶液（１０ｍＭトリス−塩酸（ｐＨ７．５）、１ｍＭ ＥＤＴＡ）に溶解後、ＲＮａｓｅ処理、フェノール−クロロホルム（１：１）処理を施し、エタノール沈殿を行なう。ペレットを再度ＴＥ溶液に溶解し、ＮａＣｌ、ポリエチレングリコール３０００をそれぞれ０．６３Ｍ、７．５％になるように加えて遠心する。
最後に、ペレットはＴＥ溶液に溶解後エタノール沈殿を行なう。これにより約３００μｇのプラスミドＤＮＡを得た。このＤＮＡ１００μｇを制限酵素ＥｃｏＲＩおよびＮｏｔＩで完全に消化後、０．８％のアガロースゲル（ＦＭＣ ＢｉｏＰｒｏｄｕｃｔｓ社製）で泳動し、ベクター断片を回収後プレップ−Ａ−ジーンＤＮＡ精製キット（バイオラッド社製）で精製しおよそ５５μｇのプラスミドＤＮＡを得た。このＤＮＡを以下のｃＤＮＡライブラリー作製に使用した。
＜実施例６＞
ＭｃＡ−ＲＨ８９９４細胞からのｍＲＮＡの精製
実施例４のコロニーアッセイの結果より比較的活性の高かったＭｃＡ−ＲＨ８９９４細胞をラットＴＰＯｃＤＮＡクローニングの材料に選び以下の実験に供した。
全ＲＮＡの単離は本質的にＭｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ［Ｓａｍｂｒｏｏｋら、Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ Ｐｒｅｓｓ（１９８９）］に記載されているようにして実施した。ＭｃＡ−ＲＨ８９９４細胞を直径９０ｍｍのシャーレ１５枚に完全に密に増殖させた後、シャーレから培養液を除き、１枚のシャーレ当り０．８ｍｌの５Ｍグアニジン溶液（５Ｍグアニジンチオシアナート、５ｍＭクエン酸ナトリウム （ｐＨ７．０）、０．１Ｍβ−メルカプトエタノール、０．５％ザルコシル硫酸ナトリウム）を加え、よく懸濁した後１本のチューブに混合液を集め、グアニジン溶液を加えて全量を２０ｍｌとした。この細胞が壊れて粘稠になった混合液は、１８Ｇさらに２１Ｇの注射針を装填した２０ｍｌ容の注射器を用い、粘性がほとんどなくなるまでおよそ２０回吸入排出を繰り返した。ベックマン社製ＳＷ２８ローターに合うポリアロマー製の遠心チューブに１８ｍｌの５．７Ｍ ＣｓＣｌ−０．１ＭＥＤＴＡ（ｐＨ７．５）をクッションとして先に加えておき、チューブがほぼ満たされるように上述の混合液約２０ｍｌを層が乱れないように静かに重層した。このようにして調製された遠心チューブを２０℃で２５０００ｒ．ｐ．ｍ．、２０時間遠心した後、得られたペレットを少量の８０％エタノールを用いて２回洗浄した。ペレットはＴＥ溶液に溶解せしめ、フェノール−クロロホルム（１：１）にて抽出後、１／１０量の３Ｍ酢酸ナトリウムと２．５倍量のエタノールを加えてエタノール沈殿を行い全ＲＮＡを得た（約１０^８個の細胞より全ＲＮＡ約２．５ｍｇを得た）。
全ＲＮＡからのポリ （Ａ）^＋ＲＮＡの精製はＯｌｉｇｏｔｅｘ^ＴＭ−ｄＴ３０（Ｓｕｐｅｒ）（日本合成ゴム／日本ロッシュ社製；ラテックス粒子の表面にオリゴｄＴが共有結合で固定してあり、ポリ （Ａ）^＋ＲＮＡ精製に使われるオリゴｄＴカラムと同様にポリ （Ａ）^＋ＲＮＡの精製ができる）を用いて行った。全ＲＮＡ約５００μｇより２０μｇのポリ （Ａ）^＋ＲＮＡを得た。
＜実施例７＞
ラットｃＤＮＡライブラリーの構築
実施例５で得られた５μｇのポリ （Ａ）^＋ＲＮＡからＴｉｍｅＳａｖｅｒ^ＴＭｃＤＮＡ ＳｙｎｔｈｅｓｉｓＫｉｔ（Ｐｈａｒｍａｃｉａ社製；Ｏｋａｙａｍａ−Ｂｅｒｇ法：Ｍｏｌ．Ｃｅｌｌ． Ｂｉｏｌ．、２、１６１−１７０、１９８２、の変法によるｃＤＮＡ合成法のキット）およびＤＩＲＥＣＴＩＯＮＡＬ ＣＬＯＮＩＮＧ ＴＯＯＬＢＯＸ （Ｐｈａｒｍａｃｉａ社製；ＮｏｔＩ配列を含むｃＤＮＡ合成のためのプライマー： ５′−ＡＡＣＴＧＧＡＡＧＡＡＴＴＣＧＣＧＧＣＣＧＣＡＧＧＡＡ（Ｔ）_１８−３′並びにＥｃｏＲＩ配列付加用アダプター：５′−ＡＡＴＴＣＧＧＣＡＣＧＡＧ−３′および５′−ＣＴＣＧＴＧＣＣＧ−３′のセット）を用いて、５’端にＥｃｏＲＩ、３’端にＮｏｔＩ認識部位を持つ２本鎖ｃＤＮＡを合成した。合成したｃＤＮＡは１．２μｇの予めＥｃｏＲＩおよびＮｏｔＩで処理した発現ベクターｐＥＦ１８Ｓ（実施例５参照）と連結させ、８．４ｍｌのコンピテント・ハイＥ．ｃｏｌｉ ＤＨ５ （東洋紡績社製）を形質転換した。その結果、５．３×１０^５個の形質転換体が得られた。＜実施例８＞
ＰＣＲ法によるラットＴＰ０ｃＤＮＡ断片の取得（クローニング）
実施例７で作製したＭｃＡ−ＲＨ８９９４ｃＤＮＡライブラリー５３万クローンを５０μｇ／ｍｌのＡｍｐｉｃｉｌｉｎを含む５０ｍｌのＬＢ培地で一夜培養した後、遠心分離により得た菌体からＱＩＡＧＥＮ−ｔｉｐ１００ （ＤＩＡＧＥＮ社製；ＤＮＡ精製用シリカゲルベースの陰イオン交換カラム）を用いてＭｃＡ−ＲＨ８９９４ｃＤＮＡライブラリープラスミドを精製し、約２００μｇのプラスミドＤＮＡを得た。
実施例２に記載のペプチド断片ＡＰ８のアミノ酸配列に対応する２種類のアンチセンスヌクレオチドプライマーＡＰ８−１Ｒ，ＡＰ８−２Ｒ、及びｃＤＮＡライブラリー作製に用いた図６に示したプラスミドベクターｐＥＦ１８Ｓのヒトエロンゲーションファクター１αの第１イントロンに対応するセンスヌクレオチドプライマーＥＦ１α−１，ＥＦ１α−２を合成した。合成にはアプライドバイオシステムズ社製３９４ＤＮＡ／ＲＮＡシンセサイザー（β−シアノエチルアミダイト法にもとづく合成機）を使用し、同社製の合成ＤＮＡ精製用ＯＰＣカラム（逆相シリカゲルを充填したカラムでトリチル基を持つ合成ＤＮＡを精製するカラム）を用いて精製した。精製した合成ＤＮＡはＴＥ溶液に５０μＭとなるように溶解し、使用時まで−２０℃に保存した。以下用いる合成オリゴヌクレオチドは全て同様に合成並びに精製して使用した。
プライマーＡＰ８−１Ｒ，ＡＰ８−２Ｒは、連続した１７個のヌクレオチドからなる混合型プライマーであり、タカハシらの研究にあるようにデオキシイノシンを使用した（Ｔａｋａｈａｓｈｉ，ｅｔ ａｌ．，Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ． （ＵＳＡ）８２，１９３１−１９３５（１９８５））。
プライマーＥＦ１α−１，ＥＦ１α−２は、Ｕｅｔｓｕｋｉ，ｅｔ ａｌ．，Ｊ．Ｂｉｏｌ．Ｃｈｅｍ． ２６４，５７９１−５７９８（１９８９）にあるゲノム配列の１４９１−１５１２、１５１３−１５３２に相当する塩基配列を基にして合成したそれぞれ２１、２０個のヌクレオチドからなるプライマーである。合成したプライマーの配列を表８に示す。
【表８】

ＭｃＡ−ＲＨ８９９４ｃＤＮＡライブラリープラスミド３μｇを鋳型とし、ＡＰ８−１Ｒ（５００ｐｍｏｌ）、ＥＦ１ α−１（１００ｐｍｏｌ）をプライマーとして、ＧｅｎｅＡｍｐ^ＴＭＰＣＲ Ｒｅａｇｅｎｔ Ｋｉｔ ｗｉｔｈ ＡｍｐｌｉＴａｑ^ＴＭ ＤＮＡ Ｐｏｌｙｍｅｒａｓｅ （宝酒造社製；ＰＣＲ用耐熱性Ｔａｑいポリメラーゼ、反応バッファー、ｄＮＴＰのセット）を用いて、ＧｅｎｅＡｍｐ^ＴＭ ＰＣＲ Ｓｙｓｔｅｍ ９６００（ＰＥＲＫＩＮ−ＥＬＭＥＲ社製；ＰＣＲ用反応機）により１００μｌの容量でＰＣＲ反応（９５℃で２分間加熱後、９５℃で１分間の変性条件、４０℃で１分間のアニール条件、７２℃で１分間の合成条件で３５回の反応を行い、さらに７分間７２℃でインキュベート）を行った。増幅されたＤＮＡ断片の特異性を上げるために、得られたＰＣＲ反応液１μｌを鋳型とし、ＥＦ１α−２（１００ｐｍｏｌ）、ＡＰ８−２Ｒ（５００ｐｍｏｌ）をプライマーとして、同様に１００μｌの容量でＰＣＲ反応（９５℃で２分間加熱後、９５℃で１分間の変性条件、４５℃で１分間のアニール条件、７２℃で１分間の合成条件で３５回の反応を行い、さらに７分間７２℃でインキュベート）を行った。
ここで得られた反応液を、２％アガロースゲル（ＦＭＣ ＢｉｏＰｒｏｄｕｃｔｓ社製）を用いた電気泳動にかけ、このＰＣＲ反応の主要産物である約３３０ｂｐのＤＮＡ断片を分離し、プレップ−Ａ−ジーンＤＮＡ精製キット（バイオラッド社製）を用いて精製した。このＤＮＡ断片をＴ４ＤＮＡリガーゼ（ライフテクノロジー社製）を用いてｐＣＲ^ＴＭＩＩ（Ｉｎｖｉｔｒｏｇｅｎ社製；ＰＣＲ産物ＴＡクローニング用ベクター：ＰＣＲに使用する耐熱性ポリメラーゼが末端トランスフェラーゼ活性を有するために、ＰＣＲで増幅したＤＮＡの３’末端にデオキシアデニル酸を１個付加する性質を利用し、５’−ｄＴ突出末端を持つベクターｐＣＲ^ＴＭＩＩにそのままサブクローニングする方法）ベクターにサブクローンした。任意に選択した２８クローンについてＱＩＡＧＥＮ−ｔｉｐ１００（ＤＩＡＧＥＮ社製）を使用してプラスミドＤＮＡを精製し、これをＴａｑ Ｄｙｅ Ｄｅｏｘｙ^ＴＭ Ｔｅｒｍｉｎａｔｅｒ Ｃｙｃｌｅ Ｓｅｑｕｅｎｉｎｇ Ｋｉｔ （アプライドバイオシステムズ社製；ＰＣＲを利用した、蛍光色素で行なうジデオキシ法：Ｓａｎｇｅｒら、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ． Ｓｃｉ．ＵＳＡ、７４、５４６３−５４６７、１９７７）を用いて、アプライドバイオシステムズ社製３７３ＡＤＮＡシークエンサー（蛍光シークエンサー）によりシークエンスし、各クローンの塩基配列を決定した。
得られたＤＮＡ断片の中で、ＡＰ８のアミノ酸のＮ末端側３個（Ｉｌｅ／Ｔｈｒ／Ｓｅｒ） −Ｖａｌ−ＰｒｏをＡＰ８−２Ｒのプライマーに隣接してコードするものを選択し、全長をシークエンスしたところ、２６ｌｂｐを有するｃＤＮＡを含んでいた。
このｃＤＮＡ断片の１７３〜１７５の塩基配列はメチオニンをコードするものでありコーディングのフレームはＡＰ８のアミノ酸のフレームと一致した。またこの配列の前後はＫｏｚａｋの配列（Ｋｏｚａｋ，Ｍ．，Ｃｅｌｌ，４４，２８３−２９２、１９８６）に適合することから翻訳の開始部位と推定され、ラットＴＰＯタンパク質のＮ末部分をコードするｃＤＮＡ断片と考えられた。このｃＤＮＡ断片をＡ１と命名した。ベクターの配列を除いたＡ１断片の塩基配列およびそれから演鐸されるアミノ酸配列を配列表（配列番号１）に示した。
＜実施例９＞
ＰＣＲ法によるラットＴＰＯｃＤＮＡのスクリーニング
前述のｃＤＮＡライブラリーを約１万クローンづつのプールに分け、５０μｇ／ｍｌのＡｍｐｉｃｉｌｉｎを含む１ｍｌのＬＢ培地中で一夜培養した後、プラスミド自動分離装置ＰＩ−１００（倉敷紡績社製、ＶＥＲ−３．０；アルカリＳＤＳ法：Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ［Ｓａｍｂｒｏｏｋら、ＣｏｌｄＳｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ Ｐｒｅｓｓ、１９８９］ ：の変法にもとづくプラスミドＤＮＡ自動抽出機）を用いてプラスミドＤＮＡを抽出した。その１／３０量を鋳型としてフラグメントＡ１をもとに設計した２種の合成オリゴヌクレオチド
５’ ＣＧＡＧＧＧＴＧＴＡＣＣＴＧＧＧＴＣＣＴＧ３’ （配列番号１の配列の１−１７のセンス配列；ＣＧＡＧはアダプターの配列）および
５’ ＣＡＧＡＧＴＴＡＧＴＣＴＴＧＣＧＧＴＧＡＧ３’ （配列番号１の配列の２１２−２３２のアンチセンス配列）
をプライマーとして合成・精製し、ＧｅｎｅＡｍｐ^ＴＭ ＰＣＲ Ｒｅａｇｅｎｔ Ｋｉｔ ｗｉｔｈ ＡｍｐｌｉＴａｑ^ＴＭＤＮＡ Ｐｏｌｙｍｅｒａｓｅ （宝酒造社製）を用いて、ＧｅｎｅＡｍｐ^ＴＭＰＣＲ Ｓｙｓｔｅｍ ９６００（ＰＥＲＫＩＮ−ＥＬＭＥＲ社製）によりＰＣＲ反応（９４℃で３０秒間の変性条件、６６℃で３０秒間のアニール条件、７２℃で１分間の合成条件で３０回の反応）を行った結果、１００プール中３プールに２３６ｂｐの特異的バンドが検出された。このうちの１プールを約９００クローンづつのプールに分けプラスミドＤＮＡを抽出し、同様のＰＣＲ反応を行ったところ、１００プール中３プールにバンドが検出された。さらにこのうちの１プールを４０クローンづつのプールに分け、同様の選別を行なった結果１００プール中３プールで特異的と考えられるバンドが観察された。これらのうち１プールを選び５０μｇ／ｍｌのＡｍｐｉｃｉｌｉｎを含むＬＢプレート（１５％アガーを含むＬＢ培地）上に蒔き、出現した１つ１つのコロニーについてプラスミドＤＮＡを抽出し同様のＰＣＲ反応を行った結果、１００クローン中２クローンで陽性バンドを検出した。
＜実施例１０＞
ラットＴＰ０ｃＤＮＡのシークエンス
プラスミドＤＮＡの精製は本質的にＭｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ［Ｓａｍｂｒｏｏｋら、Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ Ｐｒｅｓｓ（１９８９）］に記載されているようにして実施した。
実施例９で最終的に得られた２個のクローンを５０μｇ／ｍｌのＡｍｐｉｃｉｌｉｎを含む５０ｍｌのＬＢ培地で一夜培養した後、実施例５と同様に精製し、最終的に約３００μｇのプラスミドＤＮＡを得た。
ここで得られたプラスミドＤＮＡをＴａｑ Ｄｙｅ Ｄｅｏｘｙ^ＴＭＴｅｒｍｉｎａｔｅｒ Ｃｙｃｌｅ Ｓｅｑｕｅｎｉｎｇ Ｋｉｔ （アプライドバイオシステムズ社製）を用いて、実施例８と同様にシークエンスし、ｃＤＮＡ全長の塩基配列を決定した。その結果、２個のクローンの塩基配列は完全に一致し同一のクローンであることが明かとなった。この中から１クローンを選び該ｃＤＮＡを担持したプラスミドクローンをｐＥＦ１８Ｓ−Ａ２αと命名した。塩基配列およびそれから演繹されるアミノ酸配列を配列表（配列番号２）に示した。
配列表（配列番号２）に示す配列の特徴は顕著である。１７２ｂｐの５’非翻訳領域後の配列は、メチオニンに始まる２１個のタンパク質から成る分泌のためのシグナル配列と推定される、疎水性に富むアミノ酸をコードしている。このタンパク質は１２６個のアミノ酸残基を有し、停止コドン（ＴＡＡ）の後に１０２２塩基の３’非翻訳配列及びポリＡ尾部の多数のアデニンが続く。このタンパク質中には、実施例２で解析された部分アミノ酸配列ＡＰ８に対応する配列のみが含まれていた（配列番号２におけるアミノ酸番号１〜１２）。Ｎ−グリコシル化のための配列部位は存在しない。また、３’非翻訳配列末端近くの１６２４〜１６２９の塩基配列は、コンセンサス配列とは異なるが、潜在的ポリアデニル化配列と考えられる。
大腸菌ＤＨ５に担持されたベクターｐＥＦ１８Ｓ−Ａ２αは１９９４年２月１４日付で通商産業省工業技術院生命工学工業技術研究所に受託番号ＦＥＲＭ ＢＰ−４５６５として寄託した。これはまた、１９９５年３月２２日付で中華民国食品工業発展研究所（ＦＩＲＤＩ）に受託番号９４００８３として寄託した。
＜実施例１１＞
ラットＴＰＯｃＤＮＡのＣＯＳ１細胞での発現〜活性確認
プラスミドｐＥＦ１８Ｓ−Ａ２αのＣＯＳ１細胞へのトランスフェクションは、クロロキン処理を含むＤＥＡＥ−デキストラン法（Ｓｏｍｐａｙｒａｃら、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ ７８巻、７５７５−７５７８頁、（１９８１） ；Ｌｕｔｈｍａｎら、Ｎｕｃｌ．Ａｃｉｄｓ Ｒｅｓ．、１１巻、１２９５−１３０８頁、（１９８３））に若干の改変を加えた以下の方法に従い実施した。１０％ＦＣＳを含むＤＭＥＭ培養液に浮遊させたＣＯＳ１細胞（ＡＴＣＣ ＣＲＬ１６５０）を直径１００ｍｍの組織培養用プラスティックシャーレに入れ、５％炭酸ガス培養器中にて３７℃で約４０％コンフルエントになるまで培養した。一方、５００μｇ／ｍｌ（７）ＤＥＡＥ−デキストラン（ファルマシア社）、８０μＭのクロロキン（シグマ社）、８％（ｖ／ｖ）のＨＢＳ （２１ｍＭ ＨＥＰＥＳ−１４５ｍＭ ＮａＣｌ、ｐＨ７．１）及び９％（ｖ／ｖ）のＮｕ−Ｓｅｒｕｍ（コラボレイティブ社）を含む４ｍｌのＤＭＥＭ培養液に３０μｌのＨＢＳに溶解したプラスミドｐＥＦ１８Ｓ−Ａ２α（１０μｇ）を混和し、トランスフェクション直前にＤＭＥＭ培養液で２度洗浄した上記のＣＯＳ１細胞に添加した後、５％炭酸ガス培養器中にて３７℃で５時間培養した。その後、培養上清を吸引除去しＤＭＥＭ培養液でシャーレを２度洗浄した後、１０％ＦＣＳを含むＤＭＥＭ培養液を１５ｍｌ加え、５％炭酸ガス培養器中にて３７℃で３日ないしは５日間培養し、培養上清を回収した。
得られた培養上清をＩＭＤＭ培養液に対して十分に透析後、ラットＣＦＵ−ＭＫアッセイ系で評価した。その結果、プラスミドｐＥＦ１８Ｓ−Ａ２αをトランスフェクションして発現させたＣＯＳ１細胞の培養上清中に用量依存的にＴＰＯ活性が認められ（図７）、また、ラットＴＰＯの場合と同様に培養４日目に多数のｃｙｔｏｐｌａｓｍｉｃ ｐｒｏｃｅｓｓ ｆｏｒｍａｔｉｏｎ形成が認められた。一方、インサートを含まないプラスミドｐＥＦ１８ＳをトランスフェクションしたＣＯＳ１細胞培養上清ではＴＰＯ活性は検出されなかった（図７）。また、Ｍ−０７ｅアッセイ系においても、プラスミドｐＥＦ１８Ｓ−Ａ２αをトランスフェクションして発現させたＣＯＳ１細胞の培養上清中に用量依存的にＭ−０７ｅ細胞増殖促進活性が認められ、インサートを含まないプラスミドｐＥＦ１８ＳをトランスフェクションしたＣＯＳ１細胞培養上清では活性が認められなかった。これらの結果から、ｐＥＦ１８Ｓ−Ａ２αがＴＰＯ活性を有するタンパク質をコードする遺伝子を担持していることが確認された。
次に、このＴＰＯ活性の血小板増加作用を調べるため、部分精製品を調製した。
調製過程でのＴＰＯ活性測定には、ラットＣＦＵ−ＭＫアッセイ系を用いた。まず、プラスミドｐＥＦ１８Ｓ−Ａ２αをトランスフェクションしたＣＯＳ１細胞を、０．２ｍｇのＢＳＡを含む無血清培養液で３日間培養し、約５．８Ｌの無血清培養上清を得た。その無血清培養上清に、ｐ−ＡＰＭＳＦを最終濃度１ｍＭ加え、０．２２μｍの濾過フィルターで通過する濾液をとった。この体積５７９３ｍｌ（蛋白質濃度０．２２９ｍｇ／ｍｌ）総蛋白質量１３２６ｍｇ、相対活性１０００、相対活性量１３２６０８０）に対し１０００ｍｌ当り０．８５ｍｏｌｅｓのＮａＣｌ（合計２８８ｇ）を加え、最終濃度０．８２２Ｍ ＮａＣｌ，５８４９ｍｌの溶液とした後、１Ｍ ＮａＣｌ， ２０ｍＭ ＮａＰｈｏｓｐｈａｔｅ ｐＨ７．２で予め平衡化してあったＴＳＫ−ｇｅｌ ＡＦ−ＢＬＵＥ ６５０ＭＨカラム（トーソー社製、カタログ番号０８７０５ ；直径５ｃｍ、ベッド高 ６ｃｍ）に、流速７ｍｌ／ｍｉｎで添加した。添加終了後、２０ｍＭＮａ Ｐｈｏｓｐｈａｔｅ， １Ｍ ＮａＣｌ， ｐＨ７．２で溶出される素通り（約７９００ｍｌ）を集め、これを限外濾過ユニット（フィルトロン社・オメガウルトラセット 分子量８０００カット）で濃縮し、素通り画分Ｆ１（４６０ｍｌ，蛋白質濃度２．１１ｍｇ／ｍｌ，総蛋白質量９７３ｍｇ，相対活性１６．３）を得た。次に、溶出液を２Ｍ ＮａＳＣＮにかえ、溶出されたＴＳＫ−ｇｅｌ ＡＦ−ＢＬＵＥ ６５０ＭＨ吸着ＴＰＯ活性画分Ｆ２（２８４０ｍｌ）を限外濾過ユニット（アミコン社製；ＹＭ３膜、直径７６ｍｍ）を用いて、６．８１ｍｌまで濃縮した。このＴＰＯ活性画分Ｆ２の総蛋白質量は１２．５ｍｇ、このステップでのＦ２の蛋白質収量は０．６２％であった。また、ＴＰＯの相対活性は、２４０であった。次に、ＨｉＬｏａｄ２６／６０ Ｓｕｐｅｒｄｅｘ ２００ｐｇ（ファルマシア バイオテク社製、カタログ番号１７−１０７１−０１；直径 ２，６ｃｍ、ベッド高 ６０ｃｍ）カラムに注入し、流速１ｍｌ／ｍｉｎで、５０ｍＭのＮａＣｌを含む２０ｍＭ Ｎａ Ａｃｅｔａｔｅ， ｐＨ５．５で展開した。展開開始後に溶出した１９４ｍｌから２６０ｍｌまでの範囲の画分にＴＰＯ活性が確認できた。そこで、これをまとめて、Ｓｕｐｅｒｄｅｘ ２００ｐｇのＴＰＯ活性画分Ｆ２（６６ｍｌ，蛋白質濃度０．１１２ｍｇ／ｍｌ，総蛋白質量７．４１ｍｇ，相対活性１４２８６０、相対活性量１０５８６００）とした。次に、展開溶媒Ａ（２０ｍＭ Ｎａ Ａｃｅｔａｔｅ， ｐＨ５．５）および展開溶媒Ｂ（５００ｍＭのＮａＣｌを含む２０ｍＭ Ｎａ Ｐｈｏｐｈａｔｅ，ｐＨ７．２）を用意し、１００％Ａで平衡化した強陽イオン交換カラムのＲＥＳＯＵＲＣＥＳ（ファルマシア バイオテク社製、カタログ番号１７−１１７８−０１：直径 ０．６４ｃｍ、ベッド高 ３ｃｍ）カラムに、流速１ｍｌ／ｍｉｎで、注入した。次いで、流速０．３ｍｌ／ｍｉｎで、４０分かけて１００％Ａから１００％Ｂまでの直線濃度勾配にて展開した。アッセイの結果、広い範囲（５％Ｂから３２％Ｂの範囲）にＴＰＯ活性が溶出されていることが分かった。このＴＰＯ活性画分をまとめて、限外濾過ユニット（アミコン社製；ＹＭ３膜、直径２５ｍｍ）で濃縮し、ＲＥＳＯＵＲＣＥＳのＴＰＯ活性画分Ｆ２（１．６５ｍｌ，蛋白質濃度４．７４ｍｇ／ｍｌ，総蛋白質量７．８２ｍｇ，相対活性７１４００、相対活性量５５８６００）を得ることができた。
得られた標品は純粋なＴＰＯ標品ではなかったが、ラットＣＦＵ−ＭＫアッセイ系において、十分に高い活性が確認されたので、マウスへの投与実験を実施した。即ち、投与前日に血小板数を測定しておいたＩＣＲ系マウス（９週齢；♂）の皮下へ、ＴＰＯ部分精製品（４７４μｇ（１００μｌ）／マウス／日）、対照としてＢＳＡ （２００μｇ（１００μｌ）／マウス／日）、あるいは対照としてＴＰＯ部分精製品が溶解されている緩衝液と同じ組成の緩衝液（１００μｌ／マウス／日）を５日間連日投与し、６日目に心臓より採血して血小板数を測定した（Ｆ８００；東亜医用電子）。ＴＰＯ部分精製品投与マウスでは投与前に比べて平均して約２．１４倍の血小板数の増加を認めた。また、投与群の間で比較すると、ＴＰＯ部分精製品投与マウスでは、ＢＳＡ投与マウスに比べて約１．７４倍、あるいは緩衝液のみを投与したマウスに比べて約１．９０倍の血小板数の増加を認めた。この結果から、ＴＰＯが生体内において血小板増加作用を有することが明らかとなった。なお、ＴＰＯ部分精製品投与マウスにおいて、マウスの急性期蛋白質の１種であるｉｍｍｕｎｏｓｕｐｐｒｅｓｉｖｅ ａｃｉｄｉｃ ｐｒｏｔｅｉｎ （ＩＡＰ）の増加が認められなかったことから、ＴＰＯは急性期蛋白質の誘導に関してＩＬ−６やＩＬ−１１とは性格を異にすることが強く示唆された。
＜実施例１２＞
ラット各種組織でのＴＰＯｍＲＮＡの検出
ラットＴＰＯｍＲＮＡがラット体内においてどの組織で発現しているかを確認するために、ラットの各種組織よりＲＮＡを抽出した。実施例１に記載されているものと同様にＸ線照射を行なった１１日目ないし１４日目のラット６匹より各種組織（脳、胸腺、肺、肝臓、心臓、脾臓、小腸、腎臓、精巣および骨髄細胞）を摘出し速やかに液体窒素で凍結した。全ＲＮＡの抽出にはＲＮＡ単離用試薬ＩＳＯＧＥＮ（和光純薬）を使用した。凍結した組織の重量に応じた量のＩＳＯＧＥＮ試薬を加え、ホモジナイザー（ヒスコトロン；日音医理科器械製作所：ＮＳ−６０）により、組織が完全に破砕されるまで（およそ１００００ＲＰＭで４５〜６０秒）処理した後、全ＲＮＡ抽出操作を行なった（ＣｈｏｍｃｚｙｎｓｋｉらのＡｃｉｄ Ｇｕａｎｉｄｉｕｍ Ｐｈｅｎｏｌ Ｃｈｒｏｌｏｌｏｒｍ法にもとづく抽出法：Ａｎａｌ． Ｂｉｏｃｈｅｍ．、１６２：１５６−１５９、１９８７を参照）。その結果、それぞれの組織より１．１ｍｇ〜５．６ｍｇの全ＲＮＡが得られた。
全ＲＮＡからポリ （Ａ）^＋ＲＮＡの精製はＯｌｉｇｏｔｅｘ^ＴＭ−ｄＴ３０（Ｓｕｐｅｒ）（日本合成ゴム／日本ロッシュ社製）を用いて行い全ＲＮＡ約５００μｇより２０ｐｇのポリ （Ａ）^＋ＲＮＡを得た。
得られた各種組織のポリ （Ａ）^＋ＲＮＡそれぞれ１μｇよりランダムプラィマーを使用してｃＤＮＡの１本鎖目を合成した。ポリ （Ａ）^＋ＲＮＡ １μｇを１０μｌの滅菌水に溶かし７０℃で１５分間加温後急冷し、７５ｐｍｏｌｅランダムプライマー（宝酒造社製）、１０Ｕ ＲＮａｓｅ Ｉｎｈｉｂｉｔｏｒ（ベーリンガーマンハイム社製）、５０ｍＭＴｒｉｓ−ＨＣｌ（ｐＨ８．３）、７５ｍＭ ＫＣｌ、３ｍＭ ＭｇＣｌ_２、２００Ｕ ＳｕｐｅｒＳｃｒｉｐｔ^ＴＭＩＩライフテクノロジー社製逆転写酵素）となるようにそれぞれの試薬を加え（全量２０μｌ）、３７℃で１時間保温した。反応液を７０℃で１０分間加熱し酵素を失活させた後−２０℃で使用時まで保存した。
実施例１０で得られたラットＴＰＯのｃＤＮＡ配列より新たにＰＣＲ用プライマーを合成した。合成したプライマーの配列は以下の通り。
ｒＴＰＯ−Ｉ：５’−ＣＣＴＧＴＣＣＴＧＣＴＧＣＣＴＧＣＴＧＴＧ−３’ （配列番号２の３４７〜３６７）
ｒＴＰＯ−Ｎ：５’−ＴＧＡＡＧＴＴＣＧＴＣＴＣＣＡＡＣＡＡＴＣ−３’ （配列番号２の１００５〜１０２５に対応するアンチセンス）
合成したｃＤＮＡ反応液のそれぞれ０．１容を鋳型に用いここで合成したプライマー（ｒＴＰＯ−Ｉ、ｒＴＰＯ−Ｎ）をそれぞれ１μＭ使用してＰＣＲを実施した。
ＧｅｎｅＡｍｐ^ＴＭ ＰＣＲ Ｒｅａｇｅｎｔ Ｋｉｔ ｗｉｔｈ ＡｍｐｌｉＴａｑ^ＴＭＤＮＡ Ｐｏｌｙｍｅｒａｓｅ（宝酒造社製）を用いて、ＧｅｎｅＡｍｐ^ＴＭ ＰＣＲ Ｓｙｓｔｅｍ ９６００（ＰＥＲＫＩＮ−ＥＬＭＥＲ社製）により１００μｌの容量でＰＣＲ反応（９５℃で２分間加熱後、９５℃で１分間の変性条件、５７℃で１分間のアニール条件、７２℃で１分間の合成条件で３０回の反応を行い、さらに７分間７２℃でインキュベート）を行った。ここで得られた反応液を、２％アガロースゲル（ＦＭＣ ＢｉｏＰｒｏｄｕｃｔｓ社製）を用いた電気泳動にかけ増幅されたバンドを観察したところ、脳、肝臓、小腸並びに腎臓で特異的と考えられるバンドが観察された。この結果より、発現量の多少は判断できないがラットにおいてはこれらの組織でＴＰＯｍＲＮＡが発現していると考えられた。人においても同様な発現様式を示すことが示唆されるが実施例４の結果と合わせて考えると、肝臓がヒトＴＰＯｃＤＮＡ取得のための出発材料としては適当であると判断された。
＜実施例１３＞
ヒト正常肝臓由来ｃＤＮＡライブラリーの構築
実施例１２の結果より、肝臓をヒトＴＰＯｃＤＮＡクローニングのための材料に選び、市販の正常ヒト肝臓由来ポリ （Ａ）^＋ＲＮＡ （Ｃｌｏｎｔｅｃｈ社製：ＣｈｏｍｃｚｙｎｓｋｉらのＡｃｉｄ Ｇｕａｎｉｄｉｕｍ ＰｈｅｎｏｌＣｈｒｏｌｏｆｏｒｍ法にもとづいて抽出したもの）５μｇを用い実施例７と同様にＴｉｍｅＳａｖｅｒ^ＴＭ ｃＤＮＡ Ｓｙｎｔｈｅｓｉｓ Ｋｉｔ （Ｐｈａｒｍａｃｉａ社製）およびＤＩＲＥＣＴＩＯＮＡＬ ＣＬＯＮＩＮＧ ＴＯＯＬＢＯＸ （Ｐｈａｒｍａｃｉａ社製）を使用して、５’端にＥｃｏＲＩ、３’端にＮｏｔＩ認識部位を持つ２本鎖ｃＤＮＡを合成した。合成したｃＤＮＡは１．２μｇの予めＥｃｏＲＩおよびＮｏｔＩで処理した発現ベクターｐＥＦ１８Ｓと連結させ、８．４ｍｌのコンピテント・ハイＥ．ｃｏｌｉ ＤＨ５ （東洋紡績社製）を形質転換した。その結果、１．２×１０^６個の形質転換体が得られた。
＜実施例１４＞
ＰＣＲによるヒトＴＰＯｃＤＮＡ断片の取得（クローニング）
市販の正常ヒト肝臓由来ポリ （Ａ）^＋ＲＮＡ （Ｃｌｏｎｔｅｃｈ社製）１μｇよりランダムプライマーを使用してｃＤＮＡの１本鎖目を合成した。ポリ （Ａ）^＋ＲＮＡ １μｇを１０μｌの滅菌水に溶かし７０℃で１５分間加温後急冷し、７５ｐｍｏｌｅランダムプライマー（宝酒造社製）、１０Ｕ ＲＮａｓｅＩｎｈｉｂｉｔｏｒ（ベーリンガーマンハイム社製）、５０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．３）、７５ｍＭ ＫＣｌ、３ｍＭ ＭｇＣｌ_２、２００Ｕ ＳｕｐｅｒＳｃｒｉｐｔ^ＴＭＩＩ（ライフテクノロジー社製）となるようにそれぞれの試薬を加え（全量２０μｌ）、３７℃で１時間保温した。反応液を７０℃で１０分間加熱し酵素を失活させた後−２０℃で使用時まで保存した。
ラットＴＰＯｃＤＮＡ配列（配列番号２）よりＰＣＲ用プライマーを合成した。プライマーの配列は以下の通り。
ｒＴＰＯ−ＡＩＮ：５’−ＡＴＧＧＡＧＣＴＧＡＣＴＧＡＴＴＴＧＣＴＣ−３’（配列番号２の１７３〜１９３）
ｒＴＰＯ−Ｎ ：５’−ＴＧＡＡＧＴＴＣＧＴＣＴＣＣＡＡＣＡＡＴＣ−３’（配列番号２の１００５〜１０２５に対応するアンチセンス）
合成したｃＤＮＡ反応液の０．１容を鋳型に用いここで合成したプライマー（ｒＴＰＯ−ＡＩＮ、ｒＴＰＯ−Ｎ）をそれぞれ１μＭ使用してＰＣＲを実施した。ＧｅｎｅＡｍｐ^ＴＭ ＰＣＲ Ｒｅａｇｅｎｔ Ｋｉｔ ｗｉｔｈ ＡｍｐｌｉＴａｑ^ＴＭ ＤＮＡ Ｐｏｌｙｍｅｒａｓｅ （宝酒造社製）を用いて、ＧｅｎｅＡｍｐ^ＴＭ ＰＣＲ Ｓｙｓｔｅｍ ９６００（ＰＥＲＫＩＮ−ＥＬＭＥＲ社製）により１００μ１の容量でＰＣＲ反応（９５℃で２分間加熱後、９５℃で１分間の変性条件、４０℃で１分間のアニール条件、７２℃で１分間の合成条件で３５回の反応を行い、さらに７分間７２℃でインキュベート）を行った。
ここで得られた反応液を、２％アガロースゲル（ＦＭＣ ＢｉｏＰｒｏｄｕｃｔｓ社製）を用いた電気泳動にかけ、このＰＣＲ反応の主要産物である約６２０ｂｐのＤＮＡ断片を分離し、プレップ−Ａ−ジーンＤＮＡ精製キット（バイオラッド社製）を用いて精製 した。この精製したＤＮＡ断片をＴａｑ Ｄｙｅ Ｄｅｏｘｙ^ＴＭ Ｔｅｒｍｉｎａｔｅｒ Ｃｙｃｌｅ ＳｅｑｕｅｎｉｎｇＫｉｔ（アプライドバイオシステムズ社製）を用いて、アプライドバイオシステムズ社製３７３ＡＤＮＡシークエンサーにより直接シークエンスし塩基配列を決定した。
プラィマー部分を除く塩基配列およびそれから演繹されるアミノ酸配列を配列表（配列番号３）に示した。
このＤＮＡ断片はプライマー配列を除くと５８０ｂｐの長さを有し、ラットｃＤＮＡ塩基配列との比較を行なった結果、８６％の相同性を持つことが明かとなり、ヒトＴＰＯｃＤＮＡの一部をコードするＤＮＡ断片であると考えられた。
＜実施例１５＞
ＰＣＲ法によるヒトＴＰＯｃＤＮＡのスクリーニング
配列表−配列番号３をもとにヒトＴＰＯに対応するＰＣＲ用プライマーを合成した。配列は以下の通り。
ｈＴＰＯ−Ｉ：５’−ＴＴＧＴＧＡＣＣＴＣＣＧＡＧＴＣＣＴＣＡＧ−３’ （配列番号３の６０−８０）
ｈＴＰＯ−Ｊ：５’−ＴＧＡＣＧＣＡＧＡＧＧＧＴＧＧＡＣＣＣＴＣ−３’ （配列番号３の４７９−４９９に対応するアンチセンス）
実施例１３において構築したヒトｃＤＮＡライブラリーを増幅し約１０万個のクローンを１個の集団とするプールに分割し、５０μｇ／ｍｌ（７）Ａｍｐｉｃｉｌｉｎを含む１ｍｌのＬＢ培地中で一夜培養した後、プラスミド自動分離装置ＰＩ−１００（倉敷紡績社製、ＶＥＲ−３．０）を用いてプラスミドＤＮＡを抽出した。抽出したＤＮＡはＴＥ溶液に溶解した。
抽出したＤＮＡの５％を鋳型として使用し、合成したプライマー（ｈＴＰＯ−Ｉ、ｈＴＰＯ−Ｊ）をそれぞれ１μＭ使ってＰＣＲを実施した。ＧｅｎｅＡｍｐ^ＴＭ ＰＣＲ Ｒｅａｇｅｎｔ Ｋｉｔ ｗｉｔｈ ＡｍｐｌｉＴａｑ^ＴＭ ＤＮＡ Ｐｏｌｙｍｅｒａｓｅ （宝酒造社製）を用いて、ＧｅｎｅＡｍｐ^ＴＭ ＰＣＲ Ｓｙｓｌｅｍ ９６００（ＰＥＲＫＩＮ−ＥＬＭＥＲ社製）により２０μｌの容量でＰＣＲ反応（９５℃で１分間の変性条件、５９℃で１分間のアニール条件、７２℃で１分間の合成条件で３５回の反応を行い、さらに７分間７２℃でインキュベート）を行った結果、９０プール中３プールで特異的と考えられるバンドが検出された。これらのうち１プールを選び、約５０００クローンづつのプールに分け９０プールよりプラスミドＤＮＡを精製し、再度同一の条件でＰＣＲを実施した。その結果、５個のプールでバンドが検出された。これらより１プールを選択し２５０個のクローンを１プールとするサブプールに分け、９０プールについてプラスミドＤＮＡを抽出した。これらについて同様にＰＣＲを行なった処、３個のプールでバンドが観察された。
これらより１プールを選択し３０個のクローンを１プールとしてサブプールを作り、９０プールからプラスミドＤＮＡを精製しＰＣＲを実施した結果３個のプールでバンドが観察された。これらのうち１プールを選び５０μｇ／ｍｌのＡｍｐｉｃｉｌｉｎを含むＬＢプレート上にまき、９０個のコロニーについて同様にプラスミドＤＮＡを抽出しＰＣＲによる確認を行なった結果、最終的にクローンＨＬ３４を得た。
＜実施例１６＞
ヒトＴＰＯｃＤＮΛのシークエンス
プラスミドＤＮＡの精製は本質的にＭｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ［Ｓａｍｂｒｏｏｋら、Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ Ｐｒｅｓｓ （１９８９）］に記載されているようにして実施した。
クローンＨＬ３４を５０μｇ／ｍｌ（７）Ａｍｐｉｃｉｌｉｎを含む５０ｍｌのＬＢ培地で一夜培養した後、遠心分離により得た菌体を４ｍｌのＴＥＧ−ｌｙｓｏｚｙｍｅ溶液に懸濁し、８ｍｌの０．２Ｎ ＮａＯＨ／１％ＳＤＳ溶液を加えてよく懸濁する。さらに３Ｍ ｐｏｔａｓｓｉｕｍ／５Ｍ ａｃｅｔａｔｅ溶液を６ｍｌ加えてよく懸濁した後遠心し、上清を得る。上清はフェノールークロロホルム（１：１）処理後、等量のイソプロパノールを加えて遠心し、ペレットを得た。ペレットはＴＥ溶液に溶解後、ＲＮＡｓｅ処理、フェノールークロロホルム（１：１）処理を施し、エタノール沈殿を行なう。ペレットを再度ＴＥ溶液に溶解し、ＮａＣｌ、ポリエチレングリコール３０００をそれぞれ０．６３Ｍ、 ７．５％になるように加えて遠心する。最後に、ペレットはＴＥ溶液に溶解後エタノール沈殿を行なう。これにより約３００μｇのプラスミドＤＮＡ ｐＥＦ１８Ｓ−ＨＬ３４を得た。
精製したプラスミドＤＮＡについてＴａｑ Ｄｙｅ Ｄｅｏｘｙ^ＴＭ Ｔｅｒｍｉｎａｔｅｒ Ｃｙｃｌｅ Ｓｅｑｕｅｎｉｎｇ Ｋｉｔ（アプライドバイオシステムズ社製）を用い、アプライドバイオシステムズ社製３７３ＡＤＮＡシークエンサーによりシークエンスし、全長の塩基配列を決定した。塩基配列およびそれから演鐸されるアミノ酸配列を配列表（配列番号４）に示した。塩基配列決定に必要なプライマーは配列番号３の配列をもとに合成したプライマー並びに、それらのプライマーによるシークエンス反応で解析された内部配列をもとに設計した合成プライマーを使用した。
その結果、得られたプラスミドクローンｐＥＦ１８Ｓ−ＨＬ３４は８６１塩基対のｃＤＮＡ断片を含み、実施例２で分析されたラットＴＰＯの部分アミノ酸配列ＡＰ８（配列番号４：アミノ酸番号１〜１２）及びＴＰ２／ＴＰ３（配列番号４：アミノ酸番号１５７〜１６２）と相同性の高い配列が確認された。このＤＮＡ断片は、２５塩基目から始まるオープンリーディングフレームをコードしていると考えられたが、終止コドンは無く３’端には７６塩基からなるポリＡ尾部様配列を持っていた。ポリＡ尾部様配列直前までの２５３個のアミノ酸配列はラットＴＰＯｃＤＮＡのもの（配列番号２の１４７残基のアミノ酸配列部分）と比較すると８４％の相同性を持っておりラットＴＰＯに対応するヒトのｃＤＮＡの一部をコードするＤＮＡ断片と考えられた。終止コドンが無く３’端にポリＡ尾部様配列を持っていたことから、このクローンは完全なｃＤＮＡを反映するものではなく、ｃＤＮＡライブラリー作製時の人工産物であることが示唆された。
＜実施例１７＞
ヒトＴＰＯｃＤＮＡのＣＯＳ１細胞での発現〜活性確認
得られたプラスミドクローンｐＥＦ１８−ＨＬ３４のＣＯＳ １細胞へのトランスフェクションは、実施例１１に従って行なった。すなわちプラスミＤＮＡ１０μｇを使用しクロロキン処理を含むＤＥＡＥ−デキストラン法を用いたトランスフェクションを行ない、３日ないし５日後に培養上清を回収した。
得られた培養上清をＩＭＤＭ培養液に対して十分に透析後、ラットＣＦＵ−ＭＫアッセイ系で評価した。その結果、ｐＥＦ１８Ｓ−ＨＬ３４をトランスフェクションして発現させたＣＯＳ１細胞培養上清では用量依存的にＴＰＯ活性が認められ（図８）、また、培養４日目に多数の巨核球による突起形成が観察された。一方、インサートを含まない発現プラスミドをトランスフェクションしたＣＯＳ１細胞培養上清では活性が認められなかった（図８）。また、Ｍ−０７ｅアッセイ系においてもｐＥＦ１８Ｓ−ＨＬ３４をトランスフェクションして発現させたＣＯＳ１細胞培養上清にのみＭ−０７ｅ細胞増殖促進活性が認められた。これらの結果から、ｐＥＦ１８Ｓ−ＨＬ３４がＴＰＯ活性を有するタンパク質をコードする遺伝子ｃＤＮＡ断片を担持していることが判明した。また、ヒトＴＰＯがラットにも作用する（種特異性がない）ことも明らかとなった。
＜実施例１８＞
ヒトＴＰＯ欠失型ｃＤＮＡの発現〜活性確認
実施例１５で得られたクローンＨＬ３４のヒトＴＰＯｃＤＮＡは、その３’側にポリＡ尾部様の連続するアデニン配列を持っていたが、この配列はラットＴＰＯｃＤＮＡには観られず実験上の人工産物である可能性が示唆された。そこで、このポリＡ尾部様の連続するアデニン配列を欠失させたｃＤＮＡを作製し、発現させた蛋白質がＴＰＯとしての活性を示すか検討した。欠失体の作製にはＰＣＲを利用した。ＰＣＲ用プライマーの配列を表９に示す。それぞれの５’端には制限酵素認識配列を付加してある（ｈＴＰＯ５にはＥｃｏＲＩ、ｈＴＰＯ３にはＮｏｔＩ並びに２つのストップコドンＴＡＡＴＧＡを付加した）。
【表９】

実施例１６で得られたプラスミドクローンｐＥＦ１８Ｓ−ＨＬ３４のプラスミドＤＮＡ１μｇを鋳型として使用し、合成したプライマー（ｈＴＰＯ５、ｈＴＰＯ３）各１０μＭを使ってＰＣＲを実施した。ＧｅｎｅＡｍｐ^ＴＭ ＰＣＲ Ｒｅａｇｅｎｔ Ｋｉｔ ｗｉｔｈ ＡｍｐｌｉＴａｑ^ＴＭ ＤＮＡ Ｐｏｌｙｍｅｒａｓｅ（宝酒造社製）を用いて、ＧｅｎｅＡｍｐ^ＴＭ ＰＣＲ Ｓｙｓｔｅｍ９６００（ＰＥＲＫＩＮ−ＥＬＭＥＲ社製）により１００μｌの容量でＰＣＲ反応（９５℃で１分間の変性条件、６５℃で１分間のアニール条件、７２℃で１分間の合成条件で１５回の反応を行い、さらに７分間７２℃でインキュベート）を行った。得られた約８００ｂｐのバンドを制限酵素ＥｃｏＲｌ並びにＮｏｔｌで消化後精製し、同様に制限酵素処理した発現ベクターｐＥＦ１８Ｓにサブクローニングした。得られた形質転換体より約８００ｂｐのＤＮＡ断片を含むクローンを５個選択しプラスミドＤＮＡを大量調製した（方法は実施例５参照）。それぞれのプラスミドについては、ＰＣＲで増幅した部分の約８００ｂｐ全領域に関して塩基配列の決定を行ない実施例１６で解析した塩基配列（配列番号４の１−７８０）と完全に一致していることを確認した。
このプラスミドクローンをｐＨＴ１−２３１と命名した。大腸菌ＤＨ５に担持されたべクターｐＨＴ１−２３１は１９９４年２月１４日付で通商産業省工業技術院生命工学工業技術研究所に受託番号ＦＥＲＭ ＢＰ−４５６４として寄託した。これはまた、１９９５年３月２２日付で中華民国 食品工業発展研究所（ＦＩＲＤＩ）に受託番号９４００８２として寄託した。
得られたプラスミドのＣＯＳ１細胞での発現は実施例１１に従って行なった。すなわちプラスミドＤＮＡ各１０μｇを使用しクロロキン処理を含むＤＥＡＥ−デキストラン法を用いたトランスフェクションを行ない、３日ないし５日後に培養上清を回収した。
得られた培養上清をＩＭＤＭ培養液に対して透析後、ラットＣＦＵ−ＭＫアッセイ系で評価した。その結果、ｐＨＴ１−２３１をトランスフェクションして発現させたＣＯＳ１細胞培養上清ではＴＰＯ活性が認められ（図９）、また、培養４日目に多数の巨核球による突起形成が観察された。一方、ｃＤＮＡインサートを含まない発現プラスミドをトランスフェクションしたＣＯＳ１細胞培養上清では活性が認められなかった（図９）。また、Ｍ−０７ｅアッセイ系においてもｐＨＴ１−２３１をトランスフェクションして発現させたＣＯＳ１細胞培養上清にのみＭ−０７ｅ細胞増殖促進活性が認められた。これらの結果から、ｐＨＴ１−２３１に含まれるｃＤＮＡ断片はＴＰＯ活性を有するタンパク質をコードする遺伝子であることが明らかとなった。
＜実施例１９＞
ＰＣＲによるヒトＴＰＯＣ末側領域のクローニング
実施例１５で得られたクローンＨＬ３４のヒトＴＰＯｃＤＮＡはその３’末端にポリＡ尾部様配列を持っており、３’部分は不完全なクローンであることが示唆された。そこで、ＰＣＲにより完全長の３’領域を取得することを試みた。実施例１６で決定した塩基配列よりＰＣＲ用５’側プライマーを４種類合成した。
【表１０】

また、３’側プライマーとしてはポリＡ部分直後からの増幅を期待して３’端にミックスのヌクレオチドを４ベース含む以下のプライマーを合成した。また、ミックス部分を含まないアンカープライマーも合成した。
【表１１】

