JP2004000237A - New protein and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new protein having activity for inhibiting differentiation and maturation of osteoclast or an osteoclastogenesis inhibiting factor, and to provide a method for producing the protein. <P>SOLUTION: This new protein having the activity for inhibiting the differentiation and/or the maturation of the osteoclast is produced from human fetal pulmonary fibroblast, wherein the protein has a molecular weight of about 60 KD when measured under a reduction condition and about 120 KD when measured under a non-reduction condition. The method for producing the protein includes a process for isolating the protein from a culture solution of the fibroblast and purifying the isolated protein. Further, the protein is produced in a genetic engineering manner. A cDNA for producing the protein in the genetic engineering manner, an antibody having specific affinity for the protein, and a method for measuring the protein in which the antibody is used are provided in the specification, respectively. <P>COPYRIGHT: (C)2004,JPO

Description

（表中、＋＋は破骨細胞形成が８０％以上抑制される活性を、＋は破骨細胞形成が３０〜８０％抑制される活性を、−は活性が検出されないことを示す。）
【００３７】
【実施例４】
ＯＣＩＦ の分子量測定
ＯＣＩＦ活性の認められたピーク６及びピーク７各４０μｌを用い、還元条件下と非還元条件下でＳＤＳ−ポリアクリルアミドゲル電気泳動を行った。即ち、各ピークフラクション２０μｌづつを２本のチューブに分取し減圧濃縮した後、１ｍＭＥＤＴＡ、２．５　％ＳＤＳ、及び０．０１％ブロモフェノールブルーを含む１０ｍＭ　Ｔｒｉｓ−ＨＣｌ，　ｐＨ８　１．５μｌで溶解し、それぞれを非還元条件下及び還元条件下（５％　２−メルカプトエタノール存在下）で３７℃で一晩放置後、それぞれの１μｌをＳＤＳ−ポリアクリルアミドゲル電気泳動に負荷した。電気泳動は１０−１５％アクリルアミドのグラジエントゲル（ファルマシア社）を使用し、電気泳動装置Ｐｈａｓｔ　Ｓｙｓｔｅｍ（ファルマシア社）を用いて行った。分子量マーカーとして、ホスホリラーゼｂ（９４ｋＤ）　、ウシ血清アルブミン（６７ｋＤ）、オボアルブミン（４３ｋＤ）、カルボニックアンヒドラーゼ（３０ｋＤ）、トリプシンインヒビター（２０．０ｋＤ）、α−ラクトアルブミン　（１４．４ｋＤ）　を用いた。電気泳動終了後、Ｐｈａｓｔ　Ｇｅｌ　Ｓｉｌｖｅｒ　Ｓｔａｉｎ　Ｋｉｔ（ファルマシア社）を用いて銀染色を行った。結果を図４に示す。
その結果、ピーク６については還元条件下、非還元条件下で約６０ｋＤの蛋白質のバンドが検出された。又、ピーク７については、還元条件下で約６０ｋＤ、非還元条件下で約１２０ｋＤａの蛋白質のバンドが検出された。従って、ピーク７はピーク６の蛋白質のホモダイマーであると考えられる。
【００３８】
【実施例５】
ＯＣＩＦ の熱安定性試験
ブルー５ＰＷフラクション５１〜５２を混合したサンプルから２０μｌずつを取り、１０ｍＭリン酸塩緩衝生理食塩水、ｐＨ７．２　３０μｌを加えた後、７０℃及び９０℃にて１０分間、又は５６℃にて３０分間熱処理を行った。このサンプルを用い、実施例２記載の方法に従いＯＣＩＦ活性を測定した。結果を表２に示す。
【００３９】
【表２】ＯＣＩＦの熱安定性

（表中、＋＋は破骨細胞形成が８０％以上抑制される活性を、＋は破骨細胞形成が３０〜８０％抑制される活性を、−は活性が検出されないことを示す。）
【００４０】
【実施例６】
内部アミノ酸配列の決定
ブルー−５ＰＷフラクション５１〜７０について、２フラクションづつを混合して１ｍｌとし、それぞれの試料に１０μｌの２５％ＴＦＡを加えた後、１ｍｌずつ１０回にわけて０．１％ＴＦＡを含む２５％アセトニトリルで平衡化した逆相カラム　（ＢＵ−３００、Ｃ４、２．１×２２０ｍｍ　、パーキンエルマー社）にかけ、６０分間でアセトニトリルを５５％にする直線勾配、流速　０．２　ｍｌ／分にて溶出を行い、ピーク６とピーク７を集めた。得られたピーク６とピーク７の一部について、それぞれプロテインシーケンサー（プロサイス、４９４　型、パーキンエルマー社）を用い、Ｎ末端アミノ酸配列分析を行ったが、分析不能でありこれらの蛋白質のＮ末端はブロックされている可能性が示唆された。そこで、これらの蛋白質の内部アミノ酸配列を解析した。即ち、ピーク６とピーク７のそれぞれを遠心濃縮した後、それぞれに１００μｇ　ジチオスレイトール、１０ｍＭ　ＥＤＴＡ、７Ｍ塩酸グアニジン、及び１％ＣＨＡＰＳを含む　０．５Ｍ　Ｔｒｉｓ−ＨＣｌ，　ｐＨ８．５　５０μｌ　を加えて室温で４時間放置し還元した後、０．２μｌの４−ビニルピリジンを加え、室温暗所で一晩放置しピリジルエチル化した。これらのサンプルに１μｌの２５％ＴＦＡを加え、０．１％ＴＦＡを含む２０％アセトニトリルで平衡化した逆相カラム（ＢＵ−３００，　Ｃ４，　２．１×３０ｍｍ，　パーキンエルマー社）にかけ、３０分間でアセトニトリル濃度を５０％にする直線勾配、流速０．３　ｍｌ／　分で溶出を行い、還元ピリジルエチル化ＯＣＩＦサンプルを得た。還元ピリジルエチル化したサンプルのそれぞれを遠心濃縮し、８Ｍ尿素及び０．１％　Ｔｗｅｅｎ８０を含む０．１Ｍ　Ｔｒｉｓ−ＨＣｌ，　ｐＨ９　２５　μｌ　で溶解した後、７３μｌ　の０．１Ｍ　Ｔｒｉｓ−ＨＣｌ，　ｐＨ９　で希釈し、０．０２μｇ　のＡＰ１（リシルエンドプロテアーゼ、和光純薬社）を加え、３７℃で１５時間反応させた。反応液に１μｌの２５％ＴＦＡを加え、０．１％ＴＦＡで平衡化した逆相カラム（ＲＰ−３００，　Ｃ８，　２．１×２２０ｍｍ　、パーキンエルマー社）にかけ、７０分間でアセトニトリル濃度を５０％にする直線勾配、流速０．２　ｍｌ／　分で溶出を行い、ペプチドフラグメントを得た（図５）。得られたペプチドフラグメント　（Ｐ１〜Ｐ３）について、プロテインシーケンサーを用いアミノ酸配列分析を行った。結果を配列表　配列番号１〜３に示す。
【００４１】
【実施例７】
ｃＤＮＡ 配列の決定
ｉ） 　 ＩＭＲ−９０ 細胞からのポリ （Ａ） ^＋ ＲＮＡ　 の単離
ＩＭＲ−９０細胞のポリ（Ａ）^＋ＲＮＡ　は、ファストトラックｍＲＮＡアイソレーションキット（インヴィトロージェン社）を用い、そのマニュアルに準じて単離した。この方法により１Ｘ１０^８個のＩＭＲ−９０細胞より約１０μｇ　のポリ（Ａ）^＋ＲＮＡを取得した。
【００４２】
ｉｉ）　 ミックスプライマーの作製
先に得られたペプチド（配列表　配列番号２及び３）のアミノ酸配列をもとに、次の２種のミックスプライマーを合成した。即ち、ペプチドＰ２の６番目（Ｇｌｎ）から１２番目（Ｌｅｕ）　までのアミノ酸配列をコードしうるすべての塩基配列を持つオリゴヌクレオチドの混合物（ミックスプライマー，Ｎｏ．２Ｆ）を合成した。又、ペプチドのＰ３　の６番目（Ｈｉｓ）　から１２番目（Ｌｙｓ）　までのアミノ酸配列をコードしうるすべての塩基配列に対する相補的オリゴヌクレオチドの混合物（ミックスプライマー，Ｎｏ．３Ｒ）を合成した。用いたミックスプライマーの塩基配列を、表３に示す。
【００４３】
【表３】

【００４４】
ｉｉｉ） 　 ＯＣＩＦｃＤＮＡ 断片の ＰＣＲ　 による増幅
実施例７−ｉ）で得たポリ（Ａ）^＋ＲＮＡ、１Ｍｇを鋳型としてスーパースクリプトＩＩｃＤＮＡ合成キット（ギブコＢＲＬ社）　を用いて、同社のプロトコールに従って一本鎖ｃＤＮＡを合成し、このｃＤＮＡと実施例７−ｉｉ）　で示したプライマーを用いて、ＰＣＲを行い、ＯＣＩＦｃＤＮＡ断片を取得した。以下に条件を示す。
【００４５】

【００４６】
上記の溶液を微量遠心チューブ中で混合後、以下の条件でＰＣＲを行った。９５℃で３　分前処理後、９５℃３０秒、５０℃３０秒、７０℃２　分の３　段階の反応を３０回繰り返したのち、７０℃５分保温した。反応液の一部をアガロース電気泳動し約４００ｂｐ　の均一なＤＮＡ断片が得られたことを確認した。
【００４７】
【実施例８】
ＰＣＲ　 により増幅された ＯＣＩＦｃＤＮＡ 断片のクローニング及び塩基配列決定
実施例７−ｉｉｉ）で得られたＯＣＩＦｃＤＮＡ断片を、Ｍａｒｃｈｕｋ，　Ｄらの方法（Ｎｕｃｌｅｉｃ　ＡｃｉｄＲｅｓ．，　Ｖｏｌ．１９，　ｐ１１５４，　１９９１）によってプラスミドｐＢｌｕｅｓｃｒｉｐｔ　ＩＩ　ＳＫ⁻（ストラタジーン社）にＤＮＡライゲーションキット　Ｖｅｒ．２（宝酒造社）を用いて挿入し、大腸菌　ＤＨ５α（ギブコＢＲＬ社）の形質転換を行った。得られた形質転換株を増殖させ、約　４００ｂｐのＯＣＩＦｃＤＮＡ断片が挿入されたプラスミドを常法に従い精製した。このプラスミドをｐＢＳＯＣＩＦと名付け、このプラスミドに挿入されているＯＣＩＦｃＤＮＡの塩基配列をタックダイデオキシターミネーターサイクルシークエンシングキット（ＴａｑＤｙｅ　Ｄｅｏｘｙ　Ｔｅｒｍｉｎａｔｏｒ　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　ｋｉｔ；　パーキンエルマー社）を用いて決定した。このＯＣＩＦｃＤＮＡの大きさは、３９７　ｂｐであった。この塩基配列から予測される１３２　個のアミノ酸からなるアミノ酸配列中に、ミックスプライマーを設計するのに用いたＯＣＩＦの内部アミノ酸配列（配列表配列番号２及び３）をそれぞれＮ末側、Ｃ末側に見出すことができた。又、ＯＣＩＦの内部アミノ酸配列（配列番号１）を、この　１３２個のアミノ酸からなるアミノ酸配列中に見出すことができた。以上の結果より、クローニングした３９７　ｂｐのｃＤＮＡは、ＯＣＩＦｃＤＮＡ断片であることが確認された。
【００４８】
【実施例９】
　ＤＮＡ プローブの作製
実施例８で作成された３９７ｂｐ　のＯＣＩＦｃＤＮＡ断片が挿入されたプラスミドを鋳型にして実施例７−ｉｉｉ）の条件でＰＣＲを行なうことにより、このＯＣＩＦｃＤＮＡ断片を増幅した。アガロース電気泳動により３９７ｂｐ　のＯＣＩＦｃＤＮＡ断片を分離後、ＱＩＡＥＸ　ゲルエクストラクションキット（キアゲン社）を用いて精製した。このＤＮＡをメガプライムＤＮＡラベリングキット（アマシャム社）　を用いて　［α^３２Ｐ］ｄＣＴＰ　で標識し、全長のＯＣＩＦｃＤＮＡをスクリーニングするためのプローブとして用いた。
【００４９】
【実施例１０】
ｃＤＮＡ ライブラリーの作成
実施例７−ｉ）で得られたポリ（Ａ）^＋ＲＮＡ　、２．５μｇを鋳型としてグレートレングスｃＤＮＡ合成キット（クロンテック社）　を用いて同社のプロトコールに従い、ｏｌｉｇｏ（ｄＴ）ｐｒｉｍｅｒ　を用いてｃＤＮＡの合成、ＥｃｏＲＩ−ＳａｌＩ−Ｎｏｔ−Ｉアダプター付加、ｃＤＮＡサイズフラクショネーションを行いエタノール沈殿の後１０μｌのＴＥバッファーに溶解した。得られたアダプター付加ｃＤＮＡ、０．１μｇをＴ４ＤＮＡ　リガーゼを用いてあらかじめＥｃｏＲＩで切断した１μｇのλＺＡＰ　エクスプレスベクター（ストラタジーン社）に挿入した。このようにして得られたｃＤＮＡ組み換えファージＤＮＡ　溶液をギガパックゴールドＩＩ（ストラタジーン社）　を用いてインヴィトロパッケージング反応に供し、λＺＡＰ　エクスプレス組み換えファージを作成した。
【００５０】
【実施例１１】
組み換えファージのスクリーニング
実施例１０で得られた組み換えファージを３７℃で１５分間大腸菌　ＸＬ１−Ｂｌｕｅ　ＭＲＦ’（ストラタジーン社）に感染させたのち、５０℃に加温した０．７　％の寒天を含むＮＺＹ　培地に添加し、ＮＺＹ　寒天培地プレートに流しこんだ。３７℃で一晩培養後、プラークの生じたプレート上にハイボンドＮ（アマシャム社）を約３０秒密着させた。このフィルターを常法に従いアルカリ変性の後、中和し、２ＸＳＳＣ　溶液に浸したのちＵＶクロスリンク（ストラタジーン社）　によりＤＮＡ　をフィルターに固定化した。得られたフィルターを１００　μｇ／ｍｌのサケ精子ＤＮＡ　を含むハイブリダイゼーションバッファー（アマシャム社）に浸漬し６５℃で４　時間前処理した後、熱変性した上記ＤＮＡ　プローブ（２Ｘ１０^５ｃｐｍ／ｍｌ）　を添加した上記バッファ−に移し替え６５℃で一晩ハイブリダイゼーションを行った。反応後フィルターを２ＸＳＳＣ　で２　回、０．１ＸＳＳＣ，０．１％　ＳＤＳ溶液で２回それぞれ６５℃で１０分間洗浄した。得られたいくつかの陽性クローンを、さらに２　回スクリーニングを行うことにより純化した。それらの中から約１．６ｋｂ　のインサートを持つものを以下に用いた。この純化したファージをλＯＣＩＦと名付けた。純化したλＯＣＩＦをλＺＡＰ　エクスプレスクローニングキット（ストラタジーン社）のプロトコールに従い、大腸菌ＸＬ１−Ｂｌｕｅ　ＭＲＦ’　に感染させたのち、ヘルパーファージＥｘＡｓｓｉｓｔ（ストラタジーン社）　で多重感染を行い、その培養上清を大腸菌ＸＬＯＬＲ（ストラタジーン社）　に感染させたのちカナマイシン耐性株を拾うことによりｐＢＫＣＭＶ（ストラタジーン社）　に上述の１．６ｋｂ　のインサートが挿入されたプラスミドｐＢＫＯＣＩＦ　をもつ形質転換株を得た。この形質転換株はｐＢＫ／０１Ｆ１０　として、通商産業省工業技術院生命工学工業技術研究所（現独立行政法人産業技術総合研究所　特許生物寄託センター）に受託番号ＦＥＲＭ　ＢＰ−５２６７（平成７年１０月２５日にＦＥＲＭ　Ｐ−１４９９８の原寄託よりブタペスト条約に基づく寄託に移管）として寄託してある。このプラスミドをもつ形質転換株を増殖させ、常法によりプラスミドを精製した。
【００５１】
【実施例１２】
ＯＣＩＦ の全アミノ酸配列をコードする ｃＤＮＡ の塩基配列の決定
実施例１１で得られたＯＣＩＦｃＤＮＡの塩基配列をタックダイデオキシターミネーターサイクルシークエンシングキット（パーキンエルマー社）　を用いて決定した。用いたプライマーはＴ３，　Ｔ７　プライマー（ストラタジーン社）及びＯＣＩＦｃＤＮＡの塩基配列に基づいて設計された合成プライマーであり、その配列を配列表配列番号１６〜２９に示す。
決定されたＯＣＩＦの塩基配列を配列番号６に、その配列から推定されるアミノ酸配列を配列番号５にそれぞれ示す。
【００５２】
【実施例１３】
２９３／ＥＢＮＡ 細胞による組み換え型 ＯＣＩＦ の生産
ｉ） 　 ＯＣＩＦｃＤＮＡ の発現プラスミドの作製
実施例１１で得られた約１．６ｋｂ　のＯＣＩＦｃＤＮＡが挿入されたプラスミドｐＢＫＯＣＩＦ　を制限酵素ＢａｍＨＩ　及びＸｈｏＩで消化し、ＯＣＩＦｃＤＮＡを切り出し、アガロース電気泳動によって分離後、ＱＩＡＥＸ　ゲルエクストラクションキット（キアゲン社）を用いて精製した。このＯＣＩＦｃＤＮＡを、あらかじめ制限酵素ＢａｍＨＩ　及びＸｈｏＩで消化しておいた発現プラスミドｐＣＥＰ４（インヴィトロージェン社）に、ライゲーションキット　Ｖｅｒ．２（宝酒造社）を用いて挿入し、大腸菌ＤＨ５　α（ギブコＢＲＬ社）の形質転換を行った。得られた形質転換株を増殖させ、ＯＣＩＦｃＤＮＡが挿入された発現プラスミドｐＣＥＰＯＣＩＦをキアゲンカラム（キアゲン社）　を用いて精製した。ＯＣＩＦ発現プラスミドｐＣＥＰＯＣＩＦをエタノールによって沈澱させた後、無菌蒸留水に溶解し以下の操作に用いた。
【００５３】
ｉｉ）　 　 ＯＣＩＦｃＤＮＡ のトランジエントな発現及びその活性の測定
実施例１３−ｉ）で得られたＯＣＩＦ発現プラスミドｐＣＥＰＯＣＩＦを用いて、以下に述べる方法で組み換えＯＣＩＦを発現させ、その活性を測定した。８×１０^５個の２９３／ＥＢＮＡ細胞（インヴィトロージェン社）を６ウェルプレートの各ウェルに１０％牛胎児血清（ギブコＢＲＬ社）を含むＩＭＤＭ培地（ギブコＢＲＬ社）を用いて植え込み、翌日、培地を除いた後、無血清ＩＭＤＭ培地で細胞を洗った。トランスフェクション用試薬リポフェクタミン（ギブコＢＲＬ社）添付のプロトコールに従い、あらかじめＯＰＴＩ−ＭＥＭ培地（ギブコＢＲＬ社）を用いて希釈しておいたｐＣＥＰＯＣＩＦとリポフェクタミンを混合した後、この混合液を各ウェルの細胞に加えた。用いたｐＣＥＰＯＣＩＦ及びリポフェクタミンの量はそれぞれ３μｇ及び１２μｌであった。３８時間後、培地を除き１ｍｌの新しいＯＰＴＩ−ＭＥＭ培地を加え、さらに３０時間後、培地を回収し、これをＯＣＩＦ活性測定用サンプルとした。ＯＣＩＦの活性測定は以下のようにして行った。生後約１７日のマウス骨髄細胞からの活性型ビタミンＤ_３存在下での破骨細胞形成を酒石酸耐性酸性ホスファターゼ活性の誘導で試験し、その抑制活性を測定し、ＯＣＩＦの活性とした。すなわち、９６ウェルマイクロプレートに２×１０^{‐ ８}Ｍ活性型ビタミンＤ_３及び１０％牛胎児血清を含むα−ＭＥＭ培地（ギブコＢＲＬ社）で希釈したサンプル１００μｌ　を入れ、生後約１７日のマウス骨髄細胞３×１０^５個を　１００μｌ　の１０％牛胎児血清を含むα−ＭＥＭ培地に懸濁させて播種し、５　％ＣＯ_２、３７℃、湿度　１００％にて一週間培養した。培養３日目と５日目に、培養液１６０μｌ　を廃棄し、１×１０^−８Ｍ活性型ビタミンＤ_３及び１０％牛胎児血清を含むα−ＭＥＭ培地で希釈したサンプル１６０μｌを添加した。培養７日後にリン酸塩緩衝生理食塩水で洗浄した後エタノール／アセトン（１：１）溶液で細胞を室温にて１分間固定し、破骨細胞形成を酸性ホスファターゼ活性測定キット（Ａｃｉｄ　Ｐｈｏｓｐｈａｔａｓｅ，　Ｌｅｕｃｏｃｙｔｅ　、カタログ　Ｎｏ．３８７−　Ａ、シグマ社）を用いた染色で検出した。酒石酸存在下での酸性ホスファターゼ活性陽性細胞の減少をＯＣＩＦ活性とした。その結果、表４に示すように、先にＩＭＲ−９０の培養液から得られた天然型ＯＣＩＦと同様の活性をこの培養液が有することが確認された。
【００５４】
【表４】

（表中、＋＋は破骨細胞形成が８０％以上抑制される活性を、＋は破骨細胞形成が３０〜８０％抑制される活性を、−は活性が検出されないことを示す。）
【００５５】
ｉｉｉ） 　 ２９３／ＥＢＮＡ 細胞由来組み換え型 ＯＣＩＦ の精製
実施例１３−ｉｉ）に記載した２９３／ＥＢＮＡ細胞を大量培養して得た培養液１．８ｌに０．１　％になるようにＣＨＡＰＳを加え、０．２２μｍ　のフィルター（ステリベックスＧＳ、ミリポア社）で濾過した後、１０ｍＭ　Ｔｒｉｓ−ＨＣｌ，　ｐＨ７．５で平衡化させた５０ｍｌのヘパリン・セファロースＣＬ−６Ｂカラム（２．６×１０ｃｍ、ファルマシア社）にかけた。０．１　％ＣＨＡＰＳを含む１０ｍＭＴｒｉｓ−ＨＣｌ，　ｐＨ７．５で洗浄した後、１００　分間でＮａＣｌを２Ｍにする直線勾配、流速４ｍｌ／分にて溶出を行い、８ｍｌ／フラクションにて分取を行った。各フラクション１５０μｌを用いて実施例２の方法に従ってＯＣＩＦ活性を測定し、約　０．６〜１．２ＭＮａＣｌで溶出されるＯＣＩＦ活性画分　１１２ｍｌを得た。
【００５６】
得られたＯＣＩＦ活性画分　１１２ｍｌを０．１　％ＣＨＡＰＳを含む　１０ｍＭ　Ｔｒｉ　ｓ−ＨＣｌ，ｐＨ７．５　で１０００ｍｌに希釈した後、０．１　％ＣＨＡＰＳを含む　１０ｍＭ　Ｔｒｉｓ−ＨＣｌ，　ｐＨ７．５で平衡化させたアフィニティカラム（ヘパリン　−５ＰＷ，　０．８×７．５　ｃｍ、トーソー社）にかけた。０．１％ＣＨＡＰＳを含む　１０ｍＭ　Ｔｒｉｓ−ＨＣｌ，　ｐＨ７．５　で洗浄した後、６０分間でＮａＣｌを２Ｍにする直線勾配、流速０．５ｍｌ／分にて溶出を行い、０．５ｍｌ／フラクションにて分取を行った。
【００５７】
得られたフラクション各４μｌを用いて実施例４の方法に従って還元及び非還元条件下でＳＤＳ−ポリアクリルアミドゲル電気泳動を行った。その結果、フラクション３０〜３２には還元条件下で約６０ｋＤ、非還元条件下で約６０ｋＤと約　１２０ｋＤのＯＣＩＦバンドのみが検出されたので、フラクション３０〜３２を集め純化２９３／ＥＢＮＡ細胞由来組み換え型ＯＣＩＦ（　ｒＯＣＩＦ（Ｅ））　画分とした。ＢＳＡをスタンダードとして用いたローリー法による蛋白定量の結果、５３５μｇ／ｍｌのｒＯＣＩＦ（Ｅ）１．５ｍｌ　が得られたことが明らかになった。
【００５８】
【実施例１４】
ＣＨＯ 細胞による組み換え型 ＯＣＩＦ の生産
ｉ） 　 ＯＣＩＦ の発現プラスミドの作製
実施例１１で得られた約１．６ｋｂ　のＯＣＩＦｃＤＮＡが挿入されたプラスミドｐＢＫＯＣＩＦ　を制限酵素ＳａｌＩ及びＥｃｏＲＶ　で消化し、約１．４ｋｂ　のＯＣＩＦｃＤＮＡ断片を切り出し、アガロース電気泳動によって分離後、ＱＩＡＥＸ　ゲルエクストラクションキット（キアゲン社）を用いて精製した。又、発現ベクターｐｃＤＬ−ＳＲ　α２９６　（Ｍｏｌｅｃｕｌａｒ　ａｎｄ　Ｃｅｌｌｕｌａｒ　Ｂｉｏｌｏｇｙ，　Ｖｏｌ．８，　ｐｐ４６６−４７２，　１９８８）　を制限酵素ＰｓｔＩ及びＫｐｎＩで消化し、約３．４ｋｂ　の発現ベクターＤＮＡ　断片をアガロース電気泳動によって分離後、ＱＩＡＥＸゲルエクストラクションキット（キアゲン社）を用いて精製した。ＤＮＡブランティングキット（宝酒造社）を用いて、これらの精製したＯＣＩＦｃＤＮＡ断片と発現ベクターＤＮＡ断片の末端を平滑化した。次に、ライゲーションキット　Ｖｅｒ．２（宝酒造社）を用いて、平滑化された発現ベクターＤＮＡ断片にＯＣＩＦｃＤＮＡ断片を挿入し、大腸菌ＤＨ５α（ギブコＢＲＬ社）の形質転換を行い、ＯＣＩＦ発現プラスミドｐＳＲ　αＯＣＩＦをもつ形質転換株を得た。
【００５９】
ｉｉ）　 発現プラスミドの調製
実施例１３−ｉ）で得られたＯＣＩＦ発現プラスミドｐＳＲ　αＯＣＩＦをもつ形質転換株及びＷＯ９２／０１０５３号公報に示されるマウスＤＨＦＲ遺伝子発現プラスミドｐＢＡｄＤＳＶ　をもつ形質転換株をそれぞれ常法を用いて増殖させ、Ｍａｎｉａｔｉｓら（Ｍｏｌｅｃｕｌａｒ　ｃｌｏｎｉｎｇ，２ｎｄ　ｅｄｉｔｉｏｎ）　の方法に従いアルカリ法及びポリエチレングリコール法で処理し、塩化セシウム密度勾配遠心法により精製した。
【００６０】
ｉｉｉ） 　 ＣＨＯｄｈＦｒ ⁻ 細胞の蛋白質不含培地への馴化
１０％牛胎児血清（ギブコＢＲＬ社）を含むＩＭＤＭ培地（ギブコＢＲＬ社）で継代されていたＣＨＯｄｈＦｒ⁻細胞（ＡＴＣＣ−ＣＲＬ９０９６）は、無血清培地　ＥＸ−ＣＥＬＬ３０１（ＪＲＨバイオサイエンス社）で馴化後、さらに蛋白質不含培地ＥＸ−ＣＥＬＬ　ＰＦ　ＣＨＯ（ＪＲＨバイオサイエンス社）で馴化させた。
【００６１】
ｉｖ）　ＯＣＩＦ 発現プラスミド及び ＤＨＦＲ 発現プラスミドの ＣＨＯｄｈＦｒ ⁻ 細胞への導入
実施例１４−ｉｉ）　で調製したＯＣＩＦ発現プラスミド　ｐＳＲαＯＣＩＦ及びＤＨＦＲ発現プラスミドｐＢＡｄＤＳＶ　を用いて実施例１４−ｉｉｉ）で調製したＣＨＯｄｈＦｒ⁻細胞を下記に示すエレクトロポレーション法により形質転換した。ｐＳＲ　αＯＣＩＦプラスミド　２００μｇとｐＢＡｄＤＳＶ　プラスミド２０μｇを無菌的に１０％牛胎児血清（ギブコＢＲＬ社）を含むＩＭＤＭ培地（ギブコＢＲＬ社）０．８ｍｌ　に溶解後、この０．８ｍｌ　を用いて　２×１０^７個のＣＨＯｄｈＦｒ⁻細胞を浮遊させた。この細胞浮遊液をキュベット（バイオラッド社）に入れ、ジーンパルサー（バイオラッド社）を用いて、３６０Ｖ、９６０　μＦの条件でエレクトロポレーション法により形質転換を行った。１０ｍｌのＥＸ−ＣＥＬＬ　ＰＦ　ＣＨＯ培地の入った浮遊細胞用Ｔフラスコ（住友ベークライト社）にエレクトロポレーション済の細胞浮遊液を移し、ＣＯ_２インキュベーター中で２日間培養した。ＥＸ−ＣＥＬＬ　ＰＦ　ＣＨＯ培地を用いて５０００ｃｅｌｌｓ／ｗｅｌｌの濃度で９６ウェルマイクロプレートにまき、約２週間培養した。ＥＸ−ＣＥＬＬ　ＰＦ　ＣＨＯ培地は核酸を含まず、この培地では親株のＣＨＯｄｈＦｒ⁻は増殖できないので、ＤＨＦＲを発現する細胞株だけが選択されてくる。ＯＣＩＦ発現プラスミドをＤＨＦＲ発現プラスミドの１０倍量用いているので、ＤＨＦＲを発現する細胞株の大部分はＯＣＩＦを発現する。得られたＤＨＦＲを発現する細胞株から培養上清中のＯＣＩＦ活性の高い細胞株を、実施例２で示した測定法によってスクリーニングした。得られたＯＣＩＦ高生産株につきＥＸ−ＣＥＬＬ　ＰＦ　ＣＨＯ培地を用いて限界希釈法により細胞のクローニングを行い、得られたクローンについて培養上清中のＯＣＩＦ活性の高い細胞株をスクリーニングし、ＯＣＩＦ高生産クローン５５６１を得た。
【００６２】
ｖ） 　組み換え型 ＯＣＩＦ の生産
組み換えＯＣＩＦ（ｒＯＣＩＦ）　の生産するため、ＥＸ−ＣＥＬＬ　３０１　培地３ｌに形質転換ＣＨＯ細胞（５５６１）を１×１０^５ｃｅｌｌｓ／ｍｌ　となるように接種し、スピナーフラスコを用いて３７℃で４、５日培養した。細胞の濃度が約１×１０^６ｃｅｌｌｓ／ｍｌ　になったところで、約２．７ｌの培地を回収した。約２．７ｌのＥＸ−ＣＥＬＬ　３０１　培地を加え、培養を繰り返した。３基のスピナーフラスコを用い、約２０ｌ　の培養液を採取した。
【００６３】
ｖｉ）　ＣＨＯ 細胞由来組み換え型 ＯＣＩＦ の精製
実施例１４−（ｖ）　で得られた培養液１ｌ　に０．１％になるようにＣＨＡＰＳを加え、０．２２μｍ　のフィルター（ステリベックスＧＳ、ミリポア社）で濾過した後、１０ｍＭ　Ｔｒｉｓ−ＨＣｌ，　ｐＨ７．５で平衡化させた５０ｍｌのヘパリン・セファロースＦＦカラム（２．６×１０ｃｍ、ファルマシア社）にかけた。０．１％ＣＨＡＰＳを含む１０ｍＭ　Ｔｒｉｓ−ＨＣｌ，ｐＨ７．５　で洗浄した後、１００　分間でＮａＣｌを２Ｍにする直線勾配、流速４ｍｌ／分にて溶出を行い、８ｍｌ／フラクションにて分取を行った。各フラクション　１５０μｌ　を用いて実施例２の方法に従ってＯＣＩＦ活性を測定し、約　０．６〜１．２Ｍで溶出されるＯＣＩＦ活性画分　１１２ｍｌを得た。
【００６４】
得られたＯＣＩＦ活性画分　１１２ｍｌを０．１　％ＣＨＡＰＳを含む　１０ｍＭ　Ｔｒｉｓ−ＨＣｌ，ｐＨ７．５　で１２００ｍｌに希釈した後、０．１　％ＣＨＡＰＳを含む　１０ｍＭ　Ｔｒｉｓ−ＨＣｌ，　ｐＨ７．５で平衡化させたアフィニティカラム（ブルー　−５ＰＷ，　０．５×５ｃｍ　、トーソー社）にかけた。０．１　％ＣＨＡＰＳを含む　１０ｍＭ　Ｔｒｉｓ−ＨＣｌ，　ｐＨ７．５　で洗浄した後、９０分間でＮａＣｌを３Ｍにする直線勾配、流速０．５ｍｌ／分にて溶出を行い、０．５ｍｌ／フラクションにて分取を行った。
【００６５】
得られたフラクション各４μｌ　を用いて実施例４の方法に従って還元及び非還元条件下でＳＤＳ−ポリアクリルアミドゲル電気泳動を行った。その結果、フラクション３０〜３８には還元条件下で約６０ｋＤ、非還元条件下で約６０ｋＤと約　１２０ｋＤのＯＣＩＦバンドのみが検出されたので、フラクション３０〜３８を集め精製ＣＨＯ細胞由来組み換え型ＯＣＩＦ〔ｒＯＣＩＦ（Ｃ）〕画分とした。ＢＳＡをスタンダードとしたローリー法による蛋白定量の結果、１１３　μｇ／ｍｌのｒＯＣＩＦ（Ｃ）４．５ｍｌが得られたことが明らかになった。
【００６６】
【実施例１５】
組み換え型 ＯＣＩＦ のＮ末端構造解析
３μｇの精製ｒＯＣＩＦ（Ｅ）及びｒＯＣＩＦ（Ｃ）を、プロスピン（ＰｒｏＳｐｉｎ，パーキンエルマー社）　を用いてポリビニリデンジフルオリド（ＰＶＤＦ）膜に固定し、２０％メタノールで洗浄した後、プロテインシーケンサー（プロサイス、４９２　型、パーキンエルマー社）を用いてＮ末端アミノ酸配列分析を行った。結果を配列表配列番号７に示す。
ｒＯＣＩＦ（Ｅ）と　ｒＯＣＩＦ（Ｃ）　のＮ末端アミノ酸は、配列表配列番号５に記載したアミノ酸配列の翻訳開始点　Ｍｅｔから２２番目の　Ｇｌｕで、Ｍｅｔ　から　Ｇｌｎまでの２１アミノ酸はシグナルペプチドであることが明らかになった。又、ＩＭＲ−９０培養液から精製し得られた天然型ＯＣＩＦのＮ末端アミノ酸配列が分析不能であったのは、Ｎ末端のＧｌｕ　が培養中又は精製中にピログルタミン酸に変換したためと考えられた。
【００６７】
【実施例１６】
組み換え型 （ｒ）ＯＣＩＦ 及び天然型 （ｎ）ＯＣＩＦ の生物活性
ｉ） 　マウス骨髄細胞系での、ビタミン Ｄ _３ で誘導される破骨細胞形成の抑制
９６ウェルマイクロプレートに、２×１０^{‐ ８}Ｍ活性型ビタミンＤ_３及び１０％牛胎児血清を含むα−ＭＥＭ培地（ギブコＢＲＬ社）で２５０ｎｇ／ｍｌから連続的に二分の一希釈した精製ｒＯＣＩＦ（Ｅ）及び　ｎＯＣＩＦ　１００μｌ　を入れた。このウェルに生後約１７日のマウス骨髄細胞３×１０^５個を　１００μｌ　の１０％牛胎児血清を含むα−ＭＥＭ培地に懸濁させて播種し、５％　ＣＯ_２、３７℃、湿度　１００％にて一週間培養した。培養７日後に、実施例２の方法に従って酸性ホスファターゼ活性測定キット（Ａｃｉｄ　Ｐｈｏｓｐｈａｔａｓｅ，　Ｌｅｕｃｏｃｙｔｅ　、カタログＮｏ．３８７−Ａ，シグマ社）を用いた染色を行い破骨細胞形成を検出した。酒石酸存在下での酸性ホスファターゼ活性陽性細胞の減少をＯＣＩＦ活性とした。酸性ホスファターゼ活性陽性細胞の減少率は、染色した細胞の色素を可溶化し、その吸光度を測定することにより算出した。即ち、細胞を固定し染色した各ウェルに０．１Ｎ水酸化ナトリウム−ジメチルスルフォキシド混合液（１：１）　１００μｌ　を加えよく振盪した。色素を十分に溶解させた後、マイクロプレートリーダー（イムノリーダーＮＪ−２０００、インターメッド社）を用い、測定波長　５９０ｎｍ、対照波長　４９０ｎｍにて吸光度を測定した。又、吸光度を測定する際のブランクウェルとして、ビタミンＤ_３未添加のウェルを用いた。結果は、ＯＣＩＦ未添加のウェルでの吸光度値を　１００とした百分率値で表し、表５に示す。
ｎＯＣＩＦと同様にｒＯＣＩＦ（Ｅ）にも、１６ｎｇ／ｍｌ　以上の濃度で用量依存的な破骨細胞形成抑制活性が見られた。
【００６８】
【表５】

