JP3793180B2 - Novel protein and method for producing the same - Google Patents

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1X10⁸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】
10X Ex Taqバッファー（宝酒造社） 5 μl
2.5 mM dNTP 4 μl
cDNA溶液 1 μl
Ex Taq (宝酒造社） 0.25 μl
蒸留水 29.75 μl
40μM プライマーNo.2F 5 μl
40μM プライマーNo.3R 5 μl
【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(Taq Dye 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 [α³²P]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(2X10⁵cpm/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。8×10⁵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D₃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D₃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３×10⁵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 ％CO₂、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D₃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 0.6〜1.2M NaCl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 2×10⁷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、CO₂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１×10⁵cells/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１×10⁶cells/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D₃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３×10⁵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％ CO₂、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D₃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D₃ 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D₃、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) 5×10³個と生後約８週間の ddyマウス脾臓細胞１×10⁵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％CO₂、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３×10⁵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％ CO₂、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)５×10³個と生後約８週間の ddyマウス脾臓細胞１×10⁵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％ CO₂、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D₃、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 、379 Cys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】
PCR1 反応液
10X Ex Taq バッファー（宝酒造社） 10 μl
2.5 mM dNTP 溶液 8 μl
実施例１１記載のプラスミドベクター（8ng/ml） 2 μl
滅菌蒸留水 73.5μl
２０μM プライマー１ 5 μl
１００μM プライマー２（変異導入用） １ μl
Ex Taq （宝酒造社） 0.5μl
【００９４】
PCR2 反応液
10X Ex Taqバッファー（宝酒造社） 10 μl
2.5 mM dNTP 溶液 8 μl
実施例１１記載のプラスミドベクター（8ng/ml） 2 μl
滅菌蒸留水 73.5μl
20μM プライマー３ 5 μl
100μM プライマー４（変異導入用） 1 μl
Ex Taq （宝酒造社） 0.5μl
【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】
PCR3 反応液
10X Ex Taqバッファー（宝酒造社） 10 μl
2.5 mM dNTP 溶液 8 μl
PCR1により得られたDNA断片 5 μl
PCR2により得られたDNA断片 5 μl
滅菌蒸留水 61.5 μl
20μＭ プライマー 1 5 μl
20μM プライマー 3 5 μl
Ex Taq (宝酒造社) 0.5 μl
【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】
Ｃ末端ドメイン欠失変異導入用 PCR 反応液
10X Ex Taq バッファー(宝酒造社) 10 μl
2.5 mM dNTP 溶液 8 μl
実施例11記載のプラスミドベクター(8ng/ml) 2 μl
滅菌蒸留水 73.5μl
20μM プライマー OCIF Xho F 5 μl
100μM 変異導入用プライマー 1 μl
Ex Taq (宝酒造社) 0.5 μl
【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。２×10⁵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 CO₂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、１×10⁶ pfu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５×10⁵cpm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 ×10⁶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】
SEQUENCELISTING
<110> Sankyo Co., Ltd
<120> Novel protein and methods for theproduction of the same
<130> 2003-119SW
<140>
<141>
<150> JP H07-54977
<151> 1995-2-20
<150> JP H07-207508
<151> 1995-7-21
<150> PCT/JP96/00374
<151> 1996-2-20
<160> 105
<170> PatentIn version 3.1.2
<210> 1
<211> 6
<212> PRT
<213> human OCIF
<220>
<221> Xaa
<222> 1
<223> Inventor:Goto, Masaaki
Inventor:Tsuda, Eisuke
Inventor:Mochizuki, Shin'ichi
Inventor:Yano, Kazuki
Inventor:Kobayashi, Fumie
Inventor:Shima, Nobuyuki
Inventor:Yasuda,Hisataka
Inventor:Nakagawa, Nobuaki
Inventor:Morinaga, Tomonori
Inventor:Ueda, Masatsugu
Inventor:Higashio, Kanji
<400> 1
Xaa Tyr His Phe Pro Lys
1 5
<210> 2
<211> 14
<212> PRT
<213> human OCIF
<220>
<221> Xaa
<222> 1
<400> 2
Xaa Gln His Ser Xaa Gln Glu Gln Thr Phe Gln Leu Xaa Lys
1 5 10
<210> 3
<211> 12
<212> PRT
<213> human OCIF
<220>
<221> Xaa
<222> 1
<400> 3
Xaa Ile Arg Phe Leu His Ser Phe Thr Met Tyr Lys
1 5 10
<210> 4
<211> 380
<212> PRT
<213> human OCIF
<400> 4
Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr Asp Glu Glu Thr Ser
1 5 10 15
His Gln Leu Leu Cys Asp Lys Cys Pro Pro Gly Thr Tyr Leu Lys
20 25 30
Gln His Cys Thr Ala Lys Trp Lys Thr Val Cys Ala Pro Cys Pro
35 40 45
Asp His Tyr Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Cys Leu
50 55 60
Tyr Cys Ser Pro Val Cys Lys Glu Leu Gln Tyr Val Lys Gln Glu
65 70 75
Cys Asn Arg Thr His Asn Arg Val Cys Glu Cys Lys Glu Gly Arg
80 85 90
Tyr Leu Glu Ile Glu Phe Cys Leu Lys His Arg Ser Cys Pro Pro
95 100 105
Gly Phe Gly Val Val Gln Ala Gly Thr Pro Glu Arg Asn Thr Val
110 115 120
Cys Lys Arg Cys Pro Asp Gly Phe Phe Ser Asn Glu Thr Ser Ser
125 130 135
Lys Ala Pro Cys Arg Lys His Thr Asn Cys Ser Val Phe Gly Leu
140 145 150
Leu Leu Thr Gln Lys Gly Asn Ala Thr His Asp Asn Ile Cys Ser
155 160 165
Gly Asn Ser Glu Ser Thr Gln Lys Cys Gly Ile Asp Val Thr Leu
170 175 180
Cys Glu Glu Ala Phe Phe Arg Phe Ala Val Pro Thr Lys Phe Thr
185 190 195
Pro Asn Trp Leu Ser Val Leu Val Asp Asn Leu Pro Gly Thr Lys
200 205 210
Val Asn Ala Glu Ser Val Glu Arg Ile Lys Arg Gln His Ser Ser
215 220 225
Gln Glu Gln Thr Phe Gln Leu Leu Lys Leu Trp Lys His Gln Asn
230 235 240
Lys Asp Gln Asp Ile Val Lys Lys Ile Ile Gln Asp Ile Asp Leu
245 250 255
Cys Glu Asn Ser Val Gln Arg His Ile Gly His Ala Asn Leu Thr
260 265 270
Phe Glu Gln Leu Arg Ser Leu Met Glu Ser Leu Pro Gly Lys Lys
275 280 285
Val Gly Ala Glu Asp Ile Glu Lys Thr Ile Lys Ala Cys Lys Pro
290 295 300
Ser Asp Gln Ile Leu Lys Leu Leu Ser Leu Trp Arg Ile Lys Asn
305 310 315
Gly Asp Gln Asp Thr Leu Lys Gly Leu Met His Ala Leu Lys His
320 325 330
Ser Lys Thr Tyr His Phe Pro Lys Thr Val Thr Gln Ser Leu Lys
335 340 345
Lys Thr Ile Arg Phe Leu His Ser Phe Thr Met Tyr Lys Leu Tyr
350 355 360
Gln Lys Leu Phe Leu Glu Met Ile Gly Asn Gln Val Gln Ser Val
365 370 375
Lys Ile Ser Cys Leu
380
<210> 5
<211> 401
<212> PRT
<213> human OCIF
<400> 5
Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
-20 -15 -10
Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
-5 -1 1 5
Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
10 15 20
Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
25 30 35
Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
40 45 50
Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
55 60 65
Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys
70 75 80
Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys
85 90 95
His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly Thr
100 105 110
Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe Phe
115 120 125
Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn
130 135 140
Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys Gly Asn Ala Thr
145 150 155
His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr Gln Lys Cys
160 165 170
Gly Ile Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg Phe Ala
175 180 185
Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val Asp
190 195 200
Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg Ile
205 210 215
Lys Arg Gln His Ser Ser Gln Glu Gln Thr Phe Gln Leu Leu Lys
220 225 230
Leu Trp Lys His Gln Asn Lys Asp Gln Asp Ile Val Lys Lys Ile
235 240 245
Ile Gln Asp Ile Asp Leu Cys Glu Asn Ser Val Gln Arg His Ile
250 255 260
Gly His Ala Asn Leu Thr Phe Glu Gln Leu Arg Ser Leu Met Glu
265 270 275
Ser Leu Pro Gly Lys Lys Val Gly Ala Glu Asp Ile Glu Lys Thr
280 285 290
Ile Lys Ala Cys Lys Pro Ser Asp Gln Ile Leu Lys Leu Leu Ser
295 300 305
Leu Trp Arg Ile Lys Asn Gly Asp Gln Asp Thr Leu Lys Gly Leu
310 315 320
Met His Ala Leu Lys His Ser Lys Thr Tyr His Phe Pro Lys Thr
325 330 335
Val Thr Gln Ser Leu Lys Lys Thr Ile Arg Phe Leu His Ser Phe
340 345 350
Thr Met Tyr Lys Leu Tyr Gln Lys Leu Phe Leu Glu Met Ile Gly
355 360 365
Asn Gln Val Gln Ser Val Lys Ile Ser Cys Leu
370 375 380
<210> 6
<211> 1206
<212> DNA
<213> human OCIF
<400> 6
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gaaccccaga gcgaaataca 420
gtttgcaaaa gatgtccaga tgggttcttc tcaaatgaga cgtcatctaa agcaccctgt 480
agaaaacaca caaattgcag tgtctttggt ctcctgctaa ctcagaaagg aaatgcaaca 540
cacgacaaca tatgttccgg aaacagtgaa tcaactcaaa aatgtggaat agatgttacc 600
ctgtgtgagg aggcattctt caggtttgct gttcctacaa agtttacgcc taactggctt 660
agtgtcttgg tagacaattt gcctggcacc aaagtaaacg cagagagtgt agagaggata 720
aaacggcaac acagctcaca agaacagact ttccagctgc tgaagttatg gaaacatcaa 780
aacaaagacc aagatatagt caagaagatc atccaagata ttgacctctg tgaaaacagc 840
gtgcagcggc acattggaca tgctaacctc accttcgagc agcttcgtag cttgatggaa 900
agcttaccgg gaaagaaagt gggagcagaa gacattgaaa aaacaataaa ggcatgcaaa 960
cccagtgacc agatcctgaa gctgctcagt ttgtggcgaa taaaaaatgg cgaccaagac 1020
accttgaagg gcctaatgca cgcactaaag cactcaaaga cgtaccactt tcccaaaact 1080
gtcactcaga gtctaaagaa gaccatcagg ttccttcaca gcttcacaat gtacaaattg 1140
tatcagaagt tatttttaga aatgataggt aaccaggtcc aatcagtaaa aataagctgc 1200
ttataa 1206
<210> 7
<211> 15
<212> PRT
<213> human OCIF
<400> 7
Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr Asp Glu Glu Thr Ser
1 5 10 15
<210> 8
<211> 1185
<212> DNA
<213> OCIF2
<400> 8
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagtgc aatcgcaccc acaaccgcgt gtgcgaatgc 300
aaggaagggc gctaccttga gatagagttc tgcttgaaac ataggagctg ccctcctgga 360
tttggagtgg tgcaagctgg aaccccagag cgaaatacag tttgcaaaag atgtccagat 420
gggttcttct caaatgagac gtcatctaaa gcaccctgta gaaaacacac aaattgcagt 480
gtctttggtc tcctgctaac tcagaaagga aatgcaacac acgacaacat atgttccgga 540
aacagtgaat caactcaaaa atgtggaata gatgttaccc tgtgtgagga ggcattcttc 600
aggtttgctg ttcctacaaa gtttacgcct aactggctta gtgtcttggt agacaatttg 660
cctggcacca aagtaaacgc agagagtgta gagaggataa aacggcaaca cagctcacaa 720
gaacagactt tccagctgct gaagttatgg aaacatcaaa acaaagacca agatatagtc 780
aagaagatca tccaagatat tgacctctgt gaaaacagcg tgcagcggca cattggacat 840
gctaacctca ccttcgagca gcttcgtagc ttgatggaaa gcttaccggg aaagaaagtg 900
ggagcagaag acattgaaaa aacaataaag gcatgcaaac ccagtgacca gatcctgaag 960
ctgctcagtt tgtggcgaat aaaaaatggc gaccaagaca ccttgaaggg cctaatgcac 1020
gcactaaagc actcaaagac gtaccacttt cccaaaactg tcactcagag tctaaagaag 1080
accatcaggt tccttcacag cttcacaatg tacaaattgt atcagaagtt atttttagaa 1140
atgataggta accaggtcca atcagtaaaa ataagctgct tataa 1185
<210> 9
<211> 394
<212> PRT
<213> OCIF2
<400> 9
Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
-20 -15 -10
Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
-5 1 5
Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
10 15 20
Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
25 30 35
Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
40 45 50
Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Cys
55 60 65
Asn Arg Thr His Asn Arg Val Cys Glu Cys Lys Glu Gly Arg Tyr
70 75 80
Leu Glu Ile Glu Phe Cys Leu Lys His Arg Ser Cys Pro Pro Gly
85 90 95
Phe Gly Val Val Gln Ala Gly Thr Pro Glu Arg Asn Thr Val Cys
100 105 110
Lys Arg Cys Pro Asp Gly Phe Phe Ser Asn Glu Thr Ser Ser Lys
115 120 125
Ala Pro Cys Arg Lys His Thr Asn Cys Ser Val Phe Gly Leu Leu
130 135 140
Leu Thr Gln Lys Gly Asn Ala Thr His Asp Asn Ile Cys Ser Gly
145 150 155
Asn Ser Glu Ser Thr Gln Lys Cys Gly Ile Asp Val Thr Leu Cys
160 165 170
Glu Glu Ala Phe Phe Arg Phe Ala Val Pro Thr Lys Phe Thr Pro
175 180 185
Asn Trp Leu Ser Val Leu Val Asp Asn Leu Pro Gly Thr Lys Val
190 195 200
Asn Ala Glu Ser Val Glu Arg Ile Lys Arg Gln His Ser Ser Gln
205 210 215
Glu Gln Thr Phe Gln Leu Leu Lys Leu Trp Lys His Gln Asn Lys
220 225 230
Asp Gln Asp Ile Val Lys Lys Ile Ile Gln Asp Ile Asp Leu Cys
235 240 245
Glu Asn Ser Val Gln Arg His Ile Gly His Ala Asn Leu Thr Phe
250 255 260
Glu Gln Leu Arg Ser Leu Met Glu Ser Leu Pro Gly Lys Lys Val
265 270 275
Gly Ala Glu Asp Ile Glu Lys Thr Ile Lys Ala Cys Lys Pro Ser
280 285 290
Asp Gln Ile Leu Lys Leu Leu Ser Leu Trp Arg Ile Lys Asn Gly
295 300 305
Asp Gln Asp Thr Leu Lys Gly Leu Met His Ala Leu Lys His Ser
310 315 320
Lys Thr Tyr His Phe Pro Lys Thr Val Thr Gln Ser Leu Lys Lys
325 330 335
Thr Ile Arg Phe Leu His Ser Phe Thr Met Tyr Lys Leu Tyr Gln
340 345 350
Lys Leu Phe Leu Glu Met Ile Gly Asn Gln Val Gln Ser Val Lys
355 360 365
Ile Ser Cys Leu
370
<210> 10
<211> 1089
<212> DNA
<213> OCIF3
<400> 10
atgaacaagt tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gaaccccaga gcgaaataca 420
gtttgcaaaa gatgtccaga tgggttcttc tcaaatgaga cgtcatctaa agcaccctgt 480
agaaaacaca caaattgcag tgtctttggt ctcctgctaa ctcagaaagg aaatgcaaca 540
cacgacaaca tatgttccgg aaacagtgaa tcaactcaaa aatgtggaat agatgttacc 600
ctgtgtgagg aggcattctt caggtttgct gttcctacaa agtttacgcc taactggctt 660
agtgtcttgg tagacaattt gcctggcacc aaagtaaacg cagagagtgt agagaggata 720
aaacggcaac acagctcaca agaacagact ttccagctgc tgaagttatg gaaacatcaa 780
aacaaagacc aagatatagt caagaagatc atccaagata ttgacctctg tgaaaacagc 840
gtgcagcggc acattggaca tgctaacctc agtttgtggc gaataaaaaa tggcgaccaa 900
gacaccttga agggcctaat gcacgcacta aagcactcaa agacgtacca ctttcccaaa 960
actgtcactc agagtctaaa gaagaccatc aggttccttc acagcttcac aatgtacaaa 1020
ttgtatcaga agttattttt agaaatgata ggtaaccagg tccaatcagt aaaaataagc 1080
tgcttataa 1089
<210> 11
<211> 362
<212> PRT
<213> OCIF3
<400> 11
Met Asn Lys Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
-20 -15 -10
Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
-5 1 5
Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
10 15 20
Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
25 30 35
Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
40 45 50
Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
55 60 65
Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys
70 75 80
Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys
85 90 95
His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly Thr
100 105 110
Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe Phe
115 120 125
Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn
130 135 140
Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys Gly Asn Ala Thr
145 150 155
His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr Gln Lys Cys
160 165 170
Gly Ile Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg Phe Ala
175 180 185
Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val Asp
190 195 200
Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg Ile
205 210 215
Lys Arg Gln His Ser Ser Gln Glu Gln Thr Phe Gln Leu Leu Lys
220 225 230
Leu Trp Lys His Gln Asn Lys Asp Gln Asp Ile Val Lys Lys Ile
235 240 245
Ile Gln Asp Ile Asp Leu Cys Glu Asn Ser Val Gln Arg His Ile
250 255 260
Gly His Ala Asn Leu Ser Leu Trp Arg Ile Lys Asn Gly Asp Gln
265 270 275
Asp Thr Leu Lys Gly Leu Met His Ala Leu Lys His Ser Lys Thr
280 285 290
Tyr His Phe Pro Lys Thr Val Thr Gln Ser Leu Lys Lys Thr Ile
295 300 305
Arg Phe Leu His Ser Phe Thr Met Tyr Lys Leu Tyr Gln Lys Leu
310 315 320
Phe Leu Glu Met Ile Gly Asn Gln Val Gln Ser Val Lys Ile Ser
325 330 335
Cys Leu
340
<210> 12
<211> 465
<212> DNA
<213> OCIF4
<400> 12
atgaacaagt tgctgtgctg ctcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gtacgtgtca atgtgcagca 420
aaattaatta ggatcatgca aagtcagata gttgtgacag tttag 465
<210> 13
<211> 154
<212> PRT
<213> OCIF4
<400> 13
Met Asn Lys Leu Leu Cys Cys Ser Leu Val Phe Leu Asp Ile Ser
-20 -15 -10
Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
-5 1 5
Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
10 15 20
Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
25 30 35
Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
40 45 50
Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
55 60 65
Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys
70 75 80
Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys
85 90 95
His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly Thr
100 105 110
Cys Gln Cys Ala Ala Lys Leu Ile Arg Ile Met Gln Ser Gln Ile
115 120 125
Val Val Thr Val
130
<210> 14
<211> 438
<212> DNA
<213> OCIF5
<400> 14
atgaacaagt tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gatgcaggag aagacccaag 420
ccacagatat gtatctga 438
<210> 15
<211> 145
<212> PRT
<213> OCIF5
<400> 15
Met Asn Lys Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
-20 -15 -10
Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
-5 1 5
Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
10 15 20
Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
25 30 35
Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
40 45 50
Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
55 60 65
Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys
70 75 80
Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys
85 90 95
His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly Cys
100 105 110
Arg Arg Arg Pro Lys Pro Gln Ile Cys Ile
115 120 125
<210> 16
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer T3
<400> 16
aattaaccct cactaaaggg 20
<210> 17
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer T7
<400> 17
gtaatacgac tcactatagg gc 22
<210> 18
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer IF1
<400> 18
acatcaaaac aaagaccaag 20
<210> 19
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer IF2
<400> 19
tcttggtctt tgttttgatg 20
<210> 20
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer IF3
<400> 20
ttattcgcca caaactgagc 20
<210> 21
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer IF4
<400> 21
ttgtgaagct gtgaaggaac 20
<210> 22
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer IF5
<400> 22
gctcagtttg tggcgaataa 20
<210> 23
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer IF6
<400> 23
gtgggagcag aagacattga 20
<210> 24
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer IF7
<400> 24
aatgaacaac ttgctgtgct 20
<210> 25
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer IF8
<400> 25
tgacaaatgt cctcctggta 20
<210> 26
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer IF9
<400> 26
aggtaggtac caggaggaca 20
<210> 27
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer IF10
<400> 27
gagctgccct cctggatttg 20
<210> 28
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer IF11
<400> 28
caaactgtat ttcgctctgg 20
<210> 29
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer IF12
<400> 29
gtgtgaggag gcattcttca 20
<210> 30
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer C19SF
<400> 30
gaatcaactc aaaaaagtgg aatagatgtt ac 32
<210> 31
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer C19SR
<400> 31
gtaacatcta ttccactttt ttgagttgat tc 32
<210> 32
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer C20SF
<400> 32
atagatgtta ccctgagtga ggaggcattc 30
<210> 33
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer C20SR
<400> 33
gaatgcctcc tcactcaggg taacatctat 30
<210> 34
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer C21SF
<400> 34
caagatattg acctcagtga aaacagcgtg c 31
<210> 35
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer C21SR
<400> 35
gcacgctgtt ttcactgagg tcaatatctt g 31
<210> 36
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer C22SF
<400> 36
aaaacaataa aggcaagcaa acccagtgac c 31
<210> 37
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer C22SR
<400> 37
ggtcactggg tttgcttgcc tttattgttt t 31
<210> 38
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer C23SF
<400> 38
tcagtaaaaa taagcagctt ataactggcc a 31
<210> 39
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer C23SR
<400> 39
tggccagtta taagctgctt atttttactg a 31
<210> 40
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer IF 14
<400> 40
ttggggttta ttggaggaga tg 22
<210> 41
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer DCR1F
<400> 41
accacccagg aaccttgccc tgaccactac tacaca 36
<210> 42
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer DCR1R
<400> 42
gtcagggcaa ggttcctggg tggtccactt aatgga 36
<210> 43
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer DCR2F
<400> 43
accgtgtgcg ccgaatgcaa ggaagggcgc tacctt 36
<210> 44
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer DCR2R
<400> 44
ttccttgcat tcggcgcaca cggtcttcca ctttgc 36
<210> 45
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer DCR3F
<400> 45
aaccgcgtgt gcagatgtcc agatgggttc ttctca 36
<210> 46
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer DCR3R
<400> 46
atctggacat ctgcacacgc ggttgtgggt gcgatt 36
<210> 47
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer DCR4F
<400> 47
acagtttgca aatccggaaa cagtgaatca actcaa 36
<210> 48
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer DCR4R
<400> 48
actgtttccg gatttgcaaa ctgtatttcg ctctgg 36
<210> 49
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer DDD1F
<400> 49
aatgtggaat agatattgac ctctgtgaaa acagcg 36
<210> 50
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer DDD1R
<400> 50
agaggtcaat atctattcca catttttgag ttgatt 36
<210> 51
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer DDD2F
<400> 51
agatcatcca agacgcacta aagcactcaa agacgt 36
<210> 52
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer DDD2R
<400> 52
gctttagtgc gtcttggatg atcttcttga ctatat 36
<210> 53
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer XhoI F
<400> 53
ggctcgagcg cccagccgcc gcctccaag 29
<210> 54
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer IF 16
<400> 54
tttgagtgct ttagtgcgtg 20
<210> 55
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer CL F
<400> 55
tcagtaaaaa taagctaact ggaaatggcc 30
<210> 56
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer CL R
<400> 56
ggccatttcc agttagctta tttttactga 30
<210> 57
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer CC R
<400> 57
ccggatcctc agtgctttag tgcgtgcat 29
<210> 58
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer CCD2 R
<400> 58
ccggatcctc attggatgat cttcttgac 29
<210> 59
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer CCD1 R
<400> 59
ccggatcctc atattccaca tttttgagt 29
<210> 60
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer CCR4 R
<400> 60
ccggatcctc atttgcaaac tgtatttcg 29
<210> 61
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer CCR3 R
<400> 61
ccggatcctc attcgcacac gcggttgtg 29
<210> 62
<211> 401
<212> PRT
<213> OCIF-C19S
<400> 62
Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
-20 -15 -10
Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
-5 -1 1 5
Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
10 15 20
Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
25 30 35
Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
40 45 50
Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
55 60 65
Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys
70 75 80
Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys
85 90 95
His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly Thr
100 105 110
Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe Phe
115 120 125
Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn
130 135 140
Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys Gly Asn Ala Thr
145 150 155
His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr Gln Lys Ser
160 165 170
Gly Ile Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg Phe Ala
175 180 185
Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val Asp
190 195 200
Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg Ile
205 210 215
Lys Arg Gln His Ser Ser Gln Glu Gln Thr Phe Gln Leu Leu Lys
220 225 230
Leu Trp Lys His Gln Asn Lys Asp Gln Asp Ile Val Lys Lys Ile
235 240 245
Ile Gln Asp Ile Asp Leu Cys Glu Asn Ser Val Gln Arg His Ile
250 255 260
Gly His Ala Asn Leu Thr Phe Glu Gln Leu Arg Ser Leu Met Glu
265 270 275
Ser Leu Pro Gly Lys Lys Val Gly Ala Glu Asp Ile Glu Lys Thr
280 285 290
Ile Lys Ala Cys Lys Pro Ser Asp Gln Ile Leu Lys Leu Leu Ser
295 300 305
Leu Trp Arg Ile Lys Asn Gly Asp Gln Asp Thr Leu Lys Gly Leu
310 315 320
Met His Ala Leu Lys His Ser Lys Thr Tyr His Phe Pro Lys Thr
325 330 335
Val Thr Gln Ser Leu Lys Lys Thr Ile Arg Phe Leu His Ser Phe
340 345 350
Thr Met Tyr Lys Leu Tyr Gln Lys Leu Phe Leu Glu Met Ile Gly
355 360 365
Asn Gln Val Gln Ser Val Lys Ile Ser Cys Leu
370 375 380
<210> 63
<211> 401
<212> PRT
<213> OCIF-C20S
<400> 63
Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
-20 -15 -10
Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
-5 -1 1 5
Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
10 15 20
Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
25 30 35
Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
40 45 50
Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
55 60 65
Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys
70 75 80
Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys
85 90 95
His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly Thr
100 105 110
Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe Phe
115 120 125
Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn
130 135 140
Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys Gly Asn Ala Thr
145 150 155
His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr Gln Lys Cys
160 165 170
Gly Ile Asp Val Thr Leu Ser Glu Glu Ala Phe Phe Arg Phe Ala
175 180 185
Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val Asp
190 195 200
Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg Ile
205 210 215
Lys Arg Gln His Ser Ser Gln Glu Gln Thr Phe Gln Leu Leu Lys
220 225 230
Leu Trp Lys His Gln Asn Lys Asp Gln Asp Ile Val Lys Lys Ile
235 240 245
Ile Gln Asp Ile Asp Leu Cys Glu Asn Ser Val Gln Arg His Ile
250 255 260
Gly His Ala Asn Leu Thr Phe Glu Gln Leu Arg Ser Leu Met Glu
265 270 275
Ser Leu Pro Gly Lys Lys Val Gly Ala Glu Asp Ile Glu Lys Thr
280 285 290
Ile Lys Ala Cys Lys Pro Ser Asp Gln Ile Leu Lys Leu Leu Ser
295 300 305
Leu Trp Arg Ile Lys Asn Gly Asp Gln Asp Thr Leu Lys Gly Leu
310 315 320
Met His Ala Leu Lys His Ser Lys Thr Tyr His Phe Pro Lys Thr
325 330 335
Val Thr Gln Ser Leu Lys Lys Thr Ile Arg Phe Leu His Ser Phe
340 345 350
Thr Met Tyr Lys Leu Tyr Gln Lys Leu Phe Leu Glu Met Ile Gly
355 360 365
Asn Gln Val Gln Ser Val Lys Ile Ser Cys Leu
370 375 380
<210> 64
<211> 401
<212> PRT
<213> OCIF-C21S
<400> 64
Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
-20 -15 -10
Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
-5 -1 1 5
Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
10 15 20
Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
25 30 35
Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
40 45 50
Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
55 60 65
Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys
70 75 80
Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys
85 90 95
His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly Thr
100 105 110
Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe Phe
115 120 125
Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn
130 135 140
Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys Gly Asn Ala Thr
145 150 155
His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr Gln Lys Cys
160 165 170
Gly Ile Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg Phe Ala
175 180 185
Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val Asp
190 195 200
Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg Ile
205 210 215
Lys Arg Gln His Ser Ser Gln Glu Gln Thr Phe Gln Leu Leu Lys
220 225 230
Leu Trp Lys His Gln Asn Lys Asp Gln Asp Ile Val Lys Lys Ile
235 240 245
Ile Gln Asp Ile Asp Leu Ser Glu Asn Ser Val Gln Arg His Ile
250 255 260
Gly His Ala Asn Leu Thr Phe Glu Gln Leu Arg Ser Leu Met Glu
265 270 275
Ser Leu Pro Gly Lys Lys Val Gly Ala Glu Asp Ile Glu Lys Thr
280 285 290
Ile Lys Ala Cys Lys Pro Ser Asp Gln Ile Leu Lys Leu Leu Ser
295 300 305
Leu Trp Arg Ile Lys Asn Gly Asp Gln Asp Thr Leu Lys Gly Leu
310 315 320
Met His Ala Leu Lys His Ser Lys Thr Tyr His Phe Pro Lys Thr
325 330 335
Val Thr Gln Ser Leu Lys Lys Thr Ile Arg Phe Leu His Ser Phe
340 345 350
Thr Met Tyr Lys Leu Tyr Gln Lys Leu Phe Leu Glu Met Ile Gly
355 360 365
Asn Gln Val Gln Ser Val Lys Ile Ser Cys Leu
370 375 380
<210> 65
<211> 401
<212> PRT
<213> OCIF-C22S
<400> 65
Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
-20 -15 -10
Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
-5 -1 1 5
Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
10 15 20
Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
25 30 35
Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
40 45 50
Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
55 60 65
Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys
70 75 80
Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys
85 90 95
His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly Thr
100 105 110
Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe Phe
115 120 125
Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn
130 135 140
Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys Gly Asn Ala Thr
145 150 155
His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr Gln Lys Cys
160 165 170
Gly Ile Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg Phe Ala
175 180 185
Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val Asp
190 195 200
Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg Ile
205 210 215
Lys Arg Gln His Ser Ser Gln Glu Gln Thr Phe Gln Leu Leu Lys
220 225 230
Leu Trp Lys His Gln Asn Lys Asp Gln Asp Ile Val Lys Lys Ile
235 240 245
Ile Gln Asp Ile Asp Leu Cys Glu Asn Ser Val Gln Arg His Ile
250 255 260
Gly His Ala Asn Leu Thr Phe Glu Gln Leu Arg Ser Leu Met Glu
265 270 275
Ser Leu Pro Gly Lys Lys Val Gly Ala Glu Asp Ile Glu Lys Thr
280 285 290
Ile Lys Ala Ser Lys Pro Ser Asp Gln Ile Leu Lys Leu Leu Ser
295 300 305
Leu Trp Arg Ile Lys Asn Gly Asp Gln Asp Thr Leu Lys Gly Leu
310 315 320
Met His Ala Leu Lys His Ser Lys Thr Tyr His Phe Pro Lys Thr
325 330 335
Val Thr Gln Ser Leu Lys Lys Thr Ile Arg Phe Leu His Ser Phe
340 345 350
Thr Met Tyr Lys Leu Tyr Gln Lys Leu Phe Leu Glu Met Ile Gly
355 360 365
Asn Gln Val Gln Ser Val Lys Ile Ser Cys Leu
370 375 380
<210> 66
<211> 401
<212> PRT
<213> OCIF-C23S
<400> 66
Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
-20 -15 -10
Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
-5 -1 1 5
Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
10 15 20
Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
25 30 35
Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
40 45 50
Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
55 60 65
Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys
70 75 80
Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys
85 90 95
His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly Thr
100 105 110
Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe Phe
115 120 125
Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn
130 135 140
Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys Gly Asn Ala Thr
145 150 155
His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr Gln Lys Cys
160 165 170
Gly Ile Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg Phe Ala
175 180 185
Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val Asp
190 195 200
Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg Ile
205 210 215
Lys Arg Gln His Ser Ser Gln Glu Gln Thr Phe Gln Leu Leu Lys
220 225 230
Leu Trp Lys His Gln Asn Lys Asp Gln Asp Ile Val Lys Lys Ile
235 240 245
Ile Gln Asp Ile Asp Leu Cys Glu Asn Ser Val Gln Arg His Ile
250 255 260
Gly His Ala Asn Leu Thr Phe Glu Gln Leu Arg Ser Leu Met Glu
265 270 275
Ser Leu Pro Gly Lys Lys Val Gly Ala Glu Asp Ile Glu Lys Thr
280 285 290
Ile Lys Ala Cys Lys Pro Ser Asp Gln Ile Leu Lys Leu Leu Ser
295 300 305
Leu Trp Arg Ile Lys Asn Gly Asp Gln Asp Thr Leu Lys Gly Leu
310 315 320
Met His Ala Leu Lys His Ser Lys Thr Tyr His Phe Pro Lys Thr
325 330 335
Val Thr Gln Ser Leu Lys Lys Thr Ile Arg Phe Leu His Ser Phe
340 345 350
Thr Met Tyr Lys Leu Tyr Gln Lys Leu Phe Leu Glu Met Ile Gly
355 360 365
Asn Gln Val Gln Ser Val Lys Ile Ser Ser Leu
370 375 380
<210> 67
<211> 360
<212> PRT
<213> OCIF-DCR1
<400> 67
Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
-20 -15 -10
Ile Lys Trp Thr Thr Gln Glu Pro Cys Pro Asp His Tyr Tyr Thr
-5 -1 1 5
Asp Ser Trp His Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val
10 15 20
Cys Lys Glu Leu Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His
25 30 35
Asn Arg Val Cys Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu
40 45 50
Phe Cys Leu Lys His Arg Ser Cys Pro Pro Gly Phe Gly Val Val
55 60 65
Gln Ala Gly Thr Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro
70 75 80
Asp Gly Phe Phe Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg
85 90 95
Lys His Thr Asn Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys
100 105 110
Gly Asn Ala Thr His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser
115 120 125
Thr Gln Lys Cys Gly Ile Asp Val Thr Leu Cys Glu Glu Ala Phe
130 135 140
Phe Arg Phe Ala Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser
145 150 155
Val Leu Val Asp Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser
160 165 170
Val Glu Arg Ile Lys Arg Gln His Ser Ser Gln Glu Gln Thr Phe
175 180 185