実施例１４と同様にヒト正常肝臓由来ポリ（Ａ）^＋ＲＮＡ （Ｃｌｏｎｔｅｃｈ社製）１μｇよりｃＤＮＡの１本鎖目を合成した。ただし、プライマーとしてはオリゴｄＴプライマー（Ｐｈａｒｍａｃｉａ社製のＴｉｍｅＳａｖｅｒ^ＴＭｃＤＮＡ Ｓｙｎｔｈｅｓｉｓ Ｋｉｔに添付のものを０．５μｇ）を使用した。ｃＤＮＡ合成の反応液の０．１容を鋳型として１回目のＰＣＲを実施した。ＰＣＲにはＧｅｎｅＡｍｐ^ＴＭ ＰＣＲ Ｒｅａｇｅｎｔ Ｋｉｔ ｗｉｔｈ ＡｍｐｌｉＴａｑ^ＴＭＤＮＡＰｏｌｙｍｅｒａｓｅ（宝酒造社製）並びに、ＧｅｎｅＡｍｐ^ＴＭ ＰＣＲ Ｓｙｓｔｅｍ ９６００（ＰＥＲＫＩＮ−ＥＬＭＥＲ社製）を用いた。プライマーにはｈＴＰＯ−Ｈ（２０μＭ）及びｈＴＰＯ３ｍｉｘ（１０μＭ）を使用し５０μｌの容量で反応を行なった（９６℃２分間の後に、９６℃１分間／４８℃１分間／７２℃１分間を１０サイクル反応し、さらに７２℃７分間）。１回目の反応液の０．１容を鋳型として２回目のＰＣＲを行なった。プライマーにはｈＴＰＯ−Ｋ（２０μＭ）及びｈＴＰＯ３ｍｉｘ（１０μＭ）を使用し５０μｌの容量で反応を行なった（９６℃２分間の後に、９６℃１分間／６３℃１分間／７２℃１分間を１０サイクル反応し、さらに７２℃７分間）。２回目の反応液の０．１容を鋳型として３回目のＰＣＲを行なった。プライマーにはｈＴＰＯ−Ｎ（２０μＭ）及びｈＴＰＯ３ｍｉｘ（１０μＭ）を使用し５０μｌの容量で反応を行なった（９６℃２分間の後に、９６℃１分間／６３℃１分間／７２℃１分間を１０サイクル反応し、さらに７２℃７分間）。３回目の反応液の０．１容を鋳型として４回目のＰＣＲを行なった。プライマーにはｈＴＰＯ−Ｏ（２０μＭ）及びｈＴＰＯ３ｍｉｘ（１０μＭ）を使用し５０μｌの容量で反応を行なった（９６℃２分間の後に、９６℃１分間／６３℃１分間／７２℃１分間を１０サイクル反応し、さらに７２℃７分間）。４回目の反応液の０．１容を鋳型として５回目のＰＣＲを行なった。プライマーにはｈＴＰＯ−Ｏ（２０μＭ）及びｈＴＰＯ３ａｎｃｈｏｒ（２０μＭ）を使用し５０μｌの容量で反応を行なった（９６℃２分間の後に、９６℃１分間／５８℃１分間／７２℃１分間を１０サイクル反応し、さらに７２℃７分間）。ここで得られた反応液を、２％アガロースゲル（ＦＭＣ ＢｉｏＰｒｏｄｕｃｔｓ社製）を用いた電気泳動にかけ、このＰＣＲ反応の主要産物である約６００ｂｐのＤＮＡ断片を分離し、プレップ−Ａ−ジーンＤＮＡ精製キット（バイオラッド社製）を用いて精製した。この精製したＤＮＡ断片をＴａｑ Ｄｙｅ Ｄｅｏｘｙ^ＴＭ Ｔｅｒｍｉｎａｔｅｒ Ｃｙｃｌｅ Ｓｅｑｕｅｎｉｎｇ Ｋｉｔ （アプライドバイオシステムズ社製）を用いて、アプライドバイオシステムズ社製３７３ＡＤＮＡシークエンサーにより直接シークエンスし塩基配列を決定した。塩基配列およびそれから演鐸されるアミノ酸配列を配列表（配列番号５）に示した。
このＤＮＡ断片はプライマーｈＴＰＯ−Ｏに始まる１３０個のアミノ酸をコードする塩基配列を持っており、さらに３’側に１８０塩基以上の配列が続いていた（５７７塩基目以降は解読できなかった）。このＤＮＡ断片にコードされるグリシンに始まる３０個のアミノ酸配列は配列番号４で記載したアミノ酸番号２０３〜２３２のアミノ酸配列と一致した。同時に１番から９４番目までの塩基配列も配列番号４の６９２〜７８５のものと一致していた。
実施例１７のｃＤＮＡ断片の解析及び本例のＰＣＲ増幅断片の解析の結果より、配列番号６に記載するように、ヒトＴＰＯタンパク質は２１残基のシグナル配列を含む３５３個のアミノ酸からなると推定される。
なお、配列番号６に示されたヒトＴＰＯタンパク質の成熟タンパク質部分に相当するアミノ酸配列を配列番号１６として示した。
＜実施例２０＞
ヒト正常肝臓由来ｃＤＮＡライブラリーの再構築
実施例１５で得られたクローンＨＬ３４は、オープンリーディングフレームに終止コドンを持たず、その３’末端にポリＡ尾部様配列を持っておりｃＤＮＡ合成の際の人工産物であることが考えられたため、新たに市販の正常ヒト肝臓由来ポリ（Ａ）^＋ ＲＮＡ（Ｃｌｏｎｔｅｃｈ社製）５μｇを用い、ｃＤＮＡライブラリーを構築し直した。ｃＤＮＡの合成にはＳｕｐｅｒＳｃｒｉｐｔ^ＴＭ Ｌａｍｂｄａ Ｓｙｓｔｅｍ ｆｏｒｃＤＮＡ Ｓｙｎｔｈｅｓｉｓａｎｄ λＣｌｏｎｉｎｇ Ｋｉｔ並びにＳｕｐｅｒＳｃｒｉｐｔ^ＴＭＩＩ ＲＮａｓｅＨ−（ともにＬＩＦＥＴＥＣＨＮＯＬＯＧＩＥＳ社製）を使用した。ポリ（Ａ）^＋ ＲＮＡを熱変性後、Ｋｉｔに付属のＮｏｔＩ配列付きオリゴｄＴをプライマーとして含む２０μｌの反応液（５０ｍＭ Ｔｒｉｓ−ＨＣｌ、ｐＨ８．３／７５ｍＭＫＣｌ／３ｍＭ ＭｇＣｌ_２／１ｍＭ ＤＴＴ／１ｍＭ ｄＮＴＰ／２００ＵＳｕｐｅｒＳｃｒｉｐｔ^ＴＭＩＩ ＲＮａｓｅＨ−）に加え、３７℃で６０分間保温した。ｃＤＮＡの２本鎖目を合成（２５ｍＭＴｒｉｓ−ＨＣｌ、ｐＨ８．３、１００ｍＭ ＫＣｌ、５ｍＭ ＭｇＣｌ_２、各２５０ μＭ ｄＮＴＰｓ）５ｍＭ ＤＴＴ、４０Ｕ Ｅ，ｃｏｌｉ ＤＮＡ ｐｏｌｙｍｅｒａｓｅ Ｉ、２Ｕ Ｅ．ｃｏｌｉ ＲＮａｓｅＨ）Ｉ０Ｕ Ｅ．ｃｏｌｉ ＤＮＡ ｌｉｇａｓｅを含む１５０μｌの反応液中で１６℃２時間保温）後、１０Ｕ Ｔ４ ＤＮＡ ｐｏｌｙｍａｒａｓｅを加えさらに１６℃で５分間保温した。反応液を６５℃で１０分間加熱後等量のフェノール−クロロホルムで１回抽出し、Ｓ１ｚｅＳｅｐ^ＴＭ ４００ｓｐｕｎ ｃｏｌｕｍｎ（Ｐｈａｒｍａｃｉａ社製のＴｉｍｅＳａｖｅｒ^ＴＭ ｃＤＮＡ Ｓｙｎｔｈｅｓｉｓ Ｋｉｔに付属の低分子量ＤＮＡ除去用スパンカラム）により４００ｂｐより小さいｃＤＮＡを除いた。このサンプルにＥｃｏＲＩ Ａｄａｐｔｏｒ （Ｐｈａｒｍａｃｉａ社製のＴｉｍｅＳａｖｅｒ^ＴＭ ｃＤＮＡ Ｓｙｎｔｈｅｓｉｓ Ｋｉｔに付属のもの）を付加後制限酵素ＮｏｔＩで消化したものを、再度ＳｉｚｅＳｅｐ^ＴＭ ４００ ｓｐｕｎ ｃｏｌｕｍｎにかけ、低分子量のＤＮＡを除去した。これによって得られた、５’端にＥｃｏＲＩ、３’端にＮｏｔＩ認識配列を持つ２本鎖ｃＤＮＡはそれぞれ１．３μｇであり、予めＥｃｏＲＩおよびＮｏｔＩで処理した発現ベクターｐＥＦ１８Ｓと連結させ、９．２ｍｌのコンピテント・ハイＥ．ｃｏｌｉ ＤＨ５ （東洋紡績社製）を形質転換した。その結果、得られたヒト肝臓ｃＤＮＡライブラリー（ｈＴＰＯ−Ｆ１）は１．０×１０_６個の形質転換体を含むものであった。
＜実施例２１＞
ヒト肝臓ｃＤＮＡライブラリーｈＴＰＯ−Ｆ１からのＴＰＯｃＤＮＡクローンのスクリーニング
配列表−配列番号３及び６をもとにヒトＴＰＯｃＤＮＡに対応するＰＣＲ用プライマーを合成した。配列は以下の通り。
ｈＴＰＯ−Ｉ ：５’−ＴＴＧＴＧＡＣＣＴＣＣＧＡＧＴＣＣＴＣＡＧ−３’（配列番号３の６０−８０）
ｈＴＰＯ−ＫＵ：５’−ＡＧＧＡＴＧＧＧＴＴＧＧＧＧＡＡＧＧＡＧＡ−３’（配列番号６の９０１−９２１に対応するアンチセンス）
実施例２０において構築したヒト肝臓ｃＤＮＡライブラリ−ｈＴＰＯ−Ｆ１（１．０×１０^６個）を３つのプール（＃１〜３）に分割し凍結保存した。それぞれのプールよりプラスミドＤＮＡを調製し、その１％を鋳型として、合成したプライマー（ｈＴＰＯ−Ｉ及びｈＴＰＯ−ＫＵ）各１μＭを用いてＰＣＲを行なった。
ＧｅｎｅＡｍｐ^ＴＭ ＰＣＲ Ｒｅａｇｅｎｔ Ｋｉｔ ｗｉｔｈ ＡｍｐｌｉＴａｑ^ＴＭＤＮＡ Ｐｏｌｙｍｅｒａｓｅ （宝酒造社製）を使用して、ＧｅｎｅＡｍｐ^ＴＭ ＰＣＲ Ｓｙｓｔｅｍ ９６００（ＰＥＲＫＩＮ−ＥＬＭＥＲ社製）により１００μ１の容量でＰＣＲ（９５℃で１分間の変性条件、５９℃で１分間のアニール条件、７２℃で１分間の合成条件で３５回の反応を行い、さらに７分間７２℃でインキュベート）を行った結果、＃３のプール由来のプラスミドＤＮＡを用いた場合に予想される大きさのＤＮＡが増幅された。そこで、この＃３のプールを１５０００個のサブプールに分割し、５０μｇ／ｍｌのＡｍｐｉｃｉｌｌｉｎを含む１ｍｌ（７）ＬＢ培地中で１夜培養した後、プラスミド自動分離装置ＰＩ−１００を用いてプラスミドＤＮＡを抽出した。抽出したＤＮＡはＴＥ溶液に溶解し、その５％を鋳型としてＰＣＲを実施した（使用したプライマー、反応条件は上記と同一である）。その結果、９０プール中６プールで予想される大きさのＤＮＡが増幅された。これらのうち１プールを選択し１０００個のクローンを１つの集団とするサブプールに分割し、上記と同様にプラスミドＤＮＡを調製しＰＣＲを行なったがＤＮＡの増幅は観察されなかった。一連のＰＣＲで増幅されるＤＮＡの電気泳動上のバンドの濃さは、サブプールを細かくして行くに連れ、薄くなってきた。そのため、求めるクローンの増殖が良くないためプラスミドＤＮＡの回収が悪く、ＰＣＲによる増幅が弱くなり最終的に増幅されなくなったことが考えられた。そこで＃３のプールに戻り、コロニーハイブリダイゼイションによるスクリーニングを行なうことにした。
＃３のプールより、１５ｃｍのＬＢプレートあたり４１００個のコロニーとなるようにクローンをまき、１００枚のプレートを作製した。それぞれのプレートについて１枚づつレプリカプレートを作製し、それらのうち片方のプレートを３７℃でさらに６時間培養してからコロニーを回収しプラスミドＤＮＡを抽出した。これらのＤＮＡについて上記と同様のＰＣＲを実施したところ、１００プール中１プールで予想される大きさのバンドの増幅が観察された。そこでこのプールのプレートより２枚のレプリカフィルターを作製した（ＰＡＬＬ社製 ＢＩＯＤＹＮＥ^ＴＭＡ ＴＲＡＮＳＦＥＲ ＭＥＭＢＲＡＮＥを使用）。フィルターの変性は１０％ＳＤＳ１０分間、０．５Ｎ ＮａＯＨ／１．５Ｍ ＮａＣｌ １０分間、０．５Ｍ Ｔｒｉｓ−ＨＣｌ（ｐＨ８，０）／１．５Ｍ ＮａＣｌ １０分間行ない、３０分間風乾後８０℃の真空オーブン中で１時間ベーキングした。ベーキングしたフィルターは６×ＳＳＣ （２０ＸＳＳＣは１１中に１７５．３ｇＮａＣｌ、８８．２ｇを含みｐＨ７．０の溶液）／１％ＳＤＳで軽く洗浄後、プレハイブリダイゼイションを行なった。反応液の組成は５０％ホルムアミド、５×ＳＳＣ、 ５×Ｄｅｎｈａｒｄｔ’ｓ ｓｏｌｕｔｉｏｎ （５０×Ｄｅｎｈａｒｄｔ’ｓ ｓｏｌｕｔｉｏｎは５００ｍｌ中に５ｇ Ｆｉｃｏｌｌ、５ｇｐｏｌｙｖｉｎｙｌｐｙｒｒｏｌｉｄｏｎｅ、５ｇ ｂｏｖｉｎｅ ｓｅｒｕｍ ａｌｂｕｍｉｎｆｒａｃｔｉｏｎ Ｖを含む溶液）、１％ＳＤＳ、２０μｇ／ｍｌサケ精子ＤＮＡを含むもので、３０ｍｌの溶液中で４２℃３０分間振盪しながら保温した。プレハイブリダイゼイションを行なった後、同組成のハイブリダイゼイション溶液３０ｍｌと交換後、［α−^３２Ｐ］ｄＣＴＰ（アマシャム社製）で放射標識したプローブ（プラスミドｐＥＦ１８Ｓ−ＨＬ３４のＥｃｏＲＩ／ＢａｍＨＩ断片：配列番号４の５’末端から４５８塩基目までを精製し、ランダムプライマー法で標識したもの；Ａｎａｌ． Ｂｉｏｃｈｅｍ．、１３２、６−１３、１９８３に記載の方法を利用したアマシャム社製キットＭｅｇａｐｒｉｍｅ ＤＮＡ ｌａｂｅｌｌｉｎｇ ｓｙｓｔｅｍを使用）を加え、４２℃で２０時間振盪しながら保温した。フィルターは２×ＳＳＣ／０．１％ＳＤＳ溶液中で４２℃３０分間洗浄後、さらに０．２×ＳＳＣ／０．１％ＳＤＳ溶液中で４２℃３０分間洗浄した。フィルターを増強スクリーン及びＸ−ＯＭＡＴＴＭＡＲ５フィルム（イーストマンコダック社製）により−７０℃１６時間オートラジオグラフィーを行なった。その結果、陽性と考えられるシグナルが１個観察された。そこで、もとのプレートよりシグナル周辺のコロニーをかきとり、１０ｃｍのＬＢプレートにまきなおした。得られたコロニー５０個を培養しＤＮＡを調製後プライマーｈＴＰＯ−Ｉ及びｈＴＰＯ−ＫＵを用いてＰＣＲを実施した（条件等は上記と同一）。その結果、１クローンのみ予想通りのバンドが増幅された。このクローンをｐＨＴＦ１と命名した。
＜実施例２２＞
ヒトＴＰＯｃＤＮＡクローンｐＨＴＦ１のシークエンス
プラスミドＤＮＡの精製は本質的にＭｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ［Ｓａｍｂｒｏｏｋら、ＣｏｌｄＳｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ Ｐｒｅｓｓ（１９８９）に記載されているようにして実施した。クローンｐＨＴＦ１を５０μｇ／ｍｌ（７）Ａｍｐｉｃｉｌｌｉｎを含む５０ｍｌのＬＢ培地で一夜培養した後、遠心分離により得た菌体を４ｍｌのＴＥＧ−ｌｙｓｏｚｙｍｅ溶液に懸濁し、８ｍｌの０．２ＮＮａＯＨ／１％ＳＤＳ溶液を加えてよく懸濁する。さらに３Ｍ ｐｏｔａｓｓｉｕｍ／５Ｍ ａｃｅｔａｔｅ溶液を６ｍｌ加えてよく懸濁した後遠心し、上清を得る。上清はフェノールークロロホルム（１：１）処理後、等量のイソプロパノールを加えて遠心し、ペレットを得た。ペレットはＴＥ溶液に溶解後、ＲＮＡｓｅ処理、フェノールークロロホルム（１：１）処理を施し、エタノール沈殿を行なう。ペレットを再度ＴＥ溶液に溶解し、ＮａＣｌ、ポリエチレングリコール３０００をそれぞれ０．６３Ｍ、７．５％になるように加えて遠心する。最後に、ペレットはＴＥ溶液に溶解後エタノール沈殿を行なう。これにより約３００μｇのプラスミドＤＮＡｐＨＴＦ１を得た。
精製したプラスミドＤＮＡについてＴａｑ Ｄｙｅ Ｄｅｏｘｙ_ＴＭ Ｔｅｒｍｉｎａｔｅｒ Ｃｙｃｌｅ Ｓｅｑｕｅｎｉｎｇ Ｋｉｔ （アプライドバイオシステムズ社製）を用い、アプライドバイオシステムズ社製３７３ΛＤＮＡシークエンサーによりシークエンスし、全長の塩基配列を決定した。塩基配列およびそれから演鐸されるアミノ酸配列を配列表（配列番号７）に示した。塩基配列決定に必要なプライマーは配列番号６の配列をもとに合成したプライマー並びに、それらのプライマーによるシークエンス反応で解析された内部配列をもとに設計した合成プライマーを使用した。
その結果、得られたクローンｐＨＴＦ１は１７２１塩基対のｃＤＮＡ断片からなり、実施例２で分析されたアミノ酸配列ＡＰ８（配列番号７：アミノ酸番号１〜１２）及びＴＰ２／ＴＰ３（配列番号７：アミノ酸番号１５７〜１６２）と相同性の高い配列が確認された。このＤＮＡ断片は、１０１塩基目までの５’ノンコーディング領域に続く、１０２〜１０４塩基目にコードされるメチオニンに始まり１１５８〜１１６０塩基目にコードされるグリシンまで続く３５３個のアミノ酸からなるオープンリーディングフレームをコードしていると考えられ、それに続く停止コドン（ＴＡＡ）以降の３’ ノンコーディング領域を５３１塩基と３０個のポリＡ尾部配列を持っていた。このオープンリーディングフレームにコードされると考えられるタンパク質のアミノ酸配列は配列番号６に記載の、ヒトＴＰＯの予想されるアミノ酸配列と完全に一致した。塩基配列は配列番号６の配列より５’側で７７塩基分、３’側のポリＡ尾部配列直前までの領域で３４７塩基分長いｃＤＮＡをコードしていた。また塩基配列は、配列番号６と３箇所で異なっていた。異なる塩基配列は配列番号７のＡ（８４塩基目）が配列番号６ではＣ、Ａ（７４０塩基目）がＴ、Ｇ（１１９８塩基目）がＡであった。２番目の塩基（７４０塩基目）のみ、タンパク質のコーディング領域内の変異であったが、スレオニンの３番目のコドンであり、アミノ酸の変換は起こさなかった。この塩基配列の置換が何に起因するのかはこの時点では明らかでなかったが、得られたクローンｐＨＴＦ１の解析より、ヒトＴＰＯタンパク質は２１残基のシグナル配列を含む３５３個のアミノ酸からなることが確認できた。シグナル配列を除いたタンパク質の推定分子量は３５４６６であった。
大腸菌ＤＨ５に担持されたベクターｐＨＴＦ１は１９９４年３月２４日付けで通商産業省工業技術院生命工学工業技術研究所に受託番号ＦＥＲＭ ＢＰ−４６１７として寄託した。これはまた、１９９５年３月２２日付で中華民国 食品工業発展研究所（ＦＩＲＤＩ）に受託番号９４００８５として寄託した。
＜実施例２３＞
ヒトＴＰＯｃＤＮＡクローンｐＨＴＦ１のＣＯＳ１細胞での発現〜活性確認
得られたクローンｐＨＴＦ１のＣＯＳ１細胞へのトランスフェクションは、実施例１１に従って行なった。すなわちプラスミドＤＮＡ１０μｇを使用しクロロキン処理を含むＤＥＡＥ−デキストラン法を用いたトランスフェクションを行ない、３日後に培養上清を回収した。
得られた培養上清をＩＭＤＭ培養液に対して透析後、ラットＣＦＵ−ＭＫアッセイ系で評価した。その結果、ｐＨＴＦ１を発現させたＣＯＳ１細胞培養上清では用量依存的にＴＰＯ活性が認められたが、ｃＤＮＡインサートを含まない発現プラスミドをトランスフェクションしたＣＯＳ１培養上清では活性が認められなかった（図１０ａ）。類似の結果は、Ｍ−０７ｅアッセイ系においても得られ、ｐＨＴＦ１を発現させたＣＯＳ１細胞培養上清中に有意なＭ−０７ｅ細胞増殖促進活性を認めた（図１０ｂ）。
これらの結果から、ｐＨＴＦ１に含まれるｃＤＮＡはＴＰＯ活性を有するタンパク質をコードする遺伝子であることが明確になった。
＜実施例２４＞
ヒトＴＰＯ染色体ＤＮＡのクローニング
ヒトＴＰＯｃＤＮＡをプローブとしてヒトＴＰＯ染色体ＤＮＡのクローニングを行なった。クローニングに使用したゲノミックライブラリーは東北大学 遺伝子実験施設山本徳男教授よりいただいた（Ｓａｋａｉら、Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．、２６９、２１７３−２１８２、１９９４に記載のライブラリーで、ヒト染色体ＤＮＡを制限酵素Ｓａｕ３ＡＩによって部分分解したものをＳｔａｔａｇｅｎｅ社製ファージベクターＬａｍｂｄａ ＥＭＢＬ３のＢａｍＨＩ部位につないで構築したライブラリー）。このライブラリーよりヒトＴＰＯｃＤＮＡをプローブとしてスクリーニングを実施したが、用いた手法はすべて本質的にＭｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ［Ｓａｍｂｒｏｏｋら、Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏ Ｌａｂｏｒａｔｏｒｙ Ｐｒｅｓｓ（１９８９）］に記載されているようにして実施した。大腸菌ＬＥ３９２を宿主菌として、１５ｃｍのＮＺＹＭプレート（１ｌ中に１０ｇ ＮＺａｍｉｎｅ、 ５ｇ ＮａＣｌ、５ｇ ｂａｃｔｏ ｙｅａｓｔ ｅ ｘｔｒａｃｔ、 ２ｇ ＭｇＣｌ_４ ７Ｈ_２Ｏ、１５％ ａｇａｒを含むｐＨ７．０のもの）１枚あたり３万個となるようにファージをまき、１８枚のプレートを作製した。それぞれのプレートより各２枚のレプリカフィルターを作製した（ＰＡＬＬ社製 ＢＩＯＤＹＮＥ^ＴＭ Ａ ＴＲＡＮＳＦＥＲＭＥＭＢＲＡＮＥを使用）。フィルターの変性は０．５Ｎ ＮａＯＨ／１．５Ｍ ＮａＣｌ１０分間、０．５ＭＴｒｉｓ−ＨＣｌ（ｐＨ８．０）／１．５Ｍ ＮａＣｌ １０分間行ない、３０分間風乾し８０℃の真空オーブン中で１．５時間ベーキングした。プレハイブリダイゼイションは５０％ホルムアミド、５×ＳＳＣ、 ５×Ｄｅｎｈａｒｄｔ’ｓ ｓｏｌｕｔｉｏｎ、１％ＳＤＳ、２０μｇ／ｍｌサケ精子ＤＮＡを含む５００ｍｌの溶液中で４２℃１時間行なった。プローブはヒトＴＰＯｃＤＮＡ断片（配列番号７の塩基配列番号１７８−１０２５）をＰＣＲで増幅後精製したものをＲａｎｄｏｍ Ｐｒｉｍｅｒ ＤＮＡ ｌａｂｅｌｌｉｎｇ ｋｉｔ （宝酒造社製；Ａｎａｌ．Ｂｉｏｃｈｅｍ．、１３２、６−１３、１９８３に記載のランダムプライマー法を利用したＤＮＡ標識キット）を用いて_３２Ｐ標識したものを用いた。ＰＣＲに使用したプライマーの配列は以下の通り。
ｈＴＰＯ−Ｉ：５’−ＴＴＧＴＧＡＣＣＴＣＣＧＡＧＴＣＣＴＣＡＧ−３’ （配列番号７の１７８−１９８）
ｈＴＰＯ−Ｎ：５’−ＡＧＧＧＡＡＧＡＧＣＧＴＡＴＡＣＴＧＴＣＣ−３’ （配列番号７の１００５−１０２５に対応するアンチセンス）
ハイブリダイゼイションはプレハイブリダイゼイションと同じ組成の溶液を５００ｍｌ使用し、放射標識したプローブを加え４２℃で２０時間行なった。フィルターは２×ＳＳＣ／０．１％ＳＤＳ溶液中室温で５分間づつ３回洗浄後、０．１×ＳＳＣ／０．１％ＳＤＳ溶液中で６８℃１時間の洗浄を行なったのち、増強スクリーン及びＸ−ＯＭＡＴ^ＴＭ ＡＲ５フィルム（イーストマンコダック社製）により−７０℃１６時間オートラジオグラフィーを行なった。その結果、１３個の陽性シグナルが得られた。もとのプレートより１３個の陽性シグナル周辺のプラークを拾い上げ、１５ｃｍのＮＺＹＭプレート１枚あたり１０００プラークとなるようにまき直した。それぞれのプレートから各２枚のレプリカフィルターを作製し、上記と同じ条件でハイブリダイゼイションを実施した。その結果、１３組全てのフィルターで陽性シグナルが検出された。各プートより１個づつプラークを単離し、Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇに記載のＰｌａｔｅ Ｌｙｓａｔｅ法でファージＤＮＡを調製した。１３クローンのファージＤＮＡについてｃＤＮＡのコーディング領域を含んでいるかどうかをＰＣＲによってチェックした。チェックに使用したプライマーの配列は以下の通り。
ｈＴＰＯ−Ｌ：５’−ＧＧＣＣＡＧＣＣＡＧＡＣＡＣＣＣＣＧＧＣＣ−３’ （配列番号６の１−２１）
ｈＴＰＯ−Ｆ：５’−ＡＴＧＧＧＡＧＴＣＡＣＧＡＡＧＣＡＧＴＴＴ−３’ （配列番号６の１２７−１４７に対応するアンチセンス）
ｈＴＰＯ−Ｐ：５’−ＴＧＣＧＴＴＴＣＣＴＧＡＴＧＣＴＴＧＴＡＧ−３’ （配列番号６の５０３−５２３）
ｈＴＰＯ−Ｖ：５’−ＡＡＣＣＴＴＡＣＣＣＴＴＣＣＴＧＡＧＡＣＡ−３’ （配列番号６の１０７０−１０９０に対応するアンチセンス）
これらのプライマーの組み合わせにてＰＣＲを実施したところ、１３クローンのうち５クローンはｃＤＮＡから予測されるアミノ酸のコーディング領域を全て含んでいると考えられた。これら５クローンに含まれる染色体ＤＮＡの長さは約２０ｋｂｐとそろっており、予備的に検討した制限酵素解析でもほぼ同一のパターンを示した。そこでこれらのクローンのうち１個（クローンλＨＧＴ１）を選択し、Ｓｏｕｔｈｅｒｎ ｂｌｏｔ解析を行なった。すなわち、クローンλＨＧＴ１のＤＮＡ各１μｇを制限酵素ＥｃｏＲＩまたはＨｉｎｄＩＩＩで完全に消化し、０．８％アガロースゲルで泳動後、ＰＡＬＬ社製 ＢＩＯＤＹＮＥ^ＴＭ Ａ ＴＲＡＮＳＦＥＲ ＭＥＭＢＲＡＮＥに転写した。フィルターは３０分間風乾後８０℃の真空オーブン中で２時間ベーキングした。プレハイブリダイゼイションは５０％ホノレムアミド、５×ＳＳＣ、 ５×Ｄｅｎｈａｒｄｔ’ ｓ ｓｏｌｕｔｉｏｎ、１％ＳＤＳ、２０μｇ／ｍｌサケ精子ＤＮＡを含む５０ｍｌの溶液中で４２℃１時間行なった。プローブはヒトＴＰＯｃＤＮＡ断片（配列番号７の塩基配列番号１７８−１０２５）をＰＣＲで増幅後精製したものをＲａｎｄｏｍＰｒｉｍｅｒ ＤＮＡ ｌａｂｅｌｌｉｎｇ ｋｉｔ （宝酒造社製）を使用して^３２Ｐ標識したものを用いた。ハイブリダイゼイションはプレハイブリダイゼイションと同じ組成の溶液を５０ｍｌ使用し、放射標識したプローブを加え４２℃で２０時間行なった。フィルターは２×ＳＳＣ／０．１％ＳＤＳ溶液中室温で５分間づつ３回洗浄後、０．１×ＳＳＣ／０．１％ＳＤＳ溶液中で６８℃１時間の洗浄を行なったのち、増強スクリーン及びＸ−ＯＭＡＴＴＭＡＲ５フィルム（イーストマンコダック社製）により−７０℃１６時間オートラジオグラフィーを行なった。その結果、制限酵素ＨｉｎｄＩＩＩで消化した場合に約１０ｋｂｐの単一のバンドが観察された。そこで、クローンλＨＧＴＩのＤＮＡ１０μｇを制限酵素ＨｉｎｄＩＩＩで消化後０．８％アガロースゲルで泳動し、１０ｋｂｐのバンドを切り出して、プレップ−Ａ−ジーンＤＮＡ精製キット（バイオラッド社製）で精製し、同様にＨｉｎｄＩＩＩで消化したクローニングベクタ−ｐＵＣ１３（Ｐｈａｒｍａｃｉａ社製）にサブクローニングした（宿主菌は東洋紡績社製コンピテント・ハイＥ．ｃｏｌｉ ＤＨ５を使用した）。
得られたクローンのうち、１０ｋｂｐのＨｉｎｄＩＩＩ断片を含むクローンを選択しｐＨＧＴ１と命名した。
ファージクローンλＨＧＴ１のおおまかな制限酵素地図を図１１に示す。
＜実施例２５＞
ヒトＴＰＯ染色体クローンｐＨＧＴ１のシークエンス
ｐＨＧＴ１クローンを培養しプラスミドＤＮＡの精製を行なった。方法は本質的にＭｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ［Ｓａｍｂｒｏｏｋら、Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ Ｐｒｅｓｓ（１９８９）］に記載されているようにして実施した。クローンｐＨＧＴ１を５０μｇ／ｍｌのＡｍｐｉｃｉｌｌｉｎを含む５０ｍｌのＬＢ培地で一夜培養した後、遠心分離により得た菌体を４ｍｌのＴＥＧ−ｌｙｓｏｚｙｍｅ溶液に懸濁し、８ｍｌの０．２Ｎ ＮａＯＨ／１％ＳＤＳ溶液を加えてよく懸濁する。さらに３Ｍ ｐｏｔａｓｓｉｕｍ／５Ｍ ａｃｅｔａｔｅ溶液を６ｍｌ加えてよく懸濁した後遠心し、上清を得る。上清はフェノールークロロホルム（１：１）処理後、等量のイソプロパノールを加えて遠心し、ペレットを得た。ペレットはＴＥ溶液に溶解後、ＲＮａｓｅ処理、フェノールークロロホルム（１：１）処理を施し、エタノール沈殿を行なう。ペレットを再度ＴＥ溶液に溶解し、ＮａＣｌ、ポリエチレングリコール３０００をそれぞれ０．６３Ｍ、７．５％になるように加えて遠心する。最後に、ペレットはＴＥ溶液に溶解後エタノール沈殿を行なう。これにより約３００μｇのプラスミドＤＮＡｐＨＧＴ１を得た。
精製したプラスミドＤＮＡについてＴａｑ Ｄｙｅ Ｄｅｏｘｙ^ＴＭ Ｔｅｒｍｉｎａｔｅｒ Ｃｙｃｌｅ Ｓｅｑｕｅｎｉｎｇ Ｋｉｔ （アプライドバイオシステムズ社製）を用い、アプライドバイオシステムズ社製３７３ＡＤＮＡシークエンサーによりシークエンスし、ｃＤＮＡの塩基配列から予想される、タンパク質コーディング領域周辺の塩基配列を決定した。塩基配列およびそれから演鐸されるアミノ酸配列を配列表（配列番号８）に示した。
塩基配列決定に必要なプライマーは実施例２２で使用した、ｃＤＮＡの塩基配列解析に用いたもの並びに、それらのプライマーによるシークエンス反応で解析された内部配列をもとに設計した合成プライマーを使用した。
その結果、プラスミドクローンｐＨＧＴ１が担持する染色体ＤＮＡは、配列番号６で予想されたアミノ酸のコーディング領域を全て含みその領域に関しては、塩基配列は完全に一致していた。また、アミノ酸をコードするエクソンに相当する領域は４つのイントロンによって分断されており、イントロンの長さは５’側から順に２３１ｂｐ、 ２８６ｂｐ、１９３２ｂｐ、２３６ｂｐであった。配列番号７に記載のｃＤＮＡ塩基配列とは３箇所で異なっていた。異なる塩基配列は実施例２２で記載した箇所（配列番号６と７の相違点）と同一であり、配列番号７のＡ（８４塩基目）がＣ、Ａ（７４０塩基目）がＴ、Ｇ（１１９８塩基目）がＡであった。すなわち、実施例２１で取得したヒトＴＰＯｃＤＮＡクローンｐＨＴＦ１の塩基配列は、ヒト染色体の塩基配列と３箇所で異なることが明かとなった。そこで、この塩基配列の変異が本来の配列を反映するものであるか解析するために、スクリーニングで最終的に選択された５個の染色体ＤＮＡクローンのうち、塩基配列を決定していない残りの４クローンについて、配列の決定を行なった。配列の解析はＭｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇに記載のＰｌａｔｅ Ｌｙｓａｔｅ法で調製したファージＤＮＡを使用した直接塩基配列決定法で行なった。反応には上記３箇所の塩基配列変異点を解析できる位置の実施例２２で合成したシークエンスプライマー並びに、Ｔａｑ Ｄｙｅ Ｄｅｏｘｙ^ＴＭ Ｔｅｒｍｉｎａｔｅｒ Ｃｙｃｌｅ Ｓｅｑｕｅｎｉｎｇ Ｋｉｔ （アプライドバイオシステムズ社製）を用い、アプライドバイオシステムズ社製３７３ＡＤＮＡシークエンサーによりシークエンスした。その結果、４個全てのクローンで配列番号７の８４塩基目がＣ、７４０塩基目がＴであり、配列番号７のｃＤＮＡとは異なり、配列番号６のものと一致した。１１９８塩基目についてはＧのクローンが２個、Ａのクローンが２個であった。すなわち、染色体ＤＮＡ本来の塩基配列が２種類あることが明かとなった。この配列の違いが相同染色体に由来するものか、複数存在する遺伝子に由来するものかは、現時点では不明である。また、購入したＣｌｏｎｔｅｃｈ社製ポリ （Ａ）_＋ＲＮＡがＣａｕｃａｓｉａｎ由来であるのに対し、染色体ＤＮＡの由来が日本人であることから、塩基配列の変異は人種の違いに起因する可能性も示唆される。
大腸菌ＤＨ５に担持されたベクターｐＨＧＴ１は１９９４年３月２４日付けで通商産業省工業技術院生命工学工業技術研究所に受託番号ＦＥＲＭ ＢＰ−４６１６として寄託した。これはまた、１９９５年３月２２日付で中華民国 食品工業発展研究所（ＦＩＲＤＩ）に受託番号９４００８４として寄託した。
＜実施例２６＞
ヒトＴＰＯ染色体ＤＮＡのＣＯＳ１細胞での発現と活性確認
サブクローニングして得られたプラスミドクローンｐＨＧＴ１には、インサート部分に３箇所、ベクター内に１箇所、合計４箇所のＥｃｏＲＩ認識配列が存在するが、インサート内の一番５’側のＥｃｏＲＩ認識配列とベクター内のＥｃｏＲＩ認識配列にはさまれる約４．３ｋｂｐのＤＮＡ断片に、ヒトＴＰＯタンパク質のコーディング領域が全て含まれることが、塩基配列の解析から明らかになった。そこで、この断片をＥｃｏＲＩ処理した発現ベクターｐＥＦ１８Ｓにつなぎ、４個のヒトＴＰＯ発現プラスミドｐＥＦＨＧＴＥ＃１−４を得た（前述の図１１）。これらのプラスミドＤＮＡを調製し発現実験を行なった。プラスミドＤＮＡの精製は本質的にＭｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ ［Ｓａｍｂｒｏｏｋら、Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ Ｐｒｅｓｓ （１９８９）］に記載されているようにして実施し、約２５０μｇのプラスミドＤＮＡを得た。
得られたクローンｐＥＦＨＧＴＥ＃１−４のＣＯＳ１細胞へのトランスフェクションは、実施例１１に従って行なった。すなわちプラスミドＤＮＡ１０μｇを使用しクロロキン処理を含むＤＥＡＥ−デキストラン法を用いたトランスフェクションを行ない、３日後に培養上清を回収した。
得られた培養上清をＩＭＤＭ培養液に対して透析後、ラットＣＦＵ−ＭＫアッセイ系で評価した。その結果、ｐＥＦＨＧＴＥを発現させたＣＯＳ１細胞培養上清中に用量依存的にＴＰＯ活性が認められ、一方、ＤＮＡインサートを含まない発現プラスミドをトランスフェクションしたＣＯＳ１細胞培養上清では活性が認められなかった。クローン＃１〜４のいずれでもほぼ同様の結果が得られたが、代表例としてｐＥＦＨＧＴＥ＃１における結果を図１２ａに示した。また、Ｍ−０７ｅアッセイ系においても、ｐＥＦＨＧＴＥを発現させたＣＯＳ１細胞培養上清にて用量依存的に有意なＭ−０７ｅ細胞増殖促進活性が認められた。代表例として、ｐＥＦＨＧＴＥ＃１における結果を図１２ｂに示した。
これらの結果より、得られたクローンｐＥＦＨＧＴＥが担持するＤＮＡはヒトＴＰＯの機能的染色体ＤＮＡであることが、明らかとなった。
＜実施例２７＞
ヒトＴＰＯ欠失誘導体の作製とＣＯＳ１細胞での発現〜活性確認
実施例１８の結果より、ヒトＴＰＯは完全長でなくても活性を発現しうることが明かとなったが、その活性発現に必要な領域を解析する目的で、欠失誘導体の作製並びに発現を行なった。実施例１８で得たプラスミドクローンｐＨＴ１−２３１をもとにＣ末端側から順次２０個のアミノ酸を欠失させた発現プラスミド並びにＴＰ２／３の直後（１６３番目）までのアミノ酸を含む発現プラスミドを作製した。作製にはＰＣＲを利用した。ＰＣＲ用に作製したプライマーの配列は以下の通り。
ｈＴＰＯ−５：５’−ＴＴＴＧＡＡＴＴＣＧＧＣＣＡＧＣＣＡＧＡＣＡＣＣＣＣＧＧＣＣ−３’
（配列番号４の１−２１にＥｃｏＲＩ配列を付加したもの；表９に記載のものと同一）
ｈＴＰＯ−Ｓ：５’−ＴＴＴＧＣＧＧＣＣＧＣＴＣＡＴＴＡＧＣＴＧＧＧＧＡＣＡＧＣＴＧＴＧＧＴＧＧＧＴ−３’（配列番号４の５５５−５７６のアンチセンス；２個の終止コドンＴＡＡＴＧＡ及びＮｏｔＩ配列を付加；アミノ酸１−１６３をコードする欠失誘導体作製用）
ｈＴＰＯ−４：５’ −ＴＴＴＧＣＧＧＣＣＧＣＴＣＡＴＴＡＣＡＧＴＧＴＧＡＧＧＡＣＴＡＧＡＧＡＧＧＴＴＣＴＧ−３’ （配列番号４の５７６−６００のアンチセンス；２個の終止コドンＴＡＡＴＧＡ及びＮｏｔＩ配列を付加；アミノ酸１−１７１をコードする欠失誘導体作製用）
ｈＴＰＯ−３０：５’ −ＴＴＴＧＣＧＧＣＣＧＣＴＣＡＴＴＡＴＣＴＧＧＣＴＧＡＧＧＣＡＧＴＧＡＡＧＴＴＴＧＴＣ−３’ （配列番号４の６３６−６６０のアンチセンス；２個の終止コドンＴＡＡＴＧＡ及びＮｏｔＩ配列を付加；アミノ酸１−１９１をコードする欠失誘導体作製用）
ｈＴＰＯ−２：５’−ＴＴＴＧＣＧＧＣＣＧＣＴＣＡＴＴＡＣＡＧＡＣＣＡＧＧＡＡＴＣＴＴＧＧＣＴＣＴＧＡＡＴ−３’ （配列番号４の６９６−７２０のアンチセンス；２個の終止コドンＴＡＡＴＧＡ及びＮｏｔＩ配列を付加；アミノ酸１−２１１をコードする欠失誘導体作製用）
実施例１８で得たクローンｐＨＴ１−２３１のプラスミドＤＮＡ１μｇを鋳型として使用し、合成したプライマー（ｈＴＰＯ−５を５’側として使用、ｈＴＰＯ−２、−３、−４、−Ｓを３’側として使用）をそれぞれ１０μＭ使ってＰＣＲを実施した。ＧｅｎｅＡｍｐ^ＴＭＰＣＲ Ｒｅａｇｅｎｔ Ｋｉｔ ｗｉｔｈＡｍｐｌｉＴａｑ^ＴＭ ＤＮＡ Ｐｏｌｙｍｅｒａｓｅ（宝酒造社製）を用いて、ＧｅｎｅＡｍｐ^ＴＭＰＣＲ Ｓｙｓｔｅｍ ９６００（ＰＥＲＫＩＮ−ＥＬＭＥＲ社製）により１００μｌの容量でＰＣＲ（９５℃で１分間の変性条件、６６℃で１分間のアニール条件、７２℃で１分間の合成条件で２０回の反応を行い、さらに７分間７２℃でインキュベート）を行った。それぞれのＰＣＲで得られたバンドを制限酵素ＥｃｏＲＩ及びＮｏｔＩで消化後、１％アガロースゲル（ＦＭＣ ＢｉｏＰｒｏｄｕｃｔｓ社製）を用いた電気泳動にかけ、それぞれのＰＣＲ反応の予想される大きさの主要なＤＮＡ断片を分離し、プレップ−Ａ−ジーンＤＮＡ精製キット（バイオラッド社製）で精製し、同様に制限酵素処理した発現ベクターｐＥＦ１８Ｓにサブクローニングした（宿主菌としては東洋紡績社製コンピテント・ハイ Ｅ．ｃｏｌｉ ＤＨ５を使用）。得られた形質転換体のうち、それぞれ予想される長さのインサートを含むクローンをそれぞれ４個ないし５個づつ選択し、プラスミドＤＮＡを調製した。方法は本質的にＭｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ［Ｓａｍｂｒｏｏｋら、Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ ＬａｂｏｒａｔｏｒｙＰｒｅｓｓ（１９８９月に記載されているようにして実施した。一連の操作によってアミノ酸１−１６３をコードする欠失誘導体（ｐＨＴ１−１６３＃１−５）、アミノ酸１−１７１をコードする欠失誘導体（ｐＨＴ１−１７１＃１−４）、アミノ酸１−１９１をコードする欠失誘導体（ｐＨＴ１−１９１＃１−４）、アミノ酸１−２１１をコードする欠失誘導体（ｐＨＴ１−２１１＃１−４）のプラスミドＤＮＡを得た。
精製したプラスミドＤＮＡについてはＴａｑ Ｄｙｅ Ｄｅｏｘｙ^ＴＭ Ｔｅｒｍｉｎａｔｅｒ Ｃｙｃｌｅ Ｓｅｑｕｅｎｉｎｇ Ｋｉｔ （アプライドバイオシステムズ社製）を用い、アプライドバイオシステムズ社製３７３ＡＤＮＡシークエンサーによりシークエンスし、全長にわたり塩基配列の置換がなく、予想通りのＴＰＯｃＤＮΛ配列を持つことを確認した。
得られたそれぞれのクローンのＣＯＳ１細胞へのトランスフェクションは、実施例１１に従って行なった。すなわちプラスミドＤＮＡ各１０μｇを使用しクロロキン処理を含むＤＥＡＥ−デキストラン法を用いたトランスフェクションを行ない、３日後に培養上清を回収した。培養上清をＩＭＤＭ培養液に対して透析後、ラットＣＦＵ−ＭＫアッセイ系で評価した。その結果、ｐＨＴ１−２１１、ｐＨＴ１−１９１、ｐＨＴ１−１７１、およびｐＨＴ１−１６３を発現させたいずれのＣＯＳ１細胞培養上清でも用量依存的にＴＰＯ活性を認めた。代表例としてｐＨＴ１−２１１＃１、ｐＨＴ１−１９１＃１、およびｐＨＴ１−１７１＃２における結果を図１３ａに、ｐＨＴ１−１６３＃２における結果を図１３ｂに示した。類似の結果は、Ｍ−０７ｅアッセイ系においても得られ、ｐＨＴ１−２１１、ｐＨＴ１−１９１、ｐＨＴ１−１７１、およびｐＨＴ１−１６３を発現させたいずれのＣＯＳ１細胞培養上清でも用量依存的に有意なＭ−０７ｅ細胞増殖促進活性を認めた。代表例として、ｐＨＴ１−２１１＃１、ｐＨＴ１−１９１＃１、ｐＨＴ１−１７１＃２、およびｐＨＴ１−１６３＃２における結果を図１４に示した。
これらの結果より、ヒトＴＰＯタンパク質を構成するアミノ酸３５３個（２１個のシグナル配列を含む）のうち、１６４位のアルギニン以降のアミノ酸を欠失させても、ｉｎ ｖｉｔｒｏにおける活性が保持されることが明らかとなり、１６４位のアルギニンより前に、ＴＰＯ活性発現のために必須な領域が含まれていることが示唆された。
＜実施例２８＞
ヒトＴＰＯ Ｃ末側欠失体の作製とＣＯＳ１細胞での発現〜活性確認
ヒトＴＰＯ蛋白質の活性発現に必須な領域の解析を行なう目的で、実施例２７で得られた欠失体よりもさらにＣ末側アミノ酸を欠失させた誘導体を作製しＴＰＯ活性を発現しうるかどうか検討した。実施例１６で得たプラスミドクローンｐＥＦ１８Ｓ−ＨＬ３４をもとに、１６３番目のセリンを基準にＣ末端側から順次ァミノ酸を欠失させた発現プラスミドを構築した。構築にはＰＣＲを利用した。ＰＣＲ用に作製したプライマーの配列は、次の通りである。
ｈＴＰＯ−５：５’−ＴＴＴＧＡＡＴＴＣＧＧＣＣＡＧＣＣＡＧＡＣＡＣＣＣＣＧＧＣＣ−３’
（配列番号４の１−２１にＥｃｏＲＩ配列を付加したもの；表９に記載のものと同一）
ｈＴＰＯ−１５０：５’−ＴＴＴＧＣＧＧＣＣＧＣＴＣＡＴＴＡＧＡＧＧＧＴＧＧＡＣＣＣＴＣＣＴＡＣＡＡＧＣＡＴ−３’
（配列番号４の５１４−５３７のアンチセンス；２個の終止コドンＴＡＡＴＧＡ及びＮｏｔＩ配列を付加：アミノ酸１−１５０をコードする欠失体作製用）
ｈＴＰＯ−１５１：５’−ＴＴＴＧＣＧＧＣＣＧＣＴＣＡＴＴＡＧＣＡＧＡＧＧＧＴＧＧＡＣＣＣＴＣＣＴＡＣＡＡ−３’
（配列番号４の５１８−５４０のアンチセンス；２個の終止コドンＴＡＡＴＧＡ及びＮｏｔＩ配列を付加；アミノ酸１−１５１をコードする欠失体作製用）
ｈＴＰＯ−１５３：５’−ＴＴＴＧＣＧＧＣＣＧＣＴＣＡＴＴＡＣＣＴＧＡＣＧＣＡＧＡＧＧＧＴＧＧＡＣＣＣ−３’
（配列番号４の５２６−５４６のアンチセンス；２個の終止コドンＴＡＡＴＧＡ及びＮｏｔＩ配列を付加；アミノ酸１−１５３をコードする欠失体作製用）
ｈＴＰＯ−１５４：５’−ＴＴＴＧＣＧＧＣＣＧＣＴＣＡＴＴＡＣＣＧＣＣＴＧＡＣＧＣＡＧＡＧＧＧＴＧＧＡ−３’
（配列番号４の５２９−５４９のアンチセンス；２個の終止コドンＴＡＡＴＧＡ及びＮｏｔＩ配列を付加；アミノ酸１−１５４をコードする欠失体作製用）
ｈＴＰＯ−１５５：５’−ＴＴＴＧＣＧＧＣＣＧＣＴＣＡＴＴＡＧＧＣＣＣＧＣＣＴＧＡＣＧＣＡＧＡＧＧＧＴ−３’
（配列番号４の５３２−５５２のアンチセンス；２個の終止コドンＴＡＡＴＧＡ及びＮｏｔＩ配列を付加：アミノ酸１−１５５をコードする欠失体作製用）
ｈＴＰＯ−１５６：５’−ＴＴＴＧＣＧＧＣＣＧＣＴＣＡＴＴＡＴＧＧＧＧＣＣＣＧＣＣＴＧＡＣＧＣＡＧＡＧ−３’
（配列番号４の５３５−５５５のアンチセンス；２個の終止コドンＴＡＡＴＧＡ及びＮｏｔＩ配列を付加：アミノ酸１−１５６をコードする欠失体作製用）
ｈＴＰＯ−１５７：５’−ＴＴＴＧＣＧＧＣＣＧＣＴＣＡＴＴＡＧＧＧＴＧＧＧＧＣＣＣＧＣＣＴＧＡＣＧＣＡ−３’
（配列番号４の５３８−５５８のアンチセンス；２個の終止コドンＴＡＡＴＧＡ及びＮｏｔＩ配列を付加；アミノ酸１−１５７をコードする欠失体作製用）。
実施例１６で得たクローンｐＥＦ１８Ｓ−ＨＬ３４のプラスミドＤＮＡ１μｇを鋳型として使用し、合成したプライマー（ｈＴＰＯ−５を５’側として使用、ｈＴＰＯ−１５０、−１５１、−１５３、−１５４、−１５５、−１５６、−１５７を３’側として使用）をそれぞれ１０μＭ使ってＰＣＲを実施した。ＧｅｎｅＡｍｐ^ＴＭ ＰＣＲ Ｒｅａｇｅｎｔ Ｋｉｔ ｗｉｔｈ ＡｍｐｌｉＴａｑ^ＴＭＤＮＡ Ｐｏｌｙｍｅｒａｓｅ（宝酒造社製）を用いて、ＧｅｎｅＡｍｐ^ＴＭＰＣＲ Ｓｙｓｔｅｍ ９６００（ＰＥＲＫＩＮ−ＥＬＭＥＲ社製）により１００μｌの容量でＰＣＲ反応（９５℃で１分間の変性条件、６６℃で１分間のアニール条件、７２℃で１分間の合成条件で２０回の反応を行い、さらに７分間７２℃でインキュベート）を行った。それぞれのＰＣＲで得られたバンドを制限酵素ＥｃｏＲＩ及びＮｏｔＩで消化後、１％アガロースゲル（ＦＭＣ ＢｉｏＰｒｏｄｕｃｔｓ社製）を用いた電気泳動にかけ、それぞれのＰＣＲ反応の予想される大きさの主要なＤＮＡ断片を分離し、プレップ−Ａ−ジーンＤＮＡ精製キット（バイオラッド社製）で精製し、同様に制限酵素処理した発現ベクターｐＥＦ１８Ｓにサブクローニングした（宿主菌としては東洋紡績社製コンピテント・ハイＥ．ｃｏｌｉ ＤＨ５を使用）。得られた形質転換体のうち、それぞれ予想される長さのインサートを含むクローンをそれぞれ３個ないし５個づつ選択し、プラスミドＤＮＡを調製した。方法は本質的にＭｏｌｅｃｕｌａｒＣｌｏｎｉｎｇ［Ｓａｍｂｒｏｏｋら、Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ Ｐｒｅｓｓ（１９８９）］に記載されているようにして実施した。一連の操作によって配列番号４において、アミノ酸番号１−１５０をコードする欠失体（ｐＨＴ１−１５０＃２１、２２、２５）、アミノ酸番号１−１５１をコードする欠失体（ｐＨＴ１−１５１＃１６、１７、１８）、アミノ酸番号１−１５３をコードする欠失体（ｐＨＴ１−１５３＃１−５）、アミノ酸番号１−１５４をコードする欠失体（ｐＨＴ１−１５４＃１−５）、アミノ酸番号１−１５５をコードする欠失体（ｐＨＴ１−１５５＃１−５）、アミノ酸番号１−１５６をコードする欠失体（ｐＨＴ１−１５６＃１−５）、アミノ酸番号１−１５７をコードする欠失体（ｐＨＴ１−１５７＃１−５）のプラスミドＤＮＡを得た。
精製したプラスミドＤＮＡ ｐＨＴ１−１５０＃２１、２２、２５並びにｐＨＴ１−１５１＃１６、１７、１８についてはＴａｑ Ｄｙｅ Ｄｅｏｘｙ^ＴＭ Ｔｅｒｍｉｎａｔｅｒ Ｃｙｃｌｅ Ｓｅｑｕｅｎｉｎｇ Ｋｉｔ（アプライドバイオシステムズ社製）を用い、アプライドバイオシステムズ社製３７３ＡＤＮＡシークエンサーによりシークエンスし、全長にわたり塩基配列の置換がなく、予想通りのＴＰ０ｃＤＮＡ配列を持つことを確認した。
得られたそれぞれのクローンのＣＯＳ１細胞へのトランスフェクションは、実施例１１に従って行なった。すなわちプラスミドＤＮＡ各１０μｇを使用しクロロキン処理を含むＤＥＡＥ−デキストラン法を用いたトランスフェクションを行ない、３日後に培養上清を回収した。培養上清をＩＭＤＭ培養液に対して十分に透析後、Ｍ−０７ｅアッセイ系で評価した。その結果、１５１位のアミノ酸であるシステインを含む、それぞれアミノ酸１−１５１位、１−１５３位、１−１５４位、１−１５５位、１−１５６位、１−１５７位からなるＣ末側欠失誘導体をコードするクローンをトランスフェクションしたＣＯＳ１細胞培養上清中には用量依存的にＴＰＯ活性が検出された。しかしながら、１５１個目のシステインまでを欠失させたアミノ酸１−１５０位のＣ末側欠失誘導体をコードするクローンをトランスフェクションしたＣＯＳ１細胞培養上清中にはＴＰＯ活性が検出されなかった。
＜実施例２９＞
ヒトＴＰＯ Ｎ末側欠失体の作製とＣＯＳ１細胞での発現〜活性確認
ヒトＴＰＯ蛋白質の活性発現に必須な領域の解析を行なう目的で、実施例２８で得られた欠失体をもとに、Ｎ末側アミノ酸を欠失させた誘導体を作製しＴＰＯ活性を発現しうるかどうか検討した。実施例１８で得たプラスミドクローンｐＥＦ１８Ｓ−ＨＬ３４並びに、実施例２７で得たプラスミドクローンｐＨＴ１−１６３をもとに、シグナル配列に続くＮ末端側アミノ酸を欠失させた発現プラスミドを構築した。構築にはＰＣＲを利用した。ＰＣＲ用に作製したプライマーの配列は、次の通りである。
ｈＴＰＯ−５：５’−ＴＴＴＧＡＡＴＴＣＧＧＣＣＡＧＣＣＡＧＡＣＡＣＣＣＣＧＧＣＣ−３’
（配列番号４の１−２１にＥｃｏＲＩ配列を付加したもの；表９に記載のものと同一）
ｈＴＰＯ３：５’−ＴＴＴＧＣＧＧＣＣＧＣＴＣＡＴＴＡＴＴＣＧＴＧＴＡＴＣＣＴＧＴＴＣＡＧＧＴＡＴＣＣ−３’
（配列番号４の７５７−７８０のアンチセンス；２個の終止コドンＴＡＡＴＧＡ及びＮｏｔＩ配列を付加；アミノ酸１−２３１をコードする欠失体作製用に合成したものと同一）
ｈＴＰＯ−Ｓ：５’−ＴＴＴＧＣＧＧＣＣＧＣＴＣＡＴＴＡＧＣＴＧＧＧＧＡＣＡＧＣＴＧＴＧＧＴＧＧＧＴ−３’
（配列番号４の５５５−５７６のアンチセンス；２個の終止コドンＴＡＡＴＧＡ及びＮｏｔＩ配列を付加；アミノ酸１−１６３をコードする欠失体作製用）
ｈＴＰＯ−１３：５’−ＡＧＴＡＡＡＣＴＧＣＴＴＣＧＴＧＡＣＴＣＣＣＡＴＧＴＣＣＴＴＣＡＣＡＧＣＡＧＡＣＴＧＡＧＣＣＡＧＴＧ−３’
（配列番号４の１２４−１７３；アミノ酸番号１−１２を欠失する誘導体作製用）
ｈＴＰＯ−１３Ｒ：５’−ＣＡＴＧＧＧＡＧＴＣＡＣＧＡＡＧＣＡＧＴＴＴＡＣＴＧＧＡＣＡＧＣＧＴＴＡＧＣＣＴＴＧＣＡＧＴＴＡＧ−３’
（配列番号４の６４−８７並びに１２４−１４８のアンチセンス；アミノ酸番号１−１２を欠失する誘導体作製用）
ｈＴＰＯ−７：５’−ＴＧＴＧＡＣＣＴＣＣＧＡＧＴＣＣＴＣＡＧＴＡＡＡＣＴＧＣＴＴＣＧＴＧＡＣＴＣＣＣＡＴＧＴＣＣＴＴＣ−３’
（配列番号４の１０６−１５４；アミノ酸番号１−６を欠失する誘導体作製用）ｈＴＰＯ−７Ｒ：５’−ＴＴＴＡＣＴＧＡＧＧＡＣＴＣＧＧＡＧＧＴＣＡＣＡＧＧＡＣＡＧＣＧＴＴＡＧＣＣＴＴＧＣＡＧＴＴＡＧ−３’
（配列番号４の６４−８７並びに１０６−１２９のアンチセンス：アミノ酸番号１−６を失する誘導体作製用）
ｈＴＰＯ−８：５’−ＧＡＣＣＴＣＣＧＡＧＴＣＣＴＣＡＧＴＡＡＡＣＴＧＣＴＴＣＧＴＧＡＣＴＣＣＣＡＴＧＴＣＣＴＴＣＡＣＡ−３’
（配列番号４の１０９−１５７；アミノ酸番号１−７を欠失する誘導体作製用）ｈＴＰＯ−８Ｒ：５’−ＣＡＧＴＴＴＡＣＴＧＡＧＧＡＣＴＣＧＧＡＧＧＴＣＧＧＡＣＡＧＣＧＴＴＡＧＣＣＴＴＧＣＡＧＴＴＡＧ−３’
（配列番号４の６４−８７並びに１０９−１３２のアンチセンス：アミノ酸番号１−７を欠失する誘導体作製用）。
（１）アミノ酸番号１−１２を欠失する誘導体（ｐＨＴ１３−２３１）作製
実施例１８で得たクローンｐＥＦ１８Ｓ−ＨＬ３４のプラスミドＤＮＡ１．４μｇを鋳型として使用し、合成したプライマー（ｈＴＰＯ−１３及びｈＴＰＯ３を組で使用：ｈＴＰＯ−５並びにｈＴＰＯ−１３Ｒを組で使用）をそれぞれ５μＭ使ってＰＣＲを実施した。ＧｅｎｅＡｍｐ^ＴＭ ＰＣＲ Ｒｅａｇｅｎｔ Ｋｉｔ ｗｉｔｈ ＡｍｐｌｉＴａｑ^ＴＭＤＮＡＰｏｌｙｍｅｒａｓｅ（宝酒造社製）を用いて、ＧｅｎｅＡｍｐ^ＴＭ ＰＣＲ Ｓｙｓｔｅｍ ９６００（ＰＥＲＫＩＮ−ＥＬＭＥＲ社製）により１００μｌの容量でＰＣＲ反応（９５℃で５分間変性後、９５℃で１分間の変性条件、６５℃で１分間のアニール条件、７２℃で１分間の合成条件で３０回の反応を行い、さらに７分間７２℃でインキュベート）を行った。それぞれのＰＣＲ産物を１．２％アガロースゲル（ＦＭＣ ＢｉｏＰｒｏｄｕｃｔｓ社製）を用いた電気泳動にかけ、各々予想される大きさの主要なＤＮＡ断片を分離し、プレップ−Ａ−ジーンＤＮＡ精製キット（バイオラッド社製）で精製し、１５μｌのＴＥバッファーに溶解した。この溶液各１μｌを鋳型として用い２回目のＰＣＲを実施した。
合成したプライマー（ｈＴＰＯ−５並びにｈＴＰＯ３を使用）をそれぞれ５μＭ使ってＰＣＲを実施した。ＧｅｎｅＡｍｐ^ＴＭ ＰＣＲ Ｒｅａｇｅｎｔ Ｋｉｔ ｗｉｔｈ ＡｍｐｌｉＴａｑ^ＴＭＤＮＡＰｏｌｙｍｅｒａｓｅ（宝酒造社製）を用いて、ＧｅｎｅＡｍｐ^ＴＭＰＣＲ Ｓｙｓｔｅｍ ９６００（ＰＥＲＫＩＮ−ＥＬＭＥＲ社製）により１００μｌの容量でＰＣＲ反応（９５℃で５分間変性後、９５℃で１分間の変性条件、６０℃で１分間のアニール条件、７２℃で１分間の合成条件で３０回の反応を行い、さらに７分間７２℃でインキュベート）を行った。ＰＣＲ産物を１．２％アガロースゲル（ＦＭＣ ＢｉｏＰｒｏｄｕｃｔｓ社製）を用いた電気泳動にかけ、予想される大きさの主要なＤＮＡ断片を分離し、プレップ−Ａ−ジーンＤＮＡ精製キット（バイオラッド社製）で精製し、１５μｌのＴＥバッファーに溶解した。制限酵素ＥｃｏＲＩ並びにＮｏｔＩで消化後、等量のフェノール／クロロホルムで１回抽出後エタノール沈殿を行なった。遠心で得られた沈殿を１５μｌのＴＥバッファーに溶解後、同様に制限酵素処理した発現べクターｐＥＦ１８Ｓにサブクローニングした（宿主菌としては東洋紡績社製コンピテント・ハイＥ．ｃｏｌｉ ＤＨ５を使用）。得られた形質転換体のうち、予想される長さのインサートを含むクローンを４５個選択し、プラスミドＤＮＡを調製した。方法は本質的にＭｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ［Ｓａｍｂｒｏｏｋら、Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ Ｐｒｅｓｓ（１９８９）］に記載されているようにして実施した。配列番号４において、アミノ酸番号１−２３１をコードする蛋白質のうち１−１２を欠失する欠失誘導体（ｐＨＴ１３−２３１）のプラスミドＤＮＡを得た。これらについてｈＴＰＯ−５並びにｈＴＰＯ３のプライマーを用いたＰＣＲを行ない、４５クローンのうち８クローンで予想される大きさ程度のインサートが確認された。それらのうち３クローンついてはＴａｑ Ｄｙｅ Ｄｅｏｘｙ^ＴＭ Ｔｅｒｍｉｎａｔｅｒ Ｃｙｃｌｅ Ｓｅｑｕｅｎｉｎｇ Ｋｉｔ（アプライドバイオシステムズ社製）を用い、アプライドバイオシステムズ社製３７３ＡＤＮＡシークエンサーによりシークエンスし、１クローンで、予想通りの欠失が起こっており、またその他の部分も含めて全長にわたり塩基配列の置換等がなく、設計通りのＴＰ０ｃＤＮＡ配列を持っクローンｐＨＴ１３−２３１＃３が得られた。
（２）アミノ酸番号１−６を欠失する誘導体（ｐＨＴ７−１６３）の作製
上記（１）の欠失誘導体作製においては、ＴＰＯ蛋白質のＣ末端を２３１番目のアミノ酸として設計したが、Ｃ末端側はさらに欠失させてもＴＰＯ活性を発現しうることが確認されたため、今回の欠失体作製にあたってはＣ末端を１６３番目のアミノ酸までとして設計した。下記（３）においても同様に１６３番目のアミノ酸をＣ末端として設計した。実施例２７で得たクローンｐＨＴ１−１６３のプラスミドＤＮＡＩ．４μｇを鋳型として使用し、合成したプライマー（ｈＴＰＯ−７及びｈＴＰＯ−Ｓを組で使用：ｈＴＰＯ−５並びにｈＴＰＯ−７Ｒを組で使用）をそれぞれ５μＭ使ってＰＣＲを実施した。ＧｅｎｅＡｍｐ^ＴＭ ＰＣＲＲｅａｇｅｎｔ Ｋｉｔｗｉｔ ｈＡｍｐｌｉＴａｑ^ＴＭ ＤＮＡ Ｐｏｌｙｍｅｒａｓｅ（宝酒造社製）を用いて、ＧｅｎｅＡｍｐ^ＴＭＰＣＲ Ｓｙｓｔｅｍ ９６００（ＰＥＲＫＩＮ−ＥＬＭＥＲ社製）により１００μｌの容量でＰＣＲ反応（９５℃で５分間変性後、９５℃で１分間の変性条件、６５℃で１分間のアニール条件、７２℃で１分間の合成条件で３０回の反応を行い、さらに７分間７２℃でインキュベート）を行った。それぞれのＰＣＲ産物を１．２％アガロースゲル（ＦＭＣ ＢｉｏＰｒｏｄｕｃｔｓ社製）を用いた電気泳動にかけ、各々予想される大きさの主要なＤＮＡ断片を分離し、プレップ−Ａ−ジーンＤＮＡ精製キット（バイオラッド社製）で精製し、１５μｌのＴＥバッファーに溶解した。この溶液各１μｌを鋳型として用い２回目のＰＣＲを実施した。
合成したプライマー（ｈＴＰＯ−５並びにｈＴＰＯ−Ｓを使用）をそれぞれ５μＭ使ってＰＣＲを実施した。ＧｅｎｅＡｍｐ^ＴＭ ＰＣＲ Ｒｅａｇｅｎｔ Ｋｉｔ ｗｉｔｈ ＡｍｐｌｉＴａｑ^ＴＭＤＮＡ Ｐｏｌｙｍｅｒａｓｅ（宝酒造社製）を用いて、ＧｅｎｅＡｍｐ^ＴＭ ＰＣＲ Ｓｙｓｔｅｍ ９６００（ＰＥＲＫＩＮ−ＥＬＭＥＲ社製）により１００μｌの容量でＰＣＲ反応（９５℃で５分間変性後、９５℃で１分間の変性条件、６０℃で１分間のアニール条件、７２℃で１分間の合成条件で３０回の反応を行い、さらに７分間７２℃でインキュベート）を行った。ＰＣＲ産物を１．２％アガロースゲル（ＦＭＣ ＢｉｏＰｒｏｄｕｃｔｓ社製）を用いた電気泳動にかけ、予想される大きさの主要なＤＮＡ断片を分離し、プレップ−Ａ−ジーンＤＮＡ精製キット（バイオラッド社製）で精製し、１５μｌのＴＥバッファーに溶解した。制限酵素ＥｃｏＲＩ並びにＮｏｔＩで消化後、等量のフェノール／クロロホルムで１回抽出後エタノール沈殿を行なった。遠心で得られた沈殿を１５μｌのＴＥバッファーに溶解後、同様に制限酵素処理した発現ベクターｐＥＦ１８Ｓにサブクローニングした（宿主菌としては東洋紡績社製コンピテント・ハイＥ．ｃｏｌｉ ＤＨ５を使用）。得られた形質転換体のうち、予想される長さのインサートを含むクローンを３０個選択し、プラスミドＤＮＡを調製した。方法は本質的にＭｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ［Ｓａｍｂｒｏｏｋら、Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ Ｐｒｅｓｓ（１９８９）］に記載されているようにして実施した。配列番号４において、アミノ酸番号１−１６３をコードする蛋白質のうち１−６を欠失する欠失誘導体（ｐＨＴ７−１６３）のプラスミドＤＮＡを得た。これらのクローンにつてはｈＴＰＯ−５並びにｈＴＰＯ−Ｓをプライマーとして用いたＰＣＲを実施し、全てのクローンで予想される大きさ程度のインサートを含むことが確認された。これらのクローンより３個を選択し、Ｔａｑ Ｄｙｅ Ｄｅｏｘｙ^ＴＭ Ｔｅｒｍｉｎａｔｅｒ Ｃｙｃｌｅ Ｓｅｑｕｅｎｉｎｇ Ｋｉｔ（アプライドバイオシステムズ社製）を用い、アプライドバイオシステムズ社製３７３ＡＤＮＡシークエンサーによりシークエンスし、２クローンで、予想通りの欠失が起こっており、またその他の部分も含めて全長にわたり塩基配列の置換等がなく、設計通りのＴＰＯｃＤＮＡ配列を持つ２個のクローンｐＨＴ７−１６３＃４、＃２９が得られた。
（３）アミノ酸番号１−７を欠失する誘導体（ｐＨＴ８−１６３）作製
アミノ酸番号１−７を欠失する誘導体（ｐＨＴ８−１６３）の作製方法は上記、アミノ酸番号１−６を欠失する誘導体（ｐＨＴ７−１６３）の作製と本質的には同様の方法で行なった。実施例２７で得たクローンｐＨＴ１−１６３のプラスミドＤＮＡ１．４μｇを鋳型として使用し、合成したプライマー（ｈＴＰＯ−８及びｈＴＰＯ−Ｓを組で使用：ｈＴＰＯ−５並びにｈＴＰＯ−８Ｒを組で使用）をそれぞれ５μＭ使ってＰＣＲを実施した。ＧｅｎｅＡｍｐ^ＴＭ ＰＣＲ Ｒｅａｇｅｎｔ Ｋｉｔ ｗｉｔｈＡｍｐｌｉＴａｑ^ＴＭＤＮＡ Ｐｏｌｙｍｅｒａｓｅ（宝酒造社製）を用いて、ＧｅｎｅＡｍｐ^ＴＭ ＰＣＲ Ｓｙｓｔｅｍ９６００（ＰＥＲＫＩＮ−ＥＬＭＥＲ社製）により１００μｌの容量でＰＣＲ反応（９５℃で５分間変性後、９５℃で１分間の変性条件、６５℃で１分間のアニール条件、７２℃で１分間の合成条件で３０回の反応を行い、さらに７分間７２℃でインキュベート）を行った。それぞれのＰＣＲ産物を１．２％アガロースゲル（ＦＭＣ ＢｉｏＰｒｏｄｕｃｔｓ社製）を用いた電気泳動にかけ、各々予想される大きさの主要なＤＮＡ断片を分離し、プレップ−Ａ−ジーンＤＮＡ精製キット（バイオラッド社製）で精製し、１５μｌのＴＥバッファーに溶解した。この溶液各１μｌを鋳型として用い２回目のＰＣＲを実施した。
合成したプライマー（ｈＴＰＯ−５並びにｈＴＰＯ−Ｓを使用）をそれぞれ５μＭ使ってＰＣＲを実施した。ＧｅｎｅＡｍｐ^ＴＭ ＰＣＲ Ｒｅａｇｅｎｔ Ｋｉｔ ｗｉｔｈ ＡｍｐｌｉＴａｑ^ＴＭＤＮＡ Ｐｏｌｙｍｅｒａｓｅ（宝酒造社製）を用いて、ＧｅｎｅＡｍｐ^ＴＭ ＰＣＲ Ｓｙｓｔｅｍ ９６００（ＰＥＲＫＩＮ−ＥＬＭＥＲ社製）により１００μｌの容量でＰＣＲ反応（９５℃で５分間変性後、９５℃で１分間の変性条件、６０℃で１分間のアニール条件、７２℃で１分間の合成条件で３０回の反応を行い、さらに７分間７２℃でインキュベート）を行った。ＰＣＲ産物を１．２％アガロースゲル（ＦＭＣ ＢｉｏＰｒｏｄｕｃｔｓ社製）を用いた電気泳動にかけ、予想される大きさの主要なＤＮＡ断片を分離し、プレップ−Ａ−ジーンＤＮＡ精製キット（バイオラッド社製）で精製し、１５μｌのＴＥバッファーに溶解した。制限酵素ＥｃｏＲＩ並びにＮｏｔＩで消化後、等量のフェノール／クロロホルムで１回抽出後エタノール沈殿を行なった。遠心で得られた沈殿を１５μｌのＴＥバッファーに溶解後、同様に制限酵素処理した発現べクターｐＥＦ１８Ｓにサブクローニングした（宿主菌としては東洋紡績社製コンピテント・ハイＥ．ｃｏｌｉ ＤＨ５を使用）。得られた形質転換体のうち、予想される長さのインサートを含むクローンを３０個選択し、プラスミドＤＮＡを調製した。方法は本質的にＭｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ［Ｓａｍｂｒｏｏｋら、Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ Ｐｒｅｓｓ（１９８９）］に記載されているようにして実施した。配列番号４において、アミノ酸番号１−１６３をコードする蛋白質のうち１−７を欠失する欠失誘導体（ｐＨＴ８−１６３）のプラスミドＤＮＡを得た。これらのクローンについてはｈＴＰＯ−５並びにｈＴＰＯ−Ｓをプライマーとして用いたＰＣＲを実施し、全てのクローンで予想される大きさ程度のインサートを含むことが確認された。これらのクローンより３個を選択し、Ｔａｑ Ｄｙｅ Ｄｅｏｘｙ^ＴＭＴｅｒｍｉｎａｔｅｒ Ｃｙｃｌｅ Ｓｅｑｕｅｎｉｎｇ Ｋｉｔ（アプライドバイオシステムズ社製）を用い、アプライドバイオシステムズ社製３７３ＡＤＮＡシークエンサーによりシークエンスし、２クローンで、予想通りの欠失が起こって起こっており、またその他の部分も含めて全長にわたり塩基配列の置換等がなく、設計通りのＴＰＯｃＤＮＡ配列を持つ２個のクローンｐＨＴ８−１６３＃３３、＃４８が得られた。
（４）欠失誘導体のＣＯＳ１細胞での発現とＴＰＯ活性の確認
得られたそれぞれの欠失体クローンのＣＯＳ１細胞へのトランスフェクションは、実施例１１に従って行なった。すなわちプラスミドＤＮＡ各１０μｇを使用しクロロキン処理を含むＤＥＡＥ−デキストラン法を用いたトランスフェクションを行ない、３日後に培養上清を回収した。培養上清をＩＭＤＭ培養液に対して十分に透析後、Ｍ−０７ｅアッセイ系で評価した。
その結果、アミノ酸７−１６３位の欠失誘導体をコードするクローンをトランスフェクションしたＣＯＳ１細胞培養上清中に弱いながらも用量依存的にＴＰＯ活性を認めた。一方、アミノ酸８−１６３位、１３−２３１位の欠失誘導体をコードするクローンをトランスフェクションしたＣＯＳ１細胞培養上清中にはＴＰＯ活性が認められなかった。
＜実施例３０＞
ヒトＴＰＯ完全長ｃＤＮＡプラスミド（ｐＨＴＰ１）の作製
配列番号６のように予想されたヒトＴＰＯｃＤＮＡのアミノ酸コーディング領域を全て持つ、動物細胞発現べクターの構築を行なった。
ＰＣＲによりヒトＴＰＯｃＤＮＡコーディング領域を全てカバーするＤＮＡ断片を以下の様に作製した。
用いたプライマーの塩基配列は次の通りである。
ｈＴＰＯ−Ｉ：５’−ＴＴＧＴＧＡＣＣＴＣＣＧＡＧＴＣＣＴＣＡＧ−３’
（配列番号６の１０５〜１２５）
ＳＡ：５’−ＣＡＧＧＴＡＴＣＣＧＧＧＧＡＴＴＴＧＧＴＣ−３’
（配列番号６の７４５〜７６５に対応するアンチセンス）
ｈＴＰＯ−Ｐ：５’−ＴＧＣＧＴＴＴＣＣＴＧＡＴＧＣＴＴＧＴＡＧ−３’
（配列番号６の５０３〜５２３）
ｈＴＰＯ−ＫＯ：５’−ＧＡＧＡＧＡＧＣＧＧＣＣＧＣＴＴＡＣＣＣＴＴＣＣＴＧＡＧＡＣＡＧＡＴＴ−３’
（配列番号６の１０６６〜１０８６に対応するアンチセンス配列に制限酵素ＮｏｔＩの認識配列並びにＧＡＧＡＧＡ配列を付加したもの）。
実施例１６で得られたクローンｐＥＦ１８Ｓ−ＨＬ３４の３００ｎｇを鋳型として１回目のＰＣＲを行なった。プライマ−ｈＴＰＯ−Ｉ並びにＳＡ各０．５μＭを用い、Ｖｅｎｔ Ｒ^ＴＭ ＤＮＡ ｐｏｌｙｍｅｒａｓｅ（Ｎｅｗ Ｅｎｇｌａｎｄ ＢｉｏＬａｂｓ社製）１ユニットを使用して反応（９６℃１分間、６２℃１分間、７２℃１分間という反応を３０サイクル行なった後７２℃７分間）を行なった。反応溶液の組成は以下の通り。最終濃度で１０ｍＭＫＣｌ、１０ｍＭ（ＮＨ_４）_２ＳＯ_４、２０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．８）、２ｍＭ ＭｇＳＯ_４、０．１％ ＴｒｉｔｏｎＸ−１００、２００μＭ ｄＮＴＰ ｍｉｘ。
市販のヒト正常肝臓由来ポリ（Ａ）＋ＲＮＡ（Ｃｌｏｎｔｅｃｈ社製）１μｇを７０℃で１０分間加熱後氷上で急冷し、１０ｍＭ ＤＴＴ、５００μＭ ｄＮＴＰｍｉｘ、２５ｎｇ ｒａｎｄｏｍ ｐｒｉｍｅｒ（宝酒造社製）、１０ユニットＲＮａｓｅ Ｉｎｈｉｂｉｔｏｒ（ベーリンガーマンハイム社製）、２００ユニットＳｕｐｅｒＳｃｒｉｐｔ^ＴＭＩＩ ＲＮａｓｅＨ−（ＬＩＦＥ ＴＥＣＨＮＯＬＯＧＩＥＳ社製）を加え、３７℃で１時間保温しｃＤＮＡを合成した。合成したｃＤＮＡ反応液の２０分の１量を鋳型として使用して２回目のＰＣＲを行なった。プライマーｈＴＰＯ−Ｐ並びにｈＴＰＯ−ＫＯ各２．５μＭ、２．５ユニットのＡｍｐｌｉＴａｑ^ＴＭ ＤＮＡ ｐｏｌｙｍｅｒａｓｅ（宝酒造社製）を用いて反応（９５℃１分間、５８℃１分間、７２℃１分間の反応を３０サイクル）を行なった。
２回のＰＣＲ溶液を１％アガロースゲル電気泳動にかけ、それぞれ予想された大きさの主要なバンドをプレップ−Ａ−ジーンＤＮＡ精製キット（バイオラッド社製）を用いて精製した。それぞれの精製量のうち各２０分の１量を鋳型として３回目のＰＣＲを実施した。Ｖｅｎｔ Ｒ^ＴＭ ＤＮＡｐｏｌｙｍｅｒａｓｅ（Ｎｅｗ Ｅｎｇｌａｎｄ ＢｉｏＬａｂｓ社製）１ユニットを使用して反応（９６℃２分間加熱後、９６℃２分間、７２℃２分間、という反応を３サイクル行なった後７２℃７分間）を行なった。この反応液にそれぞれ１μＭとなるようにｈＴＰＯ−Ｉ並びにｈＴＰＯ−ＫＯを加えた後、９６℃２分間の加熱を行ない、そののち９６℃１分間、６２℃１分間、７２℃１分間の反応を２５サイクル行なったあと７２℃でさらに７分間反応させた。反応液を等量の水飽和フェノールークロロホルムで１回抽出後、さらに等量のクロロホルムで１回抽出したのち、エタノール沈殿（０．３Ｍ酢酸ナトリウム、０．５μｌベーリンガーマンハイム社製グリコーゲン、２．５倍量エタノール存在下）を行なってＤＮＡを回収した。
回収したＤＮＡを制限酵素ＢａｍＨＩ並びにＮｏｔＩで消化後１％アガロースゲル電気泳動にかけ、予想された大きさの主要なバンドをプレップ−Ａ−ジーンＤＮＡ精製キット（バイオラッド社製）を用いて精製したのち、予め同様に制限酵素ＢａｍＨＩ並びにＮｏｔＩで消化したｐＢｌｕｅｓｃｒｉｐｔＩＩ ＳＫ＋べクター（Ｓｔｒａｔａｇｅｎｅ社）に連結後、コンピテントハイＥ．ｃｏｌｉ ＤＨ５（東洋紡績社製）を形質転換した。得られたコロニーより４クローンを選びプラスミドＤＮＡを調製した。精製したプラスミドＤＮＡについてはＴａｑ Ｄｙｅ Ｄｅｏｘｙ^ＴＭ Ｔｅｒｍｉｎａｔｅｒ Ｃｙｃｌｅ Ｓｅｑｕｅｎｉｎｇ Ｋｉｔ（アプライドバイオシステムズ社製）を用い、アプライドバイオシステムズ社製３７３ＡＤＮＡシークエンサーによりシークエンスし、ＢａｍＨＩからＮｏｔＩにかけての領域に塩基配列の置換がなく、予想通りのＴＰＯｃＤＮＡ配列を持つことが確認できたクローンｐＢＬＴＰを得た。
ｐＢＬＴＰを制限酵素ＥｃｏＲＩ並びにＢａｍＨＩで消化後１％アガロースゲル電気泳動にかけ、高分子量のバンドをプレップ−Ａ−ジーンＤＮＡ精製キット（バイオラッド社製）を用いて精製した。同様にｐＥＦ１８Ｓ−ＨＬ３４も制限酵素処理し４５０ｂｐのバンドを精製した。それぞれのＤＮＡをＬｉｇａｔｉｏｎしコンピテントハイＥ．ｃｏｌｉ ＤＨ５（東洋紡績社製）を形質転換した。得られたコロニーよりプラスミドＤＮＡを調製し、ヒトＴＰＯｃＤＮＡのインサートを含むクローンｐＢＬＴＥＮを得た。
得られたｐＢＬＴＥＮを制限酵素ＥｃｏＲＩ並びにＮｏｔＩで消化後、１％アガロースゲル電気泳動にかけ、約１２００ｂｐのバンドをプレップ−Ａ−ジーンＤＮＡ精製キット（バイオラッド社製）を用いて精製した後、同様に制限酵素処理した発現ベクタ−ｐＥＦ１８Ｓに連結しコンピテントハイＥ．ｃｏｌｉ ＤＨ５（東洋紡績社製）を形質転換した。得られたコロニーよりプラスミドＤＮＡを調製し、ヒトＴＰＯｃＤＮＡのコーディング領域を全て含むクローンｐＨＴＰ１を得た。このクローンのプラスミドＤＮＡを大量に調製し以下の実験に使用した。プラスミドＤＮＡの調製は本質的にＭｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ（Ｓａｍｂｒｏｏｋら、Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ Ｐｒｅｓｓ、１９８９）に記載されているようにして実施した。
＜実施例３１＞
ＣＨＯ細胞用組換えベクター、ｐＤＥＦ２Ｏ２−ｈＴＰＯ−Ｐ１の構築
マウスＤＨＦＲミニ遺伝子を含むプラスミドｐＭＧ１、１μｇを制限酵素ＥｃｏＲＩとＢａｍＨＩで処理した後、アガロースゲル電気泳動しマウスＤＨＦＲミニ遺伝子を含む断片（約２．５ｋｂｐ）を回収した。回収した断片を５０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．５）、７ｍＭ ＭｇＣｌ２、１ｍＭ ｍｅｒｃａｐｔｏｅｔｈｎｏｌ、０．２ｍＭ ｄＮＴＰからなる反応液２５μｌ中に溶解し、Ｋｌｅｎｏｗフラグメント２単位を加え、室温で３０分間反応させ、ＤＮＡの末端を平滑化した。
次いで、フェノール／クロロホルム処理、エタノール沈殿後、１０ｍＭ Ｔｒｉ−ＨＣｌ（ｐＨ８．０）、１ｍＭ ＥＤＴＡからなる溶液１０μｌに溶解した。次に、得られたマウスＤＨＦＲミニ遺伝子を含む断片と動物細胞用発現ベクターｐＥＦ１８Ｓを制限酵素Ｓｍａｌで処理した後、アルカリフォスファターゼ（宝酒造製）で脱リン酸化してえられたベクターＤＮＡをＴ４ＤＮＡリガーゼ（宝酒造製）で結合させ、発現ベクターｐＤＥＦ２０２を得た。
次に、このベクターｐＤＥＦ２０２を制限酵素ＥｃｏＲＩとＳｐｅｌで処理し、アガロースゲル電気泳動で大きい方のベクターフラグメントを回収したのち、このフラグメントとヒトＴＰＯｃＤＮΛ（Ｐ１クローン）を含むプラスミドｐＨＴＰ１を制限酵素ＥｃｏＲＩとＳｐｅｌで処理して得られたヒトＴＰＯｃＤＮＡ（Ｐ１クローン）とをＴ４ＤＮＡリガーゼ（宝酒造製）で結合させ、発現ベクターｐＤＥＦ２０２−ｈＴＰＯ−Ｐ１得た。このプラスミドはＳＶ４０の複製開始領域、ヒトエロンゲーションファクタ−１−アルファプロモーター、ＳＶ４０初期ポリアデニル部位、マウスＤＨＦＲミニ遺伝子、ｐＵＣ１８の複製開始領域、β−ラクタマーゼ遺伝子（Ａｍｐ^ｒ）を含み、ヒトエロンゲーションファクタ−１−アルファプロモーター下流にヒトＴＰＯｃＤＮＡが接続されている。
＜実施例３２＞
ＣＨＯ細胞でのヒトＴＰＯの発現
ＣＨＯ細胞（ｄｈｆｒ−株、ＵｒｌａｕｂとＣｈａｓｉｎ；Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ；７７巻４２１６頁、１９８０）を６ｃｍ径のプレート（Ｆａｌｃｏｎ社製）中１０％牛胎児血清を含むα最小必須培地（α−ＭＥＭ（−）、チミジン、ヒポキサンチン添加）で培養増殖させ、これをリン酸カルシウム法（ＣｅｌｌＰｈｅｃｔ、ファルマシア社製）によって形質転換した。すなわち、実施例３１で調製したｐＤＥＦ２０２−ｈＴＰＯ−Ｐ１プラスミド１０μｇにバッファーＡ：１２０μｌおよびＨ_２Ｏ：１２０μｌを加え混合したのち、室温で１０分間放置した。つぎに、この溶液にバッファーＢ：１２０μｌを加え、再度混合したのち、室温で３０分間放置した。このＤＮＡ溶液をプレートに滴下したのち、ＣＯ_２インキュベーター中で６時間培養した。プレートから培地を除去し、α−ＭＥＭ（−）にて２回洗浄後、１０％ジメチルスルフォオキシド含有α−ＭＥＭ（−）を添加し、室温で２分間処理した。次いで、１０％透析牛胎児血清含有非選択培地（前出α−ＭＥＭ（−）、ヒポキサンチン、チミジン添加）を添加して２日間培養したのち、１０％透析牛胎児血清含有選択培地（α−ＭＥＭ（−）、ヒポキサンチン、チミジン無添加）での選択をおこなった。選択は細胞をトリプシン処理した後、６ｃｍ径プレート１枚あたりを、１０ｃｍ径プレート５枚あるいは２４ウエルプレート２０枚に分割したのち、２日ごとに選択培地にて培地交換を行いながら培養を続行する事により実施した。細胞が増殖してきたプレートあるいはウエルについてはその培養上清中のヒトＴＰＯ活性をＭ−０７ｅアッセイ及びＢａ／Ｆ３アッセイを用いて測定したところ、いずれのアッセイ系においても活性が認められた。培養上清中にヒトＴＰＯ活性の認められたものについては新しいプレートあるいはウエルに２５ｎＭのメソトレキセートを含む選択培地で１：１５に細胞を分割し、培養を続行することによりメソトレキセートに耐性の細胞を増殖させてクローニングを行った。
なお、ＣＨＯ細胞の形質転換はＣＨＯ細胞に対しｐＨＴＰ１とｐＭＧ１を同時形質転換（ｃｏ−ｔｒａｎｓｆｅｃｔｉｏｎ）することによっても行うことができる。
プラスミドｐＤＥＦ２０２−ｈＴＰＯ−Ｐ１によって形質転換されたＣＨＯ細胞株（ＣＨＯ−ＤＵＫＸＢ１１）は１９９５年１月３１日付で通商産業省工業技術院生命工学工業技術研究所に受託番号ＦＥＲＭ ＢＰ−４９８８として寄託されている。
これはまた、１９９５年３月２２日付で中華民国 食品工業発展研究所（ＦＩＲＤＩ）に受託番号９６００２３として寄託されている。
＜実施例３３＞
Ｘ６３．６．５．３．細胞用組換えベクター、ＢＭＣＧＳｎｅｏ−ｈＴＰＯ−Ｐ１の構築
動物細胞用発現ベクターＢＭＣＧＳｎｅｏ１μｇを制限酵素ＸｈｏＩとＮｏｔＩで処理した後、アガロースゲル電気ゲル電気泳動し、ベクターＤＮＡ部分を回収した。次いで得られたＤＮＡ断片とヒトＴＰＯｃＤＮＡを含むプラスミドｐＢＬＴＥＮを制限酵素ＸｈｏＩとＮｏｔＩで処理して得られたヒトＴＰＯｃＤＮＡ（Ｐ１クローン）とをＴ４ＤＮＡリガーゼで結合させ、発現プラスミドＢＭＣＧＳｎｅｏ−ｈＴＰ０−Ｐ１を得た。この発現プラスミドはサイトメガロウイルス初期プロモーター、ウサギβグロビン遺伝子由来のイントロンおよびポリアデニル部位ヒトβグロビン遺伝子の一部、ウシパピローマウイルス１遺伝子の６９％、チミジンキナーゼプロモーターおよびポリアデニル部位、ホスホトランスフェラーゼＩ（ネオマイシン耐性遺伝子）、ｐＢＲ３２２の複製開始領域およびβ−ラクタマーゼ遺伝子（Ａｍｐ^Ｒ）を含みサイトメガロウイルスプロモーター下流にヒトＴＰＯｃＤＮＡが接続されている。
＜実施例３４＞
Ｘ６３．６．５．３．細胞での発現
Ｘ６３．６．５．３．細胞は１０％牛胎児血清を含むＤｕｌｂｅｃｃｏ′ｓ ｍｉｎｉｍａｌｅｓｓｅｎｔｉａｌ （ＤＭＥ）培地中で培養増殖させ、これをエレクトロポレーション法によって形質転換した。
すなわち、実施例３３で調製したＢＭＣＧＳｎｅｏ−ｈＴＰ０−Ｐ１プラスミド２０μｇを１０^７個の細胞を含むＤＭＥ培地７５０μｌに加え、エレクトロポレーション用の４ｍｍギャップのキュベットにセットし、４℃で１０分間放置した。このキュベットをエレクトロポレーション装置（ＢＴＸ６００、ＢＴＸ社製）にセットし、３８０Ｖ）２５ｍＦ、２４オングストロームの条件で遺伝子導入した。遺伝子導入後、キュべットを再び４℃で１５分間放置したのち、細胞を１０％牛胎児血清を含むＤＭＥで一回洗浄した。次に細胞を５０ｍｌの１０％牛胎児血清を含むＤＭＥに懸濁し、９６ウエルプレート５枚に分割したのち、ＣＯ_２インキュベーター中で２日間培養した。その後、培養液を１ｍｇ／ｍｌのＧ４１８（ＧＩＢＣＯ社）、１０％牛胎児血清を含むＤＭＥに交換し、以後３日ごとに培地交換した。細胞が増殖してきたウエルについてはその培養上清中のヒトＴＰＯ活性をＣＦＵ−ＭＫアッセイ、Ｍ−０７ｅアッセイ及びＢａ／Ｆ３アッセイを用いて測定したところ、いずれのアッセイ系においても活性が認められた。培養上清中にヒトＴＰＯ活性の認められたものについては耐性の細胞を増殖させて、２回クローニングを行い、ヒトＴＰＯ産生細胞株を樹立した。
＜実施例３５＞
ヒトＴＰＯのＣＯＳ１細胞での大量発現
ＣＯＳ１細胞へのトランスフェクションは、実施例１１に記載のクロロキン処理を含むＤＥＡＥ−デキストラン法で実施した。ＣＯＳ１細胞（ＡＴＣＣ ＣＲＬ１６５０）をコラーゲンコート処理した１７５ｃｍ^２の培養フラスコ中で、１０％（ｖ／ｖ）のＦＣＳを含むＩＭＤＭを用いて、３７℃の５％炭酸ガスインキュベーター内で約１００％コンフルエントになるまで培養した。培養フラスコのコラーゲンコート処理は、１ｍＭ ＨＣｌで０．３ｍｇ／ｍｌに調製したコラーゲン溶液（イワキ社製Ｃｅｌｌｍａｔｒｉｘ ｔｙｐｅＩ−Ｃ）を１７５ｃｍ^２の培養フラスコあたり２５ｍｌ加え、室温で１時間放置後コラーゲン溶液を回収し、２０〜５０ｍｌのＰＢＳで１回洗浄することで行なった。トランスフェクションは１７５ｃｍ^２の培養フラスコ１枚あたり、２５０μｇ／ｍｌのＤＥＡＥ−デキストラン（ファルマシア社）、６０μＭのクロロキン（シグマ社）、および１０％（ｖ／ｖ）のＮｕ−Ｓｅｒｕｍ（コラボレイティブ社）を含む２０ｍｌのＩＭＤＭ溶液に５００μｌのＨＢＳに溶解したプラスミドｐＨＴＰ１（４０μｇ）を混和し、トランスフェクション直前にＩＭＤＭで１回洗浄した上記のＣＯＳ１細胞に添加した後、３７℃の５％炭酸ガスインキュベーター内で３時間培養した。その後、培養上清を吸引除去しＩＭＤＭで１回洗浄した後、５０ｍｌの無血清培地を添加し、３７℃の５％炭酸ガスインキュベーター内で培養し、５日後に培養上清を回収した。１回の操作には１７５ｃｍ^２の培養フラスコ１００から２６０枚を使用し、５〜１３１の培養上清を回収した。無血清培地の組成は、５μｇ／ｍｌ Ｉｎｓｕｌｉｎ（シグマ社製）、５μｇ／ｍｌ Ｔｒａｎｓｆｅｒｒｉｎ（シグマ社製）、１０μＭ ｍｏｎｏｅｔｈａｎｏｌａｍｉｎｅ（和光純薬）、２５ｎＭ Ｓｏｄｉｕｍ ｓｅｌｅｎｉｔｅ（シグマ社製）、２００μｇ／ｍｌのＢＳＡ（ニチレイ社製脂肪酸フリー高純度ウシアルブミン）を含むＩＭＤＭである。
＜実施例３６＞
ＣＯＳ１細胞で発現したヒト完全長ＴＰＯ（発現プラスミドｐＨＴＰ１，Ｐ１クローン由来）の精製と活性確認
ＣＯＳ１細胞で発現したヒト完全長ＴＰＯ（発現プラスミドｐＨＴＰ１由来）の活性と精製の実施例を以下に述べる。ＴＰＯの血小板増加作用を調べるため、部分精製品をまず調製し、かつ高純度のＴＰＯ標品を得るための精製を行った。以下の調製過程でのＴＰＯ活性測定には、Ｍ−０７ｅアッセイ系を主に用いたが、ラットＣＦＵ−ＭＫアッセイ系においても同様のＴＰＯ活性が示された。またこれらのアッセイに際し、最終濃度０．０２〜０．０５％のヒト血清アルブミン（ＨＳＡ）を標品に添加した。
まず、プラスミドｐＨＴＰ１を大橋、須藤の方法（Ｈｉｄｅｙａ Ｏｈａｓｈｉ ａｎｄ Ｔａｄａｓｈｉ Ｓｕｄｏ，Ｂｉｏｓｃｉ．Ｂｉｏｔｅｃｈ．Ｂｉｏｃｈｅｍ．，５８（４），７５８−７５９，１９９４）を用いてトランスフェクションしたＣＯＳ１細胞を、１０００ｍｌ当たり０．２ｇのＢＳＡ、５ｍｇの牛インシュリン、５ｍｇのヒトトランスフェリンを含みかつ、０．０２ｍＭのモノエタノールアミン、２５ｎＭのＳｏｄｉｕｍ Ｓｅｌｅｎｉｔｅを含む無血清ＩＭＤＭ培養液で５日間、５％炭酸ガス培養器中にて３７℃で培養し、約７Ｌの無血清培養上清を得た。その無血清培養上清に、蛋白質分解酵素阻害剤であるｐ−ＡＰＭＳＦ及びＰｅｆａｂｌｏｃ ＳＣ（４−（２−Ａｍｉｎｏｅｔｈｙｌ）−ｂｅｎｚｅｎｅｓｕｌｆｏｎｙｌ ｆｌｕｏｒｉｄｅ ｈｙｄｒｏｃｈｌｏｒｉｄｅ，Ｍｅｒｋ社製、カタログ番号２４８３９）を最終濃度約１ｍＭ加え、０．２２μｍの濾過フィルターの濾液をとった。これを限外濾過ユニット（フィルトロン社製・オメガウルトラセット分子量８０００カット、またはミリポア社製・ＰＬＧＣペリコンカセット 分子量１００００カット）で約１０倍濃縮し、体積７２３ｍｌ（蛋白質濃度３．３８ｍｇ／ｍｌ、総蛋白質量２４４５ｍｇ、相対活性４３０００、相対活性量１０５１０００００）に対し１０００ｍｌ当り１．６ｍｏｌｅｓのＡｍｍｏｎｉｕｍ Ｓｕｌｆａｔｅ（合計２８８ｇ）を加え、最終濃度１．５Ｍ Ａｍｍｏｎｉｕｍ Ｓｕｌｆａｔｅを含む８０４ｍｌの溶液とした後、１．２５Ｍ Ａｍｍｏｎｉｕｍ Ｓｕｌｆａｔｅを含む２０ｍＭクエン酸ナトリウム緩衝液（ｐＨ５．２）で予め平衡化してあったＭａｃｒｏ−Ｐｒｅｐ Ｍｅｔｈｙｌ ＨＩＣカラム（Ｂｉｏ−Ｒａｄ社製、カタログ番号１５６−００８０；直径５ｃｍ、ベッド高 ９ｃｍ）に、流速１０ｍｌ／ｍｉｎで添加した。添加終了後、１．２５ＭＡｍｍｏｎｉｕｍ Ｓｕｌｆａｔｅを含む２０ｍＭクエン酸ナトリウム緩衝液（ｐＨ５．２）で溶出された素通り画分Ｆ１（２３８４ｍｌ，蛋白質濃度０．８６４ｍｇ／ｍｌ，総蛋白質量２０６１ｍｇ，相対活性６０００）を得た。
次に、溶出液を２０ｍＭ Ｎａ Ｃｉｔｒａｔｅ，ｐＨ５．８にかえ、溶出された画分Ｆ２（１０９２ｍｌ，蛋白質濃度０．７７６ｍｇ／ｍｌ，総蛋白質量８４７ｍｇ，相対活性１５００００）を集めた。
さらに、Ｍａｃｒｏ−Ｐｒｅｐ Ｍｅｔｈｙｌ ＨＩＣカラム Ｆ２（１０８１ｍｌ）を、２０ｍＭクエン酸ナトリウム緩衝液（ｐＨ５．８）で予め平衡化したＳＰ Ｓｅｐｈａｒｏｓｅ Ｆａｓｔ Ｆｌｏｗ（ファルマシア バイオテク社製、カタログ番号１７−０７２９−０１；直径 ３ｃｍ、ベッド高 １０ｃｍ）カラムに注入し、流速１０ｍｌ／ｍｉｎで添加した。添加終了後、さらに１１０ｍＭ ＮａＣｌを含む２０ｍＭクエン酸ナトリウム緩衝液（ｐＨ５．６）で溶出されたものをまとめた画分Ｆ１（２２６２ｍｌ，蛋白質濃度０．２７０ｍｇ／ｍｌ，総蛋白質量６１０ｍｇ，相対活性３００００）を得た。次に、溶出液を４００ｍＭ ＮａＣｌを含む２０ｍＭクエン酸ナトリウム緩衝液（ｐＨ５．４）にかえ、溶出された画分Ｆ２（８５６ｍｌ，蛋白質濃度０．１８９ｍｇ／ｍｌ，総蛋白質量１６２ｍｇ，相対活性３０００００）を集めた。次に、溶出液を１０００ｍＭ ＮａＣｌを含む２０ｍＭクエン酸ナトリウム緩衝液（ｐＨ５．２）にかえ、溶出された画分Ｆ３（３７０ｍｌ，蛋白質濃度０．０３４ｍｇ／ｍｌ，総蛋白質量１２．６ｍｇ，相対活性１５００００）を集めた。
さらに、ＳＰ Ｓｅｐｈａｒｏｓｅ Ｆａｓｔ Ｆｌｏｗカラムにおいて主たるＴＰＯ活性画分Ｆ２（８４５ｍｌ）に、最終濃度約１０％の１−プロパノールを加え、４００ｍＭのＮａＣｌを含む２０ｍＭクエン酸ナトリウム緩衝液（ｐＨ５．４）で予め平衡化したＬＡ−ＷＧＡカラム（ホーネン社製、カタログ番号ＷＧ−００７；直径 ２ｃｍ、ベッド高 １４ｃｍ）カラムに注入し、流速３ｍｌ／ｍｉｎで添加した。添加終了後、４００ｍＭのＮａＣｌを含む２０ｍＭクエン酸ナトリウム緩衝液（ｐＨ５．４）と１−プロパノールの混液（９：１）で溶出されたものをまとめた画分Ｆ１（６４．４ｍｌ，蛋白質濃度０．０１７８ｍｇ／ｍｌ，総蛋白質量１．１５ｍｇ，相対活性１７２２０）を得た。次に、溶出液を０．４Ｍ ＧｌｃＮａｃ，１０％１−プロパノールを含む２０ｍＭクエン酸ナトリウム緩衝液（ｐＨ６．１）にかえ、溶出された画分Ｆ２（４５ｍｌ，蛋白質濃度０．０１０４ｍｇ／ｍｌ，総蛋白質量０．４７０ｍｇ，相対活性６７５０００）を集めた。
そこで、ＬＡ−ＷＧＡカラムにおいて主たるＴＰＯ活性画分Ｆ２（３４０ｍｌ）に、最終濃度約０．００５％のＴＦＡを加えた後、ＹＭＣ−Ｐａｃｋ ＣＮ−ＡＰ（ＹＭＣ社製、カタログ番号ＡＰ−５１３；直径 ６ｍｍ、ベッド高 ２５０ｍｍ）カラムにて展開した。即ち、展開溶媒Ａに０．１％ＴＦＡ、展開溶媒Ｂに０．０５％ＴＦＡを含む１−プロパノールを用い、１５％Ｂで平衡化したＹＭＣ−Ｐａｃｋ ＣＮ−ＡＰカラムに、流速０．６ｍｌ／ｍｉｎで注入した。注入終了後、１５％Ｂから２５％Ｂにプロパノール濃度を上げ、さらに２５％Ｂから５０％Ｂまで６５分の直線濃度勾配で展開し、１．５ｍｌ（２．５ｍｉｎ）ずつポリプロピレン製チューブに集めた。
それぞれ０．５μｌ（３０００分の１フラクション）を取りＨＳＡを加え、限外濾過濃縮し、最終的に０．０５％ＨＳＡを含む０．２５ｍｌのＩＭＤＭアッセイ培養液溶液とし、これをアッセイにかけ、ＴＰＯ活性画分を特定した。この結果、チューブ番号２４〜３０（プロパノール濃度で３５．０〜４２．０％の範囲）に強いＴＰＯの活性（相対活性約６３００００〜４８０００００）があったため、ＴＰＯ活性画分ＦＡ（１３．５ｍｌ）とした。
そこでさらに、ＹＭＣ−Ｐａｃｋ ＣＮ−ＡＰで得られたＦＡを、展開溶媒Ａに０，１％ＴＦＡ、展開溶媒Ｂに０．０５％ＴＦＡを含む１−プロパノールを用いたＣａｐｃｅｌｌ Ｐａｋ Ｃ１ ３００Ａ （資生堂製、カタログ番号Ｃ１ＴＹＰＥ：ＳＧ３００Ａ；直径 ４．６ｍｍ、ベッド高 １５０ｍｍに加え、直径 ４．６ｍｍ、ベッド高 ３５ｍｍのプレカラムを接続したもの）カラムにて展開した。即ち、ＹＭＣ−Ｐａｃｋ ＣＮ−ＡＰで得られたＦＡの一部（８，９ｍｌ）をとり、０．３ｍｌのグリセロールを添加後、遠心エバポレーションで濃縮後、約２．５ｍｌの１０％Ｂ液を加え、２０％Ｂで平衡化したＣａｐｃｅｌｌ Ｐａｋ Ｃ１ ３００Ａカラムに流速０．４ｍｌ／ｍｉｎで注入した。注入終了後、２０％Ｂにて５分溶出した後、２０％Ｂから４０％Ｂまで５０分の直線濃度勾配で展開し、１ｍｌ（２．５ｍｉｎ）ずつポリプロピレン製チューブに集めた。
それぞれ１μｌ（１０００分の１フラクション）を取りＨＳＡを加え、限外濾過濃縮し、最終的に０．０５％ＨＳＡを含む０．２５ｍｌのＩＭＤＭアッセイ培養液溶液とし、これをアッセイにかけ、ＴＰＯ活性画分を特定した。この結果、チューブ番号２０〜２３（プロパノール濃度で２８．５〜３２．５％の範囲）に強いＴＰＯの活性（相対活性約３００００００〜２２５０００００）を示す高活性標品を得ることができた。
＜実施例３７＞
ＣＯＳ１細胞で発現したヒトＴＰＯの分子量測定
ＣＯＳ１細胞で発現したヒト完全長ＴＰＯ（発現プラスミドｐＨＴＦ１、Ｆ１クローン由来）の分子量は、糖鎖が付加された結果、ペプチド鎖の大きさから推定しうる分子量よりも大きいことが考えられた。そこでまず、ＣＯＳ１細胞で発現したヒト完全長ＴＰＯ（発現プラスミドｐＨＴＦ１、Ｆ１クローン由来）を含む培養上清から部分精製ＴＰＯ画分を以下のように調製した。即ち、展開溶媒Ａ（０．１％トリフルオロ酢酸（ＴＦＡ））および展開溶媒Ｂ（０．０５％ ＴＦＡを含む１−プロパノール）を用いて、２５％Ｂで予め平衡化したＹＭＣ−Ｐａｃｋ ＰＲＯＴＥＩＮ−ＲＰ（ＹＭＣ社、カタログ番号Ａ−ＰＲＲＰ−３３−４６−２５；直径 ０．４６ｃｍ、ベッド高 １５ｃｍ）カラムに、培養上清０．３ｍｌを注入し、流速０，４ｍｌ／ｍｉｎで５分２５％Ｂを通液した後、５０分間の２５％Ｂから５０％Ｂまでの直線濃度勾配にて分画した。ＴＰＯ活性は３４．５％〜４３．５％１−プロパノールの範囲に溶出され、これを遠心エバポレーションで乾固し、部分精製標品を得た。
次に、実施例１に述べた非還元下ＳＤＳ−ＰＡＧＥのゲルから蛋白質抽出操作後、Ｍ−０７ｅアッセイ系にてＴＰＯ活性を調べ、あるいは実施例４５に述べるウエスターン分析により、還元処理されたＤＰＣＩＩＩマーカーに対する分子量を測定した。この結果、見かけ上の分子量約６９０００〜９４０００の広い範囲にＴＰＯ活性が存在し、分子量の不均一性を確認できた。さらにこれと同様にして、Ｎ−Ｇｌｙｃａｎａｓｅ（ジェンザイム社製、カタログ番号１４７２−００）によるＮ結合型糖鎖切断後のＴＰＯについて、還元処理されたＤＰＣＩＩＩマーカーに対する見かけ上の分子量を調べてみると、３６０００〜４００００となり、ペプチド鎖の大きさから推定しうる分子量約３５０００よりもなおも大きいことが判明し、Ｏ結合型糖鎖をも含むことが強く示唆された。
これらと同様、ＣＯＳ１細胞で発現したヒト完全長ＴＰＯ（発現プラスミドｐＨＴＰＩ、Ｐ１クローン由来）の見かけ上の分子量についても調べたところ、６３０００〜８３０００であった。
＜実施例３８＞ヒトＴＰＯの生物学的特性
主に発現プラスミドｐＨＴＦ１をＣＯＳ１細胞へトランスフェクションして得られたＴＰＯ活性を含有する培養上清を用いて、ヒトＴＰＯのヒト、およびラット血液系細胞に対する作用を調べた。
ヒト臍帯血から調製したＣＤ３４^＋、ＤＲ^＋細胞画分を用いたコロニーアッセイ系で検定したところ、ヒトＴＰＯにより有意な数の巨核球コロニーが形成された。例えば、発現プラスミドｐＨＴ１−２３１をトランスフェクション、発現させたＣＯＳ１細胞培養上清を１０％添加した条件下で、６０００個のＣＤ３４^＋、ＤＲ^＋細胞から平均１１．５個の巨核球コロニーが形成された。ヒト抹消血からＦｉｃｏｌｌ−Ｐａｑｕｅを用いた比重遠心法にて得られた白血球画分からプラスティック付着性細胞を除去し、さらにＳＢＡ（ダイズアグルチニン）に親和性を有する細胞をＡＩＳマイクロセレクターＣＤ３４（旭メディカル株）を用いたパニング法にて除去し、最終的にＡＩＳマイクロセレクターＣＤ３４（旭メディカル株）を用いたパニング法にてＣＤ３４^＋細胞画分を得た。この細胞画分にヒトＴＰＯ（発現プラスミドｐＨＴＦ１をトランスフェクション、発現させたＣＯＳ１細胞培養上清）を添加し、１０日間液体培養したところ、サイズの大きいＧｐＩＩｂ／ＩＩＩ ａ^＋細胞の選択的増殖が認められ、さらにこのＧｐＩＩｂ／ＩＩＩ ａ^＋細胞では核の倍数性（ｐｌｏｉｄｙ）が増加していた。このことから、ヒトＴＰＯがヒト巨核球系前駆細胞に特異的に作用し、その増殖・分化を促進することが強く示唆された。
ラット骨髄細胞から分離・精製したＧｐＩＩｂ／ＩＩＩ ａ^＋細胞画分、及びＧｐＩＩｂ／ＩＩＩ ａ^＋細胞画分を得る前段階のプラスティック非付着性細胞画分を用いたコロニーアッセイ系で検定したところ、ヒトＴＰＯにより有意な数の巨核球コロニーが形成された。例えば、発現プラスミドｐＨＴＦ１をトランスフェクション、発現させたＣＯＳ１細胞培養上清を２０％添加した条件下で、培養５日目に１０００個のＧｐＩＩｂ／ＩＩＩ ａ^＋細胞から平均３４．５個の巨核球コロニー、また２００００個のプラスティック非付着性細胞から平均２８．５個の巨核球コロニーが形成された。さらに、プラスチック非付着細胞画分には巨核球系以外の様々な系統の前駆細胞が存在するが、ヒトＴＰＯの添加では巨核球コロニーしか形成されず、ヒトＴＰＯが巨核球系前駆細胞に特異的に作用することが強く示唆された。また、ラット骨髄由来のＧｐＩＩｂ／ＩＩＩ ａ^＋細胞画分をヒトＴＰＯ（発現プラスミドｐＨＴＦ１をトランスフェクション、発現させたＣＯＳ１細胞培養上清を２０％添加）の存在下に３〜５日間液体培養し、核の倍数性（ｐｌｏｉｄｙ）を調べたところ、培養５日目に明らかにｐｌｏｉｄｙが増加していた。
＜実施例３９＞
グルタチォン−ｓ−トランスフェラーゼ（ＧＳＴ）とヒトＴＰＯ （アミノ酸１−１７４）の融合タンパク質（以下、この融合タンパク質を「ＧＳＴ−ＴＰＯ（１−１７４）」と称す）の大腸菌発現用ベクターの構築
大腸菌でのヒトＴＰＯの発現を容易にするために、ヒトＴＰＯをコードしかつ大腸菌優先コドンを含有する人工遺伝子を作製した。なお、このＤＮＡ塩基配列は大腸菌翻訳開始用のアミノ末端メチオニンコドン（ＡＴＧ）を−１の位置に有する。
表１２に示した１−１２の合成オリゴヌクレオチドを作製し、２−１１の合成オリゴヌクレオチドをＴ４キナーゼ（ファルマシア社製）により１ｍＭ ＡＴＰ，１０ｍＭ Ｔｒｉｓ−ａｃｅｔａｔｅ，１０ｍＭ Ｍｇ−ａｃｅｔａｔｅ，５０ｍＭ Ｋ−ａｃｅｔａｔｅの溶液中でリン酸化した。これらの合成オリゴヌクレオチドを６本の二本鎖ＤＮＡにするために１及び２；３及び４；５及び６；７及び８；９及び１０；１１及び１２それぞれの組み合わせの一本鎖ＤＮＡを１０ｍＭ Ｔｒｉｓ／ＨＣｌ（ｐＨ７．５），１０ｍＭ ＭｇＣｌ_２。，５０ｍＭ ＮａＣｌの溶液中でアニールした。次に１及び２；３及び４；５及び６の三組の二本鎖ＤＮＡと、７及び８；９及び１０；１１及び１２の三組の二本鎖ＤＮＡをそれぞれＴ４リガーゼ（ライフテクノロジー社製）を用いて反応し、更にこれら二種の反応液を同様にしてＴ４リガーゼを用いて反応した。このライゲーション反応で得られたＤＮＡをＢａｍＨＩ （べーリンガーマンハイム社製）で消化後、２％のアガロースゲルで泳動し、約３９０−４００ｂｐの大きさのフラグメントを回収してプレップ−Ａ−ジーンＤＮＡ精製キットで精製し、Ｘｂａｌ，ＢａｍＨＩで消化したｐＵＣ１８にサブクローニングした（宿主はＥ．ｃｏｌｉ ＤＨ５αを使用した）。得られたクローンのうち、表１３に示したヒトＴＰＯをコードしかつ大腸菌優先コドンを含有する人工遺伝子をもつクローンを、塩基配列の解析により選択し、これをｐＵＣ１８（ＸＢ）（１−１２３）とした。表１３のコーディング鎖のＤＮＡ配列を配列表（配列番号９）に示した。
【表１２】

【表１３】

ヒト正常肝臓由来ポリ （Ａ）^＋ＲＮＡ１μｇよりｃＤＮＡの１本鎖目をオリゴｄＴプライマーをプライマーとして合成し、ｃＤＮＡ合成の反応液の０．１容を鋳型としてＰＣＲを行った。プライマーにはｈＴＰＯ−Ｃ及びｈＴＰＯ−こ（ＥｃｏＲＩ）を使用し、１００μｌの容量で反応を行った（９６℃２分間の後に、９５℃１分間／５８℃１分間／７２℃１分間を３０サイクル反応し、さらに７２℃７分間）。これによって得られた、ヒトＴＰＯｃＤＮＡ断片をＢａｍＨＩとＥｃｏＲＩで消化後２％のアガロースゲルで泳動し、約６００ｂｐの大きさのフラグメントを回収してプレップ−Ａ−ジーンＤＮＡ精製キットで精製し、ＥｃｏＲＩ，ＢａｍＨＩで消化したｐＵＣ１８にサブクローニングした（宿主はＥ．ｃｏｌｉ ＤＨ５αを使用した）。正しいヒトＴＰＯ塩基配列をコードするクローンを、塩基配列の解析により選択し、ｐＵＣ１８（ＢＥ）（１２４−３３２）とした。ここで用いたＰＣＲ用プライマーの配列は以下の通りである。
ｈＴＰＯ−Ｃ：５’−ＧＧＡＧＧＡＧＡＣＣＡＡＧＧＣＡＣＡＧＧＡ−３’
（ｐＨＴＦ１クローンの３２９−３４９）
ｈＴＰ０−こ（ＥｃｏＲＩ）：５’−ＣＣＧＧＡＡＴＴＣＴＴＡＣＣＣＴＴＣＣＴＧＡＧＡＣＡＧＡＴＴ−３’
（ｐＨＴＦ１クローンの１１４３−１１６３に対応するアンチセンスにＥｃｏＲＩ配列を付加）。
ｐＵＣ１８（ＢＥ）（１２４−３３２）をＢａｍＨＩとＥｃｏＲＩで消化後２％のアガロースゲルで泳動し、約６００ｂｐの大きさのヒトＴＰＯｃＤＮＡのＣ末端側フラグメントを回収してプレップ−Ａ−ジーンＤＮＡ精製キットで精製し、ＥｃｏＲＩ，ＢａｍＨＩで消化したｐＵＣ１８（ＸＢ）（１−１２３）にサブクローニングした（宿主はＥ．ｃｏｌｉ ＤＨ５αを使用した）。ここで得られたクローンをｐＵＣ１８（ＸＥ）（１−３３２）とした。
ヒトＴＰＯｃＤＮＡのＣ末端側の種々のデリーションコンストラクトを作製し、それを発現させインビトロのヒトＴＰＯ活性の測定を行った実施例より、ヒトＴＰＯアミノ酸１−１６３はヒトＴＰＯ活性を保持することが明らかになったので、このぺプチド断片を含んだ発現ベクターを構築することにした。ここでは、ＧＳＴ−ＴＰＯ（１−１７４）の発現を行った。
ｐＨＴＦ１クローンの６８１−６８６（アミノ酸１７３−１７４）に相当する塩基配列は、制限酵素Ｓａｃｌにより認識されるので、この制限酵素認識部位を利用して２本の合成オリゴヌクレオチドにより終止コドンを導入した。具体的には、２本の合成オリゴヌクレオチドＳＳＥ１，ＳＳＥ２を１０ｍＭ Ｔｒｉｓ／ＨＣｌ（ｐＨ７．５），１０ｍＭ ＭｇＣｌ_２，５０ｍＭＮａＣｌの溶液中でアニールし、ここで得られた二本鎖ＤＮＡを、ＳａｃＩ，ＥｃｏＲＩで消化してヒトＴＰＯｃＤＮＡのＣ末端側約４８０ｂｐを除いたｐＵＣ１８（ＸＥ）（１−３３２）にＤＮＡ ＬｉｇａｔｉｏｎＫｉｔ（宝酒造社製）を用いて導入し、クローンｐＵＣ１８（ＸＳ）（１−１７４）を得ることができた（宿主はＥ．ｃｏｌｉ ＤＨ５αを使用した）。ここで用いた合成オリゴヌクレオチドの配列を表１４に示した。
【表１４】

ｐＵＣ１８（ＸＳ）（１−１７４）をＸｂａＩ，ＥｃｏＲＩで消化後、２％のアガロースゲルで泳動し、ヒトＴＰＯアミノ酸１−１７４をコードする約６００ｂｐの大きさのフラグメントを回収してプレップ−Ａ−ジーンＤＮＡ精製キットで精製し、ＸｂａＩ，ＥｃｏＲＩで消化したｐＢｌｕｅｓｃｒｉｐｔ ＩＩＳＫ^＋ （ストラタジーン社製）にサブクローニングした（宿主はＥ．ｃｏｌｉＤＨ５αを使用した）。ここで得られたクローンをｐＢＬ（ＸＳ）（１−１７４）とした。さらに、これと同様にしてｐＢＬ（ＸＳ）（１−１７４）をＸｂａＩ，ＨｉｎｄＩＩＩで消化後、ヒトＴＰＯアミノ酸１−１７４をコードする約５５０ｂｐの大きさのフラグメントを回収してプレップーＡ−ジーンＤＮＡ精製キットで精製し、ＸｂａＩ，ＨｉｎｄＩＩＩで消化したｐＣＦＭ５３６（特表示昭６０−５０１９８８）にクローニングした（宿主はＥ．ｃｏｌｉ ＤＨ５αを使用した）。ここで得られたクローンをｐＣＦＭ５３６／ｈＴ（１−１７４）とした。
グルタチオン−Ｓ−トランスフェラーゼ（ＧＳＴ）との融合タンパク発現ベクターであるｐＧＥＸ−２Ｔ （ファルマシア社製）によって、ＧＳＴ−ＴＰＯ（１−１７４）を発現させるために、次のことを行った。ｐＣＦＭ５３６／ｈＴ（１−１７４）を鋳型として、２本のＰＣＲ用プライマーＧＥＸ１，ＧＥＸ３を用いてＰＣＲを行った（９６℃２分間の後に、９５℃１分間／４１℃１分間／７２℃１分間を２２サイクル反応し、さらに７２℃７分間）。これによって得られた、ヒトＴＰＯをコードする断片をＮａｅＩとＥｃｏＲＩで消化後２％のアガロースゲルで泳動し、約５５０ｂｐの大きさのフラグメントを回収してプレップ−Ａ−ジーンＤＮＡ精製キットで精製し、ＥｃｏＲＩ，Ｓｍａｌで消化したｐＧＥＸ−２Ｔにクローニングし（宿主はＥ，ｃｏｌｉ ＤＨ５を使用した）、正しいヒトＴＰＯ塩基配列をコードするクローンｐＧＥＸ−２Ｔ／ｈＴ（１−１７４）を、塩基配列の解析により選択し、これをＧＳＴ−ＴＰＯ（１−１７４）発現用の形質転換体とした。ここで用いたＰＣＲ用プライマーの配列は以下の通りである。
ＧＥＸ１：５′−ＡＴＣＧＣＣＧＧＣＴＣＣＧＣＣＡＧＣＴＴＧＴＧＡＣ−３′（配列番号１０の２１−３９にＮａｅＩ配列を付加）
ＧＥＸ３：５′−ＧＣＣＧＡＡＴＴＣＴＣＡＴＴＡＧＡＧＣＴＣＧＴＴＣＡＧＴＧＴ−３′（配列番号１０の５２３−５４９のアンチセンス）。
またこの発現プラスミドは、ＧＳＴタンパクに続いてトロンビン認識配列、及びヒトＴＰＯ（アミノ酸１−１７４）をコードする配列を含んでいる。トロンビン認識配列、及びヒトＴＰＯ（アミノ酸１−１７４）をコードする配列を配列表（配列番号１０）に示した。
＜実施例４０＞
ＧＳＴ−ＴＰＯ（１−１７４）の大腸菌での発現
実施例３９で得られた形質転換株を、アンピシリン５０μｇ／ｍｌを含むＬＢ培地６０ｍｌに３７℃で一晩振盪培養し、この培養液２５ｍｌをアンピシリン５０μｇ／ｍｌを含むＬＢ培地１０００ｍｌに加えて、ＯＤはＡ_６００が０．７−０．８に至るまで３７℃で振盪培養した。次いで最終濃度が、０．１ｍＭになるようにＩＰＴＧを添加し、さらに３時間振盪培養して、ＧＳＴ−ＴＰＯ（１−１７４）の発現を誘導した。
＜実施例４１＞
大腸菌で発現した、ヒトＴＰＯ塩基配列をコードするクローンｐＧＥＸ−２Ｔ／ｈＴ（１−１７４）由来ＧＳＴ−ＴＰＯ（１−１７４）の精製と活性確認
ヒトＴＰＯ塩基配列をコードするクローンｐＧＥＸ−２Ｔ／ｈＴ（１−１７４）由来ＧＳＴ−ＴＰＯ（１−１７４）生産組換体凍結菌体５．９ｇに水１０ｍｌを加えてけん濁し、高圧破砕機で菌体を破砕した。遠心分離により、ＧＳＴ−ＴＰＯ（１−１７４）を沈殿画分に回収し、大部分の混在蛋白質、菌体成分等を除去した。次に、回収されたＧＳＴ−ＴＰＯ（１−１７４）を含む沈殿画分に水５ｍｌを加えてけん濁した後、攪拌しながら１ＭＴｒｉｓ緩衝液ｐＨ８．５を６ｍｌ、１０Ｍ尿素１２０ｍｌ、水１６ｍｌを加えた。室温にて５分間攪拌可溶化後、溶液を４等分し、各々、以下のとおり（１）〜（４）の４種の操作を行った。
（１）２０ｍＭＴｒｉｓ緩衝液ｐＨ８．５で１０倍希釈した。これに還元型グルタチオンと酸化型グルタチオンを加えて、各々、最終濃度５ｍＭ及び０．５ｍＭとし、４℃で一晩静置した。遠心分離により、上清中にＧＳＴ−ＴＰＯ（１−１７４）を回収し、これに２０ｍＭクエン酸ナトリウム緩衝液ｐＨ５．５で２倍希釈した後、酢酸を用いてｐＨ５．５に調整した。２０ｍＭクエン酸ナトリウム緩衝液ｐＨ５．５で平衡化したＳＰ Ｓｅｐｈａｒｏｓｅ Ｆａｓｔ Ｆｌｏｗ（ファルマシア バイオテク社製、カタログ番号１７−０７２９−０１）陽イオン交換カラムにＧＳＴ−ＴＰＯ（１−１７４）を吸着させた。同樹脂を２０ｍＭクエン酸ナトリウム緩衝液ｐＨ５．５で洗浄した後、５００ｍＭ塩化ナトリウムを含む２０ｍＭクエン酸ナトリウム緩衝液ｐＨ５．５を用いてＧＳＴ−ＴＰＯ（１−１７４）を溶出させた。溶出液１２９ｍｌに２．６ｍｌの１ＭＴｒｉｓ緩衝液ｐＨ８．５を加えて、ｐＨを８．１にした後、グルタチオンセファロース４Ｂ（ファルマシア バイオテク社製、カタログ番号１７−０７５６−０１）カラムに添加しＧＳＴ−ＴＰＯ（１−１７４）を吸着させた。ＰＢＳで洗浄した後、１０ｍＭ還元型グルタチオンを含む２０ｍＭＴｒｉｓ緩衝液ｐＨ８．５でＧＳＴ−ＴＰＯ（１−１７４）を溶出させた。溶出液にトロンビン３７ＮＩＨ Ｕｎｉｔを加えて室温で４時間静置した後、ＰＢＳで１０倍希釈し、グルタチオンセファロース４Ｂカラムに添加し切断されたＧＳＴを吸着させ、非吸着画分にＴＰＯ（１−１７４）を回収した。回収した非吸着画分を２０ｍＭクエン酸ナトリウム緩衝液ｐＨ５．５で３倍希釈し、同緩衝液で平衡化したＳＰ ＳｅｐｈａｒｏｓｅＦａｓｔ Ｆｌｏｗ陽イオン交換カラムに添加し、同緩衝液中で塩化ナトリウム濃度０Ｍから５００ｍＭまでの直線濃度勾配法によって溶出を行なった。
（２）（１）の操作を全て０．１％ポリソルベート８０存在下で行った。
（３）２０ｍＭＴｒｉｓ緩衝液ｐＨ８．５で１０倍希釈した。これに還元型グルタチオンと酸化型グルタチオンを加えて、各々、最終濃度５ｍＭ及び０．５ｍＭとし、４℃で一晩静置した。遠心分離により、上清中にＧＳＴ−ＴＰＯ（１−１７４）を回収し、これに２０ｍＭクエン酸ナトリウム緩衝液ｐＨ５．５で２倍希釈した後、酢酸を用いてｐＨ５．５に調整した。２０ｍＭクエン酸ナトリウム緩衝液ｐＨ５．５で平衡化したＳＰ Ｓｅｐｈａｒｏｓｅ Ｆａｓｔ Ｆｌｏｗ陽イオン交換樹脂にＧＳＴ−ＴＰＯ（１−１７４）を吸着させた。同樹脂を２０ｍＭクエン酸ナトリウム緩衝液ｐＨ５．５で洗浄した後、５００ｍＭ塩化ナトリウムを含む２０ｍＭクエン酸ナトリウム緩衝液ｐＨ５．５を用いてＧＳＴ−ＴＰＯ（１−１７４）を溶出させた。溶出液に１ＭＴｒｉｓ緩衝液ｐＨ８．５を加えてｐＨを約８とし、トロンビン３２０ＮＩＨ Ｕｎｉｔを加えて室温で４時間静置した後、２０ｍＭクエン酸ナトリウム緩衝液ｐＨ５．５で５倍希釈し、同緩衝液で平衡化したＳＰ Ｓｅｐｈａｒｏｓｅ Ｆａｓｔ Ｆｌｏｗ陽イオン交換カラムに添加し、同緩衝液中で塩化ナトリウム濃度０Ｍから５００ｍＭまでの直線濃度勾配法によって溶出を行なった。
（４）（３）の操作を全て０．１％ポリソルベート８０存在下で行った。
（１）〜（４）の操作で得られたＳＰ Ｓｅｐｈａｒｏｓｅ Ｆａｓｔ Ｆｌｏｗ陽イオン交換クロマトグラフィーの塩化ナトリウム濃度約２００ｍＭから４００ｍＭで溶出された画分を、各々、ＩＭＤＭ培養液に対して十分に透析後、ラットＣＦＵ−ＭＫ系で評価した結果、容量依存的にＴＰＯ活性を認めた。同画分を還元剤存在下でのＳＤＳ−ＰＡＧＥで分析した結果、この画分の主タンパク質バンドの一つとして分子量１９Ｋダルトンのバンドが検出された（純度１−２０％）。このバンドについて実施例１に記載された方法により、ＳＤＳ−ＰＡＧＥ後、ＰＶＤＦ膜に転写しＮ末端シークエンス分析を行なった結果、このｐＧＥＸ−２Ｔ／ｈＴ（１−１７４）由来の融合蛋白質として発現されるべきＴＰＯの配列を含むことが確認できた。
＜実施例４２＞
アミノ酸１（Ｓｅｒ→Ａｌａ）、アミノ酸３（Ａｌａ→Ｖａｌ）に変換したヒトＴＰＯ （アミノ酸１−１６３）の大腸菌用発現べクターの構築
実施例３９で作製したｐＵＣ１８（ＸＥ）（１−３３２）をＸｂａＩ，ＥｃｏＲＩで消化後、２％のアガロースゲルで泳動し、ヒトＴＰＯアミノ酸１−３３２をコードする約１０００ｂｐの大きさのフラグメントを回収してプレップ−Ａ−ジーンＤＮＡ精製キットで精製し、ＸｂａＩ，ＥｃｏＲＩで消化したｐＢｌｕｅｓｃｒｉｐｔ ＩＩ ＳＫ^＋（ストラタジーン社製）にサブクローニングした（宿主はＥ．ｃｏｌｉ ＤＨ５αを使用した）。ここで得られたクローンをｐＢＬ（ＸＥ）（１−３３２）とした。
次にこのクローンｐＢＬ（ＸＥ）（１−３３２）のＢａｍＨＩ認識配列からアミノ酸１６３（３６６−４８９）までを大腸菌優先コドンに変更することとした。表に示した１３−２０の合成オリゴヌクレオチドを作成し、１３及び１４；１５及び１６；１７及び１８の合成オリゴヌクレオチドを同チューブ中でＴ４キナーゼ（ファルマシア社製）を用いて０．１ｍＭ ＡＴＰ，１０ｍＭ Ｔｒｉｓ−ａｃｅｔａｔｅ，１０ｍＭ Ｍｇ−ａｃｅｔａｔｅ，５０ｍＭ Ｋ−ａｃｅｔａｔｅの溶液中でリン酸化した。さらに１／１０量の１００ｍＭ Ｔｒｉｓ／ＨＣｌ（ｐ７．５），１００ｍＭ ＭｇＣｌ_２，５００ｍＭ ＮａＣｌの溶液を加え、水浴中で３分間煮沸した後、放置することにより２本鎖ＤＮＡとした。次に１３及び１４；１５及び１６；１７及び１８の三組の二本鎖ＤＮＡをＤＮＡライゲーションキット（宝酒造社製）を用いて連結し、これを鋳型として１９及び２０の合成オリゴヌクレオチドをプライマーとしてＰＣＲ反応を行った。これによって得られたＰＣＲ産物をＢａｍＨＩとＨｉｄＩＩＩで消化後２％のアガロースゲルで泳動し、約１３０ｂｐの断片をプレップ−Ａ−ジーンＤＮＡ精製キットにより回収した。これをＢａｍＨＩとＨｉｄＩＩＩで消化したｐＢＬ（ＸＥ）（１−３３２）にサブクローニングした（宿主はＥ．ｃｏｌｉ ＤＨ５を使用した）。得られたクローンのうち表１５に示した塩基配列を有するものをシークエンシングにより選択し、これをｐＢＬ（ＸＨ）（１−１６３）とした。
【表１５】