【００６９】
ｉｉ）　 ストローマ細胞とマウス脾臓細胞の共培養系でのビタミン Ｄ _３ で誘導される破骨細胞形成の抑制
ビタミンＤ_３で誘導されるストローマ細胞とマウス脾臓細胞の共培養系での破骨細胞形成の試験は、宇田川らの方法（Ｅｎｄｏｃｒｉｎｏｌｏｇｙ，Ｖｏｌ．１２５，　ｐ１８０５−１８１３，１９８９）　に従って行った。即ち、９６ウェルマイクロプレートに２×１０^{‐ ８}Ｍ活性型ビタミンＤ_３、２×１０^{‐ ７}でデキサメサゾン及び１０％牛胎児血清を含むα−ＭＥＭ培地（ギブコＢＲＬ社）で、連続的に希釈した精製　ｒＯＣＩＦ（Ｅ）、　ｒＯＣＩＦ（Ｃ）　及びｎＯＣＩＦ１００μｌを入れた。このウェルにマウス骨髄由来ストローマ細胞株ＳＴ２細胞　（ＲＩＫＥＮ　Ｃｅｌｌ　Ｂａｎｋ−ＲＣＢ０２２４）　５×１０^３個と生後約８週間の　ｄｄｙマウス脾臓細胞１×１０^５個を　１００μｌ　の１０％牛胎児血清を含むα−ＭＥＭ培地に懸濁させて播種し、５％ＣＯ_２、３７℃、湿度　１００％にて５日間培養した。培養５日後にリン酸塩緩衝生理食塩水で洗浄した後、エタノール／アセトン（１：１）溶液で細胞を室温にて１分間固定し、破骨細胞形成を酸性ホスファターゼ活性測定キット（Ａｃｉｄ　Ｐｈｏｓｐｈａｔａｓｅ，　Ｌｅｕｃｏｃｙｔｅ、カタログＮｏ．３８７−Ａ，　シグマ社）を用いた染色で検出した。酒石酸存在下での酸性ホスファターゼ活性陽性細胞の減少をＯＣＩＦ活性とした。又、酸性ホスファターゼ活性陽性細胞数の減少率は実施例１６−ｉ）に記載した方法に従って染色された細胞の色素を溶解させて算出した。ｒＯＣＩＦ（Ｅ）とｒＯＣＩＦ（Ｃ）を用いて試験した結果を表６に、ｒＯＣＩＦ（Ｅ）とｎＯＣＩＦ　を用いて試験した結果を表７に、それぞれ示す。
ｎＯＣＩＦと同様にｒＯＣＩＦ（Ｅ）及びｒＯＣＩＦ（Ｃ）についても、６〜１６ｍｇ／ｍｌ以上の濃度で容量依存的な破骨細胞形成抑制活性が見られた。
【００７０】
【表６】

【００７１】

【００７２】
ｉｉｉ） 　 ＰＴＨ で誘導される破骨細胞形成の抑制
ＰＴＨで誘導される破骨細胞形成の試験は、高橋らの方法　（Ｅｎｄｏｃｒｉｎｏｌｏｇｙ，　　Ｖｏｌ．１２２，　ｐ１３７３−１３８２，　１９８８）に従って行った。即ち、９６ウェルマイクロプレートに２×１０^{‐ ８}ＭＰＴＨ及び１０％牛胎児血清を含むα−ＭＥＭ培地（ギブコ社）で、１２５ｎｇ／ｍｌから連続的に希釈したｎＯＣＩＦ及び精製ｒＯＣＩＦ（Ｅ）　１００μｌ　を入れた。このウェルに生後約１７日のマウス骨髄細胞３×１０^５個を１００μｌの１０％牛胎児血清を含むα−ＭＥＭ培地に懸濁させて播種し、５％　ＣＯ_２、３７℃、湿度１００　％にて５日間培養した。培養５日後にリン酸塩緩衝生理食塩水で洗浄した後エタノール／アセトン（１：１）溶液で細胞を室温にて１分間固定し、破骨細胞形成を酸性ホスファターゼ活性測定キット（Ａｃｉｄ　Ｐｈｏｓｐｈａｔａｓｅ，　Ｌｅｕｃｏｃｙｔｅ　、カタログ　Ｎｏ．３８７−Ａ，シグマ社）を用いた染色で検出した。酒石酸存在下での酸性ホスファターゼ活性陽性細胞の減少をＯＣＩＦ活性とした。又、酸性ホスファターゼ活性陽性細胞数の減少率は実施例１６−ｉ）に記載した方法に従って染色された細胞の色素を溶解させて算出した。結果を表８に示す。
ｎＯＣＩＦ　と同様にｒＯＣＩＦ（Ｅ）についても、１６ｎｇ／ｍｌ　以上の濃度で容量依存的な破骨細胞形成抑制活性が見られた。
【００７３】
【表８】

【００７４】
ｉｖ）　ＩＬ−１１ で誘導される破骨細胞形成の抑制
ＩＬ−１１　で誘導される破骨細胞形成の試験は、田村らの方法　（Ｐｒｏｃ．　Ｎａｔｌ．　Ａｃａｄ．Ｓｃｉ．ＵＳＡ，　Ｖｏｌ．９０，　ｐ１１９２４−１１９２８，　１９９３）に従って行った。即ち、９６ウェルマイクロプレートに　２０ｎｇ／ｍｌ　ＩＬ−１１及び１０％牛胎児血清を含むα−ＭＥＭ培地（ギブコＢＲＬ社製）で希釈したｎＯＣＩＦ　及び精製ｒＯＣＩＦ（Ｅ）　１００μｌ　を入れた。このウェルにマウス新生児頭蓋骨由来前脂肪細胞株　ＭＣ３Ｔ３−Ｇ２／ＰＡ６　細胞（ＲＩＫＥＮ　Ｃｅｌｌ　Ｂａｎｋ−ＲＣＢ１１２７）５×１０^３個と生後約８週間の　ｄｄｙマウス脾臓細胞１×１０^５個を　１００μｌの１０％牛胎児血清を含むα−ＭＥＭ培地に懸濁させて播種し、５％　ＣＯ_２、３７℃、湿度１００　％にて５日間培養した。培養５日後にリン酸塩緩衝生理食塩水で洗浄した後エタノール／アセトン（１：１）溶液で細胞を室温にて１分間固定し、破骨細胞形成を酸性ホスファターゼ活性測定キット（Ａｃｉｄ　Ｐｈｏｓｐｈａｔａｓｅ，　Ｌｅｕｃｏｃｙｔｅ　、カタログ　Ｎｏ．３８７−Ａ，　シグマ社）を用いた染色で検出した。酒石酸存在下での酸性ホスファターゼ活性陽性細胞数を計測し、その減少をＯＣＩＦ活性とした。結果を表９に示す。
ｎＯＣＩＦ　及びｒＯＣＩＦ（Ｅ）とも、２ｎｇ／ｍｌ以上の濃度で容量依存的にＩＬ−１１　で誘導される破骨細胞形成を抑制する活性が見られた。
【００７５】
【表９】

【００７６】
このように種々の標的細胞を用いた破骨細胞形成の試験系において、ＯＣＩＦはビタミンＤ_３、ＰＴＨ、及びＩＬ−１１　等の破骨細胞形成誘導因子による破骨細胞の形成をほぼ同じ濃度で抑制することが明らかになった。従って、ＯＣＩＦはこのような様々な骨吸収促進物質で誘導される異なるタイプの骨量減少症の治療に、効果的に使用出来る可能性が示唆された。
【００７７】
【実施例１７】
モノマー型及びダイマー型 ＯＣＩＦ サンプルの調製
ｒＯＣＩＦ（Ｅ）及びｒＯＣＩＦ（Ｃ）それぞれ　１００μｇを含むサンプルに、１／１００容量の２５％ＴＦＡ（トリフルオロ酢酸）を加えた後、０．１％ＴＦＡを含む３０％アセトニトリルで平衡化した逆相カラム（ＰＲＯＴＥＩＮ−ＲＰ、２．０×２５０ｍｍ、ワイエムシー社）にかけ、５０分間でアセトニトリルを５５％にする直線勾配、流速０．２ｍｌ／分にて溶出を行い、各ＯＣＩＦピークを分取した。得られたピーク画分を凍結乾燥することにより、モノマー型ＯＣＩＦ及びダイマー型ＯＣＩＦを得た。
【００７８】
【実施例１８】
組み換え型 ＯＣＩＦ の分子量測定
実施例３−ｖｉ）　の方法で逆相カラムを用いて精製したモノマー型及びダイマー型ｎＯＣＩＦと実施例１７記載の方法で精製したモノマー型及びダイマー型ｒＯＣＩＦ約１μｇを含むサンプルを減圧濃縮した。これらのサンプルにつき、実施例４の方法でＳＤＳ処理、ＳＤＳ−ポリアクリルアミド電気泳動、及び銀染色を行った。非還元条件下及び還元条件下で電気泳動した結果を、図６及び図７にそれぞれ示す。
【００７９】
その結果、非還元条件下では、何れのモノマー型サンプルでも６０ｋＤの蛋白質バンドが検出され、又、何れのダイマー型サンプルでも　１２０ｋＤの蛋白質バンドが検出された。又、還元条件下では何れのサンプルでも約６０ｋＤの蛋白質バンドのみが検出された。従って、ＩＭＲ−９０細胞由来　ｎＯＣＩＦ、２９３／ＥＢＮＡ細胞由来組み換え型ＯＣＩＦ、及びＣＨＯ細胞由来組み換え型ＯＣＩＦの各々のモノマー型とダイマー型の分子量はほぼ同一であることが示された。
【００８０】
【実施例１９】
ＩＭＲ− ９０ 細胞由来天然型 ＯＣＩＦ と組み換え型 ＯＣＩＦ のＮ−結合型糖鎖の除去と分子量測定
実施例３−ｖｉ）　の方法で逆相カラムを用いて精製したモノマー型及びダイマー型ｎＯＣＩＦと実施例１７記載の方法で精製したモノマー型及びダイマー型ｒＯＣＩＦの各々を約５μｇ含むサンプルを減圧濃縮した。これらのサンプルに１００ｍＭ　２−メルカプトエタノールを加えた５０ｍＭリン塩緩衝液、ｐＨ８．６，　９．５μｌ　を加えて溶解させ、更に２５０Ｕ／ｍｌ　Ｎ−グリカナーゼ溶液（生化学工業社）０．５　μｌ　を加え３７℃で一日放置した。これらのサンプルに２ｍＭ　ＭＥＤＴＡ、５％ＳＤＳ、及び０．０２％ブロモフェノールブルーを含む　２０ｍＭ　Ｔｒｉｓ−ＨＣｌ，　ｐＨ８．０，　１０μｌ　を加え、１００　℃で５分間加熱した。これらのサンプルの１μｌ　を実施例４の方法でＳＤＳ−ポリアクリルアミド電気泳動した後、銀染色した。結果を図８に示す。
【００８１】
その結果、Ｎ−グリカナーゼ処理によりＮ−結合糖鎖を除去したＯＣＩＦ蛋白質の還元条件下での分子量は、いずれも約４０ｋＤであることが示された。糖鎖除去の処理を行っていないＩＭＲ−９０細胞由来ｎＯＣＩＦ，２９３／ＥＢＮＡ細胞由来ｒＯＣＩＦ、及びＣＨＯ細胞由来ｒＯＣＩＦの各々の還元条件下での分子量はいずれも約６０ｋＤであることから、これらのＯＣＩＦはその分子内にＮ−結合糖鎖を含有する糖蛋白質であることが明らかになった。
【００８２】
【実施例２０】
ＯＣＩＦ 類縁体（バリアント） ｃＤＮＡ のクローニング及び塩基配列の決定
実施例１０及び１１で示したように、純化したいくつかの陽性ファージのひとつからｐＢＫＣＭＶ（ストラータジーン社）にＯＣＩＦ　ｃＤＮＡが挿入されたプラスミドｐＢＫＯＣＩＦ　を持つ形質転換株を得たが、その際、他のいくつかの陽性ファージからも長さの異なるインサートが挿入されたプラスミドを持つ形質転換株が得られた。これらのプラスミドを持つ形質転換株を増殖させ、常法によりプラスミドを精製した。これらのインサートＤＮＡ　の塩基配列をタックダイデオキシターミネーターサイクルシークエンシングキット（パーキンエルマー社）を用いて決定した。用いたプライマーはＴ３，Ｔ７プライマー（ストラータジーン社）及びＯＣＩＦｃＤＮＡの塩基配列に基づいて設計された合成プライマーを用いた。オリジナルタイプのＯＣＩＦ以外に、ＯＣＩＦバリアントは全部で４種類（ＯＣＩＦ２，　３，　４，　５）　存在した。決定されたＯＣＩＦ２ｃＤＮＡの塩基配列を配列番号８にその配列から推定されるアミノ酸配列を配列番号９に示す。決定されたＯＣＩＦ３　ｃＤＮＡの塩基配列を配列番号１０にその配列から推定されるアミノ酸配列を配列番号１１　に示す。決定されたＯＣＩＦ４　ｃＤＮＡの塩基配列を配列番号１２にその配列から推定されるアミノ酸配列を配列番号１３に示す。決定されたＯＣＩＦ５　ｃＤＮＡの塩基配列を配列番号１４にその配列から推定されるアミノ酸配列を配列番号１５に示す。これらのＯＣＩＦバリアントの構造の特徴を、図９〜１２及び以下の記載をもって、簡単に説明する。
【００８３】
ＯＣＩＦ ２
ＯＣＩＦｃＤＮＡの塩基配列（配列番号６）の　２６５番目のグアニンから２８５　番目のグアニンまでの２１ｂｐの欠失があり、アミノ酸配列ではＯＣＩＦのアミノ酸配列（配列表配列番号５）の６８番目のグルタミン酸（Ｇｌｕ）から７４番目のグルタミン（Ｇｌｎ）までの７アミノ酸の欠失がある。
【００８４】
ＯＣＩＦ ３
ＯＣＩＦｃＤＮＡの塩基配列（配列番号６）の９番目のシチジンがグアニンに変換していて、アミノ酸配列ではＯＣＩＦのアミノ酸配列（配列表配列番号５）の−１９番目のアスパラギン（Ａｓｎ）がリジン（Ｌｙｓ）に変わっている。但し、これはシグナル配列の中のアミノ酸置換であり、分泌されるＯＣＩＦ３には影響しないと思われる。
ＯＣＩＦｃＤＮＡの塩基配列（配列番号６）の８７２番目のグアニンから９８８番目のグアニンまでの　１１７ｂｐの欠失があり、アミノ酸配列ではＯＣＩＦのアミノ酸配列（配列表配列番号５）の　２７０番目のスレオニン（Ｔｈｒ）から３０８　番目のロイシン（Ｌｅｕ）までの３９アミノ酸の欠失がある。
【００８５】
ＯＣＩＦ ４
ＯＣＩＦｃＤＮＡの塩基配列（配列番号６）の９番目のシチジンがグアニンに変換していて、アミノ酸配列ではＯＣＩＦのアミノ酸配列（配列表配列番号５）の−１９番目のアスパラギン（Ａｓｎ）がリジン（Ｌｙｓ）に変わっている。又、２２番目のグアニンがチミジンに変換していて、アミノ酸配列ではＯＣＩＦのアミノ酸配列（配列表配列番号５）の−１４番目のアラニン（Ａｌａ）がセリン（Ｓｅｒ）に変わっている。但し、これらはシグナル配列の中のアミノ酸置換であり、分泌されるＯＣＩＦ４には影響しないと思われる。
ＯＣＩＦｃＤＮＡの塩基配列（配列番号６）の　４００番目と　４０１番目の間に約　４ｋｂのイントロン２の挿入があり、オープンリーリングフレームがその中で止まる。アミノ酸配列ではＯＣＩＦのアミノ酸配列（配列表配列番号５）の　１１２番目のアラニン（Ａｌａ）の後に２１アミノ酸からなる新規なアミノ酸配列が付加されている。
【００８６】
ＯＣＩＦ ５
ＯＣＩＦｃＤＮＡの塩基配列（配列番号６）の９番目のシチジンがグアニンに変換していて、アミノ酸配列ではＯＣＩＦのアミノ酸配列（配列表配列番号５）の−１９番目のアスパラギン（Ａｓｎ）がリジン（Ｌｙｓ）に変わっている。但し、これはシグナル配列の中のアミノ酸置換であり、分泌されるＯＣＩＦ５には影響しないと思われる。
ＯＣＩＦｃＤＮＡの塩基配列（配列番号６）の４００番目と４０１番目の間に約１．８　ｋｂのイントロン２の後半部分の挿入があり、オープンリーリングフレームがその中で止まる。アミノ酸配列ではＯＣＩＦのアミノ酸配列（配列表配列番号５）の　１１２番目のアラニン（Ａｌａ）の後に１２アミノ酸からなる新規なアミノ酸配列が付加されている。
【００８７】
【実施例２１】
ＯＣＩＦ 類縁体（バリアント）の生産
ｉ） 　 ＯＣＩＦ バリアント ｃＤＮＡ の発現プラスミドの作製
実施例２０で得られたＯＣＩＦバリアントｃＤＮＡのうち、ＯＣＩＦ　２，　３　のｃＤＮＡがそれぞれ挿入されたプラスミドｐＢＫＯＣＩＦ２、ｐＢＫＯＣＩＦ３を制限酵素ＸｈｏＩ及びＢａｍＨＩ（宝酒造社）で消化し、ＯＣＩＦ　２及び３　のｃＤＮＡをそれぞれ切り出し、アガロース電気泳動によって分離後、ＱＩＡＥＸ　ゲルエクストラクションキット（キアゲン社）を用いて精製した。これらのＯＣＩＦ　２及び３　のｃＤＮＡを、あらかじめ制限酵素ＸｈｏＩ及びＢａｍＨＩ　（宝酒造社）で消化しておいた発現プラスミドｐＣＥＰ４（インヴィトロージェン社）　に、ライゲーションキット　Ｖｅｒ．２（宝酒造社）　を用いて挿入し、大腸菌　ＤＨ５α（ギブコＢＲＬ社）の形質転換を行った。
又、実施例２０で得られたＯＣＩＦバリアントｃＤＮＡのうち、ＯＣＩＦ４　のｃＤＮＡをが挿入されたプラスミドｐＢＫＯＣＩＦ４を制限酵素ＳｐｅＩ及びＸｈｏＩ（宝酒造社）で消化し、アガロース電気泳動によって分離後、ＱＩＡＥＸ　ゲルエクストラクションキット（キアゲン社）を用いて精製した。この　ＯＣＩＦ４のｃＤＮＡを、あらかじめ制限酵素ＮｈｅＩ及びＸｈｏＩ　（宝酒造社）で消化しておいた発現プラスミドｐＣＥＰ４（インヴィトロージェン社）　に、ライゲーションキット　Ｖｅｒ．２（宝酒造社）を用いて挿入し、大腸菌ＤＨ５α（ギブコＢＲＬ社）の形質転換を行った。
【００８８】
又、実施例２０で得られたＯＣＩＦバリアントｃＤＮＡのうち、ＯＣＩＦ５　のｃＤＮＡをが挿入されたプラスミドｐＢＫＯＣＩＦ５を制限酵素Ｈｉｎｄ　ＩＩＩ（宝酒造社）で消化し、ＯＣＩＦ５ｃＤＮＡ　のコーディング領域の５’領域を切り出し、アガロース電気泳動によって分離後、ＱＩＡＥＸ　ゲルエクストラクションキット（キアゲン社）を用いて精製した。実施例１３‐ｉ）で得られたＯＣＩＦ発現プラスミドｐＣＥＰＯＣＩＦを制限酵素Ｈｉｎｄ　ＩＩＩ（宝酒造社）で消化し、ＯＣＩＦｃＤＮＡのコーディング領域の５’領域を取り除き、ｐＣＥＰプラスミドとＯＣＩＦｃＤＮＡの３’領域を含んだＤＮＡ断片ｐＣＥＰＯＣＩＦ−３’　をアガロース電気泳動によって分離後、ＱＩＡＥＸ　ゲルエクストラクションキット（キアゲン社）を用いて精製した。この　ＯＣＩＦ５　ｃＤＮＡ　のＨｉｎｄ　ＩＩＩ断片をｐＣＥＰＯＣＩＦ−３’　にライゲーションキット　Ｖｅｒ．２（宝酒造社）　を用いて挿入し、大腸菌ＤＨ５α（ギブコＢＲＬ社）の形質転換を行った。
得られた形質転換株を増殖させ、ＯＣＩＦ２，　３，　４，　５のｃＤＮＡが挿入された発現プラスミドｐＣＥＰＯＣＩＦ　２，　３，　４，　５　を、キアゲンカラム（キアゲン社）　を用いて精製した。ＯＣＩＦバリアント発現プラスミドをエタノールによって沈澱させた後、無菌蒸留水に溶解し以下の操作に用いた。
【００８９】
ｉｉ）　ＯＣＩＦ バリアント ｃＤＮＡ のトランジエントな発現及びその活性の測定
実施例２１−ｉ）で得られたＯＣＩＦバリアント発現プラスミドｐＣＥＰＯＣＩＦ　２，　３，　４，　５　を用いて、実施例１３−ｉｉ）　で述べた方法でＯＣＩＦバリアントをトランジエントに発現させ、それらの活性を調べた。その結果、これらのＯＣＩＦバリアントに弱い活性を認めた。
【００９０】
【実施例２２】
ＯＣＩＦ 変異体の作製
ｉ） 　 ＯＣＩＦ 変異体 ｃＤＮＡ サブクローニング用プラスミドベクターの作製
実施例１１記載のプラスミドベクター５μｇ　を、制限酵素ＢａｍＨＩ　及びＸｈｏＩ（宝酒造社）で切断した。切断したＤＮＡを調製用アガロースゲル電気泳動に供した。ＯＣＩＦｃＤＮＡ全長を含む約　１．６キロベースペア（ｋｂ）のＤＮＡ断片を単離し、ＱＩＡＥＸ　ゲルエクストラクションキット（キアゲン社）により精製し、２０μｌ　の滅菌蒸留水に溶解したＤＮＡ溶液１を得た。次に、ｐＢｌｕｅｓｃｒｉｐｔ　ＩＩＳＫ^＋（ストラータジーン社）３μｇ　を制限酵素ＢａｍＨＩ　及びＸｈｏＩ（宝酒造社）で切断した。切断したＤＮＡを調製用アガロースゲル電気泳動に供した。約３．０　ｋｂのＤＮＡ断片を単離し、ＱＩＡＥＸ　ゲルエクストラクションキット（キアゲン社）により精製し、２０μｌ　の滅菌蒸留水に溶解したＤＮＡ溶液２を得た。１μｌ　のＤＮＡ溶液２と４μｌ　のＤＮＡ溶液１を混合し、５μｌ　のＤＮＡライゲーションキットｖｅｒ．２　Ｉ液（宝酒造社）を添加し混合後、１６℃で３０分間保温し、ライゲーション反応を行った。尚、以下のライゲーション反応は全て１６℃３０分の保温条件で行った。
【００９１】
このライゲーション反応液を用い、以下の条件で大腸菌の形質転換を行った。尚、以後大腸菌の形質転換は以下の条件で行った。このライゲーション反応液５μｌ　と大腸菌ＤＨ５αコンピテント細胞（ギブコＢＲＬ社）１００　μｌ　とを１５ｍｌ用滅菌チューブ（岩城ガラス社）中で混合し、氷水中３０分放置した。４２℃４５秒保温後、２５０　μｌ　のＬ培地　（１％トリプトン、０．５　％イーストエキストラクト、１％　ＮａＣｌ）を添加し攪拌しながら３７℃で培養した。５０μｌ　の菌液を５０μｇ／ｍｌアンピシリンを含む２ｍｌのＬ寒天培地上にスプレッドした。３７℃で一晩培養し、生育してきたコロニー６種を２ｍｌのＬアンピシリン液体培地でさらに一晩培養し、各株が持つプラスミドの構造を調べた。ｐＢｌｕｅｓｃｒｉｐｔ　ＩＩＳＫ^＋のＢａｍＨＩ　ＸｈｏＩ切断部位にＯＣＩＦｃＤＮＡ全長を含む約１．６ｋｂ　のＤＮＡ断片が挿入された構造を持つプラスミド（以後　ｐＳＫ^＋−ＯＣＩＦ　と呼ぶ）を得た。
【００９２】
ｉｉ）　 ＣｙｓをＳｅｒに置換した変異体の作製
（１）　 変異の導入
配列表配列番号４に記載のアミノ酸配列中、１７４，　１８１，　２５６，　２９８及び３７９　番のＣｙｓ残基を　Ｓｅｒ残基に置換した変異体を作製した。１７４ＣｙｓをＳｅｒ　に置換した変異体をＯＣＩＦ−Ｃ１９Ｓ　、１８１ＣｙｓをＳｅｒ　に置換した変異体をＯＣＩＦ−Ｃ２０Ｓ　、２５６Ｃｙｓを　Ｓｅｒに置換した変異体をＯＣＩＦ−Ｃ２１Ｓ　、２９８ＣｙｓをＳｅｒ　に置換した変異体をＯＣＩＦ−Ｃ２２Ｓ　、３７９Ｃｙｓを　Ｓｅｒに置換した変異体をＯＣＩＦ−Ｃ２３Ｓ２と、それぞれ名付けた。変異体作製のためにまず、各Ｃｙｓ　残基をコードする塩基配列をＳｅｒ　残基をコードする塩基配列に置換した。変異導入は二段階のＰＣＲ（ｐｏｌｙｍｅｒａｓｅ　ｃｈａｉｎ　ｒｅａｃｔｉｏｎ）　により行った。以後、二段階ＰＣＲ反応と呼ぶ。第一段階は２つのＰＣＲ反応より成る（ＰＣＲ１及びＰＣＲ２）。
【００９３】

【００９４】
ＰＣＲ２ 反応液

【００９５】
各変異導入時には、プライマーの種類だけを変え、他の反応組成は同一とした。各反応で用いたプライマーを表１０に、その配列を配列表配列番号２０、２３、２７、３０〜４０に示す。ＰＣＲ１反応液及びＰＣＲ２反応液をそれぞれ別の微量遠心チューブに入れ混合後、以下の条件でＰＣＲを行った。９７℃で３分処理後、９５℃１分、５５℃１分、７２℃３分の３段階の反応を２５回繰り返したのち、７０℃５分保温した。反応液の一部をアガロース電気泳動に供し、目的の長さのＤＮＡ断片が合成されていることを確認した。第一段階ＰＣＲ反応終了後、アミコンマイクロコン（アミコン社）により反応液からプライマーを除去し、滅菌蒸留水により最終液量を５０μｌ　に調製し、得られたＤＮＡ断片を用いさらに第２段階ＰＣＲ反応（ＰＣＲ３）を行った。
【００９６】