Gln Leu Leu Lys Leu Trp Lys His Gln Asn Lys Asp Gln Asp Ile
190 195 200
Val Lys Lys Ile Ile Gln Asp Ile Asp Leu Cys Glu Asn Ser Val
205 210 215
Gln Arg His Ile Gly His Ala Asn Leu Thr Phe Glu Gln Leu Arg
220 225 230
Ser Leu Met Glu Ser Leu Pro Gly Lys Lys Val Gly Ala Glu Asp
235 240 245
Ile Glu Lys Thr Ile Lys Ala Cys Lys Pro Ser Asp Gln Ile Leu
250 255 260
Lys Leu Leu Ser Leu Trp Arg Ile Lys Asn Gly Asp Gln Asp Thr
265 270 275
Leu Lys Gly Leu Met His Ala Leu Lys His Ser Lys Thr Tyr His
280 285 290
Phe Pro Lys Thr Val Thr Gln Ser Leu Lys Lys Thr Ile Arg Phe
295 300 305
Leu His Ser Phe Thr Met Tyr Lys Leu Tyr Gln Lys Leu Phe Leu
310 315 320
Glu Met Ile Gly Asn Gln Val Gln Ser Val Lys Ile Ser Cys Leu
325 330 335
<210> 68
<211> 359
<212> PRT
<213> OCIF-DCR2
<400> 68
Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
-20 -15 -10
Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
-5 -1 1 5
Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
10 15 20
Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
25 30 35
Val Cys Ala Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe
40 45 50
Cys Leu Lys His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln
55 60 65
Ala Gly Thr Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp
70 75 80
Gly Phe Phe Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys
85 90 95
His Thr Asn Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys Gly
100 105 110
Asn Ala Thr His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr
115 120 125
Gln Lys Cys Gly Ile Asp Val Thr Leu Cys Glu Glu Ala Phe Phe
130 135 140
Arg Phe Ala Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val
145 150 155
Leu Val Asp Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val
160 165 170
Glu Arg Ile Lys Arg Gln His Ser Ser Gln Glu Gln Thr Phe Gln
175 180 185
Leu Leu Lys Leu Trp Lys His Gln Asn Lys Asp Gln Asp Ile Val
190 195 200
Lys Lys Ile Ile Gln Asp Ile Asp Leu Cys Glu Asn Ser Val Gln
205 210 215
Arg His Ile Gly His Ala Asn Leu Thr Phe Glu Gln Leu Arg Ser
220 225 230
Leu Met Glu Ser Leu Pro Gly Lys Lys Val Gly Ala Glu Asp Ile
235 240 245
Glu Lys Thr Ile Lys Ala Cys Lys Pro Ser Asp Gln Ile Leu Lys
250 255 260
Leu Leu Ser Leu Trp Arg Ile Lys Asn Gly Asp Gln Asp Thr Leu
265 270 275
Lys Gly Leu Met His Ala Leu Lys His Ser Lys Thr Tyr His Phe
280 285 290
Pro Lys Thr Val Thr Gln Ser Leu Lys Lys Thr Ile Arg Phe Leu
295 300 305
His Ser Phe Thr Met Tyr Lys Leu Tyr Gln Lys Leu Phe Leu Glu
310 315 320
Met Ile Gly Asn Gln Val Gln Ser Val Lys Ile Ser Cys Leu
325 330 335
<210> 69
<211> 363
<212> PRT
<213> OCIF-DCR3
<400> 69
Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
-20 -15 -10
Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
-5 -1 1 5
Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
10 15 20
Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
25 30 35
Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
40 45 50
Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
55 60 65
Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys
70 75 80
Arg Cys Pro Asp Gly Phe Phe Ser Asn Glu Thr Ser Ser Lys Ala
85 90 95
Pro Cys Arg Lys His Thr Asn Cys Ser Val Phe Gly Leu Leu Leu
100 105 110
Thr Gln Lys Gly Asn Ala Thr His Asp Asn Ile Cys Ser Gly Asn
115 120 125
Ser Glu Ser Thr Gln Lys Cys Gly Ile Asp Val Thr Leu Cys Glu
130 135 140
Glu Ala Phe Phe Arg Phe Ala Val Pro Thr Lys Phe Thr Pro Asn
145 150 155
Trp Leu Ser Val Leu Val Asp Asn Leu Pro Gly Thr Lys Val Asn
160 165 170
Ala Glu Ser Val Glu Arg Ile Lys Arg Gln His Ser Ser Gln Glu
175 180 185
Gln Thr Phe Gln Leu Leu Lys Leu Trp Lys His Gln Asn Lys Asp
190 195 200
Gln Asp Ile Val Lys Lys Ile Ile Gln Asp Ile Asp Leu Cys Glu
205 210 215
Asn Ser Val Gln Arg His Ile Gly His Ala Asn Leu Thr Phe Glu
220 225 230
Gln Leu Arg Ser Leu Met Glu Ser Leu Pro Gly Lys Lys Val Gly
235 240 245
Ala Glu Asp Ile Glu Lys Thr Ile Lys Ala Cys Lys Pro Ser Asp
250 255 260
Gln Ile Leu Lys Leu Leu Ser Leu Trp Arg Ile Lys Asn Gly Asp
265 270 275
Gln Asp Thr Leu Lys Gly Leu Met His Ala Leu Lys His Ser Lys
280 285 290
Thr Tyr His Phe Pro Lys Thr Val Thr Gln Ser Leu Lys Lys Thr
295 300 305
Ile Arg Phe Leu His Ser Phe Thr Met Tyr Lys Leu Tyr Gln Lys
310 315 320
Leu Phe Leu Glu Met Ile Gly Asn Gln Val Gln Ser Val Lys Ile
325 330 335
Ser Cys Leu
340
<210> 70
<211> 359
<212> PRT
<213> OCIF-DCR4
<400> 70
Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
-20 -15 -10
Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
-5 -1 1 5
Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
10 15 20
Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
25 30 35
Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
40 45 50
Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
55 60 65
Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys
70 75 80
Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys
85 90 95
His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly Thr
100 105 110
Pro Glu Arg Asn Thr Val Cys Lys Ser Gly Asn Ser Glu Ser Thr
115 120 125
Gln Lys Cys Gly Ile Asp Val Thr Leu Cys Glu Glu Ala Phe Phe
130 135 140
Arg Phe Ala Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val
145 150 155
Leu Val Asp Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val
160 165 170
Glu Arg Ile Lys Arg Gln His Ser Ser Gln Glu Gln Thr Phe Gln
175 180 185
Leu Leu Lys Leu Trp Lys His Gln Asn Lys Asp Gln Asp Ile Val
190 195 200
Lys Lys Ile Ile Gln Asp Ile Asp Leu Cys Glu Asn Ser Val Gln
205 210 215
Arg His Ile Gly His Ala Asn Leu Thr Phe Glu Gln Leu Arg Ser
220 225 230
Leu Met Glu Ser Leu Pro Gly Lys Lys Val Gly Ala Glu Asp Ile
235 240 245
Glu Lys Thr Ile Lys Ala Cys Lys Pro Ser Asp Gln Ile Leu Lys
250 255 260
Leu Leu Ser Leu Trp Arg Ile Lys Asn Gly Asp Gln Asp Thr Leu
265 270 275
Lys Gly Leu Met His Ala Leu Lys His Ser Lys Thr Tyr His Phe
280 285 290
Pro Lys Thr Val Thr Gln Ser Leu Lys Lys Thr Ile Arg Phe Leu
295 300 305
His Ser Phe Thr Met Tyr Lys Leu Tyr Gln Lys Leu Phe Leu Glu
310 315 320
Met Ile Gly Asn Gln Val Gln Ser Val Lys Ile Ser Cys Leu
325 330 335
<210> 71
<211> 326
<212> PRT
<213> OCIF-DDD1
<400> 71
Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
-20 -15 -10
Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
-5 -1 1 5
Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
10 15 20
Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
25 30 35
Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
40 45 50
Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
55 60 65
Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys
70 75 80
Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys
85 90 95
His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly Thr
100 105 110
Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe Phe
115 120 125
Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn
130 135 140
Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys Gly Asn Ala Thr
145 150 155
His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr Gln Lys Cys
160 165 170
Gly Ile Asp Ile Asp Leu Cys Glu Asn Ser Val Gln Arg His Ile
175 180 185
Gly His Ala Asn Leu Thr Phe Glu Gln Leu Arg Ser Leu Met Glu
190 195 200
Ser Leu Pro Gly Lys Lys Val Gly Ala Glu Asp Ile Glu Lys Thr
205 210 215
Ile Lys Ala Cys Lys Pro Ser Asp Gln Ile Leu Lys Leu Leu Ser
220 225 230
Leu Trp Arg Ile Lys Asn Gly Asp Gln Asp Thr Leu Lys Gly Leu
235 240 245
Met His Ala Leu Lys His Ser Lys Thr Tyr His Phe Pro Lys Thr
250 255 260
Val Thr Gln Ser Leu Lys Lys Thr Ile Arg Phe Leu His Ser Phe
265 270 275
Thr Met Tyr Lys Leu Tyr Gln Lys Leu Phe Leu Glu Met Ile Gly
280 285 290
Asn Gln Val Gln Ser Val Lys Ile Ser Cys Leu
295 300 305
<210> 72
<211> 327
<212> PRT
<213> OCIF-DDD2
<400> 72
Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
-20 -15 -10
Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
-5 -1 1 5
Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
10 15 20
Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
25 30 35
Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
40 45 50
Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
55 60 65
Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys
70 75 80
Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys
85 90 95
His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly Thr
100 105 110
Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe Phe
115 120 125
Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn
130 135 140
Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys Gly Asn Ala Thr
145 150 155
His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr Gln Lys Cys
160 165 170
Gly Ile Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg Phe Ala
175 180 185
Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val Asp
190 195 200
Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg Ile
205 210 215
Lys Arg Gln His Ser Ser Gln Glu Gln Thr Phe Gln Leu Leu Lys
220 225 230
Leu Trp Lys His Gln Asn Lys Asp Gln Asp Ile Val Lys Lys Ile
235 240 245
Ile Gln Asp Ala Leu Lys His Ser Lys Thr Tyr His Phe Pro Lys
250 255 260
Thr Val Thr Gln Ser Leu Lys Lys Thr Ile Arg Phe Leu His Ser
265 270 275
Phe Thr Met Tyr Lys Leu Tyr Gln Lys Leu Phe Leu Glu Met Ile
280 285 290
Gly Asn Gln Val Gln Ser Val Lys Ile Ser Cys Leu
295 300 305
<210> 73
<211> 399
<212> PRT
<213> OCIF-CL
<400> 73
Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
-20 -15 -10
Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
-5 -1 1 5
Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
10 15 20
Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
25 30 35
Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
40 45 50
Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
55 60 65
Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys
70 75 80
Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys
85 90 95
His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly Thr
100 105 110
Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe Phe
115 120 125
Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn
130 135 140
Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys Gly Asn Ala Thr
145 150 155
His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr Gln Lys Cys
160 165 170
Gly Ile Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg Phe Ala
175 180 185
Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val Asp
190 195 200
Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg Ile
205 210 215
Lys Arg Gln His Ser Ser Gln Glu Gln Thr Phe Gln Leu Leu Lys
220 225 230
Leu Trp Lys His Gln Asn Lys Asp Gln Asp Ile Val Lys Lys Ile
235 240 245
Ile Gln Asp Ile Asp Leu Cys Glu Asn Ser Val Gln Arg His Ile
250 255 260
Gly His Ala Asn Leu Thr Phe Glu Gln Leu Arg Ser Leu Met Glu
265 270 275
Ser Leu Pro Gly Lys Lys Val Gly Ala Glu Asp Ile Glu Lys Thr
280 285 290
Ile Lys Ala Cys Lys Pro Ser Asp Gln Ile Leu Lys Leu Leu Ser
295 300 305
Leu Trp Arg Ile Lys Asn Gly Asp Gln Asp Thr Leu Lys Gly Leu
310 315 320
Met His Ala Leu Lys His Ser Lys Thr Tyr His Phe Pro Lys Thr
325 330 335
Val Thr Gln Ser Leu Lys Lys Thr Ile Arg Phe Leu His Ser Phe
340 345 350
Thr Met Tyr Lys Leu Tyr Gln Lys Leu Phe Leu Glu Met Ile Gly
355 360 365
Asn Gln Val Gln Ser Val Lys Ile Ser
370 375
<210> 74
<211> 351
<212> PRT
<213> OCIF-CC
<400> 74
Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
-20 -15 -10
Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
-5 -1 1 5
Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
10 15 20
Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
25 30 35
Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
40 45 50
Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
55 60 65
Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys
70 75 80
Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys
85 90 95
His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly Thr
100 105 110
Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe Phe
115 120 125
Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn
130 135 140
Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys Gly Asn Ala Thr
145 150 155
His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr Gln Lys Cys
160 165 170
Gly Ile Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg Phe Ala
175 180 185
Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val Asp
190 195 200
Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg Ile
205 210 215
Lys Arg Gln His Ser Ser Gln Glu Gln Thr Phe Gln Leu Leu Lys
220 225 230
Leu Trp Lys His Gln Asn Lys Asp Gln Asp Ile Val Lys Lys Ile
235 240 245
Ile Gln Asp Ile Asp Leu Cys Glu Asn Ser Val Gln Arg His Ile
250 255 260
Gly His Ala Asn Leu Thr Phe Glu Gln Leu Arg Ser Leu Met Glu
265 270 275
Ser Leu Pro Gly Lys Lys Val Gly Ala Glu Asp Ile Glu Lys Thr
280 285 290
Ile Lys Ala Cys Lys Pro Ser Asp Gln Ile Leu Lys Leu Leu Ser
295 300 305
Leu Trp Arg Ile Lys Asn Gly Asp Gln Asp Thr Leu Lys Gly Leu
310 315 320
Met His Ala Leu Lys His
325 330
<210> 75
<211> 272
<212> PRT
<213> OCIF-CDD2
<400> 75
Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
-20 -15 -10
Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
-5 -1 1 5
Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
10 15 20
Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
25 30 35
Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
40 45 50
Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
55 60 65
Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys
70 75 80
Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys
85 90 95
His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly Thr
100 105 110
Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe Phe
115 120 125
Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn
130 135 140
Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys Gly Asn Ala Thr
145 150 155
His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr Gln Lys Cys
160 165 170
Gly Ile Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg Phe Ala
175 180 185
Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val Asp
190 195 200
Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg Ile
205 210 215
Lys Arg Gln His Ser Ser Gln Glu Gln Thr Phe Gln Leu Leu Lys
220 225 230
Leu Trp Lys His Gln Asn Lys Asp Gln Asp Ile Val Lys Lys Ile
235 240 245
Ile Gln
250
<210> 76
<211> 197
<212> PRT
<213> OCIF-CDD1
<400> 76
Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
-20 -15 -10
Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
-5 -1 1 5
Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
10 15 20
Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
25 30 35
Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
40 45 50
Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
55 60 65
Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys
70 75 80
Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys
85 90 95
His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly Thr
100 105 110
Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe Phe
115 120 125
Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn
130 135 140
Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys Gly Asn Ala Thr
145 150 155
His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr Gln Lys Cys
160 165 170
Gly Ile
175
<210> 77
<211> 143
<212> PRT
<213> OCIF-CCR4
<400> 77
Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
-20 -15 -10
Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
-5 -1 1 5
Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
10 15 20
Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
25 30 35
Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
40 45 50
Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
55 60 65
Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys
70 75 80
Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys
85 90 95
His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly Thr
100 105 110
Pro Glu Arg Asn Thr Val Cys Lys
115 120
<210> 78
<211> 106
<212> PRT
<213> OCIF-CCR3
<400> 78
Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
-20 -15 -10
Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
-5 -1 1 5
Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
10 15 20
Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
25 30 35
Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
40 45 50
Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
55 60 65
Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys
70 75 80
Glu
85
<210> 79
<211> 393
<212> PRT
<213> OCIF-CBst
<400> 79
Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
-20 -15 -10
Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
-5 -1 1 5
Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
10 15 20
Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
25 30 35
Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
40 45 50
Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
55 60 65
Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys
70 75 80
Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys
85 90 95
His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly Thr
100 105 110
Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe Phe
115 120 125
Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn
130 135 140
Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys Gly Asn Ala Thr
145 150 155
His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr Gln Lys Cys
160 165 170
Gly Ile Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg Phe Ala
175 180 185
Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val Asp
190 195 200
Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg Ile
205 210 215
Lys Arg Gln His Ser Ser Gln Glu Gln Thr Phe Gln Leu Leu Lys
220 225 230
Leu Trp Lys His Gln Asn Lys Asp Gln Asp Ile Val Lys Lys Ile
235 240 245
Ile Gln Asp Ile Asp Leu Cys Glu Asn Ser Val Gln Arg His Ile
250 255 260
Gly His Ala Asn Leu Thr Phe Glu Gln Leu Arg Ser Leu Met Glu
265 270 275
Ser Leu Pro Gly Lys Lys Val Gly Ala Glu Asp Ile Glu Lys Thr
280 285 290
Ile Lys Ala Cys Lys Pro Ser Asp Gln Ile Leu Lys Leu Leu Ser
295 300 305
Leu Trp Arg Ile Lys Asn Gly Asp Gln Asp Thr Leu Lys Gly Leu
310 315 320
Met His Ala Leu Lys His Ser Lys Thr Tyr His Phe Pro Lys Thr
325 330 335
Val Thr Gln Ser Leu Lys Lys Thr Ile Arg Phe Leu His Ser Phe
340 345 350
Thr Met Tyr Lys Leu Tyr Gln Lys Leu Phe Leu Glu Met Ile Gly
355 360 365
Asn Leu Val
370
<210> 80
<211> 321
<212> PRT
<213> OCIF-CSph
<400> 80
Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
-20 -15 -10
Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
-5 -1 1 5
Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
10 15 20
Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
25 30 35
Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
40 45 50
Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
55 60 65
Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys
70 75 80
Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys
85 90 95
His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly Thr
100 105 110
Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe Phe
115 120 125
Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn
130 135 140
Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys Gly Asn Ala Thr
145 150 155
His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr Gln Lys Cys
160 165 170
Gly Ile Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg Phe Ala
175 180 185
Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val Asp
190 195 200
Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg Ile
205 210 215
Lys Arg Gln His Ser Ser Gln Glu Gln Thr Phe Gln Leu Leu Lys
220 225 230
Leu Trp Lys His Gln Asn Lys Asp Gln Asp Ile Val Lys Lys Ile
235 240 245
Ile Gln Asp Ile Asp Leu Cys Glu Asn Ser Val Gln Arg His Ile
250 255 260
Gly His Ala Asn Leu Thr Phe Glu Gln Leu Arg Ser Leu Met Glu
265 270 275
Ser Leu Pro Gly Lys Lys Val Gly Ala Glu Asp Ile Glu Lys Thr
280 285 290
Ile Lys Ala Ser Leu Asp
295 300
<210> 81
<211> 187
<212> PRT
<213> OCIF-CBsp
<400> 81
Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
-20 -15 -10
Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
-5 -1 1 5
Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
10 15 20
Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
25 30 35
Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
40 45 50
Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
55 60 65
Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys
70 75 80
Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys
85 90 95
His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly Thr
100 105 110
Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe Phe
115 120 125
Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn
130 135 140
Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys Gly Asn Ala Thr
145 150 155
His Asp Asn Ile Cys Ser Gly
160 165
<210> 82
<211> 84
<212> PRT
<213> OCIF-CPst
<400> 82
Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
-20 -15 -10
Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
-5 -1 1 5
Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
10 15 20
Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
25 30 35
Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
40 45 50
Thr Ser Asp Glu Cys Leu Tyr Leu Val
55 60
<210> 83
<211> 1206
<212> DNA
<213> OCIF-C19S
<400> 83
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gaaccccaga gcgaaataca 420
gtttgcaaaa gatgtccaga tgggttcttc tcaaatgaga cgtcatctaa agcaccctgt 480
agaaaacaca caaattgcag tgtctttggt ctcctgctaa ctcagaaagg aaatgcaaca 540
cacgacaaca tatgttccgg aaacagtgaa tcaactcaaa aaagtggaat agatgttacc 600
ctgtgtgagg aggcattctt caggtttgct gttcctacaa agtttacgcc taactggctt 660
agtgtcttgg tagacaattt gcctggcacc aaagtaaacg cagagagtgt agagaggata 720
aaacggcaac acagctcaca agaacagact ttccagctgc tgaagttatg gaaacatcaa 780
aacaaagacc aagatatagt caagaagatc atccaagata ttgacctctg tgaaaacagc 840
gtgcagcggc acattggaca tgctaacctc accttcgagc agcttcgtag cttgatggaa 900
agcttaccgg gaaagaaagt gggagcagaa gacattgaaa aaacaataaa ggcatgcaaa 960
cccagtgacc agatcctgaa gctgctcagt ttgtggcgaa taaaaaatgg cgaccaagac 1020
accttgaagg gcctaatgca cgcactaaag cactcaaaga cgtaccactt tcccaaaact 1080
gtcactcaga gtctaaagaa gaccatcagg ttccttcaca gcttcacaat gtacaaattg 1140
tatcagaagt tatttttaga aatgataggt aaccaggtcc aatcagtaaa aataagctgc 1200
ttataa 1206
<210> 84
<211> 1206
<212> DNA
<213> OCIF-C20S
<400> 84
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gaaccccaga gcgaaataca 420
gtttgcaaaa gatgtccaga tgggttcttc tcaaatgaga cgtcatctaa agcaccctgt 480
agaaaacaca caaattgcag tgtctttggt ctcctgctaa ctcagaaagg aaatgcaaca 540
cacgacaaca tatgttccgg aaacagtgaa tcaactcaaa aatgtggaat agatgttacc 600
ctgagtgagg aggcattctt caggtttgct gttcctacaa agtttacgcc taactggctt 660
agtgtcttgg tagacaattt gcctggcacc aaagtaaacg cagagagtgt agagaggata 720
aaacggcaac acagctcaca agaacagact ttccagctgc tgaagttatg gaaacatcaa 780
aacaaagacc aagatatagt caagaagatc atccaagata ttgacctctg tgaaaacagc 840
gtgcagcggc acattggaca tgctaacctc accttcgagc agcttcgtag cttgatggaa 900
agcttaccgg gaaagaaagt gggagcagaa gacattgaaa aaacaataaa ggcatgcaaa 960
cccagtgacc agatcctgaa gctgctcagt ttgtggcgaa taaaaaatgg cgaccaagac 1020
accttgaagg gcctaatgca cgcactaaag cactcaaaga cgtaccactt tcccaaaact 1080
gtcactcaga gtctaaagaa gaccatcagg ttccttcaca gcttcacaat gtacaaattg 1140
tatcagaagt tatttttaga aatgataggt aaccaggtcc aatcagtaaa aataagctgc 1200
ttataa 1206
<210> 85
<211> 1206
<212> DNA
<213> OCIF-C21S
<400> 85
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gaaccccaga gcgaaataca 420
gtttgcaaaa gatgtccaga tgggttcttc tcaaatgaga cgtcatctaa agcaccctgt 480
agaaaacaca caaattgcag tgtctttggt ctcctgctaa ctcagaaagg aaatgcaaca 540
cacgacaaca tatgttccgg aaacagtgaa tcaactcaaa aatgtggaat agatgttacc 600
ctgtgtgagg aggcattctt caggtttgct gttcctacaa agtttacgcc taactggctt 660
agtgtcttgg tagacaattt gcctggcacc aaagtaaacg cagagagtgt agagaggata 720
aaacggcaac acagctcaca agaacagact ttccagctgc tgaagttatg gaaacatcaa 780
aacaaagacc aagatatagt caagaagatc atccaagata ttgacctcag tgaaaacagc 840
gtgcagcggc acattggaca tgctaacctc accttcgagc agcttcgtag cttgatggaa 900
agcttaccgg gaaagaaagt gggagcagaa gacattgaaa aaacaataaa ggcatgcaaa 960
cccagtgacc agatcctgaa gctgctcagt ttgtggcgaa taaaaaatgg cgaccaagac 1020
accttgaagg gcctaatgca cgcactaaag cactcaaaga cgtaccactt tcccaaaact 1080
gtcactcaga gtctaaagaa gaccatcagg ttccttcaca gcttcacaat gtacaaattg 1140
tatcagaagt tatttttaga aatgataggt aaccaggtcc aatcagtaaa aataagctgc 1200
ttataa 1206
<210> 86
<211> 1206
<212> DNA
<213> OCIF-C22S
<400> 86
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gaaccccaga gcgaaataca 420
gtttgcaaaa gatgtccaga tgggttcttc tcaaatgaga cgtcatctaa agcaccctgt 480
agaaaacaca caaattgcag tgtctttggt ctcctgctaa ctcagaaagg aaatgcaaca 540
cacgacaaca tatgttccgg aaacagtgaa tcaactcaaa aatgtggaat agatgttacc 600
ctgtgtgagg aggcattctt caggtttgct gttcctacaa agtttacgcc taactggctt 660
agtgtcttgg tagacaattt gcctggcacc aaagtaaacg cagagagtgt agagaggata 720
aaacggcaac acagctcaca agaacagact ttccagctgc tgaagttatg gaaacatcaa 780
aacaaagacc aagatatagt caagaagatc atccaagata ttgacctctg tgaaaacagc 840
gtgcagcggc acattggaca tgctaacctc accttcgagc agcttcgtag cttgatggaa 900
agcttaccgg gaaagaaagt gggagcagaa gacattgaaa aaacaataaa ggcaagcaaa 960
cccagtgacc agatcctgaa gctgctcagt ttgtggcgaa taaaaaatgg cgaccaagac 1020
accttgaagg gcctaatgca cgcactaaag cactcaaaga cgtaccactt tcccaaaact 1080
gtcactcaga gtctaaagaa gaccatcagg ttccttcaca gcttcacaat gtacaaattg 1140
tatcagaagt tatttttaga aatgataggt aaccaggtcc aatcagtaaa aataagctgc 1200
ttataa 1206
<210> 87
<211> 1206
<212> DNA
<213> OCIF-C23S
<400> 87
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gaaccccaga gcgaaataca 420
gtttgcaaaa gatgtccaga tgggttcttc tcaaatgaga cgtcatctaa agcaccctgt 480
agaaaacaca caaattgcag tgtctttggt ctcctgctaa ctcagaaagg aaatgcaaca 540
cacgacaaca tatgttccgg aaacagtgaa tcaactcaaa aatgtggaat agatgttacc 600
ctgtgtgagg aggcattctt caggtttgct gttcctacaa agtttacgcc taactggctt 660
agtgtcttgg tagacaattt gcctggcacc aaagtaaacg cagagagtgt agagaggata 720
aaacggcaac acagctcaca agaacagact ttccagctgc tgaagttatg gaaacatcaa 780
aacaaagacc aagatatagt caagaagatc atccaagata ttgacctctg tgaaaacagc 840
gtgcagcggc acattggaca tgctaacctc accttcgagc agcttcgtag cttgatggaa 900
agcttaccgg gaaagaaagt gggagcagaa gacattgaaa aaacaataaa ggcatgcaaa 960
cccagtgacc agatcctgaa gctgctcagt ttgtggcgaa taaaaaatgg cgaccaagac 1020
accttgaagg gcctaatgca cgcactaaag cactcaaaga cgtaccactt tcccaaaact 1080
gtcactcaga gtctaaagaa gaccatcagg ttccttcaca gcttcacaat gtacaaattg 1140
tatcagaagt tatttttaga aatgataggt aaccaggtcc aatcagtaaa aataagcagc 1200
ttataa 1206
<210> 88
<211> 1083
<212> DNA
<213> OCIF-DCR1
<400> 88
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaacctt gccctgacca ctactacaca gacagctggc acaccagtga cgagtgtcta 120
tactgcagcc ccgtgtgcaa ggagctgcag tacgtcaagc aggagtgcaa tcgcacccac 180
aaccgcgtgt gcgaatgcaa ggaagggcgc taccttgaga tagagttctg cttgaaacat 240
aggagctgcc ctcctggatt tggagtggtg caagctggaa ccccagagcg aaatacagtt 300
tgcaaaagat gtccagatgg gttcttctca aatgagacgt catctaaagc accctgtaga 360
aaacacacaa attgcagtgt ctttggtctc ctgctaactc agaaaggaaa tgcaacacac 420
gacaacatat gttccggaaa cagtgaatca actcaaaaat gtggaataga tgttaccctg 480
tgtgaggagg cattcttcag gtttgctgtt cctacaaagt ttacgcctaa ctggcttagt 540
gtcttggtag acaatttgcc tggcaccaaa gtaaacgcag agagtgtaga gaggataaaa 600
cggcaacaca gctcacaaga acagactttc cagctgctga agttatggaa acatcaaaac 660
aaagaccaag atatagtcaa gaagatcatc caagatattg acctctgtga aaacagcgtg 720
cagcggcaca ttggacatgc taacctcacc ttcgagcagc ttcgtagctt gatggaaagc 780
ttaccgggaa agaaagtggg agcagaagac attgaaaaaa caataaaggc atgcaaaccc 840
agtgaccaga tcctgaagct gctcagtttg tggcgaataa aaaatggcga ccaagacacc 900
ttgaagggcc taatgcacgc actaaagcac tcaaagacgt accactttcc caaaactgtc 960
actcagagtc taaagaagac catcaggttc cttcacagct tcacaatgta caaattgtat 1020
cagaagttat ttttagaaat gataggtaac caggtccaat cagtaaaaat aagctgctta 1080
taa 1083
<210> 89
<211> 1080
<212> DNA
<213> OCIF-DCR2
<400> 89
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccg aatgcaagga agggcgctac cttgagatag agttctgctt gaaacatagg 240
agctgccctc ctggatttgg agtggtgcaa gctggaaccc cagagcgaaa tacagtttgc 300
aaaagatgtc cagatgggtt cttctcaaat gagacgtcat ctaaagcacc ctgtagaaaa 360
cacacaaatt gcagtgtctt tggtctcctg ctaactcaga aaggaaatgc aacacacgac 420
aacatatgtt ccggaaacag tgaatcaact caaaaatgtg gaatagatgt taccctgtgt 480
gaggaggcat tcttcaggtt tgctgttcct acaaagttta cgcctaactg gcttagtgtc 540
ttggtagaca atttgcctgg caccaaagta aacgcagaga gtgtagagag gataaaacgg 600
caacacagct cacaagaaca gactttccag ctgctgaagt tatggaaaca tcaaaacaaa 660
gaccaagata tagtcaagaa gatcatccaa gatattgacc tctgtgaaaa cagcgtgcag 720
cggcacattg gacatgctaa cctcaccttc gagcagcttc gtagcttgat ggaaagctta 780
ccgggaaaga aagtgggagc agaagacatt gaaaaaacaa taaaggcatg caaacccagt 840
gaccagatcc tgaagctgct cagtttgtgg cgaataaaaa atggcgacca agacaccttg 900
aagggcctaa tgcacgcact aaagcactca aagacgtacc actttcccaa aactgtcact 960
cagagtctaa agaagaccat caggttcctt cacagcttca caatgtacaa attgtatcag 1020
aagttatttt tagaaatgat aggtaaccag gtccaatcag taaaaataag ctgcttataa 1080
<210> 90
<211> 1092
<212> DNA
<213> OCIF-DCR3
<400> 90
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcagatg tccagatggg ttcttctcaa atgagacgtc atctaaagca 360
ccctgtagaa aacacacaaa ttgcagtgtc tttggtctcc tgctaactca gaaaggaaat 420
gcaacacacg acaacatatg ttccggaaac agtgaatcaa ctcaaaaatg tggaatagat 480
gttaccctgt gtgaggaggc attcttcagg tttgctgttc ctacaaagtt tacgcctaac 540
tggcttagtg tcttggtaga caatttgcct ggcaccaaag taaacgcaga gagtgtagag 600
aggataaaac ggcaacacag ctcacaagaa cagactttcc agctgctgaa gttatggaaa 660
catcaaaaca aagaccaaga tatagtcaag aagatcatcc aagatattga cctctgtgaa 720
aacagcgtgc agcggcacat tggacatgct aacctcacct tcgagcagct tcgtagcttg 780
atggaaagct taccgggaaa gaaagtggga gcagaagaca ttgaaaaaac aataaaggca 840
tgcaaaccca gtgaccagat cctgaagctg ctcagtttgt ggcgaataaa aaatggcgac 900
caagacacct tgaagggcct aatgcacgca ctaaagcact caaagacgta ccactttccc 960
aaaactgtca ctcagagtct aaagaagacc atcaggttcc ttcacagctt cacaatgtac 1020
aaattgtatc agaagttatt tttagaaatg ataggtaacc aggtccaatc agtaaaaata 1080
agctgcttat aa 1092
<210> 91
<211> 1080
<212> DNA
<213> OCIF-DCR4
<400> 91
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gaaccccaga gcgaaataca 420
gtttgcaaat ccggaaacag tgaatcaact caaaaatgtg gaatagatgt taccctgtgt 480
gaggaggcat tcttcaggtt tgctgttcct acaaagttta cgcctaactg gcttagtgtc 540
ttggtagaca atttgcctgg caccaaagta aacgcagaga gtgtagagag gataaaacgg 600
caacacagct cacaagaaca gactttccag ctgctgaagt tatggaaaca tcaaaacaaa 660
gaccaagata tagtcaagaa gatcatccaa gatattgacc tctgtgaaaa cagcgtgcag 720
cggcacattg gacatgctaa cctcaccttc gagcagcttc gtagcttgat ggaaagctta 780
ccgggaaaga aagtgggagc agaagacatt gaaaaaacaa taaaggcatg caaacccagt 840
gaccagatcc tgaagctgct cagtttgtgg cgaataaaaa atggcgacca agacaccttg 900
aagggcctaa tgcacgcact aaagcactca aagacgtacc actttcccaa aactgtcact 960
cagagtctaa agaagaccat caggttcctt cacagcttca caatgtacaa attgtatcag 1020
aagttatttt tagaaatgat aggtaaccag gtccaatcag taaaaataag ctgcttataa 1080
<210> 92
<211> 981
<212> DNA
<213> OCIF-DDD1
<400> 92
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gaaccccaga gcgaaataca 420
gtttgcaaaa gatgtccaga tgggttcttc tcaaatgaga cgtcatctaa agcaccctgt 480
agaaaacaca caaattgcag tgtctttggt ctcctgctaa ctcagaaagg aaatgcaaca 540
cacgacaaca tatgttccgg aaacagtgaa tcaactcaaa aatgtggaat agatattgac 600
ctctgtgaaa acagcgtgca gcggcacatt ggacatgcta acctcacctt cgagcagctt 660
cgtagcttga tggaaagctt accgggaaag aaagtgggag cagaagacat tgaaaaaaca 720
ataaaggcat gcaaacccag tgaccagatc ctgaagctgc tcagtttgtg gcgaataaaa 780
aatggcgacc aagacacctt gaagggccta atgcacgcac taaagcactc aaagacgtac 840
cactttccca aaactgtcac tcagagtcta aagaagacca tcaggttcct tcacagcttc 900
acaatgtaca aattgtatca gaagttattt ttagaaatga taggtaacca ggtccaatca 960
gtaaaaataa gctgcttata a 981
<210> 93
<211> 984
<212> DNA
<213> OCIF-DDD2
<400> 93
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gaaccccaga gcgaaataca 420
gtttgcaaaa gatgtccaga tgggttcttc tcaaatgaga cgtcatctaa agcaccctgt 480
agaaaacaca caaattgcag tgtctttggt ctcctgctaa ctcagaaagg aaatgcaaca 540
cacgacaaca tatgttccgg aaacagtgaa tcaactcaaa aatgtggaat agatgttacc 600
ctgtgtgagg aggcattctt caggtttgct gttcctacaa agtttacgcc taactggctt 660
agtgtcttgg tagacaattt gcctggcacc aaagtaaacg cagagagtgt agagaggata 720
aaacggcaac acagctcaca agaacagact ttccagctgc tgaagttatg gaaacatcaa 780
aacaaagacc aagatatagt caagaagatc atccaagacg cactaaagca ctcaaagacg 840
taccactttc ccaaaactgt cactcagagt ctaaagaaga ccatcaggtt ccttcacagc 900
ttcacaatgt acaaattgta tcagaagtta tttttagaaa tgataggtaa ccaggtccaa 960
tcagtaaaaa taagctgctt ataa 984
<210> 94
<211> 1200
<212> DNA
<213> OCIF-CL
<400> 94
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gaaccccaga gcgaaataca 420
gtttgcaaaa gatgtccaga tgggttcttc tcaaatgaga cgtcatctaa agcaccctgt 480
agaaaacaca caaattgcag tgtctttggt ctcctgctaa ctcagaaagg aaatgcaaca 540
cacgacaaca tatgttccgg aaacagtgaa tcaactcaaa aatgtggaat agatgttacc 600
ctgtgtgagg aggcattctt caggtttgct gttcctacaa agtttacgcc taactggctt 660
agtgtcttgg tagacaattt gcctggcacc aaagtaaacg cagagagtgt agagaggata 720
aaacggcaac acagctcaca agaacagact ttccagctgc tgaagttatg gaaacatcaa 780