さらに、ヒトＴＰＯ（アミノ酸１−１６３）の発現量を増すこと、またＮ末端のプロテアーゼによる分解を防ぐことを目的として、ヒトＴＰＯ（アミノ酸１−１６３）の１位のアミノ酸をＳｅｒ→Ａｌａに、３位のアミノ酸をＡｌａ→Ｖａｌに変換し、更に−１位にＬｙｓ、−２位にＭｅｔをコードするような変異型ヒトＴＰＯ（アミノ酸１−１６３） （以下、このようなタンパク質を「ｈ６Ｔ（１−１６３）」と称す）を発現させるための発現ベクターの構築を行った。表に示した４種の合成オリゴヌクレオチドを作製し、２−９、３−３の合成オリゴヌクレオチドをＴ４キナーゼ（ファルマシア社製）により１ｍＭ ＡＴＰ，１０ｍＭＴｒｉｓ−ａｃｅｔａｔｅ，１０ｍＭＭｇ−ａｃｅｔａｔｅ，５０ｍＭ Ｋ−ａｃｅｔａｔｅの溶液中でリン酸化した。これらの合成オリゴヌクレオチドを２本の二本鎖ＤＮＡにするために１−９及び２−９；３−３及び４−３それぞれの組み合わせの一本鎖ＤＮＡを１０ｍＭ Ｔｒｉｓ／ＨＣｌ（ｐＨ７．５），１０ｍＭ ＭｇＣｌ_２，５０ｍＭ ＮａＣｌの溶液中でアニールした。次に１−９及び２−９；３−３及び４−３の二組の二本鎖ＤＮＡをＤＮＡ ＬｉｇａｔｉｏｎＫｉｔ（宝酒造社製）を用いて反応した。このライゲーション反応で得られたＤＮＡを、ＸｂａＩ，ＮｒｕＩで消化したｐＢＬ（ＸＨ）（１−１６３）にサブクローニングし、塩基配列の解析により表１６に示した合成オリゴヌクレオチドにより正しく置換されたクローンを選択し（宿主はＥ．ｃｏｌｉ ＤＨ５αを使用した）、これをｐＢＬ（ＸＨ）ｈ６Ｔ（１−１６３）とした。
【表１６】

ｐＢＬ（ＸＨ）ｈ６Ｔ（１−１６３）をＸｂａＩ，ＨｉｎｄＩＩＩで消化後、変異型のヒトＴＰＯアミノ酸１−１６３をコードする約５００ｂｐの大きさのフラグメントを回収してプレップ−Ａ−ジーンＤＮＡ精製キットで精製し、ＸｂａＩ，ＨｉｎｄＩＩＩで消化したｐＣＦＭ５３６（特表昭６０−５０１９８８）にクローニングした（宿主はｐＭＷ１（ＡＴＣＣ Ｎｏ．３９９３３）で予め形質転換されたＥ，ｃｏｌｉ ＪＭ１０９を使用した）。ここで得られたクローンをｐＣＦＭ５３６／ｈ６Ｔ（１−１６３）とし、この発現ベクターを有する大腸菌株を変異型のヒトＴＰＯ、すなわちｈ６Ｔ（１−１６３）発現用の形質転換体とした。この発現プラスミドは、配列表（配列番号１１）に示されたＤＮＡ配列を含んでいる。
＜実施例４３＞
ｈ６Ｔ（１−１６３）の大腸菌での発現
発現プラスミドｐＣＦＭ５３６の発現制御は、λＰＬプロモーターによるが、これ自体が、ｃｌ８５７リプレッサー遺伝子の制御下にある。＜実施例４２＞で得られた形質転換株を、アンピシリン５０μｇ／ｍｌ）テトラサイクリン１２．５μｇ／ｍｌを含むＬＢ培地６０ｍｌに３０℃で一晩振盪培養し、この培養液２５ｍｌをアンピシリン５０μｇ／ｍｌを含むＬＢ培地１０００ｍｌに加えて、ＯＤはＡ_６００が１．０−１．２に至るまで３０℃で振盪培養した。次いで最終温度が、４２℃になるように６５℃の約３３０ｍｌＬＢ培地を添加し、さらに３時間４２℃で振盪培養してｈ６Ｔ（１−１６３）の発現を誘導した。
＜実施例４４＞
大腸菌で発現した、ヒトＴＰＯ塩基配列をコードするクローンｐＣＦＭ５３６／ｈ６Ｔ（１−１６３）由来ｈ６Ｔ（１−１６３）の精製と活性確認
ｈ６Ｔ（１−１６３）生産組換体凍結菌体３．６ｇに水１０ｍｌを加えてけん濁し、高圧破砕機で菌体を破砕した。遠心分離により沈殿画分を回収し、大部分の混在蛋白質、菌体成分等を除去する。回収されたｈ６Ｔ（１−１６３）を含む沈殿画分に水７ｍｌを加えてけん濁した後、攪拌しながら１Ｍ Ｔｒｉｓ緩衝液ｐＨ８．５を３ｍｌを加えた後、尿素（終濃度８Ｍ）、塩酸グアニジン（終濃度６Ｍ）、Ｎ−ラウロイルサルコシンナトリウム（終濃度２％）を各々加えて室温にて５−２０分間攪拌して可溶化した。これを２０ｍＭ Ｔｒｉｓ緩衝液ｐＨ８．５で１０倍希釈し、４℃中にて一晩でタンパク質の巻き戻し（リフオールディング）操作を行なった。この際に、空気酸化の他、添加物としてグルタチオン及び硫酸銅を各々添加した。遠心分離により、上清中にｈ６Ｔ（１−１６３）を回収した。各々の回収画分について還元剤存在下でのＳＤＳ−ＰＡＧＥで分析した結果、どの方法に於ても、この画分の主タンパク質バンドとして分子量約１８Ｋダルトンのバンドが検出された（純度３０−４０％）。このバンドについて実施例１に記載された方法により、ＳＤＳ−ＰＡＧＥ後、ＰＶＤＦ膜に転写しＮ末端シークエンス分析を行なった結果、このｐＣＦＭ５３６／ｈ６Ｔ（１−１６３）由来の変異型ＴＰＯとして発現されるべきＴＰＯの配列を含むことが確認できた。
各々の画分をＩＭＤＭ培養液に対して十分に透析後、ラットＣＦＵ−ＭＫ系で評価した結果、全ての画分に於て容量依存的にＴＰＯ活性を認めた。
＜実施例４５＞
抗ＴＰＯペプチド抗体の作製と、ＳＤＳ−ＰＡＧＥ→ウエスターン分析によるＴＰＯの検出
抗ＴＰＯ抗体が作製できれば、免疫学的手法によりＴＰＯの蛋白質を検出することができる。そこで、最初に判明したラットＴＰＯのアミノ酸配列のうち、比較的抗原として適していると考えられた３カ所の領域（表１７に示す）を選び、Ｔａｍ （Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ，ＵＳＡ，８５，５４０９−５４１３，１９８８）の方法により４本鎖のＭｕｌｔｉｐｌｅ ＡｎｔｉｇｅｎＰｅｐｔｉｄｅ（ＭＡＰ）型のペプチドを合成し、１００μｇずつ８回にわたってそれぞれウサギ２把に免疫した結果、これらの抗血清を得ることができた。合成ペプチド抗原に含まれるアミノ酸配列
【表１７】

次いで、これらの抗血清のうち、まず抗ＲＴ１ペプチド、抗ＲＴ２ペプチド抗体について、プロテインＡカラム（ＰＲＯＳＥＰ−Ａ；Ｂｉｏｐｒｏｃｅｓｓｉｎｇ Ｌｔｄ社製、カタログ番号８４２７）を用いて、ＩｇＧ画分（それぞれ２３４４ｍｇ、１９２０ｍｇ）を得、これらのうちそれぞれ５４ｍｇ、３２ｍｇをとり活性型ビオチン（ＮＨＳ−ＬＣ−Ｂｉｏｔｉｎ ＩＩ、ＰＩＥＲＣＥ社製、カタログ番号２１３３６）とカップリングすることにより、ビオチン化した。。実施例１に述べた方法と同様に、組み換え体ＴＰＯを含む標品をＳＤＳ−ＰＡＧＥにかけ、次いでＰＶＤＦあるいは、ニトロセルロース膜にエレクトロブロティングし、定法によりこれらのビオチン化抗体を一次抗体として用いてウェスタン分析を行った。
即ち、ブロッティング後の膜を２０ｍＭ Ｔｒｉｓ−ＨＣｌ、０．５Ｍ ＮａＣｌ（ｐＨ７．５）（ＴＢＳ）で５分洗浄し、０．１％Ｔｗｅｅｎ２０入りＴＢＳ（ＴＴＢＳ）で５分２回洗浄後、ブロッキング剤（ＢｌｏｃｋＡｃｅ、大日本製薬社製、カタログ番号ｕｋ−Ｂ２５）で６０分処理した。次に１０μｇ／ｍｌの濃度のビオチン化抗ＴＰＯぺプチド抗体、０．０５％ＢＳＡ、１０％ＢｌｏｃｋＡｃｅを含むＴＴＢＳ溶液で６０分後処理後、ＴＴＢＳで５分２回洗浄した。次にアルカリフォスファターゼ標識アビジン（Ｌｅｉｎｃｏ Ｔｅｃｈｎｏｌｏｇｉｅｓ社製、カタログ番号Ａ１０８）を１０％ＢｌｏｃｋＡｃｅ、ＴＴＢＳ溶液で５０００倍希釈したものを含む溶液で３０分処理し、５分２回のＴＴＢＳ洗浄、５分間のＴＢＳ洗浄を行った後、アルカリフォスファターゼ基質（Ｂｉｏ−Ｒａｄ社製、カタログ番号１７０−６４３２）により発色させた。以上のウエスタン分析は室温にて実施した。
この結果、ＣＯＳ１細胞で発現した各種組み換えラットＴＰＯのみならず、ＣＯＳ１細胞及び大腸菌で発現した各種組み換えヒトＴＰＯ（具体的には、これまでの実施例に述べたＣＯＳ１細胞で発現したプラスミドｐＨＴＰ１あるいはプラスミドｐＨＴＦ１由来ヒトＴＰＯとそのＮ結合型糖鎖切断後のＴＰＯ、大腸菌で発現したＧＳＴ−ＴＰＯ（１−１７４）及びそのトロンビン消化後のＴＰＯ、大腸菌で発現した変異型ＴＰＯであるｈ６Ｔ（１−１６３））をも認識することができた。そして、これら各種組み換えヒトＴＰＯの分析に用いることが可能となった。
以上の手法と同様にして、抗ヒトＴＰＯぺプチド抗体の作製が可能であることが示された。これらの手法で得た抗体により、ウェスタン分析のみならず、抗体カラムによるＴＰＯの精製をはじめ、通常考えられる抗体を用いたあらゆる免疫学的手法への応用が可能となる。
また、配列番号６に示されるヒトＴＰＯのアミノ酸配列のうち、比較的抗原として適していると考えられた６カ所の領域（表１９に示す）を選び、Ｔａｍ（Ｐｒｏｃ．Ｎａｔｌ，Ａｃａｄ．Ｓｃｉ．ＵＳＡ，８５，５４０９−５４１３，１９８８）の方法により４本鎖のＭｕｌｔｉｐｌｅ Ａｎｔｉｇｅｎ Ｐｅｐｔｉｄｅ（ＭＡＰ）型のペプチドを合成し、１００μｇずつ８回にわたってそれぞれウサギ２把に免疫した。
表１９で示された各ペプチド領域のＣ末端にシステイン残基を結合させた１本鎖ペプチドを別途合成し、これを試験抗原として酵素免疫測定法を用いて抗体価を調べたところ、いずれの血清についても抗体価の上昇が確認されたので、これらを抗血清とした。
抗体は、１種の抗原ペプチドにつきウサギ抗血清２把ずっ免疫したため、それぞれのウサギごとに抗体を分けて調製した。具体的には、これら由来するウサギの個体ごとに区別して、抗ＨＴ１ペプチド抗体では、それぞれ、抗ＨＴ１−１ペプチド抗体、抗ＨＴ１−２ペプチド抗体の様に称する。
以下に抗ＨＴ１−１ペプチド抗体についての精製を例として示す。
まず、システイン残基を結合したＨＴ１の１本鎖ペプチド３０ｍｇを１２ｍｌのＳｕｌｆｏＬｉｎｋカップリングゲル（Ｐｉｅｒｃｅ社製、カタログ番号４４８９５）に結合させた。即ち、ゲル体積の６倍容のカップリングバッファー（５０ｍＭＴｒｉｓ）５ｍＭ ＥＤＴＡ−Ｎａ ｐＨ８．５）で平衡化したゲルに、抗原の含まれるペプチド溶液を１５分間カップリングさせた。次に３０分間静置した後、ゲル体積の３倍容のカップリングバッファーでゲルを洗浄した。次に０．０５Ｍ Ｌ−Ｃｙｓｔｅｉｎ−ＨＣｌを含むカップリングバッファーを、１ｍｌ／ｍｌゲルの割合で添加し、１５分間未反応基をブロックした。次に３０分間静置した後、ゲル体積の８倍容のカップリングバッファーでゲルを洗浄した。以上のカップリングは室温で行った。このようにして、抗原領域を含むペプチドをカップリング効率２８．３％にてゲルに共有結合させ、１ｍｌゲル当たりに結合したペプチドが０．８ｍｇである抗原ペプチド抗原カラムを調製した。次に、全採血後の抗ＨＴ１−１ペプチド抗体を含む抗血清７８．４ｍｌのうち７６．７ｍｌ（蛋白質量３６２０ｍｇ）を予め１５０ｍＭのＮａＣｌ、０．０５％アジ化ナトリウムを含む５０ｍＭリン酸緩衝液（ｐＨ８）で平衡化した抗原カラムに添加し、さらに同緩衝液で洗浄後１０５．９ｍｌの素通り画分（蛋白質量３６８０ｍｇ）を得た。次に０．１Ｍクエン酸緩衝液（ｐＨ３．０）で吸着画分を溶出し、ただちに２１．１ｍｌの０．１Ｍ炭酸緩衝液（ｐＨ９．９）を加えて中和後、限外濾過（アミコン社製ＹＭ３０膜）にて濃縮し、１１．２ｍｌ（蛋白質量７７．７ｍｇ）の１５０ｍＭのＮａＣｌ、０．０５％アジ化ナトリウムを含む５０ｍＭリン酸緩衝液（ｐＨ８）の溶液中に精製された抗ＨＴ１−１ペプチド抗体を得ることができた。
同様にして、抗ＨＴ１−２ペプチド抗体（６０．０ｍｇ）、抗ＨＴ２−１ペプチド抗体（１８．８ｍｇ）、抗ＨＴ２−２ペプチド抗体（８．２ｍｇ）などを得ることができた。抗ＨＴ３〜６ペプチド抗体も同様にして得ることができる。
アフィニティ精製した抗ＨＴ１−１ペプチド抗体、抗ＨＴ１−２ペプチド抗体、抗ＨＴ２−１ペプチド抗体、抗ＨＴ２−２ペプチド抗体をそれぞれ３ｍｇをとり活性型ビオチン（ＮＨＳ−ＬＣ−Ｂｉｏｔｉｎ ＩＩ、ＰＩＥＲＣＥ社製、カタログ番号２１３３６）とカップリングすることにより、ビオチン化した。
【表１９】

のアミノ酸配列をコードする遺伝子を導入・発現させたＣＨＯ細胞の培養上清から部分精製された組換えヒトＴＰＯ標品を定法に従い、ＳＤＳ−ＰＡＧＥにかけ、次いでＰＶＤＦあるいはニトロセルロース膜にエレクトロブロテイングし、上述の抗ＲＴペプチド抗体の場合と同様にウェスタン分析を行ったところ、それぞれの精製された抗体により、ヒトＴＰＯが認識、検出されることが確認できた。＜実施例４６＞
抗ＴＰＯペプチド抗体カラムの調製
実施例４５で得られた抗ラットＴＰＯペプチド抗体は、ラット及びヒトＴＰＯを認識することができたため、抗ＲＴＩペプチド抗体、抗ＲＴ２ペプチド抗体の免疫グロブリン（ＩｇＧ）画分を以下のようにして、クロマトグラフィー坦体に結合させ、抗ＴＰＯペプチド抗体カラムを作製した。材料となった抗体は、それぞれの抗原ペプチドにっきウサギ抗血清２把ずつから由来し、それぞれのウサギごとに抗体を分けて調製した。具体的には、抗ＲＴ１ペプチド抗体では、それぞれ、抗ＲＴ１−１ペプチド抗体と抗ＲＴ１−２ぺプチド抗体と称し、抗ＲＴ２ペプチド抗体では、抗ＲＴ２−１ペプチド抗体と抗ＲＴ２−２ペプチド抗体と称す。これらを別々にして抗体カラムを調製したため、抗ＲＴ１ペプチド抗体カラムは２種（それぞれ抗ＲＴ１−１抗体カラム、抗ＲＴ１−２抗体カラムと称す）、抗ＲＴ２ペプチド抗体カラムは２種（それぞれ抗ＲＴ２−１抗体カラム、抗ＲＴ２−２抗体カラムと称す）、さらに抗ＲＴ１−２ペプチド抗体と抗ＲＴ２−１ペプチド抗体を混合してゲルにカップリングした抗体カラム（抗ＲＴ１−２＋２−１混合抗体カラム）、の５種類を調製した。
各々の抗体を５ｍｇ／ｍｌ（抗ＲＴ１＋２ｍｉｘ抗体カラムでは抗ＲＴ１−２ペプチド抗体と抗ＲＴ２−１ペプチド抗体を等量混合した２．５ｍｇ／ｍｌ）の濃度で含む５０ｍＭ Ｎａ Ｐｈｏｓｐｈａｔｅ，０．１５Ｍ ＮａＣｌ（ｐＨ８．０）の溶液とし、これを２．３１ｍｌとり、１．５４ｍ体積の膨潤したホルミル活性化ゲル（Ｆｏｒｍｙｌ−Ｃｅｌｌｕｌｏｆｉｎｅ、チッソ株式会社製）と合わせ、４℃で２時間カップリング反応させた。次いで、１０ｍｇ／ｍｌの濃度の還元剤溶液（Ｔｒｉｍｅｔｈｙｌａｍｉｎｅ ｂｏｒａｎｅ（ＴＭＡＢ）、生化学工業製、カタログ番号６８０２４６）を１．１ｍｌ加え、さらに６時間カップリング反応させた後、遠心分離によりゲル部分のみを回収した。これに１０ｍｌの精製水を加え、遠心分離でゲル部分のみの回収といった操作を４回繰り返し、未反応の抗体を取り除いた。次に４．６ｍｌのブロッキング緩衝液（０．２Ｍ Ｎａ Ｐｈｏｓｐｈａｔｅ，１Ｍ ｅｔｈａｎｏｌ ａｍｉｎｅ（ｐＨ７．０））と１，１ｍｌの還元剤溶液を加え、４℃で２時間以上処理することにより、未反応のゲルの活性基をブロックした。最後にゲルを遠心分離を用いて精製水、ＤＰＢＳで洗浄し、小カラムチューブに充填し、３Ｍチオシアン酸カリウム溶液、０．１Ｍグリシン−ＨＣｌ（ｐＨ２．５）溶液で洗浄後、再度ＤＰＢＳで再平衡化して保存した。
抗ＲＴ１−１抗体カラム、抗ＲＴ１−２抗体カラム、抗ＲＴ２−１抗体カラム、抗ＲＴ２−２抗体カラム、抗ＲＴ１−２＋２−１混合抗体カラムの５種類それぞれの抗ＴＰＯペプチド抗体ゲルでは、各ＩｇＧ画分のカップリング効率は順に９７．４％、９５．４％、９８．４％、９８．３％、９９．４％に達した。また、それぞれのゲル体積当たりにカップリングしたＩｇＧ画分量は、５．６ｍｇ／ｍｌゲル、５．８ｍｇ／ｍｌゲル、５．７ｍｇ／ｍｌゲル、５．７ｍｇ／ｍｌゲル、２．９ｍｇ／ｍｌゲルであった。従って、抗体の由来や抗原の違いによって、カップリング効率やカップリング量に大きな差がないことが確認できたため、これらの抗体カラムを利用して、以下、実施例４７に示す実験を行った。
＜実施例４７＞
発現ベクターｐＨＴＰ１をＣＯＳ１細胞にトランスフェクションして得られた培養上清由来ＴＰＯの抗ＴＰＯ抗体カラム→逆相カラムクロマトグラフィーでの精製〜生物学的活性の確認
以下の精製サンプルの評価のためのｉｎ ｖｉｔｒｏアッセイ方法は、Ｍ−０７ｅアッセイ系を用いた。発現ベクターｐＨＴＰ１をＣＯＳ１細胞にトランスフェクションして得られた、実施例３５の培養上清由来ＴＰＯの部分精製標品（ＴＰＯが回収された主たる画分ではない画分、即ち、Ｍａｃｒｏ−Ｐｒｅｐ Ｍｅｔｈｙｌ ＨＩＣカラムの素通り画分Ｆ１と、Ｆ２の後に２０ｍＭ Ｎａ Ｃｉｔｒａｔｅ，ｐＨ５．８でカラム洗浄し溶出したＦ３、ＳＰ Ｓｅｐｈａｒｏｓｅ Ｆａｓｔ ＦｌｏｗカラムのＦ１とＦ３をまとめてＴＰＯサブプール画分（５４６３．７９ｍｌ，蛋白質濃度０．４９０ｍｇ／ｍｌ，総蛋白質量２６７６ｍｇ，相対活性１２１００，相対活性量３２３８００００）とし、限外濾過ユニット（フィルトロン社製・オメガウルトラセット 分子量８０００カット）でまず濃縮し、さらに溶媒をＤＰＢＳに置換し、小体積になってからはＹＭ−１０膜付き限外濾過ユニット（アミコン社製）を用いて最終的に１２０．２ｍｌの０．０５％アジ化ナトリウムを含むＤＰＢＳ溶液にした。次に実施例４６で調製した５種類の抗ＴＰＯペプチド抗体ゲルを全て混合し、１本の抗体カラム（直径１．６ｃｍ，ベッド高４．８ｃｍ，抗ＲＴ１＋２混合抗体カラムと称す）にまとめ、室温にて流速０．０３３ｍｌ／ｍｉｎでＴＰＯサブプール画分を添加した。添加終了後、ＤＰＢＳで溶出液の紫外線吸収が十分下がるまでを集め、さらにＹＭ−１０膜付き限外濾過ユニット（アミコン社製）で濃縮した素通り画分Ｆ１（８２．６２ｍｌ，蛋白質濃度１４．３ｍｇ／ｍｌ，総蛋白質量１１８４ｍｇ，相対活性６７５００，相対活性量７９９０００００）を集めた。次に酸性溶離液（０．１Ｍグリシン−ＨＣｌ（ｐＨ２．５））で溶出されるカラム吸着画分Ｆ２（９２．９７ｍｌ，蛋白質濃度０．１２ｍｇ／ｍｌ，総蛋白質量１１．１ｍｇ，相対活性２５７１００，相対活性量２８６００００）を溶出した。素通り画分Ｆ１には相当量のＴＰＯ活性があったが、抗体カラムに吸着したＴＰＯについては約２０倍の相対活性の上昇が認められたため、さらに精製を行った。即ち、展開溶媒Ａに０．１％ＴＦＡ、展開溶媒Ｂに０．０５％ＴＦＡを含む１−プロパノールを用いたＣａｐｃｅｌｌ Ｐａｋ Ｃ１ ３００Ａ（資生堂製、カタログ番号Ｃ１ ＴＹＰＥ：ＳＧ３００Ａ；直径４．６ｍｍ、ベッド高１５０ｍｍに加え、直径４．６ｍｍ）ベッド高３５ｍｍのプレカラムを接続したもの）カラムにて、抗体カラムで得られたＦ２に１０分の１体積の展開溶媒Ｂを加え、２０％Ｂで平衡化したＣａｐｃｅｌｌ Ｐａｋ Ｃ１ ３００Ａカラムに流速０．４ｍｌ／ｍｉｎで注入した。注入終了後、２０％Ｂにて５分溶出した後、２０％Ｂから４０％Ｂまで５０分の直線濃度勾配で展開し、１ｍｌ（２．５ｍｉｎ）ずつポリプロピレン製チューブに集めた。
それぞれ２μｌ（５００分の１フラクション）を取りＨＳＡを加え、限外濾過濃縮し、最終的に０．０２％ＨＳＡを含む０．２５ｍｌのＩＭＤＭアッセイ培養液溶液とし、これをアッセイにかけ、ＴＰＯ活性画分を特定した。この結果、チューブ番号２０〜２３（プロパノール濃度で２８．５〜３２．５％の範囲）に強いＴＰＯの活性を示す標品を得ることができた。またこれらの標品を＜実施例４５＞に述べた方法によりウエスターン分析をしたところ、ＤＰＣＩＩＩ分子量マーカーに対し、還元下において見かけ上の分子量が６００００〜７００００のＴＰＯが確かに存在し、これとは別に、３２０００〜４３０００、２００００〜３００００の分子量をもっ分子も存在していることが判明した。
＜実施例４８＞
発現ベクターｐＨＴＰ１をＣＯＳ１細胞にトランスフェクションして得られた培養上清由来で、Ｃａｐｃｅｌｌ Ｐａｋ Ｃ１ ３００Ａカラムの段階まで精製されたＴＰＯの生物学的活性の確認
実施例３６で精製されたＴＰＯ活性画分、即ちＣａｐｃｅｌｌ Ｐａｋ Ｃ１３００Ａカラムのチューブ番号２０〜２３（プロパノール濃度で２８．５〜３２．５％の範囲）までを集め、０．２１ｍｌのグリセロールを添加後遠心エバポレーションで濃縮した。これに６Ｍ塩酸グアニジン溶液０．２１ｍｌを加えたものをＤＰＢＳで１ｍｌに希釈後、Ｓｅｐｈａｄｅｘ Ｇ２５カラム（ＮＡＰ−１０，ファルマシア バイオテック社製、カタログ番号１７−０８５４−０１）で０．０１％ＨＳＡを含むＤＰＢＳ溶液に置換し、さらに１．１ｍｌの０．０１％ＨＳＡを含むＤＰＢＳを加えて、最終的にＴＰＯ活性画分ＦＡ（２．６ｍｌ）を調製した。この標品の生物学的ＴＰＯ活性をについて調べるために、ｉｎ ｖｉｖｏアッセイを行った。即ち、１群４匹のＩＣＲ系雄性マウス（８週齢）に、活性画分１００μｌ（Ｍ−０７ｅアッセイ系での相対活性量８７４００を含む）を１日１回、５日間連日皮下投与した。対照として、０．０１％ＨＳＡを含むＤＰＢＳ１００μｌを同様のスケジュールで皮下投与した。採血は、投与開始直前及び投与終了翌日に眼底より行い、血球測定装置（東亜医用電子製、Ｆ８００）を用いて血小板数を測定した。ＴＰＯ投与群では、投与終了後において投与前と比較して平均で１．４２倍の血小板の増加を認め、対照群の血小板数と比較しても１．２３倍の高値を示し、両者の間に有意差（ｐ＜０．０５，Ｓｔｕｄｅｎｔ ｔ−ｔｅｓｔ）を認めた。この結果から、動物細胞で生産された上記のヒトＴＰＯが、生体内において血小板増加作用を有することが明らかとなった。
＜実施例４９＞
発現ベクターｐＨＴＰ１をＣＯＳ１細胞にトランスフェクションして得られた、培養上清３３Ｌを出発材料にし、陽イオン交換カラムで得た粗精製ＴＰＯ画分の生物学的活性の確認
（１）以下の精製サンプルの評価のためのｉｎ ｖｉｔｒｏアッセイ方法は、Ｍ−０７ｅアッセイ系を用いた。実施例３６で記載された方法と同様にして、３３Ｌの発現ベクターｐＨＴＰ１をＣＯＳ１細胞にトランスフェクションして得られた無血清培養上清を得て、０．２２μｍの濾過フィルターの濾液をとった。これを限外濾過ユニット（ミリポア社製・ＰＬＧＣペリコンカセット 分子量１００００カット）で約１０倍濃縮し、蛋白質分解酵素阻害剤であるｐ−ＡＰＭＳＦを最終濃度約１ｍＭ加え、体積２０１８ｍｌ（蛋白質濃度３．２２ｍｇ／ｍｌ、総蛋白質量６５０２ｍｇ、相対活性６６０００、相対活性量４２９１０００００）を得た。次に、限外濾過ユニット（フィルトロン社製・オメガウルトラセット 分子量３００００カット）で濃縮工程を繰り返し、分子量３００００以上の画分（体積１１９０ｍｌ）蛋白質濃度２．５４ｍｇ／ｍｌ、総蛋白質量３０２０ｍｇ、相対活性８２５００、相対活性量２４９００００００）と、分子量３００００以下の画分（体積２９７５ｍｌ、蛋白質濃度０．４７１ｍｇ／ｍｌ、総蛋白質量１４０２ｍｇ、相対活性４５００、相対活性量６３１００００）を得ることができた。分子量３００００以下の画分にＴＰＯ活性が存在することは、ＴＰＯの動物細胞での発現・分泌の過程において、最終的に培養上清中に、低分子化したＴＰＯが含まれている可能性を示す。一方、分子量３００００以上の画分をさらに２０ｍＭクエン酸ナトリウム緩衝液（ｐＨ６．１）に置換し、２０ｍＭクエン酸ナトリウム緩衝液（ｐＨ６．１）で予め平衡化したＳＰ Ｓｅｐｈａｒｏｓｅ Ｆａｓｔ Ｆｌｏｗ（ファルマシア バイオテク社製、カタログ番号１７−０７２９−０１；直径２．６ｃｍ、ベッド高２９ｃｍ）カラムに注入し、流速５ｍｌ／ｍｉｎで添加した。添加終了後、さらに２０ｍＭクエン酸ナトリウム緩衝液（ｐＨ６．１）で洗浄溶出した。次に展開溶媒Ａに２０ｍＭクエン酸ナトリウム緩衝液（ｐＨ６．１）、展開溶媒Ａ中に１Ｍ ＮａＣｌを含む溶液を展開溶媒Ｂとして用い、流速３ｍｌ／ｍｉｎで０％Ｂから５０％Ｂまで２１５分間、さらに５０％Ｂから１００％Ｂまで２０分の直線濃度勾配で展開し、３０ｍｌ（１０ｍｉｎ）ずつポリプロピレン製チューブに集めた。これらの一部を取り、Ｍ−０７ｅアッセイ系で活性の分布を調べたところ、広範囲にわたりＴＰＯ活性が溶出されたことが判明した。そこで素通りを含め、５０ｍＭ以下のＮａＣｌで溶出された画分をＦ１、ＴＰＯの主画分として５０〜１０００ｍＭのＮａＣｌで溶出された画分をＦ２としてまとめた。Ｆ１は体積１９５１ｍｌ（蛋白質濃度２．０５ｍｇ／ｍｌ、総蛋白質量３９９４ｍｇ、相対活性１３５００、相対活性量５３９０００００）、Ｆ２は体積６４９．８ｍｌ（蛋白質濃度１．１１ｍｇ／ｍｌ、総蛋白質量７２１ｍｇ、相対活性２６８０００、相対活性量１９３００００００）であった。
（２）ＳＰ Ｓｅｐｈａｒｏｓｅ Ｆａｓｔ Ｆｌｏｗ Ｆ２の生物学的活性の確認 （１）で分取したＳＰ Ｓｅｐｈａｒｏｓｅ Ｆａｓｔ ＦｌｏｗのＦ２を２．５ｍｌ取り、Ｓｅｐｈａｄｅｘ Ｇ２５カラム（ＮＡＰ−２５，ファルマシア バイオテック社製、カタログ番号１７−０８５２−−０１）で０．０１％ＨＳＡを含むＤＰＢＳ溶液３．５ｍｌに置換し、このサンプル（蛋白質濃度１．４６ｍｇ／ｍｌ）の生物学的ＴＰＯ活性について調べるために、ｉｎｖｉｖｏアッセイを行った。即ち、１群４匹のＩＣＲ系雄性マウス（８週齢）に、活性画分１００μ１（Ｍ−０７ｅアッセイ系での相対活性量３１５０を含む）を１日１回、５日間連日皮下投与した。対照として、０．０１％ＨＳＡを含むＤＰＢＳ１００μｌを同様のスケジュールで皮下投与した。採血は、投与開始直前及び投与終了翌日に眼底より行い、血球測定装置（東亜医用電子製、Ｆ８００）を用いて血小板数を測定した。ＴＰＯ投与群では、投与終了後において投与前と比較して平均で１．２９倍の血小板の増加を認め、対照群の血小板数と比較しても１．１２倍の高値を示し、両者の間に有意差（ｐ＜０．０５，Ｓｔｕｄｅｎｔ ｔ−ｔｅｓｔ）を認めた。この結果から、動物細胞で生産された上記のヒトＴＰＯが、生体内において血小板増加作用を有することが明らかとなった。
＜実施例５０＞
ヒトＴＰＯ染色体ＤＮＡのＣＨＯ細胞用組換え発現ベクター、ｐＤＥＦ２０２−ｇｈＴＰＯの構築
ベクターｐＤＥＦ２０２を制限酵素ＫｐｎｌとＳｐｅｌで処理し、アガロースゲル電気泳動で小さい方のマウスＤＨＦＲミニ遺伝子とＳＶ４０ポリアデニル化シグナルを含むフラグメントを回収したのち、このフラグメントとヒトＴＰＯ染色体ＤＮＡを含むプラスミドｐＥＦＨＧＴＥを制限酵素ＫｐｎＩとＳｐｅＩで処理しＳＶ４０ポリアデニル化シグナルを含む領域を除去したべクターＤＮＡとをＴ４ＤＮＡリガーゼ（宝酒造製）で結合させ、発現ベクターｐＤＥＦ２０２−ｇｈＴＰＯを得た。このプラスミドはＳＶ４０の複製開始領域、ヒトエロンゲーションファクタ−１−アルファプロモーター、ＳＶ４０初期ポリアデニル部位、マウスＤＨＦＲミニ遺伝子、ｐＵＣ１８の複製開始領域、β−ラクタマーゼ遺伝子（Ａｍｐ^ｒ）を含み、ヒトエロンゲーションファクタ−１−アルファプロモーター下流にヒトＴＰＯ染色体ＤＮＡが接続されている。
＜実施例５１＞
ＣＨＯ細胞によるヒトＴＰＯ染色体ＤＮＡの発現
ＣＨＯ細胞（ｄｈｆｒ−株、ＵｒｌａｕｂとＣｈａｓｉｎ；Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ；７７巻４２１６頁、１９８０）を６ｃｍ径のプレート（Ｆａｌｃｏｎ社製）中１０％牛胎児血清を含むα最小必須培地（α−ＭＥＭ（−）、チミジン、ヒポキサンチン添加）で培養増殖させ、これをリン酸カルシウム法（ＣｅｌｌＰｈｅｃｔ）ファルマシア社製）によって形質転換した。すなわち、実施例５０で調製したｐＤＥＦ２０２−ｇｈＴＰ０プラスミド１０μｇにバッファーＡ：１２０μｌおよびＨ２０：１２０μｌを加え混合したのち、室温で１０分間放置した。つぎに、この溶液にバッファーＢ：１２０μｌを加え、再度混合したのち、室温で３０分間放置した。このＤＮＡ溶液をプレートに滴下したのち、ＣＯ_２インキュベーター中で６時間培養した。プレートから培地を除去し、α−ＭＥＭ（−）にて２回洗浄後、１０％ジメチルスルフォオキシド含有α−ＭＥＭ（−）を添加し、室温で２分間処理した。次いで、１０％透析牛胎児血清含有非選択培地（前出α−ＭＥＭ（−）、ヒポキサンチン、チミジン添加）を添加して２日間培養したのち、１０％透析牛胎児血清含有選択培地（α−ＭＥＭ（−）、ヒポキサンチン、チミジン無添加）での選択をおこなった。選択は細胞をトリプシン処理した後、６ｃｍ径プレート１枚あたりを、１０ｃｍ径プレート５枚あるいは２４ウエルプレート２０枚に分割したのち、２日ごとに選択培地にて培地交換を行いながら培養を続行する事により実施した。細胞が増殖してきたプレートあるいはウエルについてその培養上清中のヒトＴＰＯ活性をＢａ／Ｆ３アッセイを用いて測定した結果、ヒトＴＰＯ活性が認められた。
なお、ＣＨＯ細胞の形質転換はＣＨＯ細胞に対しｐＥＦＨＧＴＥとｐＭＧ１を同時形質転換（ｃｏ−ｔｒａｎｓｆｅｃｔｉｏｎ）することによっても行うことができる。
＜実施例５２＞
−１位にＬｙｓ、−２位にＭｅｔが付加されたヒトＴＰＯ（アミノ酸１−１６３）（以下、このタンパク質を「ｈＭＫＴ（１−１６３）」と称す）の大腸菌用発現ベクターの構築〜発現・発現の確認
配列番号６のアミノ酸配列（１−１６３位）の−１位にＬｙｓ）−２位にＭｅｔが付加されたタンパク質を発現するため、実施例４２と同様にして、以下の表１８に示した１−１３、２−１３、３−３、４−３の合成オリゴヌクレオチドを用いて、ｈＭＫＴ（１−１６３）の大腸菌用発現べクターｐＣＦＭ５３６／ｈＭＫＴ（１−１６３）を構築した。
【表１８】

この発現プラスミドは、配列表（配列番号１２）に示されたＤＮＡ配列を含んでいる。さらに、実施例４３と同様にして、ｈＭＫＴ（１−１６３）の発現を誘導した。
ここで得られた発現タンパクをＳＤＳ−ＰＡＧＥ後ＰＶＤＦ膜に転写し、Ｎ末端アミノ酸配列分析を行った結果、発現されるべきｈＭＫＴ（１−１６３）のアミノ酸配列を含むことが確認できた。
＜実施例５３＞
大腸菌で発現した、ヒトＴＰＯ塩基配列をコードするクローンｐＣＦＭ５３６／ｈ６Ｔ（１−１６３）由来変異型ヒトＴＰＯ、ｈ６Ｔ（１−１６３）の塩酸グアニジンとグルタチオンを用いたリフォールディング〜精製〜生物学的活性の確認実施例４３で調製されたｈ６Ｔ（１−１６３）生産組換体凍結菌体１．２ｇに水３ｍｌを加えてけん濁し、高圧破砕機で菌体を破砕した。遠心分離により沈殿画分を回収し、大部分の混在蛋白質、菌体成分等を除去する。回収されたｈ６Ｔ（１−１６３）を含む沈殿画分に水を加えてけん濁し、最終４ｍｌとした。攪拌しながら１ｍｌの１ＭＴｒｉｓ緩衝液ｐＨ８．５を加えた後、２０ｍｌの８Ｍ塩酸グアニジンを加えて室温にて５分間攪拌して可溶化した。これを２０ｍＭＴｒｉｓ緩衝液ｐＨ８．５で１０倍希釈し、５ｍＭ還元型グルタチオンと０．５ｍＭの酸化型グルタチオンを加えて攪拌し溶かした後、４℃中にて一晩静置した。遠心分離により、上清中にｈ６Ｔ（１−１６３）を回収した。得られた上清のうち１６０ｍｌをＹＭ１０限外濾過膜（アミコン社製）を用いて濃縮し、ＤＰＢＳにて緩衝液を置換して最終容量３．４ｍｌとした（蛋白濃度２．１８ｍｇ／ｍｌ）。この画分をＩＭＤＭ培養液に対して十分に透析後、ラットＣＦＵ−ＭＫアッセイ系で評価した結果、強いＴＰＯの活性（相対活性５８０００）を示した。
上記の方法により調製したＴＰＯ活性画分についてｉｎ ｖｉｖｏアッセィを行った。１群４匹のＩＣＲ系雄性マウス（７週齢）に、活性画分１７０μｌ（ラットＣＦＵ−ＭＫアッセイ系での相対活性量２２０００を含む）を１日１回、５日間連日皮下投与した。対照として、１群６匹の同様のマウスにＤＰＢＳ１７０ｐｌを同様のスケジュールで皮下投与した。採血は、投与開始直前及び投与終了翌日、終了３日後に眼底より行い、血球測定装置（東亜医用電子製、Ｆ８００）を用いて血小板数を測定した。ＴＰＯ投与群では、投与終了翌日において投与前と比較して平均で２．１５倍の血小板の増加を認め、また投与終了３日後においてもその効果は持続しており２．１３倍の高値を維持した。投与終了翌日及び３日後のいずれの日においても、血小板数は、それぞれの日の対照群のそれと比較して１．７３倍、１．８０倍と高値を示し、両者の間に有意差（ｐ＜０．００１，Ｓｔｕｄｅｎｔ ｔ−ｔｅｓｔ）を認めた。
この結果から、大腸菌より生産され、上記の方法で調製したヒトＴＰＯが、生体内において血小板増加作用を有することが明らかとなった。
＜実施例５４＞
大腸菌で発現した、ヒトＴＰＯ塩基配列をコードするクローンｐＣＦＭ５３６／ｈ６Ｔ（１−１６３）由来変異型ヒトＴＰＯＮｈ６Ｔ（１−１６３）のＮ−ラウロイルサルコシンナトリウムと硫酸銅を用いたリフォールディング〜精製〜生物学的活性の確認
実施例４３で調製されたｈ６Ｔ（１−１６３）生産組換体凍結菌体０．６ｇに水３ｍｌを加えてけん濁し、高圧破砕機で菌体を破砕した後、遠心分離により沈殿画分を回収した。沈殿画分に３．１ｍｌの水を加えて懸濁した後、０．１９ｍｌの１ＭＴｒｉｓ緩衝液ｐＨ９．２、１１．２５μｌの１Ｍ ＤＴＴ、３８μｌの０．５ＭＥＤＴＡ、０．３８ｍｌの１０％デオキシコール酸ナトリウムを攪拌しながら加え、室温にて４０分間攪拌した。遠心分離により沈殿画分を回収し、大部分の混在蛋白質、菌体成分等を除去した。沈殿に水４ｍｌを加えてけん濁した後、遠心分離によってｈ６Ｔ（１−１６３）を含む沈殿画分を回収した。得られた沈殿画分に水３．８ｍｌを加え懸濁した後、攪拌しながら０．２ｍｌの１ＭＴｒｉｓ緩衝液ｐＨ８と１ｍｌの１０％Ｎ−ラウロイルサルコシンナトリウムを加えて室温にて２０分間攪拌して可溶化した。これに５μｌの１％硫酸銅を加え、室温にて一晩（約２０時間）攪拌した。遠心分離によりｈ６Ｔ（１−１６３）を含む上清画分を回収した後、上清５ｍｌに５ｍｌの水、１０ｍｌの２０ｍＭＴｒｉｓ緩衝液ｐＨ７．７加えた後、２．６ｇのＤｏｗｅｘ １−ｘ４，２０−５０ｍｅｓｈ，ｃｈｌｏｒｉｄｅ ｆｏｒｍイオン交換樹脂を加え、９０分間撹拌した。グラスフィルターを用いてイオン交換樹脂非吸着画分を回収した後、遠心分離によって上清を回収した。得られた上清を２０ｍＭＴｒｉｓ緩衝液ｐＨ７．７で平衡化したＳＰ Ｓｅｐｈａｒｏｓｅ Ｆａｓｔ Ｆｌｏｗ陽イオン交換カラムに添加し、同緩衝液中でＮａＣｌ濃度０Ｍから５００ｍＭまでの直線濃度勾配法によって溶出を行なった。溶出画分をＩＭＤＭ培養液に対して十分に透析後、ラットＣＦＵ−ＭＫアッセイ系で評価した結果、ＳＰ Ｓｅｐｈａｒｏｓｅ Ｆａｓｔ Ｆｌｏｗ陽イオン交換クロマトグラフイーのＮａＣｌ濃度約１００ｍＭ付近で溶出された画分に強いＴＰＯの活性（相対活性約１９００００００）を示した。この画分をウルトラフリーＣＬ分画分子量５０００限外濾過ユニット（ミリポア社製、型番ＵＦＣ４ＬＣＣ２５）を用いて１．６倍濃縮した（２．５ｍｌ、蛋白濃度２５μｇ／ｍｌ）。同画分を還元剤存在下でＳＤＳ−ＰＡＧＥで分析した結果、ＴＰＯのバンドが主バンドとして検出された（純度７０−８０％）。
上記の方法により調製したＴＰＯ活性画分についてｉｎ ｖｉｖｏアッセイを行った。１群４匹のＩＣＲ系雄性マウス（８週齢）に、活性画分１００μｌ（ラットＣＦＵ−ＭＫアッセイ系での相対活性量４７５００を含む）を１日１回、５日間連日皮下投与した。対照として、１００ｍＭＮａＣｌを含む２０ｍＭＴｒｉｓ緩衝液ｐＨ７．７１００μｌ．を同様のスケジュールで皮下投与した。採血は、投与開始直前及び投与終了翌日に眼底より行い、血球測定装置（東亜医用電子製、Ｆ８００）を用いて血小板数を測定した。ＴＰＯ投与群では、投与終了後において投与前と比較して平均で１．７３倍の血小板の増加を認め、対照群の血小板数と比較しても１．５９倍の高値を示し、両者の間に有意差（ｐ＜０．０１，Ｓｔｕｄｅｎｔ ｔ−ｔｅｓｔ）を認めた。この結果から、大腸菌より生産され、上記の方法で調製したヒトＴＰＯが、生体内において血小板増加作用を有することが明らかとなった。
＜実施例５５＞
ＣＨＯ細胞の大量培養
実施例３２にぉぃてヒトＴＰＯ発現プラスミドｐＤＥＦ２０２−ｈＴＰ０−Ｐ１をＣＨＯ細胞にトランスフェクションして得られたヒトＴＰＯ産生ＣＨＯ細胞株（ＣＨＯ２８−３０細胞、２５ｎＭ ＭＴＸ耐性）の大量培養は以下のようにして実施した。細胞を２５ｎＭ ＭＴＸおよび１０％ＦＣＳを含むＤＭＥＭ／Ｆ−１２培地（ＧＩＢＣＯ社）を用いて培養増殖させた。この細胞をトリプシン溶液を用いて剥離したのち、２００ｍｌの同培地を含むＦａｌｃｏｎ社製ローラーボトル（Ｆａｌｃｏｎ３０００）に１×１０^７個の細胞を接種し、３７℃で１ｒｐｍの回転速度で３日間培養した。３日後、培養液を吸引除去し、１００ｍｌのＰＢＳで細胞培養表面をリンスしたのち、２５ｎＭ ＭＴＸおよび１０％ＦＣＳを含まないＤＭＥＭ／Ｆ−１２培地（ＧＩＢＣＯ社）を２００ｍｌ加え、３７℃、１ｒｐｍの回転速度で７日間培養し、７日後その培養上清を回収し、次の精製操作の出発材料にした。上記の操作をローラーボトル５００本分実施し、培養上清１００Ｌを得た。
＜実施例５６＞
ヒトＴＰＯ産生ＣＨＯ細胞株からヒトＴＰＯの精製
（１）実施例５５より無血清培養上清約１００Ｌを得て、０．２２μｍの濾過フィルターの濾液をとった。これを限外濾過ユニット（ミリポア社製・ＰＬＴＫペリコンカセット 分子量３００００カット）で濃縮かつ溶媒を精製水に置換し、蛋白質分解酵素阻害剤であるｐ−ＡＰＭＳＦ（和光純薬社製）を最終濃度１ｍＭ及びＰｅｆａｂｌｏｃ ＳＣ （Ｍｅｒｋ社製）を最終濃度０．３５ｍＭ加え、体積３６２８ｍｌ（蛋白質濃度１．６０ｍｇ／ｍｌ）総蛋白質量５８０５ｍｇ、相対活性１２３００００、相対活性量７１４９００００００）の分子量３００００以上の画分を得た。この画分を実施例４５で述べたウエスターン分析で調べたところ、分子量６６０００〜１０００００の範囲にＴＰＯ蛋白質が存在することが明らかとなった。さらによく調べてみると、これとは別に、より低分子のＴＰＯをも含まれていることが判明した。
分子量３００００以下の限外濾液はこれとは別に限外濾過ユニット（ミリポア社製・ＰＬＧＣペリコンカセット 分子量１００００カット）で濃縮したところ、体積１９０１ｍｌ（蛋白質濃度０．３６ｍｇ／ｍｌ、総蛋白質量６８４ｍｇ、相対活性２４５５００、相対活性量１６７９０００００）であったため、低分子化したＴＰＯ分子が培養工程において生じたことが確認された。
次に、分子量３００００以上の画分３６１４ｍｌに７６４ｇの硫酸アンモニウム、及び１４４．５ｍｌの０．５Ｍクエン酸ナトリウム緩衝液（ｐＨ５．５）を加え、最終濃度１．４１Ｍ硫酸アンモニウム、１７．７ｍＭのクエン酸ナトリウム緩衝液を含む４０８９ｍｌの溶液とし、生じた不溶物を遠心分離後可溶物を得た。
これを１．２Ｍ硫酸アンモニウムを含む２０ｍＭクエン酸ナトリウム緩衝液（ｐＨ５．５）で予め平衡化してあったＭａｃｒｏ−Ｐｒｅｐ Ｍｅｔｈｙｌ ＨＩＣカラム（Ｂｉｏ−Ｒａｄ社製、カタログ番号１５６−００８０；直径５ｃｍ、ベッド高 ２４．５ｃｍ）に、流速約２５ｍｌ／ｍｉｎで添加した。添加終了後、１．２Ｍ硫酸アンモニウムを含む２０ｍＭクエン酸ナトリウム緩衝液（ｐＨ５．５）で溶出されたものを限外濾過ユニット（フィルトロン社製・オメガウルトラセット 分子量８０００カット）で濃縮し、素通り画分Ｆ１（４４５５ｍｌ，蛋白質濃度０．４００ｍｇ／ｍｌ，総蛋白質量１７８０ｍｇ，相対活性１８３１０００）を得た。
次に、溶出液を２０ｍＭ Ｎａ Ｃｉｔｒａｔｅ，ｐＨ６．０にかえ、溶出されたものを限外濾過ユニット（フィルトロン社製・オメガウルトラセット 分子量８０００カット）で濃縮し、画分Ｆ２（１４５７ｍｌ，蛋白質濃度０．９６９ｍｇ／ｍｌ，総蛋白質量１４１１ｍｇ，相対活性１７１５０００）を集めた。ＳＤＳ−ＰＡＧＥにより、このＦ２には分子量６６０００〜１０００００の蛋白質が存在することが確認された。さらにウエスターン分析により、この蛋白質がＴＰＯであることが明らかとなった。
次に、Ｍａｃｒｏ−Ｐｒｅｐ Ｍｅｔｈｙｌ ＨＩＣカラム Ｆ２（１４４３ｍｌ）を、２０ｍＭクエン酸ナトリウム緩衝液（ｐＨ６．０）で予め平衡化したＳＰ Ｓｅｐｈａｒｏｓｅ Ｆａｓｔ Ｆｌｏｗ（ファルマシア バイオテク社製、カタログ番号１７−０７２９−０１；直径 ５ｃｍ、ベッド高 １２ｃｍ）カラムに注入し、流速１５ｍｌ／ｍｉｎで添加した。添加終了後、さらに５０ｍＭ ＮａＣｌを含む２０ｍＭクエン酸ナトリウム緩衝液（ｐＨ６．０）で溶出されたものまでをまとめ、画分Ｆ１（３００７ｍｌ，蛋白質濃度０．２２６ｍｇ／ｍｌ，総蛋白質量６７９ｍｇ，相対活性８８８３０）を得た。次に、溶出液を７５０ｍＭ ＮａＣｌを含む２０ｍＭクエン酸ナトリウム緩衝液（ｐＨ５．４）にかえ、溶出された画分Ｆ２（９３１ｍｌ，蛋白質濃度０．７６３ｍｇ／ｍｌ，総蛋白質量７１０ｍｇ，相対活性５５５８０００）を集めた。これを限外濾過ユニット（フィルトロン社製・オメガウルトラセット 分子量８０００カット、及びアミコン社製ＹＭ３膜）で２０２ｍｌまで濃縮した。
次に、ＳＰ Ｓｅｐｈａｒｏｓｅ Ｆａｓｔ ＦｌｏｗカラムのＴＰＯ活性画分Ｆ２の濃縮液（１９７ｍｌ）をＳｅｐｈａｃｒｙｌ Ｓ−２００ＨＲカラム（ファルマシア・バイオテク社製、カタログ番号１７−０５８４−０５；直径 ７．５ｃｍ、ベッド高 １００ｃｍ）に注入し、流速３ｍｌ／ｍｉｎでゲル濾過を行った。溶離液体積１２００〜１７８５ｍｌ、１７８５〜２０１０ｍｌ、２０１０〜２２８０ｍｌ、２２８０〜３０００ｍｌの各範囲をまとめ、それぞれ画分Ｆ１（５８５ｍｌ，蛋白質濃度１．００ｍｇ／ｍｌ，総蛋白質量５８９ｍｇ，相対活性４１１８０００）、画分Ｆ２（２２５ｍｌ，蛋白質濃度０．２６３／ｍｌ，総蛋白質量５９．２ｍｇ，相対活性２５０９０００）、画分Ｆ３（２７０ｍｌ，蛋白質濃度０．１１９／ｍｌ，総蛋白質量３２．１ｍｇ，相対活性２５３５０００）、画分Ｆ４（７２０ｍｌ， 蛋白質濃度０．０４６７／ｍｌ， 総蛋白質量３３．６ｍｇ，相対活性１１５５０００）を得た。
このように、分画されたＴＰＯ活性はゲル濾過法での分子量範囲が広いことが判明したため、ＳＤＳ−ＰＡＧＥ後、ウエスターン分析を実施したところ、Ｆ１ではほとんどが分子量６６０００〜１０００００のＴＰＯであったのに対し、Ｆ２、Ｆ３ではそれぞれ分子量３２０００〜６００００、分子量３２０００〜４２０００のＴＰＯ分子種が存在していた。いずれの分子量のＴＰＯ分子も全てＴＰＯ活性をもっていた。
なお、分子量６６０００〜１０００００のＴＰＯ分子についてＮ末端アミノ酸配列分析を実施したところ、ヒトＴＰＯ遺伝子でコードされるタンパク質のアミノ酸配列を含むことが確認できた。
また、分子量６６０００〜１０００００（７）ＴＰＯ分子について、Ｎ−グリカナーゼ（Ｎ−ｇｌｙｃａｎａｓｅ：Ｇｅｎｚｙｍｅ社製、カタログ番号１４７２−００）、ノイラミニダーゼ（Ｎｅｕｒａｍｉｎｉｄａｓｅ：ナカライテスク社製、カタログ番号２４２−２９ ＳＰ）、エンド−α−Ｎ−アセチルガラクトサミニダーゼ（Ｅｎｄｏ−α−Ｎ−ａｃｅｔｙｌｇａｌａｃｔｏｓａｍｉｎｉｄａｓｅ：生化学工業製、カタログ番号１００４５３）、およびＯ−グリコシダーゼ（Ｏ−Ｇｌｙｃｏｓｉｄａｓｅ：Ｂｏｅｈｒｉｎｇｅｒ Ｍａｎｎｈｅｉｍ Ｂｉｏｃｈｅｍｉｃａ社製、カタログ番号１３４７１０１）の糖鎖切断酵素を単独あるいは組み合わせて酵素消化実験を行いＳＤＳ−ＰＡＧＥで調べたところ、ＴＰＯのポリペプチド部分は理論値から推定される分子量約３６０００であり、Ｎ結合型糖鎖とＯ結合型糖鎖の両方を持つ糖タンパク質であることが判明した。＜実施例５７＞
ヒトＴＰＯ産生ＣＨＯ細胞株からヒトＴＰＯの精製
（１）実施例５５と同様にして得られたＣＨＯ細胞無血清培養上清１Ｌに２１１．４ｇの硫酸アンモニウムを加え、０．２μｍフイルター（Ｇｅｌｍａｎ Ｓｃｉｅｎｃｅ社製、カタログ番号１２９９２）で濾過し、予め１．２Ｍ硫酸アンモニウム，２０ｍＭ酢酸ナトリウム緩衝液（ｐＨ５．６）で平衡化しておいたＭａｃｒｏ−Ｐｒｅｐ Ｍｅｔｈｙｌ ＨＩＣカラム（Ｂｉｏ−Ｒａｄ社製，カタログ番号１５６−００８１，直径５０ｍｍ、ベッド高９０ｍｍ）に流速１５ｍｌ／ｍｉｎで添加した。添加終了後、２０ｍＭ酢酸ナトリウム緩衝液（ｐＨ５．６）４５０ｍｌで溶出した。この溶出液をＶｙｄａｃ 逆相Ｃ４カラム（Ｔｈｅ Ｓｅｐａｒａｔｉｏｎｓ Ｇｒｏｕｐ社製、カタログ番号２１４ＢＴＰ５４，直径４．６ｍｍ，ベッド高２５０ｍｍ）に流速０．７５ｍｌ／ｍｉｎで添加した。添加終了後、１５分間５％エタノールを含む１０ｍＭトリス緩衝液（ｐＨ６．４）（展開溶媒Ａ）で洗浄後、展開溶媒Ａから１０ｍＭトリス緩衝液（ｐＨ６．４）を含む９４％エタノール（展開溶媒Ｂ）まで６６分間の直線グラジエントで溶出した。このクロマトグラムを図１５に示した。ＴＰＯと思われる分子量約６５０００−１０００００の分子は、ＳＤＳ−ＰＡＧＥによる分析の結果、サンプル添加後の保持時間６８−７２分付近の画分に得ることができ、単一なバンドであることが確認できた（図１６参照）。さらにウエスターン分析により、このタンパク質がＴＰＯであることを確認した。
このサンプルの一部を取り、Ｎ末端アミノ酸分析を行った結果、ヒトＴＰＯ遺伝子でコードされるタンパク質のアミノ酸配列を含むことが確認できた。
＜実施例５８＞
ヒトＴＰＯの昆虫細胞での発現用組換えウイルスの作製
実施例３０で作製したｐＨＴＰ１を制限酵素ＥｃｏＲＩ並びにＮｏｔＩで消化後１％アガロースゲル電気泳動にかけ、約１２００ｂｐのバンドをプレップ−Ａ−ジーンＤＮＡ精製キットを用いて精製した後、同様に制限酵素処理したトランスファーベクターｐＶＬ１３９３（インビトローゲン社製）に連結しコンピテントハイＥ．ｃｏｌｉ ＤＨ５（東洋紡績社製）を形質転換した。得られたコロニーよりプラスミドＤＮＡを調製し、ヒトＴＰＯｃＤＮＡのコーディング領域を全て含むクローンｐＶＬ１３９３／ｈＴＰＯを得た。このクローンのプラスミドＤＮＡをＭｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ（Ｓａｍｂｒｏｏｋら、Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ Ｐｒｅｓｓ，１９８９）に記載されているようにして調製し、ＢａｃｕｌｏＧｏｌｄ^ＴＭ Ｔｒａｎｓｆｅｃｔｉｏｎ Ｋｉｔ（ファーミンゲン社製）を用いて昆虫細胞Ｓｆ２１（インビトローゲン社製）にトランスフェクション後、Ｓｆ−９００培地（ライフテクノロジー社製）中で２７℃で４日間培養し、そのウイルスを含んだ上清を回収した。この上清を１０^９〜１０^５倍に希釈してその１ｍｌを用いて、３５ｍｍ径のシャーレ中で約７×１０^５個のＳｆ２１細胞に２７℃で１時間感染させた。上清を抜きとりＳｆ−９００培地を含む１％アガロースを流し込みアガロースが固まった後、加湿下で２７℃で６日間培養した。ここで形成されたシングルプラークをピックアップし、２００μｌのＳｆ−９００培地中でウイルスを溶出させた。このウイルスクローンを２４ウエルプレート中でＳｆ２１細胞に感染させウイルスの増幅を行った。得られたシングルプラーク由来のウイルス液の一部をフェノール／クロロホルム処理後、エタノール沈殿してウイルスＤＮＡを回収した。このウイルスＤＮＡを鋳型としてヒトＴＰＯｃＤＮＡに特異的なプライマーを用いてＰＣＲを行い、特異的なＤＮＡ断片が増幅されるか否かにより、ヒトＴＰＯｃＤＮＡを含む組換え体ウイルスを選択した。ここで得られたヒトＴＰＯｃＤＮＡを含む組換え体ウイルスを含む上清を用いてＳｆ２１細胞に感染させ、さらにウイルスの増幅を行った。
＜実施例５９＞
Ｓｆ２１昆虫細胞でのヒトＴＰＯの発現と活性確認
１７５ｃｍ２の培養フラスコ中に、Ｓｆ２１細胞を約８０％コンフルエントになるまで培養し、実施例５８で作製したヒトＴＰＯｃＤＮＡを含む組換え体ウイルスを２７℃で１時間感染させた後、Ｓｆ−９００培地中で２７℃で４日間培養し、その上清を回収した。得られた培養上清をＮＡＰ^ＴＭ−５カラム（ファルマシア社製）を用いてＩＭＤＭ培養液に置換し、これをラットＣＦＵ−ＭＫアッセイ系及びＭ−０７ｅアッセイ系にかけたところ、用量依存的に有意なＴＰＯ活性及びＭ−０７ｅ細胞増殖促進活性が認められた。また、実施例４５に記載したのと同様のウエスタン分析により、Ｓｆ２１細胞で発現した組み換えヒトＴＰＯが確認された。
＜実施例６０＞
大腸菌で発現した、ヒトＴＰＯ塩基配列をコードするクローンｐＣＦＭ５３６／ｈ６Ｔ（１−１６３）由来変異型ヒトＴＰＯ、ｈ６Ｔ（１−１６３）のＮ−ラウロイルサルコシンナトリウムと硫酸銅を用いたリフォールディング法とリフォールディング〜精製
実施例４３で調製されたｈ６Ｔ（１−１６３）生産組換体凍結菌体３０ｇに水３００ｍｌを加えてけん濁し、高圧破砕機（１００００ｐｓｉ、Ｒａｎｎｉｅ Ｈｉｇｈ ＰｒｅｓｓｕｒｅＬａｂｏｒａｔｏｒｙ）で菌体を破砕した後、遠心分離により沈殿画分を回収した。沈殿画分に９０ｍｌの水を加えて懸濁し、さらに撹拌しながら水を加えて液量を１５０ｍｌにした後、９ｍｌの１ＭＴｒｉｓ緩衝液ｐＨ９．２、５４０μｌの１ＭＤＴＴ、１．８ｍｌの０．５ＭＥＤＴＡ、１８ｍｌの１０％デオキシコール酸ナトリウムを攪拌しながら加え、室温にて３０分間攪拌した。遠心分離により沈殿画分を回収し、大部分の混在蛋白質、菌体成分等を除去した。沈殿に１８０ｍｌの５ｍＭＤＴＴを加えてけん濁した後、遠心分離によってｈ６Ｔ（１−１６３）を含む沈殿画分を回収した。得られた沈殿画分に水３００ｍｌを加え懸濁し、さらに撹拌しながら水を加えて液量を５７０ｍ１にした後、攪拌しながら３０ｍｌの１ＭＴｒｉｓ緩衝液ｐＨ８と１５０ｍｌの１０％Ｎ−ラウロイルサルコシンナトリウムを加えて室温にて２０分間攪拌して可溶化した。これに７５０μｌの１％硫酸銅を加え、室温にて一晩（約２０時間）攪拌した。遠心分離によりｈ６Ｔ（１−１６３）を含む上清画分を回収した後、上清７５０ｍｌに７５０ｍｌの水、１５００ｍｌの２０ｍＭＴｒｉｓ緩衝液ｐＨ７．７加えた後、撹拌しながら３ｍｌのポリソルベート８０（日光ケミカルズ）を加えた。同溶液に６００ｇのＤｏｗｅｘ １−ｘ４，２０−５０ｍｅｓｈ，ｃｈｌｏｒｉｄｅｆｏｒｍイオン交換樹脂を加え、室温にて９０分間撹拌した。グラスフィルターを用いてイオン交換樹脂非吸着画分を回収した後、７５０ｍｌの２０ｍＭＴｒｉｓ緩衝液ｐＨ７．７でイオン交換樹脂を洗浄した。イオン交換樹脂非吸着画分と洗浄液を合わせた溶液に２Ｎ水酸化ナトリウム加えてｐＨを９．２に調整し、０．１％ポリソルベート８０を含む２０ｍＭＴｒｉｓ緩衝液ｐＨ９．２で平衡化したＱ Ｓｅｐｈａｒｏｓｅ Ｆａｓｔ Ｆｌｏｗ陰イオン交換カラム（内径５ｃｍ ｘ １０ｃｍ）に添加し、非吸着画分を回収した。塩酸を用いて得られた非吸着画分のｐＨを７．２に調整し、０．１％ポリソルベート８０を含む２０ｍＭＴｒｉｓ緩衝液ｐＨ７．２で平衡化したＳＰ Ｓｅｐｈａｒｏｓｅ Ｆａｓｔ Ｆｌｏｗ陽イオン交換カラム（内径５ｃｍ ｘ １０ｃｍ）に添加し、同緩衝液中で塩化ナトリウム濃度０Ｍから５００ｍＭまでの直線濃度勾配法によって溶出を行なった。溶出液についてＳＤＳ−ＰＡＧＥによって分析を行い、ＴＰＯ溶出画分を集めた。ＴＰＯ溶出画分にトリフルオロ酢酸を０．１％になる様に加え、カプセルパック５μｍ３００Ａ Ｃ１カラム（内径２．１ｃｍｘ ５ｃｍ ｘ ２本、資生堂）に添加した後、０．１％トリフルオロ酢酸中で１−プロパノール濃度を上昇させる直線濃度勾配溶離法によって逆相ＨＰＬＣを行なった。溶出液について還元剤非存在下でのＳＤＳ−ＰＡＧＥにより分析した結果及び逆相ＨＰＬＣでの溶出位置によって、３画分に分画し、各々について０．１％トリフルオロ酢酸を用いて３倍希釈した後、上記の逆相ＨＰＬＣカラムに添加し、同様の方法で再クロマトグラフィーを行なった。得られたＴＰＯ３画分を逆相ＨＰＬＣの溶出順にＦｒ．Ｓ−ａ、Ｆｒ．Ｓ−ｂ、 Ｆｒ，Ｓ−ｃ （図１７参照）として還元剤非存在下でのＳＤＳ−ＰＡＧＥで分析した結果、各々、分子量１８Ｋダルトン（Ｆｒ．Ｓ−ａ）、１９Ｋダルトン（Ｆｒ．Ｓ−ｂ）、１８Ｋダルトン（Ｆｒ．Ｓ−ｃ）付近に単一のバンドとして検出された（図１８参照）。各々についてアミノ酸組成分析を行った結果、得られたアミノ酸組成はシークエンス情報からの理論値とほぼ一致し，またＮ末端アミノ酸配列分析の結果、予想される配列のみが得られた。またアミノ酸分析の結果から得られた各画分の蛋白質量は、各々、０．６４ｍｇ（Ｆｒ．Ｓ−ａ）１．８１ｍｇ（Ｆｒ．Ｓ−ｂ）、３．４９ｍｇ（Ｆｒ．Ｓ−ｃ）であった。各画分をＩＭＤＭ培養液に対して十分に透析後、Ｍ−０７ｅアッセイを行った結果、相対活性約１６２００００（Ｆｒ．Ｓ−ａ）、相対活性約２３５０００００（Ｆｒ．Ｓ−ｂ）、相対活性約７４６００００００（Ｆｒ．Ｓ−ｃ）のＴＰＯ活性を示した。
＜実施例６１＞
大腸菌で発現した、ヒトＴＰＯ塩基配列をコードするクローンｐＣＦＭ５３６／ｈ６Ｔ（１−１６３）由来変異型ヒトＴＰＯ）ｈ６Ｔ（１−１６３）の塩酸グアニジンとシステインーシスチンを用いたリフォールディング〜精製
実施例４３で調製されたｈ６Ｔ（１−１６３）生産組換体凍結菌体５０ｇに水５００ｍｌを加えてけん濁し、高圧破砕機（１００００ｐｓｉ、Ｒａｎｎｉｅ Ｈｉｇｈ ＰｒｅｓｓｕｒｅＬａｂｏｒａｔｏｒｙ）で菌体を破砕した後、遠心分離により沈殿画分を回収した。沈殿画分に水を加えて懸濁し、さらに撹拌しながら水を加えて液量を２５０ｍｌにした後、１５ｍｌの１ＭＴｒｉｓ緩衝液ｐＨ９．２、９００μｌの１ＭＤＴＴ、３ｍｌの０．５ＭＥＤＴＡ、３０ｍｌの１０％デオキシコール酸ナトリウムを攪拌しながら加え、室温にて３０分間攪拌した。遠心分離により沈殿画分を回収し、大部分の混在蛋白質、菌体成分等を除去した。沈殿に３００ｍｌの５ｍＭＤＴＴを加えてけん濁した後、遠心分離によってｈ６Ｔ（１−１６３）を含む沈殿画分を回収した。回収されたｈ６Ｔ（１−１６３）を含む沈殿画分に水を加えてけん濁し、最終１０４ｍ１とした。攪拌しながら２０ｍｌの１ＭＴｒｉｓ緩衝液ｐＨ８．５を加えた後、３７６ｍｌの８Ｍ塩酸グアニジンを加えて室温にて１０分間攪拌して可溶化した。これに撹拌しながら２５００ｍｌの０．１％ポリソルベート８０を含む２０ｍＭＴｒｉｓ緩衝液ｐＨ８．５を加え、さらに１Ｍ塩酸グアニジンを含む２０ｍＭＴｒｉｓ緩衝液ｐＨ８．５を２０００ｍｌ加えた後、５ｍＭシステインと０ ５ｍＭシスチンを加えて攪拌し溶かした。４℃中にて一晩静置した後、遠心分離によって上清中にｈ６Ｔ（１−１６３）を回収した。得られた上清をプレップスケールＵＦカートリッジＰＬＤＣ限外濾過膜（ミリポア）を用いて濃縮し、０．１％ポリソルベート８０を含む２０ｍＭＴｒｉｓ緩衝液ｐＨ９．２にて緩衝液を置換して最終容量約１０００ｍＬとした。これを０．１％ポリソルベート８０を含む２０ｍＭＴｒｉｓ緩衝液ｐＨ９．２で平衡化したＱ Ｓｅｐｈａｒｏｓｅ Ｆａｓｔ Ｆｌｏｗ陰イオン交換カラム（内径５ｃｍ ｘ１０ｃｍ）に添加し、同緩衝液中で塩化ナトリウム濃度０Ｍから５００ｍＭまでの直線濃度勾配法によって溶出を行ない、塩化ナトリウム濃度２０ｍＭから１５０ｍＭ付近までの塩濃度で溶出された画分２４０ｍＬを回収した。得られた画分を２０ｍＭＴＴｉｓ緩衝液ｐＨ７．２で４倍に希釈して９６０ｍＬとし、酢酸を用いてｐＨを７．２に調整した。これを０．１％ポリソルベート８０を含む２０ｍＭＴｒｉｓ緩衝液ｐＨ７．２で平衡化したＳＰ Ｓｅｐｈａｒｏｓｅ Ｆａｓｔ Ｆｌｏｗ陽イオン交換カラム（内径５ｃｍｘ １０ｃｍ）に添加し、同緩衝液中で塩化ナトリウム濃度０Ｍから５００ｍＭまでの直線濃度勾配法によって溶出を行なった。溶出液についてＳＤＳ−ＰＡＧＥによって分析を行い、ＴＰＯ溶出画分を集めた。ＴＰＯ溶出画分にトリフルオロ酢酸を０．１％になる様に加え、カプセルパック５μｍ３００ＡＣ１カラム（内径２．１ｃｍ ｘ ５ｃｍ ｘ ２本、資生堂）に添加した後、０．１％トリフルオロ酢酸中で１−プロパノール濃度を上昇させる直線濃度勾配溶離法によって逆相ＨＰＬＣを行なった。溶出液について還元剤非存在下のＳＤＳ−ＰＡＧＥにより分析した結果及び逆相ＨＰＬＣでの溶出位置によって２画分に分画した。得られたＴＰＯ溶出２画分を逆相ＨＰＬＣの溶出順にＦｒ．Ｇ−ａ、Ｆｒ．Ｇ−ｄ（図１７参照）として各々について還元剤非存在下でのＳＤＳ−ＰＡＧＥで分析した結果、各々、分子量１８Ｋダルトン（Ｆｒ．Ｇ−ａ）、３２Ｋダルトン（Ｆｒ．Ｇ−ｄ）付近に単一のバンドとして検出された（図１８参照）。また、還元剤存在下でのＳＤＳ−ＰＡＧＥ分析を行った結果、どちらの画分も分子量２０Ｋダルトン付近に単一のバンドとして検出された。各々にっいてアミノ酸組成分析を行った結果、得られたアミノ酸組成はシークエンス情報からの理論値とほぼ一致し，またＮ末端アミノ酸配列分析の結果、予想される配列のみが得られた。またアミノ酸分析の結果から得られた各画分の蛋白質量は、各々、２．５６ｍｇ（Ｆｒ．Ｇ−ａ）、１．１６ｍｇ（Ｆｒ．Ｇ−ｄ）であった。各画分をＩＭＤＭ培養液に対して十分に透析後、Ｍ−０７ｅアッセイを行った結果、相対活性約３９６００００（Ｆｒ．Ｇ−ａ）、相対活性約７７６００００（Ｆｒ．Ｇ−ｄ）のＴＰＯ活性を示した。
＜実施例６２＞
部分長ヒトＴＰＯ（アミノ酸１−１６３） （以下、このタンパク質を「ｈＴＰＯ１６３」と称す）のＣＨＯ細胞用組換えベクター、ｐＤＥＦ２０２−ｈＴＰＯ１６３の構築 実施例３１で得られたべクターｐＤＥＦ２０２を制限酵素ＥｃｏＲＩとＳｐｅＩで処理し、アガロースゲル電気泳動で大きい方のべクターフラグメントを回収したのち、このフラグメントと−２１から１６３位のアミノ酸までをコードするｈＴＰＯ１６３ｃＤＮＡ （配列表 配列番号１３参照）を含むプラスミドｐＥＦ１８Ｓ−ｈＴＰ０１６３を制限酵素ＥｃｏＲＩとＳｐｅＩで処理して得られたｈＴＰＯ１６３ｃＤＮＡとをＴ ４ＤＮＡリガーゼ（宝酒造製）で結合させ、発現べクターｐＤＥＦ２０２−ｈＴＰＯ１６３を得た。このプラスミドはＳＶ４０の複製開始領域、ヒトエロンゲーションファクター１−アルファプロモーター、ＳＶ４０初期ポリアデニル部位、マウスＤＨＦＲミニ遺伝子、ｐＵＣ１８の複製開始領域、β−ラクタマーゼ遺伝子（Ａｍｐ^ｒ）を含み、ヒトエロンゲーションファクター１−アルファプロモーター下流にｈＴＰＯ１６３ｃＤＮＡが接続されている。
＜実施例６３＞
ＣＨＯ細胞によるｈＴＰＯ１６３の発現
ＣＨＯ細胞（ｄｈｆｒ−株、ＵｒｌａｕｂとＣｈａｓｉｎ；Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ；７７巻４２１６頁、１９８０）を６ｃｍ径のプレート（Ｆａｌｃｏｎ社製）中１０％牛胎児血清を含むα最小必須培地（α−ＭＥＭ（−）、チミジン、ヒポキサンチン添加）で培養増殖させ、これをトランスフェクタム法（生化学工業社製）によって形質転換した。
すなわち、実施例６２で調製したｐＤＥＦ２０２−ｈＴＰＯ１６３プラスミド１０μｇに０．３Ｍ ＮａＣｌ２４０μｌを加え混合したのち、トランスフェクタム２０μｌとＨ_２Ｏ２２０μｌとの混合液を加え、再度混合した。このＤＮＡ溶液をプレートに滴下したのち、ＣＯ_２インキュベーター中で６時間培養した。プレートから培地を除去し、α−ＭＥＭ（−）にて２回洗浄後、１０％ジメチルスルフォオキシド含有α−ＭＥＭ（−）を添加し、室温で２分間処理した。次いで、１０％透析牛胎児血清含有非選択培地（前出α−ＭＥＭ（−）、ヒポキサンチン、チミジン添加）を添加して２日間培養したのち、１０％透析牛胎児血清含有選択培地（α−ＭＥＭ（−）、ヒポキサンチン、チミジン無添加）での選択をおこなった。選択は細胞をトリプシン処理した後、６ｃｍ径プレート１枚あたりを、１０ｃｍ径プレート５枚あるいは２４ウエルプレート２０枚に分割したのち、２日ごとに選択培地にて培地交換を行いながら培養を続行する事により実施した。細胞が増殖してきたプレートあるいはウエルについてはその培養上清中のＴＰＯ活性をＢａ／Ｆ３アッセイ法を用いて測定したところ、ＴＰＯ活性が認められた。培養上清中にＴＰＯ活性の認められたものについては新しいプレートあるいはウエルに２５ｎＭのメソトレキセートを含む選択培地で１：１５に細胞を分割し、培養を続行することによりメソトレキセートに耐性の細胞を増殖させてクローニングを行った。
なお、ＣＨＯ細胞の形質転換はＣＨＯ細胞に対しｐＥＦ１８Ｓ−ｈＴＰＯ１６３とｐＭＧＩを同時形質転換（ｃｏ−ｔｒａｎｓｆｅｃｔｉｏｎ）することによっても行うことができる。
プラスミドｐＤＥＦ２０２−ｈＴＰＯ１６３によって形質転換されたＣＨＯ細胞株（ＣＨＯ−ＤＵＫＸＢ１１）は１９９５年１月３１日付で通商産業省工業技術院生命工学工業技術研究所に受託番号ＦＥＲＭ ＢＰ−４９８９として寄託されている。
これはまた、１９９５年３月２２日付で中華民国 食品工業発展研究所（ＦＩＲＤＩ）に受託番号９６００２４として寄託されている。
＜実施例６４＞
ＣＨＯ細胞の大量培養
実施例６３においてｈＴＰＯ１６３発現プラスミドｐＤＥＦ２０２−ｈＴＰＯ１６３をＣＨＯ細胞にトランスフェクションして得られたｈＴＰＯ１６３産生ＣＨＯ細胞株（ＣＨＯ１０９細胞、０ｎＭ ＭＴＸ耐性）の大量培養は以下のようにして実施した。細胞を１０％ＦＣＳを含むＤＭＥＭ／Ｆ−１２培地（ＧＩＢＣＯ社）を用いて培養増殖させた。この細胞をトリプシン溶液を用いて剥離したのち、２００ｍｌの同培地を含むＦａｌｃｏｎ社製ローラーボトル（Ｆａｌｃｏｎ３０００）に１０００万個の細胞を接種し、３７℃で１ｒｐｍの回転速度で３日間培養した。３日後、培養液を吸引除去し、１００ｍｌのＰＢＳで細胞培養表面をリンスしたのち、１０％ＦＣＳを含まないＤＭＥＭ／Ｆ−１２培地（ＧＩＢＣＯ社）を２００ｍｌ加え、３７℃、１ｒｐｍの回転速度で７日間培養し、７日後その培養上清を回収し、次の精製操作の出発材料にした。上記の操作をローラーボトル３００本分実施し、無血清培養上清６０Ｌを得た。
＜実施例６５＞
ｈＴＰＯ１６３産生ＣＨＯ細胞株からｈＴＰＯ１６３の精製
（１）実施例６４より得られた無血清培養上清６０Ｌを得て、０．２２μｍの濾過フィルターの濾液をとった。さらに限外濾過ユニット（フィルトロン社製・分子量１００００カット）を用いて濃縮画分（６００ｍｌ、蛋白質濃度１１．２ｍｇ／ｍｌ、総蛋白質量６４３０ｍｇ）を得た。この画分を抗ＨＴ１ペプチド抗体を用いてウエスターン分析で調べたところ、見かけの分子量２００００〜２６０００の範囲に発現されたｈＴＰ０１６３蛋白質が存在することが明らかとなった。
この濃縮培養上清５７３ｍｌを流速約２０ｍｌ／ｍｉｎにて、１０ｍＭリン酸ナトリウム緩衝液（ｐＨ６．８）で予め平衡化してあったＳｅｐｈａｄｅｘ Ｇ−２５ Ｆｉｎｅカラム（ファルマシア・バイオテク社製、カタログ番号１７−００３２−０２；直径１０ｃｍ、ベッド高３０ｃｍ）にて処理することにより、１０ｍＭリン酸ナトリウム緩衝液（ｐＨ６．８）溶液となった蛋白質画分Ｆ１（９３８ｍｌ，蛋白質濃度４．９ｍｇ／ｍｌ，総蛋白質量４５９４ｍｇ）を得た。このＳｅｐｈａｄｅｘ Ｇ−２５ Ｆｉｎｅカラム蛋白質画分９２９ｍｌを、予め１０ｍＭリン酸ナトリウム緩衝液（ｐＨ６．８）平衡化したＳＰ Ｓｅｐｈａｒｏｓｅ Ｆａｓｔ Ｆｌｏｗ（ファルマシア・バイオテク社製、カタログ番号１７−０７２９−０１；直径５ｃｍ、ベッド高 １２ｃｍ）カラムに流速１５ｍｌ／ｍｉｎで添加した。添加終了後、さらに１０ｍＭリン酸ナトリウム緩衝液（ｐＨ６．８）、次いで１０％エタノールを含む１０ｍＭリン酸ナトリウム緩衝液（ｐＨ６．８）で溶出されたものまでをまとめ、画分Ｆ１（１６０８ｍｌ，蛋白質濃度２．１３ｍｇ／ｍｌ，総蛋白質量３４２６ｍｇ）を得た。次に、溶出液を１０ｍＭリン酸ナトリウム（ｐＨ６．８）中に７５０ｍＭ ＮａＣｌ、２５％エタノールを含む緩衝液にかえ、溶出されたｈＴＰＯ１６３の主たる溶出画分Ｆ２（６５１ｍｌ，蛋白質濃度１．６７ｍｇ／ｍｌ，総蛋白質量１０８７ｍｇ）を集めた。
ＳＰ Ｓｅｐｈａｒｏｓｅ Ｆａｓｔ ＦｌｏｗカラムのＴＰＯ活性画分Ｆ２のうち２００ｍｌをとり、エタノール及び精製水を加え、最終エタノール濃度４５％の溶液３００ｍｌを調製した。ここでエタノール添加の結果生じた不溶物を遠心分離により取り除いた後、展開溶媒Ａ（１０ｍＭ酢酸ナトリウム緩衝液（ｐＨ６．７））、展開溶媒Ｂ（９０％エタノールを含む１０ｍＭ酢酸ナトリウム緩衝液（ｐＨ６．７））を用意し、予め５０％Ｂ液にて平衡化したＳＯＵＲＣＥ １５ＲＰＣカラム（ファルマシア・バイオテク社製、カタログ番号１７−０７２７−０２；直径２ｃｍ、ベッド高２０ｃｍ）に流速２ｍｌ／ｍｉｎで注入した。注入後、カラム非吸着物質がほぼ溶出するまで４５％Ｂ液にて洗浄した後、流速を１．５ｍｌ／ｍｉｎにし、まず５分間５０％Ｂ）次いで５０％Ｂから１００％Ｂまで１４０分の直線濃度勾配の後、１００％Ｂ液にて３５分間展開した。この間５分（７．５ｍｌ）ごとに集められた溶出画分をＳＤＳ−ＰＡＧＥ及びウエスターン分析にかけ、ｈＴＰ０１６３の溶出範囲を調べたところ、エタノール濃度６６％〜８７％の範囲に見かけの分子量約２００００から２６０００の高度に精製されたｈＴＰ０１６３が溶出されていることが判明した。これらのｈＴＰＯ１６３のうち分子量の高いものほどより早く逆相カラムから溶出したことから、糖鎖がより多く結合したｈＴＰＯ１６３分子ほど親水性が増していることが示唆された。
ＳＯＵＲＣＥ １５ＲＰＣカラムのｈＴＰＯ１６３溶出画分（エタノール濃度６８％〜８６．５％の範囲に溶出したｈＴＰＯ１６３画分９０ｍｌ）のうち、８８．８ｍｌにＣＨＡＰＳを添加後、限外濾過法（アミコン社製、ＹＭ−３膜付き限外濾過ユニット）により濃縮、洗浄を行い、最終的に約５％エタノール、４ｍＭＣＨＡＰＳを含む２．５ｍｌの濃縮標品とした。この標品を予め１０％エタノールを含む１０ｍＭリン酸ナトリウム緩衝液（ｐＨ６．８）で平衡化したＳｕｐｅｒｄｅｘ ７５ｐｇカラム（ファルマシア・バイオテク社製、カタログ番号１７−１０７０−０１；直径２．６ｃｍ）ベッド高６０ｃｍ）に流速１．５ｍｌ／ｍｉｎで注入した。注入後６０分後から、６ｍｌ（４分）ごとに溶出画分を集めたところ、ＳＤＳ−ＰＡＧＥにより試験管番号１６番以降の画分にｈＴＰＯ１６３が溶出しはじめ、少なくとも３１番目までの広い範囲にｈＴＰＯ１６３が溶出していることを確認した。これは、ゲルろ過の標準分子量マーカー（Ｂｉｏ−Ｒａｄ社製Ｇｅｌ Ｆｉｌｔｒａｔｉｏｎ Ｓｔａｎｄａｒｄ；カタログ番号１５１−１９０１及びＣａｌｂｉｏｃｈｅｍ社製インシュリン；カタログ番号４０７６９６の混合物）に対して、分子量約４４０００から６０００の範囲に相当する。しかもこれらのｈＴＰ０１６３は糖がより多く付加された分子量の大きいものから順に溶出していると考えられた。試験管番号１６〜３１（溶出体積１８０〜２７６ｍｌ）の溶出範囲にあるすべてのｈＴＰＯ１６３の分子種をまとめたものとして画分ＦＡを調製した。画分ＦＡとは別に、試験管番号１６〜１８（溶出体積１８０〜１９８ｍｌ）、試験管番号１９〜２４（溶出体積１９８〜２３４ｍｌ）、試験管番号２５〜３１（溶出体積２３４〜２７６ｍｌ）の各々の一部をとり、それぞれ画分ＦＨ、ＦＭ、ＦＬとした。つまり画分ＦＡは、実質的に画分ＦＨ、ＦＭ、ＦＬを合わせたものとなる。以上、図１９に示す。
（２）次に（１）で述べたＳｕｐｅｒｄｅｘ ７５ｐｇカラムのｈＴＰＯ１６３溶出画分ＦＨ）ＦＭ）ＦＬ及びＦＡについて詳細に述べる。このようにして得られたｈＴＰＯ１６３のＮ末端側のアミノ酸配列を実施例１に述べた方法と同様にして調べたところ、Ｎ末端のセリンの大部分は同定不能であったことより、Ｎ末端のセリンにＯ結合型糖鎖が付加されていることが強く示唆された。また、Ｎ末端のセリンに続く配列は遺伝子配列から期待されたアミノ酸配列であることが確認できた。アミノ酸組成分析（ＡｃｃＱ．Ｔａｇ法、ウォターズ社）の結果、ＦＨ、ＦＭ、ＦＬ及びＦＡのｈＴＰＯ１６３蛋白質（糖鎖を含まないペプチド部分）の濃度がそれぞれ１０．２ｎｇ／ｍｌ、６．２ｎｇ／ｍｌ、０．８４ｎｇ／ｍｌ、３．２ｎｇ／ｍｌであった。これらの画分１００ｎｇをマルチゲル１５／２５（第一化学薬品社製１５〜２５％プレキャストポリアクリルアミドゲル）を用いて非還元条件或いはＤＴＴにより還元後、ＳＤＳ−ＰＡＧＥを実施し銀染色（第一化学薬品社製）したところ、それぞれ極めて高純度なｈＴＰＯ１６３標品であることが確認できた。還元条件下においてはＤＰＣＩＩＩ分子量マーカー（第一化学薬品社製）を標準として算出されたＦＨ、ＦＭ、ＦＬ及びＦＡのｈＴＰＯ１６３の見かけの分子量はそれぞれ２４０００〜２１５００、２３０００〜２１０００、２３０００〜２０５００、２３５００〜２０５００であった（図２０を参照）。またウエスターン分析により還元条件下においてはビオチン標識ＳＤＳ−ＰＡＧＥスタンダード（Ｂｉｏ−Ｒａｄ社製Ｂｉｏｔｙｎｙｌａｔｅｄ ＳＤＳ−ＰＡＧＥ Ｓｔａｎｄａｒｄｓ，Ｂｒｏａｄ Ｒａｎｇｅ：カタログ番号１６１−０３１９）を標準として算出されたＦＨ、ＦＭ、ＦＬ及びＦＡのｈＴＰＯ１６３の見かけの分子量はそれぞれ２６０００〜２２０００、２５５００〜２２０００、２６０００〜２１０００、２６０００〜２１０００であった。ＦＨ、ＦＭ、ＦＬ及びＦＡの分子量の不均一性は、付加されたＯ結合型糖鎖の不均一性によることと推定できる。そこでＦＨ、ＦＭ、ＦＬ及びＦＡの各画分をノイラミニダーゼ（Ｎｅｕｒａｍｉｎｉｄａｓｅ：ナカライテスク社製、カタログ番号２４２−２９ ＳＰ）で酵素消化し、ＤＴＴで還元後ＳＤＳ−ＰＡＧＥで分析したところ、いずれの画分も見かけの分子量が１９０００付近となったことから、ｈＴＰＯ１６３蛋白質に付加された糖鎖のシアル酸の量の不均一性が確認されたのと同時に、ここで得られたｈＴＰＯ１６３が糖蛋白質としてＣＨＯ細胞で発現されたものであることが明らかとなった。
（３）（２）で得られたＦＨ、ＦＭ、ＦＬ及びＦＡの各画分をＭ−０７ｅアッセイ系でｉｎ ｖｉｔｒｏ活性を調べたところ、相対比活性は１ｍｇｈＴＰＯ１６３蛋白質（糖鎖重量を含まないペプチド部分の重量）当たり、それぞれ５１１００００００、７７５００００００、１１５０００００００、７１５００００００であった。
＜実施例６６＞
−１位にＬｙｓ、−２位にＭｅｔが付加されたヒトＴＰＯ（アミノ酸１−３３２）（以下「ｈＭＫＴ（１−３３２）」と称す）の大腸菌用発現ベクターの構築及び発現
ヒトＴＰＯ全長アミノ酸を大腸菌で発現させるためアミノ酸１６４以降のアミノ酸３３２までの使用コドンを以下に述べるように大腸菌優先コドンに変更した。表２０に示した２１−４０の合成オリゴヌクレオチドを作成した。
【表２０】