【００９７】
【表１０】

【００９８】
上記の溶液を微量遠心チューブに入れ混合後、ＰＣＲ１、ＰＣＲ２と同一の条件でＰＣＲを行った。反応液の一部をアガロース（１％或いは１．５　％）電気泳動に供し、目的の長さのＤＮＡ断片が合成されていることを確認した。ＰＣＲにより得られたＤＮＡをエタノールにより沈殿させ、真空中で乾燥させ、４０μｌ　の滅菌蒸留水に溶解した。Ｃ１９Ｓ変異ＤＮＡ断片を含む溶液を溶液Ａ、Ｃ２０Ｓ変異ＤＮＡ断片を含む溶液を溶液Ｂ、Ｃ２１Ｓ変異ＤＮＡ断片を含む溶液を溶液Ｃ、Ｃ２２Ｓ変異ＤＮＡ断片を含む溶液を溶液Ｄ、Ｃ２３Ｓ変異ＤＮＡ断片を含む溶液を溶液Ｅと名付けた。
【００９９】
溶液Ａ２０μｌ　中のＤＮＡ断片を制限酵素ＮｄｅＩ及びＳｐｈＩ（宝酒造社）により切断した。調製用電気泳動により約４００ｂｐ　のＤＮＡ断片を分離・精製し２０μｌ　の蒸留水に溶解した（ＤＮＡ溶液３）。次に、２μｇ　のｐＳＫ^＋−ＯＣＩＦを制限酵素ＮｄｅＩ及びＳｐｈＩ（宝酒造社）により切断し、調製用電気泳動により約４．２ｋｂ　のＤＮＡ断片を分離・精製し２０μｌ　の滅菌蒸留水に溶解した（ＤＮＡ溶液４）。２μｌ　のＤＮＡ溶液３と３μｌ　のＤＮＡ溶液４を混合し、さらにＤＮＡライゲーションキットｖｅｒ．２　Ｉ液５μｌ　を添加しライゲーション反応を行った。反応後のライゲーション溶液５μｌ　を用い、大腸菌ＤＨ５αを形質転換した。得られたアンピシリン耐性形質転換細胞から、ＤＮＡ構造の解析により目的のプラスミドＤＮＡを持つ株を選びだした。ＤＮＡ構造は、制限酵素切断により得られる断片の長さの測定及び塩基配列の決定により解析した。得られた目的のプラスミドＤＮＡをｐＳＫ−ＯＣＩＦ−Ｃ１９Ｓ　と名付けた。
【０１００】
溶液Ｂ２０μｌ　中のＣ２０Ｓ変異ＤＮＡ断片を制限酵素ＮｄｅＩ及びＳｐｈＩ（宝酒造社）により切断した。調製用電気泳動により約４００ｂｐ　のＤＮＡ断片を分離・精製し２０μｌの蒸留水に溶解した（ＤＮＡ溶液５）。２μｌ　のＤＮＡ溶液５と３μｌ　のＤＮＡ溶液４を混合し、さらにＤＮＡライゲーションキットｖｅｒ．２　Ｉ液５μｌ　を添加しライゲーション反応を行った。反応後のライゲーション溶液５μｌ　を用い、大腸菌ＤＨ５αを形質転換した。得られたアンピシリン耐性形質転換細胞から、ＤＮＡ構造の解析により目的のプラスミドＤＮＡを持つ株を選びだした。ＤＮＡ構造は、制限酵素切断により得られる断片の長さの測定及び塩基配列の決定により解析した。得られた目的のプラスミドＤＮＡをｐＳＫ−ＯＣＩＦ−Ｃ２０Ｓ　と名付けた。
【０１０１】
溶液Ｃ２０μｌ　中のＤＮＡ断片を制限酵素ＮｄｅＩ及びＳｐｈＩ（宝酒造社）により切断した。調製用電気泳動により約　４００ｂｐのＤＮＡ断片を分離・精製し２０μｌ　の蒸留水に溶解した（ＤＮＡ溶液６）。２μｌ　のＤＮＡ溶液６と３μｌ　のＤＮＡ溶液４を混合し、さらにＤＮＡライゲーションキットｖｅｒ．２　Ｉ液５μｌ　を添加しライゲーション反応を行った。反応後のライゲーション溶液５μｌ　を用い、大腸菌ＤＨ５αを形質転換した。得られたアンピシリン耐性形質転換細胞から、ＤＮＡ構造の解析により目的のプラスミドＤＮＡを持つ株を選びだした。ＤＮＡ構造は、制限酵素切断により得られる断片の長さの測定及び塩基配列の決定により解析した。得られた目的のプラスミドＤＮＡをｐＳＫ−ＯＣＩＦ−Ｃ２１Ｓ　と名付けた。
【０１０２】
溶液Ｄ２０μｌ　中のＤＮＡ断片を制限酵素ＮｄｅＩ及びＢｓｔＰＩ（宝酒造社）により切断した。調製用電気泳動により約６００ｂｐ　のＤＮＡ断片を分離・精製し２０μｌの蒸留水に溶解した（ＤＮＡ溶液７）。次に、２μｇのｐＳＫ^＋−ＯＣＩＦ　を制限酵素ＮｄｅＩ及びＢｓｔＰＩ　（宝酒造社）により切断し、調製用電気泳動により約４．０ｋｂ　のＤＮＡ断片を分離・精製し２０μｌの蒸留水に溶解した（ＤＮＡ溶液８）。２μｌ　のＤＮＡ溶液７と３μｌ　のＤＮＡ溶液８を混合し、さらにＤＮＡライゲーションキットｖｅｒ．２　Ｉ液５μｌ　を添加しライゲーション反応を行った。反応後のライゲーション溶液５μｌ　を用い、大腸菌ＤＨ５αを形質転換した。得られたアンピシリン耐性形質転換細胞から、ＤＮＡ構造の解析により目的のプラスミドＤＮＡを持つ株を選びだした。ＤＮＡ構造は、制限酵素切断により得られる断片の長さの測定及び塩基配列の決定により解析した。得られた目的のプラスミドＤＮＡをｐＳＫ−ＯＣＩＦ−Ｃ２２Ｓ　と名付けた。
【０１０３】
溶液Ｅ２０μｌ　中のＤＮＡ断片を制限酵素ＢｓｔＰＩ　及びＥｃｏＲＶ　（宝酒造社）により切断した。調製用電気泳動により約１２０ｂｐ　のＤＮＡ断片を分離・精製し２０μｌ　の滅菌蒸留水に溶解した（ＤＮＡ溶液９）。次に、２μｇ　のｐＳＫ^＋−ＯＣＩＦ　を制限酵素ＢｓｔＥＩＩ及びＥｃｏＲＶ　（宝酒造社）により切断し、調製用電気泳動により約４．５ｋｂのＤＮＡ断片を分離・精製し２０μｌ　の蒸留水に溶解した（ＤＮＡ溶液１０）。２μｌのＤＮＡ溶液９と３μｌ　のＤＮＡ溶液１０を混合し、さらにＤＮＡライゲーションキットｖｅｒ．２　Ｉ液５μｌ　を添加しライゲーション反応を行った。反応後のライゲーション溶液５μｌ　を用い、大腸菌ＤＨ５αを形質転換した。得られたアンピシリン耐性形質転換細胞から、ＤＮＡ構造の解析により目的のプラスミドＤＮＡを持つ株を選びだした。ＤＮＡ構造は、制限酵素切断により得られる断片の長さの測定及び塩基配列の決定により解析した。得られた目的のプラスミドＤＮＡをｐＳＫ−ＯＣＩＦ−Ｃ２３Ｓ　と名付けた。
【０１０４】
（２）　 変異体発現ベクターの構築
得られた目的のプラスミドＤＮＡ（ｐＳＫ−ＯＣＩＦ−Ｃ１９Ｓ，　ｐＳＫ−ＯＣＩＦ−Ｃ２０Ｓ　ｐＳＫ−ＯＣＩＦ−Ｃ２１Ｓ，ｐＳＫ−ＯＣＩＦ−Ｃ２２Ｓ，ｐＳＫ−ＯＣＩＦ−Ｃ２３Ｓ）を制限酵素ＢａｍＨＩ　及びＸｈｏＩ（宝酒造社）で切断し、ＯＣＩＦｃＤＮＡ全長を含む約１．６ｋｂ　のＤＮＡ断片（目的の変異も含む）　を分離・精製し、滅菌蒸留水２０μｌ　に溶解した。それぞれＣ１９ＳＤＮＡ　溶液、Ｃ２０ＳＤＮＡ　溶液、Ｃ２１ＳＤＮＡ　溶液、Ｃ２２ＳＤＮＡ　溶液、Ｃ２３ＳＤＮＡ　溶液と名付けた。次に、発現ベクターｐＣＥＰ４（インヴィトロージェン社）５μｇ　を制限酵素ＢａｍＨＩ　及びＸｈｏＩ（宝酒造社）で切断し、約１０　ｋｂ　のＤＮＡを分離・精製し滅菌蒸留水４０μｌ　に溶解した（ｐＣＥＰ４ＤＮＡ溶液）。ｐＣＥＰ４ＤＮＡ溶液１μｌ　と各６μｌ　のＣ１９ＳＤＮＡ　溶液、Ｃ２０ＳＤＮＡ　溶液、Ｃ２１ＳＤＮＡ溶液、Ｃ２２ＳＤＮＡ　溶液、Ｃ２３ＳＤＮＡ　溶液を別々に混合し、各混合液に７μｌ　のＤＮＡライゲーションキット　Ｖｅｒ．２　Ｉ液を添加し、ライゲーション反応を行った。反応終了後、７μｌ　の反応液を用い、大腸菌ＤＨ５αコンピテント細胞液１００ｍｌ　を形質転換した。得られたアンピシリン耐性形質転換細胞から、ｐＣＥＰ４のＸｈｏＩ、ＢａｍＨＩ部位に約１．６ｋｂ　の各ＤＮＡ断片が挿入された目的の構造のプラスミドＤＮＡを持つ株計５種を選びだし、それぞれ、ｐＣＥＰ４−ＯＣＩＦ−Ｃ１９Ｓ，　ｐＣＥＰ４−ＯＣＩＦ−Ｃ２０Ｓ，ｐＣＥＰ４−ＯＣＩＦ−Ｃ２１Ｓ，ｐＣＥＰ４−ＯＣＩＦ−Ｃ２２Ｓ，ｐＣＥＰ４−ＯＣＩＦ−Ｃ２３Ｓ　と名付けた。
【０１０５】
ｉｉ）　 ドメイン欠失変異体の作製
（１）　 ドメイン欠失変異の導入
配列番号４に記載したアミノ酸中、２番のＴｙｒ　から４２番のＡｌａ　まで、４３番のＰｒｏから８４番の　Ｃｙｓまで、８５番のＧｌｕ　から　１２２番のＬｙｓ　まで、１２３番のＡｒｇ　から　１６４番の　Ｃｙｓまで、１７７番のＡｓｐ　から　２５１番のＧｌｎ　まで、２５３番のＩｌｅ　から３２６　番のＨｉｓ　までを、それぞれ欠失させた変異体を作製した。２番のＴｈｒ　から４２番のＡｌａまでを欠失させた変異体をＯＣＩＦ−ＤＣＲ１　、４３番のＰｒｏ　から８４番の　Ｃｙｓまでを欠失させた変異体をＯＣＩＦ−ＤＣＲ２　、８５番のＧｌｕ　から１２２番のＬｙｓ　までを欠失させた変異体をＯＣＩＦ−ＤＣＲ３　、１２３　番のＡｒｇ　から１６４番のＣｙｓまでを欠失させた変異体をＯＣＩＦ−ＤＣＲ４、１７７番のＡｓｐから２５１番のＧｌｎ　までを欠失させた変異体をＯＣＩＦ−ＤＤＤ１、２５３番Ｉｌｅ　から　３２６番のＨｉｓ　までを欠失させた変異体をＯＣＩＦ−ＤＤＤ２　と、それぞれ名付けた。ドメイン欠失変異の導入も、実施例２２−ｉｉ）　に記載の二段階ＰＣＲ法によって行った。各変異導入反応時に用いたプライマーを表１１に、その配列を配列表配列番号１９、２５、４０〜５３、及び５４に示す。
【０１０６】
【表１１】

【０１０７】
ＰＣＲにより得られたＤＮＡをエタノールにより沈殿させ真空中で乾燥させ、４０μｌの滅菌蒸留水に溶解した。　ＤＣＲ１変異ＤＮＡ断片を含む溶液を溶液Ｆ、ＤＣＲ２変異ＤＮＡ断片を含む溶液を溶液Ｇ、ＤＣＲ３変異ＤＮＡ断片を含む溶液を溶液Ｈ、ＤＣＲ４変異ＤＮＡ断片を含む溶液を溶液Ｉ、ＤＤＤ１変異ＤＮＡ断片を含む溶液を溶液Ｊ、ＤＤＤ２変異ＤＮＡ断片を含む溶液を溶液Ｋと名付けた。
【０１０８】
溶液Ｆ２０μｌ　中のＤＮＡ断片を制限酵素ＮｄｅＩ及びＸｈｏＩ（宝酒造社）により切断した。調製用電気泳動により約５００ｂｐ　のＤＮＡ断片を分離・精製し２０μｌ　の滅菌蒸留水に溶解した（ＤＮＡ溶液１１）。次に、２μｇ　のｐＳＫ^＋−ＯＣＩＦ　を制限酵素ＮｄｅＩ及びＸｈｏＩ（宝酒造社）により切断し、調製用電気泳動により約４．０ｋｂ　のＤＮＡ断片を分離・精製し２０μｌ　の滅菌蒸留水に溶解した（ＤＮＡ溶液１２）。２μｌ　のＤＮＡ溶液１１と３μｌのＤＮＡ溶液１２を混合し、さらにＤＮＡライゲーションキットｖｅｒ．２　Ｉ液５μｌ　を添加しライゲーション反応を行った。反応後のライゲーション溶液５μｌ　を用い、大腸菌ＤＨ５αを形質転換した。得られたアンピシリン耐性形質転換細胞から、ＤＮＡ構造の解析により、ＯＣＩＦｃＤＮＡに目的の変異の導入されたプラスミドＤＮＡを持つ株を選びだした。ＤＮＡ構造は、制限酵素切断により得られる断片の長さの測定及び塩基配列の決定により解析した。得られた目的のプラスミドＤＮＡをｐＳＫ−ＯＣＩＦ−ＤＣＲ１と名付けた。
【０１０９】
溶液Ｇ２０μｌ　中のＤＮＡ断片を制限酵素ＮｄｅＩ及びＸｈｏＩ（宝酒造社）により切断した。調製用電気泳動により約５００ｂｐ　のＤＮＡ断片を分離・精製し２０μｌ　の滅菌蒸留水に溶解した（ＤＮＡ溶液１３）。２μｌ　のＤＮＡ溶液１３と３μｌ　のＤＮＡ溶液１２を混合し、さらにＤＮＡライゲーションキットｖｅｒ．２　Ｉ液を５μｌ　添加し、ライゲーション反応を行った。反応後のライゲーション溶液５μｌ　を用い、大腸菌ＤＨ５αを形質転換した。得られたアンピシリン耐性形質転換細胞から、ＤＮＡ構造の解析により目的のプラスミドＤＮＡを持つ株を選びだした。ＤＮＡ構造は、制限酵素切断により得られる断片の長さの測定及び塩基配列の決定により解析した。得られた目的のプラスミドＤＮＡをｐＳＫ−ＯＣＩＦ−ＤＣＲ２　と名付けた。
【０１１０】
溶液Ｈ２０μｌ　中のＤＮＡ断片を制限酵素ＮｄｅＩ及びＸｈｏＩ（宝酒造社）により切断した。調製用電気泳動により約５００ｂｐ　のＤＮＡ断片を分離・精製し２０μｌ　の滅菌蒸留水に溶解した（ＤＮＡ溶液１４）。２μｌ　のＤＮＡ溶液１４と３μｌ　のＤＮＡ溶液１２を混合し、さらにＤＮＡライゲーションキットｖｅｒ．２　Ｉ液を５μｌ　添加し、ライゲーション反応を行った。反応後のライゲーション溶液５μｌ　を用い、大腸菌ＤＨ５αを形質転換した。得られたアンピシリン耐性形質転換細胞から、ＤＮＡ構造の解析により、ＯＣＩＦｃＤＮＡに目的の変異の導入されたプラスミドＤＮＡを持つ株を選びだした。ＤＮＡ構造は、制限酵素切断により得られる断片の長さの測定及び塩基配列の決定により解析した。得られた目的のプラスミドＤＮＡをｐＳＫ−ＯＣＩＦ−ＤＣＲ３　と名付けた。
【０１１１】
溶液Ｉ２０μｌ　中のＤＮＡ断片を制限酵素ＸｈｏＩ及びＳｐｈＩ（宝酒造社）により切断した。調製用電気泳動により約９００ｂｐ　のＤＮＡ断片を分離・精製し２０μｌ　の滅菌蒸留水に溶解した（ＤＮＡ溶液１５）。次に、２μｇ　のｐＳＫ^＋−ＯＣＩＦ　を制限酵素ＸｈｏＩ及びＳｐｈＩ（宝酒造社）により切断し、調製用電気泳動により約３．６ｋｂ　のＤＮＡ断片を分離・精製し２０μｌ　の滅菌蒸留水に溶解した（ＤＮＡ溶液１６）。２μｌのＤＮＡ溶液１５と３μｌのＤＮＡ溶液１６を混合し、さらにＤＮＡライゲーションキットｖｅｒ．２　Ｉ液５μｌ　を添加し、ライゲーション反応を行った。反応後のライゲーション溶液５μｌ　を用い、大腸菌ＤＨ５αを形質転換した。得られたアンピシリン耐性形質転換細胞から、ＤＮＡ構造の解析により目的のプラスミドＤＮＡを持つ株を選びだした。ＤＮＡ構造は、制限酵素切断により得られる断片の長さの測定及び塩基配列の決定により解析した。得られた目的のプラスミドＤＮＡをｐＳＫ−ＯＣＩＦ−ＤＣＲ４　と名付けた。
【０１１２】
溶液Ｊ２０μｌ　中のＤＮＡ断片を制限酵素ＢｓｔＰＩ　及びＮｄｅＩ（宝酒造社）により切断した。調製用電気泳動により約　４００ｂｐのＤＮＡ断片を分離・精製し２０μｌの滅菌蒸留水に溶解した（ＤＮＡ溶液１７）。２μｌのＤＮＡ溶液１７と３μｌ　のＤＮＡ溶液８を混合し、さらにＤＮＡライゲーションキットｖｅｒ．２　Ｉ液を５μｌ　添加し、ライゲーション反応を行った。反応後のライゲーション溶液５μｌ　を用い、大腸菌ＤＨ５αを形質転換した。得られたアンピシリン耐性形質転換細胞から、ＤＮＡ構造の解析により目的のプラスミドＤＮＡを持つ株を選びだした。ＤＮＡ構造は、制限酵素切断により得られる断片の長さの測定及び塩基配列の決定により解析した。得られた目的のプラスミドＤＮＡをｐＳＫ−ＯＣＩＦ−ＤＤＤ１　と名付けた。
【０１１３】
溶液Ｋ２０μｌ　中のＤＮＡ断片を制限酵素ＢｓｔＰＩ　及びＮｄｅＩ（宝酒造社）により切断した。調製用電気泳動により約４００ｂｐのＤＮＡ断片を分離・精製し２０μｌの滅菌蒸留水に溶解した（ＤＮＡ溶液１８）。２μｌのＤＮＡ溶液１８と３μｌのＤＮＡ溶液８を混合し、さらにＤＮＡライゲーションキットｖｅｒ．２　Ｉ液を５μｌ添加し、ライゲーション反応を行った。反応後のライゲーション溶液５μｌ　を用い、大腸菌ＤＨ５αを形質転換した。得られたアンピシリン耐性形質転換細胞から、ＤＮＡ構造の解析により目的のプラスミドＤＮＡを持つ株を選びだした。ＤＮＡ構造は、制限酵素切断により得られる断片の長さの測定及び塩基配列の決定により解析した。得られた目的のプラスミドＤＮＡをｐＳＫ−ＯＣＩＦ−ＤＤＤ２　と名付けた。
【０１１４】
（２）　 変異体発現ベクターの構築
得られた目的のプラスミドＤＮＡ（ｐＳＫ−ＯＣＩＦ−ＤＣＲ１，　ｐＳＫ−ＯＣＩＦ−ＤＣＲ２，ｐＳＫ−ＯＣＩＦ−ＸＲ３，ｐＳＫ−ＯＣＩＦ−ＤＣＲ４，ｐＳＫ−ＯＣＩＦ−ＤＤＤ１，ｐＳＫ−ＯＣＩＦ−ＤＤＤ２）を制限酵素ＢａｍＨＩ　及びＸｈｏＩ（宝酒造社）で切断しＯＣＩＦｃＤＮＡ全長を含む約１．４−１．５　ｋｂのＤＮＡ断片（目的の変異も含む）を分離・精製し、滅菌蒸留水２０μｌ　に溶解した。それぞれをＤＣＲ１ＤＮＡ溶液、ＤＣＲ２ＤＮＡ　溶液、ＤＣＲ３ＤＮＡ　溶液、ＤＣＲ４ＤＮＡ　溶液、ＤＤＤ１ＤＮＡ　溶液、ＤＤＤ２ＤＮＡ　溶液と名付けた。実施例２２−ｉｉ）　に記載のｐＣＥＰ４　ＤＮＡ　溶液１μｌ　と各６μｌ　のＤＣＲ１ＤＮＡ　溶液、ＤＣＲ２ＤＮＡ　溶液、ＤＣＲ３ＤＮＡ　溶液、ＤＣＲ４ＤＮＡ　溶液、ＤＤＤ１ＤＮＡ　溶液、ＤＤＤ２ＤＮＡ溶液を別々に混合し、各混合液に７μｌ　のＤＮＡライゲーションバッファーを添加し、ライゲーション反応を行った。反応終了後、７μｌ　の反応液を用い、大腸菌ＤＨ５αを形質転換した。得られたアンピシリン耐性形質転換細胞からｐＣＥＰ４ＢａｍＨＩ　ＸｈｏＩ部位に各１．４−１．５ｋｂ　断片が挿入された構造のプラスミドＤＮＡを持つ株計６種を選びだした。目的の構造を持つプラスミドをそれぞれｐＣＥＰ４−ＯＣＩＦ−ＤＣＲ１　、ｐＣＥＰ４−ＯＣＩＦ−ＤＣＲ２　、ｐＣＥＰ４−ＯＣＩＦ−ＤＣＲ３　、ｐＣＥＰ４−ＯＣＩＦ−ＤＣＲ４　、ｐＣＥＰ４−ＯＣＩＦ−ＤＤＤ１　、ｐＣＥＰ４−ＯＣＩＦ−ＤＤＤ２　と名付けた。
【０１１５】
ｉｉｉ） 　Ｃ末端ドメイン欠失変異体の作製
（１）　 Ｃ末端ドメイン欠失変異の導入
配列番号４に記載したアミノ酸中、３７９　番の　Ｃｙｓと３８０　番のＬｅｕ　、３３１番のＳｅｒから３８０番のＬｅｕ　まで、２５２番のＡｓｐから３８０番のＬｅｕ　まで、１７７番のＡｓｐ　から　３８０番のＬｅｕ　まで、１２３　番のＡｒｇ　から　３８０番のＬｅｕ　まで、８６番のＣｙｓから３８０番のＬｅｕ　までを、それぞれ欠失させた変異体を作製した。３７９番のＣｙｓと３８０　番のＬｅｕ　を欠失させた変異体をＯＣＩＦ−ＣＬ、３３１番のＳｅｒから３８０番のＬｅｕ　までを欠失させた変異体をＯＣＩＦ−ＣＣ　、２５２番のＡｓｐ　から　３８０番のＬｅｕ　までを欠失させた変異体をＯＣＩＦ−ＣＤＤ２、１７７番のＡｓｐ　から３８０番のＬｅｕ　までを欠失させた変異体をＯＣＩＦ−ＣＤＤ１、１２３番のＡｒｇ　から３８０番のＬｅｕ　までを欠失させた変異体をＯＣＩＦ−ＣＣＲ４　、８６番の　Ｃｙｓから　３８０番のＬｅｕ　までを欠失させた変異体をＯＣＩＦ−ＣＣＲ３　と、それぞれ名付けた。
【０１１６】
変異体ＯＣＩＦ−ＣＬ　の作製用の変異導入は、実施例２２−ｉｉ）　に記載の二段階ＰＣＲ法によって行った。変異導入反応時に用いたプライマーを表１２に、その塩基配列を配列表配列番号２３、４０、５５及び５６に示す。ＰＣＲにより得られたＤＮＡをエタノールにより沈殿させ、真空中で乾燥させ、４０μｌ　の滅菌蒸留水に溶解した　（溶液Ｌ）。
【０１１７】
溶液Ｌ２０μｌ　中のＤＮＡ断片を制限酵素ＢｓｔＰＩ　及びＥｃｏＲＶ　（宝酒造社）により切断した。調製用電気泳動により約１００ｂｐ　のＤＮＡ断片を分離・精製し２０μｌの滅菌蒸留水に溶解した（ＤＮＡ溶液１９）。次に、２μｌ　のＤＮＡ溶液９と３μｌの実施例２２−ｉｉ）　記載のＤＮＡ溶液１０を混合し、さらにＤＮＡライゲーションキットｖｅｒ．２　Ｉ液を５μｌ　添加し、ライゲーション反応を行った。反応後のライゲーション溶液５μｌ　を用い、大腸菌ＤＨ５αを形質転換した。得られたアンピシリン耐性形質転換細胞から、ＤＮＡ構造の解析により目的のプラスミドＤＮＡを持つ株を選びだした。ＤＮＡ構造は、制限酵素切断により得られる断片の長さの測定及び塩基配列の決定により解析した。得られた目的のプラスミドＤＮＡをｐＳＫ−ＯＣＩＦ−ＣＬと名付けた。変異体ＯＣＩＦ−ＣＣ　、変異体ＯＣＩＦ−ＣＤＤ２　、変異体ＯＣＩＦ−ＣＤＤ１　、変異体をＯＣＩＦ−ＣＣＲ４　、変異体ＯＣＩＦ−ＣＣＲ３　作製用の変異導入には、一段階のＰＣＲ法を用いた。以下に反応条件を示す。
【０１１８】

【０１１９】
【表１２】

【０１２０】
各変異導入時には、プライマーの種類だけを変え、他の反応組成は同一とした。
各反応での変異導入用プライマーを表１３に、その配列を配列表配列番号５７〜６１に示す。ＰＣＲ反応液を微量遠心チューブに入れ混合後、以下の条件でＰＣＲを行った。９７℃で３分処理後、９５℃３０秒、５０℃３０秒、７０℃３分の３段階の反応を２５回繰り返したのち、７０℃５分保温した。反応液の一部をアガロース電気泳動に供し、目的の長さのＤＮＡ断片が合成されていることを確認した。反応液からアミコン・マイクロコンによりプライマーを除去し、ＤＮＡをエタノールにより沈殿させ真空中で乾燥させ、４０μｌ　の滅菌蒸留水に溶解した。各変異ＤＮＡ断片を含む溶液２０μｌ　中のＤＮＡ断片を制限酵素ＸｈｏＩ及びＢａｍＨＩ　によりＤＮＡを切断した。酵素切断終了後、ＤＮＡをエタノールにより沈殿させ真空中で乾燥させ、２０μｌ　の滅菌蒸留水に溶解した。溶液をそれぞれＣＣＤＮＡ　溶液、ＣＤＤ２ＤＮＡ　溶液、ＣＤＤ１ＤＮＡ　溶液、ＣＣＲ４ＤＮＡ　溶液、ＣＣＲ３ＤＮＡ　溶液と名付けた。
【０１２１】
【表１３】

【０１２２】
（２）　 変異体発現ベクターの構築
ｐＳＫ−ＯＣＩＦ−ＣＬ　を制限酵素ＢａｍＨＩ　及びＸｈｏＩ（宝酒造社）で切断し、ＯＣＩＦｃＤＮＡを含む約１．５　ｋｂのＤＮＡ断片（目的の変異も含む）を分離・精製し、滅菌蒸留水２０μｌに溶解した（ＣＬＤＮＡ　溶液）。実施例２２−ｉｉ）　に記載のｐＣＥＰ４　ＤＮＡ　溶液１μｌ　と各６μｌ　のＣＬＤＮＡ　溶液、ＣＣＤＮＡ　溶液、ＣＤＤ２ＤＮＡ　溶液、ＣＤＤ１ＤＮＡ　溶液、ＣＣＲ４ＤＮＡ　溶液、ＣＣＲ３ＤＮＡ　溶液を別々に混合し、７μｌ　のＤＮＡライゲーションキット　Ｖｅｒ．２　Ｉ液を添加し、ライゲーション反応を行った。反応終了後、７μｌ　の反応液を用い、大腸菌ＤＨ５αを形質転換した。得られたアンピシリン耐性形質転換細胞から目的の変異を持つＯＣＩＦ　ｃＤＮＡ断片がｐＣＥＰ４　のＸｈｏＩ−ＢａｍＨＩ部位に挿入された構造のプラスミドＤＮＡを持つ株計６種を選びだした。目的の構造を持つプラスミドをそれぞれ、ｐＣＥＰ４−ＯＣＩＦ−ＣＬ，　ｐＣＥＰ４−ＯＣＩＦ−ＣＣ，ｐＣＥＰ４−ＯＣＩＦ−ＣＤＤ２，ｐＣＥＰ４−ＯＣＩＦ−ＣＤＤ１，ｐＣＥＰ４−ＯＣＩＦ−ＣＣＲ４，ｐＣＥＰ４−ＯＣＩＦ−ＣＣＲ３と名付けた。
【０１２３】
ｉｖ）　 Ｃ末端欠失変異体の作製
（１）　 Ｃ末端欠失変異の導入
配列番号４に記載したアミノ酸中、３７１　番Ｇｌｎ　から　３８０番Ｌｅｕ　までを欠失させＬｅｕ−Ｖａｌ　の２残基を付加した変異体（ＯＣＩＦ−ＣＢｓｔ）、２９８番　Ｃｙｓから　３８０番Ｌｅｕまでを欠失させＳｅｒ−Ｌｅｕ−Ａｓｐ　の残基を付加した変異体（ＯＣＩＦ−ＣＳｐｈ）、１６７番Ａｓｎから　３８０番Ｌｅｕ　までを欠失させた変異体（ＯＣＩＦ−ＣＢｓｐ）、６２番　Ｃｙｓから　３８０番Ｌｅｕまでを欠失させＬｅｕ−Ｖａｌ　の２残基を付加した変異体（ＯＣＩＦ−ＣＰｓｔ）を作製した。各２μｇ　のｐＳＫ^＋−ＯＣＩＦ　を制限酵素ＢｓｔＰＩ　、ＳｐｈＩ、ＰｓｔＩ（宝酒造社）、及びＢｓｐＥＩ（Ｎｅｗ　Ｅｎｇｌａｎｄ　Ｂｉｏｌａｂ社）で切断し、フェノール処理、エタノール沈殿によりＤＮＡを精製し、１０μｌ　の滅菌蒸留水に溶解した。各２μｌ　の溶液を用いＤＮＡブランティングキット（宝酒造社）により各ＤＮＡの末端を平滑化した（最終容量５μｌ）。この反応液に、アンバーコドンを含むＸｂａＩリンカー（５’−ＣＴＡＧＴＣＴＡＧＡＣＴＡＧ−３’）１μｇ（１μｌ）と、６μｌ　のＤＮＡライゲーションキットｖｅｒ．２　Ｉ液を添加し、ライゲーション反応を行った。反応後のライゲーション溶液６μｌ　を用い、大腸菌ＤＨ５αを形質転換した。得られたアンピシリン耐性形質転換細胞から、ＤＮＡ構造の解析により目的のプラスミドＤＮＡを持つ株を選びだした。ＤＮＡ構造は、制限酵素切断により得られる断片の長さの測定及び塩基配列の決定により解析した。得られた目的のプラスミドＤＮＡをｐＳＫ−ＯＣＩＦ−ＣＢｓｔ、ｐＳＫ−ＯＣＩＦ−ＣＳｐｈ、ｐＳＫ−ＯＣＩＦ−ＣＢｓｐ　、ｐＳＫ−ＯＣＩＦ−ＣＰｓｔ　と名付けた。
【０１２４】
（２）　 変異体発現ベクターの構築
得られたプラスミドＤＮＡ（ｐＳＫ−ＯＣＩＦ−ＣＢｓｔ、ｐＳＫ−ＯＣＩＦ−ＣＳｐｈ、ｐＳＫ−ＯＣＩＦ−ＣＢｓｐ、ｐＳＫ−ＯＣＩＦ−ＣＰｓｔ）を制限酵素ＢａｍＨＩ　及びＸｈｏＩ（宝酒造社）で切断し、ＯＣＩＦｃＤＮＡ全長を含む約１．５　キロベースペア（ｋｂ）のＤＮＡ断片（目的の変異も含む）を分離・精製し、滅菌蒸留水２０μｌ　に溶解した（それぞれＣＢｓｔＤＮＡ　溶液、ＣＳｐｈＤＮＡ　溶液、ＣＢｓｐＤＮＡ　溶液、ＣＰｓｔＤＮＡ　溶液と名付けた）。
実施例２２−ｉｉ）に記載のｐＣＥＰ４　ＤＮＡ　溶液１μｌ　と各６μｌ　のＣＢｓｔＤＮＡ　溶液、ＣＳｐｈＤＮＡ　溶液、ＣＢｓｐＤＮＡ　溶液、ＣＰｓｔＤＮＡ　溶液を別々に混合し、各混合液に７μｌ　のＤＮＡライゲーションキット　Ｖｅｒ．２　Ｉ液を添加し、ライゲーション反応を行った。反応終了後、７μｌ　の反応液を用い、大腸菌ＤＨ５αを形質転換した。得られたアンピシリン耐性形質転換細胞から目的の変異を持つＯＣＩＦｃＤＮＡ断片がｐＣＥＰ４　のＸｈｏＩ　ＢａｍＨＩ部位間に挿入された構造のプラスミドＤＮＡを持つ株計５種を選びだした。目的の構造を持つプラスミドをそれぞれ、ｐＣＥＰ４−ＯＣＩＦ−ＣＢｓｔ，　ｐＣＥＰ４−ＯＣＩＦ−ＣＳｐｈ，ｐＣＥＰ４−ＯＣＩＦ−ＣＢｓｐ，ｐＣＥＰ４−ＯＣＩＦ−ＣＰｓｔと名付けた。
【０１２５】
ｖ） 　変異体発現ベクターの調製
変異体発現ベクターを持つ大腸菌（計２１種類）を増殖させ、各種変異体発現ベクターをキアゲンカラム（キアゲン社）を用いて精製した。各発現ベクターはエタノールによって沈殿させた後、滅菌蒸留水に溶解し以下の操作に用いた。
【０１２６】
ｖｉ ）　変異体 ｃＤＮＡ のトランジェントな発現及びその活性の測定
実施例２２−ｖ）で精製した各種ＯＣＩＦ変異体発現プラスミドを用い、実施例１３の方法に従いＯＣＩＦ変異体を発現させた。以下に変更した点のみを記する。ＤＮＡ導入には２４ウェルプレートを用いた。２×１０^５個の２９３／ＥＢＮＡ細胞を１０％牛胎児血清を含むＩＭＤＭ培地を用いて各ウェルに植え込んだ。ＤＮＡ導入の際用いた変異体発現ベクターとリポフェクタミンの量は、それぞれ１μｇ　及び４μｌ　であった。ＯＰＴＩ−ＭＥＭ培地（ギブコＢＲＬ社）で希釈し最終容量を０．５ｍｌ　とした。変異体発現ベクターとリポフェクタミンの混合液を細胞に添加し、２４時間３７℃で　ＣＯ_２インキュベーター中で培養した後混合液を除去し、０．５ｍｌ　のＥｘ−ｃｅｌｌ　３０１　培地（ＪＳＲ社）を加え、さらに４８時間３７℃で　ＣＯ_２インキュベーター中で培養した。培地を回収し、これを変異体活性測定用サンプルとした。得られた各変異体の塩基配列を配列表配列番号８３〜１０３　に、その配列から推定されるアミノ酸配列を配列表配列番号６２〜８２に、それぞれ示す。ＯＣＩＦの活性測定は実施例１３に従った。また、実施例２４に記
載のＥＩＡ法により、ＯＣＩＦの抗原量を定量した。表１４に未改変ＯＣＩＦと比較した抗原量当たりの活性を示す。
【０１２７】
【表１４】