aacaaagacc aagatatagt caagaagatc atccaagata ttgacctctg tgaaaacagc 840
gtgcagcggc acattggaca tgctaacctc accttcgagc agcttcgtag cttgatggaa 900
agcttaccgg gaaagaaagt gggagcagaa gacattgaaa aaacaataaa ggcatgcaaa 960
cccagtgacc agatcctgaa gctgctcagt ttgtggcgaa taaaaaatgg cgaccaagac 1020
accttgaagg gcctaatgca cgcactaaag cactcaaaga cgtaccactt tcccaaaact 1080
gtcactcaga gtctaaagaa gaccatcagg ttccttcaca gcttcacaat gtacaaattg 1140
tatcagaagt tatttttaga aatgataggt aaccaggtcc aatcagtaaa aataagctaa 1200
<210> 95
<211> 1056
<212> DNA
<213> OCIF-CC
<400> 95
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gaaccccaga gcgaaataca 420
gtttgcaaaa gatgtccaga tgggttcttc tcaaatgaga cgtcatctaa agcaccctgt 480
agaaaacaca caaattgcag tgtctttggt ctcctgctaa ctcagaaagg aaatgcaaca 540
cacgacaaca tatgttccgg aaacagtgaa tcaactcaaa aatgtggaat agatgttacc 600
ctgtgtgagg aggcattctt caggtttgct gttcctacaa agtttacgcc taactggctt 660
agtgtcttgg tagacaattt gcctggcacc aaagtaaacg cagagagtgt agagaggata 720
aaacggcaac acagctcaca agaacagact ttccagctgc tgaagttatg gaaacatcaa 780
aacaaagacc aagatatagt caagaagatc atccaagata ttgacctctg tgaaaacagc 840
gtgcagcggc acattggaca tgctaacctc accttcgagc agcttcgtag cttgatggaa 900
agcttaccgg gaaagaaagt gggagcagaa gacattgaaa aaacaataaa ggcatgcaaa 960
cccagtgacc agatcctgaa gctgctcagt ttgtggcgaa taaaaaatgg cgaccaagac 1020
accttgaagg gcctaatgca cgcactaaag cactga 1056
<210> 96
<211> 819
<212> DNA
<213> OCIF-CDD2
<400> 96
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gaaccccaga gcgaaataca 420
gtttgcaaaa gatgtccaga tgggttcttc tcaaatgaga cgtcatctaa agcaccctgt 480
agaaaacaca caaattgcag tgtctttggt ctcctgctaa ctcagaaagg aaatgcaaca 540
cacgacaaca tatgttccgg aaacagtgaa tcaactcaaa aatgtggaat agatgttacc 600
ctgtgtgagg aggcattctt caggtttgct gttcctacaa agtttacgcc taactggctt 660
agtgtcttgg tagacaattt gcctggcacc aaagtaaacg cagagagtgt agagaggata 720
aaacggcaac acagctcaca agaacagact ttccagctgc tgaagttatg gaaacatcaa 780
aacaaagacc aagatatagt caagaagatc atccaatga 819
<210> 97
<211> 594
<212> DNA
<213> OCIF-CDD1
<400> 97
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gaaccccaga gcgaaataca 420
gtttgcaaaa gatgtccaga tgggttcttc tcaaatgaga cgtcatctaa agcaccctgt 480
agaaaacaca caaattgcag tgtctttggt ctcctgctaa ctcagaaagg aaatgcaaca 540
cacgacaaca tatgttccgg aaacagtgaa tcaactcaaa aatgtggaat atga 594
<210> 98
<211> 432
<212> DNA
<213> OCIF-CCR4
<400> 98
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gaaccccaga gcgaaataca 420
gtttgcaaat ga 432
<210> 99
<211> 321
<212> DNA
<213> OCIF-CCR3
<400> 99
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg a 321
<210> 100
<211> 1182
<212> DNA
<213> OCIF-CBst
<400> 100
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gaaccccaga gcgaaataca 420
gtttgcaaaa gatgtccaga tgggttcttc tcaaatgaga cgtcatctaa agcaccctgt 480
agaaaacaca caaattgcag tgtctttggt ctcctgctaa ctcagaaagg aaatgcaaca 540
cacgacaaca tatgttccgg aaacagtgaa tcaactcaaa aatgtggaat agatgttacc 600
ctgtgtgagg aggcattctt caggtttgct gttcctacaa agtttacgcc taactggctt 660
agtgtcttgg tagacaattt gcctggcacc aaagtaaacg cagagagtgt agagaggata 720
aaacggcaac acagctcaca agaacagact ttccagctgc tgaagttatg gaaacatcaa 780
aacaaagacc aagatatagt caagaagatc atccaagata ttgacctctg tgaaaacagc 840
gtgcagcggc acattggaca tgctaacctc accttcgagc agcttcgtag cttgatggaa 900
agcttaccgg gaaagaaagt gggagcagaa gacattgaaa aaacaataaa ggcatgcaaa 960
cccagtgacc agatcctgaa gctgctcagt ttgtggcgaa taaaaaatgg cgaccaagac 1020
accttgaagg gcctaatgca cgcactaaag cactcaaaga cgtaccactt tcccaaaact 1080
gtcactcaga gtctaaagaa gaccatcagg ttccttcaca gcttcacaat gtacaaattg 1140
tatcagaagt tatttttaga aatgataggt aacctagtct ag 1182
<210> 101
<211> 966
<212> DNA
<213> OCIF-CSph
<400> 101
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gaaccccaga gcgaaataca 420
gtttgcaaaa gatgtccaga tgggttcttc tcaaatgaga cgtcatctaa agcaccctgt 480
agaaaacaca caaattgcag tgtctttggt ctcctgctaa ctcagaaagg aaatgcaaca 540
cacgacaaca tatgttccgg aaacagtgaa tcaactcaaa aatgtggaat agatgttacc 600
ctgtgtgagg aggcattctt caggtttgct gttcctacaa agtttacgcc taactggctt 660
agtgtcttgg tagacaattt gcctggcacc aaagtaaacg cagagagtgt agagaggata 720
aaacggcaac acagctcaca agaacagact ttccagctgc tgaagttatg gaaacatcaa 780
aacaaagacc aagatatagt caagaagatc atccaagata ttgacctctg tgaaaacagc 840
gtgcagcggc acattggaca tgctaacctc accttcgagc agcttcgtag cttgatggaa 900
agcttaccgg gaaagaaagt gggagcagaa gacattgaaa aaacaataaa ggctagtcta 960
gactag 966
<210> 102
<211> 564
<212> DNA
<213> OCIF-CBsp
<400> 102
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gaaccccaga gcgaaataca 420
gtttgcaaaa gatgtccaga tgggttcttc tcaaatgaga cgtcatctaa agcaccctgt 480
agaaaacaca caaattgcag tgtctttggt ctcctgctaa ctcagaaagg aaatgcaaca 540
cacgacaaca tatgttccgg ctag 564
<210> 103
<211> 255
<212> DNA
<213> OCIF-CPst
<400> 103
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatacctag tctag 255
<210> 104
<211> 1317
<212> DNA
<213> human OCIF genome
<220>
<221> exon
<222> 1173..1193
<400> 104
ctggagacat ataacttgaa cacttggccc tgatggggaa gcagctctgc agggactttt 60
tcagccatct gtaaacaatt tcagtggcaa cccgcgaact gtaatccatg aatgggacca 120
cactttacaa gtcatcaagt ctaacttcta gaccagggaa ttaatggggg agacagcgaa 180
ccctagagca aagtgccaaa cttctgtcga tagcttgagg ctagtggaaa gacctcgagg 240
aggctactcc agaagttcag cgcgtaggaa gctccgatac caatagccct ttgatgatgg 300
tggggttggt gaagggaaca gtgctccgca aggttatccc tgccccaggc agtccaattt 360
tcactctgca gattctctct ggctctaact accccagata acaaggagtg aatgcagaat 420
agcacgggct ttagggccaa tcagacatta gttagaaaaa ttcctactac atggtttatg 480
taaacttgaa gatgaatgat tgcgaactcc ccgaaaaggg ctcagacaat gccatgcata 540
aagaggggcc ctgtaatttg aggtttcaga acccgaagtg aaggggtcag gcagccgggt 600
acggcggaaa ctcacagctt tcgcccagcg agaggacaaa ggtctgggac acactccaac 660
tgcgtccgga tcttggctgg atcggactct cagggtggag gagacacaag cacagcagct 720
gcccagcgtg tgcccagccc tcccaccgct ggtcccggct gccaggaggc tggccgctgg 780
cgggaagggg ccgggaaacc tcagagcccc gcggagacag cagccgcctt gttcctcagc 840
ccggtggctt ttttttcccc tgctctccca ggggacagac accaccgccc cacccctcac 900
gccccacctc cctgggggat cctttccgcc ccagccctga aagcgttaat cctggagctt 960
tctgcacacc ccccgaccgc tcccgcccaa gcttcctaaa aaagaaaggt gcaaagtttg 1020
gtccaggata gaaaaatgac tgatcaaagg caggcgatac ttcctgttgc cgggacgcta 1080
tatataacgt gatgagcgca cgggctgcgg agacgcaccg gagcgctcgc ccagccgccg 1140
cctccaagcc cctgaggttt ccggggacca ca atg aac aag ttg ctg tgc tgc 1193
Met Asn Lys Leu Leu Cys Cys
-20 -15
gcg ctc gtg gtaagtccct gggccagccg acgggtgccc ggcgcctggg 1242
ala leu val
gaggctgctg ccacctggtc tcccaacctc ccagcggacc ggcggggaaa aaggctccac 1302
tcgctccctc ccaag 1317
<210> 105
<211> 10190
<212> DNA
<213> human OCIF genome
<220>
<221> exon
<222> 131..499
<220>
<221> exon
<222> 4504..4694
<220>
<221> exon
<222> 5043..6939
<220>
<221> exon
<222> 8960..9347
<400> 105
gcttactttg tgccaaatct cattaggctt aaggtaatac aggactttga gtcaaatgat 60
actgttgcac ataagaacaa acctattttc atgctaagat gatgccactg tgttcctttc 120
tccttctag ttt ctg gac atc tcc att aag tgg acc acc cag gaa acg ttt 171
Phe Leu Asp Ile Ser Ile Lys Trp Thr Thr Gln Glu Thr Phe
-10 -5 -1 +1
cct cca aag tac ctt cat tat gac gaa gaa acc tct cat cag ctg ttg 219
Pro Pro Lys Tyr Leu His Tyr Asp Glu Glu Thr Ser His Gln Leu Leu
5 10 15
tgt gac aaa tgt cct cct ggt acc tac cta aaa caa cac tgt aca gca 267
Cys Asp Lys Cys Pro Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala
20 25 30 35
aag tgg aag acc gtg tgc gcc cct tgc cct gac cac tac tac aca gac 315
Lys Trp Lys Thr Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp
40 45 50
agc tgg cac acc agt gac gag tgt cta tac tgc agc ccc gtg tgc aag 363
Ser Trp His Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys
55 60 65
gag ctg cag tac gtc aag cag gag tgc aat cgc acc cac aac cgc gtg 411
Glu Leu Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val
70 75 80
tgc gaa tgc aag gaa ggg cgc tac ctt gag ata gag ttc tgc ttg aaa 459
Cys Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys
85 90 95
cat agg agc tgc cct cct gga ttt gga gtg gtg caa gct g gtacgtgtca 509
His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala
100 105 110
atgtgcagca aaattaatta ggatcatgca aagtcagata gttgtgacag tttaggagaa 569
cacttttgtt ctgatgacat tataggatag caaattgcaa aggtaatgaa acctgccagg 629
taggtactat gtgtctggag tgcttccaaa ggaccattgc tcagaggaat actttgccac 689
tacagggcaa tttaatgaca aatctcaaat gcagcaaatt attctctcat gagatgcatg 749
atggtttttt tttttttttt taaagaaaca aactcaagtt gcactattga tagttgatct 809
atacctctat atttcacttc agcatggaca ccttcaaact gcagcacttt ttgacaaaca 869
tcagaaatgt taatttatac caagagagta attatgctca tattaatgag actctggagt 929
gctaacaata agcagttata attaattatg taaaaaatga gaatggtgag gggaattgca 989
tttcattatt aaaaacaagg ctagttcttc ctttagcatg ggagctgagt gtttgggagg 1049
gtaaggacta tagcagaatc tcttcaatga gcttattctt tatcttagac aaaacagatt 1109
gtcaagccaa gagcaagcac ttgcctataa accaagtgct ttctcttttg cattttgaac 1169
agcattggtc agggctcatg tgtattgaat cttttaaacc agtaacccac gttttttttc 1229
tgccacattt gcgaagcttc agtgcagcct ataacttttc atagcttgag aaaattaaga 1289
gtatccactt acttagatgg aagaagtaat cagtatagat tctgatgact cagtttgaag 1349
cagtgtttct caactgaagc cctgctgata ttttaagaaa tatctggatt cctaggctgg 1409
actccttttt gtgggcagct gtcctgcgca ttgtagaatt ttggcagcac ccctggactc 1469
tagccactag ataccaatag cagtccttcc cccatgtgac agccaaaaat gtcttcagac 1529
actgtcaaat gtcgccaggt ggcaaaatca ctcctggttg agaacagggt catcaatgct 1589
aagtatctgt aactatttta actctcaaaa cttgtgatat acaaagtcta aattattaga 1649
cgaccaatac tttaggttta aaggcataca aatgaaacat tcaaaaatca aaatctattc 1709
tgtttctcaa atagtgaatc ttataaaatt aatcacagaa gatgcaaatt gcatcagagt 1769
cccttaaaat tcctcttcgt atgagtattt gagggaggaa ttggtgatag ttcctacttt 1829
ctattggatg gtactttgag actcaaaagc taagctaagt tgtgtgtgtg tcagggtgcg 1889
gggtgtggaa tcccatcaga taaaagcaaa tccatgtaat tcattcagta agttgtatat 1949
gtagaaaaat gaaaagtggg ctatgcagct tggaaactag agaattttga aaaataatgg 2009
aaatcacaag gatctttctt aaataagtaa gaaaatctgt ttgtagaatg aagcaagcag 2069
gcagccagaa gactcagaac aaaagtacac attttactct gtgtacactg gcagcacagt 2129
gggatttatt tacctctccc tccctaaaaa cccacacagc ggttcctctt gggaaataag 2189
aggtttccag cccaaagaga aggaaagact atgtggtgtt actctaaaaa gtatttaata 2249
accgttttgt tgttgctgtt gctgttttga aatcagattg tctcctctcc atattttatt 2309
tacttcattc tgttaattcc tgtggaatta cttagagcaa gcatggtgaa ttctcaactg 2369
taaagccaaa tttctccatc attataattt cacattttgc ctggcaggtt ataattttta 2429
tatttccact gatagtaata aggtaaaatc attacttaga tggatagatc tttttcataa 2489
aaagtaccat cagttataga gggaagtcat gttcatgttc aggaaggtca ttagataaag 2549
cttctgaata tattatgaaa cattagttct gtcattctta gattcttttt gttaaataac 2609
tttaaaagct aacttaccta aaagaaatat ctgacacata tgaacttctc attaggatgc 2669
aggagaagac ccaagccaca gatatgtatc tgaagaatga acaagattct taggcccggc 2729
acggtggctc acatctgtaa tctcaagagt ttgagaggtc aaggcgggca gatcacctga 2789
ggtcaggagt tcaagaccag cctggccaac atgatgaaac cctgcctcta ctaaaaatac 2849
aaaaattagc agggcatggt ggtgcatgcc tgcaacccta gctactcagg aggctgagac 2909
aggagaatct cttgaaccct cgaggcggag gttgtggtga gctgagatcc ctctactgca 2969
ctccagcctg ggtgacagag atgagactcc gtccctgccg ccgcccccgc cttccccccc 3029
aaaaagattc ttcttcatgc agaacatacg gcagtcaaca aagggagacc tgggtccagg 3089
tgtccaagtc acttatttcg agtaaattag caatgaaaga atgccatgga atccctgccc 3149
aaatacctct gcttatgata ttgtagaatt tgatatagag ttgtatccca tttaaggagt 3209
aggatgtagt aggaaagtac taaaaacaaa cacacaaaca gaaaaccctc tttgctttgt 3269
aaggtggttc ctaagataat gtcagtgcaa tgctggaaat aatatttaat atgtgaaggt 3329
tttaggctgt gttttcccct cctgttcttt ttttctgcca gccctttgtc atttttgcag 3389
gtcaatgaat catgtagaaa gagacaggag atgaaactag aaccagtcca ttttgcccct 3449
ttttttattt tctggttttg gtaaaagata caatgaggta ggaggttgag atttataaat 3509
gaagtttaat aagtttctgt agctttgatt tttctctttc atatttgtta tcttgcataa 3569
gccagaattg gcctgtaaaa tctacatatg gatattgaag tctaaatctg ttcaactagc 3629
ttacactaga tggagatatt ttcatattca gatacactgg aatgtatgat ctagccatgc 3689
gtaatatagt caagtgtttg aaggtattta tttttaatag cgtctttagt tgtggactgg 3749
ttcaagtttt tctgccaatg atttcttcaa atttatcaaa tatttttcca tcatgaagta 3809
aaatgccctt gcagtcaccc ttcctgaagt ttgaacgact ctgctgtttt aaacagttta 3869
agcaaatggt atatcatctt ccgtttacta tgtagcttaa ctgcaggctt acgcttttga 3929
gtcagcggcc aactttattg ccaccttcaa aagtttatta taatgttgta aatttttact 3989
tctcaaggtt agcatactta ggagttgctt cacaattagg attcaggaaa gaaagaactt 4049
cagtaggaac tgattggaat ttaatgatgc agcattcaat gggtactaat ttcaaagaat 4109
gatattacag cagacacaca gcagttatct tgattttcta ggaataattg tatgaagaat 4169
atggctgaca acacggcctt actgccactc agcggaggct ggactaatga acaccctacc 4229
cttctttcct ttcctctcac atttcatgag cgttttgtag gtaacgagaa aattgacttg 4289
catttgcatt acaaggagga gaaactggca aaggggatga tggtggaagt tttgttctgt 4349
ctaatgaagt gaaaaatgaa aatgctagag ttttgtgcaa cataatagta gcagtaaaaa 4409
ccaagtgaaa agtctttcca aaactgtgtt aagagggcat ctgctgggaa acgatttgag 4469
gagaaggtac taaattgctt ggtattttcc gtag ga acc cca gag cga aat aca 4523
gly thr pro glu arg asn thr
115
gtt tgc aaa aga tgt cca gat ggg ttc ttc tca aat gag acg tca tct 4571
Val Cys Lys Arg Cys Pro Asp Gly Phe Phe Ser Asn Glu Thr Ser Ser
120 125 130 135
aaa gca ccc tgt aga aaa cac aca aat tgc agt gtc ttt ggt ctc ctg 4619
Lys Ala Pro Cys Arg Lys His Thr Asn Cys Ser Val Phe Gly Leu Leu
140 145 150
cta act cag aaa gga aat gca aca cac gac aac ata tgt tcc gga aac 4667
Leu Thr Gln Lys Gly Asn Ala Thr His Asp Asn Ile Cys Ser Gly Asn
155 160 165
agt gaa tca act caa aaa tgt gga ata g gtaattacat tccaaaatac 4715
Ser Glu Ser Thr Gln Lys Cys Gly Ile
170 175
gtctttgtac gattttgtag tatcatctct ctctctgagt tgaacacaag gcctccagcc 4775
acattcttgg tcaaacttac attttccctt tcttgaatct taaccagcta aggctactct 4835
cgatgcatta ctgctaaagc taccactcag aatctctcaa aaactcatct tctcacagat 4895
aacacctcaa agcttgattt tctctccttt cacactgaaa tcaaatcttg cccataggca 4955
aagggcagtg tcaagtttgc cactgagatg aaattaggag agtccaaact gtagaattca 5015
cgttgtgtgt tattactttc acgaatgtct gtattattaa ctaaagtata tattggcaac 5075
taagaagcaa agtgatataa acatgatgac aaattaggcc aggcatggtg gcttactcct 5135
ataatcccaa cattttgggg ggccaaggta ggcagatcac ttgaggtcag gatttcaaga 5195
ccagcctgac caacatggtg aaaccttgtc tctactaaaa atacaaaaat tagctgggca 5255
tggtagcagg cacttctagt accagctact cagggctgag gcaggagaat cgcttgaacc 5315
caggagatgg aggttgcagt gagctgagat tgtaccactg cactccagtc tgggcaacag 5375
agcaagattt catcacacac acacacacac acacacacac acacattaga aatgtgtact 5435
tggctttgtt acctatggta ttagtgcatc tattgcatgg aacttccaag ctactctggt 5495
tgtgttaagc tcttcattgg gtacaggtca ctagtattaa gttcaggtta ttcggatgca 5555
ttccacggta gtgatgacaa ttcatcaggc tagtgtgtgt gttcaccttg tcactcccac 5615
cactagacta atctcagacc ttcactcaaa gacacattac actaaagatg atttgctttt 5675
ttgtgtttaa tcaagcaatg gtataaacca gcttgactct ccccaaacag tttttcgtac 5735
tacaaagaag tttatgaagc agagaaatgt gaattgatat atatatgaga ttctaaccca 5795
gttccagcat tgtttcattg tgtaattgaa atcatagaca agccatttta gcctttgctt 5855
tcttatctaa aaaaaaaaaa aaaaaaatga aggaaggggt attaaaagga gtgatcaaat 5915
tttaacattc tctttaatta attcattttt aattttactt tttttcattt attgtgcact 5975
tactatgtgg tactgtgcta tagaggcttt aacatttata aaaacactgt gaaagttgct 6035
tcagatgaat ataggtagta gaacggcaga actagtattc aaagccaggt ctgatgaatc 6095
caaaaacaaa cacccattac tcccattttc tgggacatac ttactctacc cagatgctct 6155
gggctttgta atgcctatgt aaataacata gttttatgtt tggttatttt cctatgtaat 6215
gtctacttat atatctgtat ctatctcttg ctttgtttcc aaaggtaaac tatgtgtcta 6275
aatgtgggca aaaaataaca cactattcca aattactgtt caaattcctt taagtcagtg 6335
ataattattt gttttgacat taatcatgaa gttccctgtg ggtactaggt aaacctttaa 6395
tagaatgtta atgtttgtat tcattataag aatttttggc tgttacttat ttacaacaat 6455
atttcactct aattagacat ttactaaact ttctcttgaa aacaatgccc aaaaaagaac 6515
attagaagac acgtaagctc agttggtctc tgccactaag accagccaac agaagcttga 6575
ttttattcaa actttgcatt ttagcatatt ttatcttgga aaattcaatt gtgttggttt 6635
tttgtttttg tttgtattga atagactctc agaaatccaa ttgttgagta aatcttctgg 6695
gttttctaac ctttctttag at gtt acc ctg tgt gag gag gca ttc ttc agg 6747
Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg
180 185
ttt gct gtt cct aca aag ttt acg cct aac tgg ctt agt gtc ttg gta 6795
Phe Ala Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val
190 195 200
gac aat ttg cct ggc acc aaa gta aac gca gag agt gta gag agg ata 6843
Asp Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg Ile
205 210 215
aaa cgg caa cac agc tca caa gaa cag act ttc cag ctg ctg aag tta 6891
Lys Arg Gln His Ser Ser Gln Glu Gln Thr Phe Gln Leu Leu Lys Leu
220 225 230 235
tgg aaa cat caa aac aaa gac caa gat ata gtc aag aag atc atc caa g 6940
Trp Lys His Gln Asn Lys Asp Gln Asp Ile Val Lys Lys Ile Ile Gln
240 245 250
gtaattacat tccaaaatac gtctttgtac gattttgtag tatcatctct ctctctgagt 7000