２１及び２２；２３及び２４；２５及び２６；２７及び２８；２９及び３０；３１及び３２；３３及び３４；３５及び３６；３７及び３８；３９および４０の合成オリゴヌクレオチドを同チューブ中でＴ４キナーゼ（ファルマシア社製）を用いて０．１ｍＭ ＡＴＰ，１０ｍＭＴｒｉｓ−ａｃｅｔａｔｅ，１０ｍＭ Ｍｇ−ａｃｅｔａｔｅ，５０ ｍＭ Ｋ−ａｃｅｔａｔｅの溶液中でリン酸化した。さらに１／１０量の１００ｍＭ Ｔｒｉｓ／ＨＣｌ（ｐＨ７．５），１００ｍＭ ＭｇＣｌ_２，５００ｍＭ ＮａＣｌの溶液を加え、水浴中で３分間煮沸した後、放置することにより二本鎖ＤＮＡとした。次に２１及び２２；２３及び２４（組合わせＡ）、２５及び２６：２７及び２８；２９及び３０（組合わせＢ）、３１及び３２；３３及び３４（組合わせＣ）、３５及び３６；３７及び３８；３９及び４０（組合わせＤ）の四組の二本鎖ＤＮＡをＤＮＡライゲーションキット（宝酒造社製）を用いて連結後、さらに（組合わせＡ）及び（組合わせＢ）、（組合わせＣ）及び（組合わせＤ）をそれぞれ同様に連結し、得られた反応液を連結１及び連結２とした。これらの反応液を鋳型として、連結１については４１および４２、連結２については４３及び４４の合成オリゴヌクレオチドをプライマーとしてＰＣＲ反応を行った。連結１のＰＣＲ反応で得られたＰＣＲ産物についてはＳａｃｌ，ＥｃｏＲＶ、連結２のＰＣＲ反応で得られたＰＣＲ産物についてはＥｃｏＲＶ，ＨｉｎｄＩＩＩで消化後２％のアガロースゲルで泳動し、それぞれ約２４０ｂｐ及び２５０ｂｐの断片をプレップ−Ａ−ジーンＤＮＡ精製キットにより回収した。これら二本の断片ををＳａｃｌとＨｉｎｄＩＩＩで消化したｐＢｌｕｅｓｃｒｉｐｔ ＩＩＫＳ＋（ストラタジーン社製）にサブクローニングした（宿主はＥ．ｃｏｌｉ ＤＨ５を使用した）。得られたクローンのうち表２１に示した塩基配列を有するものをシークエンシングにより選択し、ここで得られたクローンをｐＢＬ（ＳＨ）（１７４−３３２）とした。
【表２１】

次に実施例４２で作成したｐＢＬ（ＸＨ）ｈ６Ｔ（１−１６３）を鋳型とし、以下の４５及び４６の合成オリゴヌクレオチドをプライマーとしてＰＣＲ反応を行い、ここで得られたＰＣＲ産物についてはＢａｍＨＩ，Ｓａｃｌで消化後６％のポリアクリルアミドゲルで泳動し、約１６０ｂｐの断片をゲルより回収した。またｐＢＬ（ＳＨ）（１７４−３３２）をＳａｃＩ，ＨｉｎｄＩＩＩで消化して得られた約４８０ｂｐの断片をプレップ−Ａ−ジーンＤＮＡ精製キットにより回収し、これら二本の断片をＢａｍＨＩとＨｉｎｄＩＩＩで消化したｐＢＩｕｅｓｃｒｉｐｔ ＩＩＫＳ＋ （ストラタジーン社製）にサブクローニングした（宿主はＥ．ｃｏｌｉＤＨ５を使用した）。
４５：５’−ＡＡＧＧＡＴＣＣＧＡＡＣＧＣＴＡＴＣＴＴＣＣＴＧ−３’
４６：５’−ＧＧＧＡＧＣＴＣＧＴＴＣＡＧＧＧＴＣＡＧＡＡＣＣＡＧＡＧＡＧＧＴＡＣＧＡＧＡＣＧＧＡＡＣＡＧＣＡＧＴＧＧＴＴＧＧ−３’
得られたクローンのうち表２２に示した塩基配列を有するものをシークエンシングにより選択し、ここで得られたクローンをｐＢＬ（ＢＨ）（１２３−３３２）とした。
【表２２】

実施例４２で作成したｐＢＬ（ＸＨ）ｈ６Ｔ（１−１６３）をＢａｍＨＩとＨｉｎｄＩＩＩで消化し、ｐＢＬ（ＢＨ）（１２３−３３２）を同じ制限酵素で消化して得られた約６４０ｂｐの断片を連結してクローンｐＢＬ（ＸＨ）ｈ６Ｔ（１−３３４）を作成した。さらに実施例５２で作成したＰＣＦＭ５３６／ｈＭＫＴ（１−１６３）をＸｂａｌ，Ｓｆｉｌで消化して得られた約２７０ｂｐの断片を、同じ制限酵素で消化したｐＢＬ（ＸＨ）ｈ６Ｔ（１−３３４）に連結してクローンｐＢＬ（ＸＨ）ｈＭＫＴ（１−３３４）を作成した。ｐＢＬ（ＸＨ）ｈＭＫＴ（１−３３４）をＸｂａｌ，ＨｉｎｄＩＩＩで消化後、変異型のヒトＴＰＯアミノ酸１−３３２をコードする約１０４０ｂｐの大きさのフラグメントを回収して精製後、Ｘｂａｌ，ＨｉｎｄＩＩＩで消化したｐＣＦＭ５３６ （特表昭６０−５０１９８８）にクローニングした（宿主はｐＭＷ１（ＡＴＣＣ Ｎｏ．３９９３３）で予め形質転換されたＥ．ｃｏｌｉ ＪＭ１０９を使用した）。ここで得られたクローンをｐＣＦＭ５３６／ｈＭＫＴ（１−３３２）とし、この発現ベクターを有する大腸菌株を変異型のｈＭＫＴ（１−３３２）タンパク発現用の形質転換体とした。この発現プラスミドは、配列表（配列番号１４）に示されたＤＮＡ配列を含んでいる。
ここで得られた形質転換株を、アンピシリン５０μｇ／ｍｌ）テトラサイクリン１２．５μｇ／ｍｌを含むＬＢ培地６０ｍｌに３０℃で一晩振盪培養し、この培養液２５ｍｌをアンピシリン５０μｇ／ｍｌを含むＬＢ培地１０００ｍｌに加えて、ＯＤはＡ_６００が１．０−１．２に至るまで３０℃で振盪培養した。次いで最終温度が、４２℃になるように６５℃の約３３０ｍｌＬＢ培地を添加し、さらに３時間４２℃で振盪培養して変異型のヒトＴＰＯ、ｈＭＫＴ（１−３３２）タンパクの発現を誘導した。
この培養菌体を直接サンプルとして用いてＳＤＳ−ＰＡＧＥを行った。ＳＤＳ−ＰＡＧＥには第一化学社製のマルチゲル１５／２５を用い、泳動後にクマシーブルー染色を行ったところｈＭＫＴ（１−３３２）タンパク質の発現を誘導したものに関しては、分子量約３５ｋＤのところに発現誘導に特異的なタンパク質が検出された。一方、ＳＤＳ−ＰＡＧＥ後にゲル上のタンパク質をニトロセルロース膜にエレクトロブロッティングし、実施例４５で作製した抗ＨＴ１ペプチド抗体と反応させ発色させたところ、分子量約３５ｋＤのところにバンドが検出されたことから、ｈＭＫＴ（１−３３２）の発現が確認された。
＜実施例６７＞
ヒトＴＰＯ置換誘導体の作製とＣＯＳ７細胞での発現〜活性確認
ヒトＴＰＯのアミノ酸の一部を置換した誘導体が活性を有するかどうかを以下のとおり検討した。
本実施例で用いた動物細胞発現ベクターｐＳＭＴ２０１は以下のように作製した。まず、マウスエリスロポエチンレセプタ−ｃＤＮＡを含む発現プラスミドｐＸＭ−ｍＥＰ０Ｒｎ（Ｄａｎａ Ｆａｒｂｏｒ癌研究所Ｄ’Ａｎｄｒｅａ博士より入手、Ｃｅｌｌ．５７巻、２７７−２８５頁（１９８９））を制限酵素ＫｐｎＩおよびＥｃｏＲＩで処理した後、アガロースゲル電気泳動しマウスエリスロポエチンレセプターｃＤＮＡ部分を除くｐＸＭベクター断片を回収した。次に回収したｐＸＭ発現ベクターに種々のクローニング用制限酵素部位を導入するための２本の合成オリゴヌクレオチドをＡＢＩ社製ＤＮＡ合成機を用いて作製した。合成したオリゴヌクレオチドの塩基配列を以下に示す。
プライマー１：

プライマー２：

この２本のオリゴヌクレオチドを混合し、アニーリングさせ２本鎖オリゴヌクレオチドとした後、上記で回収したｐＸＭベクター断片とをＴ４ＤＮＡリガーゼ（宝酒造製）で結合させ、発現ベクターｐＤＭＴ２０１を得た。次にこの発現ベクターｐＤＭＴ２０１に含まれるマウスＤＨＦＲ ｃＤＮＡを除去するためにｐＤＭＴ２０１ベクター及びｐＥＦ１８Ｓベクターを制限酵素ＮｏｔＩおよびＨｐａＩで処理後アガロースゲル電気泳動し、ｐＤＭＴ２０１ベクターＤＮＡ由来の大きい方の断片とｐＥＦ１８ＳベクターＤＮＡ由来の小さい方の断片を回収し、Ｔ４ＤＮＡリガーゼ（宝酒造製）で結合させることにより発現ベクターｐＳＭＴ２０１を得た。このベクターは、図２１に示したとおりＳＶ４０の複製開始領域およびエンハンサー配列、アデノウィルス主要後期プロモーター配列、アデノウィルストリパータイトリーダー配列、スプライス信号配列、ＳＶ４０初期ポリアデニル部位配列、アデノウィルスＶＡＲＮＡ遺伝子配列、ｐＵＣ１８の複製開始領域、β−ラクタマーゼ遺伝子（Ａｍｐ^ｒ）を含み、目的とする遺伝子を連結するための制限酵素配列として、ＢｇｌＩＩ、ＰｓｔＩ、ＫｐｎＩ、ＸｈｏＩ、ＥｃｏＲＩ、ＳｍａＩ、ＳｐｅＩ、およびＮｏｔＩ部位を有する。実施例３０で示した完全長ヒトＴＰＯｃＤＮＡを含むｐＨＴＰ１を制限酵素ＥｃｏＲＩおよびＳｐｅＩを用いて酵素消化し得られた完全長ヒトＴＰＯｃＤＮＡ断片を、同様に制限酵素消化したベクターｐＳＭＴ２０１にサブクローニングすることによりプラスミドｐＳＭＴ２０１−ｈＴＰＯを得た。
以上のようにして得たプラスミドｐＳＭＴ２０１−ｈＴＰＯを鋳型にして、まず誘導体作製用の鋳型となるプラスミドβＧＬ−ＴＰＯ／ｐＢｌｕｅを以下のように作製した。
βＧＬ−ＴＰＯ／ｐＢｌｕｅではヒトＴＰＯの５’非翻訳側に、Ａｎｎｗｅｉｌｅｒらの方法に従いウサギのβ−グロビンリーダー配列の付加を試みた（Ａｎｎｗｅｉｌｅｒら、ＮｕｃｌｅｉｃＡｃｉｄｓＲｅｓｅａｒｃｈ、１９巻、３７５０頁）。まず以下に示す化学合成した２本のＤＮＡ鎖、そしてベクターＢｌｕｅｓｃｒｉｐｔＩＩＳＫ＋（東洋紡績社製）を制限酵素ＥｃｏＲＩ及びＳｍａＩで消化した断片とをライゲーションすることによりリーダー配列が挿入されたベクターβＧＬ／ｐＢｌｕｅを作製した。

ＰＣＲに用いたプライマーの配列は以下の通り。

（配列番号７の１０２−１２２にＳｍａＩ配列を付加したもの）
ｅｎｄ：５’−ＴＣＡＡＧＣＴＴＡＣＴＡＧＴＣＣＣＴＴＣＣＴＧＡＧＡＣＡＧＡＴＴＣＴＧ−３’（配列番号７の１１４０−１１６０のアンチセンス、ＳｐｅＩ及びＨｉｎｄＩＩＩ配列を付加） ＰＣＲはプラスミドｐＳＭＴ２０１−ｈＴＰＯ ＤＮＡ１ｎｇを鋳型として使用し、合成したプライマーをそれぞれ１０μＭ使って実施した。ＴａＫａＲａ ＰＣＲ Ａｍｐｌｉｆｉｃａｔｉｏｎ Ｋｉｔ（宝酒造社製）を用いて、Ｐｒｏｇｒａｍｍａｂｌｅ Ｔｈｅｒｍａｌ Ｃｏｎｔｒｏｌｌｅｒ（ＭＪ Ｒｅｓｅａｒｃｈ社製）により１００μｌの容量でＰＣＲ（９４℃１分間、５５℃２分間、７２℃２分間を３サイクル、続けて９４℃４５秒間、５５℃１分間、７２℃１分間を２０サイクル）を行った。ＰＣＲ産物をクロロホルム抽出し、エタノール沈殿を２回行った後１００μｌのＴＥバッファーに溶解した。これを制限酵素ＳｍａＩ、ＨｉｎｄＩＩＩで消化後、フェノール／クロロホルム抽出、エタノール沈殿を行った。得られた沈殿を１０μｌ（７）ＴＥバッファーに溶解後、同様に制限酵素消化したベクターβＧＬ／ｐＢｌｕｅにサブクローニングした（宿主菌は東洋紡績社製コンピテント・ハイＥ．ｃｏｌｉ ＪＭ１０９を使用）。得られた形質転換体のうち２０クローン選択し、プラスミドＤＮＡを調製した。方法は本質的にＭｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ ［Ｓａｍｂｏｏｋら、Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ ＬａｂｏｒａｔｏｒｙＰｒｅｓｓ（１９８９）］に記載されているようにして実施した。精製したプラスミドＤＮＡについてはＴａｑ Ｄｙｅ Ｄｅｏｘｙ^ＴＭＴｅｒｍｉｎａｔｅｒ Ｃｙｃｌｅ Ｓｅｑｕｅｎｃｉｎｇ Ｋｉｔ（アプライドバイオシステムズ社製）を用い、アプライドバイオシステムズ社製３７３ＡＤＮＡシークエンサーにより塩基配列の確認を行った。βＧＬ−ＴＰＯ／ｐＢｌｕｅをコードするプラスミドは全長にわたり塩基配列の置換がなく予想通りのＴＰＯｃＤＮＡ配列を持っことを確認した。さらにβＧＬ−ＴＰＯ／ｐＢ１ｕｅを制限酵素ＢｇＩＩＩＮＳｐｅＩで消化後、フェノール／クロロホルム抽出、エタノール沈殿を行った。得られた沈殿を１０μｌのＴＥバッファーに溶解し、同様に制限酵素消化したベクターｐＳＭＴ２０１にサブクローニングした（宿主菌は東洋紡績社製コンピテント・ハイＥ．ｃｏｌｉ ＪＭ１０９を使用）。本質的にＭｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ［Ｓａｍｂｏｏｋら、Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ Ｐｒｅｓｓ（１９８９）］に記載されている方法に従い、形質転換体からプラスミドＤＮＡを調製した。得られた発現ベクターｐＳＭＴ／βＧＬ−ＴＰＯは、図２１に示したｐＳＭＴ２０１ベクターのスプライス信号配列の下流にある制限酵素配列ＢｇｌＩＩ／ＳｐｅＩ部位にβ−グロビンリーダー配列及びヒトＴＰＯｃＤＮＡが接続された構造を有している。このｐＳＭＴ／βＧＬ−ＴＰＯベクターをＣＯＳ７細胞へのトランスフェクション実験に用いた。
ヒトＴＰＯ置換誘導体の作製にはＰＣＲを利用し、Ｉｔｏらの方法に従った（Ｉｔｏら、Ｇｅｎｅ、１０２巻、６７−７０頁 １９９１年）。ここでは、ヒトＴＰＯの２５位のＡｒｇ残基をＡｓｎ残基に置換した誘導体、並びに３３位のＨｉｓ残基をＴｈｒ残基に置換した誘導体の作製を試みた。ＰＣＲに用いたプライマーの配列は以下の通り。
Ｔ７：５’−ＴＡＡＴＡＣＧＡＣＴＣＡＣＴＡＴＡＧＧＧＣＧ−３’（ＢｌｕｅｓｃｒｉｐｔＩＩＳＫ＋のＴ７プロモ一タ一領域に対応）
ΔＢｇＩＩＩ ：５’−ＡＡＴＴＣＣＡＡＧＡＴＣＡＣＡＣＡＣＴＴＧＣ−３’（制限酵素ＢｇＩＩＩ認識配列置換用） ｅｎｄ ：５’−ＴＣＡＡＧＣＴＴＡＣＴＡＧＴＣＣＣＴＴＣＣＴＧＡＧＡＣＡＧＡＴＴＣＴＧ−３’（配列番号７の１１４０−１１６０のアンチセンス、ＳｐｅＩ及びＨｉｎｄＩＩＩ配列を付加）Ｎ３：５’−ＴＧＧＧＣＡＣＴＧＧＣＴＣＡＧＧＴＴＧＣＴＧＴＧＡＡＧＧＡＣＡＴＧＧＧ−３’（配列番号７のＡｒｇ２５→Ａｓｎ）
０９：５’−ＴＧＴＡＧＧＣＡＡＡＧＧＧＧＴＡＡＣＣＴＣＴＧＧＧＣＡ−３’（配列番号７のＨｉｓ３３→Ｔｈｒ） 第１段階のＰＣＲはプラスミドβＧＬ−ＴＰＯ／ｐＢｌｕｅ ＤＮＡ１ｎｇを鋳型として使用し、合成したプライマーをそれぞれ１０μＭ使って実施した（プライマーの組み合わせは［１］ΔＢｇＩＩＩとｅｎｄ、［２］Ｔ７とＮ３、［３］Ｔ７と０９）。ＴａＫａＲａ ＰＣＲ ＡｍｐｌｉｆｉｃａｔｉｏｎＫｉｔ（宝酒造社製）を用いて、Ｐｒｏｇｒａｍｍａｂｌｅ Ｔｈｅｒｍａｌ Ｃｏｎｔｒｏｌｌｅｒ（ＭＪ Ｒｅｓｅａｒｃｈ社製）により１００μｌの容量でＰＣＲ（９４℃１分間、５５℃２分間、７２℃２分間を３サイクル、続けて９４℃４５秒間、５５℃１分間、７２℃１分間を１７サイクル）を行った。それぞれのＰＣＲ産物をクロロホルム抽出し、エタノール沈殿を２度行った後１００μｌのＴＥバッファーに溶解した。このＰＣＲ産物を２種類１μｌずつ用いて第２段階のＰＣＲを行った。鋳型の組み合わせは［１］と［２］のＰＣＲ産物、［１］と［３］のＰＣＲ産物である。用いたプライマーはすべてＴ７とｅｎｄの組み合わせである。９４℃で１０分間インキュベートした後ＴａＫａＲａ Ｔａｑを加え、９４°Ｃ１分間、５５℃２分間、７２℃２分間を３サイクル、続けて９４℃４５秒間、５５℃１分間、７２℃１分間を９サイクル行った。それぞれのＰＣＲ産物に５ｍｇ／ｍｌ ｐｒｏｔｅｉｎａｓｅ Ｋを１μｌ）０．５Ｍ ＥＤＴＡを２μｌ、２０％ＳＤＳを２μｌ加え３７℃で３０分間保温しＴａｑを失活させた後、フェノール／クロロホルム抽出、エタノール沈殿し２０μｌの滅菌水に溶解した。これを制限酵素ＢｇＩＩＩ、ＳｐｅＩで消化後、フェノール／クロロホルム抽出、エタノール沈殿を行った。得られた沈殿を１０μｌのＴＥバッファーに溶解後、同様に制限酵素消化しアルカリホスファターゼ（仔ウシ腸、ベーリンガー・マンハイム社製）処理した発現ベクターｐＳＭＴ２０１にサブクローニングした（宿主菌は東洋紡績社製コンピテント・ハイＥ．ｃｏｌｉ ＪＭ１０９を使用）。得られた形質転換体のうち、それぞれ２クローンずつ選択し、プラスミドＤＮＡを調製した。方法は本質的にＭｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ［Ｓａｍｂｏｏｋら、Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ Ｐｒｅｓｓ （１９８９）］に記載されているようにして実施した。精製したプラスミドＤＮＡについてはＴａｑＤｙｅ Ｄｅｏｘｙ^ＴＭＴｅｒｍｉｎａｔｅｒ Ｃｙｃｌｅ Ｓｅｑｕｅｎｃｉｎｇ Ｋｉｔ（アプライドバイオシステムズ社製）を用い、アプライドバイオシステムズ社製３７３ＡＤＮＡシークエンサーにより塩基配列の確認を行った。
［１］と［３］のＰＣＲ産物に由来するプラスミドには意図したアミノ酸置換［Ｈｉｓ３３（ＣＡＣ）→Ｔｈｒ（ＡＣＣ）］をもち、かつＣ末端（Ｇｌｙ３３２）に以下のアミノ酸配列からなるペプチドが付加されたＴＰＯ置換誘導体（０９／ＴＰＯ）をコードするｃＤＮＡが含まれていることが確認された。
一方、［１］と［２］のＰＣＲ産物に由来するプラスミドは意図したアミノ酸置換［Ａｒｇ２５（ＡＧＡ）→Ａｓｎ（ＡＡＣ）］以外に更に１カ所のアミノ酸置換［Ｇｌｕ２３１（ＧＡＡ）→Ｌｙｓ（ＡＡＡ）］もち、かつＣ末端（Ｇ１ｙ３３２）に以下のアミノ酸配列からなるペプチドが付加されたＴＰＯ置換誘導体（Ｎ３／ＴＰＯ）をコードするｃＤＮＡを含んでいることが判明した。
ペプチド：
ＴＳＩＧＹＰＹＤＶＰＤＹＡＧＶＨＨＨＨＨＨ
得られたそれぞれのクローンのＣＯＳ７細胞へのトランスフェクションは、実施例３５に従いクロロキン処理を含むＤＥＡＥ−デキストラン法で行った。すなわちプラスミドＤＮＡ４０μｇを使用し、トランスフェクション５日後に培養上清を回収した。回収した培養上清をセントリコン−３０（アミコン社製）で２０倍に濃縮しＭ−０７ｅアッセイ系で評価した。その結果、ヒトＴＰＯ置換誘導体Ｎ３／ＴＰＯ、０９／ＴＰＯをコードするプラスミドでトランスフェクトしたＣＯＳ７細胞培養上清中に、いずれも用量依存的にＴＰＯ活性が検出された（図２２参照）。
＜実施例６８＞
ヒトＴＰＯ挿入・欠失誘導体の作製とＣＯＳ７細胞での発現〜活性確認
ｈＴＰＯ１６３にアミノ酸を挿入した誘導体またはｈＴＰＯ１６３からアミノ酸を欠失した誘導体が活性を有するかどうか検討した。作製した誘導体は配列番号１３の旧ｓ３３欠失体（ｄＨ３３）、Ｇｌｙ１１６欠失体（ｄＧ１１６）、Ａｒｇ１１７欠失体（ｄＲ１１７）、Ｈｉｓ３３とＰｒｏ３４の間へのＴｈｒ挿入体（Ｔ３３’）、Ａｌａ挿入体（Ａ３３’）、Ｇｌｙ挿入体（Ｇ３３’）およびＧｌｙ１１６とＡｒｇ１１７の間へのＡｓｎ挿入体（Ｎ１１６’）、Ａｌａ挿入体（Ａ１１６’）、Ｇｌｙ挿入体（Ｇ１１６’）である。これらのうちＴ３３’、Ｎ１１６’はそれぞれムチン型糖鎖、Ａｓｎ結合型糖鎖の導入を意図した誘導体である。
誘導体の作製にはＰＣＲを利用し、Ｉｔｏらの方法に従った（Ｉｔｏら、Ｇｅｎｅ、１０２巻、６７−７０頁、１９９１）。ＰＣＲに用いたプライマーの配列は以下の通り。
ｄＨ３３：５’−ＴＧＴＡＧＧＣＡＡＡＧＧＡＡＣＣＴＣＴＧＧＧＣＡ−３’（配列番号７の旧ｓ３３欠失体作製用）
Ｔ３３’：５’−ＴＧＴＡＧＧＣＡＡＡＧＧＡＧＴＧＴＧＡＡＣＣＴＣＴＧＧ−３’（配列番号７の旧ｓ３３とＰｒｏ３４の間へのＴｈｒ挿入体作製用）
Ａ３３’：５’−ＴＧＴＡＧＧＣＡＡＡＧＧＡＧＣＧＴＧＡＡＣＣＴＣＴＧＧ−３’（配列番号７のＨｉｓ３３とＰｒｏ３４の間へのＡｌａ挿入体作製用）
Ｇ３３’：５’−ＴＧＴＡＧＧＣＡＡＡＧＧＴＣＣＧＴＧＡＡＣＣＴＣＴＧＧ−３’（配列番号７の旧ｓ３３とＰｒｏ３４の間へのＧｌｙ挿入体作製用）
ｄＧ１１６：５’−ＡＧＣＴＧＴＧＧＴＣＣＴＣＴＧＴＧＧＡＧＧＡＡＧ−３’（配列番号７のＧｌｙ１１６欠失体作製用）
ｄＲ１１７：５’−ＧＴＧＡＧＣＴＧＴＧＧＴＧＣＣＣＴＧＴＧＧＡＧＧ−３’（配列番号７のＡｒｇ１１７欠失体作製用）
Ｎ１１６’：５’−ＡＧＣＴＧＴＧＧＴＣＣＴＧＴＴＧＣＣＣＴＧＴＧＧＡＧＧ−３’（配列番号７のＧＩｙ１１６とＡｒｇ１１７の間へのＡｓｎ挿入体作製用）
Ａ１１６：５’−ＡＧＣＴＧＴＧＧＴＣＣＴＡＧＣＧＣＣＣＴＧＴＧＧＡＧＧ−３’（配列番号７のＧｌｙ１１６とＡｒｇ１１７の間へのＡｌａ挿入体作製用）
Ｇ１１６’：５’−ＡＧＣＴＧＴＧＧＴＣＣＴＴＣＣＧＣＣＣＴＧＴＧＧＡＧＧ−３’（配列番号７のＧＩｙ１１６とＡｒｇ１１７の間へのＧｌｙ挿入体作製用）
ｄＲＩ：５’−ＣＧＧＧＣＴＧＣＡＧＧＡＴＡＴＣＣＡＡＧＡＴＣＴＣＡ−３’（制限酵素ＥｃｏＲＩ認識配列置換用）Ｔ７：５’−ＴＡＡＴＡＣＧＡＣＴＣＡＣＴＡＴＡＧＧＧＣＧ−３’（ＢｌｕｅｓｃｒｉｐｔＩＩＳＫ＋のＴ７プロモーター領域に対応）
ｅｎｄＮｏｔＩ：５’−ＧＧＧＣＧＧＣＣＧＣＴＣＡＧＣＴＧＧＧＧＡＣＡＧＣＴＧＴＧＧＴＧＧＧ−３’（配列番号７の６３３−６５３のアンチセンス、終止コドンとＮｏｔＩ認識配列を付加）
第１段階のＰＣＲは実施例６７で作製したプラスミドβＧＬ−ＴＰＯ／ｐＢｌｕｅ ＤＮＡ１ｎｇを鋳型として使用し、合成したプライマーをそれぞれ１０ｐＭ使って実施した。プライマーの組み合わせは［１］ｄＲＩとｅｎｄＮｏｔＩ、［２］Ｔ７と変異導入プライマー（ｄＨ３３，Ａ３３’，Ｇ３３’，Ｔ３３’，ｄＧ１１６，ｄＲ１１７，Ａ１１６’，Ｇ１１６’，Ｎ１１６’）である。ＴａＫａＲａ ＰＣＲ Ａｍｐｌｉｆｉｃａｔｉｏｎ Ｋｉｔ（宝酒造社製）を用いて、Ｐｒｏｇｒａｍｍａｂｌｅ Ｔｈｅｒｍａｌ Ｃｏｎｔｒｏｌｌｅｒ （ＭＪＲｅｓｅｒｃｈ社製）により１００μｌの容量でＰＣＲを行った。［１］の反応は、９４℃１分間、４５℃２分間、７２℃２分間を３サイクル、続けて９４℃４５秒間、４５℃１分間、７２℃１分間を１７サイクル。［２］の反応は、９４℃１分間、５５℃２分間、７２℃２分間を３サイクル、続けて９４℃４５秒間、５５℃１分間、７２℃１分間を１７サイクル。それぞれのＰＣＲ産物をクロロホルム抽出し、エタノール沈殿を２度行った後１００μｌのＴＥバッファーに溶解した。
次に、［１］と［２］のＰＣＲ産物をそれぞれ１μｌずつ用いて第２段階のＰＣＲを行った。用いたプライマーはすべてＴ７とｅｎｄＮｏｔＩの組み合わせである。９４℃で１０分間インキュベートした後ＴａＫａＲａ Ｔａｑを加え、９４℃１分間、５５℃２分間、７２℃２分間を３サイクル、続けて９４℃４５秒間、５５℃１分間、７２℃１分間を９サイクル行った。それぞれのＰＣＲ産物に５ｍｇ／ｍｌ ｐｒｏｔｅｉｎａｓｅ Ｋを１μｌ、０．５Ｍ ＥＤＴＡを２μｌ、２０％ＳＤＳを２μｌ加え３７℃で３０分間保温しＴａｑを失活させた後、フェノール／クロロホルム抽出、エタノール沈殿し２０ｐｌの滅菌水に溶解した。これを制限酵素ＥｃｏＲＩ、ＮｏｔＩで消化後、フェノール／クロロホルム抽出、エタノール沈殿を行った。得られた沈殿を１０μｌ（７）ＴＥバッファーに溶解後、同様に制限酵素消化しアルカリホスファターゼ（仔ウシ腸、ベーリンガー・マンハイム社製）処理した発現べクターｐＥＦ１８Ｓにサブクローニングした（宿主菌は東洋紡績社製コンピテント・ハイＥ．ｃｏｌｉ ＪＭ１０９を使用）。得られた形質転換体からプラスミドＤＮＡを調製した。方法は本質的にＭｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ［Ｓａｍｂｏｏｋら、Ｃｏｌｄ Ｓｐｒｉｎｇ ＨａｒｂｏｒＬａｂｏｒａｔｏｒｙ Ｐｒｅｓｓ（１９８９）］に記載されているようにして実施した。精製したプラスミドＤＮＡについてはＴａｑ Ｄｙｅ Ｄｅｏｘｙ^ＴＭＴｅｒｍｉｎａｔｅｒ Ｃｙｃｌｅ Ｓｅｑｕｅｎｃｉｎｇ Ｋｉｔ（アプライドバイオシステムズ社製）を用い、アプライドバイオシステムズ社製３７３ＡＤＮＡシークエンサーにより塩基配列の確認を行った。その結果、ｄＨ３３、Ａ３３’、Ｔ３３’、ｄＧ１１６、ｄＲ１１７、Ａ１１６’、Ｇ１１６’、Ｎ１１６’をコードするプラスミドは全長にわたり意図した部位以外に塩基配列の置換がなく予想通りのＴＰＯｃＤＮＡ配列を持っことを確認した。また、Ｇ３３’では意図した部位以外に１カ所のアミノ酸置換をもたらす塩基置換［Ｐｒｏ３８（ＣＣＴ）→Ｓｅｒ（ＴＣＴ）］が認められた。
得られたそれぞれのクローンのＣＯＳ７細胞へのトランスフェクションは、実施例１１に従って行った。すなわちプラスミドＤＮＡ１０μｇを使用し、クロロキン処理を含むＤＥＡＥ−デキストラン法を用いたトランスフェクションを行い、４−５日後に培養上清を回収した。回収した培養上清をＭ−０７ｅアッセイ系で評価した。その結果、アミノ酸挿入・欠失誘導体をコードするプラスミドでトランスフェクトしたＣＯＳ７細胞培養上清中に、いずれも用量依存的にＴＰＯ活性が検出された（図２３参照）。ＴＰＯ誘導体をコードするｃＤＮＡを含まない発現プラスミドｐＥＦ１８ＳをトランスフェクションしたＣＯＳ７細胞培養上清には活性が認められなかった。
＜実施例６９＞
ＴＰＯによる血小板増加作用
あらかじめ眼窩静脈より採血し、マイクロセルカウンター（東亜医用電子製、Ｆ−８００）にて血小板数を測定した正常な８週齢のＩＣＲ系雄性マウス２０匹を無作為に４群に分けた。そのうちの１群（コントロールーｉＶ群）には１００μｌのＰＢＳを１日１回５日間連続で静脈内に投与し、他の１群（ＴＰＯ−ｉｖ群）には実施例５６で得られたＳＰ Ｓｅｐｈａｒｏｓｅ Ｆａｓｔ ＦｌｏｗのヒトＴＰＯ活性画分Ｆ２の濃縮液をＮＡＰ−２５カラム（ファルマシア・バイオテク社製、カタログ番号１７−０８５２−０２）でＰＢＳに置換し、さらにＰＢＳで希釈したもののうち１００μｌ（Ｍ−０７ｅアッセイ系での相対活性量２１１，９００を含む）を１日１回５日間連続で静脈内に投与した。また、別の１群（コントロール−ｓｃ群）には１００μｌのＰＢＳを１日１回５日間連続で皮下に投与し、他の１群（ＴＰＯ−ｓｃ群）にはＴＰＯ−ｉｖ群と同様に１００μｌを１日１回５日間連続で皮下に投与した。
投与開始後６、８、１０、１３、１５日目にそれぞれのマウスの眼窩静脈より経日的に採血し、マイクロセルカウンター（東亜医用電子製、Ｆ−８００）にて血小板数を測定した。
血小板数の推移を図２４に示した。
すなわち、コントロールーｉｖ群では血小板数は最も増加した６日目においても投与前と比較して４４％の増加であり、コントロール−ｓｃ群では最も増加した８日目において４７％増加が観察されたにすぎなかった。その一方で、ＴＰＯ−ｉｖ群では６日目には投与前と比較して１７７％の増加がみられ、その後も増加を続け１０日目には４６９％という最大の増加が観察された。また、ＴＰＯ−ｓｃ群では６日目にすでに３４７％の増加が見られ、８日後には最大の４９３％の増加が観察された。
以上の結果より、ヒトＴＰＯは種差、投与ルートに関係なく血小板の増加作用を有することが確認された。
＜実施例７０＞
ＴＰＯによる血小板増加作用
あらかじめ眼窩静脈より採血し、マイクロセルカウンター（東亜医用電子製、Ｆ−８００）にて血小板数を測定した正常な１１週齢のＣ３Ｈ／ＨｅＪ系雄性マウス１６匹を無作為に４群に分けた。そのうちの１群（コントロール群）には１００μｌのＰＢＳを１日１回５日間連続で皮下に投与した。他の１群（ＴＰＯ−１群）には実施例５６で得られたＳｅｐｈａｃｒｙｌ Ｓ−２００ＨＲのヒトＴＰＯ活性画分Ｆ１をＮＡＰ−２５でＰＢＳに置換し、さらにＰＢＳで希釈したもののうち１００μｌ（Ｍ−０７ｅアッセイ系での相対活性量８３，０００を含む）を１日１回５日間連続で皮下に投与した。別の１群（ＴＰＯ−２群）にはＴＰＯ−１群で投与したＴＰＯ活性画分をさらにＰＢＳで２倍に希釈したもののうち１００μｌ（Ｍ−０７ｅアッセイ系での相対活性量４１，５００を含む）を１日１回５日間連続で皮下に投与した。また最後の１群（ＴＰＯ−３群）にはＴＰＯ−２群で投与したＴＰＯ活性画分をさらにＰＢＳで２倍に希釈したもののうち１００μｌ（Ｍ−Ｏ７ｅアッセイ系での相対活性量２０，７５０を含む）を１日１回５日間連続で皮下に投与した。
投与開始後６、８、１０、１２日目にそれぞれのマウスの眼窩静脈より経日的に採血し、マイクロセルカウンター（東亜医用電子製、Ｆ−８００）にて血小板数を測定した。
血小板数の推移を図２５に示した。
すなわち、コントロール群では血小板数にほとんど変動が見られなかった。ＴＰＯ投与群では、いずれの群においても投与開始８日目に最大となるような血小板数の増加が認められた。また、各投与群の間には用量依存性が認められた。すなわち、ＴＰＯ−１群では、６日目に既に約２００％の増加が見られ、８日目には約２７０％にまで増加した。その後減少したが、１２日目においても約６５の増加が見られコントロール群との間に有意差（ｐ＜０．０１：ダネットの多重比較検定：Ｄｕｎｎｅｔ ｍｕｌｔｉｐｌｅ ｃｏｍｐａｒｉｓｏｎ ｔｅｓｔ）が認められた。ＴＰＯ−２群においても同様に増加が見られたが、増加作用はＴＰＯ−１群には及ばないものの６、８日目にはそれぞれ約１４０％、約１６０％の増加を認めた。また、１２日目にはコントロール群とほぼ同様のレベルにまで減少した。ＴＰＯ−３群における増加作用はＴＰＯ−２群よりもさらに弱かったが、最高値に達した８日目には約１１０％の増加が見られており、コントロール群との間に有意差（ｐ＜０．０１）も認められた。
以上の結果より、ヒトＴＰＯは、マウスの系統に関係なく血小板数を増加させること、またその作用には用量依存性があることが確認された。
＜実施例７１＞
ＴＰＯによる制がん剤投与による血小板減少症阻止効果
８週齢のＩＣＲ雄性マウス３０匹に５−ＦＵを２００ｍｇ／ｋｇ体重の割合で静脈内投与した後、無作為に各々１５匹の２群に分け、その翌日（Ｄａｙ１）より一方の群（コントロール群）には１００μｌのＰＢＳを１日１回５日間連続で皮下に投与した。またもう一方の群（ＴＰＯ群）には、実施例６９で用いたヒトＴＰＯ活性画分Ｆ２１００μｌ（Ｍ−０７ｅアッセイ系での相対活性量２１１，９００を含む）を１日１回５日間連続で皮下に投与した。
投与開始後Ｄａｙ４、６、８にそれぞれの群より各々５匹のマウスを無作為に抽出し、眼窩静脈より採血し、マイクロセルカウンター（東亜医用電子製、Ｅ−２５００）にて血小板数を測定した。
血小板数の推移を図２６に示した。
すなわち、コントロール群では５−ＦＵ投与後血小板数は経日的に減少し、Ｄａｙ６には最低値（５−ＦＵ投与前値の約２８％）に達したが、Ｄａｙ８には減少の反動で約１．３倍にまで増加した。ＴＰＯ群においても５−ＦＵ投与後血小板数は経日的に減少し、Ｄａｙ６には最低値（５−ＦＵ投与前値の約５２％）に達したがＤａｙ４と６の血小板数にほとんど差はなく、また、Ｄａｙ６においてはコントロール群に比して有意（ｐ＜０．０５：ダネットの多重比較検定）に高値を維持した。Ｄａｙ８においては、５−ＦＵ投与前値の約２００％にまで増加した。
以上の結果より、ヒトＴＰＯには、制がん剤による血小板減少を阻止する効果があることが確認された。
＜実施例７２＞
ＴＰＯによる血小板減少症治療効果
７週齢のＩＣＲ雄性マウス３６匹に塩酸ニムスチン（ＡＣＮＵ）を５０ｍｇ／ｋｇ体重の割合で静脈内投与した後、無作為に各々１８匹の２群に分け、その翌日（Ｄａｙｌ）より一方の群（コントロール群）には２００μｌのＰＢＳを１日２回連日皮下投与し、またもう一方の群（ＴＰＯ群）には、実施例７０で用いたＴＰＯ活性画分Ｆ１にＰＢＳで希釈せずに０．０４％の割合でＴｗｅｅｎ−８０を添加したもの２００μｌ（Ｍ−０７ｅアッセイ系での相対活性量３６０，０００を含む）を１日２回連日皮下投与した。
投与開始後各々の群でマウスを無作為に３群に分け、それぞれをＤａｙ５、１２に採血する群、Ｄａｙ８、１４に採血する群そしてＤａｙ１０に採血する群とした。採血は眼窩静脈より行い、マイクロセルカウンター（東亜医用電子製、Ｆ−８００）にて血小板数を測定した。
血小板数の推移を図２７に示した。
すなわち、コントロール群ではＡＣＮＵ投与後に血小板数は経日的に減少し、Ｄａｙ８〜１０で最低（各々ＡＣＮＵ投与前値の約２９％）となり、Ｄａｙ１２に約４９％、Ｄａｙ１４に約７４％に回復したにすぎなかった。一方ＴＰＯ群では、Ｄａｙ５にはＡＣＮＵ投与前値の約３８％にまで減少した。その後は増加に転じ、ＡＣＮＵ投与前値に比してＤａｙ８では約６３％にまで回復し、Ｄａｙ１０以降はＡＣＮＵ投与前値以上の血小板数を示し、Ｄａｙ１２では約３００％、Ｄａｙ１４では約４００％にまで増加した。
以上のように、コントロール群では減少の過程にあるＤａｙ８〜１０において既にＴＰＯ群では増加に転じており、Ｄａｙ１０で既にＡＣＮＵ投与前値以上の血小板数まで増加していることから、ヒトＴＰＯは制ガン剤により惹起される血小板減少からの回復を促進する効果を有することが確認された。
なお、実施例７１の結果を併せてもヒトＴＰＯには制ガン剤の種類に関係なく血小板数の減少を阻止する効果および血小板減少からの回復を促進する効果を有することが期待される。
＜実施例７３＞
ＴＰＯによるＢＭＴ施行後の血小板減少症治療効果
７週齢のＣ３Ｈ／ＨｅＮ系の雄性マウス４８匹に１０Ｇｙの放射線を全身照射した後、同系のマウスの骨髄細胞１×１０^６個を直ちに静脈内投与した。そこで無作為に２群に分け、翌日（Ｄａｙ１）より一方の群（コントロール群）にはＰＢＳを、他方の群（ＴＰＯ群）には実施例７０で用いたＴＰＯ活性画分Ｆ１（Ｍ−０７ｅアッセイ系での相対活性量４４，０００を含む）をそれぞれ１日１回１００μｌずつ２０日間連日で皮下投与した。
投与開始後Ｄａｙ５、１０、１４、２１にそれぞれの群より各々６匹のマウスを無作為に抽出し、眼窩静脈より採血し、マイクロセルカウンター（東亜医用電子製、Ｅ−２５００）にて血小板数を測定した。
血小板数の推移を図２８に示した。
すなわち、いずれの群においてもＢＭＴ施行後に血小板数は経日的に減少し、Ｄａｙ１０に最低（ＢＭＴ施行前の約３％）となった。その後は徐々に回復し、Ｄａｙ１４にはコントロール群で約１１％、ＴＰＯ群で約１３％となった。Ｄａｙ２１にはコントロール群では３７％に回復したにすぎなかったが、ＴＰＯ群では約６５％にまで回復し有意な回復促進効果が認められた。
以上の結果より、実施例５６で精製されたヒトＴＰＯには、ＢＭＴ施行後の血小板数の回復を促進する効果があることが確認された。
＜実施例７４＞
ＴＰＯによる放射線照射後の血小板減少症治療効果
８週齢のＩＣＲ系の雄性マウス３６匹に５ＧｙのＸ線を全身照射した後、無作為に２群に分け、翌日（Ｄａｙ１）より一方の群（コントロール群）にはＰＢＳを、他方の群（ＴＰＯ群）には実施例７０で用いたＴＰＯ活性画分Ｆ１（Ｍ−０７ｅアッセイ系での相対活性量１，４４０，０００を含む）をそれぞれ１日１回３日間連日で皮下投与し、翌日よりＭ−０７ｅアッセイ系での相対活性量３６０，０００を１日１回７日間連日で皮下投与した。投与開始後各々の群でマウスを無作為に３群に分け、それぞれをＤａｙ４、１１、２１に採血する群、Ｄａｙ７、１３に採血する群そしてＤａｙ９、１５に採血する群とした。採血は眼窩静脈より行い、マイクロセルカウンター（東亜医用電子製、Ｆ−８００）にて血小板数を測定した。図２９に血小板数の推移を示した。
すなわち、コントロール群ではＸ線照射後に血小板数は経日的に減少し、Ｄａｙ９で最低（Ｘ線照射前値の約２４％）となり、Ｄａｙ１１に約３８％、Ｄａｙ１３に約６７％に回復し、Ｄａｙ１５にＸ線照射前値にまで回復した。一方ＴＰＯ群では、Ｄａｙ７にはＸ線照射前値の約２４％にまで減少した。その後は増加に転じ、ｘ線照射前値に比してＤａｙ９では約８２％にまで回復し、Ｄａｙ１１以降はＸ線照射前値以上の血小板数を示し、Ｄａｙ２１までその傾向は維持された。
以上のように、コントロール群では減少の過程にあるＤａｙ９において既にＴＰＯ群では増加に転じており、Ｄａｙ１１で既にＸ線照射前値以上の血小板数まで増加し、その後も高値を維持した。一方、コントロール群ではＸ線照射前値以上の血小板数まで回復するために１５日を要している。この結果より、実施例５６で精製されたヒトＴＰＯは放射線照射後の血小板減少からの回復期間を短縮することが認められ、放射線照射後の血小板減少症治療効果が認められた。
＜実施例７５＞
ｈＴＰＯ１６３による血小板増加作用
あらかじめ眼窩静脈より採血し、マイクロセルカウンター（東亜医用電子製、Ｆ−８００）にて血小板数を測定した正常なＣ３Ｈ／ＨｅＮ系雄性マウス１５匹を無作為に４群（コントロール群、Ａ，Ｂ，Ｃ群）に分けた。１群（コントロール群）には０．１％のマウス血清を含むＰＢＳを１日１回５日間連続で皮下に投与した。Ａ群には、実施例６５で得られたＳＰＳｅｐｈａｒｏｓｅ Ｆａｓｔ ＦｌｏｗのＴＰＯ活性画分Ｆ２を０．１％のマウス血清を含むＰＢＳで希釈しｈＴＰＯ１６３を１日あたり、Ｍ−０７ｅアッセイ系での相対活性量として約４０，０００，０００／ｋｇ体重、Ｂ群には同様にＭ−０７ｅアッセイ系での相対活性量として約８，０００，０００／ｋｇ体重、そしてＣ群にも同様にＭ−０７ｅアッセイ系としての相対活性量として約１，６００，０００／ｋｇ体重の割合で５日間連続で皮下に投与した。
投与開始後６、８、１０、１２日目にそれぞれのマウスの眼窩静脈より経日的に採血し、マイクロセルカウンター（東亜医用電子製、Ｆ−８００）にて血小板数を測定した。血小板数の推移を図３０に示した。
すなわち、コントロール群では血小板数にほとんど変動が見られなかった。ＴＰＯ投与群では、いずれの群においても投与開始８日目に最大となるような血小板数の増加が認められた。また、各投与群の間には用量依存性が認められた。すなわち、Ａ群では、６日目に約８８％、８日目に約１００％の増加が見られ、１０日目においても約９７％の増加を維持し、いずれもコントロール群との間に有意差（ｐ＜０．０１：ダネットの多重比較検定）が認められた。Ｂ群においても同様に増加が見られたが、増加作用はＡ群には及ばないものの６、８日目にはそれぞれ約６５％、約８４％の増加を認め、コントロール群との間に有意差（ｐ＜０．０１もしくはｐ＜０．０５：ダネットの多重比較検定）が認められた。Ｃ群における増加作用はＢ群よりもさらに弱かったが、最高値に達した８日目には約３１％の増加が見られた。
以上の結果より、実施例６５で精製されたｈＴＰＯ１６３には、血小板数を増加させること、またその作用には用量依存性があることが確認された。
＜実施例７６＞
ｈＴＰＯ１６３による血小板減少症治療効果
８週齢のＣ３Ｈ／ＨｅＮ雄性マウス１００匹に塩酸ニムスチン（ＡＣＮＵ）を５０ｍｇ／ｋｇ体重の割合で静脈内投与した後、無作為に各々２５匹の４群（コントロール，Ａ，Ｂ，Ｃ群）に分け、その翌日（Ｄａｙ１）よりコントロール群にはＰＢＳを５ｍｌ／ｋｇ体重の割合で１日１回連日皮下投与し、Ａ群には実施例６５で得られたＳＰ Ｓｅｐｈａｒｏｓｅ Ｆａｓｔ ＦｌｏｗのＴＰＯ活性画分Ｆ２をＰＢＳで希釈し、ｈＴＰＯ１６３をＭ−０７ｅアッセィ系における相対活性量として約１６，０００，０００／ｋｇ体重の割合で１日１回連日皮下投与した。Ｂ群には同様にＭ−０７ｅアッセイ系における相対活性量として約４８，０００，０００／ｋｇ体重の割合で、またＣ群にはＭ−０７ｅアッセイ系における相対活性量として約１６０，０００，０００／ｋｇ体重の割合で１日１回連日皮下投与した。
投与開始後各々の群でマウスを無作為に５群に分け、それぞれをＤａｙ５、８、１０、１２そして１４に採血した。採血は眼窩静脈より行い、マイクロセルカウンター（東亜医用電子製、Ｅ−２５００）にて血小板数を測定した。血小板数の推移を図３１に示した。
すなわち、コントロール群ではＡＣＮＵ投与後に血小板数は経日的に減少し、Ｄａｙ８〜１０で最低（ＡＣＮＵ投与前値の約１５〜１６％）となり、Ｄａｙ１２に約２８％、Ｄａｙ１４に約５１％に回復したにすぎなかった。一方Ａ群では、Ｄａｙ８にはＡＣＮＵ投与前値の約２５％にまで減少したが、その後は増加に転じ、Ｄａｙ１０には約３４％に、Ｄａｙ１２には約８９％に、そしてＤａｙ１４にはＡＣＮＵ投与前値以上にまで回復した。
Ｂ群では、Ｄａｙ８にはＡＣＮＵ投与前値の約２３％にまで減少したが、その後は増加に転じ、Ｄａｙ１０にはすでに約６４％にまで回復し、Ｄａｙ１４にはＡＣＮＵ投与前値以上にまで回復した。また、Ｃ群においても同様にＤａｙ１０以降に増加に転じ、Ｄａｙ１２以降にはＡＣＮＵ投与前以上にまで回復した。
以上のように、いずれの群においても血小板数の最低値はＤａｙ８に観察されたが、ヒトＴＰＯ投与群ではコントロール群に比して減少が緩やかであり、最低値の底上げが見られた。また、コントロール群ではＤａｙ１０においても血小板数の回復はほとんど見られなかったが、ヒトＴＰＯ投与群ではＤａｙ１０には程度の差はあるが全ての群で血小板数の回復が観察され、５０μｇ／ｋｇ投与群ではＤａｙ１２にはすでにＡＣＮＵ投与前置以上にまで回復した。コントロール群ではＤａｙ１４においてもＡＣＮＵ投与前置の約５１％に回復したにすぎなかったことと比較すると、参考例２で生産されたｈＴＰＯ１６３は、血小板減少からの回復を促進する効果を有することが明らかとなった。以上、実施例６５で精製されたｈＴＰＯ１６３は制ガン剤により惹起される血小板減少症に対する治療効果が認められた。
＜実施例７７＞
ヒトＴＰＯ誘導体の大腸菌における作製
大腸菌で発現されるＴＰＯの生物活性、安定性、および溶解性の向上を試みて、ＴＰＯ誘導体を設計し、作製した。作製した誘導体は、配列番号１１のＬｅｕ１２９のＡｒｇ置換体（Ｌ１２９Ｒ）、Ｈｉｓ１３３のＡｒｇ置換体（Ｈ１３３Ｒ）、Ｍｅｔ１４３のＡｒｇ置換体（Ｍ１４３Ｒ）、Ｇｌｙ８２のＬｅｕ置換体（Ｇ８２Ｌ）、Ｇｌｙ１４６のＬｅｕ置換体（Ｇ１４６Ｌ）、Ｓｅｒ１４８のＰｒｏ置換体（Ｓ１４８Ｐ）、Ｌｙｓ５９のＡｒｇ置換体（Ｋ５９Ｒ）およびＧｌｎ１１５のＡｒｇ置換体（Ｑ１１５Ｒ）である。
ＴＰＯ誘導体作製のための鋳型としては、実施例４２に記載のｈ６Ｔ（１−１６３）を用い、変異導入のため以下に示すオリゴＤＮＡを合成した。
Ｌ１２９Ｒ；５’−ＡＴＣＴＴＣ ＣＧＴ ＴＣＴＴＴＣＣＡＧＣＡＣＣＴ−３’（配列番号１１のＬｅｕ１２９のＡｒｇ置換体作製用）
Ｈ１３３Ｒ；５’−ＴＣＴＴＴＣＣＡＧＣＧＴ ＣＴＧＣＴＧＣＧＴ−３’（配列番号１１のＨｉｓ１３３のＡｒｇ置換体作製用）
Ｍ１４３Ｒ；５’−ＣＧＴＴＴＣＣＴＧＣＧＴ ＣＴＧＧＴＴＧＧＣ−３’（配列番号１１のＭｅｔ１４３のＡｒｇ置換体作製用）
Ｇ８２Ｌ；５’−ＧＧＣＣＡＧＣＴＴＣＴＧ ＣＣＧＡＣＣＴＧＣＣＴ−３’（配列番号１１のＧｌｙ８２のＬｅｕ置換体作製用）
Ｇ１４６Ｌ：５’−ＡＴＧＣＴＧＧＴＴＣＴＧ ＧＧＴＴＣＴＡＣＣＣＴ−３’（配列番号１１のＧｌｙ１４６のＬｅｕ置換体作製用）
Ｓ１４８Ｐ ；５’−ＧＴＴＧＧＣＧＧＴＣＣＧ ＡＣＣＣＴＧＴＧＣＧ−３’（配列番号１１のＳｅｒ１４８のＰｒｏ置換体作製用）
Ｋ５９Ｒ；５’−ＧＡＡＧＡＧＡＣＣＣＧＣ ＧＣＴＣＡＧＧＡＣＡＴＣＣ−３’（配列番号１１のＬｙｓ５９のＡｒｇ置換体作製用）
Ｑ１１５Ｒ：５’−ＣＴＧＣＣＧＣＣＡＣＧＴ ＧＧＣＣＧＴＡＣＣＡＣ−３’（配列番号１１のＧｌｎ１１５のＡｒｇ置換体作製用）
以下に示す変異プラスミドはすべてＳｃｕｌｐｔｏｒインビトロミュータジェネシスキツト（Ａｍｅｒｓｈａｍ社製）を用いて作製した。
実施例４２に記載のプラスミドｐＣＦＭ５３６／ｈ６Ｔ（１−１６３）から得られるＸｂａ Ｉ−Ｈｉｎｄ ＩＩＩ断片をＢｌｕｅｓｃｒｉｐｔ ＩＩ ＳＫ− （東洋紡績社製）のＸｂａ Ｉ−Ｈｉｎｄ ＩＩＩ切断部位間に挿入して、プラスミドｐＳＫＴＰＯを作製した。プラスミドｐＳＫＴＰＯは大腸菌ＪＭ１０９に保持させた。ヘルパーファージＭ１３Ｋ０７（宝酒造社製）を用いてｐＳＫＴＰＯから一本鎖ＤＮＡを調整し、上記の変異用合成オリゴＤＮＡを用いて、キットに添付のプロトコールに従い、変異プラスミドを作製した。得られた候補プラスミドについて挿入断片の塩基配列を決定し、目的とする部位が変異されていることを確認した。
上記で作製したすべてのｐＳＫＴＰＯの誘導体よりＸｂａ Ｉ−Ｈｉｎｄ ＩＩＩ断片を抽出し、プラスミドｐＣＦＭ５３６のＸｂａ Ｉ−Ｈｉｎｄ ＩＩＩ切断部位間に挿入した後、大腸菌２６１に形質転換し、目的とするＴＰＯ誘導体を発現させる形質転換体を完成させた。
目的とするＴＰＯ誘導体をコードする遺伝子を含む形質転換体の培養は、実施例４３に従って実施した。得られたｈ６Ｔ（１−１６３）生産組換体の凍結菌体（約３ｇ）に、１０ｍＭ ＥＤＴＡ、１０ｍＭ ＤＴＴ、１ｍＭ ＰＭＳＦを含む２０ｍＭトリス緩衝液（ｐＨ８，５）を約３０ｍＬ加え、懸濁した後、氷冷下１分間の間隔をおいて１分間のソニケーション（超音波破砕機を使用）を５回行なった。破砕した菌体を遠心チューブに移し、４℃、１５０００ｒｐｍで１０分間遠心し、ペレットを集めた。
このペレットに、８Ｍグアニジン塩酸塩、５ｍＭ ＥＤＴＡ、１ｍＭ ＰＭＳＦを含む１０ｍＭトリス緩衝液（ｐＨ８．７）約３０ｍＬを加えて懸濁し、さらに約５０ｍｇのＤＴＴを加え、室温にて１〜２時間攪拌し、蛋白質試料を還元した。還元後、２Ｍ塩酸溶液を用いて試料のｐＨを５に調整し、４℃に放置した。還元した蛋白質試料を、３０％グリセロール、３Ｍ尿素、３ｍＭシスタミン、１ｍＭＬ−システインを含む１０ｍＭ ＣＡＰＳ緩衝液（ｐＨ１０．５、約１．５Ｌ）に対して希釈した。
希釈は、室温で一晩かけて実施した。希釈後の試料溶液を室温で２日間攪拌し、試料蛋白質を充分に酸化した後、８０００ｒｐｍで４５分間遠心し、不溶物を除去した。遠心後の上澄みを６Ｍリン酸を用いてｐＨ６．８に調整し、イオン交換水で２倍に希釈した後、不溶物をフィルターペーパー（東洋濾紙製、＃２、直径９０ｍｍ）２枚を用いてろ過した。ろ液をイオン交換樹脂（ＣＭ−Ｓｅｐｈａｒｏｓｅ ｆａｓｔ ｆｌｏｗ、Ｐｈａｒｍａｃｉａ製）のカラム（２．６ｘ１０ｃｍ）に吸着させた後、１５％グリセロール、１Ｍ尿素を含む１０ｍＭリン酸緩衝液（ｐＨ６．８）約４００ｍＬで洗った後、１５％グリセロールを含む１０ｍＭリン酸緩衝液（ｐＨ７．２）約３００ｍＬでカラムを平衡化した。目的試料の溶出には、１５％グリセロールを含む１０ｍＭリン酸緩衝液から０．５Ｍ食塩を含む同緩衝液までの直線濃度勾配を用い、流速１ｍＬ／ｍｉｎで行なった。カラムからの蛋白質の溶出を２８０ｎｍにおける紫外吸収で追跡し、目的蛋白質を含むフラクションを集めた（約３０〜４０ｍＬ）。このフラクションを遠心濃縮器（Ｃｅｎｔｒｉｐｒｅｐ １０、Ａｍｉｃｏｎ社製）を用いて約２ｍＬに濃縮した後、逆相ＨＰＬＣ（Ｗａｔｅｒｓ社製）を用いて精製した。この時、カラムにはＷａｔｅｒｓ社製μＢｏｎｄａｓｐｈｅｒｅ Ｃ４（３．９ｘ１５０ｍｍ）を用い、蛋白質の溶出には、０．０５％ＴＦＡ溶液である溶離液Ａと、７０％２−Ｐｒｏｐａｎｏｌ， ３０％ＣＨ３ＣＮおよび０．０２％ＴＦＡを含む溶離液Ｂを用い、Ｂ溶離液の濃度を４０分間で１％から１００％まで変化させ、目的蛋白質を溶出させた。ＴＰＯ誘導体は、この条件では、いずれも約３０分の位置に溶出された。
精製されたＴＰＯ誘導体の活性測定を行なうため、溶出された誘導体を集め、２Ｌの１０ｍＭリン酸緩衝液に対して２日間透析した後、遠心濃縮器（Ｃｅｎｔｒｉｐｒｅｐ １０、Ａｍｉｃｏｎ社製）を用いて濃縮し、Ｍ−０７ｅアッセイに供した。調製された各ＴＰＯ誘導体は、いずれも対照として用いたｈ６Ｔ（１−１６３）と同様の生物活性を示した（図３２、図３３参照）。
以下、製剤例を示す。
＜製剤実施例１＞
実施例５６で得られたヒトＴＰＯ画分を濃縮し、無菌処理した後−２０℃で凍結された凍結物を用いて注射剤とした。
＜製剤実施例２＞
実施例５６で得られたヒトＴＰＯ画分を濃縮し、これを無菌操作で１０ｍｌバイアル瓶に５ｍｌ充填し、−２０℃で凍結乾燥後、ゴム栓に施栓した凍結乾燥物を用いて注射剤とした。
＜製剤実施例３＞
実施例５６で得られたヒトＴＰＯ画分を濃縮し、これを無菌濾過した後１０ｍ１バイアル瓶に充填し注射剤とした。
＜製剤実施例４＞
実施例６５で得られたｈＴＰＯ１６３画分を濃縮し、これを無菌処理した後−２０℃で凍結された凍結物を用いて注射剤とした。
＜製剤実施例５＞
実施例５６で得られたｈＴＰＯ１６３画分を濃縮し、これを無菌操作で１０ｍｌバイアル瓶に５ｍｌ充填し、−２０℃で凍結乾燥後、ゴム栓に施栓した凍結乾燥物を用いて注射剤とした。
＜製剤実施例６＞
実施例６５で得られたｈＴＰＯ１６３画分を濃縮し、これを無菌濾過した後１０ｍ１バイアル瓶に充填し注射剤とした。
＜製剤実施例７＞
参考例実施例５７で得られたヒトＴＰＯ画分を濃縮し、これを無菌処理した後−２０℃で凍結された凍結物を用いて注射剤とした。
＜製剤実施例８＞
実施例５７で得られたヒトＴＰＯ画分を濃縮し、これを無菌操作で１０ｍｌバイアル瓶に５ｍｌ充填し、−２０℃で凍結乾燥後、ゴム栓に施栓した凍結乾燥物を用いて注射剤とした。
＜製剤実施例９＞
実施例５７で得られたヒトＴＰＯ画分を濃縮し、これを無菌濾過した後１０ｍｌバイアル瓶に充填し注射剤とした。
【配列表】
配列番号：１
配列の長さ：２６１
配列の型：核酸
鎖の数：二本鎖
トポロジー：直鎖状
配列の種類：ｃＤＮＡ ｔｏ ｍＲＮＡ
起源：ラット（Ｒａｔｔｕｓ ｎｏｒｖｅｇｉｃｕｓ）
セルライン：ＭｃＡ−ＲＨ８９９４
配列：

配列番号：２
配列の長さ：１６６３
配列の型：核酸
鎖の数：二本鎖
トポロジー：直鎖状
配列の種類：ｃＤＮＡ ｔｏ ｍＲＮＡ
起源：ラット（Ｒａｔｔｕｓ ｎｏｒｖｅｇｉｃｕｓ）
セルライン：ＭｃＡ−ＲＨ８９９４
配列：

配列番号：３
配列の長さ：５８０
配列の型：核酸
鎖の数：二本鎖
トポロジー：直鎖状
配列の種類：ｃＤＮＡ ｔｏ ｍＲＮＡ
起源：ホモ サピエンス（Ｈｏｍｏ Ｓａｐｉｅｎｓ）
組織の種類：肝臓
配列：

配列番号：４
配列の長さ：８６１
配列の型：核酸
鎖の数：二本鎖
トポロジー：直鎖状
配列の種類：ｃＤＮＡ ｔｏ ｍＲＮＡ
起源：ホモ サピエンス（Ｈｏｍｏ Ｓａｐｉｅｎｓ）
組織の種類：肝臓
配列：

配列番号：５
配列の長さ：５７６
配列の型：核酸
鎖の数：二本鎖
トポロジー：直鎖状
配列の種類：ｃＤＮＡ ｔｏ ｍＲＮＡ
起源：ホモ サピエンス（Ｈｏｍｏ Ｓａｐｉｅｎｓ）
組織の種類：肝臓
配列：

配列番号：６
配列の長さ：１２６７
配列の型：核酸
鎖の数：二本鎖
トポロジー：直鎖状
配列の種類：ｃＤＮＡ ｔｏ ｍＲＮＡ
起源：ホモ サピエンス（Ｈｏｍｏ Ｓａｐｉｅｎｓ）
組織の種類：肝臓
配列：

配列番号：７
配列の長さ：１７２１
配列の型：核酸
鎖の数：二本鎖
トポロジー：直鎖状
配列の種類：ｃＤＮＡ ｔｏ ｍＲＮＡ
起源：ホモ サピエンス（Ｈｏｍｏ Ｓａｐｉｅｎｓ）
組織の種類：肝臓
配列：

配列番号：８
配列の長さ：４５０６
配列の型：核酸
鎖の数：二本鎖
トポロジー：直鎖状
配列の種類：Ｇｅｎｏｍｉｃ ＤＮＡ
起源：ホモ サピエンス（Ｈｏｍｏ Ｓａｐｉｅｎｓ）
配列：

配列番号：９
配列の長さ：４１６
配列の型：核酸
鎖の数：二本鎖
トポロジー：直鎖状
配列の種類：合成ＤＮＡ
配列：

配列番号：１０
配列の長さ：５４９
配列の型：核酸
鎖の数：二本鎖
トポロジー：直鎖状
配列の種類：合成ＤＮＡ
配列：

配列番号：１１
配列の長さ：５３５
配列の型：核酸
鎖の数：二本鎖
トポロジー：直鎖状
配列の種類：合成ＤＮＡ
起源：ヒト（Ｈｏｍｏ Ｓａｐｉｅｎｓ）

配列番号：１２
配列の長さ：５３５
配列の型：核酸
鎖の数：二本鎖
トポロジー：直鎖状
配列の種類：合成ＤＮＡ
起源：ヒト（Ｈｏｍｏ Ｓａｐｉｅｎｓ）

配列番号：１３
配列の長さ：５５５
配列の型：核酸
鎖の数：二本鎖
トポロジー：直鎖状
配列の種類：ｃＤＮＡ ｔｏ ｍＲＮＡ
起源：ホモ サピエンス（Ｈｏｍｏ Ｓａｐｉｅｎｓ）
組織の種類：肝臓
配列：