【０１２８】
ｖｉ）　 ウェスタンブロッティング解析
活性測定に用いたサンプルの１０μｌ　をウェスタンブロット解析に供した。サンプル１０μｌ　に１０μｌ　のＳＤＳ‐ＰＡＧＥ用サンプルバッファー（０．５Ｍ　Ｔｒｉｓ−ＨＣｌ、２０％グリセロール、４％ＳＤＳ、２０μｇ／ｍｌブロムフェノール　ブルー（ｐＨ　６．８））を加え、１００　℃で３分煮沸し非還元状態で１０％ＳＤＳポリアクリルアミド電気泳動を行った。泳動終了後、セミドライブロッティング装置（バイオラッド社）によりＰＶＤＦメンブレン（ＰｒｏＢｌｏｔｔＲ、パーキンエルマー社）に蛋白質をブロッティングした。そのメンブレンをブロッキング後、実施例２４に記載のＥＩＡ用西洋ワサビパーオキシダーゼ標識抗ＯＣＩＦ抗体とともに、３７℃で２時間保温した。
洗浄後ＥＣＬシステム（アマシャム社）により抗ＯＣＩＦ抗体に結合する蛋白質を検出した。ＯＣＩＦでは、約１２０　キロダルトン（ｋＤ）及び６０ｋＤのバンドが検出された。
一方、ＯＣＩＦ−Ｃ２３Ｓ　、ＯＣＩＦ−ＣＬ　、ＯＣＩＦ−ＣＣ　では、ほとんど６０ｋＤのバンドのみが検出された。また、ＯＣＩＦ−ＣＤＤ２　及びＯＣＩＦ−ＣＤＤ１　ではそれぞれ約４０−５０ｋＤ及び３０−４０ｋＤ　のバンドが主要なバンドとして検出された。以上の結果より、ＯＣＩＦでは、配列表配列番号４のアミノ酸配列にける　３７９番目のＣｙｓ残基が二量体形成に係わっていること、単量体でも活性を保持していること、及び１７７　番Ａｓｐ
から　３８０番Ｌｅｕ　までの残基を欠失させても活性を保持してることが明らかとなった。
【０１２９】
【実施例２３】
ヒト ＯＣＩＦ ゲノム ＤＮＡ の分離
Ｉ） 　ヒトゲノム ＤＮＡ ライブラリーのスクリーニング
ヒト胎盤の染色体ＤＮＡとλＦＩＸ　ＩＩベクターを用いて作製されたゲノム・ライブラリーをストラタジーン社から購入し、これをＯＣＩＦｃＤＮＡをプローブとしてスクリーニングした。スクリーニングは、基本的にはゲノム・ライブラリーに添付されているプロトコールに従って実施したが、ファージ、大腸菌、ＤＮＡを扱う一般的方法はＭｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ：Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ　に従って行った。
購入したゲノムＤＮＡライブラリーのタイターを検定したのち、１×１０^６ｐｆｕのファージを大腸菌ＸＬ１−Ｂｌｕｅ　ＭＲＡに感染させ、２０枚のプレート（９×１３ｃｍ）　にプレート当たり９ｍｌのトップ・アガロースとともに蒔いた。プレートを一夜３７℃でインキュベートしたのち、Ｈｙｂｏｎｄ−Ｎナイロン膜（アマシャム社）をアガープレート上に乗せてファージを転写した。ファージの転写したナイロン膜を１．５Ｍ　ＮａＣｌ／０．５Ｍ　ＮａＯＨ溶液で湿らせた濾紙上に１分間乗せ、その後１Ｍ　Ｔｒｉｓ−ＨＣｌ（ｐＨ７．５）と１．５Ｍ　ＮａＣｌ／０．５Ｍ　Ｔｒｉｓ−ＨＣｌ　（ｐＨ７．５）でそれぞれ１分ずつ処理して中和したのち、最後に２ＸＳＳＣで湿らせた濾紙の上に移した。その後、このナイロン膜にストラタリンカー（ストラタジーン社）を用いて１２００マイクロジュールのＵＶ　を照射することによってファージＤＮＡを膜に固定した。次に、このナイロン膜をラピッドハイブリダイゼーション・バッファー（アマシャム社）に浸漬してプレハイブリダイゼーションを行った。１時間のプレハイブリダイゼーションの後、^３２Ｐ標識したＯＣＩＦｃＤＮＡを加え、６５℃にて一夜ハイブリダイゼーションを行った。このｃＤＮＡプローブは、実施例１１で得られた１．６ｋｂ　のＯＣＩＦｃＤＮＡを有するプラスミドｐＢＫＯＣＩＦを、制限酵素ＢａｍＨＩ　及びＸｈｏＩを用いて切断し、ＯＣＩＦｃＤＮＡをアガロースゲル電気泳動によって単離したのち、このＯＣＩＦｃＤＮＡをメガプライムＤＮＡラベリングシステム（アマシャム社）を用いて^３２Ｐで標識することによって作製した。標識は、ラベリングシステムに添付されたプロトコールに従って行った。
【０１３０】
ハイブリダイゼーションには、ハイブリダイゼーション・バッファー１ｍｌ当たりおよそ５×１０^５ｃｐｍのプローブを使用した。ハイブリダイゼーションの後、ナイロン膜を室温にて２ＸＳＳＣで５分間洗浄し、その後６５℃において０．５　ＸＳＳＣ／０．１％ＳＤＳで４回、それぞれ２０分ずつ洗浄した。４回目の洗浄ののちナイロン膜を乾燥させ、富士フィルム社製Ｘ腺フィルム、スーパーＨＲ−Ｈと増感スクリーンとを用いて−８０℃にてオートラジオグラフィーを行った。オートラジオグラム上に６個のシグナルが検出されたので、それぞれのシグナルに相当するアガープレート上の位置からトップ・アガロースを切り出し、１％のクロロホルムを添加した０．５ｍｌ　のＳＭバッファーにそれぞれ浸漬して一夜放置し、ファージを抽出した。それぞれのファージ抽出液をＳＭバッファーで１０００倍に希釈し、その中から１μｌ　と２０μｌ　を取り、再び上記大腸菌に感染させ、トップ・アガロースとともに上記の方法でアガープレートに蒔いた。ファージをナイロン膜に転写後、上記の方法でプレハイブリダイゼーション、ハイブリダイゼーション、洗浄、乾燥、オートラジオグラフィーを行った。このファージ純化の操作を当初オートラジオグラフィーで検出された６個のシグナル全部について行い、アガープレート上のすべてのファージプラークがｃＤＮＡプローブとハイブリダイズするまで繰り返した。純化されたファージのプラークを切り出し、１％クロロホルムを含むＳＭバッファー０．５ｍｌ　に浸漬し、４℃で保存した。こうして得られた６種の純化ファージを、それぞれλＯＩＦ３，λＯＩＦ８，　λＯＩＦ９，　λＯＩＦ１１，λＯＩＦ１２，λＯＩＦ１７　と名付けた。
【０１３１】
ＩＩ）　 制限酵素消化及びサザンブロット・ハイブリダイゼーションによるヒト ＯＣＩＦ ゲノム ＤＮＡ クローンの分析
純化された６種のファージのＤＮＡを、Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ：Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌに書かれた方法に従ってプレートリシス法によって精製した。これらのＤＮＡを制限酵素によって消化し、得られたフラグメントをアガロース電気泳動によって分離した。またアガロース・ゲルで分離されたフラグメントを、一般的な方法でナイロン膜に転移させたのち、ＯＣＩＦｃＤＮＡをプローブとしてサザンブロット・ハイブリダイゼーションを行った。これらの分析の結果、それぞれ純化された６種のファージは異なったクローンであることが判明した。制限酵素消化によって得られたＤＮＡフラグメントのうち、ＯＣＩＦｃＤＮＡとハイブリダイズするものについては、プラスミドベクターにサブクローンした後に下記の方法で塩基配列の分析を行った。
【０１３２】
ｉｉｉ） 　ゲノム ＤＮＡ クローンから制限酵素消化によって得られた ＤＮＡ フラグメントのプラスミド・ベクターへのサブクローニングと塩基配列の決定
λＯＩＦ８　ＤＮＡを制限酵素ＥｃｏＲＩ　とＮｏｔＩによって消化し、生じたフラグメントを０．７％アガロースゲルに供与して分離した。５．８ｋｂ　のＥｃｏＲＩ／ＮｏｔＩフラグメントをＱＩＡＥＸＩＩ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　Ｋｉｔ（キアゲン社）を用いて添付されたプロトコールに従ってゲルから抽出した。このフラグメントを、前もってＥｃｏＲＩ　とＮｏｔＩによって切断しておいたｐＢｌｕｅｓｃｒｉｐｔＩＩ　ＳＫ＋　ベクター（ストラタジーン社）とＲｅａｄｙ−Ｔｏ−Ｇｏ　Ｔ４Ｌｉｇａｓｅ（ファルマシア社）を用いて添付のプロトコールに従ってライゲーションした。得られたリコンビナント・プラスミドを、コンピテントＤＨ５α大腸菌（アマシャム社）に導入した後、５０μｇ／ｍｌのアンピシリンを含有するアガロースプレート上に蒔いてプラスミドを有する大腸菌を選択した。以上のようにして作製された５．８ｋｂ　ＥｃｏＲＩ／ＮｏｔＩフラグメントを有するリコンビナント・プラスミドを、ｐＢＳＧ８−５．８　と命名した。次に、ｐＢＳＧ８−５．８　を制限酵素ＨｉｎｄＩＩＩ　で消化して生ずる０．９　ｋｂのＤＮＡフラグメントをアガロースゲルで分離し、上記の方法にしたがって抽出した後、ＨｉｎｄＩＩＩ　で前もって切断しておいたｐＢｌｕｅｓｃｒｉｐｔＩＩ　ＳＫ−（ストラタジーン社）に挿入して、上記の方法に従ってクローニングした。この０．９ｋｂのＨｉｎｄＩＩＩ　フラグメントを有するリコンビナント・プラスミドを、ｐＢＳ８Ｈ０．９と命名した。一方、λＯＩＦ１１のＤＮＡをＥｃｏＲＩを用いて消化して生ずる６ｋｂ、３．６ｋｂ、及び２．６ｋｂのフラグメントをそれぞれ単離したのち、上記と同様の方法に従ってｐＢｌｕｅｓｃｒｉｐｔＩＩ　ＳＫ＋ベクターに挿入してクローニングした。こうして作製した６ｋｂ、３．６　ｋｂ、及び２．６ｋｂ　のＥｃｏＲＩ　フラグメントを有するリコンビナント・プラスミドを、それぞれｐＢＳＧ１１−６、ｐＢＳＧ１１−３．６、ｐＢＳＧ１１−２．６と命名した。
【０１３３】
さらに、ｐＢＳＧ１１−６を制限酵素ＨｉｎｄＩＩＩ　によって消化することによって生ずる、２．２ｋｂ、１．１ｋｂ、１．０５ｋｂの３種のフラグメントをアガロースゲル電気泳動によって分離し、それぞれｐＢｌｕｅｓｃｒｉｐｔＩＩ　ＳＫ−のＨｉｎｄＩＩＩ　サイトに挿入してクローニングした。これら２．２ｋｂ　、１．１ｋｂ　、１．０５　ｋｂ　のＨｉｎｄＩＩＩ　フラグメントを有するリコンビナント・プラスミドを、それぞれｐＢＳ６Ｈ２．２、ｐＢＳ６Ｈ１．１、ｐＢＳ６Ｈ１．０５　と命名した。ゲノムＤＮＡの塩基配列の分析には、ＡＢＩ　Ｄｙｅｄｅｏｘｙ　Ｔｅｒｍｉｎａｔｏｒ　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　Ｒｅａｄｙ　Ｒｅａｃｔｉｏｎ　Ｋｉｔ　（パーキンエルマー社）と３７３　ＤＮＡ　Ｓｅｑｕｅｎｃｉｎｇ　Ｓｙｓｔｅｍ（アプライドバイオシステムズ社）を使用した。Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ：Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ　に書かれた方法に従ってｐＢＳＧ８−５．８　、ｐＢＳ８Ｈ０．９、ｐＢＳＧ１１−６、ｐＢＳＧ１１−３．６、ｐＢＳＧ１１−２．６、ｐＢＳ６Ｈ２．２、ｐＢＳ６Ｈ１．１、ｐＢＳ６Ｈ１．０５　を調製し、塩基配列決定用の鋳型として用いた。ヒトＯＣＩＦゲノムＤＮＡの塩基配列を配列表配列番号１０４　及び１０５　に示す。エクソン１とエクソン２の間に介在する塩基の配列は必ずしも全部は決定されておらず、配列表配列番号１０４　及び１０５　に示された塩基配列の間に、およそ１７ｋｂのヌクレオチドが介在することが確認されている。
【０１３４】
【実施例２４】
ＥＩＡ による ＯＣＩＦ の定量
ｉ） 　ウサギ抗 ＯＣＩＦ 抗体の調製
雄性日本白色ウサギ（体重２．５　〜３．０ｋｇ　、北山ラベス社より入手）３羽に、ｒＯＣＩＦ２００　μｇ／ｍｌをフロイント完全アジュバント（ＤＩＦＣＯ社）と等量混合してエマルジョンとしたものを、１回１ｍｌずつ皮下免疫した。免疫は１週間隔で合計６回行い、最終免疫後１０日目に全採血を行った。分離した血清から抗体を以下の様に精製した。即ち、ＰＢＳにて２倍希釈した抗血清に最終濃度４０ｗ／ｖ　％となるように硫酸アンモニウムを添加して４℃１時間放置後、８０００×ｇで２０分間遠心分離を行い、沈殿を得た。沈殿を少量のＰＢＳに溶解し、ＰＢＳに対して４℃で透析した後、Ｐｒｏｔｅｉｎ　Ｇ−Ｓｅｐｈａｒｏｓｅ　カラム（ファルマシア社）に負荷した。ＰＢＳにて洗浄後、０．１Ｍグリシン塩酸緩衝液（ｐＨ３．０）　にて吸着した免疫グロブリンＧを溶出し、直ちに１．５　Ｍトリス塩酸緩衝液（ｐＨ８．７）　で中性ｐＨとした。溶出蛋白質画分をＰＢＳに対して透析後、２８０ｎｍ　における吸光度を測定し、その濃度を決定した（Ｅ^１％１３．５）。西洋ワサビパーオキシダーゼ標識した抗ＯＣＩＦ抗体は、マレイミド活性化パーオキシダーゼキット（ピアス社）を用いて作製した。即ち、１ｍｇの精製抗体に８０μｇのＮ−スクシンイミド−Ｓ−アセチルチオ酢酸を添加し、室温で３０分間反応させた。これに５ｍｇのヒドロキシルアミンを添加して脱アセチル化した後、修飾された抗体をポリアクリルアミド脱塩カラムにて分画した。蛋白質画分を１ｍｇのマレイミド活性化パーオキシダーゼと混合し、室温で１時間反応させ酵素標識抗体を得た。
【０１３５】
ｉｉ）　 サンドイッチ ＥＩＡ による ＯＣＩＦ の定量
９６ウェルのマイクロタイタープレート（ＭａｘｉＳｏｒｐ　Ｉｍｍｕｎｏｐｌａｔｅ，　Ｎｕｎｃ社）の各ウェルに、１００μｌ　のウサギ抗ＯＣＩＦ抗体（２μｇ／ｍｌ、５０ｍＭ　炭酸緩衝液（ｐＨ９．６））を添加し４℃にて一晩静置して、抗体を固相化した。ＰＢＳにて調製した２５％ブロックエース（雪印乳業社）を３００μｌずつ各ウェルに添加し、３７℃で１時間放置してブロッキングした後、検体（１００μｌ／ウェル）を添加し室温で２時間反応させた。０．０５％　Ｔｗｅｅｎ２０を含むＰＢＳ（ＰＢＳＴ）にて３回洗浄した後、１００００　倍希釈した西洋ワサビパーオキシダーゼ標識抗ＯＣＩＦ抗体を　１００μｌ　ずつ添加し室温で２時間インキュベートした。ＰＢＳＴにて３回洗浄した後、１００μｌ　の酵素基質溶液（ＴＭＢ、ＳｃｙＴｅｋ社）を加え室温で発色させた後、反応を停止した。　４５０ｎｍにおける吸光度をマイクロプレートリーダー（イムノリーダー　ＮＪ２０００、日本インターメッド社）を用いて測定し、精製した組み換えＯＣＩＦを標準とした検量線から、検体のＯＣＩＦ濃度を定量した。ＯＣＩＦの検量線を図１３に示す。
【０１３６】
【実施例２５】
抗 ＯＣＩＦ モノクローナル抗体
ｉ） 　ヒト ＯＣＩＦ 抗体産生ハイブリドーマの調製
ヒト線維芽細胞ＩＭＲ−９０を培養し、その培養液から実施例１１記載の方法でＯＣＩＦを精製した。精製ＯＣＩＦを１０μｇ／１００μｌ　の濃度になるようにＰＢＳに溶解し、この溶液を２週間おきにＢＡＬＢ／ｃマウスに腹腔内投与し免疫した。初回及び２回目の免疫においては、等量のフロインド完全アジュバントの混合物を投与した。最終の免疫から３日目に脾臓を摘出し、Ｂリンパ球を分離し、マウスミエローマ細胞Ｐ３ｘ６３−ＡＧ８．６５３　とを通常用いられているポリエチレングリコール法により細胞融合させた。ついで融合細胞を選択するためにＨＡＴ培地で培養を行うことにより、ハイブリドーマ細胞をセレクションした。次に、セレクションされた細胞がＯＣＩＦ特異的抗体を産生しているか否かを確認するために、０．１Ｍ　重曹溶液に溶解したＯＣＩＦ溶液　（１０μｇ／ｍｌ）１００μｌ　を、９６穴マイクロプレート（Ｎｕｎｃ社）　に加えて作製したソリッドフェーズＥＬＩＳＡを用いて、ハイブリドーマ培養液中のＯＣＩＦ特異的抗体の測定を行った。抗体生産が認められたハイブリドーマを限界希釈法によりクローニングを３−５回繰り返し行い、その都度上記ＥＬＩＳＡにより抗体産生量をチェックした。得られた抗体生産株の中から、抗体生産量の高いクローンを選別した。
【０１３７】
ｉｉ）　 モノクローナル抗体の生産　
実施例２５−ｉ）で得た抗体生産株を、それぞれ１　×１０^６を予めプリスタン　（アルドリッチケミカル社）　を接種しておいたＢＡＬＢ／ｃ系マウスの腹腔内に移植した。移植２週間後、蓄積した腹水を採取し、本発明のモノクローナル抗体を含む腹水を得た。この腹水より、アフィゲルプロテインＡセファロース（　バイオラッド社製）　を用いたアフィニティクロマトグラフィーにより精製抗体を得た。即ち、腹水を等量のバインディングバッファー（バイオラッド社）　で希釈し、プロテインＡカラムに負荷した後、充分量の同バッファーで洗浄した。ＩｇＧの溶出は、エリューションバッファー（バイオラッド社）で行った。得られた溶出液を水で透析した後、凍結乾燥を行った。得られた精製抗体をＳＤＳ‐ＰＡＧＥにより純度検定を行ったところ、分子量約１５０，０００　の位置に均一なバンドを認めた。
【０１３８】
ｉｉｉ） 　 ＯＣＩＦ に対して高親和性を有するモノクローナル抗体の選択
実施例２５−ｉｉ）　で得た抗体をＰＢＳに溶解し、ローリー法により蛋白定量を行った。ついで、各抗体を蛋白濃度が一定になるようにＰＢＳに溶解し、この溶液を段階希釈法により希釈した。実施例２５−ｉｉ）　に記載のソリッドフェーズＥＬＩＳＡを用いて、高い希釈段階までＯＣＩＦと反応するモノクローナル抗体を選別した。その結果、Ａ１Ｇ５、Ｅ３Ｈ８、及びＤ２Ｆ４の３種の抗体が得られた。
【０１３９】
ｉｖ）　 抗体のサブクラスの検定
実施例２５−ｉｉｉ）で選択した本発明の抗体のクラス及びサブクラスを、イムノグロブリンクラス及びサブクラス分析キット　（アマシャム社）　を用いて検定した。検定は、キットに指示されているプロトコールに従って実施した。結果を表１５に示す。Ｅ３Ｈ８、Ａ１Ｇ５、及びＤ２Ｆ４は、それぞれＩｇＧ_１、ＩｇＧ_２ａ、及びＩｇＧ_２ｂ　であった。
【０１４０】
【表１５】

【０１４１】
ｖ） 　 ＯＣＩＦ の ＥＬＩＳＡ による測定方法
実施例２５−ｉｖ）　で得たＡ１Ｇ５、Ｅ３Ｈ８、及びＤ２Ｆ４の３種のモノクローナル抗体を、それぞれ固相抗体と標識抗体とした。それぞれの組み合わせにより、サンドイッチＥＬＩＳＡを構築した。抗体の標識は、マレイミド活性化パーオキシダーゼキット（ピアス社）を用いて行った。各々の抗体を１０μｇ／ｍｌの濃度になるように０．１Ｍ　重曹溶液に溶解し、９６穴イムノプレート（Ｎｕｎｃ　社）の各ウエル当たり　１００μｌづつそれぞれ分注し、室温で一晩放置した。次いで、各々のプレートを１／２　濃度のブロックエース　（雪印乳業社）　でブロックし、０．１　％のＴｗｅｅｎ２０　を含むＰＢＳ（洗浄バッファー）で３回洗浄した。各濃度のＯＣＩＦを第一次反応バッファー（１／２．５濃度のブロックエース及び０．１　％Ｔｗｅｅｎ２０　を含む０．２Ｍトリス塩酸緩衝液、ｐＨ　７．４）で調製した。
【０１４２】
調製した各濃度のＯＣＩＦ溶液　１００μｌ　づつ各ウエルに加え、３７℃で３時間放置し、次いで洗浄バッファーで３回洗浄した。標識抗体の希釈には、第二次反応バッファー　（１／４　濃度のブロックエース及び　０．１％の　Ｔｗｅｅｎ２０を含む０．１Ｍトリス塩酸緩衝液、ｐＨ　７．４）　を用いた。各標識抗体を第２次反応バッファーで４００　倍に希釈し、その各々　１００μｌ　づつを各ウエルにそれぞれ添加した。各々のプレートを３７℃で２時間放置し、次いで３回洗浄した後、基質溶液（０．４ｍｇ／ｍｌのオルトフェニレンジアミン塩酸、０．００６　％過酸化水素を含む０．１Ｍクエン酸−リン酸バッファー、ｐＨ　４．５）　１００　μｌ　を各ウエルに添加した。３７℃で１５分間暗室に放置した後、６Ｎ硫酸５０μｌ　を各ウエルに添加することにより酵素反応を停止させ、イムノリーダー　（ＮＪ２０００，日本インターメッド社）を用いて　４９２ｎｍの吸光度を測定した。３種の抗体をそれぞれ固相抗体或いは標識抗体としたいずれの組み合わせにおいても良好な測定結果が得られ、３種の抗体はそれぞれＯＣＩＦの異なるエピトープを認識することを認めた。代表例として、Ａ１Ｇ５を固相抗体としＥ３Ｈ８を標識抗体としたときの検量線を図１４に示す。
【０１４３】
ｖｉ）　 ヒト血清中の ＯＣＩＦ の測定
健常人５名の血清中のＯＣＩＦを実施例２５−（ｖ）　の図１４のＥＬＩＳＡ系で測定した。即ち、Ａ１Ｇ５を実施例２５−（ｖ）　と同様にイムノプレートに固相化し、各ウエルに第１次反応バッファーを５０μｌ　加え、次いで各ヒト血清５０μｌ　を加えて３７℃で３時間放置した。洗浄バッファーで３回洗浄した後、第２次反応バッファーで４００　倍に希釈したＥ３Ｈ８の標識抗体１００　μｌ　を各ウエルに加えて、３７℃で２時間放置した。プレートを洗浄バッファーで３回洗浄後、上記基質溶液　１００μｌ　を各ウエルに添加し、３７℃で１５分間反応させた。各ウエルに６Ｎ硫酸５０μｌ　づつ添加して酵素反応を停止させ、イムノリーダーで４９２ｎｍ　の吸光度を測定した。既知量のＯＣＩＦを含む第１次反応バッファーについても同様に操作し、図１４に示すようなＯＣＩＦの検量線を作成し、血清試料の吸光度から血清中のＯＣＩＦ量を求めた。結果を表１６に示す。
【０１４４】
【表１６】

【０１４５】
【実施例２６】
骨粗鬆症に対する治療効果
神経切除による不動性の骨萎縮モデルに対するＯＣＩＦの治療効果を確認した。
Ｆｉｓｃｈｅｒ　系雄ラットを用い、６週齢（体重約１２０ｇ）で左上腕神経叢を切除することにより、左前肢の不動化を惹起して骨萎縮モデルを作成した。ＯＣＩＦは０．０１％Ｔｗｅｅｎ８０　を含むＰＢＳ（−）で調整し、翌日から５μｇ／ｋｇ及び５０μｇ／ｋｇの用量で１２時間間隔で１日２回、２週間連日静脈内投与した。正常群には偽手術を施し、対照群には０．０１％Ｔｗｅｅｎ８０　を含むＰＢＳ（−）を同様に投与した。投与終了後、左上腕を摘出し骨強度を測定した。結果を図１５に示す。
この結果、正常群に比べ対照群では骨強度の低下が観察されたが、ＯＣＩＦ５０μｇ／ｋｇ投与群において改善が認められた。
【０１４６】
【発明の効果】
本発明により、新規な破骨細胞形成抑制活性を有する蛋白質及びその効率的な製造方法が提供される。本発明の蛋白質は破骨細胞形成抑制活性を有し、骨粗鬆症等各種の骨量減少性疾患の治療剤として或いはこれらの疾患の免疫学的診断のための抗原等として利用することができる。
【配列表】