tgaacacaag gcctccagcc acattcttgg tcaaacttac attttccctt tcttgaatct 7060
taaccagcta aggctactct cgatgcatta ctgctaaagc taccactcag aatctctcaa 7120
aaactcatct tctcacagat aacacctcaa agcttgattt tctctccttt cacactgaaa 7180
tcaaatcttg cccataggca aagggcagtg tcaagtttgc cactgagatg aaattaggag 7240
agtccaaact gtagaattca cgttgtgtgt tattactttc acgaatgtct gtattattaa 7300
ctaaagtata tattggcaac taagaagcaa agtgatataa acatgatgac aaattaggcc 7360
aggcatggtg gcttactcct ataatcccaa cattttgggg ggccaaggta ggcagatcac 7420
ttgaggtcag gatttcaaga ccagcctgac caacatggtg aaaccttgtc tctactaaaa 7480
atacaaaaat tagctgggca tggtagcagg cacttctagt accagctact cagggctgag 7540
gcaggagaat cgcttgaacc caggagatgg aggttgcagt gagctgagat tgtaccactg 7600
cactccagtc tgggcaacag agcaagattt catcacacac acacacacac acacacacac 7660
acacattaga aatgtgtact tggctttgtt acctatggta ttagtgcatc tattgcatgg 7720
aacttccaag ctactctggt tgtgttaagc tcttcattgg gtacaggtca ctagtattaa 7780
gttcaggtta ttcggatgca ttccacggta gtgatgacaa ttcatcaggc tagtgtgtgt 7840
gttcaccttg tcactcccac cactagacta atctcagacc ttcactcaaa gacacattac 7900
actaaagatg atttgctttt ttgtgtttaa tcaagcaatg gtataaacca gcttgactct 7960
ccccaaacag tttttcgtac tacaaagaag tttatgaagc agagaaatgt gaattgatat 8020
atatatgaga ttctaaccca gttccagcat tgtttcattg tgtaattgaa atcatagaca 8080
agccatttta gcctttgctt tcttatctaa aaaaaaaaaa aaaaaaatga aggaaggggt 8140
attaaaagga gtgatcaaat tttaacattc tctttaatta attcattttt aattttactt 8200
tttttcattt attgtgcact tactatgtgg tactgtgcta tagaggcttt aacatttata 8260
aaaacactgt gaaagttgct tcagatgaat ataggtagta gaacggcaga actagtattc 8320
aaagccaggt ctgatgaatc caaaaacaaa cacccattac tcccattttc tgggacatac 8380
ttactctacc cagatgctct gggctttgta atgcctatgt aaataacata gttttatgtt 8440
tggttatttt cctatgtaat gtctacttat atatctgtat ctatctcttg ctttgtttcc 8500
aaaggtaaac tatgtgtcta aatgtgggca aaaaataaca cactattcca aattactgtt 8560
caaattcctt taagtcagtg ataattattt gttttgacat taatcatgaa gttccctgtg 8620
ggtactaggt aaacctttaa tagaatgtta atgtttgtat tcattataag aatttttggc 8680
tgttacttat ttacaacaat atttcactct aattagacat ttactaaact ttctcttgaa 8740
aacaatgccc aaaaaagaac attagaagac acgtaagctc agttggtctc tgccactaag 8800
accagccaac agaagcttga ttttattcaa actttgcatt ttagcatatt ttatcttgga 8860
aaattcaatt gtgttggttt tttgtttttg tttgtattga atagactctc agaaatccaa 8920
ttgttgagta aatcttctgg gttttctaac ctttctttag at att gac ctc tgt 8974
Asp Ile Asp Leu Cys
255
gaa aac agc gtg cag cgg cac att gga cat gct aac ctc acc ttc gag 9022
Glu Asn Ser Val Gln Arg His Ile Gly His Ala Asn Leu Thr Phe Glu
260 265 270
cag ctt cgt agc ttg atg gaa agc tta ccg gga aag aaa gtg gga gca 9070
Gln Leu Arg Ser Leu Met Glu Ser Leu Pro Gly Lys Lys Val Gly Ala
275 280 285
gaa gac att gaa aaa aca ata aag gca tgc aaa ccc agt gac cag atc 9118
Glu Asp Ile Glu Lys Thr Ile Lys Ala Cys Lys Pro Ser Asp Gln Ile
290 295 300
ctg aag ctg ctc agt ttg tgg cga ata aaa aat ggc gac caa gac acc 9166
Leu Lys Leu Leu Ser Leu Trp Arg Ile Lys Asn Gly Asp Gln Asp Thr
305 310 315 320
ttg aag ggc cta atg cac gca cta aag cac tca aag acg tac cac ttt 9214
Leu Lys Gly Leu Met His Ala Leu Lys His Ser Lys Thr Tyr His Phe
325 330 335
ccc aaa act gtc act cag agt cta aag aag acc atc agg ttc ctt cac 9262
Pro Lys Thr Val Thr Gln Ser Leu Lys Lys Thr Ile Arg Phe Leu His
340 345 350
agc ttc aca atg tac aaa ttg tat cag aag tta ttt tta gaa atg ata 9310
Ser Phe Thr Met Tyr Lys Leu Tyr Gln Lys Leu Phe Leu Glu Met Ile
355 360 365
ggt aac cag gtc caa tca gta aaa ata agc tgc tta taactggaaa 9356
Gly Asn Gln Val Gln Ser Val Lys Ile Ser Cys Leu
370 375 380
tggccattga gctgtttcct cacaattggc gagatcccat ggatgagtaa actgtttctc
9416aggcacttga ggctttcagt gatatctttc tcattaccag tgactaattt tgccacaggg
9476tactaaaaga aactatgatg tggagaaagg actaacatct cctccaataa accccaaatg
9536gttaatccaa ctgtcagatc tggatcgtta tctactgact atattttccc ttattactgc
9596ttgcagtaat tcaactggaa attaaaaaaa aaaaactaga ctccactggg ccttactaaa
9656tatgggaatg tctaacttaa atagctttgg gattccagct atgctagagg cttttattag
9716aaagccatat ttttttctgt aaaagttact aatatatctg taacactatt acagtattgc
9776tatttatatt cattcagata taagatttgg acatattatc atcctataaa gaaacggtat
9836gacttaattt tagaaagaaa attatattct gtttattatg acaaatgaaa gagaaaatat
9896atatttttaa tggaaagttt gtagcatttt tctaataggt actgccatat ttttctgtgt
9956ggagtatttt tataatttta tctgtataag ctgtaatatc attttataga aaatgcatta 10
016tttagtcaat tgtttaatgt tggaaaacat atgaaatata aattatctga atattagatg 100
76ctctgagaaa ttgaatgtac cttatttaaa agattttatg gttttataac tatataaatg 1013
6acattattaa agttttcaaa ttatttttta ttgctttctc tgttgctttt attt 1019
0
【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]
BACKGROUND OF THE INVENTION
The present invention relates to a novel protein exhibiting an activity of suppressing osteoclast differentiation and / or maturation, ie, osteoclastogenesis inhibitory factor (OCIF) and a method for producing the same.
[0002]
[Prior art]
Human bones are constantly resorbing and remodeling, and the cells that play a central role in this process are osteoblasts responsible for bone formation and osteoclasts responsible for bone resorption. Osteoporosis is a representative example of a disease caused by abnormal bone metabolism, which is in charge of these cells. This disease is a disease that occurs when bone formation by osteoblasts exceeds bone resorption by osteoclasts. Although the mechanism of occurrence of this disease has not yet been fully elucidated, this disease is a disease that causes bone pain and causes fractures due to bone weakening. As the elderly population increases, this disease, which causes the generation of bedridden elderly people due to fractures, has also become a social problem, and the development of therapeutic drugs is urgently needed. Such bone loss due to abnormal bone metabolism is expected to be treated by suppressing bone resorption, promoting bone formation, or improving the balance thereof.
[0003]
Bone formation is expected to be promoted by promoting the growth, differentiation and activation of cells responsible for bone formation, or by inhibiting the growth, differentiation and activation of cells responsible for bone resorption. In recent years, interest in bioactive proteins (cytokines) having such activity has increased, and energetic research has been conducted. As cytokines that promote the proliferation or differentiation of osteoblasts, fibroblast growth factor family (FGF, Non-Patent Document 1), 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, Non-patent document 5), Vasculotropin (Non-patent document 6), and ectopic bone morphogenetic protein family (bone morphogenetic protein; BMP: BMP-2, Non-patent document 7, OP-1, Non-patent documents 8 and 9) 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 (non-patent document 10) and interleukin-4 (interleukin-4) 4, Non-Patent Document 11) etc. have been reported. Moreover, as cytokine which suppresses bone resorption by osteoclast, calcitonin (calcitonin, nonpatent literature 12), macrophage colony-stimulating factor (macrophage colony-stimulating factor ;, nonpatent literature 13), interleukin-4 (nonpatent literature) Reference 14), interferon-γ (non-patent reference 15) and the like have been reported.
[0005]
These cytokines are expected to improve bone loss by promoting bone formation and suppressing bone resorption. Insulin-like growth factor-I and ectopic bone morphogenetic family cytokines Some cytokines are undergoing clinical trials as bone metabolism improving agents. Calcitonin is already on the market as a therapeutic agent for osteoporosis and a pain relieving agent.
[0006]
Currently, it is active vitamin D in the clinical setting as a medicine to treat bone-related diseases and shorten the treatment period._Three, Calcitonin and its derivatives, hormone preparations such as estradiol, ipriflavone, vitamin K₂(Menatetrenone) or calcium preparations are used. However, treatment methods using these drugs are not always satisfactory in terms of their effects and treatment results, and the development of new therapeutic agents to replace them has been desired. As mentioned 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 osteopenia and other osteopenia. The
[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, p469, 1986
[0008]
[Problems to be solved by the invention]
The present invention has been made from such a viewpoint, and an object of the present invention is to provide a novel osteoclast formation inhibitor (OCIF) and an efficient production method thereof.
[0009]
[Means for Solving the Problems]
As a result of diligent search in view of such a current situation, the present inventors have found that osteoclast formation inhibitory activity in the culture medium of human fetal lung fibroblast IMR-90 (ATCC deposit-deposit number CCL186), We have found OCIF, a protein that has the activity of inhibiting differentiation and maturation.
Further, it has been found that when an alumina ceramic piece is used as a cell culture carrier, the osteoclast formation inhibitor OCIF of the present invention accumulates in a high concentration in the medium and can be purified efficiently.
Furthermore, the present inventors established a method for efficiently purifying the protein OCIF by treating the culture broth sequentially with an ion exchange column, an affinity column, and a reverse phase column and repeating adsorption and elution.
[0010]
Next, the present inventors succeeded in cloning a cDNA encoding this protein based on the amino acid sequence information of the obtained natural OCIF protein. Furthermore, the present inventors have established a method for producing a protein having osteoclast differentiation and / or maturation inhibitory activity by genetic engineering techniques using this cDNA.
[0011]
The present invention is derived from human fetal lung fibroblasts, has a molecular weight of about 60 kD on SDS-PAGE under reducing conditions, and a molecular weight of about 60 kDa and about 120 kD on SDS-PAGE under non-reducing conditions. It has affinity for the column, and the activity to suppress osteoclast differentiation and maturation is reduced by heat treatment at 70 ° C for 10 minutes or 56 ° C for 30 minutes. The present invention relates to a protein characterized in that its differentiation / maturation inhibitory activity is lost. The amino acid sequence of the protein OCIF of the present invention is clearly different from known osteoclast formation inhibitors.
[0012]
The present invention also cultivates human fibroblasts, treats the culture solution with a heparin column, elutes the adsorbed fraction, adsorbs and elutes this fraction through a cation exchange column, and further affinity column, reverse phase column It is related with the manufacturing method of protein OCIF which collect | recovers said protein after refine | purifying by. The column treatment in the present invention is not limited to simply flowing the culture solution or the like down to a heparin sepharose column or the like, but also includes those that exhibit the same effect as when the culture solution is mixed with heparin sepharose or the like by a batch method and column treatment is performed. To 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. Examples of the packing material for the Cibacron Blue column include those in which a hydrophilic synthetic polymer is used as a carrier and the pigment Cibacron Blue F3GA is bound, and this column is usually called a blue column.
Furthermore, the present invention relates to a method for efficiently producing the protein by performing cell culture using an alumina ceramic piece as a carrier.
[0013]
The protein OCIF of the present invention can be isolated and purified efficiently from human fibroblast culture broth with high yield. The isolation and purification of the protein OCIF of the present invention from this raw material utilized the physical and chemical properties of the target protein OCIF using the usual methods commonly used for the purification of proteinaceous substances from biological samples. It can be carried out according to various purification operations. Examples of the concentration means include ordinary biochemical treatment means such as ultrafiltration, lyophilization, and salting out. As purification means, various ion exchange chromatography, affinity chromatography, gel filtration chromatography, hydrophobic chromatography, reverse phase chromatography, various types used for purification of ordinary protein substances using preparative electrophoresis, etc. These methods can be used in combination. It is particularly preferable to use human fetal lung fibrocyte IMR-90 (ATCC-CCL 186) as a human fibroblast used as a raw material. Then, the human fetal lung fibroblast IMR-90 as a raw material was cultured using a DMEM medium containing human fetal lung fibroblast IMR-90 attached to an alumina ceramic piece and supplemented with 5% bovine neonatal serum as a culture solution. It is good to use what was obtained by carrying out stationary culture for about 10 days from a week in a roller bottle. Further, it is desirable to add 0.1% CHAPS (3-[(3-cholamidopropyl) -dimethylammonio] -1-propanesulfonate) as a surfactant during the purification treatment.
[0014]
The protein OCIF of the present invention is prepared by first applying the culture solution to a heparin column (heparin-Sepharose CL-6B, Pharmacia) and eluting with a 10 mM Tris-HCl buffer solution containing 2M NaCl, pH 7.5, and the 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 basic OCIF fraction. it can. The obtained OCIF active fractions were 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 to a phase column (BU-300 C4, Perkin Elmer), and this material is specified by the above-mentioned properties.
[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 this cDNA to differentiate osteoclasts and / or 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 treated with an endoprotease (eg, 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 prepared. .
Next, using the prepared oligonucleotide mixture as a primer, an OCIF cDNA fragment is obtained by PCR (preferably RT-PCR). Using this OCIF cDNA fragment as a probe, OCIF full-length cDNA is cloned from a cDNA library. Recombinant OCIF can be produced by inserting the obtained OCIF cDNA into an expression vector to prepare an OCIF expression plasmid, which is introduced into various cells or strains and expressed.
[0017]
The present invention also relates to novel proteins OCIF2, OCIF3, OCIF4, OCIF5, which are analogs (variants) of the OCIF protein of the present invention having the above-mentioned activity.
These analogs are poly (A) of IMR-90 cells.⁺A cDNA library prepared using RNA can be obtained by hybridizing using an OCIF cDNA fragment as a probe. By inserting the cDNA of these OCIF analogs into an expression vector, expressing the OCIF analog expression vector in a normal host, and purifying it by a conventional method, the target analog protein can be obtained.
[0018]
The present invention also relates to OCIF mutants.
These mutants are obtained by substituting Ser residues for Cys residues that may be involved in OCIF dimer formation, or by introducing deletion mutations into natural OCIF. Substitution or deletion mutations are introduced into OCIF cDNA by PCR or restriction enzyme digestion. This cDNA is inserted into a vector having an appropriate expression promoter, transfected into a eukaryotic cell such as a mammalian cell, this cell is cultured, and purified from the culture solution by a conventional method to obtain the target 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.
The anti-OCIF polyclonal antibody is prepared by a conventional method using OCIF as an immunogen. Antigens (immunogens) used at this time include natural OCIF obtained from IMR-90 culture, and recombinant OCIF produced using microorganisms and eukaryotic cells as hosts using OCIF cDNA, or the amino acid sequence of OCIF. Synthetic peptides designed based on these and OCIF hydrolyzed 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 immunoadjuvant, and purifying the serum by a conventional method. By labeling the obtained anti-OCIF polyclonal antibody with an isotope or 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 OCIF concentrations in biological samples such as blood and ascites and cell culture fluid.
[0020]
The present invention also relates to an anti-OCIF monoclonal antibody and a method for measuring OCIF using the same.