配列番号：１４
配列の長さ：１０４３
配列の型：核酸
鎖の数：二本鎖
トポロジー：直鎖状
配列の種類：合成ＤＮＡ
起源：ヒト（Ｈｏｍｏ Ｓａｐｉｅｎｓ）

配列番号：１５
配列の長さ：３２６
配列の型：アミノ酸
トポロジー：直鎖状
配列の種類：ペプチド
起源：ラット（Ｒａｔｔｕｓ ｎｏｒｖｅｇｉｃｕｓ）
配列：

配列番号：１６
配列の長さ：３３２
配列の型：アミノ酸
トポロジー：直鎖状
配列の種類：ペプチド
起源：ヒト（Ｈｏｍｏ ｓａｐｉｅｎｓ）
配列：

【図面の簡単な説明】
【図１】ＸＲＰからのラットＴＰＯ精製における、ゲル濾過クロマトグラフィー（Ｓｅｐｈａｃｒｙｌ Ｓ−２００ ＨＲカラム）のクロマトグラム及びラットＣＦＵ−ＭＫアッセイにおけるＴＰＯ活性を示すグラフ。
【図２】ＸＲＰ由来のラット低分子ＴＰＯ標品精製における逆相クロマトグラフィー（Ｃａｐｃｅｌｌ Ｐａｋ Ｃｌ ３００Ａカラム）のクロマトグラム、及びラットＣＦＵ−ＭＫアッセイにおけるＴＰＯ活性を示すグラフ。
【図３】ＸＲＰ由来のラット低分子ＴＰＯ標品精製における逆相クロマトグラフィー（Ｃａｐｃｅｌｌ Ｐａｋ Ｃｌ ３００Ａカラム）のＴＰＯ活性画分ＦＡ（チューブ番号３６〜４２）のＳＤＳ−ポリアクリルアミドゲル電気泳動による分離を示す写真、及びラットＣＦＵ−ＭＫアッセイにおけるＴＰＯ活性を示すグラフ。
【図４】ＸＲＰ由来のラット低分子ＴＰＯ標品精製における逆相クロマトグラフィー（Ｃａｐｃｅｌｌ Ｐａｋ Ｃｌ ３００Ａカラム）のＴＰＯ活性画分ＦＡの、３段階消化によるペプチドマップを示すグラフ。
【図５】ラットＣＦＵ−ＭＫアッセイにおけるラット血漿由来ＴＰＯ活性を示すグラフ。
【図６】発現ベクターｐＥＦ１８Ｓの構築を示す概念図。
【図７】ラットＣＦＵ−ＭＫアッセイにおけるｐＥＦ１８Ｓ−Ａ２αを導入・発現させたＣＯＳ１細胞の培養上清中のＴＰＯ活性を示すグラフ。
【図８】ラットＣＦＵ−ＭＫアッセイにおけるｐＥＦ１８Ｓ−ＨＬ３４を導入・発現させたＣＯＳ１細胞の培養上清中のＴＰＯ活性を示すグラフ。
【図９】ラットＣＦＵ−ＭＫアッセイにおけるｐＨＴ１−２３１を導入・発現させたＣＯＳ１細胞の培養上清中のＴＰＯ活性を示すグラフ。
【図１０ａ】ラットＣＦＵ−ＭＫアッセイにおけるｐＨＴＦ１を導入・発現させたＣＯＳ１細胞の培養上清中のＴＰＯ活性を示すグラフ。
【図１０ｂ】Ｍ−０７ｅアッセイにおけるｐＨＴＦ１を導入・発現させたＣＯＳ１細胞の培養上清中のＴＰＯ活性を示すグラフ。
【図１１】ファージクローンλＨＧＴ１の制限酵素地図の概略。（図中、Ｅ：ＥｃｏＲＩ、Ｈ：ＨｉｎｄＩＩＩ、Ｓ：Ｓａｌ Ｉを示す。）
【図１２ａ】ラットＣＦＵ−ＭＫアッセイにおけるｐＥＦＨＧＴＥを導入・発現させたＣＯＳ１細胞培養上清中のＴＰＯ活性を示すグラフ。
【図１２ｂ】Ｍ−０７ｅアッセイにおけるｐＥＦＨＧＴＥを導入・発現させたＣＯＳ１細胞培養上清中のＴＰＯ活性を示すグラフ。
【図１３ａ】ラットＣＦＵ−ＭＫアッセイにおける、およびｐＨＴ１−２１１＃１、およびｐＨＴ１−１９１＃１、およびｐＨＴ１−１７１＃２を導入・発現させたＣＯＳ１細胞培養上清中のＴＰＯ活性を示すグラフ。
【図１３ｂ】ラットＣＦＵ−ＭＫアッセイにおけるｐＨＴ１−１６３＃２を導入・発現させたＣＯＳ１細胞培養上清中のＴＰＯ活性を示すグラフ。
【図１４】Ｍ−０７ｅアッセイにおけるｐＨＴ１−２１１＃１、ｐＨＴ１−１９１＃１、ｐＨＴ１−１７１＃２、およびｐＨＴ１−１６３＃２を導入・発現させたＣＯＳ１細胞培養上清中のＴＰＯ活性を示すグラフ。
【図１５】ｐＤＥＦ２Ｏ２−ｈＴＰＯ−Ｐ１を導入・発現させたＣＨＯ細胞の培養上清からのヒトＴＰＯ精製における、逆相クロマトグラフィー（Ｖｙｄａｃ Ｃ４カラム）のクロマトグラム。
【図１６】ｐＤＥＦ２０２−ｈＴＰＯ−Ｐ１を導入・発現させたＣＨＯ細胞の培養上清から精製されたヒトＴＰＯのＳＤＳ−ポリアクリルアミドゲル電気泳動による分離を示す写真。
【図１７】ｐＣＦＭ５３６／ｈ６Ｔ（１−１６３）を導入・発現させた大腸菌からのヒトＴＰＯの精製における、逆相クロマトグラフィーのクロマトグラム。
【図１８】ｐＣＦＭ５３６／ｈ６Ｔ（１−１６３）を導入・発現させた大腸菌から分離・精製された変異型ヒトＴＰＯ、ｈ６Ｔ（１−１６３）のＳＯＳ−ポリアクリルアミドゲル電気泳動による分離を示す写真。
【図１９】ヒトＴＰＯ発現プラスミドｐＤＥＦ２０２−ｈＴＰＯ１６３をＣＨＯ細胞にトランスフェクションして得られた培養上清を出発材料にしたｈＴＰＯ１６３の精製におけるＳｕｐｅｒｄｅｘ ７５ｐｇカラムでのｈＴＰＯ１６３の溶出。蛋白質は２２０ｎｍの紫外吸収で測定した。
【図２０】ヒトＴＰＯ発現プラスミドｐＤＥＦ２０２−ｈＴＰＯ１６３をＣＨＯ細胞にトランスフェクションして得られた培養上清を出発材料にしたｈＴＰＯ１６３の精製において、Ｓｕｐｅｒｄｅｘ ７５ｐｇカラムで得られたｈＴＰＯ１６３標品のＳＤＳ−ポリアクリルアミドゲル電気泳動写真。各ｈＴＰＯ１６３は銀染色された。
【図２１】発現ベクターｐＳＭＴ２０１の構造を示す図。
【図２２】Ｍ−０７ｅアッセイにおけるβＧＬ−ＴＰＯ、Ｎ３／ＴＰＯ、０９／ＴＰＯを導入・発現させたＣＯＳ７細胞培養上清中のＴＰＯ活性を示すグラフ。
【図２３】Ｍ−０７ｅアッセイにおけるヒトＴＰＯ挿入・欠失誘導体を導入・発現させたＣＯＳ７細胞培養上清中のＴＰＯ活性を示すグラフ。
【図２４】ヒトＴＰＯを正常マウスに静脈内投与、または皮下投与した場合の血小板数の推移を示すグラフ。
【図２５】ヒトＴＰＯを正常マウスに投与量を変えて皮下投与した場合の血小板数の推移を示すグラフ。
【図２６】マウスに制ガン剤（５−ＦＵ）を投与した後にヒトＴＰＯを投与した場合の血小板数の推移を示すグラフ。
【図２７】マウスに制ガン剤（ＡＣＮＵ）を投与した後にヒトＴＰＯを投与した場合の血小板数の推移を示すグラフ。
【図２８】マウスにＢＭＴ（骨髄移植）を施行した後にヒトＴＰＯを投与した場合の血小板数の推移を示すグラフ。
【図２９】放射線照射後のマウスにヒトＴＰＯを投与した場合の血小板数の推移を示すグラフ。
【図３０】ｈＴＰＯ１６３を正常マウスに皮下投与した場合の血小板数の推移を示すグラフ。
【図３１】マウスに制ガン剤（ＡＣＮＵ）を投与した後にｈＴＰＯ１６３を投与した場合の血小板数の推移を示すグラフ。
【図３２】大腸菌で発現されたヒトＴＰＯ誘導体（Ｈ１３３Ｒ、Ｓ１４８Ｐ、Ｑ１１５Ｒ）のＭ−０７ｅアッセイにおけるＴＰＯ活性を示すグラフ。
【図３３】大腸菌で発現されたヒトＴＰＯ誘導体（Ｍ１４３Ｒ、Ｌ１２９Ｒ、Ｇ８２Ｌ、Ｇ１４６Ｌ、Ｋ５９Ｒ）のＭ−０７ｅアッセイにおけるＴＰＯ活性を示すグラフ。[Industrial application fields]
The present invention relates to a novel protein having an activity of promoting proliferation and differentiation of megakaryocyte progenitor cells, or specifically stimulating or enhancing the production of platelets in vivo, and use of such a protein.
[Prior art]
Megakaryocytes are large, multinucleated, cytoplasm-rich cells that are observed primarily in the bone marrow and produce platelets. The source of megakaryocytes is pluripotent stem cells in the bone marrow. Pluripotent stem cells differentiate to some extent to become megakaryocyte progenitor cells (CFU-MK) directed only to the megakaryocyte system, and further proliferate and differentiate into megakaryocytes. Megakaryocytes further undergo nucleopolyploidization and cytoplasmic maturation, and finally release platelets, which are anucleated cytoplasmic fragments, into the blood circulation. On average, 2000-4000 platelets are produced from one mature megakaryocyte. Although there are many unclear points about the mechanism of platelet production, megakaryocytes are located in the subendothelium of the venous sinus of the bone marrow, and the cytoplasm penetrates the endothelium and forms a string-like projection on the inner wall of the sinus. It is thought to release and release platelets.
Regarding such megakaryocyte hematopoiesis and platelet production, the existence of specific production regulation mechanisms has been suggested. In healthy individuals and normal animals, effective platelet counts are maintained.For example, when anti-platelet antibodies are administered to normal animals, only platelets rapidly decrease in a short period of time, and then start to increase. It is known that the normal value is transiently exceeded, but eventually returns to the normal value again. In clinical practice, it is known that the platelet count is decreased (thrombocytopenia) and thrombocytosis despite the normal red blood cell count and white blood cell count. However, to date, the isolation and identification of specific regulators responsible for such production mechanisms (for example, the presence of erythropoietin in erythropoiesis) has not been successful.
The most important function of platelets is the formation of a hemostatic thrombus in the hemostatic mechanism. If the hemostasis mechanism fails to work normally due to thrombocytopenia, it tends to bleed. In cancer radiotherapy and chemotherapy, thrombocytopenia due to bone marrow suppression is a fatal complication, and such cancer patients are given platelet transfusions to prevent bleeding tendency. Platelet transfusions are also given to patients who have undergone bone marrow transplants and patients with aplastic anemia.
Platelets for such platelet transfusions are prepared from healthy donor blood by plateletpheresis, but transfused platelets have a short shelf life and can be contaminated by bacterial infections. In addition, patients are exposed to dangerous viruses such as human immunodeficiency virus (HIV) or various hepatitis viruses, patients are induced with antibodies against histocompatibility antigens (HLA) on the surface of transfused platelets, or platelets for transfusion are used. There is a risk of contaminating lymphocytes causing graft-versus-host disease (GVHD).
Therefore, it would be extremely beneficial if a patient with thrombocytopenia could stimulate endogenous platelet production and at the same time reduce platelet transfusion dependence. In addition, if patients undergoing radiotherapy or chemotherapy for cancer can correct or prevent thrombocytopenia, they will be able to make their treatment safer and more focused, and further control will be possible. A cancer effect can be expected.
For this reason, a great deal of research has been conducted to isolate and identify specific regulatory factors involved in the regulation of megakaryocyte and platelet production. According to in vitro studies, the regulatory factors involved in the process of proliferation and differentiation of megakaryocyte cells are considered to be roughly classified into the following two types (for example, Williams et al., J. Cell. Physiol. 110, pages 101-104, (1982)). Megakaryocyte colony-stimulating factor (Meg-CSF) is a regulatory factor that promotes the growth and differentiation of CFU-MK and experimentally induces the formation of clumps (colony) consisting of megakaryocytes in a semi-solid culture solution. Has activity. Another regulatory factor is called megakaryocyte amplifying factor (Meg-Pot), megakaryocyte stimulating factor, platelet-stimulating factor, etc., mainly acting on megakaryocytes in the differentiation stage after CFU-MK to promote differentiation and maturation. Promote. Experimentally, it is often detected as an activity that amplifies Meg-CSF activity. In addition, when serum or plasma in the thrombocytopenia of an experimentally thrombocytopenic animal is injected into another normal animal, platelets increase, suggesting the presence of a humoral factor that has a platelet-increasing action in vivo. It is called thrombopoietin (TPO).
In recent years, some of the cytokines whose genes have been cloned have been examined for effects on megakaryocyte cells and platelet-increasing action in vitro. Human IL-3 stimulates the formation of human megakaryocyte colonies (Bruno et al., Exp. Hematol., 16, 371-377, (1988)), leading to an increase in platelet count in at least monkeys (Donahue et al., Science). 241, 1820-page (1988)). However, IL-3 is a factor that affects the proliferation and differentiation of all hematopoietic cells, and can be distinguished from megakaryocyte hematopoiesis and specific regulators of platelet production. Human IL-6 does not show Meg-CSF activity but acts on immature megakaryocytes to promote differentiation into mature megakaryocytes (Williams et al., Exp. Hematol., 18, 69-page, (1990)). ). In mice and monkeys, it exhibits platelet-increasing action and increases bone marrow megakaryocyte size and nuclear ploidy, but side effects such as weight loss and induction of acute phase proteins have also been observed (Asano et al., Blood). 75, 1602-1605, (1990); Stahl et al., Blood 78, 1467-1475, (1991). Human IL-11 also does not show Meg-CSF activity, exerts Meg-Pot activity, and has been reported to increase platelets using mice (Neven et al., Blood, Vol. 81, pages 901-908, (1993). ). In addition, human LIF significantly increases the platelet count in monkeys (Mayer et al., Blood, 81, 3226-3233, (1993)), but its effect on megakaryocytes in vitro is weak (Burstein) Et al., J. Cell. Physiol., 153, 305-312 (1992)).
These cytokines are expected to be clinically applicable as platelet-increasing factors, but they do not act specifically on the megakaryocyte system and have side effects. Therefore, in the clinic, a platelet increasing factor specific to the megakaryocyte-platelet system with fewer side effects is desired.
The presence of Meg-CSF, Meg-Pot, or TPO activity has been reported in the serum, plasma, urine of humans and animals with thrombocytopenia for a long time, or in the supernatant of certain human cell lines. Whether such factors exist alone or in a plurality and their activity is exerted, and differences with known factors remain largely unclear. Regarding factors derived from the human body, Hoffman et al. In addition to platelet reduction, sera from patients with aplastic anemia with reduced bone marrow megakaryocytes or a megakaryocyte thrombocytopenic purpura, compared to that of healthy individuals And found significantly higher Meg-CSF activity (Hoffman et al., N. Eng. J. Med., 305, 533-538, (1981)), and Mazur et al. Further in the serum of patients with aplastic anemia. This Meg-CSF activity was shown to be different from IL-3 or GM-CSF (Mazur et al., Blood) 76, 290-297, (1990). Similar Meg-CSF activity has also been detected in the serum of cancer patients who have undergone intensive chemotherapy and who have undergone bone marrow transplantation (Mazur et al., Exp. Hematol., 12, 624-628). (1984); de Alarcon and Schmieder, Prog. Clin. Bio. Res., 215, 335-340 (1986)). Hoffman et al. Reported that Meg-CSF having an apparent molecular weight of 46000 was purified from the plasma of patients with hypoplastic thrombocytopenia with bone marrow megakaryocytes (Hoffman et al., J. Clin. Invest., 75, 1174- 1182, (1985)) and subsequent studies have shown that the purity of the purified preparation is insufficient to determine the amino acid sequence (Hoffman, Blood) 74, 1196-1212, (1989). )). In mice from plasma from similar thrombocytopenia patients or from urine from patients with idiopathic thrombocytopenic purpura (ITP)⁷⁵se-selenomethionine (⁷⁵It has been shown that TPO-like activity that promotes the uptake of se-selenomethionine) into neoplastic platelets has been partially purified and that plasma-derived activity has an apparent molecular weight of 40000 (Grossi et al., Hematologicala) Vol. 72, 291- 295, (1987): Vannucci et al., Leukemia), Vol. 2, pages 236-240 (1988)).
Meg-CSF activity and TPO-like activity have also been detected in the urine of patients with aplastic anemia and severe ITP (Kawakita et al., Br. J. Haematol., 48, 609-615, (1981). Kawakita et al., Blood) 61, 556-560, (1983)). Furthermore, Kawakita et al. Report that the Meg-CSF activity contained in the urine extract of patients with aplastic anemia has an apparent molecular weight of 45000 by gel filtration under dissociated conditions (Kawakita et al., Br. J). Haematol., 62, 715-722, (1986)). Erikson-Miller et al. Also reported purification of Meg-CSF from a similar sample, but the structure has not been elucidated (Erikson-Miller et al., “Blood Cell Growth Factors; and oncology "ed. by Murphy, AlphaMed Press, Dayton, Ohio) pages 204-220, (1992)). Turner et al. Purified megakaryocyte stimulating factor (MSF) with Meg-CSF activity from the urine of patients undergoing bone marrow transplantation and cloned the gene (Turner et al., Blood, 78, 1106 279a, ( 1991) (abstr., Suppl. 1)). This MSF is a dimer of a protein having a molecular weight of 28000 to 35000. It is unclear whether this factor is the same as Meg-CSF that has been detected in the serum and plasma of thrombocytopenic patients and whether it has a platelet-increasing action in vivo. In addition, a TPO-like activity having a molecular weight of 32000 has been purified from the culture supernatant of a human fetal kidney-derived cell line (HEK cell), and various properties have been investigated, but the structure has not yet been clarified (McDonald et al., J. Lab. Clin. Med., 106, 162-174, (1985); McDonald, Int. J. Cell Cloning, 7, 139-155, (1989)).
On the other hand, another investigator has shown that the main activity that promotes maturation of megakaryocytes in vitro in the conditioned medium of this HEK cell is the known cytokine produced by this cell, namely IL- 6 and EPO are also reported (Withy et al., J. Cell. Physiol., 15, 3362-372, (1992)).
For animal-derived factors, Evatt et al. In rabbits and mice in thrombocytopenic plasma of rabbits injected with anti-platelet antibodies.⁷⁵A TPO-like activity that promotes the uptake of se-selenomethionine into new platelets has been reported (Evatt et al., J. Lab. Clin. Med., 83, 364-371, (1974)). There are several other similar reports from the 1960s to the 1970s (eg, Odell et al., Proc. Soc. Biol. Med., 108, 428-431; (1961); Evatt and Levin. J. Clin. Invest., 48, 1615-1626, (1969); Harker, Am. J. Physiol., 218, 1373-1380, (1970); Sheiner and Levin, J. Clin. Invest., 49, 1709-1713, (1970); Penington, Br. Med. J., 1, 606-608, (1970)). Evatt et al. And Hill and Levin partially purified TPO-like activity from thrombocytopenic rabbit plasma induced by antiplatelet antibody administration (Evatt et al., Blood, 54, 377-388, (1979); Hill and Levin, Exp. Hematol., Vol. 14, pp. 752-759, (1986)), and further, this factor with the activity of promoting the differentiation and maturation of megakaryocytes in vitro, ie, Meg-Pot activity as an index. However, it has been clarified that it has an apparent molecular weight of 40000-46000 on gel filtration, but has not yet been completely purified (Keller et al., Exp. Hematol., 16: 262-267, (1988). Hill et al., Exp. Hematol., 20, 354-360, (19 2)). No IL-6 activity was detected in the plasma of rabbits with severe acute thrombocytopenia induced by antiplatelet antibody administration, indicating that this TPO-like activity is carried by a molecule different from IL-6. (Hill et al., Blood, 80, 346-351, (1992)).
Tayrien and Rosenberg also purified a factor having an apparent molecular weight of 15000 that stimulates the production of platelet factor 4 in a megakaryocyte cultured cell line from the same rabbit plasma or HEK cell culture supernatant. Its structure has not been elucidated (Tayrien and Rosenberg, J. Biol. Chem., 262, 3262-3268, (1987)).
Nakeff has also found Meg-CSF activity in the serum of mice whose platelets have been decreased by administration of anti-platelet antibodies (Nakeff, “Experimental Health Today” ed. By Baum and Ledney, Springer-Verlag, NY, 111- 123, (1977)).
On the other hand, in the plasma of rabbits that have been thrombocytopenic by similar treatment, maturation of megakaryocytes is promoted in vitro (Keller et al., Exp. Hematol., 16, 262-267, (1988); Hill et al., Exp. Hematol., 17, 903-907 (1989)), stimulating megakaryocytic morphological changes to platelets (Leven and Yee, Blood, 69, 1046-1052, (1987)). Activity is detected, but Meg-CSF activity is not observed. The reason for this difference is unknown.
Miura et al. Detected Meg-CSF activity in thrombocytopenic plasma of rats systemically irradiated with sublethal doses of radiation (Miura et al., Blood, 63, 1060-1066, (1984)). Is not reduced by platelet transfusion, inducing Meg-CSF activity in vivo requires the reduction of megakaryocytes instead of platelets (Miura et al., Exp. Hematol., 16). 139-144, (1988). Mazur and South have detected Meg-CSF activity in the serum of dogs with aplastic anemia by irradiation and reported an apparent molecular weight of 175000 on gel filtration (Mazur and South, Exp. Hematol., 13 (1164-1172) (1985)). In addition, factors derived from serum, plasma, or urine have also been reported by Straneva et al. (Straneva et al., Exp. Hematol., Vol. 15, pages 657-663, (1987)).
Thus, despite the existence of factors that promote megakaryocyte hematopoiesis and platelet formation in biological samples of humans and animals with thrombocytopenia, only such factors have been identified to date. Isolation, biochemical and biological identification, and characterization has not been successful because such factors are present in extremely small amounts in natural sources such as blood and urine.
[Problems to be solved by the invention]
Accordingly, an object of the present invention is to promote the proliferation and differentiation of megakaryocyte progenitor cells and / or a protein (TPO) having an activity (TPO activity) that specifically stimulates or enhances the production of platelets in vivo. To provide a means for producing large quantities of the protein homogeneously using recombinant DNA technology by isolating and identifying the protein from a natural source and further isolating the gene encoding such protein. is there. If that is possible, it can replace or reduce the frequency of currently employed platelet transfusions, or it can be used to treat and diagnose platelet disorders.
[Means for Solving the Problems]
According to the present invention, a novel DNA (hereinafter referred to as “the DNA of the present invention”) encoding an amino acid sequence of a protein having TPO activity is provided. The DNA of the present invention includes DNA encoding the amino acid sequence shown in SEQ ID NO: 16. The DNA of the present invention has the amino acid sequence shown in SEQ ID NO: 16 and a part of the amino acid sequence modified (substitution, deletion, insertion, and / or addition) as long as it retains TPO activity That is, DNA encoding a TPO derivative is included.
In other words, the DNA of the present invention includes a DNA whose amino acid sequence substantially encodes the amino acid sequence shown in SEQ ID NO: 16. As used herein, “substantially the amino acid sequence shown in SEQ ID NO: 16” refers to the amino acid sequence shown in SEQ ID NO: 16 in addition to “the amino acid sequence shown in SEQ ID NO: 16” as long as it retains TPO activity. A part of the amino acid sequence includes an amino acid sequence having a substitution, deletion, insertion and / or addition.
As used herein, “DNA encoding an amino acid sequence” means DNA having all base sequences in a degenerate relationship.
Furthermore, the DNA of the present invention can be used to copy the amino acid sequence of a protein having TPO activity.
DNAs selected from the following (a) to (c).
(A) DNA shown in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 or a complementary strand thereof
(B) DNA that hybridizes (under stringent conditions) with the DNA of (a) or a fragment thereof
(C) DNA that can hybridize with the DNAs of (a) and (b) without the degeneracy of the genetic code.
In other words, the DNA of the present invention includes the DNA shown in either of the following (a) and (b).
(A) DNA encoding the amino acid sequence of a protein having TPO activity incorporated in the vector pEF18S-A2α (accession number FERM BP-4565) carried in E. coli DH5, or the vector pHT1-231 carried in E. coli DH5 DNA encoding the amino acid sequence of a protein having TPO activity incorporated in (Accession No. FERM BP-4564)
(B) DNA encoding the amino acid sequence of a protein having TPO activity, which hybridizes (under stringent conditions) with the DNA of (a) or a fragment thereof.
The expression “strict” hybridization conditions here refers to a series of implementations of PCR amplification of DNA in the present invention, which are performed by using degenerate and / or single sequence oligonucleotide primers (probes). Used in the examples.
For example, Sambrook et al., “Molecular Cloning” (Cold Spring Harbor Laboratory Press, 1989), Chapters 11 and 14, or Ausubel et al., “Current Protocols in Molecular Biology” (Cur. 1993), unit 2.10.
The DNA of the present invention includes a DNA containing a base sequence encoding the amino acid sequence from the 1st position to the 163rd position among the amino acid sequence shown in SEQ ID NO: 16 and encoding a protein having TPO activity.
Such DNA may include the provision of a cleavage site by a restriction enzyme and / or additional donation of DNA to the start, stop or intermediate for construction of an expression vector to facilitate expression. it can. In addition, when a non-mammalian host is selected, a codon (preferred codon) preferable for expression in the host may be incorporated.
The DNA of the present invention includes cDNA obtained by preparing mRNA from mammalian cells such as humans and the like and then screening from a cDNA library prepared by a known method by a known method. Examples of mRNA supply sources in this case include rat liver cell-derived cell lines McA-RH8994 cells, HTC cells, H4-II-E cells, rat liver, kidney, brain, small intestine, human liver and the like.
Moreover, the DNA of the present invention may be genomic DNA screened by a known method from a genomic library prepared by a known method from mammalian cells including humans. Examples of the genomic DNA supply source in this case include chromosomal DNA of human, rat, mouse and the like.
In addition, a cDNA encoding a protein having TPO activity obtained in this way is modified by using a well-known site-directed mutagenesis method, so that a part of the amino acid sequence is modified, that is, a TPO derivative. Can be obtained.
Further, based on the amino acid sequence / base sequence of a protein having TPO activity disclosed in the present specification, a DNA encoding a protein in which a part of the amino acid sequence is modified is chemically synthesized partly or entirely. This can be easily obtained by those skilled in the art.
The DNA of the present invention is a useful substance for mass-producing a protein having TPO activity by various gene recombination techniques.
Furthermore, the DNΛ of the present invention is a substance suitable for use as a labeled probe when isolating genes encoding TPO-related proteins, as well as cDNAs and genomic DNAs of other mammalian TPOs. It is also useful in gene therapy in humans and other mammalian species. The DNA of the present invention is useful for the development of transgenic mammalian species that can be used as large quantities of TPO and eukaryotic hosts for TPO production (Palmiter et al., Science, 222, 809-814, (1983)).
In addition, according to the present invention, a vector incorporating a DNA encoding a protein having TPO activity, a host cell transformed with the vector, and culturing the host cell to isolate the produced protein having TPO activity A method for producing a protein having TPO activity characterized by purification is provided.
As a host cell in this case, a prokaryotic (eg, bacteria, preferably E. coli) or eukaryotic (eg, yeast, insect, or mammal) cell can be used. Examples of mammalian cells include COS cells, Chinese hamster ovary cells, X63.3.6.5.3. Examples include cells, C-127 cells, BHK (Baby Hamster Kidney) cells, and human-derived cells (for example, HeLa cells). Examples of yeast include baker's yeast (Saccharomyces cerevisiae) and methanol-utilizing yeast (Pichia pastoris). Examples of insect cells include spider culture cells (eg, Sf21 cells).
Vectors used to transform these host cells include pKC30 (Shimatake H. and M. Rosenberg, Nature, 292, 128-132, 1981), pTrc99A (Amann E. et al., Gene 6, 69) for E. coli. 301-315, 1988).
For mammalian cells, pSV2-neo (Southern and Berg; J. Mol. Appl. Genet., 1,327-341, 1982), pCAGGS (Niwa et al .; Gene, 108, 193-200, 1991), or pcDL -SR α296 (Takebe et al .; Mol. Cell. Biol., 8, 466-472, 1988). For yeast, pG-1 (Schena M. and Yamamoto KR; Science) 241, 965-967, 1988) is available. For sputum cells, there are transfer vectors pAc373 (Luckow et al., Bio / Technology) 6, 47-55, 1988) for producing recombinant viruses.
These vectors contain an origin of replication, a selection marker, and a promoter as necessary. Furthermore, RNA splice sites, polyadenylation signals, and the like are added to vectors for eukaryotic cells as necessary.
As a replication origin, vectors derived from SV40, adenovirus, bovine papilloma virus, etc. can be used as mammalian cell vectors. As the vector for E. coli, those derived from ColE1, R factor, F factor and the like can be used. For yeast, 2 μm DNA, those derived from ARS1 and the like can be used.
As a vector for mammalian cells as a gene expression promoter, a virus-derived retrovirus, polyomavirus, adenovirus, SV40-derived one or the like, a chromosome-derived one (for example, EF1-α), etc. may be used. it can. As the vector for E. coli, those derived from bacteriophage λ, trp, lpp, lac, tac promoters and the like can be used. ADH, PH05, GPD, PGK, MAF α promoter can be used for baker's yeast, and AOX1 promoter can be used for methanol-assimilating yeast. A vector derived from a nuclear polyhedrosis virus or the like can be used as a sputum cell vector.
As a selectable marker, neomycin (neo) resistance gene, thymidine kinase (TK) gene, dihydrofolate reductase (DHFR) gene, E. coli xanthine guanine phosphoribosyltransferase (Ecogpt) gene, etc. may be used as a mammalian cell vector. it can. As a vector for E. coli, a kanamycin resistance gene, an ampicillin resistance gene, a tetracycline resistance gene, or the like can be used. For yeast, Leu2, Trpl, Ura3 genes and the like can be used.
In order to obtain a protein having TPO activity using the host-vector system as described above, it is obtained after transforming a host cell with a recombinant DNA body in which the gene is incorporated at an appropriate site of the vector. The transformant may be cultured, and the polypeptide may be further separated and purified from the cell or the culture solution. The means and methods used for these can be performed in combination with known ones.
When expressing using a host, the original signal sequence may be modified or a signal sequence of another protein may be used in order to make the N-terminus of the expression product more uniform. Further, by modifying (substituting or adding) the amino acid residues at and near the N-terminus (for example, adding lysine residues in addition to methionine residues when expressed using E. coli), N It is possible to make the ends uniform.
Also, glutathione-S-transferase (GST) and TPO are expressed in a suitable host as a fusion protein via a thrombin recognition peptide and isolated, and then the fusion protein is treated with thrombin. To remove the GST moiety and add a glycine residue at position -1.^-1TPO protein can be obtained.
The novel protein having the TPO activity of the present invention (hereinafter referred to as “protein of the present invention”) includes a protein consisting of the amino acid sequence shown in SEQ ID NO: 16. In addition, the present invention includes those in which a part of the amino acid sequence is modified (substitution, deletion, insertion, and / or addition), that is, a TPO derivative as long as it retains TPO activity.
In other words, the protein of the present invention includes a protein whose amino acid sequence is substantially the amino acid sequence shown in SEQ ID NO: 16. As used herein, “substantially the amino acid sequence shown in SEQ ID NO: 16” means “in addition to the“ amino acid sequence shown in SEQ ID NO: 16 ”, as well as“ as long as it retains TPO activity ”. The amino acid sequence includes a substitution, deletion, insertion and / or addition in a part of the amino acid sequence.
The protein of the present invention also includes a protein comprising the amino acid sequence at positions 7 to 151 of the amino acid sequence shown in SEQ ID NO: 16 and having TPO activity.
More specifically, among the amino acid sequence shown in SEQ ID NO: 6, the 1st to 231st positions, the 1st to 211th positions, the 1st to 191st positions, the 1st to 171st positions, the 1st to 163rd positions, the 1st position Human TPO protein consisting of ˜157, 1-156, 1-155, 1-154, 1-153, 1-151 and 7-163 is the protein of the present invention. Can be mentioned.
Furthermore, a protein having at least one amino acid substitution, deletion, insertion and / or addition within the above-mentioned 7- to 151-position sequence or outside thereof within a range not impairing the TPO activity. include.
As another example of the TPO derivative of the present invention, for example, a sugar chain that is improved in stability and persistence in the body by modifying amino acids (substitution, deletion, insertion and / or addition) One or more potential sites to be deleted or added are altered, one or more cysteine residues are deleted, or the cysteine is replaced with another amino acid residue (for example, an alanine or serine residue). And the like substituted with a group).
In general, it has been theoretically shown that introduction of proline residues and removal of glycine residues are effective as methods for improving the thermodynamic stability of proteins. (Matthews et al., Proc. Natl. Acad. Sci. USA 84, 6663-6667.1987). Therefore, when designing a TPO derivative for the purpose of improving stability, a TPO derivative into which a proline residue has been introduced and a TPO derivative from which a glycine residue has been removed can be considered.
For selection of a site for introducing a proline residue into human TPO, for example, TPO derived from other species having high homology to this (mouse TPO: Si Lok et al., Proc. Natl. Cad. Sci. The amino acid sequence of S.A. 369, 565-568 (1994), canine TPO: TD Bartley et al., Cell 77, 1117-124 (1994)) can be used.
For example, human type and rat type amino acid sequences (SEQ ID NO: 15) are compared, and human type is not proline but rat type has proline, or human type has glycine but rat type has amino acids other than glycine. You can choose the part.
For example, as such a site, the leucine residue at position 9, the glycine residue at position 82, the glycine residue at position 146, the serine residue at position 148 in the amino acid sequence of human TPO shown in SEQ ID NO: 16 and the like Derivatives in which those glycine residues are substituted with other amino acid residues, derivatives in which serine residues are substituted with proline residues, and the like are conceivable.
In general, proteins form a three-dimensional structure with hydrophobic amino acids on the inside and hydrophilic amino acids on the outside. By replacing amino acids present on the protein surface with charged amino acids having higher hydrophilicity, An improvement in solubility can be expected. Even in the case of human TPO, a derivative can be designed from such a viewpoint. For example, the lysine residue at position 14, the lysine residue at position 52, the lysine residue at position 59, the glutamine residue at position 115, the threonine residue at position 119, and the lysine residue at position 138 are replaced with arginine residues, respectively. And the like.
Furthermore, in Erythropoietin (EPO), whose similarity in amino acid sequence is pointed out with TPO, the fourth helix located in the primary structure (D helix) was predicted, and when a positive charge was introduced, the biological activity was improved. Since it is recognized (Japanese Patent Laid-Open No. 3-72885), TPO derivatives can also be designed by predicting the D helix from the amino acid sequence of TPO.
In general, for example, a secondary structure prediction program IDEAS (IDEAS user's manual) by Kanahisa et al. Can be used to predict a D helix of a protein.
Next, in the region where the D helix is predicted to be located, amino acids located outside are estimated, and amino acid residues that are candidates for introducing a positive charge are selected. Such a candidate can be selected, for example, by creating a wheel model of a target area (Shiffer and Edmundson, Biophys. J. 7, 121-135.1967).
In the case of human TPO, the position of the D helix is predicted to be the 126th to 142nd region. As amino acid residues selected in this region, for example, leucine residue at position 129, histidine residue at position 133, 143 Methionine residue at the position and the like. Therefore, derivatives that introduce positive charges by substituting these amino acid residues with arginine residues or lysine residues are conceivable.
More specifically exemplifying the TPO derivative of the present invention, at least a protein in which the serine residue at position 1 of human TPO is substituted with an alanine residue and the alanine residue at position 3 is substituted with a valine residue, position 25 A protein in which the arginine residue is replaced with an asparagine residue, a protein in which the histidine residue at position 33 is replaced with a threonine residue, an arginine residue at position 25 is an asparagine residue, and a glutamic acid residue at position 231 is Examples include proteins substituted with lysine residues, and proteins in which ThrSerleGlyTyrProTyrAspValProAspTyrAlaGlyValHisHisHisHisHis polypeptide is added to the C-terminus of these proteins.
Furthermore, at least a protein lacking a histidine residue at position 33, a protein lacking a glycine residue at position 116, a protein lacking an arginine residue at position 117, a histidine residue at position 33, and a position 34 A protein in which a threonine residue is inserted between the proline residues of, a protein in which an alanine residue is inserted between the histidine residue at position 33 and the proline residue at position 34, a histidine residue at position 33 and the position 34 A protein in which a glycine residue is inserted between the proline residues, a glycine residue is inserted between the histidine residue at position 33 and the proline residue at position 34, and the proline residue at position 38 is a serine residue A protein in which an asparagine residue is inserted between the glycine residue at position 116 and the arginine residue at position 117, and the glycine residue at position 116 17 of the protein which alanine residues were inserted between the arginine residue include a protein glycine residue is inserted between the arginine residue at position 116 glycine residue and position 117.
Also, at least a protein in which the leucine residue at position 129 is replaced with an arginine residue, a protein in which the histidine residue at position 133 is replaced with an arginine residue, and a methionine residue at position 143 is replaced with an arginine residue A protein, a protein in which the glycine residue at position 82 is replaced with a leucine residue, a protein in which the glycine residue in position 146 is replaced with a leucine residue, a protein in which the serine residue at position 148 is replaced with a proline residue, Examples include a protein in which the lysine residue at position 59 is substituted with an arginine residue, and a protein in which glutamine at position 115 is replaced with an arginine residue.
The TPO protein of the present invention includes a human TPO having the amino acid sequence shown in SEQ ID NO: 16, and a protein having a methionine residue added at the -2 position and a lysine residue at the -1 position of the aforementioned derivative, Proteins with a methionine residue added at the -1 position are also included.
Preferably, the protein of the present invention is a recombinant vector containing cDNA, genomic DNA, or DNA obtained by chemical synthesis, and is obtained by separation and purification from a transformed host cell. To do.
When bacterial expression is performed using bacteria (for example, Escherichia coli) as a host, a protein having an initiation methionine residue added to the N-terminal side of a protein having TPO activity is obtained. It is included in the present invention. Depending on the host to be used, the produced protein having TPO activity may be glycosylated or may not be glycosylated, and both are included in the protein of the present invention.
The protein of the present invention also includes a natural protein having TPO activity purified and isolated from a natural source (eg, a conditioned medium containing TPO activity, or human urine, serum, plasma).
The invention also includes a method for purifying TPO from such natural sources. Such purification methods generally include steps used for protein purification (ion exchange chromatography, lectin affinity chromatography, dye adsorption chromatography, hydrophobic mutual chromatography, gel filtration chromatography, reverse phase chromatography, heparin affinity chromatography). , Sulfated gel chromatography, hydroxyapatite chromatography, metal chelating chromatography, isoelectric focusing, preparative electrophoresis, isoelectric focusing, etc.) . In addition, a method that can be combined using the physicochemical properties of TPO that can be estimated from the examples described later is also included in the present invention. Furthermore, an antibody column using an antibody capable of recognizing TPO can also be used. Since TPO was found to be a ligand of Mp1 (de Sauvage et al., Nature 369: 533-538 (1994), Bartley et al., Cell 77, 1171-1124 (1994), Kaushansky et al., Nature 369. 565-568 (1994)), an affinity gel column is prepared by coupling Mp1 to an activated gel, and TPO can also be purified by using this. More specifically, an Mp1-X column produced and prepared by a gene recombination method using an CHO cell as a host for the extracellular region of Mp1 (Mp1-X) can be mentioned (the aforementioned Bartley et al. ( 1994)).
The present invention further provides a protein of the type encoded by a portion of DNA complementary to the protein coding strand of the human cDNA or genomic DNA of TPO, ie Tramontano et al. (Nucl. Acids Res., 12, 5049-5059 (1984)). Includes "complementary reverse proteins" as described.
The present invention is labeled with a detectable marker substance (eg, to provide reagents useful for the detection and quantification of TPO or its receptor-bearing cells in solid tissues and fluid samples such as blood or urine (eg,¹²⁵Also included are proteins of the invention that have been radiolabeled with I or biotinylated.
Biotinylated proteins of the invention are useful when combined with immobilized streptavidin to remove megakaryoblastic cells from bone marrow in autologous bone marrow transplantation. Furthermore, it is useful when combined with immobilized streptavidin to concentrate autologous or allogeneic megakaryocyte cells in the case of autologous or allogeneic bone marrow transplantation. Toxin conjugates of toxins such as ricin, diphtheria toxin and TPO, and radioisotopes are useful for anti-cancer therapy or as a conditioning regimen for bone marrow transplantation.
The present invention also provides for the labeling of human TPO genes and / or the position of any related gene family in a chromosomal map when labeled with a detectable marker (eg, a radiolabel or a nonisotopic label such as biotin). Nucleic acid materials useful for use in hybridization methods to locate are also provided. They are also useful for confirming human TPO gene disorders at the DNA level, and are also used as gene markers for confirming adjacent genes and their disorders.
The present invention further includes pharmaceutical compositions containing a therapeutically effective amount of the protein of the present invention together with useful and suitable diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or carriers. The term “therapeutically effective amount” as used herein refers to an amount that provides a therapeutic effect for a specified condition and method of administration. Such compositions are liquid or lyophilized or otherwise dried dosage forms that are of various pH and buffer strengths (eg, Tris-HCl, acetate, phosphate). ) More selected diluents, additives such as albumin or gelatin to prevent adsorption on the surface, surfactants (eg Tween 20, Tween 80, Pluronic F68, bile salts), solubilizers (eg glycerol, polyethylene) Glycol), antioxidants (eg ascorbic acid, sodium metabisulfite), preservatives (eg thimerosal, benzyl alcohol, parabens), excipients or isotonic agents (eg lactose, mannitol) . In addition, a covalent bond with a polymer such as polyethylene glycol to a protein, a complex with a metal ion, or a granular compound of a polymer compound such as polylactic acid, polyglycolic acid, hydrogel, etc. or on the surface thereof, or Incorporation of the substance into liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts. Since such compositions are likely to affect the physical state, solubility, stability, in vivo release rate, and in vivo clearance of TPO, the choice of composition will depend on the physical properties of the protein having TPO activity. And by chemical properties. In addition, a particulate coated with a polymer (eg, poloxamer or poloxamine) and TPO that binds to a tissue specific receptor, an antibody to a ligand or antigen, or binds to a ligand of a tissue specific receptor. Compositions are also encompassed by the present invention. Other dosage forms of the compositions of the present invention include granular forms, protective coatings, protease inhibitors, or absorption enhancers for various routes of administration including parenteral, pulmonary, nasal, and oral. To do.
The pharmaceutical composition containing the protein of the present invention can be administered usually at a dose of 0.05 μg / kg body weight to 1 mg / kg body weight as an active ingredient several times a day depending on the disease state, sex, route of administration and the like.
The pharmaceutical composition containing the protein of the present invention has, as an active ingredient, a relative activity amount of 25,000 to 500,000,000 / kg body weight in the M-07e assay, which will be described later, depending on the disease state, sex, administration route, and the like. It can be administered once to several times a day for about 1 to 7 days per week.
Furthermore, the present invention includes EPO, G-CSF, GM-CSF, M-CSF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, in addition to the protein of the present invention. A composition comprising one or more factors such as IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, LIF, SCF as additional hematopoietic factors. Include.
The proteins of the invention are useful for the treatment of a number of thrombocytopenia, alone or in combination with other additional hematopoietic factors. Other additional hematopoietic factors include EPO, G-CSF, GM-CSF, M-CSF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7. , IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, LIF and SCF.
According to the present invention, there is provided a therapeutic agent for thrombocytopenia in chemotherapy or radiotherapy or bone marrow transplantation (BMT) by administration of an anticancer agent or an immunosuppressant, comprising the protein of the present invention as an active ingredient. In addition, there are numerous diseases that are characterized by platelet disorders, such as platelet production disorders and platelet reduction due to shortened platelet life (increased platelet destruction or increased platelet consumption) and can be treated with the protein of the present invention. Examples include congenital Fanconi anemia, aplastic anemia associated with chemotherapy and radiation therapy, myelodysplastic syndrome, acute myelogenous leukemia, or thrombocytopenia due to myelogenesis failure such as bone marrow transplantation. Can be used to promote platelet recovery in healthy patients. It is also useful for thrombocytopenia due to abnormal TPO production. Examples of thrombocytopenia due to shortened life span of platelets and megakaryocytes include idiopathic thrombocytopenic purpura, acquired immune deficiency syndrome (AIDS), disseminated intravascular coagulation syndrome, thrombotic thrombocytopenia, etc. It is also useful for promoting platelet recovery in healthy patients. Furthermore, it is useful as an application to administer TPO before surgery to increase its own platelets and use the platelets as blood transfusion platelets during their surgery (autologous platelet transfusion).
Yet another use of the proteins of the invention is in the treatment of disorders caused by transient platelet loss or damage, for example by other chemicals or pharmaceuticals, or therapeutic measures. TPO can be used to facilitate the release of new “intact” platelets in such patients.
The present invention further provides an antibody that specifically binds to TPO, and an antigen for obtaining the same. The protein of the present invention or a partial fragment of the protein having an antigenic determinant can be used as an antigen. Such antibodies include both monoclonal and polyclonal antibodies, as well as chimeric antibodies made by known methods, ie “recombinant” antibodies and the like. In contrast to conventional antibody (polyclonal) formulations that generally contain different antibodies to different antigenic determinants (epitopes), each monoclonal antibody is an antibody against a single antigenic determinant on the antigen. Specific antibodies against TPO are useful for improving the selectivity and specificity of diagnostic and analytical assays using antigen-antibody reactions, and for isolating and purifying TPO. In addition, they are used to neutralize or remove TPO from serum. The advantage of monoclonal antibodies is that they can be synthesized by hybridoma cells in a medium that is not contaminated with other immunoglobulins. Monoclonal antibodies are prepared from the culture supernatant of hybridoma cells or from ascites induced by intraperitoneal inoculation of mice with hybridoma cells. The hybridoma technology first described by Kohler and Milstein (Eur. J. Immunol. 6, 511-519 (1976)) is widely used to generate hybrid cell lines carrying high levels of monoclonal antibodies against a number of specific antigens. Can be used.
Hereinafter, the present invention will be described in more detail.
(A) Purification of rat TPO-Analysis of partial amino acid sequence of rat purified TPO-Analysis of biological characteristics of rat purified TPO
The present inventors first attempted to purify a protein (rat TPO) having a proliferation / differentiation promoting activity of rat CFU-MK. In this purification, a number of trials and errors were repeated for the assembly of purification, such as selection of various natural sources, selection of chromatographic support, selection of separation means, and the like. As a result, the present inventors have used TPO activity based on rat CFU-MK assay described in <Reference Example> described below as an index from plasma of thrombocytopenic rats induced by X-ray or γ-ray irradiation. A protein having activity was purified and its partial amino acid sequence was determined <Examples 1-2>.
In addition, the biological properties of the plasma-derived rat TPO were tested <Example 3>.
The outline from purification to determination of the partial amino acid sequence of purified rat TPO is as follows.
(1) Plasma of about 1100 rats with thrombocytopenia caused by X-ray or γ-ray irradiation was collected and separated by Sephadex G-25 chromatography, anion exchange chromatography (Q-Sepharose FF), and lectin chromatography (WGA- (Agarose) step to obtain WGA-Agarose-adsorbed TPO active fraction.
(2) Next, this WGA-Agarose-adsorbed TPO activity fraction was subjected to dye adsorption affinity chromatography (TSK AF-BLUE 650MH), hydrophobic interaction chromatography (Phenyl Sepharose 6FF / LS), gel filtration chromatography (Sephacry S-). Up to 200HR). In this Sephacryl S-200HR, the TPO activity was divided into four peaks (from those having a high molecular weight to F1, F2, F3, F4). Therefore, the TPO active fractions F2 and F3 were concentrated, respectively. The sample F2 and the low-molecular-weight TPO sample F3 were individually advanced to the next purification step.
(3) The low-molecular-weight TPO sample F3 is separated by preparative reverse phase chromatography (YMC-Pack PROTEIN-RP), reverse phase chromatography (YMC-Pack CN-AP), and further reverse phase chromatography (Capcell Pack C1). did. When the obtained TPO activity fraction was subjected to SDS gel electrophoresis and TPO activity was extracted from the electrophoresis gel, it was confirmed that TPO activity was present in a band having an apparent molecular weight of about 17,000-19000 under non-reduction. did it.
(4) Therefore, all TPO active fractions were subjected to SDS gel electrophoresis under non-reduction and transferred to a PVDF membrane. Subsequently, peptide fragmentation by systematic limited enzymatic degradation of proteins on the PVDF membrane was performed, and the partial amino acid sequence of rat TPO protein could be determined (based on the amino acid sequence information of the two fragments, the gene of rat TPO). Cloning was performed.)
(5) On the other hand, the high-molecular-weight TPO sample F2 from Sephacryl S-200HR was also refined in the same manner as the low-molecular-weight TPO sample F3, and the TPO obtained by reverse phase chromatography (Capcell Pack C1) at the final step. The active fraction was subjected to SDS gel electrophoresis to extract TPO activity. When the activity was examined, it was confirmed that TPO activity was present in a band having an apparent molecular weight of about 17000 to 22000 under non-reduction, similar to F3-derived TPO.
(B) Specification of rat TPO-producing cells-Preparation of mRNA-Construction of rat cDNA library
Since purified rat TPO is derived from plasma, it was necessary to select an organ or a cell as a source of mRNA for cDNA cloning. Therefore, based on the biochemical properties and biological properties of rat plasma-derived TPO, TPO activity in the culture supernatants of various organs or cells was screened. As a result, rat plasma-derived TPO was also found in the culture supernatant of McA-RH8994 cells, HTC cells, and H4-II-E cells, which are cell lines derived from rat liver cells, and in the culture supernatant of rat primary liver cells. An almost equivalent TPO activity was found <Example 4>.
Meanwhile, a cDNA expression vector pEF18S in monkey cells (COS1 cells) was constructed by combining two expression vectors pME18S and pEFBOS <Example 5>. By constructing this vector, it has become easy to clone cDNA using a vector with high expression efficiency that has many types of cloning sites that can be used for insertion of the insert.
In addition to the above-described expression vectors, pUC and pBR plasmid vectors, λ phage vectors, and the like are mainly used for the construction of cDNA libraries.
The inventors cultured McA-RH8994 cells, added guanidine thiocyanate solution and homogenized, and then obtained total RNA by CsCl density gradient centrifugation <Example 6>.
RNA can also be prepared by the hot phenol method or the acidic guanidine phenol chloroform method.
Poly (A) is obtained from this total RNA by oligo dT-immobilized latex particles.⁺After the RNA was purified, single-stranded cDNA was synthesized by reverse transcriptase using oligo dT to which the restriction enzyme NotI cleavage sequence was added as a primer, and RNase H and E. coli. Double-stranded cDNA was obtained using E. coli DNA polymerase I. An EcoRI linker was added to the resulting double-stranded cDNA, and this was combined with the cDNA expression vector pEF18S constructed in Example 5 cut with NotI and EcoRI to transform competent E. coli DH5. A cDNA library was constructed <Example 7>.
(C) Acquisition (cloning) of rat TPO cDNA fragment by PCR method
Based on the partial amino acid sequence of rat TPO purified from rat plasma, a DNA sequence considered to encode the sequence was predicted, and degenerate primers used for polymerase chain reaction (PCR) were synthesized. The primer to be used may be based on the amino acid sequence at a position other than the primer used in the present invention. A highly degenerate primer can also be used without using inosine. Furthermore, primers with reduced degeneracy can be designed using codons frequently used in rats (see Wada et al. Nucleic Acids Res., 18, 2367-2411, 1990).
When plasmid DNA was extracted from the entire cDNA library prepared previously and PCR was performed using this DNA as a template, a band of about 330 bp was detected. When the nucleotide sequence was determined, it encoded a part of the rat TPO gene. It was found to be a DNA fragment (A1 fragment) <Example 8>.
(D) Screening of rat TPO cDNA by PCR method—Sequence of rat TPO cDNA—Expression confirmation
The previously constructed cDNA library was divided into pools of about 10,000 clones, and plasmid DNA was extracted from 100 pools. When PCR was performed using these DNAs as templates and primers newly synthesized based on the base sequence of the A1 fragment, bands considered to be specific to 3 pools out of 100 pools were detected. Therefore, one of these pools was selected, and this pool was further divided into sub-pools of about 900 clones, and when 100 PCRs of 100 pools were subjected to similar PCR, bands were detected in three sub-pools. Of these, one pool was selected, further divided into sub-pools of 40 clones, and finally, selection by one PCR was performed for each clone. As a result, plasmid pEF18S-A2α carrying the rat TPO gene cDNA fragment was obtained. Clones were successfully isolated <Examples 9 to 10>.
When the nucleotide sequence of this cDNA fragment was determined, it was revealed that it was encoded from a partially determined amino acid sequence purified from rat plasma, and considered to be a rat TPO gene <Example 10>.
When plasmid DNA was purified from the clone obtained as described above and transfected into COS1 cells, TPO activity could be detected in the culture supernatant. As a result, it was confirmed that the obtained pEF18S-A2α carries a cDNA encoding rat TPO <Example 11>.
(E) Detection of TPO mRNA in various rat tissues
TPO mRNA expression tissue in the rat body was analyzed by PCR, and specific expression was detected in the brain, liver, small intestine, and kidney <Example 12>.
(F) Construction of human cDNA library
Based on the results of Examples 4 and 12, liver was selected as the starting tissue for human TPO cDNA cloning. Therefore, a cDNA library was constructed from commercially available mRNA derived from normal human liver. PEF18S was used as the vector in the same manner as in the rat library, cDNA was synthesized by the same operation as in the rat, and a library having directionality using restriction enzyme NotI and EcoRI sites was obtained. The number of library clones introduced into E. coli strain DH5 and produced was about 1.2 million <Example 13>.
(G) Acquisition (cloning) of human TPO cDNA fragment by PCR method
Based on the nucleotide sequence of cDNA encoding rat TPO carried on pEF18S-A2α, several types of PCR primers were synthesized. When cDNA was synthesized from commercially available mRNA derived from normal human liver and PCR was performed using these primers, a band around 620 bp was observed. When the nucleotide sequence was determined, it was revealed that it was a DNA fragment having about 86% homology with rat TPO and considered to be a part of the gene encoding human TPO <Example 14>.
(H) Screening of human TPO cDNA by PCR method-Sequence of human TPO cDNA-Expression confirmation
The previously prepared human cDNA library was amplified and divided into pools of about 100,000 clones, and plasmids were extracted from 90 pools. When PCR was performed using these DNAs as templates and primers newly synthesized based on the base sequence of the human-TPO fragment obtained in Example 14, bands were observed in three pools.
One pool was selected from these, and 5,000 clones were divided into sub-pools with one pool, and plasmid DNA was purified from 90 pools. When similar PCR was carried out using these as templates, bands were detected in 5 subpools. Therefore, one pool was selected from these, and 250 clones were further divided into sub-pools, and plasmid DNA was purified from 90 pools of them, and detection was performed again by PCR. Was observed. Therefore, one pool was selected, and 30 clones were divided into sub-pools, one of which was purified. Plasmid DNA was purified from 90 pools and PCR was performed. As a result, bands were observed in three pools. It was. Of these, 90 colonies were picked from one pool, plasmid DNA was prepared, and PCR was performed to finally obtain clone HL34 <Example 15>.
When the nucleotide sequence of plasmid DNA possessed by this clone was determined, it was revealed that it carries cDNA having about 84% homology with the nucleotide sequence of rat TPO <Example 16>.
When the plasmid DNA of this clone was purified and transfected into COS1 cells, TPO activity was detected in the culture supernatant, and it was found that the plasmid DNA possessed by this clone carries the gene cDNA fragment encoding human TPO. <Example 17> confirmed.
However, this cDNA fragment had no stop codon and had a poly A tail-like sequence at the 3 'end, which was considered to be an artificial product during cloning. Thus, when an expression vector encoding an amino acid immediately before the poly A tail-like sequence was constructed and expressed in COS1 cells, TPO activity was detected in the culture supernatant <Example 18>.
In order to analyze the structure of the full-length cDNA, PCR was used to obtain a DNA fragment on the human TPO 3 'end side.
When the nucleotide sequence of this fragment was determined, it was found that it overlapped with the cDNA of clone HL34 obtained in Example 15, and the open reading frame also extended and was predicted to encode a protein consisting of 353 amino acids in total. . That is, it was suggested that human TPO protein consists of 353 amino acids containing 21 signal sequences <Example 19>.
In addition to the cloning method described above, human TPO cDNA cloning uses a library constructed using a pUC or pBR plasmid vector, a λ phage vector, etc., and a rat TPO cDNA fragment as a probe. It can also be carried out by colony hybridization or plaque hybridization. In designing a degenerate probe, inosine can also be used to reduce the degree of degeneracy. When an assay that can detect TPO activity specifically and with high sensitivity is available, expression cloning using an expression library as used in the present invention can also be performed.
However, as described in Example 15, since the content of RNA encoding human TPO is considered to be extremely low in normal human liver (the content calculated from this example was 1 in 3 million). In the screening method by hybridization using a synthesized oligonucleotide probe or rat or human TPO cDNA fragment as a probe, the number of clones and plaques to be processed becomes enormous, Since sensitivity and specificity do not reach the PCR method, it is expected to be extremely difficult. The present inventors actually performed colony hybridization using the rat TPO cDNA fragment as a probe for 2 million clones of the normal human liver cDNA library prepared in Example 13, but a human TPO cDNA clone could not be obtained. It was.
(I) Reconstruction of human TPO cDNA library
Since clone HL34 obtained in Example 15 was considered to be a clone containing incomplete cDNA, for the purpose of obtaining a full-length human TPO cDNA, a commercially available poly (A) derived from normal human liver⁺A human TPO cDNA library (hTPO-F1) was reconstructed using RNA. The number of library clones introduced into E. coli strain DH5 was about 1 million. <Example 20>
(J) Re-screening of human TPO cDNA by colony hybridization-Sequence of human TPO cDNA-Expression confirmation
Primers for PCR based on the base sequence (SEQ ID NO: 3) of the partial-length human TPO cDNA obtained in Example 14 and the base sequence (SEQ ID NO: 6) of the full-length human TPO cDNA estimated in Example 19 Was synthesized.
The cDNA library (hTPO-F1) constructed in Example 20 was divided into three pools (# 1 to 3), plasmid DNA was prepared from each pool, and primers synthesized using these DNAs as templates were used. PCR was performed. Since DNA of the expected size was amplified when DNA from the # 3 pool was used, the # 3 pool was divided into 15000 sub-pools, and PCR was performed with the previous synthetic primer. The expected size of DNA in 6 pools was amplified. One pool was selected from these, and 1,000 clones were divided into sub-pools with one pool. Plasmid DNA was prepared and PCR was performed, but no DNA amplification was observed. This was thought to be because the recovery rate of the plasmid DNA deteriorated because the desired clones grew slower than the other clones.
Therefore, the pool was returned to the # 3 pool, and clones were seeded so that there were 4100 colonies per LB plate, and 100 plates and respective replica plates were prepared. When DNA was extracted from the colonies on the plate in the same manner as described above, amplification of a band was observed in 1 of 100 pools. Two replica filters were prepared from the pool plate, and colony hybridization was performed using a radiolabeled probe (EcoRI / BamHI fragment of plasmid pEF18S-HL34). As a result, since one positive signal was observed, a colony was picked up from the original plate, and plasmid DNA was prepared from 50 colonies obtained by re-spreading on the LB plate. pHTF1 was obtained. <Example 21>
Thus, in the screening of DNA clones, hybridization, PCR) expression cloning, etc. are mainly used, but the efficiency and sensitivity of screening can be increased or labor can be reduced by appropriately combining them. .
When the nucleotide sequence of the clone pHTF1 obtained here was determined, an open reading frame exists, and the amino acid sequence of the protein considered to be encoded by this open reading frame is the amino acid sequence of human TPO estimated in Example 19 Completely matched the sequence (SEQ ID NO: 6). Although the nucleotide sequence was different from SEQ ID NO: 6 at 3 positions, no amino acid conversion occurred. This confirmed that the human TPO protein consists of 353 amino acids including a 21-residue signal sequence. <Example 22>
When plasmid DNA was prepared from the clone pHTF1 obtained as described above and transfected into COS1 cells, TPO activity could be detected in the culture supernatant. <Example 23>
(K) Screening of human TPO chromosomal DNA by plaque hybridization-Sequence of human TPO chromosomal DNA-Expression confirmation
Tohoku University Genetic Experiment Facility From a human genomic library provided by Prof. Tokuo Yamamoto, phages were plated at 30,000 per NZYM plate, and 18 plates and their respective replica filters were prepared. A human TPO cDNA fragment (base sequence number 178-1025 of SEQ ID NO: 7) contained in clone pHTF1 was amplified by PCR and purified.³²Plaque hybridization was performed using P-labeled one as a probe. As a result, since 13 positive signals were detected, plaques were picked up from the original plate, re-rolled to 1000 plaques per NZY plate, and plaque hybridization was performed again on the replica filters prepared from each. did. As a result, since positive signals were detected in all 13 sets of filters, plaques were isolated, phage DNA was prepared, and each of the 13 clones was checked by PCR to determine whether it contained the coding region of human TPO cDNA.
Since 5 out of 13 clones were considered to contain all of the coding region predicted from cDNA, 1 clone (λHGT1) was selected and Southern blot analysis (the probe used was the same as above) was performed. When digested with the restriction enzyme HindIII, a single band of about 10 kbp was observed. Therefore, the DNA of the clone λHGT1 was digested with the restriction enzyme HindIII and then electrophoresed on an agarose gel, and the 10 kbp band was excised and purified. Subcloning finally yielded clone pHGT1. <Example 24>
When the nucleotide sequence of this clone was determined, the carried chromosomal DNA contained all of the coding region of human TPO protein shown in SEQ ID NO: 6, and the nucleotide sequence was completely identical for that region. Further, the region corresponding to the exon is divided by four introns, and it was found that the region differs from the base sequence of the full-length human TPO cDNA carried in the clone pHTF1 obtained in Example 21 at three places. Of the 5 clones finally selected, the remaining 4 clones were also subjected to nucleotide sequence determination. As a result, 2 sites were consistent with clone pHGT1, 2 clones were consistent with pHGT1, It was consistent with clone pHTF1. <Example 25>
The EcoRI fragment of the plasmid clone pHGT1 obtained as described above was ligated to the expression vector pEF18S to prepare a human TPO expression plasmid pEFHGTE and transfected into COS1 cells, and TPO activity was detected in the culture supernatant. I was able to. <Example 26>
(L) Preparation of human TPO-deficient derivative-expression in COS1 cells-activity confirmation
From the results of Examples 18 and 22, it was predicted that the human TPO protein does not require a full-length amino acid for its activity expression. Therefore, for the purpose of analyzing the region necessary for the expression of the activity of human TPO protein, PCR was carried out using the plasmid DNA of clone pHT1-231 obtained in Example 18 as a template, and the C-terminal of human TPO protein. An expression plasmid with deleted sides, ie, plasmid DNA of deletion derivatives encoding amino acids 1-211, 1-191, 1-171, and 1-163 was obtained. When the obtained plasmid DNAs were transfected into COS1 cells, TPO activity was detected in any culture supernatant. <Example 27>
In order to examine the region responsible for TPO activity in more detail, a derivative was sequentially deleted from the C-terminal to the 151st amino acid, and a derivative was deleted to the N-terminal 6, 7, and 12th amino acids. However, when the activity was detected by expressing in COS1 cells, the activity could not be detected when the amino acid was deleted up to the seventh amino acid at the N-terminal side and the amino acid at the C-terminal side was deleted up to the 151st amino acid. . <Examples 28 and 29>
By preparing a derivative (deletion, substitution, insertion, addition, etc.) based on the cDNA thus obtained, a modified protein responsible for the activity of the original protein can be obtained. As a method used in this case, there are a PCR method, a Site directed mutationage method, a chemical synthesis method, and the like.
(M) Expression-purification of human TPO cDNA in CHO cells
A cDNA animal cell expression plasmid, pHTP1, encoding the amino acid sequence of SEQ ID NO: 6 was constructed. <Example 30>
A CHO cell expression vector pDEF202-hTPO-P1 was constructed using the TTP cDNA region of pHTP1. <Example 31>
As a result of transfection and selection of this vector into CHO cells, transformed cells in which an expression vector encoding human TPO was incorporated into the chromosome were obtained. When this cell clone was cultured, TPO activity was detected in the culture supernatant. <Example 32>
In Example 32, a large amount of a human TPO-producing CHO cell line (CHO28-30 cells, 25 nM MTX resistant) obtained by transfecting CHO cells with the human TPO expression plasmid pDEF202-hTPO-P1 <Example 55> .
Human TPO was purified from 100 L of the culture supernatant. <Example 56>
Further, TPO was purified from the culture supernatant obtained in Example 55 by another method. <Example 57>
(N) X63.3.6.5.3 of human TPO cDNA. Cell expression to activity confirmation
Using the TPO cDNA region of the plasmid pBLTEN prepared in Example 30, an expression vector for X63.6.5.3 cells, BMCGSneo-hTPO-P1, was constructed. <Example 33>
When this vector was transfected into X63.6.6.3 cells, transformed cells in which an expression vector encoding human TPO was incorporated into the chromosome were obtained. When these cells were cultured, TPO activity was detected in the culture supernatant. <Example 34>
(O) Mass expression of human TPO in COS1 cells-purification-molecular weight measurement, biological properties
A large amount (about 401 in total) of the culture supernatant in which COS1 cells were transfected with the expression vector prepared in Example 30, pHTP1, was expressed. <Example 35>
TPO was purified from about 71 serum-free culture supernatant of COS1 cells containing the expression vector, pHTP1-derived TPO, prepared by the method of Example 35. Through each step of hydrophobic interaction chromatography, cation exchange column, WGA column and reverse phase column, highly active TPO could be obtained. <Example 36>
Thus, TPO partially purified from the COS1 cell culture supernatant was subjected to molecular weight measurement and analysis of biological characteristics. <Examples 37 and 38>
(P) Expression of human TPO in E. coli
A vector for expression of E. coli, pGEX-2T / hT (1-174), of a fusion protein of glutathione-S-transferase and human TPO (amino acids 1-174) ("GST-TPO (1-174)") was constructed. At this time, a part of the base sequence of human TPO cDNA (a region approximately half on the 5 'side) was converted to an Escherichia coli preferential codon. <Example 39>
GST-TPO (1-174) was expressed in E. coli, and after disrupting the cells, GST-TPO (1-174) contained in the precipitate fraction was solubilized. Next, as a result of combining TPO unwinding (refolding) conditions, purification conditions (glutathione affinity column, cation exchange column, etc.) and cleavage of the GST protein region by thrombin digestion, A protein containing the amino acid sequence of TPO could be partially purified. This protein was confirmed to have TPO activity in the rat CFU-MK assay system. <Examples 40 and 41>
In addition, the Ser residue at the 1st position of human TPO (amino acid positions 1 to 163) is converted to an Ala residue, the Ala residue at the 3rd position is converted to a Val residue, and a Lys residue at the -1 position; A vector for expression of Escherichia coli of a mutant human TPO protein (referred to as “h6T (1-163)”) having a Met residue added at the position, pCFM536 / h6T (1-163) was constructed. The base sequence of the amino acid (1-163) encoded by the human TPO cDNA carried by this vector was all converted to a preferential codon in E. coli. <Example 42>
After h6T (1-163) is expressed in E. coli, the cells are disrupted, the solubilization of h6T (1-163) contained in the precipitate fraction is examined, and the refolding conditions are examined. As a result, TPO as designed A protein containing the amino acid sequence was partially purified. This protein was confirmed to have TPO activity in the rat CFU-MK assay system. <Examples 43 and 44>
Furthermore, for the expression of Escherichia coli of a mutant human TPO protein (referred to as “hMKT (1-163)”) in which a Lys residue is added to the −1 position and a Met residue is added to the −2 position of human TPO cDNA (amino acids 1-163) The vector pCFM536 / hMKT (1-163) was constructed. hMKT (1-163) was expressed in E. coli as in Example 43, and the resulting expressed protein was transferred to a PVDF membrane after SDS-PAGE. As a result of N-terminal amino acid sequence analysis, the amino acid sequence as designed was obtained. It was confirmed that it included. <Example 52>
E. coli expression of a mutant human TPO protein (referred to as “hMKT (1-332)”) having a Lys residue at position −1 and a Met residue at position −2 of human TPO (amino acids 1-332). The vector pCFM536 / hMKT (1-332) was constructed. hMKT (1-332) was expressed in E. coli as in Example 42, and expression was confirmed by Western blotting using an anti-human TPO peptide antibody prepared in Example 45 described later. <Example 66>
(Q) Preparation of anti-TPO peptide antibody to preparation of anti-TPO peptide antibody column
Of the amino acid sequence of rat TPO identified in Example 10, peptides corresponding to three partial regions were synthesized to prepare rabbit polyclonal anti-TPO peptide antibodies. It was confirmed that these antibodies recognize rat and human TPO. Further, a peptide corresponding to 6 partial regions of the amino acid sequence of human TPO shown in SEQ ID NO: 6 (or SEQ ID NO: 7) was synthesized to prepare a rabbit polyclonal anti-TPO peptide antibody. It was confirmed that these antibodies recognize human TPO. <Example 45>
A method of purifying TPO by affinity column chromatography by binding a molecule having binding affinity for TPO, that is, an anti-TPO antibody, a TPO receptor, or the like to a column carrier. Therefore, first, an anti-TPO antibody column in which the anti-TPO peptide antibody obtained in Example 45 was bound to a gel carrier was prepared. <Example 46>
(R) Purification of human TPO expressed in COS1 cells using an anti-TPO peptide antibody column to molecular weight measurement, biological characteristics
Partially purified TPO was obtained using a culture supernatant obtained by transfecting COS1 cells with the expression vector pHTP1 and applied to an anti-TPO antibody column. Since it was confirmed that the adsorbed fraction had TPO activity, it was further purified by reverse phase column chromatography, and its molecular weight and biological activity were examined. <Example 47>
(S) Activity confirmation of partially purified preparation of human TPO expressed in COS1 cells
The TPO activity fraction purified in Example 36, ie, the biological activity of TPO purified from the culture supernatant obtained by transfecting COS1 cells with the expression vector pHTP1 and purified to the stage of the CapcellPak C1 300A column. As a result, it was found that it has a platelet increasing action in vivo. <Example 48>
In addition, as a result of examining the biological activity of the crude purified TPO fraction obtained with a cation exchange column using 33 L of culture supernatant obtained by transfection of COS1 cells with the expression vector pHTP1, It was found to have platelet-increasing action in the body. <Example 49>
(T) Expression of human TPO chromosomal DNA in CHO cells-activity confirmation
A human TPO chromosome expression vector, DEF202-ghTPO, in CHO cells was constructed. <Example 50>
When this vector was introduced into CHO cells, transformed cells in which an expression vector carrying human TPO chromosomal DNA was incorporated into the chromosome were obtained. When these cells were cultured, TPO activity was detected in the culture supernatant. <Example 51>
(U) Partial purification to activity confirmation of mutant human TPO expressed in E. coli
The clone pCFM536 / h6T (1-163) -derived mutant human TPO expressed in Escherichia coli and encoding the human TPO base sequence was subjected to a refolding operation using guanidine hydrochloride and glutathione, and the obtained h6T (1-163) was obtained. ) Was confirmed to have a platelet-increasing action in vivo. <Example 53>
Clone pCFM536 / h6T (1-163) -derived mutant human TPO expressed in Escherichia coli and encoding human TPO base sequence is subjected to refolding operation using sodium N-lauroyl sarcosine and copper sulfate, and cation exchange chromatography. It was confirmed that h6T (1-163) purified by means of chromatography has a platelet-increasing action in vivo. <Example 54>
Furthermore, refolding to purification of mutant human TPO, h6T (1-163) was performed using another method. <Examples 60 and 61>
(V) Expression of human TPO cDNA in insect cells to confirmation of activity
A recombinant virus for expression of human TPO in insect cells was prepared <Example 58> and expressed in insect cells Sf21, and the activity in the culture supernatant was confirmed. <Example 59>
(W) Expression to purification of human TPO (amino acids 1 to 163) in CHO cells
Among the amino acid sequences of human TPO shown in SEQ ID NO: 6, a vector DEF202-hTPO163 for expression of CHO cells of human TPO protein (referred to as “hTPO163”) having an amino acid sequence at positions 1 to 163 was constructed. The hTPO163-producing CHO cell line obtained by transfecting CHO cells with the expression vector pDEF202-hTPO163 was cultured in large quantities, and hTPO163 was purified from the culture supernatant. <Examples 62 to 65>
(X) Preparation of human TPO derivative
Derivative (referred to as “N3 / TPO”) in which Arg residue at position 25 of human TPO is substituted with Asn residue and Glu residue at position 231 is substituted with Lys residue, and additionally has an additional peptide at the C-terminus, And the His residue at position 33 was replaced with a Thr residue, and TPO was transfected into a COS7 cell culture supernatant transfected with a plasmid encoding a derivative having an additional peptide at the C-terminus (referred to as “O9 / TPO”). Activity was detected. <Example 67>
TPO activity was detected in COS7 cell culture supernatant transfected with a plasmid encoding a derivative in which an amino acid was inserted into hTPO163 or a derivative in which a part of the amino acid of hTPO163 was deleted. <Example 68>
A TPO derivative was prepared using E. coli using h6T (1-163) as a template, and its TPO activity was confirmed. <Example 77>
(Y) Pharmacological action by TPO
The effect of increasing platelets in normal mice by intravenous administration of human TPO obtained in Example 56 and subcutaneous administration was confirmed. <Examples 69 and 70>
In addition, the human TPO obtained in Example 56 has an inhibitory effect on platelet reduction by the administration of an anticancer drug and an effect of promoting recovery of platelet count from platelet reduction, and further, the platelet count after BMT (bone marrow transplantation) and after irradiation. It was confirmed to have a recovery promoting effect. <Examples 71 to 74>
In addition, the platelet-increasing action of human TPO obtained in Example 65 in normal mice and the platelet-number recovery promoting effect from platelet reduction by administration of an anticancer drug were confirmed. <Examples 75 and 76>
(Z) Preparation of human TPO preparation
An example of preparation of a preparation containing human TPO as an active ingredient was shown. <Formulation Examples 1 to 9>
The method for measuring TPO activity (in vitro assay system) used in the present invention will be described below as <Reference Example>.
<Reference example>
A. Rat megakaryocyte progenitor cell (CFU-MK) assay (rat CFU-MK assay) system (liquid culture system)
Megakaryocytes take up extracellular serotonin in an energy-dependent manner and accumulate in dark-stained granules (Fedorko, Lab. Invest., 36, 310-320, (1977)). This phenomenon has already been observed in small, mononuclear acetylcholine positive cells located at least in the differentiation stage between CFU-MK and recognizable megakaryocytes (Bricker and Zuckerman, Exp. Hematol., Vol. 12, 672-675). (1984)), and then the uptake of serotonin increases with increasing megakaryocyte size (Sick and Weinstein, J. Lab. Clin. Med., 98, 607-615, (1981)) . Moreover, it is known that the bone marrow cells are specific only for megakaryocyte cells (Sick and Weinstein, J. Lab. Clin. Med., 98, 607-615, (1981)). The assay system consists of highly enriched rat CFU-MK (GpIIb / IIIa⁺  CFU-MK fraction (described later) is cultured in the presence of the test specimen to increase proliferation and differentiation of megakaryocytes¹⁴C-serotonin (¹⁴C-5-hydroxy trypamine creatine sulfate:¹⁴C-5HT)).
The advantage of this assay system is that the proportion of CFU-MK contained in the cells used is extremely high (see “Assay Method” described later), and on the contrary, there are few cells other than TPO target cells that are contaminated. To reduce indirect effects (for example, some substance other than this factor acts on contaminating cells to induce Meg-CSF activity, or mixed cells produce some factor that cooperates with this factor). In addition, since the total number of cells to be cultured in one well is small, a good culture environment can be maintained for a relatively long period. In addition, many large mature megakaryocytes generated from CFU-MK by the active preparation during the culture period can be observed under a phase-contrast microscope, and the presence or degree of activity can be qualitatively determined. is there. The result of this qualitative judgment is¹⁴Corresponds well with the quantitative results from C-serotonin incorporation. Therefore, the reliability of the quantitative result can be improved by using the qualitative determination together.
"Assay Method"
First, the highly concentrated rat CFU-MK ("GpIIb / IIIa"⁺CFU-MK fraction ") (Miyazaki et al., Exp, Hematol., 20, 855-861, (1992)) was prepared according to a slightly improved method. The outline is as follows.
The femur and tibia of Wistar rats (ラ ッ ト, 8-12 weeks old) are removed and a bone marrow cell suspension is prepared according to a conventional method. For the preparation of bone marrow cell suspension, a medium (13.6 mM citric acid) slightly improved from a medium for separating megakaryocytes (Levine and Fedoko, Blood), Volume 50, pages 713-725, (1977) reported by Levine and Fedoko. Trisodium, 11.1 mM glucose, 1 mM adenosine, 1 mM theophylline, 10 mM HEPES (pH 7.25), 0.5% bovine serum albumin (hereinafter abbreviated as BSA) (Path-0-Cyte4; Seikagaku Corporation), and Ca²+ And Mg²⁺A solution composed of Hanks balanced salt solution not containing (hereinafter abbreviated as HATCH solution). A bone marrow cell suspension was prepared by a Percoll discontinuous density gradient solution (density: 1.050 g / ml / 1.063 g / ml / 1.082 g / ml) prepared by diluting Percoll stock solution (Pharmacia) with HATCH solution. Overlay and centrifuge at 20O 0 C at 400 xg for 20 minutes. After centrifugation, the cells gathered at the interface of density 1.063 g / ml and 1.082 g / ml are collected. The cells were washed, suspended in Iscove modified Dulbecco medium (hereinafter abbreviated as IMDM medium) containing 10% fetal calf serum (hereinafter abbreviated as FCS), placed in a 100 mm diameter tissue culture plastic dish, and 5% carbonic acid. Incubate for 1 hour at 37 ° C. in a gas incubator. After culturing, the non-adherent cell fraction is collected, placed in a plastic dish having a diameter of 100 mm, and further cultured at 37 ° C. for 1 hour, and then the non-adherent cell fraction is collected. The collected cells were resuspended in a HATCH solution, and the mouse monoclonal anti-rat platelet GpIIb / IIIa antibody P55 antibody (Miyazaki et al., Thromb. Res, 59, 941-953, (1990)) was adsorbed in advance. Put into a 100 mm Petri dish (Falcon 1005; manufactured by Becton-Dickinson) for bacterial inspection, and let stand at room temperature for 1 hour. Thereafter, the non-adsorbed cells are sufficiently washed and removed with a HATCH solution, and then the cells adsorbed to the immobilized P55 antibody are removed by pipetting and collected. Usually from 3 to 4 x 10 rats⁵Cells are obtained. Rat CFU-MK is highly concentrated in the obtained cell fraction (hereinafter referred to as “GpIIb / IIIa”).⁺It is known that a CFU-MK fraction is usually contained in an amount of about 5 to 10% by an assay in the presence of a saturating concentration of rat IL-3 in a colony assay system described later. In addition, the hybridoma (p55 cell) which produces the said P55 antibody was deposited on February 14, 1994 to the Ministry of International Trade and Industry, Institute of Industrial Science, Biotechnology Institute of Technology as accession number FERM BP-4563.
Next, the obtained GpIIb / IIIa⁺The CFU-MK fraction is resuspended in IMDM medium containing 10% FCS, distributed to a 96-well flat-bottom plate for tissue culture so that 104 cells per well are contained, and dialyzed sufficiently against the IMDM medium. Standard samples (to be described in detail later) and test samples are added to make the final culture volume 200 μl / well. Place the plate in a carbon dioxide incubator and incubate at 37 ° C. for 4 days. 0.1 μCi (3.7 KBq) per well 3 hours before the end of culture on the 4th day¹⁴C-serotonin is added followed by incubation at 37 ° C. After completion of the culture, the plate is centrifuged at 1000 rpm for 3 minutes, and the supernatant is removed by aspiration. Subsequently, 200 μl of PBS containing 0.05% EDTA is added and centrifuged, the supernatant is removed by aspiration, and the cells are washed. This washing operation is repeated once more. 200 μl of 2% Triton X-100 is added to the resulting cell pellet, and the plate is shaken with a plate mixer for about 5 to 10 minutes to sufficiently lyse the cells. 150 μl of the obtained cell lysate is transferred into a commercially available cup-shaped solid scintillator (Ready Cap; manufactured by Beckman), and left to stand in a dryer at 50 ° C. overnight to dry. The next day, put Readycap in a glass vial and use a liquid scintillation counter.¹⁴The radioactivity of C is measured.
The acetylcholinesterase activity in the cell lysate described above may be measured according to the method of Ishibashi and Burstein (Ishibashi and Burstein, Blood) Vol. 67, pages 1512-1514 (1986))¹⁴A result very similar to the quantitative result obtained by incorporation of C-serotonin is obtained.
"Standard goods"
First, thrombocytopenic rat plasma used for preparation of a standard product was prepared by the following method.
Seven to eight weeks old Wistar normal rats (♂) were injected intravenously twice at intervals of about 24 hours with the above-mentioned P55 antibody at a single dose of 0.5 mg, and about 24 of the second administration. The laparotomy was performed under ether anesthesia in the thrombocytopenic phase after a lapse of time, and the abdominal aorta was removed from the abdominal aorta using a 10 ml syringe that had previously sucked 1 ml of 3.8% (v / v) trisodium citrate solution as an anticoagulant Blood was collected. The blood was transferred to a plastic centrifuge tube and centrifuged at 1200 × g for 10 minutes to collect a plasma fraction. This plasma fraction was centrifuged again at 1200 × g for 10 minutes, and after centrifugation, the plasma fraction was collected and pooled while taking care not to suck up pellets made of cells, platelets, etc. The plasma thus obtained is referred to as “TRP”).
Subsequently, the Ca-treated TRP (standard product C), WGA-Agarose column active fraction (standard product W), or Phenyl Sepharose 6 FF / LS column active fraction (standard) prepared from TRP according to the method described in Example 1 was used. The product P) was thoroughly dialyzed against a sufficient amount of IMDM culture medium to obtain a standard product for activity assay.
In the purification process of rat TPO shown in Example 1, standard product C was used in the initial stage, but standard product W was used from the middle, and standard product P was used thereafter. Arbitrarily, the activity of standard product C was defined as 1, and based on this, the relative activities of standard product w and standard product P were determined. To determine the relative activity of a test sample, draw a dose-response curve between the standard and the test sample. When the activity of the test sample is n times the activity of the standard C, calculate the relative activity of the sample. n.
B. Colony assay system
Meg-CSF activity is measured by culturing bone marrow cells in a semi-solid culture medium in the presence of a test sample and calculating the number of megakaryocyte colonies formed by proliferation and differentiation of CFU-MK. Assay method.
"Assay Method"
(A) When using rat non-isolated bone marrow cells
Rat non-isolated bone marrow cells, A. GpIIb / IIIa⁺Cells obtained at each stage of CFU-MK separation / concentration operation, or GpIIb / IIIa⁺A final volume 1 ml IMDM culture solution containing a CFU-MK fraction, 10% FCS, 2 mM glutamine, 1 mM sodium pyruvate, 50 μM 2-mercaptoethanol, 0.3% agar (AGAR NOBLE, manufactured by DIFCO) is 35 mm in diameter. In a tissue culture plastic dish and solidified at room temperature, and then cultured at 37 ° C. in a carbon dioxide incubator. Typically, the number of engrafted cells per dish is determined as non-segregated bone marrow cells, Percoll centrifugation stage and plastic dish adherent cell removal stage cells, and GpIIb / IIIa⁺CFU-MK fraction, 2-4x10 respectively⁵2-5 × 10⁴0.5-2 × 10³It is a piece. On day 6-7, the agar gel is removed from the dish and placed on a glass slide (76 mm x 52 mm). A nylon mesh with a pore size of 50 μm is placed on the filter, followed by filter paper to absorb the moisture. Let After heat-fixing on a hot plate at 50 ° C. for 5 minutes, the cells were immersed in an acetylcholinesterase staining solution prepared according to the method of Jackson (Jackson, Blood, Vol. 42, pages 413-421, (1973)) for 2 to 4 hours. After confirming that the dye is sufficiently dyed, it is taken out, washed with water, dried, then post-stained with Harris hematoxylin solution for 30 seconds, washed with water and air-dried. The number of megakaryocyte colonies is calculated using a conglomerate of 3 or more acetylcholinesterase-positive megakaryocytes as one colony.
(B) When using mouse non-isolated bone marrow cells
2-4x10 number of cells per dish⁵It can carry out by the method similar to said (a) as a piece.
(C) When using human bone marrow cells or human cord blood cells
Although human bone marrow cells and human cord blood cells can be used as they are, the CFU-MK fraction concentrated from them can be used as follows.
First, bone marrow fluid or cord blood is layered on Lymphoprep (Daiichi Kagaku Co., Ltd.), and after centrifugation, the leukocyte fraction collected at the interface is collected. From this cell fraction, cells to which biotinylated monoclonal antibodies against biotinylated human cell surface antigens (CD2, CD11c, and CD19) bind are removed using magnetic beads to which avidin is bound. Cells that can be removed by this magnetic bead method are mainly B cells, T cells, macrophages, and some granulocytes. The remaining cells were stained with FITC-labeled anti-CD34 antibody and PE-labeled anti-HLA-DR antibody, followed by CD34-positive and HLA-DR using a cell sorter (for example, ELITE; manufactured by COULTER). Collect the positive cell fraction. CFU-MK is concentrated in this fraction (CD34⁺DR⁺Abbreviated as CFU-MK fraction). The human colony assay is performed in substantially the same manner as the colony assay using rat bone marrow cells described above, but CD34 per plastic dish.⁺DR⁺3-5 × 10 for the number of CFU-MK fractions³Use a mixture of 12.5% human AB plasma and 12.5% FCS instead of 10% FCS. Further, the culture period until the megakaryocyte colony is formed is 12 days to 14 days. For detection of human megakaryocytes, megakaryocytes are immunostained by an alkaline phosphatase-anti-alkaline phosphatase antibody method using a mouse monoclonal antibody against human GpIIb / IIIa, which is a surface antigen of megakaryocytes (see, eg, Teramara et al., Exp. Hematol., 16, 843-848 (1988)), a colony composed of 3 or more megakaryocytes is counted as a megakaryocyte colony.
C. Assay system using human megakaryoblastic cell line (M-07e assay)
M-07e cells, which are human megakaryoblastic cell lines, are known to proliferate in response to GM-CSF, IL-3, SCF, IL-2, etc. (Avanzi et al., J. Cell. Physiol., 145, 458-464, (1990), Kiss et al., Leukemia), 7)), which was also found to respond to TPO, and as an alternative assay for the rat CFU-MK assay system Can be used.
"Assay Method"
M-07e cells subcultured in the presence of GM-CSF are collected, washed thoroughly, and resuspended in IMDM culture medium containing 10% FCS. 10 cells per well in a 96-well flat bottom plate for tissue culture⁴M-07e cells are added so that they become individual, and a standard product and a test sample are added to make the final volume 200 μl / well. The plate is placed in a 5% carbon dioxide incubator and incubated at 37 ° C. for 3 days. 1 μCi (37 KBq) / well 4 hours before the end of the culture on the third day³H-thymidine was added, and after culturing, the cells were collected on a glass fiber filter with a cell harvester.³The radioactivity of H is measured with a liquid scintillation counter (for example, a beta plate; manufactured by Pharmacia).
In addition, the definition of the relative activity amount in the M-07e assay is as described in In the rat CFU-MK assay.
D. Assay system using mouse pro B cell line (Ba / F3 assay)
The mouse pro B cell line Ba / F3 cell is known to be a cell line that proliferates in response to IL-3, IL-4, etc. (Palacios et al., Cell, 41, 727-734).
Sub-strain BF-TE22 cells were found to respond to TPO as well as IL-3 and IL-4 and proliferate, and assay using rat CFU-MK assay or human megakaryoblastic cell line It can be used as an alternative assay method for (M-07e assay). The outline of this assay system is as follows. First, BF-TE22 cells subcultured in the presence of 1 ng / ml mouse IL-3 are collected, washed 3 times with Iskov modified DME medium (IMDM; GIBCO), and then contain 10% FCS. Resuspend in IMDM medium. Next, the number of cells per well is set to 1 × 10 6 on a 96-well flat bottom plate for tissue culture.⁴Cells are inoculated so that they become individual, and a TPO standard or test sample is added to make the final volume 200 ml / well, and the plate is cultured in a 5% carbon dioxide incubator for 2-3 days. . 1 mCi (37KBq) / well 4 hours before the end of the culture on the 2nd or 3rd day³H-thymidine is added and the culture is continued. After culturing, the cells were collected on a glass fiber filter using a cell harvester and taken up by the cells.³The radioactivity of H is measured with a liquid scintillation counter. In this assay system, cells are almost killed in the test sample that does not contain TPO activity.³In the test sample containing TPO activity, cells show active proliferation in a TPO concentration-dependent manner, whereas H-thymidine hardly takes up.³Uptake of H-thymidine is observed. Moreover, this assay shows parallel results with the M-07e assay and the CFU-MK assay.
【Example】
Hereinafter, the present invention will be described in detail with reference to examples.
<Example 1-1>
Purification of rat TPO from plasma of thrombocytopenic rats by administration of antiplatelet antibody
"Preparation of plasma of thrombocytopenic rats by administration of antiplatelet antibody"
<Reference Example> A. above. Approximately 1000 rats of TRP were prepared by the method described in the rat megakaryocyte progenitor cell (CFU-MK) assay system and used as a source of purification.
"Purification of rat TPO from TRP"
Initially, TRP content in TRP was estimated to be at most about 1 / 3,000,000, and as a result of purifying TRP for about 1000 rats as a material, partially purified preparation of rat TPO of less than 1 pmole Got. Next, analysis of the partial amino acid sequence of this preparation was tried, and three partial amino acid sequence results were obtained. These were identical or similar to the amino acid sequence of serine protease inhibitor (SPI) produced in the liver. Therefore, the possibility that TPO has a structure similar to a serine protease inhibitor and the possibility that it is a sequence derived from a contaminating protein other than TPO cannot be denied.
Although TPO gene cloning from a rat cDNA library was performed based on these uncertain sequences, no candidate gene could be obtained after all. This was thought to be due to the lack of purity and quantity of the specimens that were analyzed. In order to obtain a final purified sample necessary for amino acid sequence analysis, the purification described in the following Example 1-2 was performed. It should be noted that the amount of TPO present in TRP or XRP (described later) is surprisingly from 1/100 million to 1 / 1.0 billion of the total plasma protein based on the results of Example 1-2. It was estimated that the content was so small that it was usually very difficult to purify it.
<Example 1-2>
Purification of rat TPO from plasma of thrombocytopenic rats by X-ray or γ-irradiation
“Preparation of plasma of thrombocytopenic rats by X-ray or γ-ray irradiation”
Sub-lethal dose (6-7 Gy) of X-rays or γ-rays were irradiated to 7-8 weeks old normal Wistar rats (♂), blood was collected on the 14th day of thrombocytopenia, and similar to TRP described above A plasma fraction was prepared by the above procedure (hereinafter, this plasma is referred to as “XRP”).
Here, a total of about 1100 rats of XRP (about 8 L) was used as a purification source.
"Purification of rat TPO from XRP"
Since the total protein content of plasma for about 1100 rats reached 493,000 mg, it could not be processed at one time. Therefore, in the purification steps described below, in (1) to (4), approximately 100 animals were divided into 11 lots. Next, (5) to (7) were divided into six batches. (8) After that, the crudely purified products from about 1100 animals were collected together.
Among these, a purification example in one lot (XW9) and batch (XB6) is given as a representative for explanation, and each step of purification is also described.
In all purification steps, TPO activity was measured using the rat CFU-MK assay system described in the above <Reference Example>.
Purification was carried out at 4 ° C unless otherwise stated except that reverse phase chromatography was performed at room temperature.
The protein was quantified using the Coomassie dye binding method (PIERCE reagent, catalog number 23236X) or the bicinchoninic acid method (PIERCE reagent catalog number 23225).
A summary of the purification is shown in Table 1.
[Table 1]

(1) Calcium chloride treatment / centrifugation / proteolytic enzyme inhibitor treatment of rat plasma Thawed about 100 XRPs (742 ml, protein concentration 54.8 mg / ml, total protein mass 40686 mg) stored at −80 ° C. And transferred to a polypropylene centrifuge tube (Nalgen). To this, calcium chloride powder was added to a final concentration of 100 mM, and the mixture was allowed to stand at 4 ° C. overnight. Next, after centrifugation at 8000 RPM for 60 minutes, the supernatant was recovered. To this supernatant containing TPO activity (742 ml, protein concentration 54.9 mg / ml, total protein mass 40740 mg), p-APMSF (p-aminodiphenylmethanesulfonyl fluoride hydrochloride, which is a protease inhibitor with a final concentration of 1 mM, Wako Pure Chemical Industries, catalog number 010-10393) was added, and the process was advanced to the buffer exchange step using the Sephadex G-25 column described below.
In this way, about 100 animals were combined into one lot, treated with calcium chloride and p-APMSF, and a total of 11 lots, about 1100 animals with XRP (total volume 8184 ml) total protein mass 493,007 mg) were repeated. , Respectively, advanced to Sephadex G-25 column.
(2) Sephadex G-25 <Buffer exchange>
Sephadex G-25M column obtained by pre-equilibrating the supernatant after treatment with calcium chloride (742 ml, protein concentration 54.9 mg / ml, total protein mass 40740 mg) obtained in (1) with 20 mM Tris-HCI, pH 8 (Pharmacia Biotech, catalog number 17-0033-03; diameter 11.3 cm, bed height 47 cm) was added at a flow rate of 40 to 70 ml / min, and 1300 ml until the protein was eluted was discarded. Next, from the point of time when ultraviolet absorption started up until the electric conductivity reached 500 μS / cm, the protein fraction containing TPO activity substituted with 20 mM Tris-HCl pH8 solution (1377 ml, protein concentration 27.56 mg) was collected. / Ml) was recovered. The total protein amount of the TPO active fraction was 37882 mg, and the protein yield at this step was 93%. The relative activity of TPO was 2.3.
As a result of performing Sephadex G-25 column for all lots in this way, the total amount was 21117 ml, total protein mass was 480306 mg, average relative activity was 1.8, and relative activity was 864600 TPO activity of Sephadex G-25 A fraction was obtained.
(3) Q-Sepharose FF <strong anion exchange chromatography>
The fraction of TPO activity (1375 ml, protein concentration 27.5 mg / ml, total protein mass 37841 mg, relative activity 2.3) of Sephadex G-25M obtained in (2) at a flow rate of 40 ml / min and Q-Sepharose FF ( Fraction F1 (3949 ml, protein concentration 0.98 mg / ml, total protein mass) added to Pharmacia Biotech, catalog number 17-0510-01; diameter 5 cm, bed height 27 cm) and passing through 20 mM Tris-HCl, pH 8 3870 mg, relative activity 0) was eluted.
Next, the TPO active fraction F2 (4375 ml, protein concentration 5.36 mg / ml) was eluted in place of 20 mM Tris-HCl, pH 8 buffer containing 175 mM NaCl.
Finally, F3 (1221 ml, protein concentration 3.9 mg / ml, total protein mass 4783 mg, relative activity 3.8) was eluted with 20 mM Tris-HCl pH 8 buffer containing 1000 mM NaCl. The total protein amount of the TPO active fraction F2 was 23440 mg, and the protein yield of F2 at this step was 61.9%. In addition, the relative activity of TPO increased to 6.8.
In this way, as a result of subjecting all lots of the TPO active fraction of Sephadex G-25 to Q-SepharoseFF, the total volume was 35842 ml, the total protein mass was 314384 mg, the average relative activity was 8.6, and the relative activity amount was 2704000. A TPO-active fraction F2 of Sepharose FF was obtained.
(4) Wheat Germ Agglutinin (WGA) -Agarose <Lectin Affinity Chromatography>
The Q-Sepharose FF (7) TPO active fraction F2 obtained in (3) was divided into three times and WGA-Agarose (manufactured by Honen, catalog number 8000027; diameter 5 cm, bed height 22.5 cm) with a flow rate of 5 ml. Fraction F1 (9336 ml, protein concentration 2.30 mg / ml, total protein mass 21407 mg, relative activity 6.9) passed through with Dulbecco's phosphate isotonic buffer (DPBS).
Next, 0.2 MN-acetyl-D-glucosamine (GlcNAc, manufactured by Nacalai, catalog number 005-20), 150 mM NaCl) eluted with 20 mM Na Phosphate, pH 7.2 buffer containing 0.02% sodium azide. The resulting pool was concentrated with an ultrafiltration unit (manufactured by Filtron, omega ultraset molecular weight 8000 cut) to obtain WGA-Agarose-adsorbed TPO active fraction F2 (2993 ml, protein concentration 0.376 mg / ml).
The total protein amount of this TPO active fraction F2 was 1125 mg, and the protein yield of F2 at this step was 4.8%. In addition, the relative activity of TPO increased to 101. The F2 obtained here was stored at -80 ° C.
As described above, the whole lot of the TPO active fraction F2 of Q-Sepharose FF was repeatedly subjected to WGA-Agarose. As a result, the total volume was 33094 ml, the total protein mass was 15030 mg, the average relative activity was 132, and the relative activity was 1987000 WGA-Agarose. Of TPO active fraction F2.
(5) TSK-gel AF-BLUE 650 MH <dye adsorption affinity chromatography>
The WGA-Agarose-adsorbed TPO active fraction of lot XW8 and the WGA-Agarose-adsorbed TPO active fraction F2 of lot XW9 obtained from (4) starting from a total of 215 XRPs were collected as batch XB6 (5974 ml, Protein concentration 0.388 mg / ml, total protein mass 2319 mg, relative activity 150).
To this volume 5974 ml, 0.85 moles NaCl (296.76 g) was added to make a solution with a final concentration of 0.822 M NaCl, 6132 ml, and then pre-equilibrated with IM NaCl, 20 mM Na Phosphate, pH 7.2. It added to TSK-gel AF-BLUE650MH column (The Tosoh company make, catalog number 08705; diameter 5cm, bed height 23cm) at the flow rate of 7 ml / min.
After completion of the addition, a flow-through (approximately 8470 ml) eluted with 20 mM Na Phosphate, 1M NaCl, pH 7.2 was collected at a flow rate of 10 ml / min, and this was collected into an ultrafiltration unit (manufactured by Filtron, omega ultraset molecular weight 8000 cut) ) To obtain a flow-through fraction F1 (543 ml, protein concentration 2.05 mg / ml, total protein mass 1112 mg, relative activity 31).
Next, the eluate was changed to 2M NaSCN to obtain an eluted TSK-gel AF-BLUE 650MH adsorbed TPO active fraction F2 (1427 ml, protein concentration 0.447 mg / ml).
The total protein amount of the TPO active fraction F2 was 638 mg, and the protein yield of F2 at this step was 27.5%. In addition, the relative activity of TPO increased to 1500.
Thus, as a result of subjecting all batches of WGA-Agarose-adsorbed TPO active fraction F2 to TSK AF-BLUE 650MH, the total volume 10655 ml, total protein mass 4236 mg, average relative activity 905, relative activity amount 3834000 TSK- A TPO active fraction F2 of gelΛF-BLUE 650MH was obtained.
(6) Phenyl Sepharose 6FF / LS <Hydrophobic interaction chromatography>
The TSK-gel AF-BLUE650MH obtained in (5) has a TPO activity fraction F2 (1424 ml, protein concentration 0.447 mg / ml), a total protein mass of 638 mg) and a relative activity of 1500) with a volume of 1424 ml, 1.5 moles of Ammonium. Sulfate (282.2 g) powder was added to give a final concentration of 1.35 MMA Sulfate, 1581 ml solution.
A Phenyl Sepharose 6FF (Low Sub) column (manufactured by Pharmacia Biotech, catalog number 17-0965-05; diameter 5 cm, bed height 10 cm), which had been equilibrated in advance with 1.5 M Ammonium Sulfate, 50 mM Na Phosphate, pH 7.2. ) At a flow rate of 7 ml / min. After the addition is completed, the eluate is changed to 0.8 M Ammonium Sulfate, 36 mM Na Phospho {e, up to a fraction (about 3160 ml) eluted at a flow rate of 10 ml / min. This was collected and concentrated with an ultrafiltration unit (manufactured by Filtron, omega ultraset molecular weight 8000 cut) to obtain F1 (485 ml, protein concentration 0.194 mg / ml, total protein mass 94.2 mg, relative activity 0). .
Next, the eluate was changed to 20 mM Na Phosphate, pH 7.2 to obtain an eluted TPO active fraction F2 (about 3500 ml). This was concentrated with an ultrafiltration unit (manufactured by Filtron, omega ultraset molecular weight 8000 cut), and once sampled. The protein concentration of TPO active fraction F2 (220 ml) at this stage was 1.45 mg / ml, and the total protein mass was 319 mg. The protein yield of F2 at this step was 50.0%. The relative activity of TPO was 1230.
Thus, as a result of repeatedly subjecting all batches of TPO-active fraction F2 of TSK-gel AF-BLUE 650MH to Phenyl Sepharose FF / LS, total volume 1966 ml, total protein mass 2762 mg, average relative activity 847, relative activity amount 2339000 Phenyl Sepharose FF / LS TPO active fraction F2 was obtained.
(7) Sephacryl S-200HR <Gel filtration chromatography>
144.8 ml of 5M NaCl solution was added to Phenyl Sepharose 6FF / LS obtained in (6) (7) TPO active fraction F2 (217 ml, protein concentration 1.45 mg / ml) total protein mass 315 mg, relative activity 1230) 362 ml of 2M NaCl, and further concentrated to about 50 ml with an ultrafiltration unit (Amicon; YM3 membrane, diameter 76 mm).
An equal volume (50 ml) of 8M urea was added thereto to make a final concentration of about 100 ml of 1M NaCl, 4M urea solution. Further, the solution was concentrated to about 80 ml, finally converted to a 88.78 ml sample, and injected into a Sephacryl S-200HR column (Pharmacia Biotech, catalog number 17-058-01; diameter 7.5 cm, bed height 100 cm).
Subsequently, the plate was developed with DPBS at a flow rate of 3 ml / min, and collected in 60 polypropylene tubes each 45 ml after the void volume (1200 ml). This elution pattern is shown in FIG. Duplicates were subjected to the assay, and the rest were stored frozen at -85 ° C until all 1100 Sephacryl S-200HR processes were completed. From the assay results, the fractions of XB6 were summarized as follows (FIG. 1).
(F1) Tube numbers 1 to 15 (fraction having a molecular weight of 94000 or more near the void volume)
(F2) Tube number 16-26 (molecular weight 94000-33000)
(F3) Tube number 27-44 (molecular weight 33,000-3000)
(F4) Tube number 45-55 (molecular weight 3000 or less)
In this way, all batches of the TPO active fraction F2 obtained with Phenyl Sepharose 6FF / LS were each subjected to Sephacryl S-200HR, each fraction was assayed, and stored frozen at -85 ° C. After completion of Sephacryl S-200HR for all batches, thawing was performed immediately before the next reverse phase chromatography (YMC-Pack PROTEIN-RP) was performed, and ultrafiltration unit (Amicon; YM3 membrane, diameter 76 mm) was used. Concentration gave the following two samples. Hereinafter, the concentrated preparation of the TPO active fraction F2 of Sephacryl S-200HR is referred to as “polymer TPO preparation F2,” and the concentrated preparation of the TPO active fraction F3 of Sephacryl S-200HR is referred to as “low molecular TPO preparation F3”. Say. For the sake of convenience, the high molecular TPO sample F2 and the low molecular TPO sample F3 described here refer to a collection of fractions with different elution positions by gel filtration chromatography, and necessarily represent the true molecular weight. is not.
[Table 2]

Thereafter, the low-molecular TPO sample F3 and the high-molecular TPO sample F2 were respectively advanced to the next purification step.
The steps of purification of the low molecular weight TPO sample F3 are described below in (8) to (11).
(8) YMC-Pack PROTEIN-RP <Reverse Phase Chromatography>
The low-molecular-weight TPO preparation F3 obtained in (7) (total protein mass 50.3 mg, protein concentration 0.184 mg / ml, relative activity 20000, relative activity 1007000, total volume 274 ml) and developing solvent A (0.025 % Trifluoroacetic acid (TFA)) and developing solvent B (1-propanol containing 0.025% TFA), final volume 508.63 ml, final propanol concentration about 20%, TFA concentration 0.012%, protein concentration 0 Adjusted to 0.089 mg / ml. Since insoluble matter was generated here, this was centrifuged, and only the supernatant was divided into two portions of 254.3 ml (25.2 mg) and YMC-Pack PROTEIN- previously equilibrated with 30% B. It added to the RP (YMC company, catalog number A-PRRP-33-03-15; diameter 3cm, bed height 7.5cm) column at the flow rate of 2 ml / min. The precipitate was 20 mM sodium acetate containing 5 mM CHAPS (3-[(3-colamidopropyl) dimethylammonio] -1-propanesulfonate; manufactured by Dojindo Laboratories, catalog number 75621-03-3), pH 5 .5 was added and solubilized, and combined and fed to the column.
After the sample was added, about 50 ml of solvent (developing solvent A: developing solvent B = 3: 1) was passed through, and first the flow-through fraction was collected. Next, a development program (linear concentration gradient from 30% B to 120% B / 120%) was started, and a total of 36 fractions of 10 ml each were collected in a polypropylene tube. Since this was repeated and collected in the same tube, a total of 36 fractions, 20 ml each, were finally obtained. The flow-through fraction was directly concentrated to 20 ml with an ultrafiltration unit (Amicon: YM3 membrane, diameter 76 mm).
Take 0.1 ml from the flow-through fraction and 20 ml fractions of tube numbers 1 to 36, add 20 μl of 5% BSA, dry to dryness by centrifugal evaporation, and finally dissolve in 0.25 ml of IMDM assay culture medium. The assay was performed to identify the TPO active fraction. As a result, tube numbers 17 to 27 (range of 36.0 to 43.0% in propanol concentration) have TPO activity, which is a TPO activity image of YMC-Pack PROTEIN-RP derived from low molecular TPO preparation F3. Minute F2. It was stored at −85 ° C. until immediately before proceeding to the next YMC-Pack CN-AP. TPO active fraction F2 of YMC-Pack PROTEIN-RP derived from low molecular TPO preparation F3
Total volume 220ml
Protein concentration 0.0130mg / ml
Total protein mass 2.85mg
Relative activity 130000
Relative activity 371000
(9) YMC-Pack CN-AP <Reverse Phase Chromatography>
214.9 ml (total protein mass 2.79 mg, protein concentration 0.0130 mg / ml, relative activity) of the TPO activity fraction F2 of the YMC-Pack PROTEIN-RP derived from the low molecular weight TPO preparation F3 obtained in (8) 130,000, relative activity 36300), 0.6 ml of 50% glycerol was added and concentrated to 1.8 ml. Finally, the volume was 5 ml, the propanol concentration was 20% or less, and glycerol was about 6%.
This was performed by dividing into 5 times (injected protein mass of 0.555 mg, volume of 1 ml each time). YMC-Pack CN-AP (manufactured by YMC Co., Ltd.) equilibrated with 15% B using 1-propanol containing 0.05% TFA in developing solvent B every time, 0.1% TFA in developing solvent A No. AP-513; diameter 6 mm, bed height 250 mm) was injected at a flow rate of 0.6 ml / min.
After completion of the injection, the propanol concentration was increased from 15% B to 25% B, and further developed from a linear concentration gradient of 65 minutes from 25% B to 50% B. In the last round, 1 ml of a solution having the same composition containing no protein was injected and developed, and the TPO activity remaining in the column was recovered. Since the same polypropylene tube was collected 6 times in total, each fraction became 44 pieces of 7.2 ml.
Take 30 μl (1/240 fraction) of this, add 20 μl of 5% BSA, dry to dryness by centrifugal evaporation, and finally dissolve in 0.24 ml of IMDM assay culture medium. Identified. As a result, since tube numbers 28 to 33 (range of 37.0 to 42.0% in propanol concentration) had strong TPO activity, this was used as the main component of the YMC-Pack CN-AP derived from low molecular weight TPO preparation F3. TPO active fraction FA was designated.
TPO activity fraction FA of YMC-Pack CN-AP derived from low molecular TPO preparation F3
Total volume 43.20ml
Protein concentration 0.00863 mg / ml
Total protein mass 0.373mg
Relative activity 800,000
Relative activity 298400
(10) Capcell Pak C1 300A <final reverse phase chromatography> 43.12 ml of the TPO active fraction FA 43.20 ml derived from the low molecular TPO preparation F3 obtained in (9) (total protein mass 0.372 mg, 0.2 ml of 50% glycerol was added to a protein concentration of 0.00863 mg / ml, a relative activity of 800,000, and a relative activity of 297600), and concentrated to a 0.1 ml glycerol solution.
To this, 2 ml of a solution of developing solvent A (0.1% TFA): developing solvent B (1-propanol containing 0.05% TFA) = 85; 15 (15% B) was added. 0.1 ml) A sample having a propanol concentration of about 14%, glycerol of about 4.8%, and a protein concentration of 0.177 mg / ml was prepared. This was injected into a Capcell Pak C1 300A (Shiseido, catalog number C1 TYPE; SG300A; diameter 4.6 mm, bed height 250 mm) column equilibrated with 15% B, and a straight line of 65 minutes from 27% B to 38% B was made. The concentration gradient was developed at a flow rate of 0.4 ml / min, and 0.6 ml was collected in 72 polypropylene tubes.
From each fraction, 3 μl (1/200 fraction) was taken, 20 μl of 5% BSA was added, and finally replaced with 225 μl of IMDM assay medium and diluted 75-fold from the original volume to the assay.
From each fraction, 1 μl (1/600 fraction) was taken for electrophoresis, centrifuged and 10 μl of SDS gel electrophoresis sample buffer without reducing agent was added, followed by treatment at 95 ° C. for 5 minutes. This was subjected to SDS gel electrophoresis using 15-25% SDS-polyacrylamide precast gel (manufactured by Daiichi Chemical Co., Ltd.), and 2D-silver staining reagent / “first” silver staining kit (manufactured by Daiichi Chemical Co., Ltd., This was stained with catalog number 167997, hereinafter referred to as “silver staining kit”. As the molecular weight marker, a “first” / III low molecular weight marker (manufactured by Daiichi Chemicals, catalog number 181061, hereinafter referred to as “DPCIII”) was used.
As a result of the above analysis, the tube numbers 35 to 43 (propanol concentration range of 30.0 to 32.5%) clearly had TPO activity. Among these, tube numbers 36 to 42 (range of propanol concentration of 30.5 to 32.0%) were used as the main TPO active fraction FA. The above results are shown in FIG.
When the protein mass was estimated from the chromatogram and evaluated together with the results of the assay, the total protein mass was 39.6 μg, the protein concentration was 9.4 μg / ml, the relative activity was 490,000, and the relative activity was 193600. Examination of the SDS gel electrophoresis images of TPO active fraction tube numbers 36 to 42 revealed that there was a band in which the intensity of activity correlated with the stained intensity. Moreover, the molecular weight of this band apparently, that is, at a position of 17000 to 19000 with respect to the standard molecular weight protein in the reduced state, was found to be a strong band that is a candidate for TPO.
(11) Extraction of TPO activity from electrophoresis gel <15% SDS polyacrylamide gel electrophoresis>
Analysis example of TPO active fraction FA
Among the TPO activity fraction FA 4200 μl (total protein mass 39.6 μg, protein concentration 9.4 μg / ml, relative activity 490,000, relative activity 193600) derived from the low molecular weight TPO sample F3 obtained in (10), 5 .5 μl (fraction 1/764) for active extraction and 2.5 μl (fraction 1680) for silver staining, respectively, in a sample tube, centrifuge evaporate, and SDS gel electrophoresis without reducing agent. 10 μl of electrophoresis sample buffer was added, treated at 37 ° C. for 1 hour, and allowed to stand at room temperature for 18 hours to form SDS.
As the molecular weight marker, a prestained low-range marker (Bio-Rad 161-0305) and a DPCIII marker were used. These samples were subjected to 15% SDS polyacrylamide gel electrophoresis using a micro slab gel at 4 ° C. according to a conventional method (Laemmli Nature 227, 680-685, (1970)). Immediately after the electrophoresis, the portion to be subjected to silver staining was cut with a knife and placed in a fixative, and silver stained using a silver staining kit.
On the other hand, the part where the activity is to be cut out is sliced into 34 gels having a width of 1.5 to 2.5 mm using a knife over the whole molecular weight, and the method of Kobayashi (Mikihiko Kobayashi, Biochemistry, Vol. 59). , No. 9 (1987)). 0.3 ml of extraction buffer (20 mM Tris-HCl, pH 8,500 mM NaCl, 0.05% BSA) was added to each gel crushed into fine fragments, and the mixture was shaken at 4 ° C. for 6 hours for extraction.
Next, a final concentration of 20 mM of 500 mM potassium phosphate, pH 6.8 was added, shaken at 4 ° C. for 1 hour, and a filtration unit with an ultra-free C3GV 0.22 μm filter (model number UFC3 OGV, manufactured by Millipore) to remove precipitated SDS. 0S) and centrifuged at 1000 × g (4000 RPM) for 15 minutes to collect the filtrate. This was transferred to an ultra-free C3-LGC molecular weight 10,000 cut ultrafiltration unit (Millipore, model number UFC3 LGC 00) and centrifuged at 3000 × g (7000 RPM). When the concentrated solution reached about 50 μl, 300 μl of 20 mM Na Phosphate, pH 7.2 buffer was added, and ultrafiltration was performed again.
This was repeated twice to remove the remaining SDS. Further, the same operation was repeated for the assay culture medium, and finally 300 μl was prepared. This was sterilized and TPO activity was measured.
As a result of such experiments, the proteins that could be clearly detected by silver staining were three kinds of apparent molecular weights of about 17000 to 19000, 14000, and 11000 with respect to the DPCIII marker.
In the electrophoresis of the TPO active fraction of the Capcell Pak C1 column, a band with an apparent molecular weight of about 17,000 to 19000, which correlates the intensity of activity with the intensity of the stained band, was observed. The apparent molecular weight at which TPO activity was detected was about 17000-19000. The above results are shown in FIG.
As a result, it was confirmed that the protein exhibiting TPO activity was purified in the active fraction of the Capcell Pak C1 300A column until it could be finally confirmed on the electrophoresis gel.
From the density of the silver-stained band obtained by 15% SDS polyacrylamide gel electrophoresis (under non-reduction) of this sample, the amount of the TPO candidate protein having an apparent molecular weight of about 17,000-19000 in the total TPO active fraction is , About 1.7 μg.
In the following, (12) to (15), each step of purification of the polymer TPO sample F2 will be described.
(12) YMC-Pack PROTEIN-RP <Reverse Phase Chromatography>
The developing solvent A (0.025% TFA) was added to the polymer TPO sample F2 obtained in (7) (total protein mass 257 mg, protein concentration 0.894 mg / ml, relative activity 7840, relative activity 2015000, total volume 287 ml). ) And 95.8 ml of developing solvent B (1-propanol containing 0.025% TFA) in one third of the sample, final volume 383 ml, final propanol concentration about 25%, TFA concentration 0.006%, The protein concentration was 0.671 mg / ml. Since insoluble matter was generated here, only the supernatant after centrifugation was divided into 62.3 ml (42.8 mg) 6 times, and YMC-Pack PROTEIN-RP (equilibrated with 30% B in advance) YMC, catalog number A-PRRP-33-03-15; diameter 3 cm, bed height 7.5 cm) was injected at a flow rate of 2 ml / min. Since the precipitate could be solubilized by adding 10 ml of 20 mM sodium acetate, pH 5.5 containing 5 mM CHAPS, it was fed together into the column.
After injecting each sample, about 50 ml of solvent (developing solvent A: developing solvent B = 3: 1) was passed through to collect the flow-through fraction, and then the developing program (from 30% B / 120% to 45% B) A total of 24 fractions of 15 ml each were collected in a polypropylene tube. It repeated in the same way from the 1st time to the 6th time, and finally the fraction of 90 ml each became 24. The flow-through fraction and tube number 1 were directly concentrated to 90 ml with an ultrafiltration unit (Amicon; YM3 membrane, diameter 76 mm).
Take 0.3 ml from the fractions containing tube numbers 1 to 24, including the flow-through, add 10 μl of 5% BSA, dry to dryness by centrifugal evaporation, and finally dissolve in 0.3 ml of IMDM assay medium and apply to the assay. The TPO active fraction was identified. As a result, tube numbers 10 to 15 (in the range of 34.0 to 39.5% in propanol concentration) have TPO activity, which is a TPO activity image of YMC-Pack PROTEIN-RP derived from high molecular TPO sample F2. Minute F2. It was stored at -85 ° C until just before proceeding to the next YMC-Pack CN-AP.
TMC active fraction F2 of YMC-Pack PROTEIN-RP derived from high molecular TPO preparation F2
Total volume 540ml
Protein concentration 0.021mg / ml
Total protein mass 11.4mg
Relative activity 227000
Relative activity 2588000
(13) Superdex 75pg <gel filtration chromatography in the presence of CHAPS>
Of the TPO active fraction F2 of the YMC-Pack PROTEIN-RP derived from the polymer TPO preparation F2 obtained in (12), 538.2 ml (total protein mass 11.3 mg, protein concentration 0.021 mg / ml, relative After adding 0.6 ml of 50% glycerol to the activity 227,000 (relative activity 256565), the solution was concentrated by centrifugal evaporation. Next, 6 ml of 20 mM CHAPS was added. Further, 18 ml of 20 mM CHAPS was added, stirred, transferred to 4 ° C., and after 41 hours, the first sample was HiLoad 26/60 Superdex 75 pg (manufactured by Pharmacia Biotech, catalog number 17-1070-01; diameter 2.6 cm, bed height 60 cm) ) It was injected into the column and developed with DPBS containing 5 mM CHAPS at a flow rate of 1 ml / min. A sample of 4 ml (protein concentration 0.466 mg / ml, protein mass 1.86 mg) was injected into the column at a time.
The column was developed on the sixth round, and the TPO active fraction of all YMC-Pack PROTEIN-RP was fractionated on a Superdex 75 pg column. Fractions were divided into 6 portions of 5 ml, that is, 45 fractions totaling 30 ml.
Take 0.1 ml from each fraction, add 10 μl of 5% BSA, dry to dryness by centrifugal evaporation, and finally dissolve in 0.25 ml of IMDM assay culture medium, apply to assay, and identify TPO activity fraction did. As a result, since tube numbers 13 to 31 (molecular weight range of 78000 to 3000) had TPO activity, this was designated as Superdex 75 pg TPO active fraction F2 derived from high molecular TPO sample F2.
Superdex 75pg TPO active fraction F2 derived from high molecular TPO preparation F2
Total volume 540ml
Protein concentration 0.00216mg / ml
Total protein mass 1.17mg
Relative activity 1750000
Relative activity 2041000
(14) YMC-Pack CN-AP <Reverse Phase Chromatography>
Of 540 ml of Superdex 75 pg TPO active fraction F2 (molecular weight 78000-3000) derived from the polymer TPO preparation F2 obtained in (13), 513.2 ml (total protein mass 1.11 mg, protein concentration 0.00216 mg / 1 ml of developing solution B (1-propanol containing 0.05% TFA) was added to ml, relative activity 1750000, relative activity 1943000), and then developed with developing solvent A (0.1% TFA). It was injected at a flow rate of 0.6 ml / min into a YMC-Pack CN-AP (manufactured by YMC, catalog number AP-513; diameter 6 mm, bed height 250 mm) equilibrated with liquid B at 15% B. After completion of the injection, the propanol concentration was increased from 15% B to 25% B, and further developed with a 65-minute linear concentration gradient from 25% B to 50% B.
In proceeding to the YMC-Pack CN-AP column, one-twentieth of all input samples was first piloted, confirming that the activity could be recovered well. Therefore, the remaining 19/20 was divided into two portions. In other words, a total of three deployments were performed. The fractions collected in a total of three times were collected in the same 44 tubes made of polypropylene, so that the total amount was 3.6 ml.
Of this, 5 μl (1/720 fraction) was taken and finally replaced with 0.25 ml of IMDM assay medium. As a result of the assay, tube No. 24 to 30 (range of 36.0 to 42.0% in propanol concentration) had extremely strong TPO activity, and this was designated as YMC-Pack CN-AP derived from high molecular TPO sample F2. The main TPO-active fraction FA.
Superdex 75pg TPO active fraction FA derived from high molecular TPO preparation F2
Total volume 25.20ml
Protein concentration 0.0246mg / ml
Total protein mass 0.620mg
Relative activity 700,000
Relative activity 434,000
(15) Capcell Pak C1 300A <final reverse phase chromatography>
24.66 ml (total protein mass 0.606 mg, protein concentration 0.0246 mg / ml) of TPO activity fraction FA25.20 ml of YMC-Pack CN-AP derived from high molecular TPO preparation F2 obtained in (14) , Relative activity 700,000, relative activity 424,000), 0.4 ml of 50% glycerol was added and concentrated by centrifugal evaporation.
The final sample was a 2 ml sample with a propanol concentration of several percent, glycerol of 10%, and a protein concentration of 0.303 mg / ml. Capcell Pak C1300A (Shiseido catalog number C1 TYPE: SG300A; diameter equilibrated with 15% B using developing solvent A (0.1% TFA) and developing solvent B (1-propanol containing 0.05% TFA) 4.6 mm, bed height 250 mm), developed from 27% B to 38% B with a linear concentration gradient of 65 minutes at a flow rate of 0.4 ml / min, and collected 0.6 ml in 72 polypropylene tubes. .
From each fraction, 0.75 μl (1/800 fraction) was taken, 20 μl of 5% BSA was added, and finally replaced with 225 μl of IMDM assay media, diluted 300-fold from the original volume to the assay. .
From each fraction, 2 μl (1/300 fraction) was taken for electrophoresis, centrifuged, 10 μl of SDS electrophoresis sample buffer without reducing agent was added, and treated at 95 ° C. for 5 minutes. This was subjected to SDS gel electrophoresis using a 15-25% SDS-polyacrylamide precast gel (Daiichi Kagaku) and stained with a silver staining kit. A DPCIII marker was used as the molecular weight marker.
As a result of the above analysis, tube numbers 33 to 39 (range of 29.5 to 31.5% in propanol concentration) clearly had TPO activity. Among these, tube numbers 34 to 39 (range of propanol concentration: 30.0 to 31.5%) were used as the main TPO active fraction FA. When the SDS gel electrophoresis image of the main TPO active fraction was examined, the strength of activity was apparently in the molecular weight range of 17000 to 22000, as in the case of starting from the low molecular TPO sample F3 described in (10). It was revealed that there was a band in which the stained intensity correlated.
<Example 2>
Analysis of partial amino acid sequence of rat purified TPO
Iwamatsu's method (Iwamatsu et al., New Basic Biochemical Experimental Method, Vol. 4, pages 33-84 (Maruzen); Iwamatsu Akihiko, Biochemistry, Vol. 63, No. 2, pages 139-143, (1991); Akihiro Iwamasu, Electrophoresis, Vol. 13, pp. 142-147, (1992)), amino acid sequence of rat TPO candidate protein in TPO fraction FA of Capcell Pak C1 300A column obtained in (10) of Example 1 Was analyzed. That is, the sample was subjected to SDS gel electrophoresis and electrically transferred to a polyvinylidene difluoride (PVDF) membrane. Subsequently, the protein on the PVDF membrane was reduced S-alkylated, and then systematically and stepwise limited enzymatic digestion with three kinds of proteases to obtain peptide fragments, which were separated and purified by reverse phase chromatography. The peptides were analyzed by sensitive amino acid sequencing. Details are described below.
Analysis example of TPO candidate protein of TPO fraction FA of Capcell PakC1 300A derived from low molecular TPO preparation F3
(1) Concentration of TPO fraction FA (tube numbers 36-42) on Capcell Pak C1 300A column
4200 μl (total protein mass 39.6 μg, protein concentration 9.4 μg) of the TPO active fraction FA (tube number 36 to 42) of the CapcellPak C1300A column derived from the low molecular weight TPO sample F3 obtained in Example 10 (10). / Ml, relative activity 490,000, relative activity 193600), 4151 μl (98.8% of all fractions) was advanced for analysis for amino acid sequence. The amount of protein estimated from the chromatogram is 39.1 μg. Among these, the amount of TPO candidate protein having an apparent molecular weight of about 17,000 to 19000 stained with SDS gel electrophoresis was about 1.6 μg. .
Glycerol was added to this sample and concentrated by centrifugal evaporation to give a 5 μl glycerol solution. To this, an SDS electrophoresis sample buffer containing no reducing agent, and 1 M Tris-HCl, pH 8 were added to adjust the pH, and finally 200 mM Tris-HCl, pH 8.0, 50 mM Tris-HCl, pH 6.8. About 25 μl of sample containing 1.1% SDS, 2 mM EDTA, 0.02% BPB, 30% glycerol.
In order to fully convert this sample into SDS without excessive heating, it was first placed at room temperature for 14 hours and then treated at 60 ° C. for 5 minutes.
(2) Electrophoresis
According to a conventional method, a micro slab gel (4.0% acrylamide concentration gel, 15% acrylamide separation gel) was prepared, and SDS gel electrophoresis was performed at room temperature for 12.5 mA) and then at a constant current of 17.5 mA for 2 hours. Carried out. As the molecular weight marker, a prestained low-range marker (Bio-Rad 161-0305) and a DPCIII marker were used. Immediately after the completion of the electrophoresis, it was transferred to a PVDF membrane (next item).
In addition, a part of the sample subjected to analysis was reduced with dithiothreitol (DTT) as it was without reduction, and 15-25% polyacrylamide precast gel (Daiichi Chemical Co., Ltd .; Multigel 15/25, catalog) No. 211072). When this was silver-stained using a silver staining kit, the band expected to be TPO had a molecular weight of about 19000 under reduction, and the purity of the TPO candidate protein in the TPO active fraction on the Capcell Pak C1 300A column was It was confirmed that it was about several percent. Moreover, since the mobility of non-reduction and reduction was different, it was suggested that the molecule has at least one S—S bond.
(3) Transcription and band detection on PVDF membrane by electroblotting
A PVDF membrane (ProBlott, Applied Biosystems, catalog) over a period of 1 hour at a constant current of 160 mA (11 to 17 V) using a semi-dry transfer device (Marisol, wet for transfer device model KS-8460) according to a conventional method. No. 400994) was transferred. The anolyte is 0.3 M Tris, 20% methanol, pH 10.4, the transfer membrane solution is 25 mM Tris, 20% methanol, pH 10.4, the catholyte is 25 mM Tris, 40 mM aminocaproic acid, 20% methanol, pH 10. 4 was used.
When the transferred membrane was stained with a Ponceau S staining solution (containing 0.1 g of Ponceau S and 1 ml of acetic acid in 100 ml), a plurality of bands were stained, and a band having a molecular weight of about 19000 expected to be TPO was included therein. I was able to confirm. This was cut out and proceeded to peptide fragmentation. (Next section)
(4) Peptide fragmentation and peptide mapping / amino acid sequence analysis
In order to systematically fragment the TPO candidate protein transcribed / reduced S-alkylated on the PVDF membrane, limited enzymatic degradation was performed step by step with the following three proteases.
Primary digestion Lysyl endopeptidase (Achromobacterium lyticus m497-1, manufactured by Wako Pure Chemical Industries, catalog number 129-02541) Secondary digestion endoproteinase Asp-N (produced by Boehringer Mannheim, catalog number 1054589)
Tertiary digestion Trypsin-TPCK (manufactured by Worthington Biochemical, catalog number 3740)
Peptide fragments obtained by each enzyme digestion were recovered, and using a mixed solution of isopropanol: acetonitrile = 7: 3 containing 0.05% TFA in developing solvent A and 0.02% TFA in developing solvent B, Wakosil- II 5C18 C18 reverse phase column (manufactured by Wako Pure Chemical Industries, Ltd .; diameter, 20 mm, length 150 mm), column temperature 30 ° C., flow rate 0.25 ml / min, linear concentration gradient from 1% B to 50% B for 30 minutes The resulting peptide fragment was recovered by mapping (Fig. 4). Each peptide fragment was subjected to Edman degradation with a gas phase amino acid sequencer (manufactured by Shimadzu Corporation, PPSQ-2), and the N-terminal PTH amino acids recovered in sequence were identified by C18 reverse phase column chromatography by isocratic elution method. Went. The results are summarized below.
Primary digestion Amino acid sequence of peptide fragment by lysyl endopeptidase
Fragment name Amino acid sequence
[Table 3]