【図面の簡単な説明】
【図１】ＨｉＬｏａｄ−Ｑ／ＦＦ　非吸着画分粗精製製品（試料３）をＨｉＬｏａｄ−Ｓ／ＨＰ　カラムにかけた時の溶出プロファイルを示す。
【図２】ヘパリン−５ＰＷ粗精製製品　（試料５）をブルー−５ＰＷカラムにかけた時の溶出プロファイルを示す。
【図３】ブルー−５ＰＷ溶出フラクション４９〜５０を逆相カラムにかけた時の溶出プロファイルを示す。
【図４】最終精製品の還元条件下と非還元条件下におけるＳＤＳ‐ＰＡＧＥの結果を示す。
【符号の説明】
レーン１、４；分子量マーカー
レーン２、５；ピーク６
レーン３、６；ピーク７
【図５】還元ピリジルエチル化後、リシルエンドプロテアーゼ処理したピーク７を逆相カラムにかけた時の溶出プロファイルを示す。
【図６】天然（ｎ）　及び組み換え型（ｒ）　ＯＣＩＦの、非還元条件下におけるＳＤＳ‐ＰＡＧＥの結果を示す。又、（Ｅ）　は２９３／ＥＢＮＡ細胞で生産したものを、（Ｃ）　はＣＨＯ細胞で生産したものをそれぞれ示す。
【符号の説明】
レーン１；分子量マーカー
レーン２；モノマー型ｎＯＣＩＦ
レーン３；ダイマー型ｎＯＣＩＦ
レーン４；モノマー型ｒＯＣＩＦ（Ｅ）
レーン５；ダイマー型ｒＯＣＩＦ（Ｅ）
レーン６；モノマー型ｒＯＣＩＦ（Ｃ）
レーン７；ダイマー型ｒＯＣＩＦ（Ｃ）
【図７】天然型（ｎ）及び組み換え型（ｒ）ＯＣＩＦの、還元条件下におけるＳＤＳ‐ＰＡＧＥの結果を示す。又、（Ｅ）は２９３／ＥＢＮＡ細胞で生産したものを、（Ｃ）　ＣＨＯ細胞で生産したものをそれぞれ示す。
【符号の説明】
レーン８；分子量マーカー
レーン９；モノマー型ｎＯＣＩＦ
レーン１０；ダイマー型ｎＯＣＩＦ
レーン１１；モノマー型ｒＯＣＩＦ（Ｅ）
レーン１２；ダイマー型ｒＯＣＩＦ（Ｅ）
レーン１３；モノマー型ｒＯＣＩＦ（Ｃ）
レーン１４；ダイマー型ｒＯＣＩＦ（Ｃ）
【図８】Ｎ−結合型糖鎖を除去した天然型（ｎ）　及び組み換え型（ｒ）　ＯＣＩＦの、還元条件下におけるＳＤＳ‐ＰＡＧＥの結果を示す。又、（Ｅ）　は２９３／ＥＢＮＡ細胞で生産したものを、（Ｃ）　はＣＨＯ細胞で生産したものをそれぞれ示す。
【符合の説明】
レーン１５；分子量マーカー
レーン１６；モノマー型ｎＯＣＩＦ
レーン１７；ダイマー型ｎＯＣＩＦ
レーン１８；モノマー型ｒＯＣＩＦ（Ｅ）
レーン１９；ダイマー型ｒＯＣＩＦ（Ｅ）
レーン２０；モノマー型ｒＯＣＩＦ（Ｃ）
レーン２１；ダイマー型ｒＯＣＩＦ（Ｃ）
【図９】ＯＣＩＦとＯＣＩＦ２の、アミノ酸配列の比較を示す。
【図１０】ＯＣＩＦとＯＣＩＦ３の、アミノ酸配列の比較を示す。
【図１１】ＯＣＩＦとＯＣＩＦ４の、アミノ酸配列の比較を示す。
【図１２】ＯＣＩＦとＯＣＩＦ５の、アミノ酸配列の比較を示す。
【図１３】抗ＯＣＩＦポリクローナル抗体を用いた時の、ＯＣＩＦの検量線を示す。
【図１４】抗ＯＣＩＦモノクローナル抗体を用いた時の、ＯＣＩＦの検量線を示す。
【図１５】ＯＣＩＦの骨粗鬆症に対する治療効果を示す。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a novel protein having an activity of inhibiting osteoclast differentiation and / or maturation, that is, an osteoclastogenesis inhibitor (Osteoclastogenesis Inhibitory Factor; OCIF) and a method for producing the same.
[0002]
[Prior art]
Human bones constantly undergo resorption and remodeling, and the cells that play a central role in this process are osteoblasts, which are responsible for bone formation, and osteoclasts, which are responsible for bone resorption. Osteoporosis is a representative disease caused by abnormal bone metabolism that these cells are responsible for. This disease occurs when bone formation by osteoblasts exceeds bone formation by osteoblasts. The mechanism of development of this disease has not yet been fully elucidated, but it is a disease that causes bone pain and causes fractures due to bone weakness. With the increase in the elderly population, the disease, which causes bedridden elderly people due to fractures, has become a social problem, and the development of therapeutics for it is urgently needed. It is expected that such bone loss caused by abnormal bone metabolism will be treated by suppressing bone resorption, promoting bone formation, or improving the balance between them.
[0003]
Bone formation is expected to be promoted by promoting proliferation, differentiation and activation of cells responsible for bone formation, or by suppressing proliferation, differentiation and activation of cells responsible for bone resorption. In recent years, interest in bioactive proteins (cytokines) having such activities has increased, and intensive research has been conducted. As cytokines that promote osteoblast proliferation or differentiation, fibroblast growth factor family (FGF, Non-patent Document 1), insulin-like growth factor-I (insulin-like growth factor-I; IGF-I, non- Patent Document 2), Insulin-like growth factor-II (IGF-II, Non-patent Document 3), Activin A (Activin A, Non-patent Document 4), Transforming Growth Factor-β (Transforming growth factor-β, Non-patent Document) 5), Vasculotropin (Non-patent Document 6), and the ectopic bone morphogenetic protein family (bone morphogenetic protein; BMP): BMP-2 , Non-Patent Document 7, {OP-1, Non-Patent Documents 8 and 9)} and the like have been reported.
[0004]
On the other hand, as cytokines that suppress osteoclast formation, that is, differentiation and / or maturation of osteoclasts, transforming growth factor-β (Transforming growth factor-β, Non-patent Document 10) and interleukin-4 (interleukin- 4, Non-Patent Document 11) and the like have been reported. Examples of cytokines that suppress bone resorption by osteoclasts include calcitonin (calcitonin, Non-patent Document 12), macrophage colony-stimulating factor (macrophage colony-stimulating factor; Non-patent Document 13), and interleukin-4 (Non-patent Document 13). Reference 14), and interferon-γ (interferon-γ, Non-Patent Reference 15)} and the like have been reported.
[0005]
These cytokines are expected to be ameliorating agents for bone loss due to their promotion of bone formation and suppression of bone resorption, and include the above-mentioned cytokines such as insulin-like growth factor-I and cytokines of the ectopic bone formation factor family. Clinical trials have been conducted on some of the cytokines as bone metabolism improving agents. Calcitonin has already been marketed as a therapeutic drug for osteoporosis and a pain relieving drug.
[0006]
Currently, active vitamin D is used as a medicine to treat bone-related diseases and to shorten the treatment period.₃, Calcitonin and its derivatives, hormone preparations such as estradiol, ipriflavone, vitamin K₂(Menatetrenone) or a calcium preparation or the like is used. However, the therapeutic methods using these drugs are not always satisfactory in their effects and therapeutic results, and development of new therapeutic agents to replace them has been desired. As described above, bone metabolism is regulated by the balance between bone formation and bone resorption, and cytokines that suppress osteoclast differentiation and maturation are expected to be therapeutic agents for bone loss such as osteoporosis. You.
[0007]
[Prior art]
[Non-Patent Document 1] Rodan @ S. B. {Et} al. , {Endocrinology} vol. $ 121, $ p1917, $ 1987
[Non-Patent Document 2] Hock @ J. M. {Et} al. , {Endocrinology} vol. $ 122, $ p254, $ 1988
[Non-Patent Document 3] McCarthy @ T. {Et} al. , {Endocrinology} vol. 124, $ p301, $ 1989
[Non-patent Document 4] Centrella @ M. {Et} al. , {Mol. {Cell. {Biol. Vol. 11, p250, 1991
[Non-Patent Document 5] Noda @ M. , The Bone, vol. $ 2, $ p29, $ 1988
[Non-Patent Document 6] Varonique @ M. {Et} al. , @Biochem. {Biophys. Res. {Commun. Vol. 199, $ p380, 1994
[Non-Patent Document 7] Yamaguchi, {A} et al. , @J. {Cell} Biol. Vol. 113, p682, 1991
[Non-Patent Document 8] Sampath @ T. K. {Et} al. , @J. {Biol. {Chem. Vol. 267, p20532, 1992
[Non-Patent Document 9] Knutsen @ R. {Et} al. , @Biochem. {Biophys. Res. {Commun. Vol. 194, $ p1352, $ 1993
[Non-Patent Document 10] Chenu @ C. {Et} al. , @Proc. {Natl. {Acad. {Sci. USA, vol. 85, $ p5683, $ 1988
[Non-Patent Document 11] Kasano @ K. {Et} al. , @ Bone-Miner. , @Vol. 21, p179, 1993
[Non-Patent Document 12] Bone-Miner. , @Vol. 17, @ p347, 1992
[Non-Patent Document 13] Hattersley @ G. {Et} al. {J. Cell. {Physiol. Vol. 137, $ p199, $ 1988
[Non-Patent Document 14] Watanabe, K. {Et} al. , @Biochem. {Biophys. Res. {Commun. Vol. 172, p1035, 1990
[Non-Patent Document 15] Gowen @ M. {Et} al. , @J. << Bone >> Miner. Res. , @Vol. $ 1, $ 469, $ 1986
[0008]
[Problems to be solved by the invention]
The present invention has been made from such a viewpoint, and it is an object of the present invention to provide a novel osteoclast formation inhibitory factor (OCIF) and an efficient method for producing the same.
[0009]
[Means for Solving the Problems]
The present inventors have conducted intensive searches in view of such a situation, and found that the culture solution of human fetal lung fibroblast IMR-90 (ATCC deposition-accession number CCL186) has an inhibitory activity on osteoclast formation, that is, The inventors have found a protein OCIF having an activity of suppressing differentiation and maturation.
It has also been found that when an alumina ceramic piece is used as a carrier for cell culture, the osteoclast formation inhibitory factor OCIF of the present invention is accumulated at a high concentration in a medium and can be purified efficiently.
Furthermore, the present inventors have established a method for efficiently purifying the protein OCIF by sequentially treating the culture solution with an ion exchange column, an affinity column, and a reverse phase column and repeating adsorption and elution.
[0010]
Next, based on the amino acid sequence information of the obtained native OCIF protein, the present inventors succeeded in cloning cDNA encoding this protein. Furthermore, the present inventors have established a method for producing a protein having osteoclast differentiation and / or maturation inhibitory activity by using this cDNA by genetic engineering techniques.
[0011]
The present invention is based on human fetal lung fibroblasts, and has a molecular weight of about 60 kD in SDS-PAGE under reducing conditions, about 60 kDa and about $ 120 kD in SDS-PAGE under non-reducing conditions, and includes a cation exchanger and heparin. Has an affinity for the column, and the heat treatment at 70 ° C. for 10 minutes or at 56 ° C. for 30 minutes reduces the activity of inhibiting osteoclast differentiation / maturation. The present invention relates to a protein characterized by a loss of the activity of inhibiting the differentiation and maturation of E. coli. The amino acid sequence of the protein OCIF of the present invention is clearly different from known osteoclastogenesis inhibitors.
[0012]
Also, the present invention provides a method for culturing human fibroblasts, treating the culture solution with a heparin column, eluting an adsorbed fraction, applying this fraction to a cation exchange column to adsorb and elute, and further purifying an affinity column, a reverse phase column. And a method for producing the protein OCIF. The column treatment in the present invention includes not only a method in which a culture solution is simply caused to flow down to a heparin sepharose column or the like, but also a method in which a culture solution is mixed with heparin sepharose or the like by a batch method and has the same effect as when column treatment is performed. I do. The affinity column used in the present invention includes a heparin column and a blue column. The blue column is particularly preferably a Cibacron blue column. As a filler for the Cibacron Blue column, a filler having a hydrophilic synthetic polymer as a carrier and a dye Cibacron Blue F3GA bound thereto is exemplified, and this column is usually called a blue column.
Further, the present invention relates to a method for producing the protein efficiently by culturing cells using alumina ceramic pieces as a carrier.
[0013]
The protein OCIF of the present invention can be isolated and purified efficiently and with high yield from a culture solution of human fibroblasts. The protein OCIF of the present invention is isolated and purified from this raw material by utilizing the physical and chemical properties of the target protein OCIF using a general method widely used for purifying a proteinaceous substance from a biological sample. It can be carried out according to various purification operations. Examples of the concentration means include ordinary biochemical treatment means such as ultrafiltration, freeze-drying, and salting out. Further, as a purification means, various kinds of materials used for purification of ordinary proteinaceous substances using ion exchange chromatography, affinity chromatography, gel filtration chromatography, hydrophobic chromatography, reverse phase chromatography, preparative electrophoresis, etc. Can be used in combination. It is particularly preferable to use human fetal lung fibroblast IMR-90 (ATCC-CCL # 186) as the human fibroblast used as a raw material. Culture of human fetal lung fibroblast IMR-90 as a raw material is performed by attaching human fetal lung fibroblast IMR-90 to an alumina ceramic piece and using a DMEM medium supplemented with 5% bovine neonatal serum as a culture solution. It is good to use what was obtained by standing and culturing for about one week to ten days in a roller bottle. In addition, when performing the purification treatment, it is preferable to add 0.1% CHAPS (3-[(3-cholamidopropyl) -dimethylammonio] -1-propanesulfonate)} as a surfactant for purification.
[0014]
The protein OCIF of the present invention is obtained by first applying a culture solution to a heparin column (Heparin-Sepharose CL-6B, Pharmacia) and eluting it with a 10 mM Tris-HCl buffer (pH 7.5) containing 2 M NaCl, and elutes with a heparin-adsorbing OCIF fraction. This fraction is applied to a Q • anion exchange column (HiLoad-Q / FF, Pharmacia), and the non-adsorbed fraction is collected to obtain a heparin-adsorbing and basic OCIF fraction. it can. The obtained OCIF active fraction was subjected to S-cation exchange column (HiLoad-S / HP, Pharmacia), heparin column (Heparin-5PW, Tosoh), Cibacron blue column (Blue-5PW, Tosoh), reverse It can be isolated and purified by applying it to a phase column (BU-300 {C4}, PerkinElmer), and this substance is identified by the properties described above.
[0015]
Furthermore, the present invention clones a cDNA encoding this protein based on the amino acid sequence of the natural OCIF protein thus obtained, and uses the cDNA to differentiate and / or differentiate osteoclasts by genetic engineering techniques. Alternatively, the present invention relates to a method for obtaining a protein OCIF having maturation inhibitory activity.
[0016]
That is, the OCIF protein purified according to the method of the present invention is
After treatment with, for example, lysyl endopeptidase, the amino acid sequence of the resulting peptide is determined and a mixture of oligonucleotides capable of encoding the resulting internal amino acid sequence is made.
Next, an OCIF cDNA fragment is obtained using a PCR method (preferably an RT-PCR method) using the prepared oligonucleotide mixture as a primer. Using this OCIF cDNA fragment as a probe, a full-length cDNA of OCIF is cloned from a cDNA library. The obtained OCIF cDNA is inserted into an expression vector to prepare an OCIF expression plasmid, which is then introduced into various cells or strains for expression, whereby a recombinant OCIF can be produced.
[0017]
The present invention also relates to a novel protein {OCIF2, {OCIF3, {OCIF4, {OCIF5}}, which is an analog (variant) of the OCIF protein of the present invention having the above-mentioned activity.
These analogs are derived from the poly (A) of IMR-90 cells.⁺It can be obtained by hybridizing a cDNA library prepared using RNA with an OCIF cDNA fragment as a probe. The desired analog protein can be obtained by inserting the cDNA of these OCIF analogs into an expression vector, expressing the OCIF analog expression vector in an ordinary host, and purifying it by a conventional method.
[0018]
The invention also relates to OCIF variants.
These mutants have a Cys residue at Ser that may be involved in OCIF dimerization.
It is a residue-substituted or natural OCIF with a deletion mutation introduced. A substitution or deletion mutation is introduced into the OCIF cDNA by PCR or cleavage with a restriction enzyme. This cDNA is inserted into a vector having an appropriate expression promoter, transfected into eukaryotic cells such as mammalian cells, and the cells are cultured and purified from the culture by a conventional method to obtain the desired OCIF. Mutants are obtained.
[0019]
The present invention also relates to an anti-OCIF polyclonal antibody and a method for measuring OCIF using the same.
An anti-OCIF polyclonal antibody is prepared by a conventional method using OCIF as an immunogen. The antigen (immunogen) used at this time may be natural OCIF obtained from the IMR-90 culture solution, recombinant OCIF produced using microorganisms or eukaryotic cells as hosts using OCIF cDNA, or amino acid sequence of OCIF. Synthetic peptides designed on the basis of these and OCIF hydrolyzing partial peptides can be used. An anti-OCIF polyclonal antibody can be obtained by immunizing a suitable mammal using these antigens and, if necessary, in combination with an immunological adjuvant, and purifying the immunized serum by a conventional method. By labeling the obtained anti-OCIF polyclonal antibody with an isotope or an enzyme, it can be used in a measurement system for radioimmunoassay (RIA) or enzyme immunoassay (EIA). By using this measurement system, it is possible to easily measure the OCIF concentration of a biological sample such as blood or ascites or a cell culture solution.
[0020]
The present invention also relates to an anti-OCIF monoclonal antibody and a method for measuring OCIF using the same.
An anti-OCIF monoclonal antibody is prepared by a conventional method using OCIF as an immunogen. As antigens, natural OCIF obtained from the IMR-90 culture solution, recombinant OCIF produced using microorganisms and eukaryotic cells as hosts using OCIF cDNA, or synthetic peptides designed based on the amino acid sequence of OCIF, , OCIF hydrolysis partial peptides. A mammal is immunized with these antigens, or cells immunized by an in vitro method are fused with a myeloma cell of a mammal (myeloma) to produce a hybridoma, and an antibody recognizing OCIF is produced from the hybridoma. The desired antibody is obtained by selecting a clone to be cloned and culturing this clone. When mammals are used for producing hybridomas, examples using small animals such as mice and rats are common.
Immunization is performed by diluting OCIF to an appropriate concentration with physiological saline or the like, administering this solution intravenously or intraperitoneally, and optionally administering an immune adjuvant thereto, and giving the animal 2-10 days every 2-20 days. Administer -5 times. The animal immunized in this manner is dissected, the spleen is removed, and spleen cells are used as immune cells.
[0021]
Examples of mouse-derived myeloma to be fused with immune cells include P3 / x63-Ag8, p3-U1, NS-1, MPC-11, SP-2 / 0, FO, P3x63Ag8. $ 653 and $ S194 can be exemplified.
Examples of rat-derived cells include cell lines such as R-210. When producing a human antibody, a human B lymphocyte is immunized by an in vitro method, and a human myeloma cell or a cell line transformed with an EB virus is used as a parent strain to obtain a hybridoma producing a human antibody. be able to.
[0022]
The fusion of the immune cell and the myeloma cell line can be carried out by a known method, for example, the method of Koehler and Milstein et al. (Koehle, G. et al. Nature vol. 256, 495-497, 1975), or the electric pulse method. Immune cells and myeloma cell lines are mixed with the medium used for cell culture (without FBS) at the ratio of the number of cells normally used, and polyethylene glycol is added to perform fusion treatment. And the fused cells can be selected.
[0023]
In order to select an anti-OCIF antibody-producing strain, a method commonly used for antibody detection such as an ELISA method, a plaque method, an Ouchterlony method, or an agglutination method can be used. The hybridomas thus selected can be subcultured by a usual culture method, and can be cryopreserved if necessary. The antibody can be produced by culturing the hybridoma by a conventional method or by transplanting the hybridoma into the peritoneal cavity of a mammal. Antibodies can be purified by conventional methods such as salting out, gel filtration, and affinity chromatography.
[0024]
The obtained antibody specifically reacts with OCIF and can be used for measuring and purifying OCIF. When used for OCIF measurement, radioimmunoassay (RIA) や or enzyme immunoassay (EIA) can be performed by labeling antibodies with isotopes or enzymes.
Can be used for the measurement system. In particular, the antibodies obtained according to the present invention have the characteristic that they can be used in a sandwich immunoassay because their antigen recognition sites are different from each other. By using this measurement system, it is possible to easily measure the OCIF concentration of a biological sample such as blood or ascites or a cell culture solution.
[0025]
The OCIF activity was determined by the method of Masayoshi Kumegawa et al. (Protein / Nucleic Acid / Enzyme, Vol.34, 及 び p999 (1989)) and Takahashi N. {Et} al. （(Endocrinology, Vol. 122, p1373 (1988)). That is, using the mouse bone marrow cells of about 17 days after birth as target cells,₃The inhibition of osteoclast formation in the presence of (Calcitriol) can be tested by inhibiting the induction of tartrate-resistant acid phosphatase activity.
[0026]
The protein of the present invention, the osteoclast formation inhibitory factor OCIF, is used for treating bone loss such as osteoporosis, bone metabolism disorders such as rheumatism or osteoarthritis, and bone metabolism disorders such as multiple myeloma. It is useful as a pharmaceutical composition for the purpose of improvement or as an antigen for establishing an immunological diagnosis of such a disease. The protein of the present invention can be formulated and administered orally or parenterally. That is, the preparation containing the protein of the present invention is safely administered to humans and animals as a pharmaceutical composition containing the osteoclast formation inhibitory factor OCIF as an active ingredient.
[0027]
Examples of the form of the pharmaceutical composition include an injection composition, an infusion composition, a suppository, a nasal agent, a sublingual agent, a transdermal absorbent, and the like. In the case of an injectable composition, it is a mixture of a pharmacologically effective amount of the osteoclastogenesis inhibitor of the present invention and a pharmaceutically acceptable carrier, among which amino acids, saccharides, cellulose derivatives, and other Excipients / activators generally added to injectable compositions, such as organic / inorganic compounds, can also be used. When an injection is prepared using the osteoclast formation inhibitory factor OCIF of the present invention and these excipients / activators, a pH adjuster, a buffer, a stabilizing agent, a solubilizing agent, etc., if necessary. To give various injections by a conventional method.
[0028]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in more detail with reference to Examples. However, these are merely examples, and the present invention is not limited thereto.
Embodiment 1
Human fibroblast IMR- 90 Preparation of culture solution
Human fetal lung fibroblasts IMR-90 (ATCC-CCL186) are available in roller bottles (490 cm).²80 g of alumina ceramic pieces (99.5% alumina, Toshiba Ceramic Co., Ltd.) and cultured. For the culture, 60 roller bottles were used, and 500 ml of a 10 mM HEPES buffer-supplemented DMEM medium (Gibco BRL) supplemented with 5% calf serum per roller bottle was used.₂The culture was allowed to stand still for 7 to 10 days in the presence. After the culture, the culture was recovered and a new culture medium was added to obtain a # 301 IMR-90 culture in a single culture. The obtained culture solution was used as Sample 1.
[0029]
Embodiment 2
Method for measuring osteoclast formation inhibitory activity
The activity of the proteinaceous osteoclastogenesis inhibitor of the present invention was measured by the method of Masayoshi Kumegawa (Protein, Nucleic Acid, Enzyme {Vol. 34 {p999 (1989))} and Takahashi {N. {Et} al. (Endocrinology, vol. 122, p1373 (1988)). That is, using active bone vitamin D using bone marrow cells isolated from a mouse about 17 days after birth.₃Osteoclast formation in the presence was tested by using the induction of tartrate-resistant acid phosphatase activity as an index and measuring its inhibitory activity. That is, 2 × 10^-8M active vitamin D₃And 100% of a sample diluted with an α-MEM medium (Gibco BRL) containing 10% fetal calf serum, and 3 × 10 3 bone marrow cells obtained from a mouse about 17 days old⁵The cells were suspended and seeded in 100 μl of α-MEM medium containing 10% fetal bovine serum, and 5% CO 2₂At 37 ° C. and 100% humidity for one week. On the 3rd and 5th days of the culture, the culture solution (160 μl) was discarded, and 1 × 10^-8M active vitamin D₃And 160 μl of a sample diluted with α-MEM medium containing 10% fetal calf serum. After 7 days of culture, the cells were washed with a phosphate buffered saline, fixed with an ethanol / acetone (1: 1) solution at room temperature for 1 minute, and the formation of osteoclasts was measured using an acid phosphatase activity assay kit (Acid @ Phosphatase, Leucocyte). , Catalog No. 387-A, Sigma). The decrease in cells positive for acid phosphatase activity in the presence of tartaric acid was defined as OCIF activity.
[0030]
Embodiment 3
OCIF Purification
i) Heparin Sepharose CL-6B Purification by
About 90 l of the IMR-90 culture solution (sample 1) was applied to a 0.22 μm０．２ filter (hydrophilic Millidisk, 2,000 cm).²Heparin-Sepharose CL-6B column equilibrated with {0.3 M} NaCl in 10 mM {Tris-HCl} buffer (hereinafter referred to as Tris-HCl), pH 7.5} after filtration through Millipore Corporation. (5 × 4.1 cm, gel volume 80 ml). After washing with 10 mM Tris-HCl, pH 7.5 at a flow rate of 500 ml / hr, elution was carried out with 10 mM Tris-HCl, pH 7.5, containing 2 M NaCl, and heparin-Sepharose CL-6B adsorption fraction 900 mL
Was obtained, and the obtained fraction was used as Sample 2.
[0031]
ii ) HiLoad-Q / FF Purification by
The heparin-Sepharose-adsorbed fraction (sample 2) was dialyzed against {10 mM {Tris-HCl, pH 7.5}, CHAPS was added to a concentration of 0.1%, and the mixture was allowed to stand at 4 ° C. overnight and divided into two portions. And applied to an anion exchange column (HiLoad-Q / FF, 2.6 mm × 10 cm, Pharmacia) equilibrated with 50 mM {Tris-HCl, pH 7.5} containing 0.1% CHAPS to obtain 1000 ml of a non-adsorbed fraction. . The obtained fraction was designated as Sample 3.
[0032]
iii) 　 HiLoad-S / HP Purification by
A cation exchange column (HiLoad-S / HP, 2.6 mm × 10 cm, Pharmacia) in which the HiLoad-Q non-adsorbed fraction (sample 3) was equilibrated with 50 mM Tris-HCl, pH 7.5 containing 0.1% CHAPS. Company). After washing with 50 mM Tris-HCl, pH 7.5 containing 0.1% CHAPS, elution was performed at a linear gradient of 1 M NaCl over 100 minutes, at a flow rate of 8 ml / min, and fractionation was performed at 12 ml / fraction. Was. Fractions 1 to 40 were combined into four fractions of 10 fractions, and OCIF activity was measured using 100 μl each. OCIF activity was observed in fractions 11 to 30 (FIG. 1: In the figure, ++ indicates an activity in which osteoclast formation is suppressed by 80% or more, and + indicates an activity in which osteoclast formation is suppressed by 30 to 80%). ,-Indicate that no activity was detected, respectively). Fractions 21 to 30 having higher specific activities were designated as Sample 4.
[0033]
iv) Affinity column (heparin -5PW ) Purification
After diluting 120 ml of sample 4 with 240 ml of 50 mM Tris-HCl, pH 7.5 containing 0.1% CHAPS, affinity column (heparin) equilibrated with 50 mM Tris-HCl, pH 7.5 containing 0.1% CHAPS -5PW, 0.8). After washing with 50 mM Tris-HCl, pH 7.5 containing 0.1% CHAPS, elution was performed at a linear gradient of 2 M NaCl over 60 minutes, at a flow rate of 0.5 ml / min, and at 0.5 ml / fraction. A fraction was taken. The OCIF activity was measured using 50 μl of each fraction, and 10 ml of an OCIF active fraction eluted with about 0.7 to 1.3 M {NaCl} was obtained.
[0034]
v) Affinity column (blue -5PW ) Purification
After diluting 10 ml of sample 5 with 190 ml of 50 mM Tris-HCl, pH 7.5 containing 0.1% CHAPS, an affinity column (blue) equilibrated with 50 mM Tris-HCl, pH 7.5 containing 0.1% CHAPS −5 PW, 0.5 × 5.0 cm, Tosoh Corporation). After washing with 50 mM Tris-HCl, pH 7.5 containing 0.1% CHAPS, elution was performed at a linear gradient of 2 M NaCl for 60 minutes, at a flow rate of 0.5 ml / min, and at 0.5 ml / fraction. A fraction was taken. The OCIF activity was measured using 25 μl of each fraction to obtain OCIF active fractions 49 to 70 eluted at about 1.0 to 1.6 M NaCl (in FIG. 2, ++ indicates that osteoclast formation was 80% or more) + Indicates an activity in which osteoclast formation is suppressed by 30 to 80%).
[0035]
vi) Purification by reversed-phase column
After adding 10 μl of 25% TFA (trifluoroacetic acid) to 49 to 50 ml of the obtained fraction, a reversed-phase column {(BU-300}, C4, 2...) Equilibrated with 25% acetonitrile containing 0.1% TFA. (1 × 220 mm, Perkin Elmer), elution was performed at a linear gradient of acetonitrile of 55% in 60 minutes, at a flow rate of 0.2 ml / min, and each peak was collected (FIG. 3). Using 100 μl of each peak fraction, OCIF activity was measured, and peaks 6 and 7 were detected in a concentration-dependent manner. Table 1 shows the results.
[0036]
[Table 1]

(In the table, ++ indicates an activity in which osteoclast formation is suppressed by 80% or more, + indicates an activity in which osteoclast formation is suppressed by 30 to 80%, and-indicates that no activity is detected.)
[0037]
Embodiment 4
OCIF Molecular weight measurement
SDS-polyacrylamide gel electrophoresis was performed under reducing and non-reducing conditions using 40 μl each of peak 6 and peak 7 in which OCIF activity was observed. That is, 20 μl of each peak fraction was collected in two tubes, concentrated under reduced pressure, and dissolved in 1.5 mM of 10 mM Tris-HCl, pH 8 containing 1 mM EDTA, 2.5% SDS, and 0.01% bromophenol blue. Then, each was allowed to stand at 37 ° C. overnight under non-reducing conditions and reducing conditions (in the presence of 5% Δ2-mercaptoethanol), and 1 μl of each was loaded on SDS-polyacrylamide gel electrophoresis. The electrophoresis was carried out using a gradient gel of 10-15% acrylamide (Pharmacia) and an electrophoresis apparatus Phast @ System (Pharmacia). As a molecular weight marker, phosphorylase b (94 kD) {, bovine serum albumin (67 kD), ovalbumin (43 kD), carbonic anhydrase (30 kD), trypsin inhibitor (20.0 kD), and α-lactalbumin {(14.4 kD)} are used. Was. After completion of the electrophoresis, silver staining was carried out using Past \ Gel \ Silver \ Stain \ Kit (Pharmacia). FIG. 4 shows the results.
As a result, about peak 6, a protein band of about 60 kD was detected under reducing and non-reducing conditions. For peak 7, a protein band of about 60 kD was detected under reducing conditions, and a protein band of about 120 kDa was detected under non-reducing conditions. Therefore, peak 7 is considered to be a homodimer of the protein of peak 6.
[0038]
Embodiment 5
OCIF Thermal stability test
Take 20 μl each from the sample mixed with Blue 5PW fractions 51-52, add 10 mM phosphate buffered saline, pH 7.2 30 μl, and then 10 minutes at 70 ° C. and 90 ° C., or 30 minutes at 56 ° C. Heat treatment was performed for minutes. Using this sample, the OCIF activity was measured according to the method described in Example 2. Table 2 shows the results.
[0039]
Table 2 Thermal stability of OCIF

(In the table, ++ indicates an activity in which osteoclast formation is suppressed by 80% or more, + indicates an activity in which osteoclast formation is suppressed by 30 to 80%, and-indicates that no activity is detected.)
[0040]
Embodiment 6
Determination of internal amino acid sequence
For the Blue-5 PW fractions 51 to 70, mix two fractions to 1 ml, add 10 μl of 25% TFA to each sample, and add 1 ml of each of the 10 times to 25% acetonitrile containing 0.1% TFA. And then eluted with a linear gradient of acetonitrile of 55% in 60 minutes at a flow rate of {0.2} ml / min. (BU-300, C4, 2.1 × 220 mm, Perkin Elmer). , Peak 6 and peak 7 were collected. N-terminal amino acid sequence analysis was performed on each of the obtained peaks 6 and 7 using a protein sequencer (Procise 494 ° type, Perkin Elmer), but analysis was not possible. It was suggested that it might have been blocked. Therefore, the internal amino acid sequences of these proteins were analyzed. That is, after each of the peaks 6 and 7 was concentrated by centrifugation, 100 μg of {0.5 M {Tris-HCl, pH 8.5} 50 μl containing 100 μg of dithiothreitol, 10 mM of EDTA, 7 M of guanidine hydrochloride, and 1% of CHAPS were added to each, followed by room temperature addition. For 4 hours, and 0.2 μl of 4-vinylpyridine was added. The mixture was allowed to stand overnight at room temperature in a dark place to undergo pyridylethylation. To each of these samples, 1 μl of 25% TFA was added, and the mixture was applied to a reverse phase column (BU-300, ΔC4, Δ2.1 × 30 mm, PerkinElmer) equilibrated with 20% acetonitrile containing 0.1% TFA for 30 minutes. Elution was performed with a linear gradient of 50% acetonitrile concentration at a flow rate of 0.3 ml / min to obtain a reduced pyridylethylated OCIF sample. Each of the reduced pyridylethylated samples was concentrated by centrifugation, dissolved in 0.1 M Tris-HCl containing 8 M urea and 0.1% Tween80, pH 9 25 μl, and then diluted with 73 μl 0.1 M Tris-HCl, pH 9 Then, 0.02 μg of AP1 (lysyl endoprotease, Wako Pure Chemical Industries, Ltd.) was added and reacted at 37 ° C. for 15 hours. 1 μl of 25% TFA was added to the reaction solution, and the mixture was applied to a reverse phase column (RP-300, {C8, {2.1 × 220 mm}, Perkin Elmer) equilibrated with 0.1% TFA, and the concentration of acetonitrile was reduced to 50% for 70 minutes. Elution was performed at a linear gradient of 0.2 ml / min to obtain a peptide fragment (FIG. 5). The obtained peptide fragment (P1 to P3) was subjected to amino acid sequence analysis using a protein sequencer. The results are shown in Sequence Listing II SEQ ID NOs: 1 to 3.
[0041]
Embodiment 7
cDNA Sequence determination
i) 　 IMR-90 Poly from cells (A) ⁺ RNA Isolation
Poly (A) of IMR-90 cells⁺RNA was isolated using a Fast Track mRNA Isolation Kit (Invitrogen) according to the manual. 1X10 by this method⁸About 10 μg of poly (A) from one IMR-90 cell⁺RNA was obtained.
[0042]
ii) Preparation of mixed primer
The following two types of mixed primers were synthesized based on the amino acid sequence of the peptide (SEQ ID NO: 2 and 3) previously obtained. That is, a mixture of oligonucleotides (mix primer, No. 2F) having all base sequences capable of encoding the amino acid sequence from the sixth (Gln) to the twelfth (Leu) of peptide P2 was synthesized. In addition, a mixture (mix primer, No. 3R) of oligonucleotides complementary to all nucleotide sequences capable of encoding the amino acid sequence from the 6th (His) １２ to the 12th (Lys) Ｐ of P3 of the peptide was synthesized. Table 3 shows the base sequences of the used mix primers.
[0043]
[Table 3]

[0044]
iii) 　 OCIF cDNA Fragmentary PCR Amplification by
Poly (A) obtained in Example 7-i)⁺Using a Superscript II cDNA synthesis kit (Gibco BRL) with RNA and 1Mg as a template, a single-stranded cDNA was synthesized according to the company's protocol, and PCR was performed using this cDNA and the primers shown in Example 7-ii). Was performed to obtain an OCIF cDNA fragment. The conditions are shown below.
[0045]

[0046]
After mixing the above solutions in a microcentrifuge tube, PCR was performed under the following conditions. After pretreatment at 95 ° C. for 3 minutes, the reaction in a 3 ° step of 95 ° C. for 30 seconds, 50 ° C. for 30 seconds, and 70 ° C. for 2 minutes was repeated 30 times, and the temperature was maintained at 70 ° C. for 5 minutes. A part of the reaction solution was subjected to agarose electrophoresis, and it was confirmed that a uniform DNA fragment of about 400 bp was obtained.
[0047]
Embodiment 8
PCR Amplified by OCIF cDNA Cloning and sequencing of fragments
The OCIF cDNA fragment obtained in Example 7-iii) was ligated with the plasmid pBluescript {II} SK by the method of Marchuk, D et al. (Nucleic Acid Res., １９Vol.19, p1154, 1991).⁻(Stratagene) and DNA Ligation Kit Ver. 2 (Takara Shuzo) to transform Escherichia coli @ DH5α (Gibco BRL). The obtained transformant was grown, and a plasmid into which an approximately 400 bp OCIF cDNA fragment was inserted was purified by a conventional method. This plasmid was named pBSOCIF, and the nucleotide sequence of the OCIF cDNA inserted into this plasmid was determined using a Tack Dide Oxyterminator Cycle Sequencing Kit (TaqDye \ Deoxy \ Terminator \ Cycle \ Sequencing \ kit; PerkinElmer). The size of this OCIF cDNA was 397 bp. In the amino acid sequence composed of 132 amino acids predicted from this nucleotide sequence, the internal amino acid sequence of OCIF (SEQ ID NOS: 2 and 3) used to design the mix primer was N-terminal and C-terminal, respectively. Could be found. Also, the internal amino acid sequence of OCIF (SEQ ID NO: 1) was found in the amino acid sequence consisting of $ 132 amino acids. From the above results, it was confirmed that the cloned cDNA of 397 bp was an OCIF cDNA fragment.
[0048]
Embodiment 9
DNA Preparation of probe
This OCIF cDNA fragment was amplified by performing PCR under the conditions of Example 7-iii) using the plasmid into which the 397 bp # OCIF cDNA fragment prepared in Example 8 was inserted as a template. The 397 bp OCIF cDNA fragment was separated by agarose electrophoresis and purified using QIAEX gel extraction kit (Qiagen). This DNA was purified using the Megaprime DNA Labeling Kit (Amersham) [α³²P] dCTP} and used as a probe to screen for full-length OCIF cDNA.
[0049]
Embodiment 10
cDNA Creating a library
Poly (A) obtained in Example 7-i)⁺Synthesis of cDNA using oligo (dT) primer, addition of EcoRI-SalI-Not-I adapter, cDNA size fragment, using RNA, 2.5 μg as a template, and a Great Strength cDNA Synthesis Kit (Clontech) according to the company's protocol. After precipitation and ethanol precipitation, it was dissolved in 10 μl of TE buffer. 0.1 μg of the obtained adapter-added cDNA was inserted into 1 μg of λZAP express vector (Stratagene) previously digested with EcoRI using T4 DNA ligase. The thus obtained cDNA recombinant phage DNA solution was subjected to an in vitro packaging reaction using Gigapack Gold II (Stratagene) to produce λZAPＺexpress recombinant phage.
[0050]
Embodiment 11
Screening of recombinant phage
The recombinant phage obtained in Example 10 was infected with Escherichia coli {XL1-Blue} MRF '(Stratagene) at 37 ° C. for 15 minutes, and then added to an NZY medium containing 0.7% agar heated to 50 ° C. Then, the mixture was poured into an NZY @ agar medium plate. After culturing overnight at 37 ° C., Hybond N (Amersham) was adhered to the plaque-formed plate for about 30 seconds. The filter was denatured with an alkali according to a conventional method, neutralized, immersed in 2X SSC {solution}, and then immobilized on the filter with UV crosslink (Stratagene). The obtained filter was immersed in a hybridization buffer (Amersham) containing 100 μg / ml of salmon sperm DNA, pretreated at 65 ° C. for 4 hours, and then heat-denatured DNA probe (2 × 10 5⁵(cpm / ml) was transferred to the above buffer to which hybridization was carried out at 65 ° C overnight. After the reaction, the filter was washed twice with 2 × SSC and twice with 0.1 × SSC, 0.1% SDS solution at 65 ° C. for 10 minutes. Some of the obtained positive clones were further purified by performing screening twice. Among them, those having an insert of about 1.6 kb were used below. This purified phage was named λOCIF. The purified λOCIF was infected with Escherichia coli XL1-Blue {MRF ′} according to the protocol of λZAP {Express Cloning Kit (Stratagene), followed by multiple infections with helper phage ExAssist (Stratagene), and the culture supernatant was subjected to Escherichia coli XLOLR. After infection with (Stratagene) 社, a kanamycin-resistant strain was picked up to obtain a transformant having a plasmid pBKOCIF in which the above 1.6 kb insert was inserted into pBKCMV (Stratagene). This transformant was designated as pBK / 01F10 at the National Institute of Advanced Industrial Science and Technology (now the National Institute of Advanced Industrial Science and Technology at the Patent Organism Depositary) under the accession number FERM @ BP-5267 (October 1995). (Transferred from the original deposit of FERM @ P-14998 to a deposit based on the Budapest Treaty) on the 25th. A transformant having this plasmid was grown, and the plasmid was purified by a conventional method.
[0051]
Embodiment 12
OCIF Encodes the entire amino acid sequence of cDNA Of the nucleotide sequence of
The nucleotide sequence of the OCIF cDNA obtained in Example 11 was determined using Tack Dide Oxyterminator Cycle Sequencing Kit (Perkin Elmer). The primers used were T3, {T7} primer (Stratagene) and a synthetic primer designed based on the nucleotide sequence of OCIF cDNA, and the sequences are shown in SEQ ID NOs: 16 to 29 in the sequence listing.
SEQ ID NO: 6 shows the determined nucleotide sequence of OCIF, and SEQ ID NO: 5 shows the amino acid sequence deduced from the sequence.
[0052]
Embodiment 13
293 / EBNA Recombinant with cells OCIF Production of
i) 　 OCIF cDNA Of expression plasmid
The plasmid pBKOCIF into which about 1.6 kb OCIF cDNA obtained in Example 11 was inserted was digested with restriction enzymes BamHI and XhoI, OCIF cDNA was cut out, separated by agarose electrophoresis, and then QIAEX gel extraction kit (Qiagen). And purified. The OCIF cDNA was ligated to the expression plasmid pCEP4 (Invitrogen), which had been digested with the restriction enzymes BamHI and XhoI in advance, using the ligation kit {Ver. 2 (Takara Shuzo) to transform E. coli DH5Δα (Gibco BRL). The resulting transformant was grown and the expression plasmid pCEPOCIF into which OCIF cDNA was inserted was purified using Qiagen column (Qiagen). After the OCIF expression plasmid pCEPOCIF was precipitated with ethanol, it was dissolved in sterile distilled water and used for the following procedure.
[0053]
ii) 　 OCIF cDNA Of Transient Expression and Its Activity
Using the OCIF expression plasmid pCEPOCIF obtained in Example 13-i), recombinant OCIF was expressed by the method described below, and its activity was measured. 8 × 10⁵293 / EBNA cells (Invitrogen) were inoculated into each well of a 6-well plate using IMDM medium (Gibco BRL) containing 10% fetal bovine serum (Gibco BRL), and the medium was removed the next day. Thereafter, the cells were washed with a serum-free IMDM medium. According to the protocol attached to the transfection reagent Lipofectamine (Gibco BRL), pCEPOCIF previously diluted with OPTI-MEM medium (Gibco BRL) and lipofectamine were mixed, and the mixture was added to the cells in each well. added. The amounts of pCEPOCIF and lipofectamine used were 3 μg and 12 μl, respectively. After 38 hours, the medium was removed and 1 ml of a new OPTI-MEM medium was added. After another 30 hours, the medium was recovered and used as a sample for OCIF activity measurement. The OCIF activity was measured as follows. Activated vitamin D from mouse bone marrow cells about 17 days after birth₃Osteoclast formation in the presence was tested by induction of tartrate-resistant acid phosphatase activity, and its inhibitory activity was measured and determined as OCIF activity. That is, 2 × 10^{- 8}M active vitamin D₃And 100 μl of a sample diluted with α-MEM medium (Gibco BRL) containing 10% fetal calf serum, and 3 × 10 3 mouse bone marrow cells about 17 days old⁵The cells were suspended and seeded in {100 μl} of α-MEM medium containing 10% fetal bovine serum, and 5% CO 2₂And cultured at 37 ° C. and 100% humidity for one week. On the third and fifth days of the culture, 160 μl of the culture solution was discarded, and 1 × 10^-8M active vitamin D₃And 160 μl of a sample diluted with α-MEM medium containing 10% fetal calf serum. After 7 days of culture, the cells were washed with phosphate-buffered saline, and fixed with an ethanol / acetone (1: 1) solution at room temperature for 1 minute. , Catalog No. 387-ΔA, Sigma). The decrease in cells positive for acid phosphatase activity in the presence of tartaric acid was defined as OCIF activity. As a result, as shown in Table 4, it was confirmed that this culture solution had the same activity as that of the natural OCIF previously obtained from the culture solution of IMR-90.
[0054]
[Table 4]