The anti-OCIF monoclonal antibody is prepared by a conventional method using OCIF as an immunogen. Antigens include natural OCIF obtained from IMR-90 culture, and 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, It may be a hydrolyzed partial peptide of OCIF. Immunize mammals with these antigens or fuse immunized cells with mammalian myeloma cells (myeloma) to produce hybridomas, and produce antibodies that recognize OCIF from these hybridomas The desired antibody is obtained by selecting a clone to be cultured and culturing this clone. In producing hybridomas, when mammals are used, examples using small animals such as mice and rats are common.
For immunization, OCIF is diluted to an appropriate concentration with physiological saline, etc., and this solution is administered intravenously or intraperitoneally. -5 doses. The immunized animal is dissected, the spleen is removed, and the 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, S194, and the like.
Examples of rat-derived cells include cell lines such as R-210. When producing human antibodies, human B lymphocytes are immunized by an in vitro method, and a hybridoma producing human antibodies is obtained by using a cell line transformed with human myeloma cells or EB virus as a parent strain. be able to.
[0022]
Fusion of immune cells and myeloma cell lines includes known methods such as 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) in the ratio of the usual number of cells, added with polyethylene glycol, subjected to fusion treatment, and HAT selection medium. Culturing can be performed to select fused cells.
[0023]
In order to select an anti-OCIF antibody-producing strain, it can be selected using a method commonly used for antibody detection, such as an ELISA method, a plaque method, an octalony method, and an agglutination method. The hybridoma thus selected can be subcultured by a normal culture method, and can be stored frozen as necessary. The antibody can be produced by culturing the hybridoma by a conventional method or transplanting it into the abdominal cavity of a mammal. The antibody can be purified by a conventional method such as salting out, gel filtration or affinity chromatography.
[0024]
The obtained antibody reacts specifically with OCIF and can be used for the measurement and purification of OCIF. When used for OCIF measurement, the antibody can be labeled with an isotope or enzyme to be used in a radioimmunoassay (RIA) or enzyme immunoassay (EIA) measurement system. In particular, the antibody obtained by the present invention has a feature that it can be used for sandwich immunoassay because the antigen recognition sites thereof are different. By using this measurement system, it is possible to easily measure OCIF concentrations in biological samples such as blood and ascites and cell culture fluid.
[0025]
OCIF activity should be measured according to Masayoshi Kumegawa et al. (Protein, nucleic acid, enzyme, Vol. 34, p999 (1989)) and Takahashi N. et al. (Endocrinology, Vol. 122, p1373 (1988)). Can do. Namely, about 17 days old mouse bone marrow cells are used as target cells, and active vitamin D₃Inhibition of osteoclast formation in the presence of (Calcitriol) can be tested by inhibiting induction of tartrate-resistant acid phosphatase activity.
[0026]
Osteoclast formation inhibitory factor OCIF, which is a protein of the present invention, is used to treat bone loss disorders such as osteoporosis, bone metabolic disorders such as rheumatism or osteoarthritis, or bone metabolism disorders such as multiple myeloma and It is useful as a pharmaceutical composition for the purpose of improvement or as an antigen for establishing immunological diagnosis of such diseases. 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 inhibitor OCIF as an active ingredient.
[0027]
Examples of the form of the pharmaceutical composition include an injectable composition, a drip composition, a suppository, a nasal agent, a sublingual agent, a transdermal absorption agent, and the like. In the case of an injectable composition, it is a mixture of a pharmacologically effective amount of the osteoclast formation inhibitory factor of the present invention and a pharmaceutically acceptable carrier, among which amino acids, saccharides, cellulose derivatives, and others Excipients / activators generally added to injectable compositions such as organic / inorganic compounds can also be used. In addition, when preparing an injection using the osteoclast formation inhibitor OCIF of the present invention and these excipients / activators, a pH adjuster, a buffer, a stabilizer, a solubilizer, etc., if necessary Can be made into various injections by conventional methods.
[0028]
DETAILED DESCRIPTION OF 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.
[Example 1]
Human fibroblast IMR- 90 Preparation of culture solution
Human fetal lung fibroblast IMR-90 (ATCC-CCL186) is a roller bottle (490cm², 110 x 171 mm, Corning) and attached to 80 g of alumina ceramic pieces (alumina 99.5%, Toshiba Ceramics) and cultured. For culture, 60 roller bottles are used, and 500 ml of DMEM medium (Gibco BRL) supplemented with 10 mM HEPES buffer supplemented with 5% calf serum per roller bottle is used at 37 ° C and 5% CO.₂In the presence, the culture was stationary for 7 to 10 days. After the culture, the culture broth was collected, and a new culture medium was added to obtain 301 IMR-90 culture broth in one culture. The obtained culture broth was designated as Sample 1.
[0029]
[Example 2]
Method for measuring osteoclast formation inhibitory activity
The activity of the protein osteoclast formation inhibitory factor of the present invention is measured by Masayoshi Kumegawa et al. (Protein / Nucleic Acid / Enzyme Vol.34 p999 (1989)) and Takahashi N. et al. (Endocrinology vol.122 p1373 ( 1988)). That is, using bone marrow cells isolated from about 17 days old mice, active vitamin D_ThreeOsteoclast formation in the presence was tested by using the induction of tartrate-resistant acid phosphatase activity as an indicator and measuring its inhibitory activity. That is, 2 x 10 in a 96-well microplate^-8M active vitamin D_Three100 μl of a sample diluted with α-MEM medium (Gibco BRL) containing 10% fetal bovine serum and 3 × 10 bone marrow cells obtained from a mouse about 17 days after birth^FiveAre suspended in α-MEM medium containing 100 μl of 10% fetal calf serum, seeded, and 5% CO₂The cells were cultured at 37 ° C. and 100% humidity for 1 week. On the 3rd and 5th day of culture, 160 μl of culture broth was discarded and 1 × 10^-8M active vitamin D_Three And 160 μl of a sample diluted with α-MEM medium containing 10% fetal bovine serum was added. After 7 days of culturing, the cells were washed with phosphate buffered saline, fixed with ethanol / acetone (1: 1) solution for 1 minute at room temperature, and osteoclast formation was measured using an acid phosphatase activity measurement kit (Acid Phosphatase, Leucocyte). , Catalog No. 387-A, Sigma). The decrease in acid phosphatase activity positive cells in the presence of tartaric acid was defined as OCIF activity.
[0030]
[Example 3]
OCIF Purification
i) Heparin Sepharose CL-6B Purification by
About 90 liters of IMR-90 culture solution (Sample 1) was added to a 0.22 μm filter (hydrophilic millidisc, 2,000 cm², Millipore), and heparin-Sepharose CL-6B column equilibrated with 10 mM Tris-HCl buffer solution (hereinafter referred to as Tris-HCl), pH 7.5 containing 0.3 M NaCl in three portions. 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 performed with 10 mM Tris-HCl, pH 7.5 containing 2 M NaCl to obtain 900 ml of heparin / sepharose CL-6B adsorption fraction, The obtained fraction was designated 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 0.1%, and the mixture was allowed to stand at 4 ° C. overnight. The mixture was applied to an anion exchange column (HiLoad-Q / FF, 2.6 × 10 cm, Pharmacia) equilibrated with 50 mM Tris-HCl, pH 7.5 containing% 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
The HiLoad-Q non-adsorbed fraction (sample 3) was applied to a cation exchange column (HiLoad-S / HP, 2.6 × 10 cm, Pharmacia) equilibrated with 50 mM Tris-HCl, pH 7.5 containing 0.1% CHAPS. . After washing with 50 mM Tris-HCl, pH 7.5 containing 0.1% CHAPS, elution was carried out with a linear gradient of NaCl of 1 M for 100 minutes and a flow rate of 8 ml / min, and fractionation was carried out at 12 ml / fraction. Fractions 1 to 40 were collected into 4 fractions of 10 fractions, and OCIF activity was measured using 100 μl of each. OCIF activity was observed in fractions 11-30 (Fig. 1: In the figure, ++ is an activity that inhibits osteoclast formation by 80% or more, and + is an activity that inhibits osteoclast formation by 30 to 80%. ,-Indicates that no activity is detected). Samples 4 were fractions 21 to 30 having higher specific activity.
[0033]
iv) Affinity column (heparin -5PW Purification by
120 ml of sample 4 was diluted with 240 ml of 50 mM Tris-HCl, pH 7.5 containing 0.1% CHAPS, and then equilibrated with 50 mM Tris-HCl, pH 7.5 containing 0.1% CHAPS (heparin-5PW, 0.8 ) After washing with 50 mM Tris-HCl, pH 7.5 containing 0.1% CHAPS, elution was performed at a flow rate of 0.5 ml / min with a linear gradient of 2M NaCl over 60 minutes and fractionated at 0.5 ml / fraction. It was. OCIF activity was measured using 50 μl of each fraction to obtain 10 ml of an OCIF active fraction eluted with about 0.7 to 1.3 M NaCl.
[0034]
v) Affinity column (blue) -5PW Purification by
A 10 ml sample 5 was diluted with 190 ml of 50 mM Tris-HCl, pH 7.5 containing 0.1% CHAPS, and then equilibrated with 50 mM Tris-HCl, pH 7.5 containing 0.1% CHAPS (Blue-5PW, 0.5 × 5.0 cm, Tosoh Corporation). After washing with 50 mM Tris-HCl, pH 7.5 containing 0.1% CHAPS, elution was performed with a linear gradient of 2M NaCl over 60 minutes and a flow rate of 0.5 ml / min, and fractionation was carried out at 0.5 ml / fraction. It was. OCIF activity was measured using 25 μl of each fraction, and OCIF activity fractions 49 to 70 eluted with about 1.0 to 1.6 M NaCl were obtained (in FIG. 2, ++ suppresses osteoclast formation by 80% or more). Activity, + indicates activity of inhibiting osteoclast formation by 30-80%).
[0035]
vi) Purification by reverse phase column
A reverse phase column (BU-300, C4, 2.1 × 220 mm) equilibrated with 25% acetonitrile containing 0.1% TFA after adding 10 μl of 25% TFA (trifluoroacetic acid) to 49-50 ml of the obtained fraction. Elution was carried out with a linear gradient of 55% acetonitrile over 60 minutes and a flow rate of 0.2 ml / min, and each peak was collected (FIG. 3). OCIF activity was measured using 100 μl of each peak fraction, and the activity was detected in peaks 6 and 7 in a concentration-dependent manner. The results are shown in Table 1.
[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]
[Example 4]
OCIF Molecular weight measurement
Using 40 μl each of peak 6 and peak 7 in which OCIF activity was observed, SDS-polyacrylamide gel electrophoresis was performed under reducing conditions and non-reducing conditions. That is, 20 μl of each peak fraction was collected in two tubes, concentrated under reduced pressure, dissolved in 10 mM Tris-HCl, pH 8 1.5 μl containing 1 mM EDTA, 2.5% SDS, and 0.01% bromophenol blue. After standing overnight at 37 ° C. under reducing conditions and reducing conditions (in the presence of 5% 2-mercaptoethanol), 1 μl of each was loaded onto SDS-polyacrylamide gel electrophoresis. Electrophoresis was performed using a 10-15% acrylamide gradient gel (Pharmacia) and an electrophoresis apparatus Phast System (Pharmacia). As molecular weight markers, phosphorylase b (94 kD), bovine serum albumin (67 kD), ovalbumin (43 kD), carbonic anhydrase (30 kD), trypsin inhibitor (20.0 kD), α-lactalbumin (14.4 kD) were used. After the completion of electrophoresis, silver staining was performed using Phast Gel Silver Stain Kit (Pharmacia). The results are shown in FIG.
As a result, for 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 under reducing conditions and about 120 kDa under non-reducing conditions was detected. Therefore, peak 7 is considered to be a homodimer of the protein of peak 6.
[0038]
[Example 5]
OCIF Thermal stability test
Take 20 μl of each sample mixed with Blue 5PW fractions 51-52, add 30 μl of 10 mM phosphate buffered saline, pH 7.2, and then 30 minutes at 70 ° C. and 90 ° C. for 10 minutes or 56 ° C. Heat treatment was performed for a minute. Using this sample, OCIF activity was measured according to the method described in Example 2. The results are shown in Table 2.
[0039]
[Table 2]
OCIF thermal stability

(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]
[Example 6]
Determination of internal amino acid sequence
For Blue-5PW fractions 51-70, mix 2 fractions to make 1 ml, add 10 μl of 25% TFA to each sample, then equilibrate with 25% acetonitrile containing 0.1% TFA in 10 ml portions. Applied to a reversed-phase column (BU-300, C4, 2.1 x 220 mm, PerkinElmer) and eluted with a linear gradient of 55% acetonitrile over 60 minutes at a flow rate of 0.2 ml / min. Peak 6 and Peak 7 Collected. About the obtained peak 6 and a part of peak 7, N-terminal amino acid sequence analysis was performed using a protein sequencer (Procise, type 494, PerkinElmer), but analysis was impossible and the N-terminal of these proteins was The possibility of being blocked was suggested. Therefore, the internal amino acid sequences of these proteins were analyzed. Specifically, after peak 6 and peak 7 were each concentrated by centrifugation, 100 μg dithiothreitol, 10 mM EDTA, 7 M guanidine hydrochloride, and 0.5 M Tris-HCl containing 1% CHAPS, pH 8.5 50 μl were added at room temperature. After reducing by standing for 4 hours, 0.2 μl of 4-vinylpyridine was added, and the mixture was left overnight in the dark at room temperature to pyridylethylate. Add 1 μl of 25% TFA to these samples, apply to a reverse phase column (BU-300, C4, 2.1 × 30 mm, PerkinElmer) equilibrated with 20% acetonitrile containing 0.1% TFA, and adjust the acetonitrile concentration in 30 minutes. Elution was performed with a linear gradient of 50% and a flow rate of 0.3 ml / min to obtain a reduced pyridylethylated OCIF sample. Each reduced pyridylethylated sample is centrifuged and dissolved in 0.1 M Tris-HCl, pH 9 25 μl containing 8 M urea and 0.1% Tween 80, then diluted with 73 μl 0.1 M Tris-HCl, pH 9 and 0.02 μg AP1 (lysyl endoprotease, Wako Pure Chemical Industries, Ltd.) was added and reacted at 37 ° C. for 15 hours. Add 1 μl of 25% TFA to the reaction mixture, apply to a reverse phase column (RP-300, C8, 2.1 × 220 mm, PerkinElmer) equilibrated with 0.1% TFA, and linearly adjust the acetonitrile concentration to 50% in 70 minutes. The peptide fragment was obtained by elution at a flow rate of 0.2 ml / min (FIG. 5). The obtained peptide fragments (P1 to P3) were subjected to amino acid sequence analysis using a protein sequencer. The results are shown in Sequence Listing SEQ ID NOs: 1-3.
[0041]
[Example 7]
cDNA Determination of sequence
i) IMR-90 Poly from cells (A) ⁺ RNA Isolation of
Poly (A) in IMR-90 cells⁺RNA was isolated using a fast track mRNA isolation kit (Invitrogen) according to the manual. 1X10 by this method⁸Approximately 10 μg of poly (A) from a single IMR-90 cell⁺RNA was obtained.
[0042]
ii) Preparation of mixed primer
Based on the amino acid sequence of the peptide obtained previously (SEQ ID NO: 2 and 3), the following two kinds of mixed primers were synthesized. That is, a mixture of oligonucleotides (mixed primer, No. 2F) having all base sequences capable of encoding the amino acid sequence from the 6th (Gln) to the 12th (Leu) of peptide P2 was synthesized. In addition, a mixture of complementary oligonucleotides (mixed primer, No. 3R) for all base sequences capable of encoding the amino acid sequence from the 6th (His) to the 12th (Lys) of P3 of the peptide was synthesized. Table 3 shows the base sequences of the mixed primers used.
[0043]
[Table 3]

[0044]
iii) OCIF cDNA Fragment PCR Amplification by
Poly (A) obtained in Example 7-i)⁺Using Superscript II cDNA synthesis kit (Gibco BRL) with RNA and 1Mg as a template, 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). To obtain an OCIF cDNA fragment. The conditions are shown below.
[0045]
10X Ex Taq buffer (Takara Shuzo) 5 μl
2.5 mM dNTP 4 μl
cDNA solution 1 μl
Ex Taq (Takara Shuzo) 0.25 μl
Distilled water 29.75 μl
40 μM Primer No.2F 5 μl
40 μM Primer No.3R 5 μl
[0046]
After mixing the above solution in a microcentrifuge tube, PCR was performed under the following conditions. After pretreatment at 95 ° C. for 3 minutes, a three-step reaction of 95 ° C. for 30 seconds, 50 ° C. for 30 seconds, and 70 ° C. for 2 minutes was repeated 30 times, and then incubated 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]
[Example 8]
PCR Amplified by OCIF cDNA Fragment cloning and sequencing
The OCIF cDNA fragment obtained in Example 7-iii) was obtained by the method of Marchuk, D et al. (Nucleic Acid Res., Vol. 19, p1154, 1991).^-The DNA ligation kit Ver.2 (Takara Shuzo) was inserted into (Stratagene) to transform E. coli DH5α (Gibco BRL). The obtained transformant was grown and the plasmid inserted with about 400 bp OCIF cDNA fragment was purified according to a conventional method. This plasmid was named pBSOCIF, and the base sequence of the OCIF cDNA inserted in this plasmid was determined using a Taq Dye Deoxy Terminator Cycle Sequencing kit (Perkin Elmer). The size of this OCIF cDNA was 397 bp. In the amino acid sequence consisting of 132 amino acids predicted from this base sequence, the internal amino acid sequences of OCIF used to design the mix primer (SEQ ID NOs: 2 and 3) are N-terminal and C-terminal, respectively. I was able to find it. The internal amino acid sequence of OCIF (SEQ ID NO: 1) could be found in this 132 amino acid sequence. From the above results, it was confirmed that the cloned 397 bp cDNA was an OCIF cDNA fragment.
[0048]
[Example 9]
DNA Probe preparation
This OCIF cDNA fragment was amplified by PCR under the conditions of Example 7-iii) using the plasmid inserted with the 397 bp OCIF cDNA fragment prepared in Example 8 as a template. A 397 bp OCIF cDNA fragment was separated by agarose electrophoresis and purified using a QIAEX gel extraction kit (Qiagen). [Α] Using this DNA, a megaprime DNA labeling kit (Amersham)³²Labeled with P] dCTP and used as a probe to screen full-length OCIF cDNA.
[0049]
[Example 10]
cDNA Creating a library
Poly (A) obtained in Example 7-i)⁺RNA, 2.5 μg as a template, using a gray-length cDNA synthesis kit (Clontech) according to the company's protocol, cDNA synthesis using oligo (dT) primer, EcoRI-SalI-Not-I adapter addition, cDNA size fractionation Nation was performed and ethanol precipitation was performed, followed by dissolution 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 cut with EcoRI using T4DNA ligase. The cDNA recombinant phage DNA solution thus obtained was subjected to in vitro packaging reaction using Gigapack Gold II (Stratagene) to prepare λZAP express recombinant phage.