[Table 4]

[Table 5]

Among the above sequences, those shown with () are amino acid residues that can be deduced from systematic enzyme digestion.
(5) Similarity analysis of the obtained amino acid sequences <homology search>
Whether the obtained amino acid sequence is contained in a known protein that has already been reported, or whether there is a protein having a similar sequence, Mac Vector (Kodak International Biotechnology, Inc.), which is sequence analysis software. Was used for analysis. Information on known proteins or known genes was used using the Entrez Release 6 database (issued August 15, 1993, US National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health). The various databases included in this are as follows.
Entrez Release 6 database
NCBI-GenBank, August 15, 1993 (Release 78.0) EMBL, July 15, 1993 (Release 35.0 plus updates) DDBJ, July 15, 1993
SWISS-PROT, April, 1993 (Release 25.0)
PIR, June 30, 1993 (Release 37, 0)
PDB, April, 1993
PRF, May, 1993
dbEST, July 15, 1993 (Release 1.10)
U. S. and European Patents (part)
As a result, the sequence (K) DSFLADVK of AP12 is the rat Corticosteroid-binding globulin (CBG) precursor [PIR database registration number A40066; under differentialphysical conditions. "Mol. Endocrinol. . (1989), 3, 420-426,] and completely matched with the internal sequence KDSFLADVK.
Upon further examination, the sequence called KQYYESE, which is highly similar to the sequence of AP3 (K) XYYESZ (where X is A, S, G, M, or Q) (Z is E or K)), It was found to be contained in the amino acid sequence of rat CBG. These sequences corresponding to AP12 and AP3 are continuous in rat CBG and correspond to the internal amino acid sequence KDSFLADVKQYYESE.
However, regarding the amino acid sequences of fragments other than AP12 and AP3, no known proteins or genes that should be considered for similarity were found.
<Example 3>
Biological characterization of TPO derived from thrombocytopenic rat plasma
(1) In the rat CFU-MK assay system (liquid culture system)
As a representative example, FIG. 5 shows a dose-response curve in the case of using a TPO partially purified preparation (YMC Pack Protein-RP column TPO active fraction F2 described in (8) of Example 1) from thrombocytopenic rat plasma. It was shown to. When the culture was observed over time under a microscope, it was observed that megakaryocyte differentiation and maturation progressed, that is, an increase in cell size over time, and that an increase in cells probably occurred. As a particularly remarkable change, protrusion formation by many megakaryocytes was observed on the 4th day of the last day of culture (mostly not observed on the 3rd day of culture). This protrusion formation is performed by cytoplasmic process formation (Leven and Yee, Blood, Vol. 69, pp. 1046-1052, (1987)) or propagated process formation (Topp et al., Blood, Vol. 7, 76, 912-924, 1992). ) And the like, and is a precursor structure of platelet that has been further differentiated from megakaryocytes, and is considered to be the final form of megakaryocyte differentiation that can be observed in vitro so far. Since such a morphological change was observed at a high frequency with the TPO preparation alone, this factor alone promotes the proliferation and differentiation of CFU-MK, generates mature megakaryocytes, and finally produces platelets. There is a possibility of progress.
(2) In the colony assay system
For TPO partially purified preparations from thrombocytopenic rat plasma, rat non-separated bone marrow cells, isolated / enriched cells, or GpIIb / IIIa⁺When assayed by a colony assay system using the CFU-MK fraction, TPO derived from rat plasma formed megakaryocyte colonies. When comparing megakaryocyte colonies formed by TPO with other known cytokines, ie, rat IL-3, mouse GM-CSF, or human EPO, megakaryocyte colonies formed by TPO Although the number of megakaryocytes constituting each colony is small, the size of each megakaryocyte is large, that is, the maturity is advanced. Furthermore, colonies of other cell lines are hardly formed, and the Meg-CSF activity exhibited by TPO is considered to be a megakaryocyte-specific activity. From these facts, TPO has different biological characteristics from other known cytokines that exert Meg-CSF activity in this colony assay system, namely rat IL-3, mouse GM-CSF, or human EPO. It has become clear that it exhibits unique Meg-CSF activity.
CD34 derived from human bone marrow cells or human blink blood cells⁺DR⁺The TPO partially purified preparation from thrombocytopenic rat plasma also showed Meg-CSF activity and formed a significant number of human megakaryocyte colonies against the cell fraction. This indicates that this factor has no species specificity.
<Example 4>
Specification of rat TPO-producing cells
(1) Search for rat TPO producing organs
First, for the purpose of securing a source of mRNA for cloning or expression cloning of the rat TPO gene based on the partial amino acid sequence of rat TPO, a rat TPO-producing organ was searched and specified. Initially, bone marrow, lung, liver, and spleen were removed from rats that had been thrombocytopenic by administration of P55 antibody, and the culture supernatant of the cells (in the case of lung, liver, organ section) was collected, and rat CFU- The activity in the supernatant was evaluated by the MK assay system, but no clear result was obtained. Subsequently, P55 antibody administration was performed in consideration of a report (Siemensma et al., J. Lab. Clin. Med., 86, 817-833, (1975)) suggesting an association between liver and TPO production in rats. Hepatocytes prepared by the collagenase perfusion method were cultured from the liver of a thrombocytopenic rat, and the fraction adsorbed on the WGA-Agarose column from the supernatant was developed on a Vydac phenyl reverse phase column. -In the MK assay system, very similar activity was observed at the same position as rat plasma-derived TPO activity. A weak but active activity was also observed in the culture supernatant of normal rat hepatocytes. These results strongly suggested that the liver may be one of TPO-producing organs.
(2) Screening of rat TPO-producing cell line
Based on the above results, a rat TPO-producing cell line was screened. First, after culturing 20 types of rat liver-derived cell lines in each subculture medium until they are almost confluent, the serum contained in the culture medium is replaced with each culture medium unified to 5% FCS. Then, the culture was further continued for 3 days, and each culture supernatant was collected. The supernatant was partially purified by the method described in (1) and examined for the presence or absence of TPO production. As a result, three types of rat hepatocyte-derived cell lines, namely McA-RH8994 cells (ATCC deposit number CRL1602, Becker) “Oncodevelopmental Gene Expression” ed. By Fishman and Sell, Academic Press, NY, pp. 259-270, (1976), purchased from Dainippon Pharmaceutical Co., Ltd.), H4-II-E cells (ATCC deposit number CRL1548, Pitot et al., Nat. Cancer Inst. Monogr., 13, 229-245, (1964), purchased from Dainippon Pharmaceutical), and HTC cells (Thompson et al., Proc. Natl. Acad. Sci. USA, 56, 96-303 pp., (1966), the production of apparently TPO activity from than purchase) Dainippon Pharmaceutical Co., Ltd. has been confirmed.
(3) Detailed analysis of TPO activity produced by McA-RH8994 cells, H4-II-E cells, and HTC cells
The rat plasma-derived TPO activity, which was being purified in parallel with the TPO activity secreted from these three types of rat cell lines, was compared in detail in terms of both biochemical and biological properties.
McA-RH8994 cells were suspended in an alpha-MEM (−) culture solution containing 10% FCS, and the bottom area was 175 cm.²1 x 10 in a culture plastic flask for tissue culture⁶The cells were placed in a flask / flask and cultured for 3 days at 37 ° C. in a 5% carbon dioxide incubator. After replacement with IMDM culture medium containing 5% FCS, the cells were further cultured for 3 days, and the supernatant was collected. H4-II-E cells were suspended in Dulbecco's modified Eagle medium (containing 4.5 g / l glucose) (hereinafter, DMEM medium) containing 10% FCS, and the bottom area was 175 cm.²5 x 10 in a plastic flask for tissue culture⁵The cells were placed in a flask / flask and cultured for 3 days at 37 ° C. in a 5% carbon dioxide incubator. After replacement with IMDM culture medium containing 5% FCS, the cells were further cultured for 3 days, and the supernatant was collected. In addition, HTC cells were suspended in a DMEM culture solution containing 5% FCS, and the bottom area was 175 cm.²2.5 x 10 in a tissue culture plastic flask⁵The cells were placed in a flask / flask and cultured for 3 days at 37 ° C. in a 5% carbon dioxide incubator. After replacement with IMDM culture medium containing 5% FCS, the cells were further cultured for 3 days, and the supernatant was collected.
According to the purification method of TPO from XRP described in Example 1-2, partial purification of cell line-derived TPO was performed from 2 liters of each of the culture supernatants of the three types of cell lines thus obtained. The outline is described below.
First, the culture supernatant was concentrated about 6 times with an ultrafilter, and then buffer exchanged to 20 mM Tris-HCl (pH 8.0) using a Sephadex G-25 column. The eluate was added to a Q-Sepharose FF column, washed with 20 mM Tris-HCl (pH 8.0), and the adsorbed fraction was eluted with 20 mM Tris-HCl (pH 8.0) containing 175 mM NaCl. This fraction was added to a WGA-Agarose column, washed with PBS, and then the adsorbed fraction was eluted with 20 mM Na Phosphate (pH 7.2) containing 0.2 M GlcNAc and 0.15 M NaCl. This eluate was added to a TSK-gel AF-BLUE 650MH column, washed with 20 mM Na Phosphate (pH 7.2) containing IM NaCl, and then eluted with 2M NaSCN. This was added to a Phenyl-Sepharose 6 FF / LS column, washed with 50 mM Na Phosphate (pH 7.2) containing 1.5 M ammonium sulfate (Ammonium Sulfate), and then with 36 mM Na Phosphate containing 0.8 M ammonium sulfate, followed by 20 mM Na Phosphate ( Elution with pH 7.2). This adsorbed fraction was concentrated and then fractionated on a reverse-phase Vydac Protein C4 column (The Separations Group catalog number 214TP51015; diameter 1 cm, bed height 15 cm). Using 1-propanol containing 0.1% TFA for developing solvent A and 0.05% TFA for developing solvent B, the column was equilibrated in advance at 20% B, and after injecting the sample, 20% B at a flow rate of 1 ml / min. Was eluted with a linear gradient from 90 to 40% B for 90 minutes. As a result, TPO activity derived from any cell line was eluted with 1-propanol at a concentration of 30% to 43%.
As a result of measuring the activity of the preparations at each purification stage in the rat CFU-MK assay system, the TPO activities derived from the three cell lines all behaved very similar to the TRP activity derived from XRP (Example 1- (See 2) (Table 6 describes the relative specific activity, activity yield, etc. at each stage in the rat CFU-MK assay system). Furthermore, as a result of measuring the activity of the elution fraction from the reverse phase column in the final stage using the rat CFU-MK assay system, the TPO activity derived from the three cell lines and the TPO activity derived from the XRP were eluted at the same peak position. It was. Focusing on the active fraction of this reverse phase column, rat GpIIb / IIIa⁺When colony assays were performed using non-adherent cells obtained at the removal of adherent cells in the CFU-MK separation / concentration process, all of them corresponded to the elution pattern of activity in the rat CFU-MK assay system. The formation of spherical colonies was observed (Table 7). Similarly to the XRP-derived TPO activity, the activities derived from the three cell lines all formed megakaryocyte colonies, and the colonies of other strains were mostly formed. I did not let it.
In accordance with the method described in Example 1-2, the active fractions of the pooled reverse-phase column were subjected to SDS polyacrylamide gel electrophoresis, and after the electrophoresis, proteins were extracted from the gel, and the activity was measured using a rat CFU-MK assay system. As a result, XRP-derived TPO has an apparent molecular weight of 17000-22000, McA-RH8994 cell-derived TPO has an apparent molecular weight of 33,000-39000, and H4-II-E cell-derived TPO has an apparent molecular weight of 31000-38000, derived from HTC cells. The TPO had an apparent molecular weight of 17000-22000 and a molecular weight of 28000-35000.
As described above, TPO activity produced by McA-RH8994 cells, H4-II-E cells, and HTC cells is slightly different in biochemical properties in terms of apparent molecular weight compared to XRP-derived TPO. However, it was found to be equivalent in biological properties. In the present invention, it should be noted that a molecule having TPO activity present in blood is cleaved at a specific or unspecific position in the molecule in the production cell or after secretion from the production cell. It suggests the possibility of being. In addition, there is a possibility that TPO genes (mRNA and cDNA) may have various lengths.
[Table 6]

[Table 7]

<Example 5>
Construction of cDNA library preparation expression vector (pEF18S)
A vector that facilitates the incorporation of the cDNA fragment into the vector and has a high expression efficiency of the cloned cDNA was prepared for the cloning and expression of TPO cDNA. That is, the expression vector pEF18S was constructed by replacing the promoter of elongation factor 1α (EF1α), which is known as a promoter with high expression efficiency, with the SRα promoter of the easy-to-use expression vector pME18S (see FIG. 6). The promoter of elongation factor 1α is the expression vector pEF-BOS (Mizushima et al., Nucleic Acids Res.,18, 5322, 1990) 1 μg was partially digested with restriction enzymes HindIII and EcoRI, and then subjected to electrophoresis using 2% agarose gel (manufactured by FMC BioProducts) to separate a DNA fragment of about 1200 bp, and prep-A -Gene DNA purification kit (Bio-Rad; Willis et al., BioTechniques,9, 92-99, 1990, and was purified using a kit that selectively purifies DNA by adsorption using a porous silica-based matrix. A 50 ng expression vector pME18S (Liu et al., Proc. Natl. Acad. Sci. USA, similarly digested with 100 ng of this DNA fragment with HindIII and EcoRI,90, 8957-8961, 1993). The host fungus is competent high E. coli. E. coli DH5 (manufactured by Toyobo; Hanahan et al., J. Mol. Biol.,166Competent host fungi prepared by a modification of the method of 557-580, 1983), 12 colonies obtained were randomly selected to purify the plasmid DNA, and the target was determined by the digestion pattern of the DNA with restriction enzymes. One of 10 clones containing the plasmid (pEF18S) was selected, and a large amount of plasmid DNA was prepared.
Purification of the plasmid DNA was performed essentially as described in Molecular Cloning [Sambrook et al., Cold Spring Harbor Laboratory Press (1989)].
That is, the clone pME18S obtained as described above was cultured overnight in 50 ml of LB medium (1% Bacto-tryptone, 0.5% Baculo-yeast extract, 0.5% NaCl) containing 50 μg / ml Ampicillin. Thereafter, the cells obtained by centrifugation were suspended in 4 ml of TEG-lysozyme (25 mM Tris-Cl (pH 8), 10 mM EDTA, 50 mM Glucose, 0.5% lysozyme) solution, and 8 ml of 0.2N NaOH / 1% SDS. Add the solution and suspend well. Further, 6 ml of 3M potassium / 5M acetate solution is added and well suspended, and then centrifuged to obtain a supernatant. The supernatant was treated with phenol-chloroform (1: 1), added with an equal amount of isopropanol, and centrifuged to obtain a pellet. The pellet is dissolved in a TE solution (10 mM Tris-hydrochloric acid (pH 7.5), 1 mM EDTA), then subjected to RNase treatment and phenol-chloroform (1: 1) treatment, followed by ethanol precipitation. The pellet is again dissolved in the TE solution, and NaCl and polyethylene glycol 3000 are added to 0.63M and 7.5%, respectively, and centrifuged.
Finally, the pellet is dissolved in TE solution and ethanol precipitated. As a result, about 300 μg of plasmid DNA was obtained. 100 μg of this DNA was completely digested with restriction enzymes EcoRI and NotI, and then electrophoresed on a 0.8% agarose gel (FMC BioProducts), and the vector fragment was recovered and prep-A-gene DNA purification kit (Biorad) To obtain about 55 μg of plasmid DNA. This DNA was used for the preparation of the following cDNA library.
<Example 6>
Purification of mRNA from McA-RH8994 cells
McA-RH8994 cells, which were relatively more active than the results of the colony assay of Example 4, were selected as the material for rat TPO cDNA cloning and subjected to the following experiment.
Total RNA isolation was performed essentially as described in Molecular Cloning [Sambrook et al., Cold Spring Harbor Laboratory Press (1989)]. After McA-RH8994 cells were grown completely and densely on 15 petri dishes having a diameter of 90 mm, the culture medium was removed from the petri dish, and 0.8 ml of 5M guanidine solution (5M guanidine thiocyanate, 5 mM citrate per petri dish). Sodium (pH 7.0), 0.1 M β-mercaptoethanol, 0.5% sodium sarcosyl sulfate) was added and suspended well, and then the mixture was collected in one tube, and the guanidine solution was added to make a total volume of 20 ml. . The mixed solution in which the cells were broken and became viscous was repeatedly inhaled and discharged approximately 20 times using a 20 ml syringe loaded with 18G and 21G injection needles until the viscosity almost disappeared. Add 18 ml of 5.7 M CsCl-0.1 MEDTA (pH 7.5) as a cushion to a polyallomer centrifuge tube that fits a Beckman SW28 rotor, and about 20 ml of the above mixture so that the tube is almost full. The layers were layered gently so as not to disturb the layers. The centrifuge tube prepared in this way was treated at 25,000 r. p. m. After centrifugation for 20 hours, the resulting pellet was washed twice with a small amount of 80% ethanol. The pellet was dissolved in TE solution, extracted with phenol-chloroform (1: 1), 1/10 volume of 3M sodium acetate and 2.5 volumes of ethanol were added, and ethanol precipitation was performed to obtain total RNA ( About 10⁸About 2.5 mg of total RNA was obtained from a single cell).
Poly from total RNA (A)⁺RNA purification is Oligotex^TM-DT30 (Super) (manufactured by Nippon Synthetic Rubber / Nippon Roche; oligo dT is covalently fixed to the surface of the latex particles,⁺Poly (A) as well as oligo dT column used for RNA purification⁺RNA can be purified). 20 μg of poly (A) from about 500 μg of total RNA⁺RNA was obtained.
<Example 7>
Construction of rat cDNA library
5 μg of poly (A) obtained in Example 5⁺RNA to TimeSaver^TMcDNA Synthesis Kit (manufactured by Pharmacia; Okayama-Berg method: Mol. Cell. Biol.,2161-170, 1982) and DIRECTIONAL CLONING TOOLBOX (manufactured by Pharmacia; primer for cDNA synthesis containing NotI sequence: 5'-AACTGGGAAGATCTGCGGCGCGCAGGAA (T)₁₈3 ′ and EcoRI sequence addition adapter: 5′-AATTCGGCCACGAG-3 ′ and 5′-CTCGGCCG-3 ′), a double strand having EcoRI at the 5 ′ end and a NotI recognition site at the 3 ′ end cDNA was synthesized. The synthesized cDNA was ligated with 1.2 μg of an expression vector pEF18S (see Example 5) previously treated with EcoRI and NotI, and 8.4 ml of competent high E. coli. E. coli DH5 (Toyobo Co., Ltd.) was transformed. As a result, 5.3 × 10⁵Individual transformants were obtained. <Example 8>
Acquisition (cloning) of rat TP0 cDNA fragment by PCR
The McA-RH8994 cDNA library 530,000 clone prepared in Example 7 was cultured overnight in 50 ml of LB medium containing 50 μg / ml of ampicillin, and then QIAGEN-tip100 (manufactured by DIAGEN; DNA purification) from the cells obtained by centrifugation. The McA-RH8994 cDNA library plasmid was purified using a silica gel-based anion exchange column for production to obtain about 200 μg of plasmid DNA.
Two types of antisense nucleotide primers AP8-1R and AP8-2R corresponding to the amino acid sequence of peptide fragment AP8 described in Example 2 and human elongation of the plasmid vector pEF18S shown in FIG. 6 used for cDNA library preparation Sense nucleotide primers EF1α-1 and EF1α-2 corresponding to the first intron of factor 1α were synthesized. For synthesis, a 394 DNA / RNA synthesizer (synthesizer based on β-cyanoethylamidite method) manufactured by Applied Biosystems was used, and a synthetic DNA purification OPC column (a column packed with reversed-phase silica gel with a trityl group) The column was purified using a column for purifying DNA. The purified synthetic DNA was dissolved in TE solution to 50 μM and stored at −20 ° C. until use. The synthetic oligonucleotides used below were all synthesized and purified in the same manner.
Primers AP8-1R and AP8-2R are mixed primers composed of 17 consecutive nucleotides, and deoxyinosine was used as described in Takahashi et al. (Takahashi, et al., Proc. Natl. Acad. Sci. (USA)821931-1935 (1985)).
Primers EF1α-1 and EF1α-2 were prepared according to Uetuki, et al. , J .; Biol. Chem.H.264, 5791-5798 (1989), and primers composed of 21 and 20 nucleotides, respectively, synthesized based on the base sequences corresponding to the genomic sequences 1491-1512 and 1513-1532. Table 8 shows the sequences of the synthesized primers.
[Table 8]

McA-RH8994 cDNA library Plasmid 3 μg as a template, AP8-1R (500 pmol), EF1 α-1 (100 pmol) as primers, GeneAmp^TMPCR Reagent Kit with AmpliTaq^TM  Using DNA Polymerase (Takara Shuzo Co., Ltd .; PCR heat-resistant Taq polymerase, reaction buffer, dNTP set), GeneAmp^TM  PCR reaction with PCR System 9600 (manufactured by PERKIN-ELMER; PCR reactor) in a volume of 100 μl (heated at 95 ° C. for 2 minutes, then denatured at 95 ° C. for 1 minute, annealed at 40 ° C. for 1 minute, 72 The reaction was performed 35 times under the synthesis conditions at 1 ° C. for 1 minute, and further incubated at 72 ° C. for 7 minutes. In order to increase the specificity of the amplified DNA fragment, PCR reaction (with 1 μl of the obtained PCR reaction solution as a template and EF1α-2 (100 pmol) and AP8-2R (500 pmol) as primers and similarly in a volume of 100 μl ( After heating at 95 ° C for 2 minutes, perform 35 reactions under denaturing conditions at 95 ° C for 1 minute, annealing conditions at 45 ° C for 1 minute, and synthesis conditions at 72 ° C for 1 minute, and further incubate for 7 minutes at 72 ° C) Went.
The reaction solution obtained here was subjected to electrophoresis using a 2% agarose gel (manufactured by FMC BioProducts), and a DNA fragment of about 330 bp, which is the main product of this PCR reaction, was separated and purified by prep-A-gene DNA purification. Purification was performed using a kit (manufactured by Bio-Rad). This DNA fragment was converted to pCR using T4 DNA ligase (Life Technology).^TMII (manufactured by Invitrogen; vector for PCR product TA cloning: use of the property of adding one deoxyadenylic acid to the 3 ′ end of DNA amplified by PCR because the thermostable polymerase used for PCR has terminal transferase activity And a vector pCR having a 5′-dT overhanging end^TSubcloning into MII as it was) Subcloning into a vector. Plasmid DNA was purified using QIAGEN-tip100 (manufactured by DIAGEN) for 28 randomly selected clones, which were then used as Taq Dye Deoxy.^TM  Terminator Cycle Sequencing Kit (Applied Biosystems, Inc .; dideoxy method using PCR with fluorescent dye: Sanger et al., Proc. Natl. Acad. Sci. USA,745463-5467, 1977) were sequenced with a 373A DNA sequencer (fluorescent sequencer) manufactured by Applied Biosystems, and the base sequence of each clone was determined.
Among the obtained DNA fragments, one encoding the N-terminal 3 amino acids of AP8 (Ile / Thr / Ser) -Val-Pro adjacent to the AP8-2R primer was selected, and the full length was sequenced. However, it contained a cDNA having 26 lbp.
The base sequence of 173 to 175 of this cDNA fragment encodes methionine, and the coding frame coincided with the amino acid frame of AP8. In addition, before and after this sequence, the Kozak sequence (Kozak, M., Cell,44, 283-292, 1986), it was presumed to be the translation initiation site, and was considered to be a cDNA fragment encoding the N-terminal part of rat TPO protein. This cDNA fragment was named A1. The base sequence of the A1 fragment excluding the vector sequence and the amino acid sequence deduced therefrom are shown in the sequence listing (SEQ ID NO: 1).
<Example 9>
Screening of rat TPO cDNA by PCR
The aforementioned cDNA library was divided into pools of about 10,000 clones and cultured overnight in 1 ml of LB medium containing 50 μg / ml of ampicillin, and then an automatic plasmid separator PI-100 (VER-3, Kurashiki Boseki Co., Ltd. 0.0; alkaline SDS method: Plasmid DNA was extracted using Molecular Cloning [Sambrook et al., ColdSpring Harbor Laboratory Press, 1989] :). Two synthetic oligonucleotides designed based on fragment A1 using 1/30 of the amount as a template
5 'CGAGGGGTGTACCTGGGTCCTG3' (1-17 sense sequence of sequence SEQ ID NO: 1; CGAG is the adapter sequence) and
5 'CAGAGTTAGTCTTGCGGTGAG3' (Antisense sequence 212-232 of the sequence of SEQ ID NO: 1)
Is synthesized and purified using as a primer, GeneAmp^TM  PCR Reagent Kit with AmpliTaq^TMUsing DNA Polymerase (Takara Shuzo), GeneAmp^TMResults of performing PCR reaction (reaction conditions of 94 ° C for 30 seconds, annealing condition at 94 ° C for 30 seconds, annealing condition at 66 ° C for 30 seconds, and synthesis conditions at 72 ° C for 1 minute) by PCR System 9600 (manufactured by PERKIN-ELMER) A specific band of 236 bp was detected in 3 out of 100 pools. One of these pools was divided into pools of about 900 clones, and plasmid DNA was extracted and subjected to the same PCR reaction. As a result, bands were detected in 3 out of 100 pools. Furthermore, one of these pools was divided into pools of 40 clones, and the same selection was performed. As a result, 3 bands out of 100 pools were considered to be specific. One of these pools was selected and plated on an LB plate (LB medium containing 15% agar) containing 50 μg / ml Ampicillin. Plasmid DNA was extracted from each colony that appeared and the same PCR reaction was performed. Positive bands were detected in 2 out of 100 clones.
<Example 10>
Sequence of rat TP0 cDNA
Purification of the plasmid DNA was performed essentially as described in Molecular Cloning [Sambrook et al., Cold Spring Harbor Laboratory Press (1989)].
The two clones finally obtained in Example 9 were cultured overnight in 50 ml of LB medium containing 50 μg / ml Ampicillin, and then purified in the same manner as in Example 5. Finally, about 300 μg of plasmid DNA was obtained. Obtained.
The plasmid DNA obtained here was designated as Taq Dye Deoxy.^TMUsing Terminator Cycle Sequencing Kit (Applied Biosystems), sequencing was performed in the same manner as in Example 8, and the base sequence of the full length cDNA was determined. As a result, it was clarified that the base sequences of the two clones were completely identical and identical. One clone was selected from these, and the plasmid clone carrying the cDNA was named pEF18S-A2α. The nucleotide sequence and the amino acid sequence deduced therefrom are shown in the sequence listing (SEQ ID NO: 2).
The characteristics of the sequences shown in the sequence listing (SEQ ID NO: 2) are remarkable. The sequence after the 172 bp 5 'untranslated region encodes a highly hydrophobic amino acid that is presumed to be a signal sequence for secretion consisting of 21 proteins starting with methionine. The protein has 126 amino acid residues, followed by a stop codon (TAA) followed by 1022 bases of 3 'untranslated sequence and a number of adenines in the poly A tail. This protein contained only a sequence corresponding to the partial amino acid sequence AP8 analyzed in Example 2 (amino acids 1 to 12 in SEQ ID NO: 2). There is no sequence site for N-glycosylation. In addition, the base sequence from 1624 to 1629 near the end of the 3 'untranslated sequence is considered to be a potential polyadenylation sequence, although it differs from the consensus sequence.
The vector pEF18S-A2α carried in Escherichia coli DH5 was deposited on February 14, 1994 at the Institute of Industrial Science and Technology of the Ministry of International Trade and Industry under the accession number FERM BP-4565. It was also deposited with the Food Industry Development Institute of China (FIRDI) on March 22, 1995 as deposit number 940083.
<Example 11>
Expression of rat TPO cDNA in COS1 cells-activity confirmation
Transfection of plasmid pEF18S-A2α into COS1 cells can be accomplished using the DEAE-dextran method (Sompayrac et al., Proc. Natl. Acad. Sci. USA 78, 7575-7578, (1981); Luthman et al., Nucl. Acids Res., Vol.11, pp.1295-1308, (1983)) was carried out according to the following method. COS1 cells (ATCC CRL1650) suspended in a DMEM culture solution containing 10% FCS are placed in a tissue culture plastic dish having a diameter of 100 mm and cultured in a 5% carbon dioxide incubator at 37 ° C. until approximately 40% confluent. did. On the other hand, 500 μg / ml (7) DEAE-dextran (Pharmacia), 80 μM chloroquine (Sigma), 8% (v / v) HBS (21 mM HEPES-145 mM NaCl, pH 7.1) and 9% (v / v) The above-described COS1 in which plasmid pEF18S-A2α (10 μg) dissolved in 30 μl of HBS is mixed with 4 ml of DMEM culture solution containing Nu-Serum (Collaborative) and washed twice with DMEM culture solution immediately before transfection. After addition to the cells, the cells were cultured at 37 ° C. for 5 hours in a 5% carbon dioxide incubator. Thereafter, the culture supernatant is removed by aspiration, and the dish is washed twice with DMEM culture solution. Then, 15 ml of DMEM culture solution containing 10% FCS is added, and the culture is carried out at 37 ° C. for 3 to 5 days in a 5% carbon dioxide incubator. After culturing, the culture supernatant was collected.
The obtained culture supernatant was sufficiently dialyzed against IMDM culture solution and then evaluated with a rat CFU-MK assay system. As a result, TPO activity was observed in a dose-dependent manner in the culture supernatant of COS1 cells transfected and expressed with the plasmid pEF18S-A2α (FIG. 7), and the fourth day of culture as in the case of rat TPO. A large number of cytoplasmic process formations were observed. On the other hand, no TPO activity was detected in the COS1 cell culture supernatant transfected with the plasmid pEF18S containing no insert (FIG. 7). In addition, in the M-07e assay system, M-07e cell growth promoting activity was observed in a dose-dependent manner in the culture supernatant of COS1 cells transfected and expressed with the plasmid pEF18S-A2α, and a plasmid containing no insert No activity was observed in the culture supernatant of COS1 cells transfected with pEF18S. From these results, it was confirmed that pEF18S-A2α carries a gene encoding a protein having TPO activity.
Next, in order to examine the platelet increasing effect of this TPO activity, a partially purified product was prepared.
A rat CFU-MK assay system was used to measure TPO activity during the preparation process. First, COS1 cells transfected with plasmid pEF18S-A2α were cultured in a serum-free culture medium containing 0.2 mg of BSA for 3 days to obtain about 5.8 L of serum-free culture supernatant. To the serum-free culture supernatant, p-APMSF was added at a final concentration of 1 mM, and a filtrate passing through a 0.22 μm filter was taken. To this volume 5793 ml (protein concentration 0.229 mg / ml), total protein mass 1326 mg, relative activity 1000, relative activity 1326080) was added 0.85 moles NaCl (total 288 g) per 1000 ml, final concentration 0.822M NaCl, 5849 ml. After being equilibrated with 1 M NaCl, 20 mM NaPhosphate pH 7.2, a TSK-gel AF-BLUE 650MH column (manufactured by Tosoh, catalog number 08705; diameter 5 cm, bed height 6 cm) was flowed at a flow rate of 7 ml / Added in min. After completion of the addition, the flow-through (about 7900 ml) eluted with 20 mM Na Phosphate, 1M NaCl, pH 7.2 was collected, concentrated with an ultrafiltration unit (Filtron, Omega Ultraset, molecular weight 8000 cut), and the flow-through fraction. F1 (460 ml, protein concentration 2.11 mg / ml, total protein mass 973 mg, relative activity 16.3) was obtained. Next, the eluate was changed to 2M NaSCN, and the eluted TSK-gel AF-BLUE 650MH adsorbed TPO active fraction F2 (2840 ml) was used using an ultrafiltration unit (Amicon; YM3 membrane, diameter 76 mm). Concentrated to 6.81 ml. The total protein amount of this TPO active fraction F2 was 12.5 mg, and the protein yield of F2 at this step was 0.62%. The relative activity of TPO was 240. Next, HiLoad26 / 60 Superdex 200 pg (Pharmacia Biotech, Catalog No. 17-1071-01; diameter 2,6 cm, bed height 60 cm) was injected into the column, and 20 mM Na Acetate containing 50 mM NaCl at a flow rate of 1 ml / min. , Developed at pH 5.5. TPO activity was confirmed in fractions ranging from 194 ml to 260 ml eluted after the start of development. Thus, this was collected and used as Superdex 200 pg TPO active fraction F2 (66 ml, protein concentration 0.112 mg / ml, total protein mass 7.41 mg, relative activity 142860, relative activity 1058600). Next, a developing solvent A (20 mM Na Acetate, pH 5.5) and a developing solvent B (20 mM Na Phophosphate, pH 7.2 containing 500 mM NaCl) were prepared, and a strong cation exchange column equilibrated with 100% A was prepared. The column was injected into a RESOURCES (Pharmacia Biotech, catalog number 17-1178-01: diameter 0.64 cm, bed height 3 cm) column at a flow rate of 1 ml / min. Subsequently, it was developed with a linear concentration gradient from 100% A to 100% B over 40 minutes at a flow rate of 0.3 ml / min. As a result of the assay, it was found that TPO activity was eluted in a wide range (ranging from 5% B to 32% B). The TPO active fractions were collected and concentrated by an ultrafiltration unit (Amicon; YM3 membrane, diameter 25 mm), and RESOURCES TPO active fraction F2 (1.65 ml, protein concentration 4.74 mg / ml, total protein). Amount 7.82 mg, relative activity 71400, relative activity 558600).
Although the obtained preparation was not a pure TPO preparation, a sufficiently high activity was confirmed in the rat CFU-MK assay system. Therefore, administration experiments to mice were performed. That is, ICR mice (9 weeks old; sputum) whose platelet count had been measured on the day before administration were subcutaneously injected with TPO partial purified product (474 μg (100 μl) / mouse / day), and BSA (200 μg (100 μl)) as a control. / Mouse / day), or as a control, a buffer solution (100 μl / mouse / day) having the same composition as the buffer solution in which the TPO partial purified product is dissolved is administered every day for 5 days. The number was measured (F800; Toa Medical Electronics). In the TPO partial purified product-administered mice, an increase of about 2.14 times the platelet count on average was observed compared with that before administration. In addition, when compared between the administration groups, the TPO partial purified product-administered mice had a platelet count that was about 1.74 times that of the BSA-administered mice or about 1.90 times that of the mice that received the buffer alone. Increased. From this result, it became clear that TPO has a platelet increasing action in vivo. In addition, since no increase in immunosuppressive acidic protein (IAP), which is one of the mouse acute phase proteins, was observed in the TPO partial purified product-administered mice, TPO is related to the induction of acute phase proteins with IL-6 and IL- It was strongly suggested that the personality was different from 11.
<Example 12>
Detection of TPO mRNA in various rat tissues
In order to confirm in which tissue rat TPO mRNA was expressed in the rat body, RNA was extracted from various rat tissues. Various tissues (brain, thymus, lung, liver, heart, spleen, small intestine, kidney, testis) from 6 rats on day 11 to day 14 subjected to X-ray irradiation in the same manner as described in Example 1 And bone marrow cells) were removed and immediately frozen in liquid nitrogen. For the extraction of total RNA, an RNA isolation reagent ISOGEN (Wako Pure Chemical Industries) was used. Add ISOGEN reagent according to the weight of the frozen tissue and treat with homogenizer (Hiscotron; Nisshin Medical Sciences Corporation: NS-60) until the tissue is completely disrupted (approximately 10000 RPM for 45-60 seconds). Then, total RNA extraction was performed (extraction method based on the Acid Guandium Phenol Chlororm method of Chomczynski et al .: Anal. Biochem.,162: 156-159, 1987). As a result, 1.1 mg to 5.6 mg of total RNA was obtained from each tissue.
Total RNA to poly (A)⁺RNA purification is Oligotex^TM-DT30 (Super) (Nippon Synthetic Rubber / Nippon Roche Co., Ltd.) and 20 pg of poly (A) from about 500 μg of total RNA⁺RNA was obtained.
Poly (A) of various tissues obtained⁺The first strand of cDNA was synthesized from 1 μg of each RNA using a random primer. Poly (A)⁺1 μg of RNA was dissolved in 10 μl of sterilized water, heated at 70 ° C. for 15 minutes and then rapidly cooled, 75 pmole random primer (Takara Shuzo), 10 U RNase Inhibitor (Boehringer Mannheim), 50 mM Tris-HCl (pH 8.3), 75 mM KCl 3 mM MgCl₂, 200U SuperScript^TMEach reagent was added so that it might become II life technology reverse transcriptase (total amount 20 microliters), and it incubated at 37 degreeC for 1 hour. The reaction solution was heated at 70 ° C. for 10 minutes to deactivate the enzyme, and then stored at −20 ° C. until use.
PCR primers were newly synthesized from the rat TPO cDNA sequence obtained in Example 10. The sequence of the synthesized primer is as follows.
rTPO-I: 5'-CCTGTCCTGCTGGCCTGCTGTG-3 '(347 to 367 of SEQ ID NO: 2)
rTPO-N: 5'-TGAAGTTCGTCTCCACAAATC-3 '(antisense corresponding to 1005 to 1025 of SEQ ID NO: 2)
PCR was carried out using 0.1 μm each of the synthesized cDNA reaction solution as a template and 1 μM each of the primers (rTPO-I and rTPO-N) synthesized here.
GeneAmp^TM  PCR Reagent Kit with AmpliTaq^TMUsing DNA Polymerase (Takara Shuzo), GeneAmp^TM  PCR reaction with PCR System 9600 (manufactured by PERKIN-ELMER) in a volume of 100 μl (after heating at 95 ° C. for 2 minutes, denaturing conditions at 95 ° C. for 1 minute, annealing conditions at 57 ° C. for 1 minute, 72 ° C. for 1 minute) The reaction was performed 30 times under the synthesis conditions, and further incubated at 72 ° C. for 7 minutes. When the amplified solution was subjected to electrophoresis using 2% agarose gel (manufactured by FMC BioProducts) and the amplified bands were observed, bands considered to be specific in the brain, liver, small intestine and kidney were observed. It was done. From this result, it was considered that TPO mRNA was expressed in these tissues in the rat although the expression level could not be determined somewhat. Although it is suggested that a similar expression pattern is exhibited in humans, when combined with the results of Example 4, it was determined that the liver was suitable as a starting material for obtaining human TPO cDNA.
<Example 13>
Construction of cDNA library derived from human normal liver
Based on the results of Example 12, liver was selected as a material for cloning human TPO cDNA, and a commercially available normal human liver-derived poly (A)⁺Time Saver in the same manner as in Example 7 using 5 μg of RNA (extracted based on Acid Guanidinium Phenol Chloroform method manufactured by Clontech, Chomczynski et al.)^TM  Using cDNA Synthesis Kit (manufactured by Pharmacia) and DIRECTIONAL CLONING TOOLBOX (manufactured by Pharmacia), double-stranded cDNA having EcoRI at the 5 'end and NotI recognition site at the 3' end was synthesized. The synthesized cDNA was ligated with 1.2 μg of the expression vector pEF18S previously treated with EcoRI and NotI, and 8.4 ml of competent high E. coli. E. coli DH5 (Toyobo Co., Ltd.) was transformed. As a result, 1.2 × 10⁶Individual transformants were obtained.
<Example 14>
Acquisition (cloning) of human TPO cDNA fragment by PCR
Commercially available normal human liver-derived poly (A)⁺The first strand of cDNA was synthesized from 1 μg of RNA (Clontech) using random primers. Poly (A)⁺1 μg of RNA was dissolved in 10 μl of sterilized water, heated at 70 ° C. for 15 minutes and then rapidly cooled, 75 pmole random primer (Takara Shuzo), 10 U RNaseInhibitor (Boehringer Mannheim), 50 mM Tris-HCl (pH 8.3), 75 mM KCl 3 mM MgCl₂, 200U SuperScript^TMEach reagent was added so that it might become II (made by Life Technology) (total amount 20 microliters), and it heat-retained at 37 degreeC for 1 hour. The reaction solution was heated at 70 ° C. for 10 minutes to deactivate the enzyme, and then stored at −20 ° C. until use.
PCR primers were synthesized from the rat TPO cDNA sequence (SEQ ID NO: 2). Primer sequences are as follows.
rTPO-AIN: 5'-ATGGAGCTGAACTGATTGCTC-3 '(173-193 of SEQ ID NO: 2)
rTPO-N: 5'-TGAAGTTCGTCTCCACAAATC-3 '(antisense corresponding to 1005 to 1025 of SEQ ID NO: 2)
PCR was carried out using 0.1 volume of the synthesized cDNA reaction solution as a template and using 1 μM each of the primers (rTPO-AIN and rTPO-N) synthesized here. GeneAmp^TM  PCR Reagent Kit with AmpliTaq^TM  Using DNA Polymerase (Takara Shuzo), GeneAmp^TM  PCR reaction with PCR System 9600 (manufactured by PERKIN-ELMER) in a volume of 100 μ1 (after heating at 95 ° C. for 2 minutes, denaturing conditions at 95 ° C. for 1 minute, annealing conditions at 40 ° C. for 1 minute, and at 72 ° C. for 1 minute) The reaction was performed 35 times under the synthesis conditions, and further incubated at 72 ° C. for 7 minutes.
The reaction solution obtained here was subjected to electrophoresis using a 2% agarose gel (manufactured by FMC BioProducts) to isolate a DNA fragment of about 620 bp, which is the main product of this PCR reaction, and purified by prep-A-gene DNA purification. Purification was performed using a kit (manufactured by Bio-Rad). This purified DNA fragment was designated as Taq Dye Deoxy.^TM  Using Terminator Cycle Sequencing Kit (Applied Biosystems), the base sequence was determined by direct sequencing with an Applied Biosystems 373A DNA sequencer.
The nucleotide sequence excluding the primer portion and the amino acid sequence deduced therefrom are shown in the Sequence Listing (SEQ ID NO: 3).
This DNA fragment has a length of 580 bp excluding the primer sequence, and as a result of comparison with the rat cDNA base sequence, it was found that it has 86% homology. DNA encoding a part of human TPO cDNA It was considered a fragment.
<Example 15>
Screening of human TPO cDNA by PCR
PCR primers corresponding to human TPO were synthesized based on SEQ ID NO: 3. The sequence is as follows.
hTPO-I: 5'-TTGTGACTCCCGAGTCCTCAG-3 '(60-80 of SEQ ID NO: 3)
hTPO-J: 5'-TGACGCAGAGGGTGGACCCTC-3 '(antisense corresponding to 479-499 of SEQ ID NO: 3)
After amplifying the human cDNA library constructed in Example 13, dividing about 100,000 clones into a pool of one population, and culturing overnight in 1 ml of LB medium containing 50 μg / ml (7) Ampicillin Plasmid DNA was extracted using an automatic plasmid separator PI-100 (Kurashikibo, VER-3.0). The extracted DNA was dissolved in TE solution.
Using 5% of the extracted DNA as a template, PCR was performed using 1 μM each of the synthesized primers (hTPO-I and hTPO-J). GeneAmp^TM  PCR Reagent Kit with AmpliTaq^TM  Using DNA Polymerase (Takara Shuzo), GeneAmp^TM  PCR reaction using PCR System 9600 (manufactured by PERKIN-ELMER) in a volume of 20 μl (35 denaturation conditions at 95 ° C. for 1 minute, annealing conditions at 59 ° C. for 1 minute, and synthesis conditions at 72 ° C. for 1 minute) And further incubated for 7 minutes at 72 ° C. As a result, 3 of 90 pools were considered to be specific. One of these pools was selected, divided into pools of about 5000 clones, plasmid DNA was purified from 90 pools, and PCR was performed again under the same conditions. As a result, bands were detected in 5 pools. One pool was selected from these, and 250 clones were divided into sub-pools each comprising one pool, and plasmid DNA was extracted from 90 pools. When PCR was similarly performed on these, bands were observed in three pools.
One pool was selected from these, 30 clones were made into one pool, a sub-pool was created, plasmid DNA was purified from 90 pools, and PCR was performed. As a result, bands were observed in three pools. One of these pools was selected and plated on an LB plate containing 50 μg / ml Ampicilin. Plasmid DNA was similarly extracted from 90 colonies and confirmed by PCR. As a result, clone HL34 was finally obtained.
<Example 16>
Sequence of human TPOcDNΛ
Purification of the plasmid DNA was performed essentially as described in Molecular Cloning [Sambrook et al., Cold Spring Harbor Laboratory Press (1989)].
Clone HL34 was cultured overnight in 50 ml of LB medium containing 50 μg / ml (7) Ampicillin, and the cells obtained by centrifugation were suspended in 4 ml of TEG-lysozyme solution, and 8 ml of 0.2 N NaOH / 1% SDS. Add the solution and suspend well. Further, 6 ml of 3M potassium / 5M acetate solution is added and well suspended, and then centrifuged to obtain a supernatant. The supernatant was treated with phenol-chloroform (1: 1), added with an equal amount of isopropanol, and centrifuged to obtain a pellet. The pellet is dissolved in a TE solution, treated with RNAse and treated with phenol-chloroform (1: 1), followed by ethanol precipitation. The pellet is dissolved again in the TE solution, and NaCl and polyethylene glycol 3000 are added to 0.63M and 7.5%, respectively, and centrifuged. Finally, the pellet is dissolved in TE solution and ethanol precipitated. As a result, about 300 μg of plasmid DNA pEF18S-HL34 was obtained.
Taq Dye Deoxy for purified plasmid DNA^TM  Using a Terminator Cycle Sequencing Kit (Applied Biosystems), sequencing was performed with a 373A DNA sequencer manufactured by Applied Biosystems, and the full-length base sequence was determined. The nucleotide sequence and the amino acid sequence deduced therefrom are shown in the Sequence Listing (SEQ ID NO: 4). Primers required for the base sequence determination were primers synthesized based on the sequence of SEQ ID NO: 3 and synthetic primers designed based on the internal sequence analyzed by the sequencing reaction using these primers.
As a result, the obtained plasmid clone pEF18S-HL34 contained a cDNA fragment of 861 base pairs, and the partial amino acid sequence AP8 (SEQ ID NO: 4: amino acid numbers 1 to 12) of rat TPO analyzed in Example 2 and TP2 / TP3. A sequence having high homology with (SEQ ID NO: 4: amino acid numbers 157 to 162) was confirmed. This DNA fragment was thought to encode an open reading frame starting from the 25th base, but had no stop codon and had a poly A tail-like sequence consisting of 76 bases at the 3 'end. The 253 amino acid sequence up to just before the poly A tail-like sequence has 84% homology compared with that of the rat TPO cDNA (the amino acid sequence portion of 147 residues of SEQ ID NO: 2), and it corresponds to the human TPO corresponding to rat TPO. It was considered as a DNA fragment encoding a part of cDNA. Since there was no stop codon and a poly A tail-like sequence at the 3 'end, this clone did not reflect the complete cDNA, suggesting that it was an artificial product at the time of cDNA library preparation.
<Example 17>
Expression of human TPO cDNA in COS1 cells-activity confirmation
Transfection of the resulting plasmid clone pEF18-HL34 into COS 1 cells was performed according to Example 11. That is, 10 μg of plasmin DNA was used for transfection using DEAE-dextran method including chloroquine treatment, and the culture supernatant was collected 3 to 5 days later.
The obtained culture supernatant was sufficiently dialyzed against IMDM culture solution and then evaluated with a rat CFU-MK assay system. As a result, TPO activity was observed in a dose-dependent manner in the COS1 cell culture supernatant expressed by transfection with pEF18S-HL34 (FIG. 8), and the formation of protrusions by many megakaryocytes was observed on the 4th day of culture. It was done. On the other hand, no activity was observed in the COS1 cell culture supernatant transfected with an expression plasmid containing no insert (FIG. 8). In addition, in the M-07e assay system, M-07e cell growth promoting activity was observed only in the COS1 cell culture supernatant in which pEF18S-HL34 was transfected and expressed. From these results, it was found that pEF18S-HL34 carries a gene cDNA fragment encoding a protein having TPO activity. It was also clarified that human TPO also acts on rats (no species specificity).
<Example 18>
Expression of human TPO-deficient cDNA to confirmation of activity
The human TPO cDNA of clone HL34 obtained in Example 15 had a polyA tail-like continuous adenine sequence on its 3 ′ side, but this sequence was not observed in rat TPO cDNA and was an experimental artificial product. One possibility was suggested. Thus, cDNA was prepared by deleting this poly A tail-like continuous adenine sequence, and it was examined whether the expressed protein exhibited activity as TPO. PCR was used to create the deletion. Table 9 shows the sequences of PCR primers. A restriction enzyme recognition sequence was added to each 5 'end (EcoRI was added to hTPO5, NotI and two stop codons TAATGA were added to hTPO3).
[Table 9]

Using 1 μg of plasmid DNA of plasmid clone pEF18S-HL34 obtained in Example 16 as a template, PCR was performed using 10 μM of each of the synthesized primers (hTPO5, hTPO3). GeneAmp^TM  PCR Reagent Kit with AmpliTaq^TM  Using DNA Polymerase (Takara Shuzo), GeneAmp^TM  PCR reaction was performed 15 times with PCR System 9600 (manufactured by PERKIN-ELMER) in a volume of 100 μl (denaturation conditions at 95 ° C. for 1 minute, annealing conditions at 65 ° C. for 1 minute, and synthesis conditions at 72 ° C. for 1 minute). And further incubated at 72 ° C. for 7 minutes). The obtained band of about 800 bp was purified after digestion with restriction enzymes EcoRl and Notl, and subcloned into an expression vector pEF18S similarly treated with restriction enzymes. Five clones containing a DNA fragment of about 800 bp were selected from the obtained transformant, and a large amount of plasmid DNA was prepared (see Example 5 for the method). For each plasmid, the base sequence was determined for the entire region of about 800 bp of the amplified portion by PCR, and it was confirmed that the plasmid completely matched with the base sequence analyzed in Example 16 (1-780 in SEQ ID NO: 4). did.
This plasmid clone was named pHT1-231. Vector pHT1-231 carried on Escherichia coli DH5 was deposited on February 14, 1994 at the Institute of Biotechnology, Institute of Industrial Science and Technology under the accession number FERM BP-4564. It was also deposited at the Food Industry Development Institute of China (FIRDI) on March 22, 1995 as deposit number 940082.
Expression of the obtained plasmid in COS1 cells was carried out according to Example 11. That is, 10 μg each of plasmid DNA was used for transfection using DEAE-dextran method including chloroquine treatment, and the culture supernatant was collected after 3 to 5 days.
The obtained culture supernatant was dialyzed against IMDM culture solution and then evaluated with a rat CFU-MK assay system. As a result, TPO activity was observed in the COS1 cell culture supernatant expressed by transfection with pHT1-231 (FIG. 9), and protrusion formation by many megakaryocytes was observed on the 4th day of culture. On the other hand, no activity was observed in the COS1 cell culture supernatant transfected with an expression plasmid containing no cDNA insert (FIG. 9). In addition, in the M-07e assay system, M-07e cell proliferation promoting activity was observed only in the COS1 cell culture supernatant in which pHT1-231 was transfected and expressed. From these results, it was revealed that the cDNA fragment contained in pHT1-231 is a gene encoding a protein having TPO activity.
<Example 19>
Cloning of human TPOC terminal region by PCR
The human TPO cDNA of clone HL34 obtained in Example 15 has a poly A tail-like sequence at its 3 'end, suggesting that the 3' portion is an incomplete clone. Therefore, an attempt was made to obtain a full-length 3 'region by PCR. Four types of 5 'primers for PCR were synthesized from the base sequence determined in Example 16.
[Table 10]

As the 3'-side primer, the following primers containing 4 bases of the mix nucleotide at the 3 'end were synthesized in anticipation of amplification immediately after the poly A portion. In addition, an anchor primer containing no mix part was also synthesized.
[Table 11]