(In the table, ++ indicates an activity in which osteoclast formation is suppressed by 80% or more, + indicates an activity in which osteoclast formation is suppressed by 30 to 80%, and-indicates that no activity is detected.)
[0055]
iii) 　 293 / EBNA Cell-derived recombinant OCIF Purification
CHAPS was added to 1.8 L of a culture solution obtained by mass-culturing 293 / EBNA cells described in Example 13-ii) to a concentration of 0.1%, and a 0.22 μm filter (Sterivex GS, Millipore) ) And applied to a 50 ml heparin-Sepharose CL-6B column (2.6 × 10 cm, Pharmacia) equilibrated with 10 mM Tris-HCl, pH 7.5. After washing with 10 mM Tris-HCl containing 0.1% CHAPS, pH 7.5, elution was performed at a linear gradient of 2 M NaCl in 100 minutes, at a flow rate of 4 ml / min, and fractionation was performed at 8 ml / fraction. . Using 150 μl of each fraction, the OCIF activity was measured according to the method of Example 2 to obtain an OCIF active fraction eluted with about 0.6 to 1.2 M NaCl # 112 ml.
[0056]
The obtained OCIF active fraction (112 ml) was diluted to 1000 ml with {10 mM Tris-HCl, pH 7.5 containing 0.1% CHAPS, and then equilibrated with {10 mM Tris-HCl, pH 7.5 containing 0.1% CHAPS. The column was applied to the affinity column (Heparin II-5PW, 0.8 × 7.5 cm, Tosoh). After washing with {10 mM} Tris-HCl containing 0.1% CHAPS, {pH 7.5}, elution was performed at a linear gradient of 2 M NaCl in 60 minutes, at a flow rate of 0.5 ml / min, and at 0.5 ml / fraction. A fraction was taken.
[0057]
Using 4 μl of each of the obtained fractions, SDS-polyacrylamide gel electrophoresis was performed under reducing and non-reducing conditions according to the method of Example 4. As a result, only the OCIF bands of about 60 kD under the reducing conditions and about 60 kD and about 120 kD under the non-reducing conditions were detected in the fractions 30 to 32. Therefore, the fractions 30 to 32 were collected and purified to obtain the purified 293 / EBNA cell-derived recombinant type. OCIF ({rOCIF (E))} fraction. As a result of protein quantification by the Lowry method using BSA as a standard, it was revealed that 1.5 ml of 535 μg / ml rOCIF (E) was obtained.
[0058]
Embodiment 14
CHO Recombinant with cells OCIF Production of
i) 　 OCIF Of expression plasmid
Plasmid pBKOCIF さ れ into which about 1.6 kb OCIF cDNA obtained in Example 11 was inserted was digested with restriction enzymes SalI and EcoRV 、, an about 1.4 kb OCIF cDNA fragment was cut out, separated by agarose electrophoresis, and then QIAEX gel extraction. Purification was performed using a kit (Qiagen). In addition, the expression vector pcDL-SR {α296} (Molecular and Cellular Biology, {Vol. 8, {pp466-472, {1988})} is digested with restriction enzymes PstI and KpnI, and an expression vector DNA fragment of about 3.4 kb is separated by agarose electrophoresis. And QIAEX gel extraction kit (Qiagen). The ends of these purified OCIF cDNA fragments and expression vector DNA fragments were blunt-ended using a DNA branding kit (Takara Shuzo). Next, Ligation Kit @ Ver. 2 (Takara Shuzo), the OCIF cDNA fragment was inserted into the blunted expression vector DNA fragment, and Escherichia coli DH5α (Gibco BRL) was transformed to obtain a transformant having the OCIF expression plasmid pSR αOCIF. .
[0059]
ii) Preparation of expression plasmid
The transformant having the OCIF expression plasmid pSR αOCIF obtained in Example 13-i) and the transformant having the mouse DHFR gene expression plasmid pBAdDSV disclosed in WO92 / 01053 were grown by a conventional method, respectively. According to the method of Maniatis et al. (Molecular cloning, 2nd edition), the mixture was treated with an alkali method and a polyethylene glycol method, and purified by a cesium chloride density gradient centrifugation method.
[0060]
iii) 　 CHOdhFr ⁻ Adaptation of cells to protein-free media
CHOdhFr subcultured in IMDM medium (Gibco BRL) containing 10% fetal calf serum (Gibco BRL)⁻The cells (ATCC-CRL9096) were conditioned with a serum-free medium EX-CELL301 (JRH Bioscience) and further conditioned with a protein-free medium EX-CELL PF CHO (JRH Bioscience).
[0061]
iv) OCIF An expression plasmid and DHFR Expression plasmid CHOdhFr ⁻ Transfection into cells
CHOdhFr prepared in Example 14-iii) using the OCIF expression plasmid {pSRαOCIF and DHFR expression plasmid pBAdDSV} prepared in Example 14-ii)⁻Cells were transformed by the electroporation method described below. 200 μg of the pSR {αOCIF plasmid} and 20 μg of the pBAdDSV plasmid are aseptically dissolved in 0.8 ml of IMDM medium (Gibco BRL) containing 10% fetal calf serum (Gibco BRL), and then used in {2 × 10⁷CHOdhFr⁻The cells were suspended. The cell suspension was placed in a cuvette (Bio-Rad) and transformed by electroporation using Gene Pulser (Bio-Rad) at 360 V and 960 μF. The electroporated cell suspension was transferred to a T-flask (Sumitomo Bakelite Co., Ltd.) for suspension cells containing 10 ml of EX-CELL @ PF @ CHO medium.₂The cells were cultured in an incubator for 2 days. The cells were spread on a 96-well microplate at a concentration of 5000 cells / well using EX-CELL / PF / CHO medium, and cultured for about 2 weeks. The EX-CELL {PF} CHO medium does not contain nucleic acids and contains the parental CHOdhFr⁻Cannot grow, so only cell lines expressing DHFR are selected. Most of the cell lines that express DHFR express OCIF because the OCIF expression plasmid is used at 10 times the amount of the DHFR expression plasmid. From the obtained DHFR-expressing cell line, a cell line having a high OCIF activity in the culture supernatant was screened by the measurement method described in Example 2. The obtained OCIF high-producing strain was cloned by limiting dilution using the EX-CELL @ PF @ CHO medium, and the obtained clone was screened for a cell line having a high OCIF activity in the culture supernatant, and the OCIF high-producing strain was screened. Clone 5561 was obtained.
[0062]
v) Recombination type OCIF Production of
To produce recombinant OCIF (rOCIF), 1 × 10 6 transformed CHO cells (5561) were placed in 3 l of EX-CELL {301} medium.⁵cells / ml, and cultured at 37 ° C. for 4 or 5 days using a spinner flask. Cell concentration of about 1 × 10⁶When the cell concentration became cells / ml, about 2.7 l of the medium was recovered. About 2.7 l of EX-CELL {301} medium was added and the culture was repeated. Using three spinner flasks, about 20 l of culture was collected.
[0063]
vi) CHO Cell-derived recombinant OCIF Purification
CHAPS was added to 1 liter of the culture solution obtained in Example 14- (v) at a concentration of 0.1%, and the mixture was filtered through a 0.22 μm filter (Sterivex GS, Millipore), and then 10 mM Tris-HCl, The mixture was applied to a 50 ml heparin-Sepharose FF column (2.6 × 10 cm, Pharmacia) equilibrated at pH 7.5. After washing with 10 mM {Tris-HCl, pH 7.5} containing 0.1% CHAPS, elution is carried out at a linear gradient of 2 M in 2 min over 100 minutes, at a flow rate of 4 ml / min, and fractionation is performed at 8 ml / fraction. Was. Using each fraction {150 μl}, the OCIF activity was measured according to the method of Example 2 to obtain an OCIF active fraction eluted at about {0.6-1.2 M}, {112 ml}.
[0064]
The obtained OCIF active fraction (112 ml) was diluted to 1200 ml with {10 mM Tris-HCl, pH 7.5 containing 0.1% CHAPS, and then equilibrated with {10 mM Tris-HCl, pH 7.5 containing 0.1% CHAPS. On an affinity column (Blue # -5 PW, {0.5 x 5 cm}, Tosoh Corporation). After washing with {10 mM Tris-HCl, pH 7.5} containing 0.1% CHAPS, elution was performed at a linear gradient of 3 M NaCl for 90 minutes, at a flow rate of 0.5 ml / min, and 0.5 ml / fraction. A fraction was taken.
[0065]
Using 4 μl of each of the obtained fractions, SDS-polyacrylamide gel electrophoresis was performed under reducing and non-reducing conditions according to the method of Example 4. As a result, only the OCIF bands of about 60 kD under the reducing conditions and about 60 kD and about 120 kD under the non-reducing conditions were detected in the fractions 30 to 38. Therefore, the fractions 30 to 38 were collected and the recombinant OCIF derived from purified CHO cells [ rOCIF (C)] fraction. As a result of protein quantification by the Lowry method using BSA as a standard, it was found that 4.5 ml of 113 μg / ml rOCIF (C) was obtained.
[0066]
Embodiment 15
Recombination type OCIF Analysis of N-terminal structure of
3 μg of purified rOCIF (E) and rOCIF (C) were immobilized on a polyvinylidene difluoride (PVDF) membrane using ProSpin (ProSpin, PerkinElmer), washed with 20% methanol, and then washed with a protein sequencer (Procise, N-terminal amino acid sequence analysis was performed using 492 ° type (Perkin Elmer). The results are shown in SEQ ID NO: 7 in the Sequence Listing.
The N-terminal amino acids of rOCIF (E) and {rOCIF (C)} are the translation start point of the amino acid sequence described in SEQ ID NO: 5, {Glu 22nd from Met, and 21 amino acids from Met} to {Gln are signal peptides. Was revealed. The N-terminal amino acid sequence of the native OCIF purified from the IMR-90 culture solution could not be analyzed because the N-terminal Glu was converted to pyroglutamic acid during culture or during purification. .
[0067]
Embodiment 16
Recombination type (R) OCIF And natural type (N) OCIF Biological activity of
i) Vitamin in mouse bone marrow cell line D ₃ Of osteoclast formation induced by osteoblasts
2x10 in 96-well microplate^{- 8}M active vitamin D₃Then, purified rOCIF (E) and {nOCIF {100 μl}, which were serially diluted by a factor of two from 250 ng / ml in α-MEM medium (Gibco BRL) containing 10% fetal calf serum, were added. In this well, 3 × 10 mouse bone marrow cells, about 17 days old, were added.⁵The cells were suspended and seeded in {100 μl} of α-MEM medium containing 10% fetal bovine serum, and 5%₂And cultured at 37 ° C. and 100% humidity for one week. Seven days after the culturing, osteoclast formation was detected by staining using an acid phosphatase activity measurement kit (Acid {Phosphatase, Leucocyte}, catalog No. 387-A, Sigma) according to the method of Example 2. The decrease in cells positive for acid phosphatase activity in the presence of tartaric acid was defined as OCIF activity. The reduction rate of the acid phosphatase activity-positive cells was calculated by solubilizing the dye of the stained cells and measuring the absorbance. That is, a 0.1 N sodium hydroxide-dimethyl sulfoxide mixed solution (1: 1) {100 μl} was added to each well in which the cells were fixed and stained, and the mixture was shaken well. After the dye was sufficiently dissolved, the absorbance was measured at a measurement wavelength of 590 nm and a control wavelength of 490 nm using a microplate reader (Immunoreader NJ-2000, Intermed). Vitamin D was used as a blank well when measuring absorbance.₃Unadded wells were used. The results are shown in Table 5 as percentage values, where the absorbance value in the wells without OCIF was defined as $ 100.
Similar to nOCIF, rOCIF (E) showed a dose-dependent osteoclast formation inhibitory activity at a concentration of 16 ng / ml or more.
[0068]
[Table 5]

[0069]
ii) Vitamin in co-culture system of stromal cells and mouse spleen cells D ₃ Of osteoclast formation induced by osteoblasts
Vitamin D₃The test for the formation of osteoclasts in a co-culture system of stromal cells and mouse spleen cells induced by the above was performed according to the method of Udagawa et al. (Endocrinology, Vol. 125, {p 1805-1813, 1989)}. That is, 2 × 10^{- 8}M active vitamin D₃, 2 × 10^{- 7}And 100 μl of purified {rOCIF (E), {rOCIF (C)} and nOCIF serially diluted in α-MEM medium (Gibco BRL) containing dexamethasone and 10% fetal calf serum. In this well, mouse bone marrow-derived stromal cell line ST2 cells (RIKEN Cell Bank-RCB0224) 5 × 10³Ddy mouse spleen cells 1 × 10 cells and 8 weeks old⁵The cells were suspended and seeded in {100 μl} of α-MEM medium containing 10% fetal bovine serum, and 5% CO 2₂The cells were cultured at 37 ° C. and 100% humidity for 5 days. After 5 days of culture, the cells were washed with a phosphate-buffered saline, and the cells were fixed with an ethanol / acetone (1: 1) solution at room temperature for 1 minute. Leucocyte, catalog No. 387-A, Sigma). The decrease in cells positive for acid phosphatase activity in the presence of tartaric acid was defined as OCIF activity. The rate of decrease in the number of cells positive for acid phosphatase activity was calculated by dissolving the dye of the stained cells according to the method described in Example 16-i). Table 6 shows the results of tests using rOCIF (E) and rOCIF (C), and Table 7 shows the results of tests using rOCIF (E) and nOCIFＯ.
Similar to nOCIF, rOCIF (E) and rOCIF (C) also showed a dose-dependent osteoclast formation inhibitory activity at a concentration of 6 to 16 mg / ml or more.
[0070]
[Table 6]

[0071]

[0072]
iii) 　 PTH Of osteoclast formation induced by osteoblasts
The test of osteoclast formation induced by PTH was performed according to the method of Takahashi et al. (Endocrinology, Vol. 122, p1373-1382, 1988). That is, 2 × 10^{- 8}In an α-MEM medium (Gibco) containing MPTH and 10% fetal bovine serum, nOCIF serially diluted from 125 ng / ml and purified rOCIF (E) {100 μl} were added. In this well, 3 × 10 mouse bone marrow cells, about 17 days old, were added.⁵The cells were suspended and seeded in 100 μl of α-MEM medium containing 10% fetal bovine serum, and 5% CO₂The cells were cultured at 37 ° C. and 100% humidity for 5 days. After 5 days of culture, the cells were washed with a phosphate-buffered saline, fixed with an ethanol / acetone (1: 1) solution at room temperature for 1 minute, and the formation of osteoclasts was measured using an acid phosphatase activity assay kit (Acid @ Phosphatase, @ Leucocyte). , Catalog No. 387-A, Sigma). The decrease in cells positive for acid phosphatase activity in the presence of tartaric acid was defined as OCIF activity. The rate of decrease in the number of cells positive for acid phosphatase activity was calculated by dissolving the dye of the stained cells according to the method described in Example 16-i). Table 8 shows the results.
Similar to nOCIF, rOCIF (E) also exhibited a dose-dependent osteoclast formation inhibitory activity at a concentration of 16 ng / ml or more.
[0073]
[Table 8]

[0074]
iv) IL-11 Of osteoclast formation induced by osteoblasts
The test for osteoclast formation induced by IL-11 was performed according to the method of Tamura et al. (Proc. Natl. Acad. Sci. USA, Vol. 90, p11924-11928, 1993). That is, a 96-well microplate was charged with {nOCIF} diluted with an α-MEM medium (manufactured by Gibco BRL) containing 20 ng / ml {IL-11 and 10% fetal bovine serum} and purified rOCIF (E) {100 μl}. In this well, a mouse neonatal skull-derived preadipocyte cell line {MC3T3-G2 / PA6} cells (RIKEN Cell Bank-RCB1127) 5 x 10³Ddy mouse spleen cells 1 × 10 cells and 8 weeks old⁵The cells were inoculated and suspended in μ100 μl of α-MEM medium containing 10% fetal bovine serum, and 5% CO₂The cells were cultured at 37 ° C. and 100% humidity for 5 days. After 5 days of culture, the cells were washed with phosphate-buffered saline, and fixed with an ethanol / acetone (1: 1) solution at room temperature for 1 minute. , Catalog No. 387-A, Sigma). The number of cells positive for acid phosphatase activity in the presence of tartaric acid was counted, and the decrease was defined as OCIF activity. Table 9 shows the results.
In both nOCIF and rOCIF (E), the activity of inhibiting osteoclast formation induced by IL-11 in a concentration-dependent manner at a concentration of 2 ng / ml or more was observed.
[0075]
[Table 9]

[0076]
Thus, in a test system for osteoclast formation using various target cells, OCIF is used for vitamin D.₃It has been revealed that the formation of osteoclasts by osteoclast formation-inducing factors such as IL, PTH, and IL-11 is suppressed at almost the same concentration. Therefore, it was suggested that OCIF may be effectively used for treating different types of bone loss induced by various bone resorption promoting substances.
[0077]
Embodiment 17
Monomer type and dimer type OCIF Sample preparation
A reverse phase equilibrated with 30% acetonitrile containing 0.1% TFA after adding 1/100 volume of 25% TFA (trifluoroacetic acid) to a sample containing rOCIF (E) and rOCIF (C) each containing 100 μg each The mixture was applied to a column (PROTEIN-RP, 2.0 × 250 mm, YMC) and eluted with a linear gradient of 55% acetonitrile in 50 minutes at a flow rate of 0.2 ml / min, and each OCIF peak was collected. The obtained peak fraction was lyophilized to obtain monomeric OCIF and dimer OCIF.
[0078]
Embodiment 18
Recombination type OCIF Molecular weight measurement
Example 3-vi) A sample containing monomer-type and dimer-type nOCIF purified using the reverse phase column according to the method of and about 1 μg of the monomer-type and dimer-type rOCIF purified by the method described in Example 17 was concentrated under reduced pressure. These samples were subjected to SDS treatment, SDS-polyacrylamide electrophoresis, and silver staining according to the method of Example 4. The results of electrophoresis under non-reducing and reducing conditions are shown in FIGS. 6 and 7, respectively.
[0079]
As a result, under the non-reducing condition, a protein band of 60 kD was detected in any of the monomer-type samples, and a protein band of 120 kD was detected in any of the dimer-type samples. Further, under the reducing conditions, only a protein band of about 60 kD was detected in each sample. Therefore, it was shown that the molecular weights of the monomer type and the dimer type of ΔnOCIF derived from IMR-90 cells, recombinant OCIF derived from 293 / EBNA cells, and recombinant OCIF derived from CHO cells were almost the same.
[0080]
Embodiment 19
IMR- 90 Cell-derived natural type OCIF And recombination type OCIF Of N-linked sugar chains and measurement of molecular weight
Example 3-vi) A sample containing about 5 μg of each of the monomer-type and dimer-type nOCIF purified using the reverse phase column according to the method of and the monomer-type and dimer-type rOCIF purified by the method described in Example 17 was concentrated under reduced pressure. . To these samples, 100 mM {50 mM phosphate buffer containing 2-mercaptoethanol, pH 8.6 {9.5 μl} was added and dissolved, and then 250 U / ml {N-glycanase solution (Seikagaku Corporation) 0.5 μl} was added. The mixture was left at 37 ° C. for one day. To these samples was added {20 mM {Tris-HCl, pH 8.0, {10 μl} containing 2 mM {MEDTA, 5% SDS, and 0.02% bromophenol blue}, and heated at 100 ° C. for 5 minutes. One microliter of each of these samples was subjected to SDS-polyacrylamide electrophoresis by the method of Example 4 and then stained with silver. FIG. 8 shows the results.
[0081]
As a result, it was shown that the molecular weight of the OCIF protein from which N-linked sugar chains had been removed by N-glycanase treatment under reducing conditions was about 40 kD. Since the molecular weights of nOCIF derived from IMR-90 cells, rOCIF derived from 293 / EBNA cells, and rOCIF derived from CHO cells, which have not been subjected to sugar chain removal treatment, are all about 60 kD under these reducing conditions, these OCIF Is a glycoprotein containing an N-linked sugar chain in its molecule.
[0082]
Embodiment 20
OCIF Analogs (variants) cDNA Cloning and nucleotide sequence determination
As shown in Examples 10 and 11, from one of several purified positive phages, a transformant having plasmid pBKOCIF in which OCIF cDNA was inserted into pBKCMV (Stratagene) was obtained. A transformant having a plasmid into which inserts of different lengths were inserted was obtained from several positive phages. Transformants having these plasmids were propagated, and the plasmids were purified by a conventional method. The base sequences of these insert DNAs were determined using Tack Dide Oxyterminator Cycle Sequencing Kit (Perkin Elmer). As primers used, T3 and T7 primers (Stratagene) and synthetic primers designed based on the nucleotide sequence of OCIF cDNA were used. In addition to the original type OCIF, there were a total of four types of OCIF variants (OCIF2, {3, {4, $ 5}}). The nucleotide sequence of the determined OCIF2 cDNA is shown in SEQ ID NO: 8, and the amino acid sequence deduced from the sequence is shown in SEQ ID NO: 9. The nucleotide sequence of the determined OCIF3 cDNA is shown in SEQ ID NO: 10, and the amino acid sequence deduced from the sequence is shown in SEQ ID NO: 11. The nucleotide sequence of the determined OCIF4 cDNA is shown in SEQ ID NO: 12, and the amino acid sequence deduced from the sequence is shown in SEQ ID NO: 13. The nucleotide sequence of the determined OCIF5 cDNA is shown in SEQ ID NO: 14, and the amino acid sequence deduced from the sequence is shown in SEQ ID NO: 15. The structural features of these OCIF variants will be briefly described with reference to FIGS.
[0083]
OCIF 2
There is a 21 bp deletion from the guanine at position # 265 to the 285th position at 285 in the nucleotide sequence of OCIF cDNA (SEQ ID NO: 6), and the amino acid sequence is glutamic acid (Glu) at position 68 in the amino acid sequence of OCIF (SEQ ID NO: 5). There is a deletion of 7 amino acids from glutamine (Gln) to the 74th glutamine.
[0084]
OCIF 3
The ninth cytidine in the nucleotide sequence of the OCIF cDNA (SEQ ID NO: 6) is converted to guanine, and the amino acid sequence of the -19th asparagine (Asn) in the amino acid sequence of the OCIF (SEQ ID NO: 5 in the sequence listing) is lysine (Lys). Has changed to However, this is an amino acid substitution in the signal sequence and does not appear to affect secreted OCIF3.
There is a deletion of 117 bp from the guanine at position 872 to the 988th position of 988 in the nucleotide sequence of the OCIF cDNA (SEQ ID NO: 6), and in the amino acid sequence, the threonine (Thr) at the 270th position of the amino acid sequence of OCIF (SEQ ID NO: 5 in the Sequence Listing). There is a deletion of 39 amino acids from the 308th leucine (Leu) to the 308th leucine (Leu).
[0085]
OCIF 4
The ninth cytidine in the nucleotide sequence of the OCIF cDNA (SEQ ID NO: 6) is converted to guanine, and the amino acid sequence of the -19th asparagine (Asn) in the amino acid sequence of the OCIF (SEQ ID NO: 5 in the sequence listing) is lysine (Lys). Has changed to In addition, the guanine at position 22 has been converted to thymidine, and in the amino acid sequence, the alanine (Ala) at position -14 in the amino acid sequence of OCIF (SEQ ID NO: 5 in the Sequence Listing) has been changed to serine (Ser). However, these are amino acid substitutions in the signal sequence and do not appear to affect secreted OCIF4.
There is an insertion of about $ 4 kb of intron 2 between the # 400 and the # 401 positions of the base sequence of the OCIF cDNA (SEQ ID NO: 6), and the open reeling frame stops therein. In the amino acid sequence, a novel amino acid sequence consisting of 21 amino acids is added after alanine (Ala) at position # 112 in the amino acid sequence of OCIF (SEQ ID NO: 5 in the Sequence Listing).
[0086]
OCIF 5
The ninth cytidine in the nucleotide sequence of the OCIF cDNA (SEQ ID NO: 6) is converted to guanine, and the amino acid sequence of the -19th asparagine (Asn) in the amino acid sequence of the OCIF (SEQ ID NO: 5 in the sequence listing) is lysine (Lys). Has changed to However, this is an amino acid substitution in the signal sequence and does not appear to affect secreted OCIF5.
There is an insertion of the latter half of intron 2 of about 1.8 kb between the 400th and 401st positions of the nucleotide sequence of the OCIF cDNA (SEQ ID NO: 6), and the open reeling frame stops therein. In the amino acid sequence, a novel amino acid sequence consisting of 12 amino acids is added after alanine (Ala) at position # 112 in the amino acid sequence of OCIF (SEQ ID NO: 5 in the Sequence Listing).
[0087]
Embodiment 21
OCIF Production of analogs (variants)
i) 　 OCIF variant cDNA Of expression plasmid
Of the OCIF variant cDNAs obtained in Example 20, plasmids pBKOCIF2 and pBKOCIF3 into which the OCIF {2} and {3} cDNAs were respectively inserted were digested with restriction enzymes XhoI and BamHI (Takara Shuzo), and the OCIF {2 and 3} cDNAs were respectively digested. The DNA was excised, separated by agarose electrophoresis, and then purified using QIAEXＥ gel extraction kit (Qiagen). These cDNAs of OCIF {2 and 3} were ligated to the expression plasmid pCEP4 (Invitrogen), which had been digested with restriction enzymes XhoI and BamHI (Takara Shuzo) in advance, and ligation kit {Ver. 2 (Takara Shuzo) was used to transform Escherichia coli {DH5α (Gibco BRL)].
Also, of the OCIF variant cDNA obtained in Example 20, plasmid pBKOCIF4 into which the OCIF4 cDNA was inserted was digested with restriction enzymes SpeI and XhoI (Takara Shuzo), separated by agarose electrophoresis, and then QIAEXＥ gel extraction. Purification was performed using a kit (Qiagen). The cDNA of OCIF4 was ligated to the expression plasmid pCEP4 (Invitrogen), which had been digested with restriction enzymes NheI and XhoI (Takara Shuzo) in advance, and ligation kit {Ver. 2 (Takara Shuzo) to transform E. coli DH5α (Gibco BRL).
[0088]
Also, of the OCIF variant cDNA obtained in Example 20, the plasmid pBKOCIF5 into which the cDNA of OCIF5 was inserted was digested with the restriction enzyme Hind III (Takara Shuzo) to cut out the 5 ′ region of the coding region of OCIF5cDNA, and the agarose was removed. After separation by electrophoresis, purification was carried out using QIAEXＥ gel extraction kit (Qiagen). The OCIF expression plasmid pCEPOCIF obtained in Example 13-i) was digested with a restriction enzyme Hind III (Takara Shuzo) to remove the 5 'region of the coding region of the OCIF cDNA, and to contain the pCEP plasmid and the 3' region of the OCIF cDNA. The fragment pCEPOCIF-3 'was separated by agarose electrophoresis, and then purified using QIAEX gel extraction kit (Qiagen). The Hind III fragment of the {OCIF5 cDNA} was ligated to the ligation kit {Ver. 2 (Takara Shuzo) was used to transform Escherichia coli DH5α (Gibco BRL).
The obtained transformant was grown and the expression plasmid pCEPOCIF {2, {3,4,5} in which cDNAs of OCIF2, 3,4,5 were inserted was purified using Qiagen column (Qiagen). After the OCIF variant expression plasmid was precipitated with ethanol, it was dissolved in sterile distilled water and used for the following procedure.
[0089]
ii) OCIF variant cDNA Of Transient Expression and Its Activity
Using the OCIF variant expression plasmid pCEPOCIF {2, {3, {4, {5}) obtained in Example 21-i), the OCIF variants are transiently expressed by the method described in Example 13-ii), and their activities are determined. Examined. As a result, weak activity was observed in these OCIF variants.
[0090]
Embodiment 22
OCIF Creating mutants
i) 　 OCIF Mutant cDNA Construction of plasmid vector for subcloning
5 μg of the plasmid vector described in Example 11 was digested with restriction enzymes BamHI and XhoI (Takara Shuzo). The cut DNA was subjected to preparative agarose gel electrophoresis. A DNA fragment of about 1.6 kilobase pairs (kb) containing the full length of the OCIF cDNA was isolated and purified by QIAEX gel extraction kit (Qiagen) to obtain a DNA solution 1 dissolved in 20 μl of sterile distilled water. Next, pBluescript @ IISK⁺(Stratagene) 3 μg was digested with restriction enzymes BamHI and XhoI (Takara Shuzo). The cut DNA was subjected to preparative agarose gel electrophoresis. A DNA fragment of about 3.0 kb was isolated and purified by QIAEX gel extraction kit (Qiagen) to obtain a DNA solution 2 dissolved in 20 μl of sterile distilled water. 1 μl of DNA solution 2 and 4 μl of DNA solution 1 were mixed, and 5 μl of DNA ligation kit ver. After adding and mixing 2 I solution (Takara Shuzo), the mixture was kept at 16 ° C. for 30 minutes to carry out a ligation reaction. In addition, the following ligation reactions were all carried out under the condition of keeping the temperature at 16 ° C. for 30 minutes.
[0091]
Using this ligation reaction solution, Escherichia coli was transformed under the following conditions. The transformation of Escherichia coli was performed under the following conditions. 5 µl of this ligation reaction solution and 100 µl of Escherichia coli DH5α competent cells (Gibco BRL) were mixed in a sterile 15 ml tube (Iwaki Glass Co., Ltd.), and allowed to stand in ice water for 30 minutes. After incubating at 42 ° C. for 45 seconds, 250 μl of L medium (1% tryptone, 0.5% yeast extract, 1% NaCl) was added, and the mixture was cultured at 37 ° C. with stirring. 50 μl of the bacterial solution was spread on 2 ml of L agar medium containing 50 μg / ml ampicillin. After culturing overnight at 37 ° C., the six growing colonies were further cultured overnight in 2 ml of L ampicillin liquid medium, and the structure of the plasmid of each strain was examined. pBluescript IISK⁺Plasmid having a structure in which a DNA fragment of about 1.6 kb including the full length of OCIF cDNA is inserted into the XhoI cleavage site of BamHI (hereinafter referred to as “pSK”).⁺-OCIF).
[0092]
ii) Preparation of mutant in which Cys is replaced with Ser
(1) Mutation introduction
In the amino acid sequence described in SEQ ID NO: 4 in the Sequence Listing, a mutant was prepared in which the Cys residues at positions 174, 181, 256, 298, and 379 were replaced with Ser residues. The mutant obtained by replacing 174Cys with Ser is OCIF-C19S, the mutant obtained by replacing 181Cys with Ser 置換 is OCIF-C20S, the mutant obtained by replacing 256Cys by {Ser} is OCIF-C21S, and the mutant obtained by replacing 298Cys by Ser is OCIF-C. Mutants obtained by substituting C22S７９ and 379Cys with Ser were named OCIF-C23S2, respectively. First, the nucleotide sequence encoding each Cys 塩 基 residue was replaced with a nucleotide sequence encoding a Ser residue. Mutagenesis was performed by two-step PCR (polymerase {chain} reaction). Hereinafter, it is referred to as a two-step PCR reaction. The first stage consists of two PCR reactions (PCR1 and PCR2).
[0093]