Poly (A) derived from human normal liver as in Example 14⁺The first strand of cDNA was synthesized from 1 μg of RNA (Clontech). However, oligo dT primer (TimeServer manufactured by Pharmacia) was used as a primer.^TM0.5 μg) attached to the cDNA Synthesis Kit was used. The first PCR was performed using 0.1 volume of the reaction solution for cDNA synthesis as a template. GeneAmp for PCR^TM  PCR Reagent Kit with AmpliTaq^TMDNA Polymerase (Takara Shuzo) and GeneAmp^TM  PCR System 9600 (manufactured by PERKIN-ELMER) was used. The reaction was performed in a volume of 50 μl using hTPO-H (20 μM) and hTPO3mix (10 μM) as primers (96 ° C. for 2 minutes, then 96 ° C. for 1 minute / 48 ° C. for 1 minute / 72 ° C. for 1 minute for 10 cycles). React, further 72 ° C. for 7 minutes). A second PCR was performed using 0.1 volume of the first reaction solution as a template. The reaction was performed in a volume of 50 μl using hTPO-K (20 μM) and hTPO3mix (10 μM) as primers (96 ° C. for 2 minutes, then 96 ° C. for 1 minute / 63 ° C. for 1 minute / 72 ° C. for 1 minute for 10 cycles). React, further 72 ° C. for 7 minutes). A third PCR was performed using 0.1 volume of the second reaction solution as a template. The reaction was performed in a volume of 50 μl using hTPO-N (20 μM) and hTPO3mix (10 μM) as primers (96 ° C. for 2 minutes, then 96 ° C. for 1 minute / 63 ° C. for 1 minute / 72 ° C. for 1 minute for 10 cycles). React, further 72 ° C. for 7 minutes). A fourth PCR was carried out using 0.1 volume of the third reaction solution as a template. The reaction was performed in a volume of 50 μl using hTPO-O (20 μM) and hTPO3mix (10 μM) as primers (96 ° C. for 2 minutes, then 96 ° C. for 1 minute / 63 ° C. for 1 minute / 72 ° C. for 1 minute for 10 cycles). React, further 72 ° C. for 7 minutes). The fifth PCR was carried out using 0.1 volume of the fourth reaction solution as a template. The reaction was performed in a volume of 50 μl using hTPO-O (20 μM) and hTPO3 anchor (20 μM) as primers (96 ° C. for 2 minutes followed by 10 cycles of 96 ° C. for 1 minute / 58 ° C. for 1 minute / 72 ° C. for 1 minute). React, further 72 ° C. for 7 minutes). The reaction solution obtained here was subjected to electrophoresis using a 2% agarose gel (manufactured by FMC BioProducts) to isolate a DNA fragment of about 600 bp which is the main product of this PCR reaction, and purified by prep-A-gene DNA purification. Purification was performed using a kit (manufactured by Bio-Rad). This purified DNA fragment was designated as Taq Dye Deoxy.^TM  Using Terminator Cycle Sequencing Kit (Applied Biosystems), the base sequence was determined by direct sequencing with an Applied Biosystems 373A DNA sequencer. The nucleotide sequence and the amino acid sequence deduced therefrom are shown in the Sequence Listing (SEQ ID NO: 5).
This DNA fragment had a base sequence encoding 130 amino acids starting from primer hTPO-O, and was further followed by a sequence of 180 bases or more on the 3 'side (the 577th base and beyond could not be decoded). The 30 amino acid sequences starting with glycine encoded by this DNA fragment matched the amino acid sequences of amino acid numbers 203 to 232 described in SEQ ID NO: 4. At the same time, the base sequences from the 1st to the 94th were also consistent with those of 692-785 of SEQ ID NO: 4.
From the results of the analysis of the cDNA fragment of Example 17 and the analysis of the PCR amplified fragment of this example, it is estimated that the human TPO protein consists of 353 amino acids including a signal sequence of 21 residues as shown in SEQ ID NO: 6. The
The amino acid sequence corresponding to the mature protein portion of the human TPO protein shown in SEQ ID NO: 6 is shown as SEQ ID NO: 16.
<Example 20>
Reconstruction of cDNA library derived from human normal liver
The clone HL34 obtained in Example 15 did not have a stop codon in the open reading frame, had a poly A tail-like sequence at its 3 ′ end, and was considered to be an artificial product during cDNA synthesis. Newly-derived normal human liver-derived poly (A)⁺  A cDNA library was reconstructed using 5 μg of RNA (Clontech). SuperScript for the synthesis of cDNA^TM  Lambda System forcDNA Synthesis and λ Cloning Kit and SuperScript^TMII RNase H- (both manufactured by LIFET ECHNOLOGIES) was used. Poly (A)⁺  After heat denaturation of RNA, 20 μl of reaction solution (50 mM Tris-HCl, pH 8.3 / 75 mM KCl / 3 mM MgCl) containing NotI sequence oligo dT attached to Kit as a primer.₂/ 1mM DTT / 1mM dNTP / 200USuperScript^TMII RNase H-) and incubated at 37 ° C. for 60 minutes. Synthesis of the second strand of cDNA (25 mM Tris-HCl, pH 8.3, 100 mM KCl, 5 mM MgCl₂Each 250 μM dNTPs) 5 mM DTT, 40 U E, coli DNA polymerase I, 2 U E. E. coli RNase H) I0U E. coli. After incubation at 150 ° C. for 2 hours in a 150 μl reaction solution containing E. coli DNA ligase), 10 U T4 DNA polymerase was added and the mixture was further incubated at 16 ° C. for 5 minutes. The reaction solution was heated at 65 ° C. for 10 minutes, extracted once with an equal amount of phenol-chloroform, and then S1zeSep.^TM  400spun column (Timeserver manufactured by Pharmacia)^TM  The cDNA smaller than 400 bp was removed by a low molecular weight DNA removing span column attached to the cDNA Synthesis Kit. EcoRI Adapter (Timeserver manufactured by Pharmacia) was added to this sample.^TM  After addition of the cDNA Synthesis Kit), digested with the restriction enzyme NotI^TM  It was subjected to 400 spin column to remove low molecular weight DNA. The double-stranded cDNA having EcoRI at the 5 ′ end and NotI recognition sequence at the 3 ′ end thus obtained was 1.3 μg, respectively, and ligated with the expression vector pEF18S previously treated with EcoRI and NotI. Competent High E. E. coli DH5 (Toyobo Co., Ltd.) was transformed. As a result, the obtained human liver cDNA library (hTPO-F1) was 1.0 × 10₆Individual transformants.
<Example 21>
Screening of TPO cDNA clones from human liver cDNA library hTPO-F1
PCR primers corresponding to human TPO cDNA were synthesized based on SEQ ID NOs: 3 and 6. The sequence is as follows.
hTPO-I: 5'-TTGTGACCTCCCAGGTCCTCAG-3 '(60-80 of SEQ ID NO: 3)
hTPO-KU: 5'-AGGATGGGGTTGGGGAAGGAGA-3 '(antisense corresponding to 901-921 of SEQ ID NO: 6)
Human liver cDNA library constructed in Example 20-hTPO-F1 (1.0 × 10⁶Were divided into three pools (# 1 to 3) and stored frozen. Plasmid DNA was prepared from each pool, and PCR was performed using 1 μM each of the synthesized primers (hTPO-I and hTPO-KU) using 1% as a template.
GeneAmp^TM  PCR Reagent Kit with AmpliTaq^TMUsing DNA Polymerase (Takara Shuzo), GeneAmp^TM  PCR was performed 35 times using PCR System 9600 (manufactured by PERKIN-ELMER) in a volume of 100 μ1 under denaturing conditions at 95 ° C. for 1 minute, annealing conditions at 59 ° C. for 1 minute, and synthesis conditions at 72 ° C. for 1 minute. As a result of further incubation for 7 minutes at 72 ° C., DNA of the expected size was amplified when plasmid DNA derived from the # 3 pool was used. Therefore, this # 3 pool was divided into 15000 sub-pools, and after overnight culture in 1 ml (7) LB medium containing 50 μg / ml Ampicillin, plasmid DNA was isolated using the automatic plasmid separator PI-100. Extracted. The extracted DNA was dissolved in a TE solution, and PCR was carried out using 5% as a template (primers used and reaction conditions were the same as above). As a result, DNA of the expected size was amplified in 6 out of 90 pools. Of these, one pool was selected and 1,000 clones were divided into sub-pools into one population. Plasmid DNA was prepared and PCR was performed in the same manner as described above, but no DNA amplification was observed. The intensity of the electrophoretic band of DNA amplified by a series of PCRs became thinner as the subpool was made finer. Therefore, it was considered that plasmid clones were poorly recovered because the clones to be sought did not grow well, and amplification by PCR was weakened so that amplification was finally impossible. Therefore, it was decided to return to the # 3 pool and perform screening by colony hybridization.
From the # 3 pool, clones were seeded so that there were 4100 colonies per 15 cm LB plate, and 100 plates were prepared. One replica plate was prepared for each plate, and one of them was further cultured at 37 ° C. for 6 hours, and then the colonies were recovered and plasmid DNA was extracted. When PCR was performed on these DNAs in the same manner as described above, amplification of a band having the expected size was observed in one of the 100 pools. Therefore, two replica filters were made from the pool plate (BIODYNE manufactured by PALL).^TMA TRANSFER MEMBRANE is used). The filter was denatured by 10% SDS for 10 minutes, 0.5N NaOH / 1.5M NaCl for 10 minutes, 0.5M Tris-HCl (pH 8, 0) /1.5M NaCl for 10 minutes, air-dried for 30 minutes, and then vacuum oven at 80 ° C. Baked in it for 1 hour. The baked filter was lightly washed with 6 × SSC (20XSSC is a solution containing 175.3 g NaCl, 88.2 g in 11 and pH 7.0) / 1% SDS, and then prehybridization was performed. The composition of the reaction solution is 50% formamide, 5 × SSC, 5 × Denhardt's solution (50 × Denhardt's solution is a solution containing 5 g Ficoll, 5 gpolyvinylpyrrolidone, 5 g bovine serum albuminfructFraction%, 1 g solution) / Ml containing salmon sperm DNA and kept warm in 30 ml solution with shaking at 42 ° C. for 30 minutes. After prehybridization, the mixture was replaced with 30 ml of a hybridization solution having the same composition, and [α-³²P] probe radiolabeled with dCTP (manufactured by Amersham) (EcoRI / BamHI fragment of plasmid pEF18S-HL34: purified from the 5 ′ end to the 458th base of SEQ ID NO: 4 and labeled by the random primer method; Anal. Biochem., 132, 6-13, 1983 (using Amersham kit Megaprime DNA labeling system) was used, and the mixture was incubated at 42 ° C. for 20 hours with shaking. The filter was washed in 2 × SSC / 0.1% SDS solution at 42 ° C. for 30 minutes, and further washed in 0.2 × SSC / 0.1% SDS solution at 42 ° C. for 30 minutes. The filter was subjected to autoradiography at −70 ° C. for 16 hours using an intensifying screen and X-OMATTMAR5 film (Eastman Kodak). As a result, one signal considered to be positive was observed. Therefore, the colony around the signal was scraped off from the original plate and re-wound onto a 10 cm LB plate. 50 colonies obtained were cultured to prepare DNA, and then PCR was performed using primers hTPO-I and hTPO-KU (conditions and the like are the same as above). As a result, only one clone was amplified as expected. This clone was named pHTF1.
<Example 22>
Sequence of human TPO cDNA clone pHTF1
Purification of plasmid DNA was performed essentially as described in Molecular Cloning [Sambrook et al., Cold Spring Harbor Laboratory Press (1989). Clone pHTF1 was cultured overnight in 50 ml of LB medium containing 50 μg / ml (7) Ampicillin, and then the cells obtained by centrifugation were suspended in 4 ml of TEG-lysozyme solution and 8 ml of 0.2N NaOH / 1% SDS solution. Suspend well. Further, 6 ml of 3M potassium / 5M acetate solution is added and well suspended, and then centrifuged to obtain a supernatant. The supernatant was treated with phenol-chloroform (1: 1), added with an equal amount of isopropanol, and centrifuged to obtain a pellet. The pellet is dissolved in a TE solution, treated with RNAse and treated with phenol-chloroform (1: 1), followed by ethanol precipitation. The pellet is again dissolved in the TE solution, and NaCl and polyethylene glycol 3000 are added to 0.63M and 7.5%, respectively, and centrifuged. Finally, the pellet is dissolved in TE solution and ethanol precipitated. As a result, about 300 μg of plasmid DNApHTF1 was obtained.
Taq Dye Deoxy for purified plasmid DNA_TM  Using a Terminator Cycle Sequencing Kit (Applied Biosystems), sequencing was performed with an Applied Biosystems 373 Λ DNA sequencer to determine the full-length base sequence. The nucleotide sequence and the amino acid sequence deduced therefrom are shown in the Sequence Listing (SEQ ID NO: 7). Primers necessary for base sequence determination were primers synthesized based on the sequence of SEQ ID NO: 6 and synthetic primers designed based on the internal sequence analyzed by the sequencing reaction using these primers.
As a result, the obtained clone pHTF1 was composed of a cDNA fragment of 1721 base pairs, and the amino acid sequence AP8 (SEQ ID NO: 7: amino acid numbers 1 to 12) and TP2 / TP3 (SEQ ID NO: 7: amino acid number) analyzed in Example 2 were analyzed. 157 to 162), a sequence having high homology was confirmed. This DNA fragment is an open reading consisting of 353 amino acids starting from methionine encoded at bases 102 to 104 and glycine encoded at bases 1158 to 1160, following the 5 ′ non-coding region up to base 101. The 3 ′ non-coding region after the stop codon (TAA) followed by 531 bases and 30 poly A tail sequences was thought to encode the frame. The amino acid sequence of the protein considered to be encoded by this open reading frame completely matched the predicted amino acid sequence of human TPO described in SEQ ID NO: 6. The base sequence encoded cDNA longer than the sequence of SEQ ID NO: 6 by 77 bases on the 5 'side and by 347 bases in the region immediately before the poly A tail sequence on the 3' side. Further, the nucleotide sequence was different from SEQ ID NO: 6 at three places. As for the different base sequences, A (84th base) of SEQ ID NO: 7 was C in SEQ ID NO: 6, A (740th base) was T, and G (1198th base) was A. Only the second base (base 740) was a mutation in the coding region of the protein, but it was the third codon of threonine, and no amino acid conversion occurred. At this point, it was not clear what caused the substitution of this nucleotide sequence, but from the analysis of the obtained clone pHTF1, the human TPO protein may consist of 353 amino acids including a signal sequence of 21 residues. It could be confirmed. The estimated molecular weight of the protein excluding the signal sequence was 35466.
The vector pHTF1 carried in Escherichia coli DH5 was deposited with the accession number FERM BP-4617 at the Institute of Biotechnology, Ministry of International Trade and Industry on March 24, 1994. It was also deposited at the Food Industry Development Institute of China (FIRDI) on March 22, 1995 as deposit number 940085.
<Example 23>
Expression of human TPO cDNA clone pHTF1 in COS1 cells-confirmation of activity
Transfection of the resulting clone pHTF1 into COS1 cells was performed according to Example 11. That is, 10 μg of plasmid DNA was used for transfection using DEAE-dextran method including chloroquine treatment, and the culture supernatant was recovered after 3 days.
The obtained culture supernatant was dialyzed against IMDM culture solution and then evaluated with a rat CFU-MK assay system. As a result, the COS1 cell culture supernatant expressing pHTF1 showed TPO activity in a dose-dependent manner, but no activity was observed in the COS1 culture supernatant transfected with an expression plasmid containing no cDNA insert (FIG. 10a). Similar results were obtained in the M-07e assay system, and significant M-07e cell proliferation promoting activity was observed in the COS1 cell culture supernatant expressing pHTF1 (FIG. 10b).
From these results, it was clarified that cDNA contained in pHTF1 is a gene encoding a protein having TPO activity.
<Example 24>
Cloning of human TPO chromosomal DNA
Human TPO chromosomal DNA was cloned using human TPO cDNA as a probe. The genomic library used for the cloning was obtained from Professor Tokuo Yamamoto of Tohoku University Gene Laboratory (Sakai et al., J. Biol. Chem., 269, 2173-2182, 1994. A library constructed by linking partly decomposed with Sau3AI to the BamHI site of the phage vector Lambda EMBL3 (Statagene). Screening was performed from this library using human TPO cDNA as a probe, but all the techniques used were performed essentially as described in Molecular Cloning [Sambrook et al., Cold Spring Harbor Press (1989)]. Escherichia coli LE392 was used as a host strain, and a 15 cm NZYM plate (10 g NZamine, 5 g NaCl, 5 g bacto yeast extract, 2 g MgCl in 1 liter)₄  7H₂O, pH 7.0 containing 15% agar) Phage was sowed that 30,000 per one plate, and 18 plates were prepared. Two replica filters were prepared from each plate (BIODYNE manufactured by PALL).^TM  A TRANSFERMEMBRANE is used). The filter was denatured by 0.5N NaOH / 1.5M NaCl for 10 minutes, 0.5M Tris-HCl (pH 8.0) /1.5M NaCl for 10 minutes, air-dried for 30 minutes and baked in a vacuum oven at 80 ° C. for 1.5 hours. did. Prehybridization was performed in a 500 ml solution containing 50% formamide, 5 × SSC, 5 × Denhardt's solution, 1% SDS, 20 μg / ml salmon sperm DNA at 42 ° C. for 1 hour. The probe is a human TPO cDNA fragment (base sequence number 178-1025 of SEQ ID NO: 7) amplified by PCR and purified, and is described in Random Primer DNA labeling kit (manufactured by Takara Shuzo; Anal. Biochem., 132, 6-13, 1983) DNA labeling kit using random primer method)₃₂P-labeled one was used. Primer sequences used for PCR are as follows.
hTPO-I: 5'-TTGTGACTCCCGAGTCCTCAG-3 '(SEQ ID NO: 7 178-198)
hTPO-N: 5'-AGGGAAGAGCGTATACTGTCC-3 '(antisense corresponding to 1005-1025 of SEQ ID NO: 7)
Hybridization was performed at 42 ° C. for 20 hours using 500 ml of a solution having the same composition as that of prehybridization, adding a radiolabeled probe. The filter was washed 3 times for 5 minutes in 2 × SSC / 0.1% SDS solution at room temperature, and then washed at 68 ° C. for 1 hour in 0.1 × SSC / 0.1% SDS solution. And X-OMAT^TM  Autoradiography was performed at -70 ° C. for 16 hours using an AR5 film (Eastman Kodak). As a result, 13 positive signals were obtained. The plaques around 13 positive signals were picked up from the original plate and re-rolled to 1000 plaques per 15 cm NZYM plate. Two replica filters were prepared from each plate, and hybridization was performed under the same conditions as described above. As a result, positive signals were detected in all 13 filters. One plaque was isolated from each pout, and phage DNA was prepared by the plate lysate method described in Molecular Cloning. It was checked by PCR whether 13 clones of phage DNA contained the coding region of cDNA. The primer sequences used for the check are as follows.
hTPO-L: 5'-GGCCAGCCAGACACCCCGGCC-3 '(1-21 of SEQ ID NO: 6)
hTPO-F: 5'-ATGGGAGTCACGAGAGAGTTTT-3 '(antisense corresponding to 127-147 of SEQ ID NO: 6)
hTPO-P: 5'-TGCGTTTCCTGATGCTTGTAG-3 '(503-523 of SEQ ID NO: 6)
hTPO-V: 5'-AACCCTACCCTTCCTGAGACA-3 '(antisense corresponding to 1070-1090 of SEQ ID NO: 6)
When PCR was carried out with a combination of these primers, it was considered that 5 out of 13 clones contained all the coding regions of amino acids predicted from cDNA. The lengths of the chromosomal DNA contained in these 5 clones were about 20 kbp, and the restriction enzyme analysis examined in advance showed almost the same pattern. Therefore, one of these clones (clone λHGT1) was selected, and Southern blot analysis was performed. That is, 1 μg of each DNA of clone λHGT1 was completely digested with restriction enzymes EcoRI or HindIII, electrophoresed on a 0.8% agarose gel, and then BIODYNE manufactured by PALL.^TM  Transcribed to A TRANSFER MEMBRANE. The filter was air-dried for 30 minutes and then baked in a vacuum oven at 80 ° C. for 2 hours. Prehybridization was performed at 42 ° C. for 1 hour in a 50 ml solution containing 50% honremamide, 5 × SSC, 5 × Denhardt 's solution, 1% SDS, 20 μg / ml salmon sperm DNA. The probe is a human TPO cDNA fragment (base sequence number 178-1025 of SEQ ID NO: 7) amplified by PCR and purified using a Random Primer DNA labeling kit (Takara Shuzo).³²P-labeled one was used. Hybridization was performed at 42 ° C. for 20 hours using 50 ml of a solution having the same composition as that of prehybridization, adding a radiolabeled probe. The filter was washed 3 times for 5 minutes in 2 × SSC / 0.1% SDS solution at room temperature, and then washed at 68 ° C. for 1 hour in 0.1 × SSC / 0.1% SDS solution. And X-OMATTMAR5 film (Eastman Kodak Co., Ltd.) was used for autoradiography at -70 ° C. for 16 hours. As a result, a single band of about 10 kbp was observed when digested with the restriction enzyme HindIII. Therefore, 10 μg of clone λHGTI DNA was digested with restriction enzyme HindIII and then electrophoresed on a 0.8% agarose gel, a 10 kbp band was excised, purified with a prep-A-gene DNA purification kit (manufactured by Bio-Rad), and similarly. It was subcloned into a cloning vector-pUC13 (manufactured by Pharmacia) digested with HindIII (competent high E. coli DH5 manufactured by Toyobo Co., Ltd. was used as the host strain).
Among the obtained clones, a clone containing a 10 kbp HindIII fragment was selected and named pHGT1.
FIG. 11 shows a rough restriction enzyme map of the phage clone λHGT1.
<Example 25>
Sequence of human TPO chromosome clone pHGT1
The pHGT1 clone was cultured and plasmid DNA was purified. The method was performed essentially as described in Molecular Cloning [Sambrook et al., Cold Spring Harbor Laboratory Press (1989)]. Clone pHGT1 was cultured overnight in 50 ml of LB medium containing 50 μg / ml Ampicillin, and then the cells obtained by centrifugation were suspended in 4 ml of TEG-lysozyme solution, and 8 ml of 0.2N NaOH / 1% SDS solution was suspended. In addition, suspend well. Further, 6 ml of 3M potassium / 5M acetate solution is added and well suspended, and then centrifuged to obtain a supernatant. The supernatant was treated with phenol-chloroform (1: 1), added with an equal amount of isopropanol, and centrifuged to obtain a pellet. The pellet is dissolved in a TE solution, and then subjected to RNase treatment and phenol-chloroform (1: 1) treatment, followed by ethanol precipitation. The pellet is again dissolved in the TE solution, and NaCl and polyethylene glycol 3000 are added to 0.63M and 7.5%, respectively, and centrifuged. Finally, the pellet is dissolved in TE solution and ethanol precipitated. As a result, about 300 μg of plasmid DNApHGT1 was obtained.
Taq Dye Deoxy for purified plasmid DNA^TM  Using Terminator Cycle Sequencing Kit (Applied Biosystems), sequencing was performed with an Applied Biosystems 373A DNA sequencer, and the base sequence around the protein coding region predicted from the base sequence of cDNA was determined. The nucleotide sequence and the amino acid sequence deduced therefrom are shown in the Sequence Listing (SEQ ID NO: 8).
The primers required for the base sequence determination were those used in Example 22, used for cDNA base sequence analysis, and synthetic primers designed based on the internal sequence analyzed by the sequencing reaction using these primers.
As a result, the chromosomal DNA carried by the plasmid clone pHGT1 contained all of the amino acid coding region predicted by SEQ ID NO: 6, and the nucleotide sequence was completely identical for that region. The region corresponding to the exon encoding the amino acid was divided by four introns, and the lengths of the introns were 231 bp, 286 bp, 1932 bp, and 236 bp in order from the 5 'side. It was different from the cDNA base sequence described in SEQ ID NO: 7 at three places. The different base sequences are the same as those described in Example 22 (difference between SEQ ID NOs: 6 and 7), A (84th base) of SEQ ID NO: 7 is C, A (740th base) is T, G ( 1198 base) was A. That is, it was revealed that the base sequence of the human TPO cDNA clone pHTF1 obtained in Example 21 is different from the base sequence of the human chromosome at three places. Therefore, in order to analyze whether the mutation of the nucleotide sequence reflects the original sequence, among the five chromosomal DNA clones finally selected in the screening, the remaining four of which the nucleotide sequence has not been determined. The clones were sequenced. The analysis of the sequence was performed by a direct base sequencing method using phage DNA prepared by the plate lysate method described in Molecular Cloning. In the reaction, the sequence primer synthesized in Example 22 at a position where the above three base sequence mutation points can be analyzed, and Taq Dye Deoxy^TM  Using a Terminator Cycle Sequencing Kit (Applied Biosystems), sequencing was performed with an Applied Biosystems 373A DNA sequencer. As a result, in all four clones, the 84th base of SEQ ID NO: 7 was C and the 740th base was T, which was different from the cDNA of SEQ ID NO: 7 and matched that of SEQ ID NO: 6. For the 1198th base, there were 2 G clones and 2 A clones. That is, it was revealed that there are two types of chromosomal DNA original base sequences. Whether this sequence difference is derived from a homologous chromosome or multiple genes is unknown at present. In addition, purchased Clontech poly (A)₊Since RNA is derived from Caucasian, the origin of chromosomal DNA is Japanese, which suggests that the variation in the base sequence may be due to the difference in race.
The vector pHGT1 carried in Escherichia coli DH5 was deposited with the accession number FERM BP-4616 at the Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry on March 24, 1994. It was also deposited at the Food Industry Development Institute (FIRDI) on March 22, 1995, with the deposit number 940084.
<Example 26>
Expression and activity confirmation of human TPO chromosomal DNA in COS1 cells
In the plasmid clone pHGT1 obtained by subcloning, there are 3 places in the insert part and 1 place in the vector, a total of 4 places, but the 5'most EcoRI recognition sequence in the insert and the vector From the analysis of the nucleotide sequence, it was revealed that the coding region of human TPO protein was contained in the approximately 4.3 kbp DNA fragment sandwiched between the EcoRI recognition sequences. Therefore, this fragment was ligated to an EcoRI-treated expression vector pEF18S to obtain four human TPO expression plasmids pEFHGTE # 1-4 (FIG. 11 described above). These plasmid DNAs were prepared and expression experiments were performed. Purification of the plasmid DNA was performed essentially as described in Molecular Cloning [Sambrook et al., Cold Spring Harbor Laboratory Press (1989)], yielding approximately 250 μg of plasmid DNA.
Transfection of the resulting clone pEFHGTE # 1-4 into COS1 cells was performed according to Example 11. That is, 10 μg of plasmid DNA was used for transfection using DEAE-dextran method including chloroquine treatment, and the culture supernatant was recovered after 3 days.
The obtained culture supernatant was dialyzed against IMDM culture solution and then evaluated with a rat CFU-MK assay system. As a result, TPO activity was observed in a dose-dependent manner in the COS1 cell culture supernatant in which pEFHGTE was expressed, whereas no activity was observed in the COS1 cell culture supernatant transfected with an expression plasmid containing no DNA insert. . Although almost the same result was obtained in any of clones # 1 to 4, the result in pEFHGTE # 1 is shown in FIG. 12a as a representative example. Also in the M-07e assay system, significant C-07e cell proliferation promoting activity was observed in a dose-dependent manner in the COS1 cell culture supernatant in which pEFHGTE was expressed. As a representative example, the result in pEFHGTE # 1 is shown in FIG. 12b.
From these results, it was revealed that the DNA carried by the obtained clone pEFHGTE is a functional chromosomal DNA of human TPO.
<Example 27>
Preparation of human TPO deletion derivative and its expression in COS1 cells-confirmation of activity
From the results of Example 18, it was clarified that human TPO can express the activity even if it is not full length. For the purpose of analyzing the region necessary for the expression of the activity, preparation and expression of the deletion derivative were performed. I did it. Based on the plasmid clone pHT1-231 obtained in Example 18, an expression plasmid in which 20 amino acids were deleted sequentially from the C-terminal side and an expression plasmid containing amino acids immediately after TP2 / 3 (163rd) were prepared. did. PCR was used for the production. Primer sequences prepared for PCR are as follows.
hTPO-5: 5'-TTTTATTGGGGCCAGCCAGACACCCCGGCC-3 '
(SEQ ID NO: 4 1-21 with EcoRI sequence added; identical to that described in Table 9)
hTPO-S: 5'-TTTGCGGCCCCGCTCATTAGCTGGGGACAGCTGTGGGTGGGT-3 '(antisense of 555-576 of SEQ ID NO: 4; addition of two stop codons TAATGA and NotI sequences; for preparation of a deletion derivative encoding amino acids 1-163)
hTPO-4: 5'-TTTGCGGCCCCTCATTAGACAGTGTGAGGACTAGAGAGGTTCTG-3 '(antisense of 576-600 of SEQ ID NO: 4; two stop codons TAATGA and NotI sequence added; for preparation of deletion derivative encoding amino acids 1-171)
hTPO-30: 5'-TTTGCGGCCCCGCTCATTATCTGGCTGAGGGCAGTGAAGTTTGTC-3 '(antisense of 636-660 of SEQ ID NO: 4; addition of two stop codons TAATGA and NotI sequences; for preparation of deletion derivatives encoding amino acids 1-191)
hTPO-2: 5'-TTTGCGGCCGCTCATTACAGACCAGGAATCTTGGCCTCTGAAT-3 '(antisense of 696-720 of SEQ ID NO: 4; addition of two stop codons TAATGA and NotI sequences; preparation of deletion derivative encoding amino acids 1-211)
Using 1 μg of plasmid DNA of clone pHT1-231 obtained in Example 18 as a template, synthesized primers (using hTPO-5 as the 5 ′ side, hTPO-2, −3, −4, and −S as the 3 ′ side) PCR was performed using 10 μM each. GeneAmp^TMPCR Reagent Kit with AmpliTaq^TM  Using DNA Polymerase (Takara Shuzo), GeneAmp^TMPCR was performed 20 times using PCR System 9600 (manufactured by PERKIN-ELMER) in a volume of 100 μl under denaturing conditions at 95 ° C. for 1 minute, annealing conditions at 66 ° C. for 1 minute, and synthesis conditions at 72 ° C. for 1 minute. And further incubated at 72 ° C. for 7 minutes). After digesting the bands obtained by each PCR with restriction enzymes EcoRI and NotI, they were subjected to electrophoresis using 1% agarose gel (manufactured by FMC BioProducts), and the main DNA fragments of the expected size of each PCR reaction Was purified with a prep-A-gene DNA purification kit (Bio-Rad) and subcloned into the expression vector pEF18S treated with the same restriction enzyme (competent high E. coli manufactured by Toyobo Co., Ltd. as the host fungus). DH5 is used). Among the obtained transformants, 4 to 5 clones each containing an insert of the expected length were selected to prepare plasmid DNA. The method was carried out essentially as described in Molecular Cloning [Sambrook et al., Cold Spring Harbor Laboratory Press (described in September 1989. A series of operations deletes a derivative (pHT1-163 # 1- 5), a deletion derivative encoding amino acids 1-171 (pHT1-171 # 1-4), a deletion derivative encoding amino acids 1-191 (pHT1-191 # 1-4), and amino acids 1-211 Plasmid DNA of the deletion derivative (pHT1-211 # 1-4) was obtained.
For purified plasmid DNA, Taq Dye Deoxy^TM  Using a Terminator Cycle Sequencing Kit (Applied Biosystems), it was sequenced with a 373A DNA sequencer manufactured by Applied Biosystems, and it was confirmed that there was no substitution of the base sequence over the entire length and it had the expected TPOcDNΛ sequence.
Transfection of each obtained clone into COS1 cells was performed according to Example 11. That is, 10 μg of each plasmid DNA was used for transfection using DEAE-dextran method including chloroquine treatment, and the culture supernatant was recovered after 3 days. The culture supernatant was dialyzed against IMDM culture medium and then evaluated with a rat CFU-MK assay system. As a result, TPO activity was observed in a dose-dependent manner in any COS1 cell culture supernatant in which pHT1-211, pHT1-191, pHT1-171, and pHT1-163 were expressed. As a representative example, the results at pHT1-211 # 1, pHT1-191 # 1, and pHT1-171 # 2 are shown in FIG. 13a, and the results at pHT1-163 # 2 are shown in FIG. 13b. Similar results were obtained in the M-07e assay system and showed a significant dose-dependent MOS in any COS1 cell culture supernatant expressing pHT1-211, pHT1-191, pHT1-171, and pHT1-163. -07e Cell growth promoting activity was observed. As representative examples, the results for pHT1-211 # 1, pHT1-191 # 1, pHT1-171 # 2, and pHT1-163 # 2 are shown in FIG.
From these results, in vitro activity is maintained even when the amino acid after arginine at position 164 is deleted from 353 amino acids (including 21 signal sequences) constituting human TPO protein. As a result, it was suggested that an essential region for TPO activity expression was included before 164th arginine.
<Example 28>
Preparation of human TPO C-terminal deletion and expression in COS1 cells-activity confirmation
Whether or not a derivative in which the amino acid at the C-terminal side is further deleted from the deletion product obtained in Example 27 can be expressed for the purpose of analyzing the region essential for the expression of the activity of human TPO protein, and whether TPO activity can be expressed. investigated. Based on the plasmid clone pEF18S-HL34 obtained in Example 16, an expression plasmid was constructed in which the amino acids were deleted sequentially from the C-terminal side based on the 163rd serine. PCR was used for the construction. Primer sequences prepared for PCR are as follows.
hTPO-5: 5'-TTTTATTGGGGCCAGCCAGACACCCCGGCC-3 '
(SEQ ID NO: 4 1-21 with EcoRI sequence added; identical to that described in Table 9)
hTPO-150: 5'-TTTGCGGCCGCTCATTAGAGGGTGGGACCCTCCTACAAGCAT-3 '
(Antisense of 514-537 of SEQ ID NO: 4; two stop codons TAATGA and NotI sequences added: for preparation of deletions encoding amino acids 1-150)
hTPO-151: 5'-TTTGCGGCCGCTCATTAGAGAGAGGGTGACCACCTCTACAA-3 '
(Antisense of 518-540 of SEQ ID NO: 4; addition of two stop codons TAATGA and NotI sequences; for preparation of deletions encoding amino acids 1-151)
hTPO-153: 5'-TTTGCGGCCGCTCATTACCTGACGCAGAGGGGTGACCC-3 '
(Antisense of 526-546 of SEQ ID NO: 4; two stop codons TAATGA and NotI sequences added; for preparation of deletions encoding amino acids 1-153)
hTPO-154: 5'-TTTGCGGCCGCTCATTACCCGCCTGACGCAGAGGGGTGGA-3 '
(Antisense of 529 to 549 of SEQ ID NO: 4; two stop codons TAATGA and NotI sequences added; for preparation of a deletion encoding amino acids 1-154)
hTPO-155: 5'-TTTGCGGCCGCTCATTAGGCCCGCCCGACGCAGAGGGT-3 '
(Antisense of 532-552 of SEQ ID NO: 4; two stop codons TAATGA and NotI sequences added: for preparation of deletions encoding amino acids 1-155)
hTPO-156: 5'-TTTGCGGGCCGCCTCATTATGGGGCCCGCCTGACGCAGAG-3 '
(Antisense of 535-555 of SEQ ID NO: 4; addition of two stop codons TAATGA and NotI sequences: for preparation of deletions encoding amino acids 1-156)
hTPO-157: 5'-TTTGCGGCCGCTCATTAGGGTGGGGCCCGCCTGACGCA-3 '
(Antisense of 538-558 of SEQ ID NO: 4; addition of two stop codons TAATGA and NotI sequences; for preparation of deletions encoding amino acids 1-157).
Using 1 μg of plasmid DNA of clone pEF18S-HL34 obtained in Example 16 as a template, synthesized primers (using hTPO-5 as the 5 ′ side, hTPO-150, −151, −153, −154, −155, − PCR was performed using 10 μM each of 156 and −157 as the 3 ′ side). GeneAmp^TM  PCR Reagent Kit with AmpliTaq^TMUsing DNA Polymerase (Takara Shuzo), GeneAmp^TMPCR reaction with PCR System 9600 (manufactured by PERKIN-ELMER) in a volume of 100 μl (20 reactions under denaturing conditions at 95 ° C. for 1 minute, annealing conditions at 66 ° C. for 1 minute, and synthesis conditions at 72 ° C. for 1 minute) And incubate at 72 ° C. for an additional 7 minutes). After digesting the bands obtained by each PCR with restriction enzymes EcoRI and NotI, they were subjected to electrophoresis using 1% agarose gel (manufactured by FMC BioProducts), and the main DNA fragments of the expected size of each PCR reaction Was purified with a prep-A-gene DNA purification kit (Bio-Rad) and subcloned into a restriction enzyme-treated expression vector pEF18S (competent high E. coli manufactured by Toyobo Co., Ltd. as a host fungus). DH5 is used). Among the obtained transformants, 3 to 5 clones each containing an insert of the expected length were selected to prepare plasmid DNA. The method was performed essentially as described in Molecular Cloning [Sambrook et al., Cold Spring Harbor Laboratory Press (1989)]. Through a series of operations, in SEQ ID NO: 4, a deletion encoding amino acid number 1-150 (pHT1-150 # 21, 22, 25), a deletion encoding amino acid number 1-151 (pHT1-151 # 16, 17, 18), a deletion coding for amino acid number 1-153 (pHT1-153 # 1-5), a deletion coding for amino acid number 1-154 (pHT1-154 # 1-5), amino acid number 1 -155 encoding a deletion (pHT1-155 # 1-5), a deletion encoding amino acid number 1-156 (pHT1-156 # 1-5), a deletion encoding amino acid number 1-157 Plasmid DNA of (pHT1-157 # 1-5) was obtained.
For purified plasmid DNAs pHT1-150 # 21, 22, 25 and pHT1-151 # 16, 17, 18 the Taq Dye Deoxy^TM  Using a Terminator Cycle Sequencing Kit (Applied Biosystems), it was sequenced with a 373A DNA sequencer manufactured by Applied Biosystems, and it was confirmed that there was no substitution of the base sequence over the entire length and it had the expected TP0 cDNA sequence.
Transfection of each obtained clone into COS1 cells was performed according to Example 11. That is, 10 μg of each plasmid DNA was used for transfection using DEAE-dextran method including chloroquine treatment, and the culture supernatant was recovered after 3 days. The culture supernatant was sufficiently dialyzed against IMDM culture solution and then evaluated by the M-07e assay system. As a result, the C-terminal deficit comprising amino acids 1-151, 1-153, 1-154, 1-155, 1-156, 1-157, including cysteine, which is the 151st amino acid, was obtained. TPO activity was detected in a dose-dependent manner in the COS1 cell culture supernatant transfected with a clone encoding the lost derivative. However, TPO activity was not detected in the culture supernatant of COS1 cells transfected with a clone encoding a C-terminal deletion derivative at amino acid positions 1-150 from which amino acids up to the 151st cysteine were deleted.
<Example 29>
Preparation of human TPO N-terminal deletion and expression in COS1 cells-activity confirmation
For the purpose of analyzing the region essential for the expression of the activity of human TPO protein, a derivative in which the N-terminal amino acid was deleted was produced based on the deletion obtained in Example 28, and the TPO activity was expressed. We examined whether it was possible. Based on the plasmid clone pEF18S-HL34 obtained in Example 18 and the plasmid clone pHT1-163 obtained in Example 27, an expression plasmid in which the N-terminal amino acid following the signal sequence was deleted was constructed. PCR was used for the construction. Primer sequences prepared for PCR are as follows.
hTPO-5: 5'-TTTTATTGGGGCCAGCCAGACACCCCGGCC-3 '
(SEQ ID NO: 4 1-21 with EcoRI sequence added; identical to that described in Table 9)
hTPO3: 5'-TTTGCGGCCCCTCATTTATTCGGTTATCCTGTTCAGGTATCC-3 '
(Antisense of 757-780 of SEQ ID NO: 4; two stop codons TAATGA and NotI sequences added; identical to that synthesized to generate a deletion encoding amino acids 1-231)
hTPO-S: 5'-TTTGCGGCCGCTCATTAGCTGGGGACAGCTGTGGTGGGGT-3 '
(Antisense of 555-576 of SEQ ID NO: 4; two stop codons TAATGA and NotI sequences added; for preparation of deletions encoding amino acids 1-163)
hTPO-13: 5'-AGTAAACTGCCTTCGTGAACTCCCATGTCCCTCACAGCAGACTGAGCCATG-3 '
(For preparation of a derivative lacking amino acid numbers 1-12, 124-173 of SEQ ID NO: 4)
hTPO-13R: 5'-CATGGGGAGTCACGAAGCAGTTTACTGGACAGCGGTTAGCTCTGCAGTTAG-3 '
(Antisenses of SEQ ID NO: 4 64-87 and 124-148; for preparation of derivatives lacking amino acid numbers 1-12)
hTPO-7: 5'-TGTGACCCTCCGAGTCCCTCAGATAAACTGCCTTCGTGACCTCCATGTCCCTTC-3 '
(For preparation of a derivative lacking amino acid numbers 1-6, SEQ ID NO: 4 106-154) hTPO-7R: 5'-TTTACTGGAGGACTCGGAGGTCACAGGACAGCGTTAGCCCTTGCAGTTAG-3 '
(Antisense of SEQ ID NO: 4 64-87 and 106-129: for preparation of derivatives that lose amino acid numbers 1-6)
hTPO-8: 5'-GACCTCCCAGGTCCCTCAGATAAACTGCCTTCGTGACCTCCATGTCCTCCACA-3 '
(For preparation of a derivative lacking amino acid numbers 1-7, SEQ ID NO: 4 109-157) hTPO-8R: 5'-CAGTTTACTGAGGACTCGGAGGTCGACACGCGTAGTAGCTTGCAGTTAG-3 '
(Antisense of 64-87 of SEQ ID NO: 4 and 109-132: for preparation of a derivative lacking amino acid numbers 1-7).
(1) Preparation of a derivative lacking amino acid numbers 1-12 (pHT13-231)
5 μM each of the synthesized primers (using hTPO-13 and hTPO3 in pairs: hTPO-5 and hTPO-13R in pairs) using 1.4 μg of plasmid DNA of clone pEF18S-HL34 obtained in Example 18 as a template PCR was performed. GeneAmp^TM  PCR Reagent Kit with AmpliTaq^TMUsing DNA Polymerase (Takara Shuzo), GeneAmp^TM  PCR reaction with PCR System 9600 (manufactured by PERKIN-ELMER) in a volume of 100 μl (denatured at 95 ° C. for 5 minutes, then denatured at 95 ° C. for 1 minute, annealed at 65 ° C. for 1 minute, and at 72 ° C. for 1 minute) The reaction was performed 30 times under the synthesis conditions, and further incubated at 72 ° C. for 7 minutes. Each PCR product was subjected to electrophoresis using a 1.2% agarose gel (manufactured by FMC BioProducts) to separate major DNA fragments of each expected size, and a prep-A-gene DNA purification kit (BioRad). And dissolved in 15 μl of TE buffer. A second PCR was performed using 1 μl of each solution as a template.
PCR was performed using 5 μM each of the synthesized primers (using hTPO-5 and hTPO3). GeneAmp^TM  PCR Reagent Kit with AmpliTaq^TMUsing DNA Polymerase (Takara Shuzo), GeneAmp^TMPCR reaction using PCR System 9600 (manufactured by PERKIN-ELMER) in a volume of 100 μl (denaturation at 95 ° C. for 5 minutes, denaturation conditions at 95 ° C. for 1 minute, annealing conditions at 60 ° C. for 1 minute, and 72 ° C. for 1 minute) The reaction was performed 30 times under the synthesis conditions, and further incubated at 72 ° C. for 7 minutes. The PCR product was subjected to electrophoresis using 1.2% agarose gel (manufactured by FMC BioProducts) to isolate a major DNA fragment of the expected size, and a prep-A-gene DNA purification kit (manufactured by BioRad). And dissolved in 15 μl of TE buffer. After digestion with restriction enzymes EcoRI and NotI, extraction was performed once with an equal amount of phenol / chloroform, followed by ethanol precipitation. The precipitate obtained by centrifugation was dissolved in 15 μl of TE buffer and then subcloned into the expression vector pEF18S similarly treated with restriction enzyme (competent high E. coli DH5 manufactured by Toyobo Co., Ltd. was used as the host fungus). Among the obtained transformants, 45 clones containing inserts of the expected length were selected to prepare plasmid DNA. The method was performed essentially as described in Molecular Cloning [Sambrook et al., Cold Spring Harbor Laboratory Press (1989)]. In SEQ ID NO: 4, a plasmid DNA of a deletion derivative (pHT13-231) in which 1-12 was deleted from the protein encoding amino acid numbers 1-231 was obtained. These were subjected to PCR using hTPO-5 and hTPO3 primers, and an insert of the expected size was confirmed in 8 out of 45 clones. Of those, 3 clones are Taq Dye Deoxy^TM  Using Terminator Cycle Sequencing Kit (Applied Biosystems), sequenced with 373A DNA sequencer manufactured by Applied Biosystems, one clone had the expected deletion, and the entire base sequence including other parts The clone pHT13-231 # 3 having the TP0 cDNA sequence as designed was obtained.
(2) Preparation of a derivative lacking amino acid numbers 1-6 (pHT7-163)
In the preparation of the deletion derivative of (1) above, the C-terminal of the TPO protein was designed as the 231st amino acid, but it was confirmed that TPO activity can be expressed even if the C-terminal is further deleted. In the preparation of the deletion product, the C-terminal was designed up to the 163rd amino acid. In the following (3), the 163rd amino acid was similarly designed as the C-terminal. Plasmid DNA of the clone pHT1-163 obtained in Example 27 PCR was carried out using 4 μg as a template and 5 μM each of the synthesized primers (hTPO-7 and hTPO-S used in pairs: hTPO-5 and hTPO-7R used in pairs). GeneAmp^TM  PCRReagent KithhAmpliaq^TM  Using DNA Polymerase (Takara Shuzo), GeneAmp^TMPCR reaction with PCR System 9600 (manufactured by PERKIN-ELMER) in a volume of 100 μl (denatured at 95 ° C. for 5 minutes, then denatured at 95 ° C. for 1 minute, annealed at 65 ° C. for 1 minute, and at 72 ° C. for 1 minute) The reaction was performed 30 times under the synthesis conditions, and further incubated at 72 ° C. for 7 minutes. Each PCR product was subjected to electrophoresis using a 1.2% agarose gel (manufactured by FMC BioProducts) to separate major DNA fragments of each expected size, and a prep-A-gene DNA purification kit (BioRad). And dissolved in 15 μl of TE buffer. A second PCR was performed using 1 μl of each solution as a template.
PCR was performed using 5 μM each of the synthesized primers (using hTPO-5 and hTPO-S). GeneAmp^TM  PCR Reagent Kit with AmpliTaq^TMUsing DNA Polymerase (Takara Shuzo), GeneAmp^TM  PCR reaction using PCR System 9600 (manufactured by PERKIN-ELMER) in a volume of 100 μl (denaturation at 95 ° C. for 5 minutes, denaturation conditions at 95 ° C. for 1 minute, annealing conditions at 60 ° C. for 1 minute, and 72 ° C. for 1 minute) The reaction was performed 30 times under the synthesis conditions, and further incubated at 72 ° C. for 7 minutes. The PCR product was subjected to electrophoresis using 1.2% agarose gel (manufactured by FMC BioProducts) to isolate a major DNA fragment of the expected size, and a prep-A-gene DNA purification kit (manufactured by BioRad). And dissolved in 15 μl of TE buffer. After digestion with restriction enzymes EcoRI and NotI, extraction was performed once with an equal amount of phenol / chloroform, followed by ethanol precipitation. The precipitate obtained by centrifugation was dissolved in 15 μl of TE buffer and then subcloned into an expression vector pEF18S similarly treated with restriction enzyme (competent high E. coli DH5 manufactured by Toyobo Co., Ltd. was used as the host strain). Of the obtained transformants, 30 clones containing an insert having the expected length were selected to prepare plasmid DNA. The method was performed essentially as described in Molecular Cloning [Sambrook et al., Cold Spring Harbor Laboratory Press (1989)]. In SEQ ID NO: 4, a plasmid DNA of a deletion derivative (pHT7-163) lacking 1-6 of the protein encoding amino acid numbers 1-163 was obtained. These clones were subjected to PCR using hTPO-5 and hTPO-S as primers, and it was confirmed that all clones contained inserts of the expected size. Three of these clones were selected and Taq Dye Deoxy^TM  Using Terminator Cycle Sequencing Kit (Applied Biosystems) and sequencing with Applied Biosystems 373A DNA sequencer, 2 clones had the expected deletion, and the entire base sequence including other parts Thus, two clones pHT7-163 # 4 and # 29 having the TPO cDNA sequence as designed were obtained.
(3) Preparation of a derivative lacking amino acid numbers 1-7 (pHT8-163)
The method for producing the derivative lacking amino acid numbers 1-7 (pHT8-163) was essentially the same as the method for producing the derivative lacking amino acid numbers 1-6 (pHT7-163). Using 1.4 μg of plasmid DNA of clone pHT1-163 obtained in Example 27 as a template, synthesized primers (hTPO-8 and hTPO-S were used in pairs: hTPO-5 and hTPO-8R were used in pairs) PCR was performed using 5 μM each. GeneAmp^TM  PCR Reagent Kit with AmpliTaq^TMUsing DNA Polymerase (Takara Shuzo), GeneAmp^TM  PCR reaction with PCR System 9600 (manufactured by PERKIN-ELMER) in a volume of 100 μl (denaturation at 95 ° C. for 5 minutes, denaturation conditions at 95 ° C. for 1 minute, annealing conditions at 65 ° C. for 1 minute, synthesis at 72 ° C. for 1 minute) The reaction was performed 30 times under the conditions, and further incubated at 72 ° C. for 7 minutes. Each PCR product was subjected to electrophoresis using a 1.2% agarose gel (manufactured by FMC BioProducts) to separate major DNA fragments of each expected size, and a prep-A-gene DNA purification kit (BioRad). And dissolved in 15 μl of TE buffer. A second PCR was performed using 1 μl of each solution as a template.
PCR was performed using 5 μM each of the synthesized primers (using hTPO-5 and hTPO-S). GeneAmp^TM  PCR Reagent Kit with AmpliTaq^TMUsing DNA Polymerase (Takara Shuzo), GeneAmp^TM  PCR reaction using PCR System 9600 (manufactured by PERKIN-ELMER) in a volume of 100 μl (denaturation at 95 ° C. for 5 minutes, denaturation conditions at 95 ° C. for 1 minute, annealing conditions at 60 ° C. for 1 minute, and 72 ° C. for 1 minute) The reaction was performed 30 times under the synthesis conditions, and further incubated at 72 ° C. for 7 minutes. The PCR product was subjected to electrophoresis using 1.2% agarose gel (manufactured by FMC BioProducts) to isolate a major DNA fragment of the expected size, and a prep-A-gene DNA purification kit (manufactured by BioRad). And dissolved in 15 μl of TE buffer. After digestion with restriction enzymes EcoRI and NotI, extraction was performed once with an equal amount of phenol / chloroform, followed by ethanol precipitation. The precipitate obtained by centrifugation was dissolved in 15 μl of TE buffer and then subcloned into the expression vector pEF18S similarly treated with restriction enzyme (competent high E. coli DH5 manufactured by Toyobo Co., Ltd. was used as the host fungus). Of the obtained transformants, 30 clones containing an insert having the expected length were selected to prepare plasmid DNA. The method was performed essentially as described in Molecular Cloning [Sambrook et al., Cold Spring Harbor Laboratory Press (1989)]. In SEQ ID NO: 4, a plasmid DNA of a deletion derivative (pHT8-163) in which 1-7 was deleted from the protein encoding amino acid numbers 1-163 was obtained. These clones were subjected to PCR using hTPO-5 and hTPO-S as primers, and it was confirmed that all clones contained inserts of the expected size. Three of these clones were selected and Taq Dye Deoxy^TMUsing Terminator Cycle Sequencing Kit (Applied Biosystems), sequenced with 373A DNA sequencer manufactured by Applied Biosystems, 2 clones had undergone the expected deletion, and the entire length including other parts Two clones pHT8-163 # 33 and # 48 having the TPO cDNA sequence as designed without base sequence substitution were obtained.
(4) Expression of deletion derivatives in COS1 cells and confirmation of TPO activity
Transfection of each of the resulting deletion clones into COS1 cells was performed according to Example 11. That is, 10 μg of each plasmid DNA was used for transfection using DEAE-dextran method including chloroquine treatment, and the culture supernatant was recovered after 3 days. The culture supernatant was sufficiently dialyzed against IMDM culture solution and then evaluated by the M-07e assay system.
As a result, TPO activity was observed in a dose-dependent manner in a COS1 cell culture supernatant transfected with a clone encoding a deletion derivative at amino acids 7-163. On the other hand, no TPO activity was observed in the COS1 cell culture supernatant transfected with a clone encoding a deletion derivative at amino acid positions 8-163 and 13-231.
<Example 30>
Construction of human TPO full-length cDNA plasmid (pHTP1)
An animal cell expression vector having all predicted amino acid coding regions of human TPO cDNA as shown in SEQ ID NO: 6 was constructed.
A DNA fragment covering the entire human TPO cDNA coding region by PCR was prepared as follows.
The base sequences of the primers used are as follows.
hTPO-I: 5'-TTGTGACTCCCGAGTCCTCAG-3 '
(105 to 125 of SEQ ID NO: 6)
SA: 5'-CAGGTATCCGGGGATTTGGGTC-3 '
(Antisense corresponding to 745 to 765 of SEQ ID NO: 6)
hTPO-P: 5'-TGCGTTTCCTGATGCTTGTAG-3 '
(503 to 523 of SEQ ID NO: 6)
hTPO-KO: 5'-GAGAGAGGCGCCCGCTTACCCCTCCTGGACAGAATT-3 '
(An antisense sequence corresponding to 1066 to 1086 of SEQ ID NO: 6 added with a restriction enzyme NotI recognition sequence and a GAGAGA sequence).
The first PCR was performed using 300 ng of the clone pEF18S-HL34 obtained in Example 16 as a template. Using Primer-hTPO-I and 0.5 μM each of SA, Vent R^TM  Reaction was carried out using 1 unit of DNA polymerase (manufactured by New England BioLabs) (96 ° C. for 1 minute, 62 ° C. for 1 minute, 72 ° C. for 1 minute followed by 72 ° C. for 7 minutes). The composition of the reaction solution is as follows. Final concentration 10 mM KCl, 10 mM (NH₄)₂SO₄20 mM Tris-HCl (pH 8.8), 2 mM MgSO₄0.1% Triton X-100, 200 μM dNTP mix.
1 μg of commercially available human normal liver-derived poly (A) + RNA (Clontech) was heated at 70 ° C. for 10 minutes and then rapidly cooled on ice. 10 mM DTT, 500 μM dNTPmix, 25 ng random primer (Takara Shuzo), 10 units RNase Inhibitor ( Boehringer Mannheim), 200 units SuperScript^TMII RNaseH- (manufactured by LIFE TECHNOLOGIES) was added and incubated at 37 ° C for 1 hour to synthesize cDNA. A second PCR was performed using 1/20 of the synthesized cDNA reaction as a template. Primer hTPO-P and hTPO-KO 2.5 μM each, 2.5 units of AmpliTaq^TM  Reaction was carried out using DNA polymerase (Takara Shuzo) (reactions at 95 ° C. for 1 minute, 58 ° C. for 1 minute, 72 ° C. for 1 minute for 30 cycles).
Two PCR solutions were subjected to 1% agarose gel electrophoresis, and major bands of the expected sizes were purified using a prep-A-gene DNA purification kit (Bio-Rad). A third PCR was carried out using one-twentieth of each purified amount as a template. Vent R^TM  The reaction was carried out using 1 unit of DNA polymerase (manufactured by New England BioLabs) (after heating at 96 ° C. for 2 minutes, followed by 3 cycles of 96 ° C. for 2 minutes and 72 ° C. for 2 minutes, then 72 ° C. for 7 minutes). After adding hTPO-I and hTPO-KO to this reaction solution to 1 μM, respectively, heating at 96 ° C. for 2 minutes, followed by reaction at 96 ° C. for 1 minute, 62 ° C. for 1 minute, and 72 ° C. for 1 minute. After 25 cycles, the reaction was further continued at 72 ° C. for 7 minutes. The reaction solution was extracted once with an equal amount of water-saturated phenol-chloroform, and further extracted once with an equal amount of chloroform, followed by ethanol precipitation (0.3 M sodium acetate, 0.5 μl glycogen, Boehringer Mannheim, 2.5 In the presence of double volume ethanol), DNA was recovered.
The recovered DNA was digested with restriction enzymes BamHI and NotI, subjected to 1% agarose gel electrophoresis, and the main band of the expected size was purified using a prep-A-gene DNA purification kit (Bio-Rad). After ligation to pBluescript II SK + vector (Stratagene) previously digested with restriction enzymes BamHI and NotI in the same manner, E. coli DH5 (Toyobo Co., Ltd.) was transformed. Four clones were selected from the obtained colonies to prepare plasmid DNA. For purified plasmid DNA, Taq Dye Deoxy^TM  Using Terminator Cycle Sequencing Kit (manufactured by Applied Biosystems), sequencing was performed with 373A DNA sequencer manufactured by Applied Biosystems, and it was confirmed that the region from BamHI to NotI had no nucleotide sequence substitution and had the expected TPO cDNA sequence. Clone pBLTP was obtained.
pBLTP was digested with restriction enzymes EcoRI and BamHI, subjected to 1% agarose gel electrophoresis, and a high molecular weight band was purified using a prep-A-gene DNA purification kit (Bio-Rad). Similarly, pEF18S-HL34 was treated with a restriction enzyme to purify a 450 bp band. Each DNA was ligated to competent high E. coli. E. coli DH5 (Toyobo Co., Ltd.) was transformed. Plasmid DNA was prepared from the obtained colonies, and a clone pBLTEN containing an insert of human TPO cDNA was obtained.
The obtained pBLTEN was digested with restriction enzymes EcoRI and NotI, subjected to 1% agarose gel electrophoresis, and a band of about 1200 bp was purified using a prep-A-gene DNA purification kit (manufactured by Bio-Rad), and similarly. Competent high E. coli ligated to the restriction enzyme-treated expression vector-pEF18S. E. coli DH5 (Toyobo Co., Ltd.) was transformed. Plasmid DNA was prepared from the obtained colonies to obtain clone pHTP1 containing all the coding region of human TPO cDNA. A large amount of plasmid DNA of this clone was prepared and used for the following experiments. Preparation of plasmid DNA was performed essentially as described in Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989).
<Example 31>
Construction of a recombinant vector for CHO cells, pDEF2O2-hTPO-P1
After treating 1 μg of the plasmid pMG1 containing the mouse DHFR minigene with restriction enzymes EcoRI and BamHI, agarose gel electrophoresis was performed to recover a fragment (about 2.5 kbp) containing the mouse DHFR minigene. The recovered fragment was dissolved in 25 μl of a reaction solution consisting of 50 mM Tris-HCl (pH 7.5), 7 mM MgCl 2, 1 mM mercaptoethanol, 0.2 mM dNTP, added with 2 units of Klenow fragment, reacted at room temperature for 30 minutes, The ends were smoothed.
Subsequently, after phenol / chloroform treatment and ethanol precipitation, it was dissolved in 10 μl of a solution consisting of 10 mM Tri-HCl (pH 8.0) and 1 mM EDTA. Next, the obtained fragment containing the mouse DHFR minigene and the animal cell expression vector pEF18S were treated with the restriction enzyme Smal, and the vector DNA obtained by dephosphorylation with alkaline phosphatase (Takara Shuzo) was subjected to T4 DNA ligase ( And the expression vector pDEF202 was obtained.
Next, this vector pDEF202 is treated with the restriction enzymes EcoRI and Spel, and the larger vector fragment is recovered by agarose gel electrophoresis. Then, the plasmid pHTP1 containing this fragment and human TPOcDNΛ (P1 clone) is converted into the restriction enzymes EcoRI and Spel. The human TPO cDNA (P1 clone) obtained by treatment with the above was ligated with T4 DNA ligase (Takara Shuzo) to obtain an expression vector pDEF202-hTPO-P1. This plasmid contains the SV40 replication initiation region, human elongation factor-1-alpha promoter, SV40 early polyadenyl site, mouse DHFR minigene, pUC18 replication initiation region, β-lactamase gene (Amp^rThe human TPO cDNA is connected downstream of the human elongation factor-1-alpha promoter.
<Example 32>
Expression of human TPO in CHO cells
Α minimum essential medium containing CHO cells (dhfr-strain, Urlaub and Chasin; Proc. Natl. Acad. Sci. USA; 77, 4216, 1980) in 6 cm diameter plate (Falcon) containing 10% fetal bovine serum (Α-MEM (−), thymidine, hypoxanthine added) was cultured and grown, and this was transformed by the calcium phosphate method (CellPhect, Pharmacia). That is, 10 μg of pDEF202-hTPO-P1 plasmid prepared in Example 31 was added to Buffer A: 120 μl and H₂O: 120 μl was added and mixed, and then allowed to stand at room temperature for 10 minutes. Next, 120 μl of buffer B was added to this solution, mixed again, and allowed to stand at room temperature for 30 minutes. After dropping this DNA solution onto the plate, CO₂The cells were cultured for 6 hours in an incubator. After removing the medium from the plate and washing twice with α-MEM (−), α-MEM (−) containing 10% dimethyl sulfoxide was added and treated at room temperature for 2 minutes. Subsequently, 10% dialyzed fetal bovine serum-containing non-selective medium (additional α-MEM (−), hypoxanthine, thymidine added) was added and cultured for 2 days. Selection was made with MEM (-), hypoxanthine, and thymidine not added. For selection, after trypsinizing the cells, each 6 cm diameter plate is divided into 5 10 cm diameter plates or 20 24-well plates, and then the culture is continued while changing the medium every 2 days. It was carried out by things. As for the plate or well in which the cells had grown, the human TPO activity in the culture supernatant was measured using the M-07e assay and the Ba / F3 assay. As a result, the activity was recognized in all assay systems. For those in which human TPO activity was observed in the culture supernatant, the cells were split 1:15 in a selective medium containing 25 nM methotrexate in a new plate or well, and the cells resistant to methotrexate were expanded by continuing the culture. Cloning.
CHO cells can also be transformed by co-transfecting CHO cells with pHTP1 and pMG1.
The CHO cell line (CHO-DUKXB11) transformed with the plasmid pDEF202-hTPO-P1 was deposited with the accession number FERM BP-4988 at the Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry on January 31, 1995. Yes.
It has also been deposited with the food industry development institute of China (FIRDI) as deposit number 960023 on March 22, 1995.
<Example 33>
X63.3.6.5.3. Construction of recombinant vector for cells, BMCGSneo-hTPO-P1
An animal cell expression vector BMCGSneo 1 μg was treated with restriction enzymes XhoI and NotI, followed by agarose gel electrophoresis and the vector DNA portion was recovered. Subsequently, the resulting DNA fragment and the human TPO cDNA (P1 clone) obtained by treating the plasmid pBLTEN containing human TPO cDNA with the restriction enzymes XhoI and NotI were ligated with T4 DNA ligase to obtain the expression plasmid BMCGSneo-hTP0-P1. . This expression plasmid contains cytomegalovirus early promoter, intron and polyadenyl site from rabbit β globin gene, part of human β globin gene, 69% of bovine papillomavirus 1 gene, thymidine kinase promoter and polyadenyl site, phosphotransferase I (neomycin resistance) Gene), the replication initiation region of pBR322 and the β-lactamase gene (Amp^R) And human TPO cDNA is connected downstream of the cytomegalovirus promoter.
<Example 34>
X63.3.6.5.3. Expression in cells
X63.3.6.5.3. The cells were cultured and grown in Dulbecco's minimalistic (DME) medium containing 10% fetal bovine serum, and transformed by electroporation.
That is, 20 μg of BMCGSneo-hTP0-P1 plasmid prepared in Example 33 was treated with 10 μg.⁷The cells were added to 750 μl of DME medium containing individual cells, set in a 4 mm gap cuvette for electroporation, and allowed to stand at 4 ° C. for 10 minutes. This cuvette was set in an electroporation apparatus (BTX600, manufactured by BTX), and the gene was introduced under conditions of 380 V) 25 mF and 24 angstroms. After gene transfer, the cuvette was again left at 4 ° C. for 15 minutes, and the cells were washed once with DME containing 10% fetal bovine serum. Next, the cells are suspended in 50 ml of DME containing 10% fetal bovine serum, divided into five 96-well plates, and then mixed with CO.₂The cells were cultured for 2 days in an incubator. Thereafter, the culture medium was replaced with DME containing 1 mg / ml G418 (GIBCO) and 10% fetal calf serum, and thereafter the medium was replaced every 3 days. For wells in which cells had grown, human TPO activity in the culture supernatant was measured using CFU-MK assay, M-07e assay and Ba / F3 assay, and activity was observed in all assay systems. . Resistant cells were grown for those in which human TPO activity was observed in the culture supernatant and cloned twice to establish a human TPO-producing cell line.
<Example 35>
Mass expression of human TPO in COS1 cells
Transfection into COS1 cells was performed by the DEAE-dextran method including chloroquine treatment described in Example 11. COS1 cells (ATCC CRL1650) treated with collagen coating 175cm²The culture flask was cultured with IMDM containing 10% (v / v) FCS in a 5% carbon dioxide incubator at 37 ° C. until it became about 100% confluent. The collagen coating treatment of the culture flask was performed by using a collagen solution (Cellmatrix type I-C manufactured by Iwaki Co.) prepared to 0.3 mg / ml with 1 mM HCl at 175 cm.²25 ml was added to each culture flask and allowed to stand at room temperature for 1 hour, and then the collagen solution was recovered and washed once with 20 to 50 ml of PBS. Transfection is 175cm²In 20 ml of IMDM solution containing 250 μg / ml DEAE-dextran (Pharmacia), 60 μM chloroquine (Sigma), and 10% (v / v) Nu-Serum (collaborative) Plasmid pHTP1 (40 μg) dissolved in 500 μl of HBS was mixed, added to the COS1 cells washed once with IMDM immediately before transfection, and then cultured in a 37 ° C. 5% carbon dioxide incubator for 3 hours. Thereafter, the culture supernatant was removed by aspiration and washed once with IMDM, 50 ml of serum-free medium was added, and the cells were cultured in a 5% carbon dioxide incubator at 37 ° C., and the culture supernatant was recovered after 5 days. 175cm for one operation²From 260 culture flasks of 100 to 260, 5 to 131 culture supernatants were collected. The composition of the serum-free medium is 5 μg / ml Insulin (manufactured by Sigma), 5 μg / ml Transferrin (manufactured by Sigma), 10 μM monoethanolamine (Wako Pure Chemical Industries), 25 nM sodium serum (manufactured by Sigma), 200 μg / ml BSA (manufactured by Sigma). IMDM containing fatty acid-free high-purity bovine albumin manufactured by Nichirei).
<Example 36>
Purification and activity confirmation of human full-length TPO expressed in COS1 cells (derived from expression plasmid pHTP1, P1 clone)
Examples of activity and purification of human full-length TPO (derived from expression plasmid pHTP1) expressed in COS1 cells are described below. In order to examine the platelet-increasing action of TPO, a partially purified product was first prepared and purified to obtain a highly pure TPO preparation. The M-07e assay system was mainly used for measuring TPO activity in the following preparation process, but similar TPO activity was also shown in the rat CFU-MK assay system. In these assays, human serum albumin (HSA) having a final concentration of 0.02 to 0.05% was added to the preparation.
First, 0.2 g per 1000 ml of COS1 cells transfected with plasmid pHTP1 using the method of Ohashi and Sudo (Hideya Ohashi and Tadashi Sudo, Biosci. Biotech. Biochem., 58 (4), 758-759, 1994). BSA, 5 mg bovine insulin, 5 mg human transferrin and in serum-free IMDM medium containing 0.02 mM monoethanolamine, 25 nM Sodium Selenite for 5 days at 37 ° C. in a 5% carbon dioxide incubator And about 7 L of serum-free culture supernatant was obtained. To the serum-free culture supernatant, p-APMSF and Pefabloc SC (4- (2-Aminoethyl) -benzoylsulfonyl fluoride, Merck, catalog number 24839), which are protease inhibitors, were added at a final concentration of about 1 mM. The filtrate of a 22 μm filter was taken. This was concentrated about 10 times with an ultrafiltration unit (manufactured by Filtron, Omega Ultraset molecular weight 8000 cut, or Millipore, PLGC pericon cassette molecular weight 10,000 cut), and volume 723 ml (protein concentration 3.38 mg / ml, total After adding 1.6 moles of Ammonium Sulfate (total 288 g) per 1000 ml to a protein mass of 2445 mg, a relative activity of 43,000, and a relative activity of 10500000), a final concentration of 1.5 M Ammonium Sulfate was added to obtain a 804 ml solution, followed by 1.25 M Ammonium. Macro-Prep Methyl HIC column (Bio-Rad, Catalog No. 1) previously equilibrated with 20 mM sodium citrate buffer (pH 5.2) containing Sulfate 56-0080; diameter 5 cm, bed height 9 cm) at a flow rate of 10 ml / min. After completion of addition, a flow-through fraction F1 (2384 ml, protein concentration 0.864 mg / ml, total protein mass 2061 mg, relative activity 6000) eluted with 20 mM sodium citrate buffer (pH 5.2) containing 1.25 Mammanium Sulfate was added. Obtained.
Next, the eluate was changed to 20 mM Na Citrate, pH 5.8, and the eluted fraction F2 (1092 ml, protein concentration 0.776 mg / ml, total protein mass 847 mg, relative activity 150,000) was collected.
Furthermore, Macro-Prep Methyl HIC column F2 (1081 ml) was pre-equilibrated with 20 mM sodium citrate buffer (pH 5.8), SP Sepharose Fast Flow (manufactured by Pharmacia Biotech, catalog number 17-0729-01; diameter 3 cm) The bed height was 10 cm) and added at a flow rate of 10 ml / min. After completion of the addition, the fraction F1 (2262 ml, protein concentration 0.270 mg / ml, total protein mass 610 mg, relative activity 30000), which was further eluted with 20 mM sodium citrate buffer (pH 5.6) containing 110 mM NaCl, was collected. ) Next, the eluate was changed to 20 mM sodium citrate buffer (pH 5.4) containing 400 mM NaCl, and the eluted fraction F2 (856 ml, protein concentration 0.189 mg / ml, total protein mass 162 mg, relative activity 300,000). Collected. Next, the eluate was changed to 20 mM sodium citrate buffer (pH 5.2) containing 1000 mM NaCl, and the eluted fraction F3 (370 ml, protein concentration 0.034 mg / ml, total protein mass 12.6 mg, relative activity). 150,000).
Furthermore, 1-propanol having a final concentration of about 10% is added to the main TPO active fraction F2 (845 ml) in the SP Sepharose Fast Flow column, and equilibrated in advance with a 20 mM sodium citrate buffer (pH 5.4) containing 400 mM NaCl. The solution was injected into an LA-WGA column (Honen, catalog number WG-007; diameter 2 cm, bed height 14 cm) and added at a flow rate of 3 ml / min. After completion of the addition, the fraction F1 (64.4 ml, protein concentration 0) was collected from the mixture eluted with a mixed solution (9: 1) of 20 mM sodium citrate buffer (pH 5.4) containing 400 mM NaCl and 1-propanol. 0178 mg / ml, total protein mass 1.15 mg, relative activity 17220). Next, the eluate was replaced with 20 mM sodium citrate buffer (pH 6.1) containing 0.4 M GlcNac, 10% 1-propanol, and the eluted fraction F2 (45 ml, protein concentration 0.0104 mg / ml, total Protein mass 0.470 mg, relative activity 675000) was collected.
Therefore, after adding TFA having a final concentration of about 0.005% to the main TPO active fraction F2 (340 ml) in the LA-WGA column, YMC-Pack CN-AP (manufactured by YMC, catalog number AP-513; diameter) 6 mm, bed height 250 mm). Specifically, 1-propanol containing 0.1% TFA in developing solvent A and 0.05% TFA in developing solvent B was used, and a flow rate of 0.6 ml / ml was applied to a YMC-Pack CN-AP column equilibrated with 15% B. Injected in min. After completion of injection, the propanol concentration is increased from 15% B to 25% B, and further developed with a linear concentration gradient from 25% B to 50% B for 65 minutes, and collected in polypropylene tubes in 1.5 ml (2.5 min) increments. It was.
Take 0.5 μl each (1/3000 fraction), add HSA, concentrate by ultrafiltration, and finally make a 0.25 ml IMDM assay culture solution containing 0.05% HSA, which is subjected to the assay, The active fraction was identified. As a result, there was strong TPO activity (relative activity of about 630000 to 480000) in tube numbers 24 to 30 (range of propanol concentration of 35.0 to 42.0%), so TPO active fraction FA (13.5 ml) It was.
Therefore, Capcell Pak C1 300A (manufactured by Shiseido Co., Ltd.) using FA obtained from YMC-Pack CN-AP, 1-propanol containing 0.1% TFA in developing solvent A and 0.05% TFA in developing solvent B , Catalog number C1TYPE: SG300A; connected to a pre-column having a diameter of 4.6 mm and a bed height of 150 mm, and a diameter of 4.6 mm and a bed height of 35 mm). That is, take a part (8,9 ml) of FA obtained with YMC-Pack CN-AP, add 0.3 ml of glycerol, concentrate by centrifugal evaporation, and then add about 2.5 ml of 10% B solution. In addition, it was injected into a Capcell Pak C1 300A column equilibrated with 20% B at a flow rate of 0.4 ml / min. After completion of the injection, the mixture was eluted with 20% B for 5 minutes, and then developed with a linear concentration gradient of 50 minutes from 20% B to 40% B, and each 1 ml (2.5 min) was collected in a polypropylene tube.
Take 1 μl (1/1000 fraction) of each, add HSA, concentrate by ultrafiltration, and finally make 0.25 ml of IMDM assay culture solution containing 0.05% HSA. Identified minutes. As a result, it was possible to obtain a highly active sample exhibiting strong TPO activity (relative activity of about 3000000 to 22500000) in tube numbers 20 to 23 (range of propanol concentration: 28.5 to 32.5%).
<Example 37>
Molecular weight measurement of human TPO expressed in COS1 cells
The molecular weight of human full-length TPO expressed in COS1 cells (derived from expression plasmid pHTF1, F1 clone) was considered to be larger than the molecular weight that can be estimated from the size of the peptide chain as a result of the addition of sugar chains. Therefore, first, a partially purified TPO fraction was prepared from the culture supernatant containing human full-length TPO expressed in COS1 cells (expression plasmid pHTF1, derived from F1 clone) as follows. That is, YMC-Pack PROTEIN- pre-equilibrated with 25% B using developing solvent A (0.1% trifluoroacetic acid (TFA)) and developing solvent B (1-propanol containing 0.05% TFA). Inject 0.3 ml of the culture supernatant into an RP (YMC, catalog number A-PRRP-33-46-25; diameter 0.46 cm, bed height 15 cm) column at a flow rate of 0.4 ml / min for 5 minutes and 25%. After passing B, fractionation was performed with a linear concentration gradient from 25% B to 50% B over 50 minutes. TPO activity was eluted in the range of 34.5% to 43.5% 1-propanol, and this was dried to dryness by centrifugal evaporation to obtain a partially purified preparation.
Next, after protein extraction from the non-reducing SDS-PAGE gel described in Example 1, the TPO activity was examined in the M-07e assay system or reduced by Western analysis as described in Example 45. The molecular weight for the DPCIII marker was measured. As a result, TPO activity was present in a wide range of an apparent molecular weight of about 6900 to 94000, and the heterogeneity of the molecular weight could be confirmed. Furthermore, in the same manner, when examining the apparent molecular weight of the reduced DPCIII marker for TPO after N-linked sugar chain cleavage by N-Glycanase (Genzyme, catalog number 1472-00), It was found that the molecular weight was 36000-40000, still larger than the molecular weight of about 35000, which can be estimated from the size of the peptide chain, and strongly suggested that it also contains an O-linked sugar chain.
Similarly to these, when the apparent molecular weight of human full-length TPO (expression plasmid pHTPI, derived from P1 clone) expressed in COS1 cells was examined, it was 63000-83000.
Example 38 Biological characteristics of human TPO
The effect of human TPO on human and rat blood cells was examined using a culture supernatant containing TPO activity obtained mainly by transfecting COS1 cells with expression plasmid pHTF1.
CD34 prepared from human umbilical cord blood⁺, DR⁺When assayed by a colony assay system using the cell fraction, a significant number of megakaryocyte colonies were formed by human TPO. For example, under the condition that 10% of COS1 cell culture supernatant transfected and expressed with expression plasmid pHT1-231 is added, 6000 CD34⁺, DR⁺An average of 11.5 megakaryocyte colonies were formed from the cells. Plastic adherent cells were removed from the leukocyte fraction obtained from human peripheral blood by specific gravity centrifugation using Ficoll-Paque, and cells having affinity for SBA (soybean agglutinin) were further removed using AIS microselector CD34 (Asahi Medical). ) Using a panning method and finally a CD34 using a panning method using AIS microselector CD34 (Asahi Medical Co., Ltd.).⁺A cell fraction was obtained. When human TPO (the COS1 cell culture supernatant in which the expression plasmid pHTF1 was transfected and expressed) was added to this cell fraction and subjected to liquid culture for 10 days, the large GpIIb / III a⁺Selective growth of cells was observed, and this GpIIb / III a⁺Cells had increased nuclear ploidy. This strongly suggested that human TPO specifically acts on human megakaryocyte progenitor cells and promotes their proliferation and differentiation.
GpIIb / III a isolated and purified from rat bone marrow cells⁺Cell fraction, and GpIIb / IIIa⁺A significant number of megakaryocyte colonies were formed by human TPO when assayed by a colony assay system using a plastic non-adherent cell fraction in the previous stage to obtain a cell fraction. For example, 1000 GpIIb / III a cells were cultured on the 5th day under the condition of adding 20% of COS1 cell culture supernatant transfected and expressed with the expression plasmid pHTF1.⁺An average of 34.5 megakaryocyte colonies formed from the cells, and an average of 28.5 megakaryocyte colonies formed from 20000 non-adherent cells. Furthermore, there are various types of progenitor cells other than megakaryocytes in the plastic non-adherent cell fraction. However, when human TPO is added, only megakaryocyte colonies are formed, and human TPO is specific to megakaryocyte progenitor cells. It was strongly suggested to act on In addition, GpIIb / IIIa derived from rat bone marrow⁺The cell fraction was subjected to liquid culture for 3 to 5 days in the presence of human TPO (transfected with the expression plasmid pHTF1 and 20% of the expressed COS1 cell culture supernatant added), and the ploidy of the nucleus was examined. The ploidy was clearly increased on the fifth day of culture.
<Example 39>
Construction of a vector for expression of Escherichia coli of a fusion protein of glutathione-s-transferase (GST) and human TPO (amino acids 1-174) (hereinafter, this fusion protein is referred to as “GST-TPO (1-174)”)
In order to facilitate the expression of human TPO in E. coli, an artificial gene encoding human TPO and containing an E. coli preferential codon was created. This DNA base sequence has an amino terminal methionine codon (ATG) for initiation of translation in E. coli at position -1.
Synthetic oligonucleotides 1-12 shown in Table 12 were prepared, and 2-11 synthetic oligonucleotides were prepared with 1 mM ATP, 10 mM Tris-acetate, 10 mM Mg-acetate, 50 mM K-acetate using T4 kinase (Pharmacia). Phosphorylated in solution. In order to make these synthetic oligonucleotides into 6 double-stranded DNAs, 1 and 2; 3 and 4; 5 and 6; 7 and 8; 9 and 10; Tris / HCl (pH 7.5), 10 mM MgCl₂. , Annealed in a solution of 50 mM NaCl. Next, three sets of double-stranded DNAs of 1 and 2; 3 and 4; 5 and 6 and 7 and 8; 9 and 10; 11 and 12 of double-stranded DNA were respectively converted to T4 ligase (Life Technologies). The two reaction solutions were further reacted using T4 ligase in the same manner. The DNA obtained by this ligation reaction is digested with BamHI (manufactured by Boehringer Mannheim), then electrophoresed on a 2% agarose gel, and a fragment of about 390-400 bp in size is recovered to prepare prep-A-gene DNA. It was purified with a purification kit and subcloned into pUC18 digested with Xbal, BamHI (the host used was E. coli DH5α). Among the obtained clones, a clone having an artificial gene encoding human TPO shown in Table 13 and containing an E. coli preferential codon was selected by analysis of the base sequence, and this was selected as pUC18 (XB) (1-123). It was. The DNA sequence of the coding strand in Table 13 is shown in the Sequence Listing (SEQ ID NO: 9).
[Table 12]

[Table 13]

Normal human liver poly (A)⁺The first strand of cDNA was synthesized from 1 μg of RNA using oligo dT primer as a primer, and PCR was carried out using 0.1 volume of the reaction solution for cDNA synthesis as a template. HTPO-C and hTPO-ko (EcoRI) were used as primers, and the reaction was performed in a volume of 100 μl (96 ° C. for 2 minutes, then 95 ° C. for 1 minute / 58 ° C. for 1 minute / 72 ° C. for 1 minute for 30 cycles). React, further 72 ° C. for 7 minutes). The human TPO cDNA fragment thus obtained was digested with BamHI and EcoRI and then electrophoresed on a 2% agarose gel. A fragment having a size of about 600 bp was recovered and purified with a prep-A-gene DNA purification kit. It was subcloned into BUCHI digested pUC18 (the host used was E. coli DH5α). A clone encoding the correct human TPO base sequence was selected by base sequence analysis and designated pUC18 (BE) (124-332). The sequences of the PCR primers used here are as follows.
hTPO-C: 5'-GGAGGAGACCAAGGCACAGGA-3 '
(329-349 of pHTF1 clone)
hTP0-ko (EcoRI): 5'-CCCGGAATTCTTACCCCTCCTGGACAGAATT-3 '
(EcoRI sequence is added to the antisense corresponding to 1143 to 1163 of pHTF1 clone).
pUC18 (BE) (124-332) was digested with BamHI and EcoRI and then run on a 2% agarose gel, and a C-terminal fragment of human TPO cDNA having a size of about 600 bp was recovered to prepare a prep-A-gene DNA purification kit. And subcloned into pUC18 (XB) (1-123) digested with EcoRI and BamHI (the host used E. coli DH5α). The clone obtained here was designated as pUC18 (XE) (1-332).
From the examples in which various deletion constructs on the C-terminal side of human TPO cDNA were prepared, expressed and measured for in vitro human TPO activity, it is clear that human TPO amino acid 1-163 retains human TPO activity. Therefore, it was decided to construct an expression vector containing this peptide fragment. Here, GST-TPO (1-174) was expressed.
Since the base sequence corresponding to 681-686 (amino acids 173-174) of the pHTF1 clone is recognized by the restriction enzyme Sacl, a stop codon was introduced by two synthetic oligonucleotides using this restriction enzyme recognition site. Specifically, two synthetic oligonucleotides SSE1 and SSE2 are mixed with 10 mM Tris / HCl (pH 7.5), 10 mM MgCl.₂, 50 mM NaCl, annealed in a solution of 50 mM NaCl, and the resulting double-stranded DNA was digested with SacI and EcoRI to remove about 480 bp of the C-terminal side of human TPO cDNA into pUC18 (XE) (1-332), DNA Ligation Kit (Takara Shuzo Co., Ltd.) was used to obtain clone pUC18 (XS) (1-174) (the host used E. coli DH5α). Table 14 shows the sequences of the synthetic oligonucleotides used here.
[Table 14]

After digesting pUC18 (XS) (1-174) with XbaI and EcoRI, the mixture was run on a 2% agarose gel, and a fragment of about 600 bp size encoding human TPO amino acid 1-174 was recovered to prepare prep-A- PBluescript IISK purified with Gene DNA Purification Kit and digested with XbaI, EcoRI⁺  It was subcloned in (Stratagene) (E. coli DH5α was used as the host). The clone obtained here was designated as pBL (XS) (1-174). Further, in the same manner as above, pBL (XS) (1-174) was digested with XbaI and HindIII, and then a fragment of about 550 bp in size encoding human TPO amino acid 1-174 was recovered to purify prep-A-gene DNA. The product was purified with the kit and cloned into pCFM536 (Japanese Patent Publication No. 60-501988) digested with XbaI and HindIII (the host used was E. coli DH5α). The obtained clone was designated as pCFM536 / hT (1-174).
In order to express GST-TPO (1-174) by pGEX-2T (Pharmacia), which is a fusion protein expression vector with glutathione-S-transferase (GST), the following was performed. PCR was performed using pCFM536 / hT (1-174) as a template and two PCR primers GEX1 and GEX3 (96 ° C. for 2 minutes followed by 95 ° C. for 1 minute / 41 ° C. for 1 minute / 72 ° C. for 1 minute) For 22 cycles and 72 ° C. for 7 minutes). The fragment encoding human TPO thus obtained was digested with NaeI and EcoRI and then electrophoresed on a 2% agarose gel. A fragment having a size of about 550 bp was recovered and purified with a prep-A-gene DNA purification kit. , Cloned into pGEX-2T digested with EcoRI, Smal (the host used E, coli DH5), and cloned pGEX-2T / hT (1-174) encoding the correct human TPO nucleotide sequence was analyzed by nucleotide sequence analysis. This was used as a transformant for expressing GST-TPO (1-174). The sequences of the PCR primers used here are as follows.
GEX1: 5'-ATCGCCGGCTCCGCCCAGCTTGGAC-3 '(added NaeI sequence to 21-39 of SEQ ID NO: 10)
GEX3: 5'-GCCGAATTCTCATTAGAGGCTCGTTCAGGTGT-3 '(antisense of 523-549 of SEQ ID NO: 10).
This expression plasmid contains a GST protein followed by a thrombin recognition sequence and a sequence encoding human TPO (amino acids 1-174). The sequence encoding the thrombin recognition sequence and human TPO (amino acids 1-174) is shown in the sequence listing (SEQ ID NO: 10).
<Example 40>
Expression of GST-TPO (1-174) in E. coli
The transformant obtained in Example 39 was shaken overnight at 37 ° C. in 60 ml of LB medium containing 50 μg / ml of ampicillin, 25 ml of this culture solution was added to 1000 ml of LB medium containing 50 μg / ml of ampicillin, and OD was obtained. Is A₆₀₀Was cultured at 37 ° C. with shaking until it reached 0.7-0.8. Next, IPTG was added so that the final concentration was 0.1 mM, and further cultured with shaking for 3 hours to induce the expression of GST-TPO (1-174).
<Example 41>
Purification and activity confirmation of GST-TPO (1-174) derived from clone pGEX-2T / hT (1-174) encoding human TPO base sequence expressed in E. coli
Clone pGEX-2T / hT (1-174) -derived GST-TPO (1-174) production recombinant recombinant cell frozen 5.9 g added with 10 ml of water and suspended in a high-pressure crusher The body was crushed. By centrifugation, GST-TPO (1-174) was recovered in the precipitate fraction, and most of the mixed proteins, bacterial cell components and the like were removed. Next, 5 ml of water was added to the collected fraction containing GST-TPO (1-174) to make it suspended, and then 6 ml of 1 M Tris buffer pH 8.5, 120 ml of 10 M urea and 16 ml of water were added with stirring. It was. After solubilization with stirring for 5 minutes at room temperature, the solution was divided into four equal parts, and each of the four operations (1) to (4) was performed as follows.
(1) Dilute 10 times with 20 mM Tris buffer pH 8.5. Reduced glutathione and oxidized glutathione were added thereto to give final concentrations of 5 mM and 0.5 mM, respectively, and allowed to stand at 4 ° C. overnight. By centrifugation, GST-TPO (1-174) was recovered in the supernatant, diluted 2-fold with 20 mM sodium citrate buffer pH 5.5, and then adjusted to pH 5.5 with acetic acid. GST-TPO (1-174) was adsorbed on an SP Sepharose Fast Flow (manufactured by Pharmacia Biotech, catalog number 17-0729-01) cation exchange column equilibrated with 20 mM sodium citrate buffer pH 5.5. The resin was washed with 20 mM sodium citrate buffer pH 5.5, and then GST-TPO (1-174) was eluted using 20 mM sodium citrate buffer pH 5.5 containing 500 mM sodium chloride. After adding 2.6 ml of 1 M Tris buffer pH 8.5 to 129 ml of the eluate to adjust the pH to 8.1, it was added to a glutathione Sepharose 4B (Pharmacia Biotech, catalog number 17-0756-01) column and added to GST- TPO (1-174) was adsorbed. After washing with PBS, GST-TPO (1-174) was eluted with 20 mM Tris buffer pH 8.5 containing 10 mM reduced glutathione. Thrombin 37NIH Unit was added to the eluate, allowed to stand at room temperature for 4 hours, diluted 10-fold with PBS, added to a glutathione sepharose 4B column to adsorb the cleaved GST, and TPO (1-174) was added to the non-adsorbed fraction. ) Was recovered. The collected non-adsorbed fraction was diluted 3-fold with 20 mM sodium citrate buffer pH 5.5 and added to an SP Sepharose Fast Flow cation exchange column equilibrated with the same buffer solution. Elution was performed by the linear concentration gradient method up to 500 mM.
(2) All the operations of (1) were performed in the presence of 0.1% polysorbate 80.
(3) Dilute 10 times with 20 mM Tris buffer pH 8.5. Reduced glutathione and oxidized glutathione were added thereto to give final concentrations of 5 mM and 0.5 mM, respectively, and allowed to stand at 4 ° C. overnight. By centrifugation, GST-TPO (1-174) was recovered in the supernatant, diluted 2-fold with 20 mM sodium citrate buffer pH 5.5, and then adjusted to pH 5.5 with acetic acid. GST-TPO (1-174) was adsorbed on SP Sepharose Fast Flow cation exchange resin equilibrated with 20 mM sodium citrate buffer pH 5.5. The resin was washed with 20 mM sodium citrate buffer pH 5.5, and then GST-TPO (1-174) was eluted using 20 mM sodium citrate buffer pH 5.5 containing 500 mM sodium chloride. 1M Tris buffer pH 8.5 was added to the eluate to adjust the pH to about 8. Thrombin 320NIH Unit was added, and the mixture was allowed to stand at room temperature for 4 hours, and then diluted 5 times with 20 mM sodium citrate buffer pH 5.5. The solution was added to an SP Sepharose Fast Flow cation exchange column equilibrated with the solution, and elution was performed in the same buffer by a linear concentration gradient method from a sodium chloride concentration of 0 M to 500 mM.
(4) All the operations of (3) were performed in the presence of 0.1% polysorbate 80.
The fractions eluted at a sodium chloride concentration of about 200 mM to 400 mM in SP Sepharose Fast Flow cation exchange chromatography obtained by the operations of (1) to (4) are each sufficiently dialyzed against IMDM culture medium. As a result of evaluation in the rat CFU-MK system, TPO activity was recognized in a dose-dependent manner. As a result of analyzing the same fraction by SDS-PAGE in the presence of a reducing agent, a band with a molecular weight of 19 KDalton was detected as one of the main protein bands of this fraction (purity 1-20%). As a result of SDS-PAGE, this band was transferred to a PVDF membrane and subjected to N-terminal sequence analysis by the method described in Example 1. As a result, this band was expressed as a fusion protein derived from pGEX-2T / hT (1-174). It was confirmed that the TPO sequence to be included was included.
<Example 42>
Construction of expression vector for E. coli of human TPO (amino acids 1-163) converted to amino acid 1 (Ser → Ala) and amino acid 3 (Ala → Val)
After digesting pUC18 (XE) (1-332) prepared in Example 39 with XbaI and EcoRI, it was run on a 2% agarose gel to recover a fragment having a size of about 1000 bp encoding human TPO amino acids 1-332. PBluescript II SK purified with Prep-A-gene DNA purification kit and digested with XbaI, EcoRI⁺Substrated into (Stratagene) (E. coli DH5α was used as the host). The clone obtained here was designated as pBL (XE) (1-332).
Next, from the BamHI recognition sequence of this clone pBL (XE) (1-332) to amino acids 163 (366-489) was changed to an E. coli preferred codon. The synthetic oligonucleotides 13-20 shown in the table were prepared, and the synthetic oligonucleotides 13 and 14; 15 and 16; 17 and 18 were added to 0.1 mM ATP, using T4 kinase (Pharmacia) in the same tube. Phosphorylation was performed in a solution of 10 mM Tris-acetate, 10 mM Mg-acetate, and 50 mM K-acetate. Furthermore, 1/10 amount of 100 mM Tris / HCl (p7.5), 100 mM MgCl₂, 500 mM NaCl solution was added, boiled in a water bath for 3 minutes, and allowed to stand to obtain double-stranded DNA. Next, three sets of double-stranded DNAs 13 and 14; 15 and 16; 17 and 18 were ligated using a DNA ligation kit (Takara Shuzo), and 19 and 20 synthetic oligonucleotides were used as primers. PCR reaction was performed. The PCR product thus obtained was digested with BamHI and HidIII and then electrophoresed on a 2% agarose gel, and a fragment of about 130 bp was recovered using a prep-A-gene DNA purification kit. This was subcloned into pBL (XE) (1-332) digested with BamHI and HidIII (the host used E. coli DH5). Among the obtained clones, a clone having the base sequence shown in Table 15 was selected by sequencing, and this was designated as pBL (XH) (1-163).
[Table 15]

Furthermore, for the purpose of increasing the expression level of human TPO (amino acids 1-163) and preventing degradation by N-terminal protease, the amino acid at position 1 of human TPO (amino acids 1-163) is changed from Ser to Ala. Mutant human TPO (amino acid 1-163) that converts the amino acid at position 3 from Ala → Val and further encodes Lys at position −1 and Met at position −2 (hereinafter referred to as “h6T ( An expression vector for expressing 1-163) ”) was constructed. Four types of synthetic oligonucleotides shown in the table were prepared, and 2-9 and 3-3 synthetic oligonucleotides were prepared with 1 mM ATP, 10 mM Tris-acetate, 10 mM Mg-acetate, and 50 mM K-acetate using T4 kinase (Pharmacia). Phosphorylated in solution. In order to make these synthetic oligonucleotides into two double-stranded DNAs, single-stranded DNAs of combinations of 1-9 and 2-9; 3-3 and 4-3 were converted to 10 mM Tris / HCl (pH 7.5). , 10 mM MgCl₂, Annealed in a solution of 50 mM NaCl. Next, two sets of double-stranded DNAs 1-9 and 2-9; 3-3 and 4-3 were reacted using DNA Ligation Kit (Takara Shuzo). The DNA obtained by this ligation reaction was subcloned into pBL (XH) (1-163) digested with XbaI and NruI, and clones correctly substituted with the synthetic oligonucleotides shown in Table 16 were selected by analyzing the nucleotide sequence. (E. coli DH5α was used as the host), and this was designated pBL (XH) h6T (1-163).
[Table 16]

After digesting pBL (XH) h6T (1-163) with XbaI and HindIII, a fragment having a size of about 500 bp encoding mutant human TPO amino acid 1-163 was recovered and purified with a prep-A-gene DNA purification kit. It was purified and cloned into pCFM536 (Japanese translations of PCT publication 60-501988) digested with XbaI and HindIII (using E, coli JM109 previously transformed with pMW1 (ATCC No. 39933)). The obtained clone was designated as pCFM536 / h6T (1-163), and an E. coli strain having this expression vector was used as a mutant human TPO, ie, a transformant for expressing h6T (1-163). This expression plasmid contains the DNA sequence shown in the sequence listing (SEQ ID NO: 11).
<Example 43>
Expression of h6T (1-163) in E. coli
The expression plasmid pCFM536 is regulated by the λPL promoter, which is itself under the control of the cl857 repressor gene. The transformed strain obtained in <Example 42> was shaken and cultured overnight at 30 ° C. in 60 ml of LB medium containing 12.5 μg / ml of ampicillin and 12.5 μg / ml of tetracycline, and 25 ml of this culture solution was mixed with 50 μg / ml of ampicillin. In addition to 1000 ml of LB medium containing, OD is A₆₀₀Was shaken at 30 ° C. until 1.0 to 1.2. Next, about 330 ml LB medium at 65 ° C. was added so that the final temperature was 42 ° C., and further cultured with shaking at 42 ° C. for 3 hours to induce the expression of h6T (1-163).
<Example 44>
Purification and activity confirmation of h6T (1-163) derived from clone pCFM536 / h6T (1-163) encoding human TPO base sequence expressed in E. coli
10 ml of water was added to 3.6 g of frozen recombinant cells produced in h6T (1-163) and suspended, and the cells were crushed with a high-pressure crusher. The precipitate fraction is collected by centrifugation, and most of the mixed proteins and bacterial cell components are removed. After adding 7 ml of water to the collected fraction containing h6T (1-163) and suspending, 3 ml of 1M Tris buffer pH 8.5 was added with stirring, and urea (final concentration 8M), hydrochloric acid was added. Guanidine (final concentration: 6M) and sodium N-lauroyl sarcosine (final concentration: 2%) were added and solubilized by stirring at room temperature for 5-20 minutes. This was diluted 10-fold with 20 mM Tris buffer pH 8.5, and the protein was unwound (refolded) overnight at 4 ° C. At this time, in addition to air oxidation, glutathione and copper sulfate were added as additives. H6T (1-163) was recovered in the supernatant by centrifugation. As a result of analyzing each collected fraction by SDS-PAGE in the presence of a reducing agent, a band having a molecular weight of about 18 KDalton was detected as the main protein band of this fraction (purity of 30-40). %). This band was expressed as a mutant TPO derived from pCFM536 / h6T (1-163) as a result of SDS-PAGE, transferred to a PVDF membrane, and subjected to N-terminal sequence analysis by the method described in Example 1. It was confirmed to contain the sequence of the power TPO.
Each fraction was sufficiently dialyzed against IMDM culture medium and then evaluated in a rat CFU-MK system. As a result, TPO activity was recognized in a dose-dependent manner in all fractions.
<Example 45>
Preparation of anti-TPO peptide antibody and detection of TPO by SDS-PAGE → Western analysis
If an anti-TPO antibody can be produced, the protein of TPO can be detected by an immunological technique. Therefore, three regions (shown in Table 17) that were considered to be relatively suitable as antigens were selected from the amino acid sequence of rat TPO that was first identified, and Tam (Proc. Natl. Acad. Sci, USA, 85, 5409-5413, 1988), a 4-chain Multiple Antigen Peptide (MAP) type peptide was synthesized, and 100 μg each of 8 rabbits were immunized 8 times. As a result, these antisera could be obtained. It was.Amino acid sequences contained in synthetic peptide antigens
[Table 17]

Next, among these antisera, first, anti-RT1 peptide and anti-RT2 peptide antibody were analyzed for IgG fractions (2344 mg and 1920 mg, respectively) using a protein A column (PROSEP-A; manufactured by Bioprocessing Ltd., catalog number 8427). Of these, 54 mg and 32 mg were taken and biotinylated by coupling with active biotin (NHS-LC-Biotin II, manufactured by PIERCE, catalog number 21336). . Similar to the method described in Example 1, the preparation containing recombinant TPO was subjected to SDS-PAGE, then electroblotted onto PVDF or nitrocellulose membrane, and these biotinylated antibodies were used as primary antibodies by a conventional method. Western analysis was performed.
That is, the membrane after blotting was washed with 20 mM Tris-HCl, 0.5 M NaCl (pH 7.5) (TBS) for 5 minutes, washed with TBS containing 0.1% Tween 20 (TTBS) twice for 5 minutes, and then the blocking agent. (BlockAce, manufactured by Dainippon Pharmaceutical Co., Ltd., catalog number uk-B25) was treated for 60 minutes. Next, after a post-treatment for 60 minutes with a TTBS solution containing a 10 μg / ml biotinylated anti-TPO peptide antibody, 0.05% BSA, 10% BlockAce, it was washed twice with TTBS for 5 minutes. Next, alkaline phosphatase-labeled avidin (manufactured by Leinco Technologies, catalog number A108) was treated with a solution containing 10% BlockAce, diluted 5,000 times with TTBS solution for 30 minutes, washed twice for 5 minutes with TTBS, and washed with TBS for 5 minutes. After washing, color was developed with an alkaline phosphatase substrate (Bio-Rad, catalog number 170-6432). The above Western analysis was performed at room temperature.
As a result, not only various recombinant rat TPO expressed in COS1 cells but also various recombinant human TPO expressed in COS1 cells and E. coli (specifically, plasmid pHTP1 or plasmid expressed in COS1 cells described in the previous examples) pHTF1-derived human TPO and its N-linked sugar chain-cut TPO, GST-TPO (1-174) expressed in Escherichia coli and TPO after its thrombin digestion, h6T (1-163), a mutant TPO expressed in Escherichia coli )). And it became possible to use for the analysis of these various recombinant human TPO.
It was shown that it is possible to produce an anti-human TPO peptide antibody in the same manner as described above. The antibodies obtained by these techniques can be applied not only to Western analysis but also to all immunological techniques using antibodies that are usually considered, including purification of TPO using an antibody column.
Further, among the amino acid sequence of human TPO shown in SEQ ID NO: 6, six regions (shown in Table 19) considered to be relatively suitable as antigens were selected, and Tam (Proc. Natl, Acad. Sci. USA, 85, 5409-5413, 1988), a 4-chain Multiple Antigen Peptide (MAP) type peptide was synthesized and immunized in two rabbits each in 100 μg over 8 times.
When a single-chain peptide in which a cysteine residue was bonded to the C-terminus of each peptide region shown in Table 19 was separately synthesized, and this was used as a test antigen, the antibody titer was examined using an enzyme immunoassay. Since an increase in antibody titer was also confirmed for sera, these were designated as antisera.
Since the antibody was immunized with 2 rabbit antisera per antigen peptide, the antibody was prepared separately for each rabbit. Specifically, the anti-HT1 peptide antibody is referred to as an anti-HT1-1 peptide antibody and an anti-HT1-2 peptide antibody, respectively, by distinguishing them from rabbits derived from these.
The following is an example of purification for anti-HT1-1 peptide antibody.
First, 30 mg of a single-chain peptide of HT1 to which a cysteine residue was bound was bound to 12 ml of SulfoLink coupling gel (Pierce, catalog number 44895). That is, the peptide solution containing the antigen was coupled for 15 minutes to a gel equilibrated with 6 volumes of coupling buffer (50 mM Tris) 5 mM EDTA-Na pH 8.5). Next, after leaving still for 30 minutes, the gel was washed with a coupling buffer having a volume three times the gel volume. Next, a coupling buffer containing 0.05 M L-Cystein-HCl was added at a rate of 1 ml / ml gel to block unreacted groups for 15 minutes. Next, after leaving still for 30 minutes, the gel was washed with a coupling buffer having a volume 8 times the gel volume. The above coupling was performed at room temperature. In this way, the peptide containing the antigen region was covalently bound to the gel at a coupling efficiency of 28.3%, and an antigen peptide antigen column having 0.8 mg peptide bound per 1 ml gel was prepared. Next, 76.7 ml (protein mass: 3620 mg) of 78.4 ml of antiserum containing anti-HT1-1 peptide antibody after whole blood collection is preliminarily 50 mM phosphate buffer containing 150 mM NaCl and 0.05% sodium azide. It was added to the antigen column equilibrated with (pH 8), and further washed with the same buffer to obtain 105.9 ml of a flow-through fraction (protein mass: 3680 mg). Next, the adsorbed fraction was eluted with 0.1 M citrate buffer (pH 3.0). Immediately after addition, 21.1 ml of 0.1 M carbonate buffer (pH 9.9) was neutralized, followed by ultrafiltration (Amicon The purified anti-antigen was concentrated in a solution of 50 mM phosphate buffer (pH 8) containing 11.2 ml (protein mass: 77.7 mg) of 150 mM NaCl and 0.05% sodium azide. An HT1-1 peptide antibody could be obtained.
Similarly, anti-HT1-2 peptide antibody (60.0 mg), anti-HT2-1 peptide antibody (18.8 mg), anti-HT2-2 peptide antibody (8.2 mg) and the like could be obtained. Anti-HT3-6 peptide antibodies can be obtained in the same manner.
Affinity-purified anti-HT1-1 peptide antibody, anti-HT1-2 peptide antibody, anti-HT2-1 peptide antibody, and anti-HT2-2 peptide antibody were each taken in 3 mg, and activated biotin (NHS-LC-Biotin II, manufactured by PIERCE, Biotinylated by coupling with catalog number 21336).
[Table 19]

A recombinant human TPO sample partially purified from the culture supernatant of CHO cells introduced and expressed with the gene encoding the amino acid sequence is subjected to SDS-PAGE according to a standard method, and then electroblotted to PVDF or nitrocellulose membrane. When Western analysis was performed in the same manner as in the case of the above-described anti-RT peptide antibody, it was confirmed that human TPO was recognized and detected by each purified antibody. <Example 46>
Preparation of anti-TPO peptide antibody column
Since the anti-rat TPO peptide antibody obtained in Example 45 was able to recognize rat and human TPO, the immunoglobulin (IgG) fraction of the anti-RTI peptide antibody and anti-RT2 peptide antibody was as follows: An anti-TPO peptide antibody column was prepared by binding to a chromatography carrier. The antibody used as the material was derived from two rabbit antisera for each antigen peptide, and was prepared by dividing the antibody for each rabbit. Specifically, anti-RT1 peptide antibodies are referred to as anti-RT1-1 peptide antibodies and anti-RT1-2 peptide antibodies, respectively, and anti-RT2 peptide antibodies are referred to as anti-RT2-1 peptide antibodies and anti-RT2-2 peptide antibodies, respectively. Call it. Since these were separately prepared, the anti-RT1 peptide antibody column was prepared in two types (referred to as anti-RT1-1 antibody column and anti-RT1-2 antibody column, respectively), and the anti-RT2 peptide antibody column was prepared in two types (respectively anti-RT2). -1 antibody column, referred to as an anti-RT2-2 antibody column), and an anti-RT1-2 peptide antibody and an anti-RT2-1 peptide antibody mixed and coupled to a gel (anti-RT1-2 + 2-1 mixed antibody column) ) And 5 types were prepared.
50 mM Na Phosphate, 0.15 M NaCl containing each antibody at a concentration of 5 mg / ml (in the case of anti-RT1 + 2mix antibody column, 2.5 mg / ml mixed with equal amounts of anti-RT1-2 peptide antibody and anti-RT2-1 peptide antibody) The solution was taken as pH 8.0), and 2.31 ml of the solution was combined with 1.54 m volume of swollen formyl activated gel (Formyl-Cellulofine, manufactured by Chisso Corporation), and subjected to a coupling reaction at 4 ° C. for 2 hours. Next, 1.1 ml of a reducing agent solution (Trimethylamine borane (TMAB), manufactured by Seikagaku Corporation, catalog number 680246) with a concentration of 10 mg / ml was added, and the mixture was further reacted for 6 hours. It was collected. An operation of adding 10 ml of purified water and collecting only the gel part by centrifugation was repeated 4 times to remove unreacted antibodies. Next, 4.6 ml of blocking buffer (0.2M Na Phosphate, 1M ethanolamine (pH 7.0)) and 1,1 ml of a reducing agent solution were added, and the mixture was treated at 4 ° C. for 2 hours or longer to react with unreacted. The active group of the gel was blocked. Finally, the gel is washed with purified water and DPBS using centrifugation, filled into a small column tube, washed with 3M potassium thiocyanate solution and 0.1M glycine-HCl (pH 2.5) solution, and then again with DPBS. Equilibrated and stored.
In each of the five anti-TPO peptide antibody gels, anti-RT1-1 antibody column, anti-RT1-2 antibody column, anti-RT2-1 antibody column, anti-RT2-2 antibody column, and anti-RT1-2 + 2-1 mixed antibody column, The coupling efficiencies of the IgG fractions reached 97.4%, 95.4%, 98.4%, 98.3%, and 99.4%, respectively. The amount of IgG fraction coupled per gel volume was 5.6 mg / ml gel, 5.8 mg / ml gel, 5.7 mg / ml gel, 5.7 mg / ml gel, and 2.9 mg / ml gel. Met. Therefore, it was confirmed that there was no significant difference in coupling efficiency and coupling amount depending on the origin of the antibody and the antigen, and therefore, the experiment shown in Example 47 was conducted using these antibody columns.
<Example 47>
Purification of culture supernatant-derived TPO obtained by transfecting COS1 cells with expression vector pHTP1 by anti-TPO antibody column → reverse phase column chromatography to confirm biological activity
For evaluation of the following purified samplesin vitroThe assay method used was the M-07e assay system. A partially purified preparation of TPO derived from the culture supernatant of Example 35 obtained by transfecting COS1 cells with the expression vector pHTP1 (a fraction that is not the main fraction from which TPO was recovered, ie, Macro-Prep Methyl HIC Column Fraction F1, F3 followed by F3 eluted with 20 mM Na Citrate, pH 5.8, and F1 and F3 of SP Sepharose Fast Flow column were combined into a TPO subpool fraction (5463.79 ml, protein concentration 0). 490 mg / ml, total protein mass 2676 mg, relative activity 12100, relative activity 323800000), and concentrated first with an ultrafiltration unit (manufactured by Filtron, Omega Ultraset molecular weight 8000 cut), and the solvent was replaced with DPBS. , Small volume Then, it was finally made into a DPBS solution containing 120.2 ml of 0.05% sodium azide using a YM-10 membrane-equipped ultrafiltration unit (Amicon) and then prepared in Example 46. All five types of anti-TPO peptide antibody gels were mixed and put together into one antibody column (diameter 1.6 cm, bed height 4.8 cm, referred to as anti-RT1 + 2 mixed antibody column), flow rate 0.033 ml / min at room temperature After completion of the addition, the TPO sub-pool fraction was collected until the ultraviolet absorption of the eluate was sufficiently reduced with DPBS, and further passed through F1 (concentrated with an ultrafiltration unit with a YM-10 membrane (Amicon)). 82.62 ml, protein concentration of 14.3 mg / ml, total protein mass of 1184 mg, relative activity of 67500, relative activity of 79900000) was collected, followed by acidic eluent ( Column adsorbed fraction F2 (92.97 ml, protein concentration 0.12 mg / ml, total protein mass 11.1 mg, relative activity 257100, relative activity 2860000) eluted with 1 M glycine-HCl (pH 2.5)) The flow-through fraction F1 had a considerable amount of TPO activity, but the TPO adsorbed on the antibody column was found to have increased by about 20 times, and thus further purified. Capcell Pak C1 300A using 1-propanol containing 0.1% TFA in A and 0.05% TFA in developing solvent B (made by Shiseido, catalog number C1 TYPE: SG300A; diameter 4.6 mm, bed height 150 mm) , Diameter 4.6 mm) with a pre-column with a bed height of 35 mm) F2 obtained with an antibody column in a column One-tenth volume of developing solvent B was added to the column and injected into a Capcell Pak C1 300A column equilibrated with 20% B at a flow rate of 0.4 ml / min. After completion of the injection, the mixture was eluted with 20% B for 5 minutes, and then developed with a linear concentration gradient of 50 minutes from 20% B to 40% B, and each 1 ml (2.5 min) was collected in a polypropylene tube.
Take 2 μl (1/500 fraction) of each, add HSA, concentrate by ultrafiltration, and finally make 0.25 ml of IMDM assay culture solution containing 0.02% HSA. Identified minutes. As a result, a sample showing strong TPO activity in tube numbers 20 to 23 (range of 28.5 to 32.5% in propanol concentration) could be obtained. Further, when these samples were subjected to Western analysis by the method described in <Example 45>, TPO having an apparent molecular weight of 60000-70000 under reduction was certainly present with respect to the DPCIII molecular weight marker. Apart from that, it was found that molecules having molecular weights of 32000-43000 and 20000-30000 were also present.
<Example 48>
Confirmation of biological activity of TPO purified from the culture supernatant obtained by transfecting COS1 cells with expression vector pHTP1 and purified to the stage of Capcell Pak C1 300A column
The fraction of TPO activity purified in Example 36, ie Capcell Pak C1300A column tube numbers 20-23 (range of propanol concentration 28.5-32.5%) was collected and 0.21 ml of glycerol was added. Concentrated by centrifugal evaporation. To this, 0.21 ml of 6M guanidine hydrochloride solution was added to dilute to 1 ml with DPBS, and 0.01% HSA was added using a Sephadex G25 column (NAP-10, Pharmacia Biotech, catalog number 17-0854-01). The DPBS solution was replaced with 1.1 ml of 0.01% HSA, and finally TPO-active fraction FA (2.6 ml) was prepared. To examine the biological TPO activity of this preparation,in  vivoThe assay was performed. That is, 100 μl of the active fraction (including a relative activity of 87400 in the M-07e assay system) was subcutaneously administered once daily for 5 days to 4 ICR male mice (8 weeks old) per group. As a control, 100 μl of DPBS containing 0.01% HSA was subcutaneously administered on the same schedule. Blood was collected from the fundus immediately before the start of administration and the next day after the end of administration, and the platelet count was measured using a blood cell measuring device (F800, manufactured by Toa Medical Electronics Co., Ltd.). In the TPO-administered group, an average increase of 1.42 times as much platelets was observed after the end of administration as compared to before administration, and it was 1.23 times as high as the number of platelets in the control group. Significant difference (p <0.05, Student t-test) was observed. From this result, it was clarified that the above-mentioned human TPO produced in animal cells has a platelet increasing action in vivo.
<Example 49>
Confirmation of biological activity of crude TPO fraction obtained by cation exchange column using 33L of culture supernatant obtained by transfecting COS1 cells with expression vector pHTP1
(1) For evaluation of the following purified samplesin vitroThe assay method used was the M-07e assay system. In the same manner as described in Example 36, a serum-free culture supernatant obtained by transfecting COS1 cells with 33 L of expression vector pHTP1 was obtained, and the filtrate of a 0.22 μm filtration filter was taken. This was concentrated about 10 times with an ultrafiltration unit (Millipore, PLGC pericone cassette, molecular weight 10,000 cuts), p-APMSF, a protease inhibitor, was added to a final concentration of about 1 mM, and a volume of 2018 ml (protein concentration 3.22 mg). / Ml, total protein mass 6502 mg, relative activity 66000, relative activity 429100000). Next, the concentration process was repeated with an ultrafiltration unit (manufactured by Filtron, Omega Ultra set, molecular weight 30000 cut), a fraction having a molecular weight of 30000 or more (volume 1190 ml), protein concentration 2.54 mg / ml, total protein mass 3020 mg, relative And a fraction having a molecular weight of 30000 or less (volume 2975 ml, protein concentration 0.471 mg / ml, total protein mass 1402 mg, relative activity 4500, relative activity 6310000). The presence of TPO activity in fractions having a molecular weight of 30000 or less indicates that in the process of expression and secretion of TPO in animal cells, the culture supernatant may eventually contain low molecular weight TPO. Show. On the other hand, a fraction having a molecular weight of 30000 or more was further replaced with 20 mM sodium citrate buffer (pH 6.1), and SP Sepharose Fast Flow (manufactured by Pharmacia Biotech) pre-equilibrated with 20 mM sodium citrate buffer (pH 6.1). , Catalog number 17-0729-01; diameter 2.6 cm, bed height 29 cm) and added at a flow rate of 5 ml / min. After completion of the addition, the product was further washed and eluted with 20 mM sodium citrate buffer (pH 6.1). Next, 20 mM sodium citrate buffer (pH 6.1) is used as the developing solvent A, and a solution containing 1 M NaCl in the developing solvent A is used as the developing solvent B, and it is 215 minutes from 0% B to 50% B at a flow rate of 3 ml / min. Further, it was developed with a linear concentration gradient of 20 minutes from 50% B to 100% B, and collected in 30 ml (10 min) portions in polypropylene tubes. Taking a part of these and examining the distribution of activity in the M-07e assay system, it was found that TPO activity was eluted over a wide range. Therefore, the fraction eluted with 50 mM or less NaCl, including the passage, was collected as F1, and the fraction eluted with 50-1000 mM NaCl as the main fraction of TPO was collected as F2. F1 has a volume of 1951 ml (protein concentration 2.05 mg / ml, total protein mass 3994 mg, relative activity 13500, relative activity 53900000), F2 has a volume of 649.8 ml (protein concentration 1.11 mg / ml, total protein mass 721 mg, relative activity) 268,000, relative activity 193000000).
(2) Confirmation of biological activity of SP Sepharose Fast Flow F2 Take 2.5 ml of SP Sepharose Fast Flow F2 fractionated in (1), and use Sephadex G25 column (NAP-25, manufactured by Pharmacia Biotech, catalog number) 17-0852--01) was replaced with 3.5 ml of DPBS solution containing 0.01% HSA, and this sample (protein concentration 1.46 mg / ml) was examined for biological TPO activity.in vivoThe assay was performed. That is, 4 ICR male mice per group (8 weeks old) were subcutaneously administered once a day for 5 days with an active fraction of 100 μ1 (including a relative activity of 3150 in the M-07e assay system). As a control, 100 μl of DPBS containing 0.01% HSA was subcutaneously administered on the same schedule. Blood was collected from the fundus immediately before the start of administration and the next day after the end of administration, and the platelet count was measured using a blood cell measuring device (F800, manufactured by Toa Medical Electronics Co., Ltd.). In the TPO-administered group, an average 1.29-fold increase in platelets was observed after the end of administration compared to before administration, and 1.12-fold higher than the control group. Significant difference (p <0.05, Student t-test) was observed. From this result, it was clarified that the above-mentioned human TPO produced in animal cells has a platelet increasing action in vivo.
<Example 50>
Construction of pDEF202-ghTPO, a recombinant expression vector for human TPO chromosomal DNA for CHO cells
The vector pDEF202 was treated with restriction enzymes Kpnl and Spel, and a fragment containing the smaller mouse DHFR minigene and SV40 polyadenylation signal was recovered by agarose gel electrophoresis, and then this fragment and plasmid pEFHGTE containing human TPO chromosomal DNA were restricted. The vector DNA from which the region containing the SV40 polyadenylation signal after treatment with the enzymes KpnI and SpeI was removed was ligated with T4 DNA ligase (Takara Shuzo) to obtain the expression vector pDEF202-ghTPO. This plasmid contains the SV40 replication initiation region, human elongation factor-1-alpha promoter, SV40 early polyadenyl site, mouse DHFR minigene, pUC18 replication initiation region, β-lactamase gene (Amp^r), And human TPO chromosomal DNA is connected downstream of the human elongation factor-1-alpha promoter.
<Example 51>
Expression of human TPO chromosomal DNA by CHO cells
Α minimum essential medium containing 10% fetal bovine serum in CHO cells (dhfr-strain, Urlaub and Chasin; Proc. Natl. Acad. Sci. USA; 77, 4216, 1980) in 6 cm diameter plate (Falcon) (Α-MEM (−), thymidine and hypoxanthine added) were cultured and grown, and this was transformed by the calcium phosphate method (CellPhect Pharmacia). That is, Buffer A: 120 μl and H20: 120 μl were added to and mixed with 10 μg of pDEF202-ghTP0 plasmid prepared in Example 50, and then left at room temperature for 10 minutes. Next, 120 μl of buffer B was added to this solution, mixed again, and allowed to stand at room temperature for 30 minutes. After dropping this DNA solution onto the plate, CO₂The cells were cultured for 6 hours in an incubator. After removing the medium from the plate and washing twice with α-MEM (−), α-MEM (−) containing 10% dimethyl sulfoxide was added and treated at room temperature for 2 minutes. Subsequently, 10% dialyzed fetal bovine serum-containing non-selective medium (additional α-MEM (−), hypoxanthine, thymidine added) was added and cultured for 2 days. Selection was made with MEM (-), hypoxanthine, and thymidine not added. For selection, after trypsinizing the cells, each 6 cm diameter plate is divided into 5 10 cm diameter plates or 20 24-well plates, and then the culture is continued while changing the medium every 2 days. It was carried out by things. As a result of measuring the human TPO activity in the culture supernatant of the plate or well in which the cells had grown using the Ba / F3 assay, human TPO activity was observed.
CHO cells can also be transformed by co-transfecting CHO cells with pEFHGTE and pMG1.
<Example 52>
Construction of expression vector for Escherichia coli of human TPO (amino acid 1-163) (hereinafter referred to as “hMKT (1-163)”) having Lys added at position −1 and Met added at position −2. Confirmation of expression
In order to express a protein in which Met was added to the -1 position of the amino acid sequence of SEQ ID NO: 6 (positions 1 to 163) at the -1 position, it was shown in Table 18 below in the same manner as in Example 42. Using the synthetic oligonucleotides -13, 2-13, 3-3, and 4-3, an expression vector pCFM536 / hMKT (1-163) for E. coli of hMKT (1-163) was constructed.
[Table 18]