[0094]
PCR2 Reaction liquid

[0095]
At the time of each mutation introduction, only the type of primer was changed, and the other reaction compositions were the same. The primers used in each reaction are shown in Table 10, and their sequences are shown in SEQ ID NOs: 20, 23, 27, and 30 to 40 in the Sequence Listing. The PCR1 reaction solution and the PCR2 reaction solution were placed in separate microcentrifuge tubes and mixed, and then PCR was performed under the following conditions. After treatment at 97 ° C. for 3 minutes, a three-step reaction of 95 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 3 minutes was repeated 25 times, and the temperature was kept at 70 ° C. for 5 minutes. A part of the reaction solution was subjected to agarose electrophoresis, and it was confirmed that a DNA fragment of a desired length was synthesized. After the completion of the first-step PCR reaction, the primer was removed from the reaction solution using Amicon Microcon (Amicon), the final volume was adjusted to 50 μl with sterile distilled water, and the second-step PCR reaction was performed using the obtained DNA fragment. (PCR3) was performed.
[0096]

[0097]
[Table 10]

[0098]
After the above solution was placed in a microcentrifuge tube and mixed, PCR was performed under the same conditions as PCR1 and PCR2. A part of the reaction solution was subjected to agarose (1% or 1.5%) electrophoresis to confirm that a DNA fragment of a desired length was synthesized. The DNA obtained by PCR was precipitated with ethanol, dried in vacuum and dissolved in 40 μl of sterile distilled water. Solution A containing the C19S mutant DNA fragment, Solution B containing the C20S mutant DNA fragment, Solution C containing the C21S mutant DNA fragment, Solution D containing the C22S mutant DNA fragment, and containing the C23S mutant DNA fragment The solution was named solution E.
[0099]
The DNA fragment in 20 μl of solution A was digested with restriction enzymes NdeI and SphI (Takara Shuzo). A DNA fragment of about 400 bp was separated and purified by preparative electrophoresis, and dissolved in 20 μl of distilled water (DNA solution 3). Next, 2 μg pSK⁺-OCIF was digested with restriction enzymes NdeI and SphI (Takara Shuzo), a DNA fragment of about 4.2 kb was separated and purified by preparative electrophoresis, and dissolved in 20 μl of sterile distilled water (DNA solution 4). 2 μl of DNA solution 3 and 3 μl of DNA solution 4 were mixed, and the DNA ligation kit ver. A ligation reaction was performed by adding 2 μl of I solution. Escherichia coli DH5α was transformed using 5 μl of the ligation solution after the reaction. From the obtained ampicillin-resistant transformed cells, a strain having the target plasmid DNA was selected by DNA structure analysis. The DNA structure was analyzed by measuring the length of the fragment obtained by restriction enzyme digestion and determining the nucleotide sequence. The obtained target plasmid DNA was named pSK-OCIF-C19S.
[0100]
The C20S mutant DNA fragment in 20 μl of solution B was digested with restriction enzymes NdeI and SphI (Takara Shuzo). A DNA fragment of about 400 bp was separated and purified by preparative electrophoresis, and dissolved in 20 μl of distilled water (DNA solution 5). 2 μl of the DNA solution 5 and 3 μl of the DNA solution 4 were mixed, and the DNA ligation kit ver. A ligation reaction was performed by adding 2 μl of I solution. Escherichia coli DH5α was transformed using 5 μl of the ligation solution after the reaction. From the obtained ampicillin-resistant transformed cells, a strain having the target plasmid DNA was selected by DNA structure analysis. The DNA structure was analyzed by measuring the length of the fragment obtained by restriction enzyme digestion and determining the nucleotide sequence. The obtained target plasmid DNA was named pSK-OCIF-C20S.
[0101]
The DNA fragment in 20 μl of solution C was digested with restriction enzymes NdeI and SphI (Takara Shuzo). A DNA fragment of about 400 bp was separated and purified by preparative electrophoresis and dissolved in 20 μl of distilled water (DNA solution 6). 2 μl of DNA solution 6 and 3 μl of DNA solution 4 were mixed, and the DNA ligation kit ver. A ligation reaction was performed by adding 2 μl of I solution. Escherichia coli DH5α was transformed using 5 μl of the ligation solution after the reaction. From the obtained ampicillin-resistant transformed cells, a strain having the target plasmid DNA was selected by DNA structure analysis. The DNA structure was analyzed by measuring the length of the fragment obtained by restriction enzyme digestion and determining the nucleotide sequence. The obtained target plasmid DNA was named pSK-OCIF-C21S.
[0102]
The DNA fragment in 20 μl of solution D was digested with restriction enzymes NdeI and BstPI (Takara Shuzo). A DNA fragment of about 600 bp was separated and purified by preparative electrophoresis and dissolved in 20 μl of distilled water (DNA solution 7). Next, 2 μg of pSK⁺-OCIF was digested with restriction enzymes NdeI and BstPI (Takara Shuzo), a DNA fragment of about 4.0 kb was separated and purified by preparative electrophoresis, and dissolved in 20 μl of distilled water (DNA solution 8). 2 μl of the DNA solution 7 and 3 μl of the DNA solution 8 were mixed, and the DNA ligation kit ver. A ligation reaction was performed by adding 2 μl of I solution. Escherichia coli DH5α was transformed using 5 μl of the ligation solution after the reaction. From the obtained ampicillin-resistant transformed cells, a strain having the target plasmid DNA was selected by DNA structure analysis. The DNA structure was analyzed by measuring the length of the fragment obtained by restriction enzyme digestion and determining the nucleotide sequence. The obtained target plasmid DNA was designated as pSK-OCIF-C22S.
[0103]
The DNA fragment in 20 μl of solution E was digested with restriction enzymes BstPI and EcoRV (Takara Shuzo). A DNA fragment of about 120 bp was separated and purified by preparative electrophoresis and dissolved in 20 μl of sterile distilled water (DNA solution 9). Next, 2 μg pSK⁺-OCIF was digested with restriction enzymes BstEII and EcoRV (Takara Shuzo), a DNA fragment of about 4.5 kb was separated and purified by preparative electrophoresis, and dissolved in 20 μ of distilled water (DNA solution 10). 2 μl of the DNA solution 9 and 3 μl of the DNA solution 10 were mixed, and the DNA ligation kit ver. A ligation reaction was performed by adding 2 μl of I solution. Escherichia coli DH5α was transformed using 5 μl of the ligation solution after the reaction. From the obtained ampicillin-resistant transformed cells, a strain having the target plasmid DNA was selected by DNA structure analysis. The DNA structure was analyzed by measuring the length of the fragment obtained by restriction enzyme digestion and determining the nucleotide sequence. The obtained target plasmid DNA was named pSK-OCIF-C23S.
[0104]
(2) Construction of mutant expression vector
The obtained target plasmid DNA (pSK-OCIF-C19S, {pSK-OCIF-C20S} pSK-OCIF-C21S, pSK-OCIF-C22S, pSK-OCIF-C23S) was cut with restriction enzymes BamHI and XhoI (Takara Shuzo). , A DNA fragment (including the target mutation) of about 1.6 kb including the full length of OCIF cDNA was separated and purified, and dissolved in 20 µl of sterile distilled water. These were named C19S DNA solution, C20S DNA solution, C21S DNA solution, C22S DNA solution, and C23S DNA solution. Next, 5 μg of the expression vector pCEP4 (Invitrogen) was cut with restriction enzymes BamHI and XhoI (Takara Shuzo), and about 10 kb of DNA was separated and purified, and dissolved in 40 μl of sterile distilled water (pCEP4 DNA solution). 1 μl of pCEP4 DNA solution and 6 μl of each of C19S DNA solution, C20S DNA solution, C21S DNA solution, C22S DNA solution and C23S DNA solution were separately mixed, and 7 μl of DNA ligation kit {Ver. A 2 I solution was added, and a ligation reaction was performed. After completion of the reaction, 100 ml of Escherichia coli DH5α competent cell solution was transformed using 7 μl of the reaction solution. From the obtained ampicillin-resistant transformed cells, a total of five strains having a plasmid DNA of the desired structure in which each DNA fragment of about 1.6 kb was inserted into the XhoI and BamHI sites of pCEP4 were selected, and pCEP4-OCIF was selected. -C19S, {pCEP4-OCIF-C20S, pCEP4-OCIF-C21S, pCEP4-OCIF-C22S, pCEP4-OCIF-C23S}.
[0105]
ii) Generation of domain deletion mutants
(1) Introduction of domain deletion mutation
Among the amino acids set forth in SEQ ID NO: 4, from Tyr # 2 to Ala # 42, from Pro # 43 to Cys # 84, from Glu # 85 to Lys # 122, from Arg # 123 to {164} Mutants were prepared in which Asp # at 177, Gln at # 251, Hele at # 253, and His at # 326 # were deleted. A mutant deleted from Thr # 2 to Ala at position 42 was deleted from OCIF-DCR1, a mutant deleted from Pro # at position 43 to {Cys at position 84 was changed from OCIF-DCR2}, and a mutant deleted from position 85 to Glu # 122. The mutant deleted from No. Lys was deleted from OCIF-DCR3, and the mutant deleted from Arg 123rd to Cys 164 was deleted from OCIF-DCR4, Asp from 177 to Gln 251. The deleted mutant was named OCIF-DDD2, and the mutant deleted from OCIF-DDD1, 253rd Ile} to {326th His} was respectively named OCIF-DDD2}. The domain deletion mutation was also introduced by the two-step PCR method described in Example 22-ii) ｉ. The primers used in each mutagenesis reaction are shown in Table 11, and their sequences are shown in SEQ ID NOs: 19, 25, 40 to 53, and 54 in the Sequence Listing.
[0106]
[Table 11]

[0107]
The DNA obtained by PCR was precipitated with ethanol, dried in vacuum, and dissolved in 40 μl of sterile distilled water. Solution F containing the DCR1 mutant DNA fragment, Solution G containing the DCR2 mutant DNA fragment, Solution H containing the DCR3 mutant DNA fragment, Solution I containing the DCR4 mutant DNA fragment, containing DDD1 mutant DNA fragment The solution was designated as solution J, and the solution containing the DDD2 mutant DNA fragment was designated as solution K.
[0108]
The DNA fragment in 20 μl of solution F was digested with restriction enzymes NdeI and XhoI (Takara Shuzo). A DNA fragment of about 500 bp was separated and purified by preparative electrophoresis, and dissolved in 20 μl of sterile distilled water (DNA solution 11). Next, 2 μg pSK⁺-OCIF was digested with restriction enzymes NdeI and XhoI (Takara Shuzo), a DNA fragment of about 4.0 kb was separated and purified by preparative electrophoresis, and dissolved in 20 µl of sterile distilled water (DNA solution 12). 2 μl of the DNA solution 11 and 3 μl of the DNA solution 12 were mixed, and the DNA ligation kit ver. A ligation reaction was performed by adding 2 μl of I solution. Escherichia coli DH5α was transformed using 5 μl of the ligation solution after the reaction. From the obtained ampicillin-resistant transformed cells, a strain having a plasmid DNA in which a desired mutation was introduced into OCIF cDNA was selected by analyzing the DNA structure. The DNA structure was analyzed by measuring the length of the fragment obtained by restriction enzyme digestion and determining the nucleotide sequence. The obtained target plasmid DNA was named pSK-OCIF-DCR1.
[0109]
The DNA fragment in 20 μl of solution G was digested with restriction enzymes NdeI and XhoI (Takara Shuzo). A DNA fragment of about 500 bp was separated and purified by preparative electrophoresis, and dissolved in 20 μl of sterile distilled water (DNA solution 13). 2 μl of the DNA solution 13 and 3 μl of the DNA solution 12 were mixed, and the DNA ligation kit ver. 5 µl of 2 I solution was added, and a ligation reaction was performed. Escherichia coli DH5α was transformed using 5 μl of the ligation solution after the reaction. From the obtained ampicillin-resistant transformed cells, a strain having the target plasmid DNA was selected by DNA structure analysis. The DNA structure was analyzed by measuring the length of the fragment obtained by restriction enzyme digestion and determining the nucleotide sequence. The obtained target plasmid DNA was named pSK-OCIF-DCR2.
[0110]
The DNA fragment in 20 μl of solution H was digested with restriction enzymes NdeI and XhoI (Takara Shuzo). A DNA fragment of about 500 bp was separated and purified by preparative electrophoresis, and dissolved in 20 μl of sterile distilled water (DNA solution 14). 2 µl of the DNA solution 14 and 3 µl of the DNA solution 12 were mixed, and the DNA ligation kit ver. 5 µl of 2 I solution was added, and a ligation reaction was performed. Escherichia coli DH5α was transformed using 5 μl of the ligation solution after the reaction. From the obtained ampicillin-resistant transformed cells, a strain having a plasmid DNA in which a desired mutation was introduced into OCIF cDNA was selected by analyzing the DNA structure. The DNA structure was analyzed by measuring the length of the fragment obtained by restriction enzyme digestion and determining the nucleotide sequence. The obtained target plasmid DNA was named pSK-OCIF-DCR3.
[0111]
The DNA fragment in 20 μl of solution I was digested with restriction enzymes XhoI and SphI (Takara Shuzo). A DNA fragment of about 900 bp was separated and purified by preparative electrophoresis and dissolved in 20 μl of sterile distilled water (DNA solution 15). Next, 2 μg pSK⁺-OCIF was digested with restriction enzymes XhoI and SphI (Takara Shuzo), a DNA fragment of about 3.6 kb was separated and purified by preparative electrophoresis, and dissolved in 20 µl of sterile distilled water (DNA solution 16). 2 μl of the DNA solution 15 and 3 μl of the DNA solution 16 were mixed, and the DNA ligation kit ver. 2 {I solution 5 μl} was added, and a ligation reaction was performed. Escherichia coli DH5α was transformed using 5 μl of the ligation solution after the reaction. From the obtained ampicillin-resistant transformed cells, a strain having the target plasmid DNA was selected by DNA structure analysis. The DNA structure was analyzed by measuring the length of the fragment obtained by restriction enzyme digestion and determining the nucleotide sequence. The obtained target plasmid DNA was named pSK-OCIF-DCR4.
[0112]
The DNA fragment in 20 μl of solution J was digested with restriction enzymes BstPI and NdeI (Takara Shuzo). A DNA fragment of about $ 400 bp was separated and purified by preparative electrophoresis and dissolved in 20 μl of sterile distilled water (DNA solution 17). 2 μl of the DNA solution 17 and 3 μl of the DNA solution 8 were mixed, and the DNA ligation kit ver. 5 µl of 2 I solution was added, and a ligation reaction was performed. Escherichia coli DH5α was transformed using 5 μl of the ligation solution after the reaction. From the obtained ampicillin-resistant transformed cells, a strain having the target plasmid DNA was selected by DNA structure analysis. The DNA structure was analyzed by measuring the length of the fragment obtained by restriction enzyme digestion and determining the nucleotide sequence. The obtained target plasmid DNA was named pSK-OCIF-DDD1.
[0113]
The DNA fragment in 20 μl of solution K was digested with restriction enzymes BstPI and NdeI (Takara Shuzo). A DNA fragment of about 400 bp was separated and purified by preparative electrophoresis and dissolved in 20 μl of sterile distilled water (DNA solution 18). 2 μl of the DNA solution 18 and 3 μl of the DNA solution 8 were mixed, and the DNA ligation kit ver. 5 μl of 2 I solution was added to carry out a ligation reaction. Escherichia coli DH5α was transformed using 5 μl of the ligation solution after the reaction. From the obtained ampicillin-resistant transformed cells, a strain having the target plasmid DNA was selected by DNA structure analysis. The DNA structure was analyzed by measuring the length of the fragment obtained by restriction enzyme digestion and determining the nucleotide sequence. The resulting plasmid DNA was named pSK-OCIF-DDD2.
[0114]
(2) Construction of mutant expression vector
The obtained target plasmid DNAs (pSK-OCIF-DCR1, {pSK-OCIF-DCR2, pSK-OCIF-XR3, pSK-OCIF-DCR4, pSK-OCIF-DDD1, pSK-OCIF-DDD2) were converted to restriction enzymes BamHI} and XhoI. (Takara Shuzo Co., Ltd.) to separate and purify a DNA fragment (including the target mutation) of about 1.4 to 1.5 kb including the full-length OCIF cDNA, which was dissolved in 20 μl of sterile distilled water. These were named DCR1 DNA solution, DCR2 DNA solution, DCR3 DNA solution, DCR4 DNA solution, DDD1 DNA solution, and DDD2 DNA solution. Example 22-ii) The pCEP4 DNA solution (1 μl) and 6 μl each of the DCR1 DNA solution, DCR2 DNA solution, DCR3 DNA solution, DCR4 DNA solution, DDD1 DNA solution, and DDD2 DNA solution described in Example 22-ii) were separately mixed, and 7 μl of the DNA ligation buffer was added to each mixture. And a ligation reaction was performed. After completion of the reaction, Escherichia coli DH5α was transformed using 7 μl of the reaction solution. From the obtained ampicillin-resistant transformed cells, a total of six strains having a plasmid DNA having a structure in which each 1.4-1.5 kb fragment was inserted into the pCEP4BamHI {XhoI site} were selected. Plasmids having the desired structure were named pCEP4-OCIF-DCR1, pCEP4-OCIF-DCR2, pCEP4-OCIF-DCR3, pCEP4-OCIF-DCR4, pCEP4-OCIF-DDD1, and pCEP4-OCIF-DDD2, respectively.
[0115]
iii) Generation of C-terminal domain deletion mutant
(1) Introduction of C-terminal domain deletion mutation
Among the amino acids described in SEQ ID NO: 4, {Cys at 379} and Leu at 380}, Ser from 331 to Leu at 380, Asp at 252 to Leu at 380, Asp at 177 to Leu at 380 , From No. 123 Arg to Leu at 380, and from Cys at 86 to Leu at 380, respectively. The mutant lacking Cys at position 379 and Leu at position 380 was deleted from OCIF-CL, the mutant from Ser at position 331 to Leu at position 380 was deleted at OCIF-CC, and the mutant at position 380 from Asp at position # 380. OCIF-CDD2, a mutant deleted from Asp at position 177 to Leu at position 380 was deleted, and a mutant deleted from Asp at position 177 to Leu at position 380 was deleted from Arg at position 123 to Leu at position 380. The mutant thus obtained was named OCIF-CCR4}, and the mutant deleted from {Cys at 86 to {Leu at 380} was named OCIF-CCR3}.
[0116]
Mutagenesis for the production of mutant OCIF-CL was performed by the two-step PCR method described in Example 22-ii). The primers used in the mutagenesis reaction are shown in Table 12, and their base sequences are shown in SEQ ID NOs: 23, 40, 55 and 56 in the Sequence Listing. The DNA obtained by PCR was precipitated with ethanol, dried in vacuo and dissolved in 40 μl of sterile distilled water (solution L).
[0117]
The DNA fragment in 20 μl of the solution L was digested with restriction enzymes BstPI and EcoRV (Takara Shuzo). A DNA fragment of about 100 bp was separated and purified by preparative electrophoresis and dissolved in 20 μl of sterile distilled water (DNA solution 19). Next, 2 µl of the DNA solution 9 and 3 µl of the DNA solution 10 described in Example 22-ii) were mixed, and the DNA ligation kit ver. 5 µl of 2 I solution was added, and a ligation reaction was performed. Escherichia coli DH5α was transformed using 5 μl of the ligation solution after the reaction. From the obtained ampicillin-resistant transformed cells, a strain having the target plasmid DNA was selected by DNA structure analysis. The DNA structure was analyzed by measuring the length of the fragment obtained by restriction enzyme digestion and determining the nucleotide sequence. The obtained target plasmid DNA was designated as pSK-OCIF-CL. A one-step PCR method was used for mutagenesis for preparing mutant OCIF-CC, mutant OCIF-CDD2, mutant OCIF-CDD1, and mutant OCIF-CCR4 and mutant OCIF-CCR3. The reaction conditions are shown below.
[0118]

[0119]
[Table 12]

[0120]
At the time of each mutation introduction, only the type of primer was changed, and the other reaction compositions were the same.
The primers for mutagenesis in each reaction are shown in Table 13, and their sequences are shown in SEQ ID NOs: 57 to 61 in the Sequence Listing. After mixing the PCR reaction solution in a microcentrifuge tube, PCR was performed under the following conditions. After treatment at 97 ° C. for 3 minutes, a three-stage reaction of 95 ° C. for 30 seconds, 50 ° C. for 30 seconds and 70 ° C. for 3 minutes was repeated 25 times, and the temperature was kept at 70 ° C. for 5 minutes. A part of the reaction solution was subjected to agarose electrophoresis, and it was confirmed that a DNA fragment of a desired length was synthesized. The primer was removed from the reaction solution with Amicon microcon, the DNA was precipitated with ethanol, dried in vacuum, and dissolved in 40 μl of sterile distilled water. The DNA fragment in 20 μl of the solution containing each mutant DNA fragment was digested with restriction enzymes XhoI and BamHIＨ. After completion of the enzyme digestion, the DNA was precipitated with ethanol, dried in vacuum, and dissolved in 20 μl of sterile distilled water. The solutions were named CCDNA solution, CDD2DNA solution, CDD1DNA solution, CCR4DNA solution, and CCR3DNA solution.
[0121]
[Table 13]

[0122]
(2) Construction of mutant expression vector
pSK-OCIF-CL was digested with restriction enzymes BamHI and XhoI (Takara Shuzo), and a DNA fragment of about 1.5 kb (including the target mutation) containing OCIF cDNA was separated and purified, and dissolved in 20 μl of sterile distilled water. (CLDNA solution). Example 22-ii) 1 μl of pCEP4 DNA solution and 6 μl of each of CL DNA solution, CCDNA solution, CDD2 DNA solution, CDD1 DNA solution, CCR4 DNA solution, and CCR3 DNA solution described in Example 22-ii) were separately mixed, and 7 μl of DNA ligation kit Ver. A 2 I solution was added, and a ligation reaction was performed. After completion of the reaction, Escherichia coli DH5α was transformed using 7 μl of the reaction solution. From the obtained ampicillin-resistant transformed cells, a total of six strains having a plasmid DNA having a structure in which an OCIF cDNA fragment having the desired mutation was inserted into the XhoI-BamHI site of pCEP4 were selected. Plasmids having the desired structure were named pCEP4-OCIF-CL, @ pCEP4-OCIF-CC, pCEP4-OCIF-CDD2, pCEP4-OCIF-CDD1, pCEP4-OCIF-CCR4, pCEP4-OCIF-CCR3, respectively.
[0123]
iv) Generation of C-terminal deletion mutant
(1) Introduction of C-terminal deletion mutation
In the amino acid described in SEQ ID NO: 4, a mutant (OCIF-CBst) in which 371th Gln} to {380 Leu} is deleted and two residues of Leu-Val} are added (OCIF-CBst), 298th Cys to {380 Leu is deleted A mutant (OCIF-CSph) having Ser-Leu-Asp residue added thereto, a mutant (OCIF-CBsp) having deletion from Asn 167 to {380 Leu}, and a mutant (OCIF-CBsp) from No. 62 {Cys to {380 Leu}. A mutant (OCIF-CPst) deleted and added with two residues of Leu-Val was prepared. 2 μg pSK each⁺-OCIF was cut with restriction enzymes BstPI, SphI, PstI (Takara Shuzo) and BspEI (New England Biolab), the DNA was purified by phenol treatment and ethanol precipitation, and dissolved in 10 μl of sterile distilled water. Using 2 μl of each solution, the ends of each DNA were blunt-ended with a DNA branding kit (Takara Shuzo) (final volume: 5 μl). To this reaction solution, 1 μg (1 μl) of an XbaI linker containing amber codon (5′-CTAGTCTAGACTAG-3 ′) and 6 μl of DNA ligation kit ver. A 2 I solution was added, and a ligation reaction was performed. Escherichia coli DH5α was transformed using 6 μl of the ligation solution after the reaction. From the obtained ampicillin-resistant transformed cells, a strain having the target plasmid DNA was selected by DNA structure analysis. The DNA structure was analyzed by measuring the length of the fragment obtained by restriction enzyme digestion and determining the nucleotide sequence. The obtained target plasmid DNAs were named pSK-OCIF-CBst, pSK-OCIF-CSph, pSK-OCIF-CBspｓ, and pSK-OCIF-CPst.
[0124]
(2) Construction of mutant expression vector
The obtained plasmid DNAs (pSK-OCIF-CBst, pSK-OCIF-CSph, pSK-OCIF-CBsp, pSK-OCIF-CPst) were cut with restriction enzymes BamHIＸ and XhoI (Takara Shuzo) to obtain about 1 fragment containing the full length OCIF cDNA. A DNA fragment (including a target mutation) of 0.5 kilobase pair (kb) was separated and purified, and dissolved in 20 µl of sterilized distilled water (named CBstDNA solution, CSphDNA solution, CBspDNA solution, and CPstDNA solution, respectively).
1 μl of the pCEP4 DNA solution described in Example 22-ii) and 6 μl each of the CBstDNA solution, CSphDNA solution, CBspDNA solution, and CPstDNA solution were separately mixed, and 7 μl of the DNA ligation kit Ver. A 2 I solution was added, and a ligation reaction was performed. After completion of the reaction, Escherichia coli DH5α was transformed using 7 μl of the reaction solution. From the resulting ampicillin-resistant transformed cells, a total of five strains having a plasmid DNA having a structure in which an OCIF cDNA fragment having a desired mutation was inserted between the XhoI and BamHI sites of pCEP4 were selected. Plasmids having the desired structure were named pCEP4-OCIF-CBst, @ pCEP4-OCIF-CSph, pCEP4-OCIF-CBsp, and pCEP4-OCIF-CPst, respectively.
[0125]
v) Preparation of mutant expression vector
Escherichia coli (21 types in total) having the mutant expression vector was grown, and various mutant expression vectors were purified using a Qiagen column (Qiagen). Each expression vector was precipitated with ethanol, dissolved in sterile distilled water, and used for the following operation.
[0126]
vi ) Mutant cDNA Of Transient Expression and Its Activity
Using the various OCIF mutant expression plasmids purified in Example 22-v), the OCIF mutant was expressed according to the method of Example 13. Only the changes are described below. A 24-well plate was used for DNA introduction. 2 × 10⁵293 / EBNA cells were seeded in each well using IMDM medium containing 10% fetal calf serum. The amounts of the mutant expression vector and lipofectamine used for DNA introduction were 1 μg and 4 μl, respectively. It was diluted with OPTI-MEM medium (Gibco BRL) to a final volume of 0.5 ml. A mixture of the mutant expression vector and lipofectamine was added to the cells, and the cells were incubated at 37 ° C. for 24 hours.₂After culturing in an incubator, the mixture was removed, and 0.5 ml of Ex-cell 301 medium (JSR) was added.₂Cultured in an incubator. The medium was recovered and used as a sample for mutant activity measurement. The nucleotide sequences of the obtained mutants are shown in SEQ ID NOs: 83 to 103 in the Sequence Listing, and the amino acid sequences deduced from the sequences are shown in SEQ ID NOs: 62 to 82, respectively. OCIF activity was measured according to Example 13. Also described in Example 24.
The antigen amount of OCIF was quantified by the EIA method described above. Table 14 shows the activity per antigen amount compared to unmodified OCIF.
[0127]
[Table 14]

[0128]
vi) Western blot analysis
10 μl of the sample used for activity measurement was subjected to Western blot analysis. To 10 μl of the sample, add 10 μl of sample buffer for SDS-PAGE (0.5 M Tris-HCl, 20% glycerol, 4% SDS, 20 μg / ml bromophenol blue (pH 6.8)) and boil at 100 ° C. for 3 minutes. 10% SDS polyacrylamide electrophoresis was performed in a non-reducing state. After completion of the electrophoresis, proteins were blotted on a PVDF membrane (ProBlott®, Perkin Elmer) using a semi-dry blotting apparatus (Bio-Rad). After blocking the membrane, the membrane was incubated with horseradish peroxidase-labeled anti-OCIF antibody for EIA described in Example 24 at 37 ° C. for 2 hours.
After washing, a protein binding to the anti-OCIF antibody was detected by an ECL system (Amersham). In OCIF, bands of about 120 ° kilodalton (kD) and 60 kD were detected.
On the other hand, in the case of OCIF-C23S, OCIF-CL, and OCIF-CC, almost only a band of 60 kD was detected. In OCIF-CDD2 and OCIF-CDD1, bands of about 40-50 kD and 30-40 kD were detected as main bands, respectively. From the above results, OCIF shows that the Cys residue at position 379 in the amino acid sequence of SEQ ID NO: 4 is involved in dimer formation, that the monomer retains activity, and that Asp
It was clarified that the activity was maintained even when the residues from to 380th Leu were deleted.
[0129]
Embodiment 23
Human OCIF genome DNA Separation
I) Human genome DNA Library screening
A genomic library prepared using human placenta chromosomal DNA and the λFIXII vector was purchased from Stratagene, and screened using OCIF cDNA as a probe. The screening was basically performed according to the protocol attached to the genomic library, but the general method for handling phage, E. coli and DNA was performed according to Molecular Cloning: A Laboratory Manual.
After assaying the titer of the purchased genomic DNA library, 1 × 10⁶The pfu phage were infected into E. coli XL1-Blue {MRA} and plated on 20 plates (9 × 13 cm) with 9 ml of top agarose per plate. After incubating the plate overnight at 37 ° C., the phage was transcribed by placing a Hybond-N nylon membrane (Amersham) on an agar plate. The nylon membrane on which the phage was transferred was placed on a filter paper moistened with a 1.5 M NaCl / 0.5 M NaOH solution for 1 minute, and then 1 M Tris-HCl (pH 7.5) and 1.5 M NaCl / 0.5 M Tris-HCl were used. (PH 7.5) for 1 minute each to neutralize, and finally transferred onto filter paper moistened with 2XSSC. Thereafter, the nylon membrane was irradiated with 1200 µJ of UV using a Stratalinker (Stratagene) to fix the phage DNA to the membrane. Next, the nylon membrane was immersed in a rapid hybridization buffer (Amersham) to perform pre-hybridization. After one hour of prehybridization,³²P-labeled OCIF cDNA was added, and hybridization was carried out at 65 ° C. overnight. This cDNA probe was obtained by cleaving the plasmid pBKOCIF having the 1.6 kb Ｏ OCIF cDNA obtained in Example 11 using restriction enzymes BamHI Ｘ and XhoI, isolating the OCIF cDNA by agarose gel electrophoresis, and separating the OCIF cDNA from Using Prime DNA Labeling System (Amersham)³²Prepared by labeling with P. Labeling was performed according to the protocol attached to the labeling system.
[0130]
For hybridization, approximately 5 x 10 per ml of hybridization buffer⁵A cpm probe was used. After hybridization, the nylon membrane was washed with 2 × SSC at room temperature for 5 minutes, and then with 65 ° C./XSSC/0.1% SDS four times at 65 ° C. for 20 minutes each. After the fourth washing, the nylon membrane was dried, and autoradiography was performed at −80 ° C. using an X gland film manufactured by Fuji Film Co., Ltd., Super HR-H, and an intensifying screen. Since six signals were detected on the autoradiogram, the top agarose was cut out from the position on the agar plate corresponding to each signal and immersed in 0.5 ml of SM buffer to which 1% chloroform was added. And left overnight to extract the phage. Each phage extract was diluted 1000-fold with SM buffer, 1 μl and 20 μl of the phage extract were taken out, and the cells were again infected with the above-mentioned Escherichia coli and plated on an agar plate together with top agarose by the above method. After the phage was transferred to a nylon membrane, prehybridization, hybridization, washing, drying, and autoradiography were performed by the methods described above. This phage purification procedure was performed for all six signals initially detected by autoradiography, and was repeated until all phage plaques on the agar plate had hybridized with the cDNA probe. Plaques of the purified phage were cut out, immersed in 0.5 ml of SM buffer containing 1% chloroform, and stored at 4 ° C. The six purified phages thus obtained were named λOIF3, λOIF8, {λOIF9, {λOIF11, λOIF12, λOIF17}, respectively.
[0131]
II) Human by restriction enzyme digestion and Southern blot hybridization OCIF genome DNA Analysis of clones
The purified DNA of the six phages was purified by a plate lysis method according to the method described in Molecular Cloning: A Laboratory Manual. These DNAs were digested with restriction enzymes, and the obtained fragments were separated by agarose electrophoresis. The fragments separated on an agarose gel were transferred to a nylon membrane by a general method, and then subjected to Southern blot hybridization using OCIF cDNA as a probe. As a result of these analyses, it was found that each of the six purified phages was a different clone. Among the DNA fragments obtained by restriction enzyme digestion, those that hybridize to OCIF cDNA were analyzed by the following method after subcloning into a plasmid vector.
[0132]
iii) genome DNA Obtained by restriction enzyme digestion from clone DNA Subcloning of fragment into plasmid vector and determination of nucleotide sequence
λOIF8 DNA was digested with restriction enzymes EcoRI and NotI, and the resulting fragment was separated on a 0.7% agarose gel. The 5.8 kb EcoRI / NotI fragment was extracted from the gel using QIAEXII Gel Extraction Kit (Qiagen) according to the attached protocol. This fragment was ligated using pBluescript II {SK +} vector (Stratagene) and Ready-To-Go @ T4Ligase (Pharmacia), which had been cut with EcoRI and NotI, according to the attached protocol. After introducing the obtained recombinant plasmid into competent DH5α E. coli (Amersham), E. coli having the plasmid was selected by plating on an agarose plate containing 50 μg / ml ampicillin. The recombinant plasmid having the 5.8 kb {EcoRI / NotI fragment prepared as described above was named pBSG8-5.8}. Next, a 0.9 kb DNA fragment produced by digesting pBSG8-5.8 with a restriction enzyme HindIII was separated on an agarose gel, extracted according to the method described above, and then pBluescript II SK previously cut with HindIII. -(Stratagene) and cloned according to the method described above. The recombinant plasmid having the 0.9 kb HindIII fragment was designated as pBS8H0.9. On the other hand, fragments of 6 kb, 3.6 kb and 2.6 kb produced by digesting the DNA of λOIF11 with EcoRI were isolated and inserted into the pBluescriptIIＢSK + vector and cloned in the same manner as described above. Recombinant plasmids having the 6 kb, 3.6 kb, and 2.6 kb EcoRI fragment were named pBSG11-6, pBSG11-3.6, and pBSG11-2.6, respectively.
[0133]
Further, three fragments of 2.2 kb, 1.1 kb, and 1.05 kb generated by digesting pBSG11-6 with a restriction enzyme HindIII were separated by agarose gel electrophoresis, and inserted into the HindIII site of pBluescriptII SK-, respectively. And cloned. The recombinant plasmids having the 2.2 kb, 1.1 kb, and 1.05 kb HindIII fragments were named pBS6H2.2, pBS6H1.1, and pBS6H1.05, respectively. For analysis of the base sequence of genomic DNA, ABI Dye Deoxy Terminator Cycle Sequencing Ready Reaction Kit (PerkinElmer) and 373 DNA Sequencing System (Applied Biosystems) were used. Molecular Cloning: A pBSG8-5.8, pBS8H0.9, pBSG11-6, pBSG11-3.6, pBSG11-2.6, pBS6H2.2, pBS6H1.1, pBS6H1.05, according to the method described in A Laboratory Manual. It was prepared and used as a template for nucleotide sequencing. The nucleotide sequence of human OCIF genomic DNA is shown in SEQ ID NOs: 104 # and 105 # in the Sequence Listing. The sequence of bases interposed between exon 1 and exon 2 has not been completely determined, and it has been confirmed that a nucleotide of about 17 kb intervenes between the base sequences shown in SEQ ID NOS: 104 # and 105 # in the Sequence Listing. Have been.
[0134]
Embodiment 24
EIA by OCIF Quantitation
i) Rabbit anti OCIF Preparation of antibodies
Three male Japanese white rabbits (weighing 2.5 kg to 3.0 kg, obtained from Kitayama Labes) were mixed with 200 μg / ml of rOCIF in an equal amount with Freund's complete adjuvant (DIFCO) to give an emulsion once. Subcutaneous immunization was performed for each 1 ml. Immunization was performed a total of 6 times at weekly intervals, and whole blood was collected 10 days after the final immunization. The antibody was purified from the separated serum as follows. That is, ammonium sulfate was added to the antiserum diluted 2-fold with PBS to a final concentration of 40 w / v%, left at 4 ° C. for 1 hour, and centrifuged at 8000 × g for 20 minutes to obtain a precipitate. The precipitate was dissolved in a small amount of PBS, dialyzed against PBS at 4 ° C., and then applied to a Protein {G-Sepharose} column (Pharmacia). After washing with PBS, the adsorbed immunoglobulin G was eluted with 0.1 M glycine hydrochloride buffer (pH 3.0) and immediately adjusted to neutral pH with 1.5 {M Tris-HCl buffer (pH 8.7)}. After the eluted protein fraction was dialyzed against PBS, the absorbance at 280 nm was measured and the concentration was determined (E^1%13.5). Horseradish peroxidase-labeled anti-OCIF antibody was prepared using a maleimide-activated peroxidase kit (Pierce). That is, 80 μg of N-succinimide-S-acetylthioacetic acid was added to 1 mg of the purified antibody and reacted at room temperature for 30 minutes. After deacetylation by adding 5 mg of hydroxylamine, the modified antibody was fractionated on a polyacrylamide desalting column. The protein fraction was mixed with 1 mg of maleimide-activated peroxidase and reacted at room temperature for 1 hour to obtain an enzyme-labeled antibody.
[0135]
ii) sandwich EIA by OCIF Quantitation
100 μl of rabbit anti-OCIF antibody (2 μg / ml, 50 mM carbonate buffer (pH 9.6)) was added to each well of a 96-well microtiter plate (MaxiSorp Immunoplate, Nunc) and allowed to stand at 4 ° C. overnight. Then, the antibody was immobilized. 300 μl of 25% Block Ace (Snow Brand Milk Products) prepared in PBS was added to each well, and left standing at 37 ° C. for 1 hour to perform blocking. Then, a sample (100 μl / well) was added and reacted at room temperature for 2 hours. Was. After washing three times with PBS containing 0.05% {Tween 20 (PBST), a 10,000-fold diluted horseradish peroxidase-labeled anti-OCIF antibody was added {100 μl} each and incubated at room temperature for 2 hours. After washing three times with PBST, 100 μl of an enzyme substrate solution (TMB, ScyTek) was added, and the color was developed at room temperature. Then, the reaction was stopped. The absorbance at 450 nm was measured using a microplate reader (Immunoleader NJ2000, Intermed Japan), and the OCIF concentration of the sample was quantified from a calibration curve using purified recombinant OCIF as a standard. The calibration curve of OCIF is shown in FIG.
[0136]
Embodiment 25
Anti OCIF Monoclonal antibody
i) Human OCIF Preparation of antibody-producing hybridoma
Human fibroblasts IMR-90 were cultured, and OCIF was purified from the culture by the method described in Example 11. Purified OCIF was dissolved in PBS to a concentration of 10 μg / 100 μl, and the solution was intraperitoneally administered to BALB / c mice every two weeks for immunization. In the first and second immunizations, a mixture of equal volumes of Freund's complete adjuvant was administered. On the third day after the final immunization, the spleen was excised, B lymphocytes were separated, and the cells were fused with mouse myeloma cells P3x63-AG8.653 by a commonly used polyethylene glycol method. Then, hybridoma cells were selected by culturing in a HAT medium to select the fused cells. Next, in order to confirm whether or not the selected cells produce an OCIF-specific antibody, a 0.1-M OCIF solution (100 μl / 10 μg / ml) dissolved in a sodium bicarbonate solution was placed in a 96-well microplate (Nunc The OCIF-specific antibody in the hybridoma culture was measured using the solid phase ELISA prepared in addition to (1). Cloning of hybridomas in which antibody production was recognized was repeated 3-5 times by the limiting dilution method, and the amount of antibody production was checked each time by the above ELISA. From the obtained antibody-producing strains, clones having high antibody production were selected.
[0137]
ii) Production of monoclonal antibodies
The antibody-producing strains obtained in Example 25-i) were each separated by 1 × 10⁶Was implanted intraperitoneally into BALB / c mice previously inoculated with Pristane {Aldrich Chemical Co., Ltd.}. Two weeks after transplantation, the accumulated ascites was collected to obtain ascites containing the monoclonal antibody of the present invention. Purified antibodies were obtained from the ascites by affinity chromatography using Affigel Protein A Sepharose (manufactured by Bio-Rad). That is, the ascites was diluted with an equal amount of binding buffer (Bio-Rad), loaded onto a protein A column, and washed with a sufficient amount of the same buffer. Elution of IgG was performed with an elution buffer (Bio-Rad). The obtained eluate was dialyzed against water, and then freeze-dried. When the purity of the obtained purified antibody was analyzed by SDS-PAGE, a uniform band was observed at a position of about 150,000 ° in molecular weight.
[0138]
iii) 　 OCIF Of Monoclonal Antibodies with High Affinity for Antibodies
The antibody obtained in Example 25-ii) was dissolved in PBS, and the protein was quantified by the Lowry method. Next, each antibody was dissolved in PBS so that the protein concentration was constant, and this solution was diluted by a serial dilution method. Monoclonal antibodies that reacted with OCIF up to high dilution steps were selected using the solid phase ELISA described in Example 25-ii). As a result, three kinds of antibodies, A1G5, E3H8, and D2F4, were obtained.
[0139]
iv) Test for antibody subclass
The class and subclass of the antibody of the present invention selected in Example 25-iii) were assayed using an immunoglobulin class and subclass analysis kit (Amersham). Assays were performed according to the protocol specified in the kit. Table 15 shows the results. E3H8, A1G5, and D2F4 were each IgG₁, IgG_2a, And IgG_2b Met.
[0140]
[Table 15]

[0141]
v) 　 OCIF of ELISA Measurement method by
The three types of monoclonal antibodies A1G5, E3H8, and D2F4 obtained in Example 25-iv) ｉ were used as solid-phase antibodies and labeled antibodies, respectively. With each combination, a sandwich ELISA was constructed. The labeling of the antibody was performed using a maleimide-activated peroxidase kit (Pierce). Each antibody was dissolved in a 0.1 M sodium bicarbonate solution to a concentration of 10 μg / ml, dispensed in a volume of 100 μl per well of a 96-well immunoplate (Nunc Corporation), and allowed to stand at room temperature overnight. Then, each plate was blocked with a 1/2 concentration of Block Ace (Snow Brand Milk Products) and washed three times with PBS (wash buffer) containing 0.1% Tween 20. OCIF at each concentration was prepared in a primary reaction buffer (0.2 M Tris-HCl buffer containing 1 / 2.5 Block Ace and 0.1% Tween 20) at pH 7.4.
[0142]
Each of the prepared OCIF solutions (100 μl) was added to each well, left at 37 ° C. for 3 hours, and then washed three times with a washing buffer. For the dilution of the labeled antibody, a secondary reaction buffer {(0.1 M Tris-HCl buffer containing 1/4 concentration of Block Ace and 0.1% of Tween 20; pH of 7.4)} was used. Each labeled antibody was diluted 400-fold with the secondary reaction buffer, and {100 μl} of each was added to each well. Each plate was left at 37 ° C. for 2 hours, then washed three times, and then washed with a substrate solution (0.4 mg / ml orthophenylenediamine hydrochloride, 0.1 M citric acid-phosphoric acid containing 0.006% hydrogen peroxide). Buffer, pH {4.5) {100 μl} was added to each well. After standing in a dark room at 37 ° C. for 15 minutes, 50 μl of 6N sulfuric acid was added to each well to stop the enzymatic reaction, and the absorbance at 492 nm was measured using Immunoleader II (NJ2000, Intermed Japan). Good measurement results were obtained with any combination of the three types of antibodies, each of which was a solid phase antibody or a labeled antibody, and it was confirmed that the three types of antibodies each recognized a different epitope of OCIF. As a representative example, FIG. 14 shows a calibration curve when A1G5 is used as a solid phase antibody and E3H8 is used as a labeled antibody.
[0143]
vi) In human serum OCIF Measurement
OCIF in the sera of five healthy subjects was measured by the ELISA system of Example 25- (v) in FIG. That is, A1G5 was immobilized on an immunoplate in the same manner as in Example 25- (v), the primary reaction buffer was added to each well in an amount of 50 µl, and then 50 µl of each human serum was added, followed by standing at 37 ° C for 3 hours. After washing three times with the washing buffer, 100 μl of the labeled antibody of E3H8 diluted 400-fold with the secondary reaction buffer was added to each well, and left at 37 ° C. for 2 hours. After the plate was washed three times with the washing buffer, the above-mentioned substrate solution {100 μl} was added to each well, and reacted at 37 ° C. for 15 minutes. The enzyme reaction was stopped by adding 50 μl of 6N sulfuric acid to each well, and the absorbance at 492 nm was measured with an immunoreader. The same procedure was used for the primary reaction buffer containing a known amount of OCIF to prepare an OCIF calibration curve as shown in FIG. 14, and the amount of OCIF in the serum was determined from the absorbance of the serum sample. Table 16 shows the results.
[0144]
[Table 16]

[0145]
Embodiment 26
Therapeutic effect on osteoporosis
The therapeutic effect of OCIF on immobilized bone atrophy model due to nerve resection was confirmed.
Using a Fischer II male rat, the left upper brachial plexus was excised at the age of 6 weeks (body weight: about 120 g) to induce immobilization of the left forelimb to create a bone atrophy model. OCIF was adjusted with PBS (-) containing 0.01% Tween80 and administered intravenously twice daily at 12-hour intervals twice daily for 5 weeks at a dose of 5 µg / kg and 50 µg / kg from the next day. A sham operation was performed for the normal group, and PBS (-) containing 0.01% Tween80 was similarly administered to the control group. After the administration, the left upper arm was excised and the bone strength was measured. The results are shown in FIG.
As a result, a decrease in bone strength was observed in the control group as compared with the normal group, but improvement was observed in the OCIF 50 μg / kg administration group.
[0146]
【The invention's effect】
According to the present invention, a protein having a novel osteoclast formation inhibitory activity and an efficient method for producing the same are provided. The protein of the present invention has osteoclast formation inhibitory activity and can be used as a therapeutic agent for various osteopenic diseases such as osteoporosis or as an antigen for immunological diagnosis of these diseases.
[Sequence list]

[Brief description of the drawings]
FIG. 1 shows an elution profile when a HiLoad-Q / FF {non-adsorbed fraction roughly purified product (sample 3) is applied to a HiLoad-S / HP} column.
FIG. 2 shows an elution profile obtained by applying a roughly purified product of heparin-5PW (sample 5) to a blue-5PW column.
FIG. 3 shows an elution profile when Blue-5PW elution fractions 49 to 50 were applied to a reversed-phase column.
FIG. 4 shows the results of SDS-PAGE under reducing and non-reducing conditions of the final purified product.
[Explanation of symbols]
Lanes 1, 4; molecular weight markers
Lanes 2, 5; peak 6
Lanes 3, 6; peak 7
FIG. 5 shows an elution profile when peak 7 treated with lysyl endoprotease after reductive pyridylethylation was applied to a reversed-phase column.
FIG. 6 shows the results of SDS-PAGE of native (n) and recombinant (r) OCIF under non-reducing conditions. (E) indicates that produced in 293 / EBNA cells, and (C) indicates that produced in CHO cells.
[Explanation of symbols]
Lane 1: molecular weight marker
Lane 2: monomeric nOCIF
Lane 3: dimer type nOCIF
Lane 4: monomeric rOCIF (E)
Lane 5: dimer rOCIF (E)
Lane 6: monomeric rOCIF (C)
Lane 7: dimer rOCIF (C)
FIG. 7 shows the results of SDS-PAGE of native (n) and recombinant (r) OCIF under reducing conditions. (E) shows the one produced in 293 / EBNA cells, and (C) shows the one produced in CHO cells.
[Explanation of symbols]
Lane 8: molecular weight marker
Lane 9: monomer type nOCIF
Lane 10: dimer type nOCIF
Lane 11: monomeric rOCIF (E)
Lane 12: dimer rOCIF (E)
Lane 13: monomeric rOCIF (C)
Lane 14: dimer rOCIF (C)
FIG. 8 shows the results of SDS-PAGE of native (n) and recombinant (r) OCIF from which N-linked sugar chains have been removed under reducing conditions. (E) indicates that produced in 293 / EBNA cells, and (C) indicates that produced in CHO cells.
[Description of sign]
Lane 15: molecular weight marker
Lane 16: monomer type nOCIF
Lane 17: dimer type nOCIF
Lane 18: monomeric rOCIF (E)
Lane 19: dimer rOCIF (E)
Lane 20: monomeric rOCIF (C)
Lane 21: dimer rOCIF (C)
FIG. 9 shows a comparison of amino acid sequences of OCIF and OCIF2.
FIG. 10 shows a comparison of amino acid sequences of OCIF and OCIF3.
FIG. 11 shows a comparison of amino acid sequences of OCIF and OCIF4.
FIG. 12 shows a comparison of amino acid sequences of OCIF and OCIF5.
FIG. 13 shows a calibration curve of OCIF when an anti-OCIF polyclonal antibody was used.
FIG. 14 shows a calibration curve of OCIF when an anti-OCIF monoclonal antibody was used.
FIG. 15 shows the therapeutic effect of OCIF on osteoporosis.

Claims (127)

A DNA that hybridizes under relatively mild conditions with the cDNA represented by the nucleotide sequence of SEQ ID NO: 6 in the Sequence Listing. A protein having an activity of inhibiting osteoclast fragmentation and / or maturation, which is obtained by expressing a cDNA encoding an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 4 in the Sequence Listing. A cDNA represented by the nucleotide sequence of SEQ ID NO: 83 in the Sequence Listing. A protein obtained by expressing the cDNA represented by the nucleotide sequence of SEQ ID NO: 83 in the Sequence Listing. A cDNA encoding the amino acid sequence represented by SEQ ID NO: 62 in the Sequence Listing. A cDNA represented by the nucleotide sequence of SEQ ID NO: 84 in Sequence Listing. A protein obtained by expressing the cDNA represented by the nucleotide sequence of SEQ ID NO: 84 in Sequence Listing. CDNA encoding the amino acid sequence represented by SEQ ID NO: 63 in the Sequence Listing. A cDNA represented by the nucleotide sequence of SEQ ID NO: 85 in the Sequence Listing. A protein obtained by expressing the cDNA represented by the nucleotide sequence of SEQ ID NO: 85 in the Sequence Listing. A cDNA encoding the amino acid sequence represented by SEQ ID NO: 64 in the Sequence Listing. A cDNA represented by the nucleotide sequence of SEQ ID NO: 86 in the Sequence Listing. A protein obtained by expressing the cDNA represented by the nucleotide sequence of SEQ ID NO: 86 in Sequence Listing. CDNA encoding the amino acid sequence represented by SEQ ID NO: 65 in the Sequence Listing. A cDNA represented by the nucleotide sequence of SEQ ID NO: 87 in the Sequence Listing. A protein obtained by expressing the cDNA represented by the nucleotide sequence of SEQ ID NO: 87 in Sequence Listing. A cDNA encoding the amino acid sequence represented by SEQ ID NO: 66 in the Sequence Listing. A cDNA represented by the nucleotide sequence of SEQ ID NO: 88 in the Sequence Listing. A protein obtained by expressing the cDNA represented by the nucleotide sequence of SEQ ID NO: 88 in the Sequence Listing. A cDNA encoding the amino acid sequence represented by SEQ ID NO: 67 in the Sequence Listing. A cDNA represented by the nucleotide sequence of SEQ ID NO: 89 in Sequence Listing. A protein obtained by expressing the cDNA represented by the nucleotide sequence of SEQ ID NO: 89 in Sequence Listing. A cDNA encoding the amino acid sequence represented by SEQ ID NO: 68 in the Sequence Listing. A cDNA represented by the nucleotide sequence of SEQ ID NO: 90 in Sequence Listing. A protein obtained by expressing the cDNA represented by the nucleotide sequence of SEQ ID NO: 90 in the sequence listing. CDNA encoding the amino acid sequence represented by SEQ ID NO: 69 in the Sequence Listing. A cDNA represented by the nucleotide sequence of SEQ ID NO: 91 in Sequence Listing. A protein obtained by expressing the cDNA represented by the nucleotide sequence of SEQ ID NO: 91 in Sequence Listing. CDNA encoding the amino acid sequence represented by SEQ ID NO: 70 in the Sequence Listing. A cDNA represented by the nucleotide sequence of SEQ ID NO: 92 in the Sequence Listing. A protein obtained by expressing the cDNA represented by the nucleotide sequence of SEQ ID NO: 92 in the Sequence Listing. A cDNA encoding the amino acid sequence represented by SEQ ID NO: 71 in the Sequence Listing. A cDNA represented by the nucleotide sequence of SEQ ID NO: 93 in Sequence Listing. A protein obtained by expressing the cDNA represented by the nucleotide sequence of SEQ ID NO: 93 in the Sequence Listing. CDNA encoding the amino acid sequence represented by SEQ ID NO: 72 in the Sequence Listing. A cDNA represented by the nucleotide sequence of SEQ ID NO: 94 in Sequence Listing. A protein obtained by expressing the cDNA represented by the nucleotide sequence of SEQ ID NO: 94 in the Sequence Listing. A cDNA encoding the amino acid sequence represented by SEQ ID NO: 73 in the Sequence Listing. A cDNA represented by the nucleotide sequence of SEQ ID NO: 95 in the Sequence Listing. A protein obtained by expressing the cDNA represented by the nucleotide sequence of SEQ ID NO: 95 in the Sequence Listing. A cDNA encoding the amino acid sequence represented by SEQ ID NO: 74 in the Sequence Listing. A cDNA represented by the nucleotide sequence of SEQ ID NO: 96 in the Sequence Listing. A protein obtained by expressing the cDNA represented by the nucleotide sequence of SEQ ID NO: 96 in the Sequence Listing. CDNA encoding the amino acid sequence represented by SEQ ID NO: 75 in the Sequence Listing. A cDNA represented by the nucleotide sequence of SEQ ID NO: 97 in Sequence Listing. A protein obtained by expressing the cDNA represented by the nucleotide sequence of SEQ ID NO: 97 in Sequence Listing. CDNA encoding the amino acid sequence represented by SEQ ID NO: 76 in the Sequence Listing. A cDNA represented by the nucleotide sequence of SEQ ID NO: 98 in the Sequence Listing. A protein obtained by expressing the cDNA represented by the nucleotide sequence of SEQ ID NO: 98 in the Sequence Listing. CDNA encoding the amino acid sequence represented by SEQ ID NO: 77. A cDNA represented by the nucleotide sequence of SEQ ID NO: 99 in the Sequence Listing. A protein obtained by expressing the cDNA represented by the nucleotide sequence of SEQ ID NO: 99 in the Sequence Listing. A cDNA encoding the amino acid sequence represented by SEQ ID NO: 78 in the Sequence Listing. A cDNA represented by the nucleotide sequence of SEQ ID NO: 100 in the Sequence Listing. A protein obtained by expressing the cDNA represented by the nucleotide sequence of SEQ ID NO: 100 in the sequence listing. CDNA encoding the amino acid sequence represented by SEQ ID NO: 79 in the Sequence Listing. A cDNA represented by the nucleotide sequence of SEQ ID NO: 101 in Sequence Listing. A protein obtained by expressing the cDNA represented by the nucleotide sequence of SEQ ID NO: 101 in the Sequence Listing. CDNA encoding the amino acid sequence represented by SEQ ID NO: 80 in the sequence listing. A cDNA represented by the nucleotide sequence of SEQ ID NO: 102 in the Sequence Listing. A protein obtained by expressing the cDNA represented by the nucleotide sequence of SEQ ID NO: 102 in the Sequence Listing. CDNA encoding the amino acid sequence represented by SEQ ID NO: 81 in the Sequence Listing. A cDNA represented by the nucleotide sequence of SEQ ID NO: 103 in the Sequence Listing. A protein obtained by expressing the cDNA represented by the nucleotide sequence of SEQ ID NO: 103 in the Sequence Listing. CDNA encoding the amino acid sequence represented by SEQ ID NO: 82 in the Sequence Listing. A protein comprising the amino acid sequence represented by SEQ ID NO: 5 in the sequence listing. A vector into which any one of the following cDNAs (1) to (4) has been inserted:
(1) cDNA encoding the amino acid sequence represented by SEQ ID NO: 4 in the sequence listing;
(2) cDNA encoding the amino acid sequence represented by SEQ ID NO: 5 in the sequence listing;
(3) cDNA represented by the nucleotide sequence of SEQ ID NO: 6;
(4) cDNA inserted into a vector carried by transformed Escherichia coli pBK / 01F10 (FERM BP-5267). The vector according to claim 67, which is carried by transformed Escherichia coli pBK / 01F10 (FERM @ BP-5267). A host cell into which the vector according to claim 67 or 68 has been introduced. A method for producing a protein having the following physicochemical properties (a) to (d) and having osteoclast differentiation and / or maturation inhibitory activity, comprising the following steps (I) and (II):
(A) Molecular weight (by SDS-PAGE); about 60 kD (under reducing conditions), about 60 kD and about 120 kD (under non-reducing conditions).
(B) affinity; having cation exchanger and heparin affinity;
(C) heat stability; heat treatment at 70 ° C. for 10 minutes or 56 ° C. for 30 minutes reduces the activity of inhibiting osteoclast differentiation and maturation, and overheating at 90 ° C. for 10 minutes causes osteoclast differentiation / Maturation inhibitory activity is lost.
(D) Amino acid sequence; has an amino acid sequence of SEQ ID NOs: 1 to 3 as an internal amino acid sequence.
(I) culturing the host cell according to claim 69;
(II) a step of recovering a protein having the above physicochemical properties (a) to (d) and having an activity of inhibiting osteoclast differentiation and / or maturation from the culture solution. A protein having the following physicochemical properties (a) to (d) obtained by the method according to claim 70 and having osteoclast differentiation and / or maturation inhibitory activity.
(A) Molecular weight (by SDS-PAGE); about 60 kD (under reducing conditions), about 60 kD and about 120 kD (under non-reducing conditions).
(B) affinity; having cation exchanger and heparin affinity;
(C) heat stability; heat treatment at 70 ° C. for 10 minutes or 56 ° C. for 30 minutes reduces the activity of inhibiting osteoclast differentiation and maturation, and overheating at 90 ° C. for 10 minutes causes osteoclast differentiation / Maturation inhibitory activity is lost.
(D) Amino acid sequence; has an amino acid sequence of SEQ ID NOs: 1 to 3 as an internal amino acid sequence. A protein represented by the amino acid sequence consisting of amino acid Nos. 1 to 373 of SEQ ID NO: 9 in the Sequence Listing. A cDNA represented by the nucleotide sequence consisting of nucleotide numbers 64 to 1185 in SEQ ID NO: 8 in the sequence listing. A protein represented by the amino acid sequence consisting of amino acid numbers 1 to 341 of SEQ ID NO: 11 in the sequence listing. A cDNA represented by the nucleotide sequence consisting of nucleotide numbers 64 to 1089 in SEQ ID NO: 10 in the sequence listing. A protein represented by the amino acid sequence consisting of amino acid Nos. 1 to 133 in SEQ ID NO: 13 in the sequence listing. A cDNA represented by the nucleotide sequence consisting of nucleotide numbers 64 to 465 in SEQ ID NO: 12 in the sequence listing. A protein represented by the amino acid sequence consisting of amino acid Nos. 1 to 124 of SEQ ID NO: 15 in the sequence listing. A cDNA represented by the base sequence consisting of base numbers 64 to 438 of SEQ ID NO: 14 in the sequence listing. A protein represented by the amino acid sequence consisting of amino acid numbers 1 to 380 of SEQ ID NO: 62 in the sequence listing. A cDNA represented by the nucleotide sequence consisting of nucleotide numbers 64 to 1206 in SEQ ID NO: 83 of the Sequence Listing. A protein represented by the amino acid sequence consisting of amino acid Nos. 1 to 380 of SEQ ID NO: 63 in the Sequence Listing. A cDNA represented by the nucleotide sequence consisting of nucleotide numbers 64 to 1206 of SEQ ID NO: 84 in the Sequence Listing. A protein represented by the amino acid sequence consisting of amino acid numbers 1 to 380 in SEQ ID NO: 64 in the sequence listing. A cDNA represented by the nucleotide sequence consisting of nucleotide numbers 64 to 1206 in SEQ ID NO: 85 of the Sequence Listing. A protein represented by the amino acid sequence consisting of amino acid Nos. 1 to 380 of SEQ ID NO: 65 in the Sequence Listing. A cDNA represented by the nucleotide sequence consisting of nucleotide numbers 64 to 1206 in SEQ ID NO: 86 of the Sequence Listing. A protein represented by the amino acid sequence consisting of amino acid Nos. 1 to 380 of SEQ ID NO: 66 in the Sequence Listing. A cDNA represented by the nucleotide sequence consisting of nucleotide numbers 64 to 1206 in SEQ ID NO: 87 of the Sequence Listing. A protein represented by the amino acid sequence consisting of amino acid Nos. 1 to 339 of SEQ ID NO: 67 in the Sequence Listing. A cDNA represented by the nucleotide sequence consisting of nucleotide numbers 64 to 1083 in SEQ ID NO: 88 in the sequence listing. A protein represented by the amino acid sequence consisting of amino acid Nos. 1 to 338 of SEQ ID No. 68 in the Sequence Listing. A cDNA represented by the nucleotide sequence consisting of nucleotide numbers 64 to 1080 of SEQ ID NO: 89 in the sequence listing. A protein represented by the amino acid sequence consisting of amino acid numbers 1 to 342 of SEQ ID NO: 69 in the sequence listing. A cDNA represented by the nucleotide sequence consisting of nucleotide numbers 64 to 1092 of SEQ ID NO: 90 in the sequence listing. A protein represented by the amino acid sequence consisting of amino acid numbers 1 to 338 of SEQ ID NO: 70 in the sequence listing. A cDNA represented by the nucleotide sequence consisting of nucleotide numbers 64 to 1080 of SEQ ID NO: 91 in the Sequence Listing. A protein represented by the amino acid sequence consisting of amino acid Nos. 1 to 305 of SEQ ID NO: 71 in the Sequence Listing. A cDNA represented by the nucleotide sequence consisting of nucleotide numbers 64 to 981 in SEQ ID NO: 92 in the sequence listing. A protein represented by the amino acid sequence consisting of amino acid Nos. 1 to 306 of SEQ ID NO: 72 in the Sequence Listing. A cDNA represented by the nucleotide sequence consisting of nucleotide numbers 64 to 984 in SEQ ID NO: 93 in the sequence listing. A protein represented by the amino acid sequence consisting of amino acid Nos. 1 to 378 of SEQ ID NO: 73 in the Sequence Listing. A cDNA represented by the nucleotide sequence consisting of nucleotide numbers 64 to 1200 of SEQ ID NO: 94 in the sequence listing. A protein represented by the amino acid sequence consisting of amino acid Nos. 1 to 330 of SEQ ID NO: 74 in the Sequence Listing. A cDNA represented by the nucleotide sequence consisting of nucleotide numbers 64 to 1056 in SEQ ID NO: 95 in the sequence listing. A protein represented by the amino acid sequence consisting of amino acid numbers 1 to 251 of SEQ ID NO: 75 in the sequence listing. A cDNA represented by the nucleotide sequence consisting of nucleotide numbers 64 to 819 of SEQ ID NO: 96 in the Sequence Listing. A protein represented by the amino acid sequence consisting of amino acid Nos. 1 to 176 of SEQ ID NO: 76 in the Sequence Listing. A cDNA represented by the nucleotide sequence consisting of nucleotide numbers 64 to 594 in SEQ ID NO: 97 in the sequence listing. A protein represented by the amino acid sequence consisting of amino acid numbers 1 to 122 of SEQ ID NO: 77 in the sequence listing. A cDNA represented by the base sequence consisting of base numbers 64 to 432 of SEQ ID NO: 98 in the sequence listing. A protein represented by the amino acid sequence consisting of amino acid Nos. 1 to 85 of SEQ ID NO: 78 in the Sequence Listing. A cDNA represented by the base sequence consisting of base numbers 64 to 321 of SEQ ID NO: 99 in the sequence listing. A protein represented by the amino acid sequence consisting of amino acid Nos. 1 to 372 of SEQ ID NO: 79 in the Sequence Listing. A cDNA represented by the nucleotide sequence consisting of nucleotide numbers 64 to 1182 in SEQ ID NO: 100 in the sequence listing. A protein represented by the amino acid sequence consisting of amino acid numbers 1 to 300 of SEQ ID NO: 80 in the sequence listing. A cDNA represented by the nucleotide sequence consisting of nucleotide numbers 64 to 966 of SEQ ID NO: 101 in the sequence listing. A protein represented by the amino acid sequence consisting of amino acid numbers 1 to 166 of SEQ ID NO: 81 in the sequence listing. A cDNA represented by the nucleotide sequence consisting of nucleotide numbers 64 to 564 of SEQ ID NO: 102 in the sequence listing. A protein represented by the amino acid sequence consisting of amino acid numbers 1 to 63 of SEQ ID NO: 82 in the sequence listing. A cDNA represented by the nucleotide sequence consisting of nucleotide numbers 64 to 255 in SEQ ID NO: 103 in the sequence listing. An osteoclast inhibitor mutant protein comprising an amino acid sequence in which at least one amino acid is substituted or deleted in SEQ ID NO: 4 in the Sequence Listing. A protein consisting of an amino acid sequence in which an integer number of amino acids selected from 1 or more and 204 or less is deleted from 380th leucine in SEQ ID NO: 4 or 5 in the sequence listing. Claims 2, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 66, 71, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, An agent for preventing or treating osteoporosis, comprising the protein according to one selected from the group consisting of 122 and 123. Claims 2, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 66, 71, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, A preventive or therapeutic agent for osteopenia, comprising the protein according to one selected from the group consisting of 122 and 123. Claims 2, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 66, 71, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, A preventive or therapeutic agent for bone metabolism disorder, comprising the protein according to one selected from the group consisting of 122 and 123. Transformed E. coli pBK / 01F10 (FERM @ BP-5267).

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