This expression plasmid contains the DNA sequence shown in the sequence listing (SEQ ID NO: 12). Further, in the same manner as in Example 43, expression of hMKT (1-163) was induced.
The expressed protein obtained here was transferred to a PVDF membrane after SDS-PAGE, and as a result of N-terminal amino acid sequence analysis, it was confirmed that it contained the amino acid sequence of hMKT (1-163) to be expressed.
<Example 53>
Refolding of mutated human TPO, h6T (1-163) derived from clone pCFM536 / h6T (1-163) encoding human TPO base sequence expressed in Escherichia coli with guanidine hydrochloride and glutathione-Purification-Biological activity 3 ml of water was added to 1.2 g of the frozen recombinant cells produced in h6T (1-163) produced in Example 43, and the cells were suspended using a high-pressure crusher. The precipitate fraction is collected by centrifugation, and most of the mixed proteins and bacterial cell components are removed. Water was added to the collected fraction containing h6T (1-163) to suspend it to make a final volume of 4 ml. 1 ml of 1M Tris buffer pH 8.5 was added while stirring, and then 20 ml of 8M guanidine hydrochloride was added and solubilized by stirring at room temperature for 5 minutes. This was diluted 10-fold with 20 mM Tris buffer pH 8.5, 5 mM reduced glutathione and 0.5 mM oxidized glutathione were added, stirred and dissolved, and then allowed to stand overnight at 4 ° C. H6T (1-163) was recovered in the supernatant by centrifugation. 160 ml of the obtained supernatant was concentrated using a YM10 ultrafiltration membrane (Amicon), and the buffer solution was replaced with DPBS to a final volume of 3.4 ml (protein concentration 2.18 mg / ml). . This fraction was sufficiently dialyzed against IMDM culture medium and then evaluated with a rat CFU-MK assay system. As a result, it showed strong TPO activity (relative activity 58000).
About the TPO active fraction prepared by the above methodin  vivoI went to Assey. One group of 4 ICR male mice (7 weeks old) was subcutaneously administered once a day for 5 days with an active fraction of 170 μl (including a relative activity of 22000 in the rat CFU-MK assay system). As a control, 6 similar mice per group were administered subcutaneously with 170 pl DPBS on a similar schedule. Blood was collected from the fundus immediately before the start of administration, the next day after the end of administration, and 3 days after the end of administration, and the platelet count was measured using a blood cell measuring device (manufactured by Toa Medical Electronics, F800). In the TPO-administered group, an average 2.15-fold increase in platelets was observed on the day following the end of administration compared to the pre-administration, and the effect persisted 3 days after the end of administration, maintaining a high value of 2.13 times. did. On both the day after the end of the administration and 3 days after the administration, the platelet count was 1.73 times and 1.80 times higher than that of the control group on each day, and there was a significant difference (p <0.001, Student t-test).
From this result, it was clarified that human TPO produced from E. coli and prepared by the above-described method has a platelet increasing action in vivo.
<Example 54>
Refolding of clone pCFM536 / h6T (1-163) -derived mutant human TPONh6T (1-163) encoding human TPO base sequence expressed in E. coli using sodium N-lauroyl sarcosine and copper sulfate-purification-biology Activity confirmation
3 ml of water was added to 0.6 g of frozen recombinant cells produced in h43T (1-163) produced in Example 43, suspended, and the cells were crushed with a high-pressure crusher, and the precipitate fraction was collected by centrifugation. did. After suspending by adding 3.1 ml of water to the precipitate fraction, 0.19 ml of 1M Tris buffer pH 9.2, 11.25 μl of 1M DTT, 38 μl of 0.5MEDTA, 0.38 ml of 10% deoxycholic acid Sodium was added with stirring and stirred at room temperature for 40 minutes. The precipitate fraction was collected by centrifugation, and most of the mixed proteins, bacterial cell components and the like were removed. After adding 4 ml of water to the precipitate and suspending, the precipitate fraction containing h6T (1-163) was collected by centrifugation. After 3.8 ml of water was added and suspended in the obtained precipitate fraction, 0.2 ml of 1M Tris buffer pH 8 and 1 ml of 10% N-lauroyl sarcosine sodium were added with stirring and stirred at room temperature for 20 minutes. Solubilized. To this, 5 μl of 1% copper sulfate was added and stirred overnight (about 20 hours) at room temperature. The supernatant fraction containing h6T (1-163) was collected by centrifugation, then 5 ml of water, 10 ml of 20 mM Tris buffer pH 7.7 was added to 5 ml of the supernatant, and 2.6 g of Dowex 1-x4,20. −50 mesh, chloride form ion exchange resin was added and stirred for 90 minutes. After collecting the non-adsorbed fraction of the ion exchange resin using a glass filter, the supernatant was collected by centrifugation. The obtained supernatant was added to an SP Sepharose Fast Flow cation exchange column equilibrated with 20 mM Tris buffer solution pH 7.7, and elution was performed in the same buffer solution by a linear concentration gradient method from NaCl concentration 0 M to 500 mM. After the dialyzed fraction was sufficiently dialyzed against the IMDM culture solution and evaluated with the rat CFU-MK assay system, the fraction eluted at a NaCl concentration of about 100 mM in SP Sepharose Fast Flow cation exchange chromatography was strong. The activity of TPO (relative activity about 19000000) was shown. This fraction was concentrated 1.6 times (2.5 ml, protein concentration 25 μg / ml) using an ultrafree CL fraction molecular weight 5000 ultrafiltration unit (manufactured by Millipore, model number UFC4LCC25). As a result of SDS-PAGE analysis of the same fraction in the presence of a reducing agent, a TPO band was detected as the main band (purity 70-80%).
About the TPO active fraction prepared by the above methodin  vivoThe assay was performed. One group of 4 ICR male mice (8 weeks old) was subcutaneously administered once a day for 5 days with 100 μl of the active fraction (including a relative activity of 47500 in the rat CFU-MK assay system). As a control, 20 mM Tris buffer pH 7.7100 μl containing 100 mM NaCl. Were administered subcutaneously on the same schedule. Blood was collected from the fundus immediately before the start of administration and the next day after the end of administration, and the platelet count was measured using a blood cell measuring device (F800, manufactured by Toa Medical Electronics Co., Ltd.). In the TPO-administered group, an average increase of 1.73 times platelets was observed after the end of administration compared to before administration, and it was 1.59 times higher than the number of platelets in the control group. A significant difference (p <0.01, Student t-test) was observed. From this result, it was clarified that human TPO produced from E. coli and prepared by the above-described method has a platelet increasing action in vivo.
<Example 55>
Mass culture of CHO cells
Mass cultivation of a human TPO-producing CHO cell line (CHO28-30 cells, 25 nM MTX resistant) obtained by transfecting CHO cells with human TPO expression plasmid pDEF202-hTP0-P1 according to Example 32 is as follows. Was carried out. The cells were grown in culture using DMEM / F-12 medium (GIBCO) containing 25 nM MTX and 10% FCS. The cells were detached using a trypsin solution and then placed in a Falcon roller bottle (Falcon 3000) containing 200 ml of the same medium at 1 × 10.⁷The cells were inoculated and cultured at 37 ° C. with a rotation speed of 1 rpm for 3 days. After 3 days, the culture medium was removed by aspiration, and the cell culture surface was rinsed with 100 ml of PBS. Then, 200 ml of DMEM / F-12 medium (GIBCO) containing no 25 nM MTX and 10% FCS was added, and the temperature was 37 ° C., 1 rpm. The culture was cultured at a rotational speed for 7 days, and after 7 days, the culture supernatant was collected and used as a starting material for the next purification operation. The above operation was performed for 500 roller bottles to obtain 100 L of culture supernatant.
<Example 56>
Purification of human TPO from human TPO-producing CHO cell line
(1) About 100 L of serum-free culture supernatant was obtained from Example 55, and the filtrate of a 0.22 μm filtration filter was taken. This was concentrated with an ultrafiltration unit (Millipore, PLTK pericone cassette, molecular weight 30000 cut), and the solvent was replaced with purified water, and p-APMSF (manufactured by Wako Pure Chemical Industries, Ltd.) as a protease inhibitor was final concentration 1 mM. And Pefabloc SC (manufactured by Merck) were added at a final concentration of 0.35 mM to obtain a fraction with a molecular weight of 30000 or more in a volume of 3628 ml (protein concentration 1.60 mg / ml), total protein mass 5805 mg, relative activity 1230000, relative activity 7149000000). It was. When this fraction was examined by Western analysis described in Example 45, it was revealed that TPO protein was present in the molecular weight range of 66000 to 100,000. Upon further examination, it was found that, apart from this, a lower molecular weight TPO was also contained.
Separately, the ultrafiltrate having a molecular weight of 30000 or less was concentrated by an ultrafiltration unit (Millipore, PLGC pericon cassette, molecular weight 10,000 cut), and the volume was 1901 ml (protein concentration 0.36 mg / ml, total protein mass 684 mg, relative Activity 245500, relative activity 1667900000), it was confirmed that low molecular weight TPO molecules were generated in the culture process.
Next, 764 g of ammonium sulfate and 144.5 ml of 0.5 M sodium citrate buffer (pH 5.5) were added to 3614 ml of a fraction having a molecular weight of 30000 or more, and a final concentration of 1.41 M ammonium sulfate, 17.7 mM sodium citrate. A 4089 ml solution containing a buffer solution was obtained, and the resulting insoluble matter was centrifuged to obtain a soluble matter.
This was a Macro-Prep Methyl HIC column (Bio-Rad, catalog number 156-0080; diameter 5 cm, bed height) which had been equilibrated in advance with 20 mM sodium citrate buffer (pH 5.5) containing 1.2 M ammonium sulfate. 24.5 cm) at a flow rate of about 25 ml / min. After the addition is complete, the eluate eluted with 20 mM sodium citrate buffer (pH 5.5) containing 1.2 M ammonium sulfate is concentrated with an ultrafiltration unit (Filtron, OMEGA Ultraset, molecular weight 8000 cut) and passed through. Minute F1 (4455 ml, protein concentration 0.400 mg / ml, total protein mass 1780 mg, relative activity 1831000) was obtained.
Next, the eluate was changed to 20 mM Na Citrate, pH 6.0, and the eluate was concentrated with an ultrafiltration unit (manufactured by Filtron, Omega Ultraset, molecular weight 8000 cut), and fraction F2 (1457 ml, protein concentration). 0.969 mg / ml, total protein mass 1411 mg, relative activity 1715000). It was confirmed by SDS-PAGE that a protein having a molecular weight of 66,000 to 100,000 was present in this F2. Furthermore, Western analysis revealed that this protein was TPO.
Next, Macro-Prep Methyl HIC column F2 (1443 ml) was pre-equilibrated with 20 mM sodium citrate buffer (pH 6.0), SP Sepharose Fast Flow (manufactured by Pharmacia Biotech, catalog number 17-0729-01; diameter) 5 cm, bed height 12 cm), and added at a flow rate of 15 ml / min. After completion of the addition, the fractions eluted with 20 mM sodium citrate buffer (pH 6.0) containing 50 mM NaCl were collected and fraction F1 (3007 ml, protein concentration 0.226 mg / ml, total protein mass 679 mg, relative activity) 88830). Next, the eluate was changed to 20 mM sodium citrate buffer (pH 5.4) containing 750 mM NaCl, and the eluted fraction F2 (931 ml, protein concentration 0.763 mg / ml, total protein mass 710 mg, relative activity 5558000). Collected. This was concentrated to 202 ml using an ultrafiltration unit (manufactured by Filtron, Omega Ultra set, molecular weight 8000 cut, and Amicon YM3 membrane).
Next, a concentrated solution (197 ml) of the TPO active fraction F2 of the SP Sepharose Fast Flow column was added to a Sephacryl S-200HR column (Pharmacia Biotech, catalog number 17-0584-05; diameter 7.5 cm, bed height 100 cm). And gel filtration was performed at a flow rate of 3 ml / min. The eluent volumes of 1200 to 1785 ml, 1785 to 2010 ml, 2010 to 2280 ml, 2280 to 3000 ml were collected, and each fraction F1 (585 ml, protein concentration 1.00 mg / ml, total protein mass 589 mg, relative activity 4118000), fraction Fraction F2 (225 ml, protein concentration 0.263 / ml, total protein mass 59.2 mg, relative activity 2509000), fraction F3 (270 ml, protein concentration 0.119 / ml, total protein mass 32.1 mg, relative activity 2535000) Fraction F4 (720 ml, protein concentration 0.0467 / ml, total protein mass 33.6 mg, relative activity 1155000) was obtained.
Thus, since the fractionated TPO activity was found to have a wide molecular weight range in the gel filtration method, Western analysis was performed after SDS-PAGE. As a result, F1 was mostly TPO having a molecular weight of 66,000 to 100,000. In contrast, in F2 and F3, TPO molecular species having molecular weights of 32000 to 60000 and molecular weights of 32000 to 42000 were present, respectively. All TPO molecules of any molecular weight had TPO activity.
When N-terminal amino acid sequence analysis was performed on TPO molecules having a molecular weight of 66,000 to 100,000, it was confirmed that the amino acid sequence of the protein encoded by the human TPO gene was included.
Moreover, about molecular weight 6600-100,000 (7) TPO molecules, N-glycanase (N-glycanase: Genzyme, catalog number 1472-00), neuraminidase (Neuraminidase: manufactured by Nacalai Tesque, catalog number 242-29 SP), end -Α-N-acetylgalactosaminidase (Endo-α-N-acetylgalactosaminidase: manufactured by Seikagaku Corporation, catalog number 1000045) and O-glycosidase (O-Glycosidase: manufactured by Boehringer Mannheim Biochemica, catalog number 1347101) When the enzyme digestion experiment was carried out with a single or a combination of cleavage enzymes and examined by SDS-PAGE, the polypeptide portion of TPO Has a molecular weight of about 36000 estimated from the theoretical value, and was found to be a glycoprotein having both an N-linked sugar chain and an O-linked sugar chain. <Example 57>
Purification of human TPO from human TPO-producing CHO cell line
(1) 211.4 g of ammonium sulfate was added to 1 L of CHO cell serum-free culture supernatant obtained in the same manner as in Example 55, and the mixture was filtered through a 0.2 μm filter (manufactured by Gelman Science, catalog number 12992). A Macro-Prep Methyl HIC column (Bio-Rad, catalog number 156-0081, diameter 50 mm, bed height 90 mm) equilibrated with 2 M ammonium sulfate, 20 mM sodium acetate buffer (pH 5.6) was supplied with a flow rate of 15 ml / Added in min. After completion of the addition, elution was performed with 450 ml of 20 mM sodium acetate buffer (pH 5.6). This eluate was added to a Vydac reverse phase C4 column (The Separations Group, catalog number 214BTP54, diameter 4.6 mm, bed height 250 mm) at a flow rate of 0.75 ml / min. After completion of the addition, after washing with 10 mM Tris buffer (pH 6.4) (developing solvent A) containing 5% ethanol for 15 minutes, 94% ethanol (developing solvent) containing 10 mM Tris buffer (pH 6.4) from developing solvent A is used. Elution with a linear gradient of 66 minutes until B). The chromatogram is shown in FIG. As a result of analysis by SDS-PAGE, a molecule having a molecular weight of about 65,000-100,000, which seems to be TPO, can be obtained in a fraction with a retention time of 68-72 minutes after the addition of the sample, and is confirmed to be a single band. (See FIG. 16). Furthermore, Western analysis confirmed that this protein was TPO.
As a result of taking a part of this sample and performing N-terminal amino acid analysis, it was confirmed that it contained the amino acid sequence of the protein encoded by the human TPO gene.
<Example 58>
Production of recombinant virus for expression of human TPO in insect cells
The pHTP1 prepared in Example 30 was digested with restriction enzymes EcoRI and NotI, subjected to 1% agarose gel electrophoresis, and a band of about 1200 bp was purified using a prep-A-gene DNA purification kit, followed by the same restriction enzyme treatment. Competent high E. coli ligated to transfer vector pVL1393 (Invitrogen). E. coli DH5 (Toyobo Co., Ltd.) was transformed. Plasmid DNA was prepared from the obtained colonies to obtain clone pVL1393 / hTPO containing all the coding region of human TPO cDNA. Plasmid DNA of this clone was prepared as described in Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989) and BaculoGold^TM  After transfection into insect cells Sf21 (manufactured by Invitrogen) using Transfection Kit (manufactured by Pharmingen), the cells were cultured in Sf-900 medium (manufactured by Life Technology) at 27 ° C. for 4 days and contained the virus. Qing was recovered. This supernatant was added to 10⁹-10⁵Dilute to double and use 1 ml of the diluted solution in a petri dish of 35 mm diameter.⁵Sf21 cells were infected for 1 hour at 27 ° C. The supernatant was extracted, 1% agarose containing Sf-900 medium was poured, and the agarose solidified, followed by culturing at 27 ° C. for 6 days under humidification. The single plaque formed here was picked up, and the virus was eluted in 200 μl of Sf-900 medium. This virus clone was infected with Sf21 cells in a 24-well plate to amplify the virus. A part of the obtained virus solution derived from a single plaque was treated with phenol / chloroform and then precipitated with ethanol to collect viral DNA. Using this viral DNA as a template, PCR was performed using primers specific to human TPO cDNA, and a recombinant virus containing human TPO cDNA was selected depending on whether or not a specific DNA fragment was amplified. Sf21 cells were infected with the supernatant containing the recombinant virus containing the human TPO cDNA obtained here, and the virus was further amplified.
<Example 59>
Confirmation of human TPO expression and activity in Sf21 insect cells
In a 175 cm 2 culture flask, Sf21 cells were cultured until they became about 80% confluent, and infected with the recombinant virus containing the human TPO cDNA prepared in Example 58 at 27 ° C. for 1 hour, and then in Sf-900 medium. And cultured at 27 ° C. for 4 days, and the supernatant was recovered. The obtained culture supernatant was NAP^TMSubstitution with IMDM culture solution using -5 column (Pharmacia) and application to rat CFU-MK assay system and M-07e assay system revealed significant TPO activity and M-07e cell proliferation in a dose-dependent manner. Promoting activity was observed. In addition, Western analysis similar to that described in Example 45 confirmed recombinant human TPO expressed in Sf21 cells.
<Example 60>
Refolding method and refolding method using clone N-lauroyl sarcosine sodium and copper sulfate derived from clone pCFM536 / h6T (1-163) derived from clone pCFM536 / h6T (1-163) expressed in Escherichia coli Folding to purification
300 g of water was added to 30 g of the frozen recombinant cell body of h6T (1-163) produced in Example 43 and suspended, and the cell body was crushed with a high-pressure crusher (10000 psi, Rannie High Pressure Laboratory), and then centrifuged. The precipitate fraction was collected. The precipitate fraction was suspended by adding 90 ml of water, and further added with stirring to make the volume 150 ml, then 9 ml of 1M Tris buffer pH 9.2, 540 μl of 1MDTT, 1.8 ml of 0.5 MEDTA, 18 ml of 10% sodium deoxycholate was added with stirring and stirred at room temperature for 30 minutes. The precipitate fraction was collected by centrifugation, and most of the mixed proteins, bacterial cell components and the like were removed. After adding 180 ml of 5 mM DTT to the precipitate and suspending, the precipitate fraction containing h6T (1-163) was collected by centrifugation. 300 ml of water was added and suspended in the resulting precipitate fraction, and water was further added while stirring to adjust the volume to 570 ml. Then, 30 ml of 1M Tris buffer pH 8 and 150 ml of 10% N-lauroyl sarcosine sodium were added while stirring. In addition, it was solubilized by stirring at room temperature for 20 minutes. 750 μl of 1% copper sulfate was added thereto, and the mixture was stirred overnight (about 20 hours) at room temperature. After collecting the supernatant fraction containing h6T (1-163) by centrifugation, 750 ml of water and 1500 ml of 20 mM Tris buffer pH 7.7 were added to 750 ml of the supernatant, and then 3 ml of polysorbate 80 (Nikko Chemicals) with stirring. ) Was added. To this solution, 600 g of Dowex 1-x4, 20-50 mesh, chlorideform ion exchange resin was added and stirred at room temperature for 90 minutes. After collecting the non-adsorbed fraction of the ion exchange resin using a glass filter, the ion exchange resin was washed with 750 ml of 20 mM Tris buffer pH 7.7. Q Sepharose Fast equilibrated with 20 mM Tris buffer pH 9.2 containing 0.1% polysorbate 80 by adding 2N sodium hydroxide to the combined solution of the ion-exchange resin non-adsorbed fraction and the washing solution to adjust the pH to 9.2. It added to the Flow anion exchange column (inner diameter 5 cm x 10 cm), and the non-adsorbed fraction was collected. The pH of the non-adsorbed fraction obtained with hydrochloric acid was adjusted to 7.2 and equilibrated with 20 mM Tris buffer pH 7.2 containing 0.1% polysorbate 80 (SP Sepharose Fast Flow cation exchange column (inner diameter 5 cm)). x 10 cm) and elution was carried out in the same buffer by a linear concentration gradient method from sodium chloride concentration of 0 M to 500 mM. The eluate was analyzed by SDS-PAGE, and the TPO elution fraction was collected. Trifluoroacetic acid was added to the TPO elution fraction to 0.1% and added to a capsule pack 5 μm 300A C1 column (inner diameter 2.1 cm × 5 cm × 2, Shiseido), then in 0.1% trifluoroacetic acid. Reversed phase HPLC was performed by a linear gradient elution method with increasing 1-propanol concentration. The eluate was fractionated into 3 fractions according to the results of SDS-PAGE analysis in the absence of a reducing agent and the elution position on reverse phase HPLC, and each was diluted 3-fold with 0.1% trifluoroacetic acid. After that, it was added to the above reverse phase HPLC column and re-chromatographed in the same manner. The obtained TPO3 fraction was subjected to Fr. Sa, Fr. As a result of analysis by SDS-PAGE in the absence of a reducing agent as Sb, Fr, Sc (see FIG. 17), molecular weights of 18 K Dalton (Fr.Sa) and 19 K Dalton (Fr.S— b) Detected as a single band in the vicinity of 18 K Dalton (Fr.Sc) (see FIG. 18). As a result of amino acid composition analysis for each, the obtained amino acid composition almost coincided with the theoretical value from sequence information, and as a result of N-terminal amino acid sequence analysis, only the expected sequence was obtained. The protein mass of each fraction obtained from the results of amino acid analysis was 0.64 mg (Fr.S-a), 1.81 mg (Fr.S-b), and 3.49 mg (Fr.S-c), respectively. Met. Each fraction was sufficiently dialyzed against IMDM culture medium and subjected to M-07e assay. As a result, the relative activity was about 1620000 (Fr.S-a), the relative activity was about 23500000 (Fr.S-b), the relative activity. TPO activity of about 746000000 (Fr.Sc) was exhibited.
<Example 61>
Refolding and purification of clone pCFM536 / h6T (1-163) -derived mutant human TPO) h6T (1-163) encoding human TPO base sequence expressed in E. coli using guanidine hydrochloride and cysteine cystine
500 g of water was added to 50 g of frozen recombinant cells produced in h43T (1-163) produced in Example 43 to suspend the cells, and the cells were disrupted with a high-pressure crusher (10000 psi, Rannie High Pressure Laboratory), and then centrifuged. The precipitate fraction was collected. Water was added to the precipitate fraction to suspend, and water was added with stirring to make the volume 250 ml, and then 15 ml of 1M Tris buffer pH 9.2, 900 μl of 1MDTT, 3 ml of 0.5 MEDTA, 30 ml of 10% Sodium deoxycholate was added with stirring, and the mixture was stirred at room temperature for 30 minutes. The precipitate fraction was collected by centrifugation, and most of the mixed proteins, bacterial cell components and the like were removed. The precipitate was suspended by adding 300 ml of 5 mM DTT, and the precipitate fraction containing h6T (1-163) was collected by centrifugation. Water was added to the collected fraction containing h6T (1-163) to suspend it, so that the final mass was 104 ml. 20 ml of 1M Tris buffer pH 8.5 was added with stirring, 376 ml of 8M guanidine hydrochloride was added, and the mixture was solubilized by stirring at room temperature for 10 minutes. While stirring, 2500 ml of 20 mM Tris buffer pH 8.5 containing 0.1% polysorbate 80 was added, and 2000 ml of 20 mM Tris buffer pH 8.5 containing 1 M guanidine hydrochloride was added, and then 5 mM cysteine and 0.5 mM cystine were added. And stirred to dissolve. After leaving still at 4 degreeC overnight, h6T (1-163) was collect | recovered in the supernatant by centrifugation. The obtained supernatant was concentrated using a prep-scale UF cartridge PLDC ultrafiltration membrane (Millipore), and the buffer solution was replaced with 20 mM Tris buffer pH 9.2 containing 0.1% polysorbate 80 to obtain a final volume of about 1000 mL. It was. This was added to a Q Sepharose Fast Flow anion exchange column (inner diameter 5 cm × 10 cm) equilibrated with 20 mM Tris buffer pH 9.2 containing 0.1% polysorbate 80, and a sodium chloride concentration of 0 M to 500 mM was added in the same buffer. Elution was performed by a linear concentration gradient method, and 240 mL fractions eluted at a salt concentration from 20 mM to around 150 mM sodium chloride were collected. The obtained fraction was diluted 4-fold with 20 mM TTis buffer pH 7.2 to 960 mL, and the pH was adjusted to 7.2 with acetic acid. This was added to an SP Sepharose Fast Flow cation exchange column (inner diameter 5 cm × 10 cm) equilibrated with 20 mM Tris buffer pH 7.2 containing 0.1% polysorbate 80, and a sodium chloride concentration of 0 M to 500 mM was added in the same buffer. Elution was performed by the linear concentration gradient method. The eluate was analyzed by SDS-PAGE, and the TPO elution fraction was collected. Trifluoroacetic acid was added to the TPO elution fraction so as to be 0.1%, added to a capsule pack 5 μm 300AC1 column (inner diameter 2.1 cm × 5 cm × 2, Shiseido), and then in 0.1% trifluoroacetic acid. Reversed phase HPLC was performed by a linear gradient elution method with increasing 1-propanol concentration. The eluate was fractionated into two fractions according to the results of SDS-PAGE analysis in the absence of a reducing agent and the elution position on reverse phase HPLC. The obtained 2 fractions of TPO elution were subjected to Fr. G-a, Fr. As a result of SDS-PAGE analysis in the absence of a reducing agent as Gd (see FIG. 17), molecular weights of 18 KDalton (Fr.Ga) and 32 KDalton (Fr.Gd) are respectively detected. It was detected as a single band (see FIG. 18). As a result of SDS-PAGE analysis in the presence of a reducing agent, both fractions were detected as a single band around a molecular weight of 20 K Dalton. As a result of amino acid composition analysis for each, the obtained amino acid composition almost coincided with the theoretical value from the sequence information, and as a result of N-terminal amino acid sequence analysis, only the expected sequence was obtained. Moreover, the protein mass of each fraction obtained from the result of amino acid analysis was 2.56 mg (Fr.G-a) and 1.16 mg (Fr.G-d), respectively. Each fraction was sufficiently dialyzed against IMDM culture and then subjected to M-07e assay. As a result, TPO activity with a relative activity of about 3960000 (Fr.Ga) and a relative activity of about 770000 (Fr.Gd) was obtained. showed that.
<Example 62>
Construction of Partial-Length Human TPO (Amino Acid 1-163) (hereinafter, this protein is referred to as “hTPO163”), a recombinant vector for CHO cells, pDEF202-hTPO163 The vector pDEF202 obtained in Example 31 is expressed by restriction enzymes EcoRI and SpeI. And the larger vector fragment was recovered by agarose gel electrophoresis, and then the plasmid pEF18S-hTP0163 containing this fragment and hTPO163 cDNA (refer to SEQ ID NO: 13 in the sequence listing) encoding amino acids -21 to 163 was obtained. The expression vector pDEF202-hTPO163 was obtained by ligating hTPO163 cDNA obtained by treatment with restriction enzymes EcoRI and SpeI with T4DNA ligase (Takara Shuzo). This plasmid contains the SV40 replication initiation region, human elongation factor 1-alpha promoter, SV40 early polyadenyl site, mouse DHFR minigene, pUC18 replication initiation region, β-lactamase gene (Amp^rHTPO163 cDNA is connected downstream of the human elongation factor 1-alpha promoter.
<Example 63>
Expression of hTPO163 by CHO cells
Α minimum essential medium containing CHO cells (dhfr-strain, Urlaub and Chasin; Proc. Natl. Acad. Sci. USA; 77, 4216, 1980) in 6 cm diameter plate (Falcon) containing 10% fetal bovine serum The cells were cultured and grown with (α-MEM (−), thymidine, hypoxanthine added), and transformed by the transferectam method (manufactured by Seikagaku Corporation).
That is, after adding 240 μl of 0.3 M NaCl to 10 μg of pDEF202-hTPO163 plasmid prepared in Example 62, 20 μl of transfectam and H₂A mixture with 220 μl of O was added and mixed again. After dropping this DNA solution onto the plate, CO₂The cells were cultured for 6 hours in an incubator. After removing the medium from the plate and washing twice with α-MEM (−), α-MEM (−) containing 10% dimethyl sulfoxide was added and treated at room temperature for 2 minutes. Subsequently, 10% dialyzed fetal bovine serum-containing non-selective medium (additional α-MEM (−), hypoxanthine, thymidine added) was added and cultured for 2 days. Selection was made with MEM (-), hypoxanthine, and thymidine not added. For selection, after trypsinizing the cells, each 6 cm diameter plate is divided into 5 10 cm diameter plates or 20 24-well plates, and then the culture is continued while changing the medium every 2 days. It was carried out by things. For the plate or well in which the cells had grown, TPO activity in the culture supernatant was measured using the Ba / F3 assay, and TPO activity was observed. For those in which TPO activity was observed in the culture supernatant, the cells were split 1:15 in a selective medium containing 25 nM methotrexate in a new plate or well, and the cells resistant to methotrexate were grown by continuing the culture. Cloning.
CHO cells can also be transformed by co-transfecting CHO cells with pEF18S-hTPO163 and pMGI.
The CHO cell line (CHO-DUKXB11) transformed with the plasmid pDEF202-hTPO163 has been deposited with the accession number FERM BP-4899 at the Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry, on January 31, 1995.
It has also been deposited with the food industry development institute of China (FIRDI) as deposit number 960024 on March 22, 1995.
<Example 64>
Mass culture of CHO cells
Mass cultivation of the hTPO163-producing CHO cell line (CHO109 cells, 0 nM MTX resistant) obtained by transfecting CHO cells with the hTPO163 expression plasmid pDEF202-hTPO163 in Example 63 was performed as follows. The cells were grown in culture using DMEM / F-12 medium (GIBCO) containing 10% FCS. After the cells were detached using a trypsin solution, 10 million cells were inoculated into a Falcon roller bottle (Falcon 3000) containing 200 ml of the same medium, and cultured at 37 ° C. at a rotation speed of 1 rpm for 3 days. After 3 days, the culture solution was removed by aspiration, and after rinsing the cell culture surface with 100 ml of PBS, 200 ml of DMEM / F-12 medium (GIBCO) without 10% FCS was added, and the rotation speed was 37 ° C. and 1 rpm. After culturing for 7 days, the culture supernatant was recovered after 7 days and used as a starting material for the next purification operation. The above operation was performed for 300 roller bottles to obtain 60 L of serum-free culture supernatant.
<Example 65>
Purification of hTPO163 from hTPO163 producing CHO cell line
(1) 60 L of serum-free culture supernatant obtained from Example 64 was obtained, and the filtrate of a 0.22 μm filtration filter was taken. Further, a concentrated fraction (600 ml, protein concentration 11.2 mg / ml, total protein mass 6430 mg) was obtained using an ultrafiltration unit (manufactured by Filtron, molecular weight 10,000 cut). When this fraction was examined by Western analysis using an anti-HT1 peptide antibody, it was found that hTP0163 protein expressed in the range of apparent molecular weight 20000 to 26000 was present.
Sephadex G-25 Fine column (manufactured by Pharmacia Biotech, catalog number 17-) in which 573 ml of this concentrated culture supernatant was previously equilibrated with 10 mM sodium phosphate buffer (pH 6.8) at a flow rate of about 20 ml / min. Protein fraction F1 (938 ml, protein concentration 4.9 mg / ml, total protein, which became a 10 mM sodium phosphate buffer (pH 6.8) solution by treatment with 10 cm in diameter and 30 cm in bed height) Amount 4594 mg). SP Sepharose Fast Flow (manufactured by Pharmacia Biotech, catalog number 17-0729-01; diameter 5 cm, 929 ml of this Sephadex G-25 Fine column protein fraction was previously equilibrated with 10 mM sodium phosphate buffer (pH 6.8). Bed height 12 cm) was added to the column at a flow rate of 15 ml / min. After completion of the addition, the fractions eluted with 10 mM sodium phosphate buffer (pH 6.8) and then with 10 mM sodium phosphate buffer (pH 6.8) containing 10% ethanol were collected and fraction F1 (1608 ml, protein Concentration 2.13 mg / ml, total protein mass 3426 mg). Next, the eluate was changed to a buffer containing 750 mM NaCl and 25% ethanol in 10 mM sodium phosphate (pH 6.8), and the main elution fraction F2 of elution hTPO163 (651 ml, protein concentration 1.67 mg / ml). , Total protein mass 1087 mg).
200 ml of the TPO-active fraction F2 of the SP Sepharose Fast Flow column was taken, ethanol and purified water were added, and 300 ml of a solution having a final ethanol concentration of 45% was prepared. Here, insoluble matters resulting from the addition of ethanol were removed by centrifugation, and then developing solvent A (10 mM sodium acetate buffer (pH 6.7)) and developing solvent B (10 mM sodium acetate buffer (pH 6 containing 90% ethanol) were added. .7)) is prepared and injected at a flow rate of 2 ml / min into a SOURCE 15RPC column (Pharmacia Biotech, catalog number 17-0727-02; diameter 2 cm, bed height 20 cm) previously equilibrated with 50% B solution. did. After the injection, the column non-adsorbed substance was washed with 45% B solution until almost eluting, and then the flow rate was adjusted to 1.5 ml / min. First, 50% B for 5 minutes) and then from 50% B to 100% B for 140 minutes After the linear concentration gradient, development was performed with 100% B solution for 35 minutes. During this period, the elution fraction collected every 5 minutes (7.5 ml) was subjected to SDS-PAGE and Western analysis, and the elution range of hTP0163 was examined. As a result, an apparent molecular weight of about 20000 was observed in the ethanol concentration range of 66% to 87%. 26000 of highly purified hTP0163 was found to be eluted. Among these hTPO163, those with higher molecular weight eluted earlier from the reverse phase column, suggesting that the hTPO163 molecules with more sugar chains bound have higher hydrophilicity.
The CHAPS was added to 88.8 ml of the hTPO163 elution fraction of the SOURCE 15 RPC column (the hTPO163 fraction eluted in the ethanol concentration range of 68% to 86.5%), followed by ultrafiltration (Amicon, YM -3 membrane ultrafiltration unit), the solution was concentrated and washed, and finally 2.5 ml of a concentrated sample containing about 5% ethanol and 4 mM CHAPS was obtained. A Superdex 75 pg column (Pharmacia Biotech, catalog number 17-1070-01; diameter 2.6 cm) in which this sample was previously equilibrated with 10 mM sodium phosphate buffer (pH 6.8) containing 10% ethanol was measured. 60 cm) at a flow rate of 1.5 ml / min. From 60 minutes after the injection, elution fractions were collected every 6 ml (4 minutes). As a result, SDS-PAGE began to elute hTPO163 in the fractions of test tube number 16 and beyond, and at least to the 31st wide range. It was confirmed that hTPO163 was eluted. This corresponds to a molecular weight range of about 44000 to 6000 relative to a standard molecular weight marker for gel filtration (Gel Filtration Standard from Bio-Rad; catalog number 151-1901 and insulin from Calbiochem; catalog number 407696). . Moreover, these hTP0163 were considered to elute in the order of increasing molecular weight to which more sugar was added. Fraction FA was prepared as a collection of all hTPO163 molecular species in the elution range of test tube numbers 16-31 (elution volume 180-276 ml). Separately from fraction FA, each of test tube numbers 16-18 (elution volume 180-198 ml), test tube numbers 19-24 (elution volume 198-234 ml), test tube numbers 25-31 (elution volume 234-276 ml) Were taken as fractions FH, FM, and FL, respectively. That is, the fraction FA is substantially the sum of the fractions FH, FM, and FL. The above is shown in FIG.
(2) Next, hTPO163 elution fraction FH) FM) FL and FA of the Superdex 75 pg column described in (1) will be described in detail. The amino acid sequence on the N-terminal side of hTPO163 thus obtained was examined in the same manner as described in Example 1. As a result, most of the N-terminal serine could not be identified. It was strongly suggested that an O-linked sugar chain was added to serine. It was also confirmed that the sequence following the N-terminal serine was the amino acid sequence expected from the gene sequence. As a result of amino acid composition analysis (AccQ.Tag method, Waters), FTP, FM, FL and FA hTPO163 protein (peptide portion not containing sugar chain) concentrations were 10.2 ng / ml, 6.2 ng / ml, 0.84 ng / ml and 3.2 ng / ml. 100 ng of these fractions were reduced by non-reducing conditions or DTT using Multigel 15/25 (15-25% precast polyacrylamide gel manufactured by Daiichi Chemicals) and then subjected to SDS-PAGE and silver staining (Daiichi Kagaku) As a result, it was confirmed that they were extremely high-purity hTPO163 preparations. Under reducing conditions, the apparent molecular weights of hTPO163 of FH, FM, FL and FA calculated with DPCIII molecular weight marker (Daiichi Chemical Co., Ltd.) as the standard are 24000 to 21500, 23000 to 21000, 23,000 to 20500, and 23500, respectively. ˜20500 (see FIG. 20). In addition, FH, FM, FL and FA calculated by Western analysis under bioreductive SDS-PAGE standard (Bio-Radiated SDS-PAGE Standards, Broad Range: Catalog No. 161-0319) manufactured by Bio-Rad under standard conditions. The apparent molecular weights of hTPO163 were 26000-22000, 25500-22000, 26000-21000, 26000-21000, respectively. It can be presumed that the molecular weight heterogeneity of FH, FM, FL and FA is due to the heterogeneity of the added O-linked sugar chain. Thus, FH, FM, FL and FA fractions were enzymatically digested with neuraminidase (Neuraminidase: Nacalai Tesque, catalog number 242-29 SP), reduced with DTT and analyzed by SDS-PAGE. Since the apparent molecular weight was around 19000, the heterogeneity of the amount of sialic acid in the sugar chain added to the hTPO163 protein was confirmed. At the same time, the obtained hTPO163 was used as a CHO cell as a glycoprotein. It was revealed that it was expressed in
(3) When the FH, FM, FL and FA fractions obtained in (2) were examined for in vitro activity using the M-07e assay system, the relative specific activity was 1 mghTPO163 protein (a peptide containing no sugar chain weight). Per unit weight) were 511,000,000, 775000000, 1150000000, and 715000000, respectively.
<Example 66>
Construction and expression of an expression vector for Escherichia coli of human TPO (amino acids 1-332) (hereinafter referred to as “hMKT (1-332)”) having Lys added at position −1 and Met added at position −2.
In order to express the full-length human TPO amino acid in E. coli, the codon used from amino acid 164 to amino acid 332 was changed to an E. coli preferred codon as described below. 21-40 synthetic oligonucleotides shown in Table 20 were prepared.
[Table 20]

Synthetic oligonucleotides 21 and 22; 23 and 24; 25 and 26; 27 and 28; 29 and 30; 31 and 32; 33 and 34; 35 and 36; 37 and 38; (Manufactured by Pharmacia) was phosphorylated in a solution of 0.1 mM ATP, 10 mM Tris-acetate, 10 mM Mg-acetate, 50 mM K-acetate. Furthermore, 1/10 amount of 100 mM Tris / HCl (pH 7.5), 100 mM MgCl₂, 500 mM NaCl solution was added, boiled in a water bath for 3 minutes, and allowed to stand to obtain double-stranded DNA. 21 and 22; 23 and 24 (combination A), 25 and 26: 27 and 28; 29 and 30 (combination B), 31 and 32; 33 and 34 (combination C), 35 and 36; 37 And 38; 39 and 40 (combination D) after four sets of double-stranded DNAs were ligated using a DNA ligation kit (manufactured by Takara Shuzo Co., Ltd.), and (combination A) and (combination B), (combination) C) and (Combination D) were connected in the same manner, and the resulting reaction solutions were named Connection 1 and Connection 2. Using these reaction solutions as templates, PCR reactions were performed using primers 41 and 42 for linkage 1 and 43 and 44 for linkage 2 as primers. PCR products obtained by PCR reaction of ligation 1 were subjected to digestion with Sacl and EcoRV, and PCR products obtained by PCR reaction of ligation 2 were digested with EcoRV and HindIII, followed by electrophoresis on 2% agarose gel. Were recovered by Prep-A-gene DNA purification kit. These two fragments were subcloned into pBluescript IIKS + (Stratagene) digested with Sacl and HindIII (the host used was E. coli DH5). Among the obtained clones, those having the base sequences shown in Table 21 were selected by sequencing, and the clone obtained here was designated as pBL (SH) (174-332).
[Table 21]

Next, PCR was performed using pBL (XH) h6T (1-163) prepared in Example 42 as a template and the following 45 and 46 synthetic oligonucleotides as primers, and the PCR product obtained here was BamHI, After digestion with Sacl, the gel was run on a 6% polyacrylamide gel, and a fragment of about 160 bp was recovered from the gel. Further, a fragment of about 480 bp obtained by digesting pBL (SH) (174-332) with SacI and HindIII was recovered by a prep-A-gene DNA purification kit, and these two fragments were digested with BamHI and HindIII. It was subcloned into pBIuescript IIKS + (manufactured by Stratagene) (the host used E. coli DH5).
45: 5'-AAGGATCCCGAACGCTCATTCTTCTG-3 '
46: 5'-GGGAGCTCGTTCAGGGTCAGACACCAGAGAGGTACGAGACGGAACAGCAGTGGTTGG-3 '
Among the obtained clones, those having the base sequences shown in Table 22 were selected by sequencing, and the obtained clone was designated as pBL (BH) (123-332).
[Table 22]

PBL (XH) h6T (1-163) prepared in Example 42 was digested with BamHI and HindIII and pBL (BH) (123-332) was digested with the same restriction enzymes, and a fragment of about 640 bp was ligated. Clone pBL (XH) h6T (1-334) was prepared. Furthermore, a fragment of about 270 bp obtained by digesting PCFM536 / hMKT (1-163) prepared in Example 52 with Xbal and Sfil was ligated to pBL (XH) h6T (1-334) digested with the same restriction enzymes. Clone pBL (XH) hMKT (1-334) was prepared. After digesting pBL (XH) hMKT (1-334) with Xbal, HindIII, a fragment having a size of about 1040 bp encoding a mutant human TPO amino acid 1-332 was recovered and purified, and then digested with Xbal, HindIII. It was cloned into pCFM536 (Japanese Patent Publication No. 60-501988) (the host used was E. coli JM109 previously transformed with pMW1 (ATCC No. 39933)). The clone obtained here was designated as pCFM536 / hMKT (1-332), and the Escherichia coli strain having this expression vector was used as a transformant for expressing mutant hMKT (1-332) protein. This expression plasmid contains the DNA sequence shown in the sequence listing (SEQ ID NO: 14).
The transformed strain obtained here was cultured overnight in 30 ml of LB medium containing 12.5 μg / ml of ampicillin and 12.5 μg / ml of tetracycline at 30 ° C., and 25 ml of this culture solution was mixed with 1000 ml of LB medium containing 50 μg / ml of ampicillin. In addition, OD is A₆₀₀Was shaken at 30 ° C. until 1.0 to 1.2. Next, about 330 ml LB medium at 65 ° C. was added so that the final temperature was 42 ° C., and further cultured with shaking at 42 ° C. for 3 hours to induce the expression of mutant human TPO, hMKT (1-332) protein.
SDS-PAGE was performed using the cultured cells directly as samples. For SDS-PAGE, multi gel 15/25 manufactured by Daiichi Kagaku Co., Ltd. was used, and Coomassie blue staining was performed after electrophoresis. As a result, hMKT (1-332) protein expression was expressed at a molecular weight of about 35 kD. A protein specific for induction was detected. On the other hand, after SDS-PAGE, the protein on the gel was electroblotted to a nitrocellulose membrane and reacted with the anti-HT1 peptide antibody prepared in Example 45 to develop a color. As a result, a band was detected at a molecular weight of about 35 kD. , Expression of hMKT (1-332) was confirmed.
<Example 67>
Preparation of human TPO-substituted derivatives and expression in COS7 cells-activity confirmation
Whether a derivative in which a part of the amino acid of human TPO was substituted had activity was examined as follows.
The animal cell expression vector pSMT201 used in this example was prepared as follows. First, an expression plasmid pXM-mEP0Rn containing mouse erythropoietin receptor cDNA (obtained from Dr. D'Andrea, Dana Farbor Cancer Institute, Cell. 57, 277-285 (1989)) was treated with restriction enzymes KpnI and EcoRI. The pXM vector fragment excluding the mouse erythropoietin receptor cDNA portion was recovered by agarose gel electrophoresis. Next, two synthetic oligonucleotides for introducing various restriction enzyme sites for cloning into the recovered pXM expression vector were prepared using an ABI DNA synthesizer. The base sequence of the synthesized oligonucleotide is shown below.
Primer 1:

Primer 2:

The two oligonucleotides were mixed and annealed to form a double-stranded oligonucleotide, and the pXM vector fragment recovered above was ligated with T4 DNA ligase (Takara Shuzo) to obtain an expression vector pDMT201. Next, in order to remove the mouse DHFR cDNA contained in this expression vector pDMT201, the pDMT201 vector and the pEF18S vector were treated with restriction enzymes NotI and HpaI and then subjected to agarose gel electrophoresis, and the larger fragment derived from the pDMT201 vector DNA and the pEF18S vector DNA The smaller fragment of origin was recovered and ligated with T4 DNA ligase (Takara Shuzo) to obtain the expression vector pSMT201. As shown in FIG. 21, this vector comprises the SV40 replication start region and enhancer sequence, adenovirus major late promoter sequence, adenovirus tripartite leader sequence, splice signal sequence, SV40 early polyadenyl site sequence, adenovirus VARNA gene sequence, and pUC18 replication. Start region, β-lactamase gene (Amp^rAnd has a BglII, PstI, KpnI, XhoI, EcoRI, SmaI, SpeI, and NotI sites as restriction enzyme sequences for ligating the target gene. By subcloning the full-length human TPO cDNA fragment obtained by enzymatic digestion of pHTP1 containing the full-length human TPO cDNA shown in Example 30 with restriction enzymes EcoRI and SpeI into a vector pSMT201 similarly digested with the restriction enzyme, plasmid pSMT201. -HTPO was obtained.
Using the plasmid pSMT201-hTPO obtained as described above as a template, a plasmid βGL-TPO / pBlue serving as a template for preparing a derivative was first prepared as follows.
In βGL-TPO / pBlue, an attempt was made to add a rabbit β-globin leader sequence to the 5 ′ untranslated side of human TPO according to the method of Annweiler et al. (Annweiler et al., Nucleic Acids Research, Vol. 19, p. 3750). First, a vector βGL / pBlue into which a leader sequence was inserted was prepared by ligating two chemically synthesized DNA strands shown below and a fragment obtained by digesting the vector BluescriptIISK + (manufactured by Toyobo) with restriction enzymes EcoRI and SmaI. did.

Primer sequences used for PCR are as follows.

(SmaI sequence added to 102-122 of SEQ ID NO: 7)
end: 5'-TCAAGCTTACTAGTCCCTCTCGAGACAGAATTCTG-3 '(antisense of 1140-1160 of SEQ ID NO: 7, SpeI and HindIII sequences added) PCR was performed using plasmid pSMT201-hTPO DNA 1ng as a template, and 10 μM each of the synthesized primers. did. Using TaKaRa PCR Amplification Kit (Takara Shuzo), Programmable Thermal Controller (MJ Research) in a volume of 100 μl (94 ° C. for 1 minute, 55 ° C. for 2 minutes, 72 ° C. for 2 minutes for 3 cycles, followed by 94 cycles) 20 cycles of 45 ° C., 55 ° C. for 1 minute, 72 ° C. for 1 minute). The PCR product was extracted with chloroform, ethanol precipitated twice, and then dissolved in 100 μl of TE buffer. This was digested with restriction enzymes SmaI and HindIII, followed by phenol / chloroform extraction and ethanol precipitation. The resulting precipitate was dissolved in 10 μl (7) TE buffer and then subcloned into the same restriction enzyme-digested vector βGL / pBlue (the host fungus used Toyobo Co., Ltd., competent high E. coli JM109). Twenty clones were selected from the obtained transformants to prepare plasmid DNA. The method was performed essentially as described in Molecular Cloning [Samboo et al., Cold Spring Harbor Laboratory Press (1989)]. For purified plasmid DNA, Taq Dye Deoxy^TMUsing Terminator Cycle Sequencing Kit (Applied Biosystems), the nucleotide sequence was confirmed by 373A DNA sequencer manufactured by Applied Biosystems. It was confirmed that the plasmid encoding βGL-TPO / pBlue has the expected TPO cDNA sequence with no base sequence substitution over the entire length. Furthermore, βGL-TPO / pB1ue was digested with the restriction enzyme BgIIINSpeI, followed by phenol / chloroform extraction and ethanol precipitation. The obtained precipitate was dissolved in 10 μl of TE buffer and subcloned into a vector pSMT201 similarly digested with restriction enzymes (the host fungus used Toyobo's competent high E. coli JM109). Plasmid DNA was prepared from the transformants essentially according to the method described in Molecular Cloning [Samboo et al., Cold Spring Harbor Laboratory Press (1989)]. The obtained expression vector pSMT / βGL-TPO has a structure in which a β-globin leader sequence and human TPO cDNA are connected to the restriction enzyme sequence BglII / SpeI site downstream of the splice signal sequence of the pSMT201 vector shown in FIG. doing. This pSMT / βGL-TPO vector was used for transfection experiments into COS7 cells.
For the preparation of the human TPO-substituted derivative, PCR was used and the method of Ito et al. Was followed (Ito et al., Gene, 102, 67-70, 1991). Here, an attempt was made to produce a derivative in which the Arg residue at position 25 of human TPO was substituted with an Asn residue, and a derivative in which the His residue at position 33 was substituted with a Thr residue. Primer sequences used for PCR are as follows.
T7: 5'-TAATACGACTCACTATAGGGG-3 '(corresponds to the T7 promoter region of BluescriptIISK +)
ΔBgIII: 5′-AATTCCAAGATCACACACTTGC-3 ′ (for restriction enzyme BgIII recognition sequence replacement) end: 5′-TCAAGCTTACTAGTCCTTCCTGAGAGAGATCTCTG-3 ′ (addition of the antisense, SpeI, and HindIII sequences of SEQ ID NO: 7) N3: 5 -TGGGCACTGGCTCAGGTTGCTGTGGAAGGACATGGGG-3 '(Arg25 → Asn of SEQ ID NO: 7)
09: 5′-TGTAGGCAAAGGGTAACCCTCTGGCA-3 ′ (His33 → Thr of SEQ ID NO: 7) The first stage PCR was performed using 1 μg of plasmid βGL-TPO / pBlue DNA as a template, and 10 μM each of the synthesized primers (primer The combinations are [1] ΔBgIII and end, [2] T7 and N3, and [3] T7 and 09). Using TaKaRa PCR Amplification Kit (Takara Shuzo), Programmable Thermal Controller (MJ Research) in a volume of 100 μl (94 ° C. for 1 minute, 55 ° C. for 2 minutes, 72 ° C. for 2 minutes for 3 cycles, followed by 94 ° C.) 17 cycles of 45 seconds, 55 ° C. for 1 minute, 72 ° C. for 1 minute). Each PCR product was extracted with chloroform, ethanol precipitated twice, and then dissolved in 100 μl of TE buffer. A second-stage PCR was performed using 2 μl of each of these PCR products. The template combination is the PCR product of [1] and [2] and the PCR product of [1] and [3]. All the primers used are a combination of T7 and end. After incubating at 94 ° C for 10 minutes, TaKaRa Taq is added, and then 94 ° C for 1 minute, 55 ° C for 2 minutes, 72 ° C for 2 minutes for 3 cycles, followed by 94 ° C for 45 seconds, 55 ° C for 1 minute, 72 ° C for 1 minute for 9 cycles went. 1 μl of 5 mg / ml proteinase K was added to each PCR product) 2 μl of 0.5 M EDTA and 2 μl of 20% SDS were added and incubated at 37 ° C. for 30 minutes to inactivate Taq, followed by phenol / chloroform extraction, ethanol precipitation and 20 μl. Dissolved in sterile water. This was digested with restriction enzymes BgIII and SpeI, followed by phenol / chloroform extraction and ethanol precipitation. The resulting precipitate was dissolved in 10 μl of TE buffer and then subcloned into the expression vector pSMT201, which was similarly digested with restriction enzymes and treated with alkaline phosphatase (calf intestine, manufactured by Boehringer Mannheim) (the host strain was competent manufactured by Toyobo Co., Ltd.). -Use high E. coli JM109). Two clones were selected from each of the obtained transformants to prepare plasmid DNA. The method was performed essentially as described in Molecular Cloning [Samboo et al., Cold Spring Harbor Laboratory Press (1989)]. For purified plasmid DNA, TaqDye Deoxy^TMUsing Terminator Cycle Sequencing Kit (Applied Biosystems), the nucleotide sequence was confirmed by 373A DNA sequencer manufactured by Applied Biosystems.
The plasmid derived from the PCR product of [1] and [3] has the intended amino acid substitution [His33 (CAC) → Thr (ACC)], and a peptide consisting of the following amino acid sequence is added to the C-terminus (Gly332). It was confirmed that cDNA encoding the TPO-substituted derivative (09 / TPO) was included.
On the other hand, the plasmid derived from the PCR product of [1] and [2] has one amino acid substitution [Glu231 (GAA) → Lys (AAA) in addition to the intended amino acid substitution [Arg25 (AGA) → Asn (AAC)]. And a cDNA encoding a TPO-substituted derivative (N3 / TPO) having a peptide having the following amino acid sequence added to the C-terminus (G1y332).
peptide:
TSIGYPYDVPDYAGVHHHHHHH
Transfection of each obtained clone into COS7 cells was performed by DEAE-dextran method including chloroquine treatment according to Example 35. That is, 40 μg of plasmid DNA was used, and the culture supernatant was collected 5 days after transfection. The collected culture supernatant was concentrated 20 times with Centricon-30 (Amicon) and evaluated with the M-07e assay system. As a result, TPO activity was detected in a dose-dependent manner in the COS7 cell culture supernatant transfected with plasmids encoding human TPO-substituted derivatives N3 / TPO and 09 / TPO (see FIG. 22).
<Example 68>
Preparation of human TPO insertion / deletion derivative and expression in COS7 cells-Activity confirmation
It was investigated whether a derivative in which an amino acid was inserted into hTPO163 or a derivative in which an amino acid was deleted from hTPO163 had activity. The prepared derivatives were the old s33 deletion (dH33), Gly116 deletion (dG116), Arg117 deletion (dR117), Thr insert between His33 and Pro34 (T33 ′), and Ala insertion. Body (A33 ′), Gly insert (G33 ′) and Asn insert (N116 ′), Ala insert (A116 ′) and Gly insert (G116 ′) between Gly116 and Arg117. Of these, T33 'and N116' are derivatives intended to introduce mucin-type sugar chains and Asn-linked sugar chains, respectively.
For the preparation of the derivative, PCR was used and the method of Ito et al. Was followed (Ito et al., Gene, 102, 67-70, 1991). Primer sequences used for PCR are as follows.
dH33: 5'-TGTAGGCAAAGGAACCCTGGGGCA-3 '(for preparing the old s33 deletion of SEQ ID NO: 7)
T33 ': 5'-TGTAGGCAAAGGAGTGTGAACCCTGG-3' (for producing a Thr insert between the old s33 of SEQ ID NO: 7 and Pro34)
A33 ': 5'-TGTAGGCAAAGGAGCGTGAACCCTGG-3' (for preparing an Ala insert between His33 and Pro34 of SEQ ID NO: 7)
G33 ': 5'-TGTAGGCAAAGGTCCGTGAACCCTGG-3' (for preparing a Gly insert between the old s33 of SEQ ID NO: 7 and Pro34)
dG116: 5'-AGCTGTGGTCCTCTGTGGAGGAAG-3 '(for preparation of Gly116 deletion of SEQ ID NO: 7)
dR117: 5'-GTGAGCTGTGGTGCCCTGTGAGG-3 '(for preparing Arg117 deletion of SEQ ID NO: 7)
N116 ': 5'-AGCTGTGGTCCTGTGCCCTGTGGAGG-3' (for producing an Asn insert between GIy116 and Arg117 of SEQ ID NO: 7)
A116: 5'-AGCTGTGGTCCTAGCGCCCTTGGAGG-3 '(for preparing an Ala insert between Gly116 and Arg117 of SEQ ID NO: 7)
G116 ': 5'-AGCTGTGGTCCTTCCGCCCTTGGAGG-3' (for preparing a Gly insert between GIy116 and Arg117 of SEQ ID NO: 7)
dRI: 5'-CGGGCTGCAGGATATCCAAGATCTCA-3 '(for restriction enzyme EcoRI recognition sequence replacement) T7: 5'-TAATACGACTCACTATAGGGG-3' (corresponding to the T7 promoter region of BluescriptIISK +)
endNotI: 5'-GGGGCGCCGCTCCAGCTGGGGACAGCTGTGGTGGGG-3 '(antisense of 633-653 of SEQ ID NO: 7, addition of stop codon and NotI recognition sequence)
The first stage PCR was performed using 1 ng of the plasmid βGL-TPO / pBlue DNA prepared in Example 67 as a template and 10 pM of each synthesized primer. The primer combinations are [1] dRI and endNotI, [2] T7 and mutagenesis primers (dH33, A33 ', G33', T33 ', dG116, dR117, A116', G116 ', N116'). Using TaKaRa PCR Amplification Kit (Takara Shuzo), PCR was performed with Programmable Thermal Controller (MJResearch) in a volume of 100 μl. The reaction of [1] was 94 ° C for 1 minute, 45 ° C for 2 minutes, 72 ° C for 2 minutes for 3 cycles, followed by 94 ° C for 45 seconds, 45 ° C for 1 minute, and 72 ° C for 1 minute for 17 cycles. In the reaction of [2], 94 ° C for 1 minute, 55 ° C for 2 minutes, and 72 ° C for 2 minutes are 3 cycles, followed by 94 ° C for 45 seconds, 55 ° C for 1 minute, and 72 ° C for 1 minute for 17 cycles. Each PCR product was extracted with chloroform, ethanol precipitated twice, and then dissolved in 100 μl of TE buffer.
Next, the second stage PCR was performed using 1 μl each of the PCR products [1] and [2]. All the primers used are a combination of T7 and endNotI. After incubating at 94 ° C for 10 minutes, TaKaRa Taq is added, and then 94 ° C for 1 minute, 55 ° C for 2 minutes, 72 ° C for 2 minutes for 3 cycles, followed by 94 ° C for 45 seconds, 55 ° C for 1 minute, 72 ° C for 1 minute for 9 cycles went. 1 μl of 5 mg / ml proteinase K, 2 μl of 0.5 M EDTA, and 2 μl of 20% SDS were added to each PCR product and incubated at 37 ° C. for 30 minutes to inactivate Taq, followed by phenol / chloroform extraction, ethanol precipitation, and 20 pl. Dissolved in sterile water. This was digested with restriction enzymes EcoRI and NotI, followed by phenol / chloroform extraction and ethanol precipitation. The resulting precipitate was dissolved in 10 μl (7) TE buffer and then subcloned into an expression vector pEF18S that had been digested with restriction enzymes and treated with alkaline phosphatase (calf intestine, manufactured by Boehringer Mannheim) in the same manner (host fungus was Toyobo Co., Ltd.) Competent high E. coli JM109 manufactured by) is used. Plasmid DNA was prepared from the obtained transformant. The method was performed essentially as described in Molecular Cloning [Samboo et al., Cold Spring Harbor Laboratory Press (1989)]. For purified plasmid DNA, Taq Dye Deoxy^TMUsing Terminator Cycle Sequencing Kit (Applied Biosystems), the nucleotide sequence was confirmed by 373A DNA sequencer manufactured by Applied Biosystems. As a result, the plasmid encoding dH33, A33 ′, T33 ′, dG116, dR117, A116 ′, G116 ′, and N116 ′ has the expected TPO cDNA sequence with no substitution of the base sequence other than the intended site over the entire length. confirmed. In addition, in G33 ', a base substitution [Pro38 (CCT) → Ser (TCT)] that results in substitution of one amino acid other than the intended site was observed.
Transfection of each obtained clone into COS7 cells was performed according to Example 11. That is, 10 μg of plasmid DNA was used for transfection using DEAE-dextran method including chloroquine treatment, and the culture supernatant was collected 4-5 days later. The collected culture supernatant was evaluated by the M-07e assay system. As a result, TPO activity was detected in a dose-dependent manner in COS7 cell culture supernatant transfected with a plasmid encoding an amino acid insertion / deletion derivative (see FIG. 23). No activity was observed in the COS7 cell culture supernatant transfected with the expression plasmid pEF18S not containing cDNA encoding the TPO derivative.
<Example 69>
Increased platelet activity by TPO
Twenty normal 8-week-old ICR male mice whose blood count was collected in advance from the orbital vein and the platelet count was measured with a microcell counter (manufactured by Toa Medical Electronics, F-800) were randomly divided into 4 groups. One group (control-iV group) was intravenously administered with 100 μl PBS once a day for 5 consecutive days, and the other group (TPO-iv group) was SP obtained in Example 56. The concentrated solution of the human TPO active fraction F2 of Sepharose Fast Flow was replaced with PBS with a NAP-25 column (Pharmacia Biotech, catalog number 17-0852-02), and further diluted with PBS (100 μl (M- (Including the relative activity amount 211,900 in the 07e assay system) was administered intravenously once a day for 5 consecutive days. Another group (control-sc group) was administered subcutaneously with 100 μl of PBS once a day for 5 consecutive days, and the other group (TPO-sc group) was administered in the same manner as the TPO-iv group. 100 μl was administered subcutaneously once a day for 5 consecutive days.
On the 6th, 8th, 10th, 13th, and 15th day after the start of administration, blood was collected daily from the orbital vein of each mouse, and the platelet count was measured with a microcell counter (manufactured by Toa Medical Electronics, F-800).
The transition of the platelet count is shown in FIG.
That is, in the control-iv group, the platelet count increased by 44% on the 6th day when the platelet count increased the most, and the control-sc group observed a 47% increase on the 8th day when the platelet count increased the most. It was only. On the other hand, in the TPO-iv group, an increase of 177% was observed on the 6th day compared to before administration, and the increase continued thereafter, and the maximum increase of 469% was observed on the 10th day. In the TPO-sc group, an increase of 347% was already observed on the 6th day, and a maximum increase of 493% was observed after 8 days.
From the above results, it was confirmed that human TPO has an effect of increasing platelets regardless of species difference and administration route.
<Example 70>
Increased platelet activity by TPO
Sixteen normal 11-week-old C3H / HeJ male mice whose blood counts were collected in advance from the orbital vein and platelet count was measured with a microcell counter (manufactured by Toa Medical Electronics, F-800) were randomly divided into 4 groups. . One group (control group) was subcutaneously administered with 100 μl of PBS once a day for 5 consecutive days. In another group (TPO-1 group), the human TPO active fraction F1 of Sephacryl S-200HR obtained in Example 56 was replaced with PBS with NAP-25, and 100 μl (M (Including a relative activity of 83,000 in the 07e assay system) was administered subcutaneously once a day for 5 consecutive days. In another group (TPO-2 group), 100 μl of the TPO active fraction administered in the TPO-1 group further diluted 2-fold with PBS (relative activity amount 41,500 in the M-07e assay system) Including) was administered subcutaneously once a day for 5 consecutive days. In the last group (TPO-3 group), 100 μl of the TPO active fraction administered in the TPO-2 group further diluted 2-fold with PBS (relative activity amount 20,750 in the M-O7e assay system) Were administered subcutaneously once a day for 5 consecutive days.
On the 6th, 8th, 10th, and 12th days after the start of administration, blood was collected daily from the orbital vein of each mouse, and the platelet count was measured with a microcell counter (manufactured by Toa Medical Electronics, F-800).
The transition of the platelet count is shown in FIG.
That is, there was almost no change in the platelet count in the control group. In the TPO-administered group, an increase in the number of platelets that was maximal on the 8th day from the start of administration in any group was observed. In addition, dose dependency was observed between each administration group. That is, in the TPO-1 group, an increase of about 200% was already observed on the 6th day, and increased to about 270% on the 8th day. Although it decreased after that, about 65 increase was seen also on the 12th day, and the significant difference (p <0.01: Dunnet's multiple comparison test: Dunnet multiple comparison test) was recognized with the control group. Although the increase was similarly observed in the TPO-2 group, the increase effect was not as much as that in the TPO-1 group, but on the 6th and 8th days, increases of about 140% and about 160% were recognized, respectively. On the 12th day, the level decreased to almost the same level as in the control group. The increasing effect in the TPO-3 group was even weaker than that in the TPO-2 group, but an increase of about 110% was observed on the 8th day when the maximum value was reached, and there was a significant difference (p <0.01) was also observed.
From the above results, it was confirmed that human TPO increases the platelet count regardless of the strain of the mouse, and that its action is dose-dependent.
<Example 71>
Prevention of thrombocytopenia by administration of anticancer drugs by TPO
Thirty 8-week-old ICR male mice were intravenously administered with 5-FU at a rate of 200 mg / kg body weight, and then randomly divided into two groups of 15 animals each, and the next day (Day 1), one group (control) Group) received 100 μl of PBS subcutaneously once a day for 5 consecutive days. In the other group (TPO group), the human TPO active fraction F2100 μl used in Example 69 (including the relative activity amount 211,900 in the M-07e assay system) was once a day for 5 consecutive days. It was administered subcutaneously.
After the start of administration, 5 mice from each group were randomly extracted on Day 4, 6, and 8, blood was collected from the orbital vein, and the platelet count was measured with a microcell counter (E-2500, manufactured by Toa Medical Electronics Co., Ltd.). did.
The transition of the platelet count is shown in FIG.
That is, in the control group, the number of platelets decreased after administration of 5-FU, and reached the lowest value for Day 6 (about 28% of the value before administration of 5-FU). It increased to 1.3 times. Even in the TPO group, the number of platelets after administration of 5-FU decreased daily, and reached the lowest value for Day 6 (about 52% of the value before administration of 5-FU), but there was almost no difference in the number of platelets for Day 4 and 6 In addition, Day 6 maintained a significantly high value (p <0.05: Dunnet's multiple comparison test) compared to the control group. In Day 8, it increased to about 200% of the value before 5-FU administration.
From the above results, it was confirmed that human TPO has an effect of preventing thrombocytopenia caused by anticancer agents.
<Example 72>
Treatment of thrombocytopenia by TPO
Nimustine hydrochloride (ACNU) was intravenously administered to 36 7-week-old ICR male mice at a rate of 50 mg / kg body weight, and then randomly divided into two groups of 18 animals each, and one group from the next day (Dayl). (Control group) 200 μl of PBS was subcutaneously administered twice daily, and the other group (TPO group) had 0% TPO activity fraction F1 used in Example 70 without dilution with PBS. 200 μl of Tween-80 added at a rate of 0.04% (including relative activity of 360,000 in the M-07e assay system) was subcutaneously administered twice daily.
The mice were randomly divided into 3 groups in each group after the start of administration, and each group was divided into a group for collecting blood on Day 5 and 12, a group for collecting blood on Day 8 and 14, and a group for collecting blood on Day 10. Blood was collected from the orbital vein, and the platelet count was measured with a microcell counter (manufactured by Toa Medical Electronics, F-800).
The transition of the platelet count is shown in FIG.
That is, in the control group, the platelet count decreased daily after ACNU administration, and reached the lowest (about 29% of the pre-ACNU administration value) at Day 8 to 10, and recovered to about 49% on Day 12 and about 74% on Day 14. It was only. On the other hand, in the TPO group, Day 5 decreased to about 38% of the value before ACNU administration. After that, it started to increase and recovered to about 63% at Day8 compared to the pre-ACNU administration value, and after Day 10 the platelet count was more than the pre-ACNU administration value, about 12% on Day 12, about 400% on Day 14 Increased to.
As described above, in the control group, Day 8 to 10, which is in the process of decrease, has already started to increase in the TPO group, and since Day 10 has already increased to the number of platelets higher than the pre-ACNU administration value, human TPO is an anticancer agent. It was confirmed that it has an effect of promoting recovery from thrombocytopenia caused by.
In addition, even when the results of Example 71 are combined, human TPO is expected to have the effect of preventing the decrease in the number of platelets and the effect of promoting the recovery from the decrease in platelets regardless of the type of anticancer agent.
<Example 73>
Treatment of thrombocytopenia after BMT with TPO
48 male mice of 7 weeks old C3H / HeN strain were irradiated with 10 Gy whole body, and then 1 × 10 bone marrow cells of the syngeneic mice.⁶Individuals were immediately administered intravenously. Therefore, it was randomly divided into two groups. From the next day (Day 1), PBS was used for one group (control group), and the TPO active fraction F1 (M-07e) used in Example 70 was used for the other group (TPO group). 100 μl of the relative activity in the assay system (including 44,000) was administered subcutaneously once a day for 20 consecutive days.
After starting administration, 6 mice were randomly extracted from each group on Day 5, 10, 14, 21 and collected from the orbital vein, and the platelet count was obtained using a microcell counter (Toa Medical Electronics, E-2500). Was measured.
The transition of the platelet count is shown in FIG.
That is, in any group, the platelet count decreased daily after BMT, and reached the lowest Day 10 (about 3% before BMT). After that, it gradually recovered, and on Day 14 it was about 11% in the control group and about 13% in the TPO group. Day 21 recovered only to 37% in the control group, but recovered to about 65% in the TPO group, and a significant recovery promoting effect was observed.
From the above results, it was confirmed that the human TPO purified in Example 56 has an effect of promoting the recovery of the platelet count after BMT.
<Example 74>
Treatment effect of thrombocytopenia after irradiation with TPO
36 ICR male mice aged 8 weeks were irradiated with 5 Gy X-rays and then randomly divided into two groups. From the next day (Day 1), one group (control group) was given PBS, the other group In the (TPO group), the TPO active fraction F1 used in Example 70 (including a relative activity of 1,440,000 in the M-07e assay system) was subcutaneously administered once a day for 3 days, From the next day, a relative activity amount of 360,000 in the M-07e assay system was subcutaneously administered once a day for 7 days. After the start of administration, the mice were randomly divided into 3 groups. The blood was collected on Day 4, 11 and 21, the group was collected on Day 7 and 13, and the group was collected on Day 9 and 15. Blood was collected from the orbital vein, and the platelet count was measured with a microcell counter (manufactured by Toa Medical Electronics, F-800). FIG. 29 shows the transition of the platelet count.
That is, in the control group, the number of platelets decreased daily after X-ray irradiation, reached the lowest at Day 9 (about 24% of the value before X-ray irradiation), recovered to about 38% on Day 11, and about 67% on Day 13. It recovered to the value before X-ray irradiation at Day 15. On the other hand, in the TPO group, Day 7 decreased to about 24% of the value before X-ray irradiation. After that, it started to increase and recovered to about 82% at Day 9 compared to the value before x-ray irradiation. After Day 11, the platelet count was higher than the value before X-ray irradiation, and this tendency was maintained until Day 21.
As described above, the Day 9 in the process of decreasing in the control group already started to increase in the TPO group, and the Day 11 already increased to the number of platelets equal to or higher than the pre-X-ray irradiation value, and then maintained the high value. On the other hand, in the control group, 15 days are required to recover to the platelet count equal to or higher than the value before X-ray irradiation. From this result, it was confirmed that human TPO purified in Example 56 shortened the recovery period from thrombocytopenia after irradiation, and the therapeutic effect on thrombocytopenia after irradiation was observed.
<Example 75>
Increased platelet activity by hTPO163
Blood was collected from the orbital vein in advance, and 15 normal C3H / HeN male mice whose platelet count was measured with a microcell counter (manufactured by Toa Medical Electronics, F-800) were randomly divided into 4 groups (control group, A, B). , C group). One group (control group) was administered subcutaneously with PBS containing 0.1% mouse serum once a day for 5 consecutive days. In group A, the TSE activity fraction F2 of SP Sepharose Fast Flow obtained in Example 65 was diluted with PBS containing 0.1% mouse serum, and hTPO163 was relative activity per day in the M-07e assay system. About 40,000,000 / kg body weight as the amount, also about 8,000,000 / kg body weight as the relative activity amount in the M-07e assay system for the group B, and the M-07e assay for the group C as well The relative activity as a system was administered subcutaneously at a rate of about 1,600,000 / kg body weight for 5 consecutive days.
On the 6th, 8th, 10th, and 12th days after the start of administration, blood was collected daily from the orbital vein of each mouse, and the platelet count was measured with a microcell counter (manufactured by Toa Medical Electronics, F-800). The transition of the platelet count is shown in FIG.
That is, there was almost no change in the platelet count in the control group. In the TPO-administered group, an increase in the number of platelets that was maximal on the 8th day from the start of administration in any group was observed. In addition, dose dependency was observed between each administration group. That is, in Group A, an increase of about 88% was observed on the 6th day, and an increase of about 100% was observed on the 8th day, and the increase of about 97% was maintained on the 10th day. A difference (p <0.01: Dunnett's multiple comparison test) was observed. Although the increase was similarly observed in the B group, the increase effect was not as much as that in the A group, but on the 6th and 8th days, increases of about 65% and about 84% were observed, respectively, which were significant between the control group and the control group. A difference (p <0.01 or p <0.05: Dunnett's multiple comparison test) was observed. The increasing effect in group C was even weaker than in group B, but an increase of about 31% was seen on day 8 when it reached the maximum.
From the above results, it was confirmed that hTPO163 purified in Example 65 increases the platelet count and its action has a dose dependency.
<Example 76>
Treatment effect of thrombocytopenia by hTPO163
Nimustine hydrochloride (ACNU) was intravenously administered at a rate of 50 mg / kg body weight to 100 8-week-old C3H / HeN male mice and then randomly divided into 4 groups of 25 mice (control, A, B, and C groups). From the next day (Day 1), PBS was subcutaneously administered once a day at a rate of 5 ml / kg body weight to the control group, and the TPO activity image of SP Sepharose Fast Flow obtained in Example 65 was given to Group A. Minute F2 was diluted with PBS, and hTPO163 was subcutaneously administered once a day at a rate of about 16,000,000 / kg body weight as a relative activity in the M-07e assay system. Similarly, group B has a relative activity amount of about 48,000,000 / kg body weight in the M-07e assay system, and group C has a relative activity amount of about 160,000,000 in the M-07e assay system. It was subcutaneously administered once a day every day at a rate of / kg body weight.
After the start of administration, mice were randomly divided into 5 groups in each group, and blood was collected on Day 5, 8, 10, 12, and 14, respectively. Blood was collected from the orbital vein, and the platelet count was measured with a microcell counter (manufactured by Toa Medical Electronics, E-2500). The transition of the platelet count is shown in FIG.
That is, in the control group, the platelet count decreased daily after ACNU administration, and reached the lowest (about 15-16% of the value before ACNU administration) at Day 8-10, and recovered to about 28% on Day 12 and about 51% on Day 14. I just did it. On the other hand, in Group A, Day 8 decreased to about 25% of the pre-ACNU dose, but then increased, Day 10 to about 34%, Day 12 to about 89%, and Day 14 to ACNU administration. It recovered to more than the previous price.
In Group B, Day 8 decreased to about 23% of the pre-ACNU value, but then began to increase, Day 10 already recovered to about 64%, and Day 14 recovered to more than the pre-ACNU value. did. Similarly, in Group C, it started to increase after Day 10 and recovered after Day 12 to the level before ACNU administration.
As described above, the minimum platelet count was observed at Day 8 in any group, but the decrease in the human TPO-administered group was slower than that in the control group, and the minimum value was raised. In the control group, recovery of the platelet count was hardly observed even in Day 10, but in the human TPO administration group, recovery of the platelet count was observed in all groups to some extent, but administration of 50 μg / kg was performed. In the group, Day 12 already recovered to more than the pre-ACNU administration. In the control group, it is clear that hTPO163 produced in Reference Example 2 has an effect of promoting recovery from thrombocytopenia, as compared with Day14, which recovered only to about 51% of the ACNU pretreatment. It became. As described above, hTPO163 purified in Example 65 was found to have a therapeutic effect on thrombocytopenia caused by anticancer drugs.
<Example 77>
Production of human TPO derivatives in E. coli
TPO derivatives were designed and made in an attempt to improve the biological activity, stability, and solubility of TPO expressed in E. coli. The prepared derivatives are Leu129 Arg substitution (L129R), His133 Arg substitution (H133R), Met143 Arg substitution (M143R), Gly82 Leu substitution (G82L), Gly146 Leu substitution of SEQ ID NO: 11 (G146L), a Pro substitution of Ser148 (S148P), an Arg substitution (K59R) of Lys59, and an Arg substitution (Q115R) of Gln115.
As a template for preparing a TPO derivative, h6T (1-163) described in Example 42 was used, and the following oligo DNA was synthesized for mutagenesis.
L129R; 5'-ATCTTCCGTTCTTTCCAGCACCT-3 '(for preparation of Arg substitution of Leu129 of SEQ ID NO: 11)
H133R; 5'-TCTTTCCCAGCGTCTGCTGCGT-3 '(for preparing an Arg substitution of His133 of SEQ ID NO: 11)
M143R; 5'-CGTTTCCTGCGTCTGGTTGGC-3 '(for preparing Arg substitution of Met143 of SEQ ID NO: 11)
G82L; 5'-GGCCAGCTTCTGCCGAC CTGCCT-3 '(for producing Leu substitution of Gly82 of SEQ ID NO: 11)
G146L: 5'-ATGCTGGGTTCTGGGTTCTACCCT-3 '(for preparation of Leu substitution of Gly146 of SEQ ID NO: 11)
S148P; 5'-GTTGGCCGGTCCGACCCTGTGCG-3 '(for producing a Pro-substitution of Ser148 of SEQ ID NO: 11)
K59R; 5'-GAAGAGACCCGCGCTCAGGACATCC-3 '(for preparing an Arg substitution of Lys59 of SEQ ID NO: 11)
Q115R: 5'-CTGCCCGCCACGT  GGCCGTACCAC-3 '(for preparing Arg substitution of Gln115 of SEQ ID NO: 11)
All the mutant plasmids shown below were prepared using Sculptor in vitro mutagenesis kit (Amersham).
The Xba I-Hind III fragment obtained from the plasmid pCFM536 / h6T (1-163) described in Example 42 was inserted between the Xba I-Hind III cleavage sites of Bluescript II SK- (Toyobo Co., Ltd.) pSKTPO was prepared. Plasmid pSKTPO was maintained in E. coli JM109. Single-stranded DNA was prepared from pSKTPO using helper phage M13K07 (manufactured by Takara Shuzo Co., Ltd.), and a mutant plasmid was prepared using the above synthetic oligo DNA for mutation according to the protocol attached to the kit. The nucleotide sequence of the inserted fragment of the obtained candidate plasmid was determined, and it was confirmed that the target site was mutated.
The Xba I-Hind III fragment was extracted from all the pSKTPO derivatives prepared above, inserted between the Xba I-Hind III cleavage sites of the plasmid pCFM536, and then transformed into Escherichia coli 261 to express the target TPO derivative. The transformant to be made was completed.
Culturing of the transformant containing the gene encoding the target TPO derivative was carried out according to Example 43. After adding about 30 mL of 20 mM Tris buffer (pH 8, 5) containing 10 mM EDTA, 10 mM DTT, and 1 mM PMSF to the frozen cells (about 3 g) of the obtained recombinant product of h6T (1-163), suspended. Then, 1-minute sonication (using an ultrasonic crusher) was performed 5 times at intervals of 1 minute under ice-cooling. The crushed cells were transferred to a centrifuge tube and centrifuged at 4 ° C. and 15000 rpm for 10 minutes to collect the pellet.
To this pellet, about 30 mL of 10 mM Tris buffer (pH 8.7) containing 8 M guanidine hydrochloride, 5 mM EDTA, and 1 mM PMSF was added and suspended, and about 50 mg of DTT was further added, followed by stirring at room temperature for 1 to 2 hours. The protein sample was reduced. After the reduction, the pH of the sample was adjusted to 5 using 2M hydrochloric acid solution and left at 4 ° C. The reduced protein sample was diluted with 10 mM CAPS buffer (pH 10.5, about 1.5 L) containing 30% glycerol, 3M urea, 3 mM cystamine, 1 mM L-cysteine.
Dilution was performed overnight at room temperature. The diluted sample solution was stirred at room temperature for 2 days to sufficiently oxidize the sample protein, and then centrifuged at 8000 rpm for 45 minutes to remove insoluble matters. The supernatant after centrifugation is adjusted to pH 6.8 using 6M phosphoric acid, diluted twice with ion-exchanged water, and then insoluble matter is removed using two filter papers (# 2, diameter 90 mm, manufactured by Toyo Roshi). Filtered. The filtrate was adsorbed on a column (2.6 × 10 cm) of an ion exchange resin (CM-Sepharose fast flow, manufactured by Pharmacia), and then 10 mM phosphate buffer (pH 6.8) containing 15% glycerol and 1M urea was added at about 400 mL. After washing, the column was equilibrated with about 300 mL of 10 mM phosphate buffer (pH 7.2) containing 15% glycerol. For elution of the target sample, a linear concentration gradient from 10 mM phosphate buffer containing 15% glycerol to the same buffer containing 0.5 M sodium chloride was used at a flow rate of 1 mL / min. The elution of the protein from the column was followed by ultraviolet absorption at 280 nm, and the fraction containing the target protein was collected (about 30 to 40 mL). This fraction was concentrated to about 2 mL using a centrifugal concentrator (Centriprep 10, Amicon) and then purified using reverse phase HPLC (Waters). At this time, μBondassphere C4 (3.9 × 150 mm) manufactured by Waters was used for the column, and eluent A as a 0.05% TFA solution, 70% 2-Propanol, 30% CH 3 CN and 0. Using the eluent B containing 02% TFA, the concentration of the B eluent was changed from 1% to 100% in 40 minutes to elute the target protein. All TPO derivatives were eluted at about 30 minutes under these conditions.
In order to measure the activity of the purified TPO derivative, the eluted derivative was collected, dialyzed against 2 L of 10 mM phosphate buffer for 2 days, and then concentrated using a centrifugal concentrator (Centriprep 10, Amicon). And subjected to the M-07e assay. Each of the prepared TPO derivatives showed the same biological activity as h6T (1-163) used as a control (see FIGS. 32 and 33).
Hereinafter, formulation examples are shown.
<Formulation Example 1>
The human TPO fraction obtained in Example 56 was concentrated, sterilized, and then frozen to −20 ° C. to prepare an injection.
<Formulation Example 2>
The human TPO fraction obtained in Example 56 was concentrated, filled with 5 ml into a 10 ml vial by aseptic operation, freeze-dried at -20 ° C, and then used as an injection with a freeze-dried product plugged into a rubber stopper. did.
<Formulation Example 3>
The human TPO fraction obtained in Example 56 was concentrated, subjected to sterile filtration, and then filled into a 10 ml vial to obtain an injection.
<Formulation Example 4>
The hTPO163 fraction obtained in Example 65 was concentrated, subjected to sterilization, and then frozen to −20 ° C. to obtain an injection.
<Formulation Example 5>
The hTPO163 fraction obtained in Example 56 was concentrated, filled with 5 ml in a 10 ml vial by aseptic operation, freeze-dried at −20 ° C., and then used as an injection using a freeze-dried product plugged into a rubber stopper. .
<Formulation Example 6>
The hTPO163 fraction obtained in Example 65 was concentrated, subjected to sterile filtration, and then filled into a 10 ml vial to obtain an injection.
<Formulation Example 7>
Reference Example Example 57 The human TPO fraction obtained in Example 57 was concentrated, sterilized, and then frozen to −20 ° C. to prepare an injection.
<Formulation Example 8>
Concentrate the human TPO fraction obtained in Example 57, aseptically fill it with 5 ml in a 10 ml vial, freeze-dry at −20 ° C., and use the freeze-dried product plugged into a rubber stopper. did.
<Formulation Example 9>
The human TPO fraction obtained in Example 57 was concentrated, subjected to sterile filtration, and filled into a 10 ml vial to obtain an injection.
[Sequence Listing]
SEQ ID NO: 1
Sequence length: 261
Sequence type: Nucleic acid
Number of strands: double strand
Topology: Linear
Sequence type: cDNA to mRNA
Origin: rat (Rattus norvegicus)
Cell line: McA-RH8994
Array:

SEQ ID NO: 2
Sequence length: 1663
Sequence type: Nucleic acid
Number of strands: double strand
Topology: Linear
Sequence type: cDNA to mRNA
Origin: rat (Rattus norvegicus)
Cell line: McA-RH8994
Array:

SEQ ID NO: 3
Sequence length: 580
Sequence type: Nucleic acid
Number of strands: double strand
Topology: Linear
Sequence type: cDNA to mRNA
Origin: Homo Sapiens
Tissue type: liver
Array:

SEQ ID NO: 4
Sequence length: 861
Sequence type: Nucleic acid
Number of strands: double strand
Topology: Linear
Sequence type: cDNA to mRNA
Origin: Homo Sapiens
Tissue type: liver
Array:

SEQ ID NO: 5
Sequence length: 576
Sequence type: Nucleic acid
Number of strands: double strand
Topology: Linear
Sequence type: cDNA to mRNA
Origin: Homo Sapiens
Tissue type: liver
Array:

SEQ ID NO: 6
Sequence length: 1267
Sequence type: Nucleic acid
Number of strands: double strand
Topology: Linear
Sequence type: cDNA to mRNA
Origin: Homo Sapiens
Tissue type: liver
Array:

SEQ ID NO: 7
Sequence length: 1721
Sequence type: Nucleic acid
Number of strands: double strand
Topology: Linear
Sequence type: cDNA to mRNA
Origin: Homo Sapiens
Tissue type: liver
Array:

SEQ ID NO: 8
Sequence length: 4506
Sequence type: Nucleic acid
Number of strands: double strand
Topology: Linear
Sequence type: Genomic DNA
Origin: Homo Sapiens
Array:

SEQ ID NO: 9
Sequence length: 416
Sequence type: Nucleic acid
Number of strands: double strand
Topology: Linear
Sequence type: synthetic DNA
Array:

SEQ ID NO: 10
Sequence length: 549
Sequence type: Nucleic acid
Number of strands: double strand
Topology: Linear
Sequence type: synthetic DNA
Array:

SEQ ID NO: 11
Sequence length: 535
Sequence type: Nucleic acid
Number of strands: double strand
Topology: Linear
Sequence type: synthetic DNA
Origin: Human (Homo Sapiens)

SEQ ID NO: 12
Sequence length: 535
Sequence type: Nucleic acid
Number of strands: double strand
Topology: Linear
Sequence type: synthetic DNA
Origin: Human (Homo Sapiens)

SEQ ID NO: 13
Sequence length: 555
Sequence type: Nucleic acid
Number of strands: double strand
Topology: Linear
Sequence type: cDNA to mRNA
Origin: Homo Sapiens
Tissue type: liver
Array:

SEQ ID NO: 14
Sequence length: 1043
Sequence type: Nucleic acid
Number of strands: double strand
Topology: Linear
Sequence type: synthetic DNA
Origin: Human (Homo Sapiens)

SEQ ID NO: 15
Sequence length: 326
Sequence type: amino acid
Topology: Linear
Sequence type: Peptide
Origin: rat (Rattus norvegicus)
Array:

SEQ ID NO: 16
Sequence length: 332
Sequence type: amino acid
Topology: Linear
Sequence type: Peptide
Origin: Homo sapiens
Array:

[Brief description of the drawings]
FIG. 1 is a chromatogram of gel filtration chromatography (Sephaacryl S-200 HR column) and a graph showing TPO activity in a rat CFU-MK assay for purification of rat TPO from XRP.
FIG. 2 is a chromatogram of reverse phase chromatography (Capcell Pak Cl 300A column) in purification of a rat small molecule TPO sample derived from XRP, and a graph showing TPO activity in a rat CFU-MK assay.
FIG. 3 shows separation of SDS-polyacrylamide gel electrophoresis of TPO active fraction FA (tube numbers 36 to 42) of reversed phase chromatography (Capcell Pak Cl 300A column) in purification of rat small molecule TPO sample derived from XRP. The photograph shown and the graph which shows TPO activity in a rat CFU-MK assay.
FIG. 4 is a graph showing a peptide map obtained by three-step digestion of TPO active fraction FA of reverse phase chromatography (Capcell Pak Cl 300A column) in purification of rat small molecule TPO sample derived from XRP.
FIG. 5 is a graph showing rat plasma-derived TPO activity in a rat CFU-MK assay.
FIG. 6 is a conceptual diagram showing the construction of an expression vector pEF18S.
FIG. 7 is a graph showing TPO activity in the culture supernatant of COS1 cells into which pEF18S-A2α has been introduced and expressed in a rat CFU-MK assay.
FIG. 8 is a graph showing TPO activity in the culture supernatant of COS1 cells into which pEF18S-HL34 has been introduced and expressed in a rat CFU-MK assay.
FIG. 9 is a graph showing TPO activity in the culture supernatant of COS1 cells into which pHT1-231 has been introduced and expressed in the rat CFU-MK assay.
FIG. 10a is a graph showing TPO activity in the culture supernatant of COS1 cells into which pHTF1 has been introduced and expressed in a rat CFU-MK assay.
FIG. 10b is a graph showing TPO activity in the culture supernatant of COS1 cells into which pHTF1 has been introduced and expressed in the M-07e assay.
FIG. 11: Outline of restriction enzyme map of phage clone λHGT1. (In the figure, E: EcoRI, H: HindIII, S: SalI are shown.)
FIG. 12a is a graph showing TPO activity in a culture supernatant of COS1 cells into which pEFHGTE has been introduced and expressed in a rat CFU-MK assay.
FIG. 12b is a graph showing TPO activity in the culture supernatant of COS1 cells into which pEFHGTE has been introduced and expressed in the M-07e assay.
FIG. 13a is a graph showing TPO activity in a COS1 cell culture supernatant in a rat CFU-MK assay and in which pHT1-211 # 1, pHT1-191 # 1, and pHT1-171 # 2 were introduced and expressed.
FIG. 13b is a graph showing TPO activity in the culture supernatant of COS1 cells into which pHT1-163 # 2 has been introduced and expressed in the rat CFU-MK assay.
FIG. 14 shows TPO activity in the culture supernatant of COS1 cells into which pHT1-211 # 1, pHT1-191 # 1, pHT1-171 # 2, and pHT1-163 # 2 have been introduced and expressed in the M-07e assay. Graph.
FIG. 15 is a chromatogram of reverse phase chromatography (Vydac C4 column) for purification of human TPO from the culture supernatant of CHO cells into which pDEF2O2-hTPO-P1 has been introduced and expressed.
FIG. 16 is a photograph showing separation by SDS-polyacrylamide gel electrophoresis of human TPO purified from the culture supernatant of CHO cells into which pDEF202-hTPO-P1 has been introduced and expressed.
FIG. 17 is a chromatogram of reverse phase chromatography in purification of human TPO from E. coli into which pCFM536 / h6T (1-163) has been introduced and expressed.
FIG. 18 is a photograph showing separation by SOS-polyacrylamide gel electrophoresis of mutant human TPO, h6T (1-163), which has been separated and purified from Escherichia coli into which pCFM536 / h6T (1-163) has been introduced and expressed.
FIG. 19 shows elution of hTPO163 on a Superdex 75 pg column in the purification of hTPO163 using as a starting material the culture supernatant obtained by transfecting CHO cells with human TPO expression plasmid pDEF202-hTPO163. Protein was measured by UV absorption at 220 nm.
FIG. 20 shows SDS-polyacrylamide of hTPO163 preparation obtained on Superdex 75 pg column in the purification of hTPO163 using the culture supernatant obtained by transfecting CHO cells with human TPO expression plasmid pDEF202-hTPO163. Gel electrophoresis photograph. Each hTPO163 was silver stained.
FIG. 21 shows the structure of the expression vector pSMT201.
FIG. 22 is a graph showing TPO activity in the culture supernatant of COS7 cells into which βGL-TPO, N3 / TPO, and 09 / TPO have been introduced and expressed in the M-07e assay.
FIG. 23 is a graph showing TPO activity in the culture supernatant of COS7 cells into which human TPO insertion / deletion derivatives have been introduced and expressed in the M-07e assay.
FIG. 24 is a graph showing the transition of the platelet count when human TPO is administered intravenously or subcutaneously to normal mice.
FIG. 25 is a graph showing the transition of the number of platelets when human TPO is subcutaneously administered to normal mice at different doses.
FIG. 26 is a graph showing the transition of the number of platelets when human TPO is administered after administering an anticancer drug (5-FU) to mice.
FIG. 27 is a graph showing the transition of the number of platelets when human TPO is administered after administering an anticancer drug (ACNU) to mice.
FIG. 28 is a graph showing changes in the number of platelets when human TPO was administered after BMT (bone marrow transplantation) was performed on mice.
FIG. 29 is a graph showing changes in the number of platelets when human TPO is administered to mice after irradiation.
FIG. 30 is a graph showing changes in the number of platelets when hTPO163 is subcutaneously administered to normal mice.
FIG. 31 is a graph showing the transition of the platelet count when hTPO163 is administered after administering an anticancer drug (ACNU) to mice.
FIG. 32 is a graph showing TPO activity in the M-07e assay of human TPO derivatives (H133R, S148P, Q115R) expressed in E. coli.
FIG. 33 is a graph showing the TPO activity in the M-07e assay of human TPO derivatives (M143R, L129R, G82L, G146L, K59R) expressed in E. coli.

Claims (1)

A method for producing an antibody having immunoreactivity with the human TPO protein consisting of the amino acid sequence represented by SEQ ID NO: 16 , using the following (a) to (d) as antigens:
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 16 (b) among the amino acid sequence shown in SEQ ID NO: 6, positions 1 to 231, positions 1 to 211, positions 1 to 191, positions 1 to 171 1st to 163rd, 1st to 157th, 1st to 156th, 1st to 155th, 1st to 154th, 1st to 151st and 7th to 163rd amino acid sequences (C) a derivative obtained by adding a methionine residue at the -2 position and a lysine residue at the -1 position, a derivative added with a methionine residue at the -1 position, or -1 of the proteins (a) to (b) above position any peptide of a peptide comprising the amino acid sequence of derivatives obtained by adding glycine residue (d) following (i) ~ (vi) ( i) to DLRVLSKLLRDSHVLHSRLSQ;
(Ii) SLGEWKTQMEETKAQD;
(Iii) LGTQLPPQGRTTAHKDPNA;
(Iv) NELPNRTSGLLETNFTASA;
(V) SLPPNLQPGISPSPTHPPTGQYT;
(Vi) PSAPTPTPTSPLLNTSYTHSQNLSQEG.

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