[0050]
Example 11
Recombinant phage screening
The recombinant phage obtained in Example 10 was infected with E. coli XL1-Blue MRF '(Stratagene) at 37 ° C for 15 minutes, and then added to NZY medium containing 0.7% agar heated to 50 ° C. Pour onto NZY agar plate. After overnight culture at 37 ° C., HiBond N (Amersham) was brought into close contact with the plaque-generated plate for about 30 seconds. This filter was alkali-denatured according to a conventional method, neutralized, immersed in 2XSSC solution, and then DNA was immobilized on the filter by UV cross linking (Stratagene). The obtained filter was immersed in a hybridization buffer (Amersham) containing 100 μg / ml salmon sperm DNA, pretreated at 65 ° C. for 4 hours, and then heat-denatured DNA probe (2X10^Fivecpm / ml) was added to the above buffer, and hybridization was carried out at 65 ° C. overnight. After the reaction, the filter was washed twice with 2XSSC and twice with 0.1XSSC, 0.1% SDS solution at 65 ° C. for 10 minutes. Some of the positive clones obtained were purified by screening twice more. Among them, those having an insert of about 1.6 kb were used below. This purified phage was named λOCIF. Purified λOCIF was infected with Escherichia coli XL1-Blue MRF 'according to the protocol of λZAP Express Cloning Kit (Stratagene), followed by multiple infection with helper phage ExAssist (Stratagene). After infection with (Stratagene), a kanamycin resistant strain was picked up to obtain a transformant having the plasmid pBKOCIF in which the above-mentioned 1.6 kb insert was inserted into pBKCMV (Stratagene). This transformed strain is pBK / 01F10, and the accession number FERM BP-5267 (October 1995) to the Biotechnology Institute of Industrial Technology, Ministry of International Trade and Industry (currently the National Institute of Advanced Industrial Science and Technology (AIST)) The deposit was transferred from the original deposit of FERM P-14998 to the deposit under the Budapest Treaty on the 25th. A transformant having this plasmid was grown and the plasmid was purified by a conventional method.
[0051]
Example 12
OCIF Encodes the entire amino acid sequence of cDNA Determination of the nucleotide sequence
The base sequence of the OCIF cDNA obtained in Example 11 was determined using a tack dideoxy terminator cycle sequencing kit (Perkin Elmer). The primers used were T3, T7 primers (Stratagene) and synthetic primers designed based on the base sequence of OCIF cDNA, and their sequences are shown in SEQ ID NOs: 16 to 29 in Sequence Listing.
The determined nucleotide sequence of OCIF is shown in SEQ ID NO: 6, and the amino acid sequence deduced from the sequence is shown in SEQ ID NO: 5, respectively.
[0052]
Example 13
293 / EBNA Recombinant cells OCIF Production of
i) OCIF cDNA Of an expression plasmid
The plasmid pBKOCIF inserted with about 1.6 kb of OCIF cDNA obtained in Example 11 was digested with restriction enzymes BamHI and XhoI, and OCIF cDNA was excised, separated by agarose electrophoresis, and then used with a QIAEX gel extraction kit (Qiagen). And purified. This OCIF cDNA was inserted into an expression plasmid pCEP4 (Invitrogen) previously digested with restriction enzymes BamHI and XhoI using Ligation Kit Ver.2 (Takara Shuzo), and Escherichia coli DH5 α (Gibco BRL) The transformation was performed. The obtained transformant was grown and the expression plasmid pCEPOCIF in which OCIF cDNA was inserted was purified using a Qiagen column (Qiagen). The OCIF expression plasmid pCEPOCIF was precipitated with ethanol, dissolved in sterile distilled water, and used for the following operations.
[0053]
ii) OCIF cDNA Of transient expression and 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 × 10^Five293 / EBNA cells (Invitrogen) were implanted in 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 serum-free IMDM medium. Follow the protocol attached to the transfection reagent Lipofectamine (Gibco BRL), mix pCEPOCIF and Lipofectamine that have been diluted in advance with OPTI-MEM medium (Gibco BRL), and add this mixture to 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 fresh OPTI-MEM medium was added. After another 30 hours, the medium was collected and used as a sample for measuring OCIF activity. The activity of OCIF was measured as follows. Active vitamin D from mouse bone marrow cells about 17 days after birth_ThreeOsteoclast formation in the presence was tested by induction of tartrate-resistant acid phosphatase activity, its inhibitory activity was measured, and OCIF activity was determined. That is, 2 x 10 in a 96-well microplate^{- 8}M active vitamin D_Three100 μl of a sample diluted with α-MEM medium (Gibco BRL) containing 10% fetal calf serum and 3 × 10 mouse bone marrow cells about 17 days after birth^FiveAre suspended in 100 μl of α-MEM medium containing 10% fetal bovine serum and seeded with 5% CO₂The cells were cultured at 37 ° C. and 100% humidity for 1 week. On the 3rd and 5th day of culture, 160 μl of the culture solution is discarded and 1 × 10^-8M active vitamin D_ThreeAnd 160 μl of sample diluted in α-MEM medium containing 10% fetal bovine serum was added. After 7 days of culturing, the cells were washed with phosphate buffered saline and fixed with ethanol / acetone (1: 1) solution at room temperature for 1 minute to measure osteoclast formation. Acid Phosphatase, Leucocyte , Catalog No.387-A, Sigma). The decrease in acid phosphatase activity positive cells 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 natural OCIF obtained from the culture solution of IMR-90 previously.
[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 type OCIF Purification
CHAPS was added to 0.1% of the culture solution obtained by mass-culturing the 293 / EBNA cells described in Example 13-ii) to a concentration of 0.1%, and filtered through a 0.22 μm filter (Steribex GS, Millipore). Then, it was 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 with a linear gradient of NaCl to 2M in 100 minutes and a flow rate of 4 ml / min, and fractionation was performed at 8 ml / fraction. OCIF activity was measured using 150 μl of each fraction according to the method of Example 2 to obtain 112 ml of an OCIF active fraction eluted with about 0.6 to 1.2 M NaCl.
[0056]
Affinity column in which 112 ml of the obtained OCIF active fraction 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 (Heparin-5PW, 0.8 × 7.5 cm, Tosoh Corporation). After washing with 10 mM Tris-HCl, pH 7.5 containing 0.1% CHAPS, elution was performed with a linear gradient of NaCl to 2 M over 60 minutes, with a flow rate of 0.5 ml / min, and fractionated at 0.5 ml / fraction. It was.
[0057]
SDS-polyacrylamide gel electrophoresis was performed under reducing and non-reducing conditions according to the method of Example 4 using 4 μl each of the obtained fractions. As a result, in fractions 30-32, only the OCIF band of about 60 kD under reducing conditions and about 60 kD and about 120 kD under non-reducing conditions was detected, so fractions 30-32 were collected and purified 293 / EBNA cell-derived recombinant type OCIF (rOCIF (E)) fraction was used. As a result of protein quantification by the Raleigh method using BSA as a standard, it was revealed that 1.5 ml of rOCIF (E) at 535 μg / ml was obtained.
[0058]
Example 14
CHO Recombinant cells OCIF Production of
i) OCIF Of an expression plasmid
The plasmid pBKOCIF inserted with the approximately 1.6 kb OCIF cDNA obtained in Example 11 was digested with restriction enzymes SalI and EcoRV, and an approximately 1.4 kb OCIF cDNA fragment was excised and separated by agarose electrophoresis, followed by QIAEX gel extraction kit ( Qiagen). In addition, the expression vector pcDL-SR α296 (Molecular and Cellular Biology, Vol. 8, pp466-472, 1988) was digested with restriction enzymes PstI and KpnI, and an about 3.4 kb expression vector DNA fragment was separated by agarose electrophoresis. Purification was performed using a QIAEX gel extraction kit (Qiagen). The ends of these purified OCIF cDNA fragments and expression vector DNA fragments were blunted using a DNA branding kit (Takara Shuzo). Next, using Ligation Kit Ver.2 (Takara Shuzo), the OCIF cDNA fragment was inserted into the blunted expression vector DNA fragment, E. coli DH5α (Gibco BRL) was transformed, and OCIF expression plasmid pSR αOCIF was A transformant having the same was obtained.
[0059]
ii) Preparation of expression plasmid
The transformant having the OCIF expression plasmid pSRαOCIF obtained in Example 13-i) and the transformant having the mouse DHFR gene expression plasmid pBAdDSV shown in WO92 / 01053 were each grown using conventional methods. The solution was treated with the alkali method and polyethylene glycol method according to the method of Maniatis et al. (Molecular cloning, 2nd edition) and purified by cesium chloride density gradient centrifugation.
[0060]
iii) CHOdhFr ^- Acclimatization of cells to protein-free medium
CHOdhFr that had been passaged in IMDM medium (Gibco BRL) containing 10% fetal bovine serum (Gibco BRL)^-The cells (ATCC-CRL9096) were conditioned with serum-free medium EX-CELL301 (JRH Biosciences) and further conditioned with protein-free medium EX-CELL PF CHO (JRH Biosciences).
[0061]
iv) OCIF Expression plasmids and DHFR Expression plasmid CHOdhFr ^- Introduction to cells
CHOdhFr prepared in Example 14-iii) using the OCIF expression plasmid pSRαOCIF prepared in Example 14-ii) and the DHFR expression plasmid pBAdDSV^-Cells were transformed by the electroporation method shown below. 200 μg of pSR αOCIF plasmid and 20 μg of pBAdDSV plasmid are aseptically dissolved in 0.8 ml of IMDM medium (Gibco BRL) containing 10% fetal calf serum (Gibco BRL).⁷CHOdhFr^-Cells were suspended. This cell suspension was placed in a cuvette (Bio-Rad) and transformed by electroporation using Gene Pulser (Bio-Rad) under conditions of 360 V and 960 μF. Transfer the electroporated cell suspension to a T-flask for suspension cells (Sumitomo Bakelite Co., Ltd.) containing 10 ml of EX-CELL PF CHO medium.₂The cells were cultured for 2 days in an incubator. Using EX-CELL PF CHO medium, the cells were seeded in a 96-well microplate at a concentration of 5000 cells / well and cultured for about 2 weeks. EX-CELL PF CHO medium does not contain nucleic acid, and the parent strain CHOdhFr^-Only cell lines that express DHFR are selected. Since the OCIF expression plasmid is used 10 times as much as the DHFR expression plasmid, most cell lines expressing DHFR express OCIF. From the obtained cell line expressing DHFR, a cell line having a high OCIF activity in the culture supernatant was screened by the measurement method shown in Example 2. The resulting OCIF high-producing strain is cloned by limiting dilution using EX-CELL PF CHO medium, and the obtained clone is screened for a cell line with high OCIF activity in the culture supernatant to produce a high OCIF product. Clone 5561 was obtained.
[0062]
v) Recombinant OCIF Production of
To produce recombinant OCIF (rOCIF), 1 x 10 transformed CHO cells (5561) were added to 3 liters of EX-CELL 301 medium.^FiveThe cells were inoculated to be cells / ml and cultured at 37 ° C for 4 to 5 days using a spinner flask. Cell concentration is about 1 × 10⁶When cells / ml were reached, approximately 2.7 l of medium was collected. About 2.7 l of EX-CELL 301 medium was added and the culture was repeated. About 3 liters of culture broth were collected using 3 spinner flasks.
[0063]
vi) CHO Cell-derived recombinant type OCIF Purification
CHAPS was added to 1 L of the culture solution obtained in Example 14- (v) to 0.1%, filtered through a 0.22 μm filter (Steribex GS, Millipore), and then 10 mM Tris-HCl, pH 7.5. On a 50 ml heparin-sepharose FF column (2.6 × 10 cm, Pharmacia) equilibrated with 1. After washing with 10 mM Tris-HCl, pH 7.5 containing 0.1% CHAPS, elution was carried out with a linear gradient of NaCl of 2 M over 100 minutes and a flow rate of 4 ml / min, and fractionation was carried out at 8 ml / fraction. Using 150 μl of each fraction, the OCIF activity was measured according to the method of Example 2 to obtain 112 ml of an OCIF active fraction eluted at about 0.6 to 1.2 M.
[0064]
112 ml of the obtained OCIF active fraction 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. Blue-5PW, 0.5 × 5 cm, Tosoh Corporation). After washing with 10 mM Tris-HCl, pH 7.5 containing 0.1% CHAPS, elution was performed with a linear gradient of NaCl to 3M in 90 minutes and a flow rate of 0.5 ml / min, and fractionation was performed at 0.5 ml / fraction. It was.
[0065]
SDS-polyacrylamide gel electrophoresis was performed under reducing and non-reducing conditions according to the method of Example 4 using 4 μl each of the obtained fractions. As a result, only fractions of about 60 kD under reducing conditions and about 60 kD and about 120 kD of OCIF bands under non-reducing conditions were detected in fractions 30 to 38. Therefore, fractions 30 to 38 were collected and purified CHO cell-derived recombinant OCIF [ rOCIF (C)] fraction. As a result of protein quantification by the Raleigh method using BSA as a standard, it was revealed that 4.5 ml of rOCIF (C) of 113 μg / ml was obtained.
[0066]
Example 15
Recombinant OCIF N-terminal structure analysis
3 μg of purified rOCIF (E) and rOCIF (C) were immobilized on polyvinylidene difluoride (PVDF) membrane using ProSpin (Perkin Elmer), washed with 20% methanol, and then protein sequencer (Procise, N-terminal amino acid sequence analysis was performed using Model 492, Perkin Elmer). The results are shown in SEQ ID NO: 7 in the sequence listing.
The N-terminal amino acid of rOCIF (E) and rOCIF (C) is the 22nd Glu from the translation start point Met of the amino acid sequence described in SEQ ID NO: 5 in the sequence listing, and 21 amino acids from Met to Gln are signal peptides Became clear. The reason why the N-terminal amino acid sequence of the natural OCIF obtained from the IMR-90 culture solution was unanalysable was thought to be that N-terminal Glu was converted to pyroglutamic acid during culture or purification. .
[0067]
Example 16
Recombinant (r) OCIF And natural type (n) OCIF Biological activity
i) Vitamins in mouse bone marrow cell lines D _Three Of osteoclast formation induced by mitochondrion
2 x 10 in 96-well microplate^{- 8}M active vitamin D_ThreePurified rOCIF (E) and 100 μl of nOCIF, which were serially diluted by half from 250 ng / ml in α-MEM medium (Gibco BRL) containing 10% fetal bovine serum, were added. In this well, about 17 days old mouse bone marrow cells 3 × 10^FiveAre suspended in 100 μl of α-MEM medium containing 10% fetal bovine serum and seeded with 5% CO₂The cells were cultured at 37 ° C. and 100% humidity for 1 week. After 7 days of culture, osteoclast formation was detected by staining with an acid phosphatase activity measurement kit (Acid Phosphatase, Leucocyte, catalog No. 387-A, Sigma) according to the method of Example 2. The decrease in acid phosphatase activity positive cells in the presence of tartaric acid was defined as OCIF activity. The decrease rate of acid phosphatase activity positive cells was calculated by solubilizing the dye of the stained cells and measuring the absorbance. That is, 100 μl of 0.1N sodium hydroxide-dimethyl sulfoxide mixture (1: 1) was added to each well where cells were fixed and stained, and shaken well. After sufficiently dissolving the dye, absorbance was measured at a measurement wavelength of 590 nm and a control wavelength of 490 nm using a microplate reader (Immuno Reader NJ-2000, Intermed). As a blank well for measuring absorbance, vitamin D_ThreeUnadded wells were used. The results are expressed as percentage values with the absorbance value in wells not added with OCIF as 100, and are shown in Table 5.
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) Vitamins in co-culture system of stromal cells and mouse spleen cells D _Three Of osteoclast formation induced by mitochondrion
Vitamin D_ThreeThe test of osteoclast formation in the co-culture system of stromal cells and mouse spleen cells induced by the above-mentioned method was performed according to the method of Udagawa et al. (Endocrinology, Vol. 125, p1805-1813, 1989). That is, 2 x 10 in a 96-well microplate^{- 8}M active vitamin D_Three2 × 10^{- 7}Purified rOCIF (E), rOCIF (C) and nOCIF (100 μl) serially diluted with α-MEM medium (Gibco BRL) containing dexamethasone and 10% fetal calf serum were added. Mouse well bone marrow-derived stromal cell line ST2 cells (RIKEN Cell Bank-RCB0224) 5 × 10^Three1 x 10 ddy mouse spleen cells about 8 weeks old^FiveAre suspended in 100 μl of α-MEM medium containing 10% fetal calf serum and seeded.₂The cells were cultured at 37 ° C. and 100% humidity for 5 days. After 5 days of culturing, the cells were washed with phosphate buffered saline, and then fixed with an ethanol / acetone (1: 1) solution at room temperature for 1 minute, and osteoclast formation was measured with an acid phosphatase activity measurement kit (Acid Phosphatase, This was detected by staining with Leucocyte, catalog No. 387-A, Sigma). The decrease in acid phosphatase activity positive cells in the presence of tartaric acid was defined as OCIF activity. The rate of decrease in the number of acid phosphatase activity positive cells was calculated by dissolving the dye of cells stained according to the method described in Example 16-i). Table 6 shows the results of testing using rOCIF (E) and rOCIF (C), and Table 7 shows the results of testing using rOCIF (E) and nOCIF.
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 mitochondrion
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 x 10 in a 96-well microplate^{- 8}In α-MEM medium (Gibco) containing MPTH and 10% fetal calf serum, 100 μl of nOCIF and purified rOCIF (E) serially diluted from 125 ng / ml were added. In this well, about 17 days old mouse bone marrow cells 3 × 10^FiveAre suspended in α-MEM medium containing 100 μl of 10% fetal bovine serum and seeded, and 5% CO₂The cells were cultured at 37 ° C. and 100% humidity for 5 days. After 5 days of culturing, the cells were washed with phosphate buffered saline, fixed with ethanol / acetone (1: 1) solution for 1 minute at room temperature, and osteoclast formation was measured for acid phosphatase activity measurement kit (Acid Phosphatase, Leucocyte , Catalog No.387-A, Sigma). The decrease in acid phosphatase activity positive cells in the presence of tartaric acid was defined as OCIF activity. The rate of decrease in the number of acid phosphatase activity positive cells was calculated by dissolving the dye of cells stained according to the method described in Example 16-i). The results are shown in Table 8.
Similarly to nOCIF, rOCIF (E) also showed 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 mitochondrion
The test of 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, nOCIF diluted with α-MEM medium (Gibco BRL) containing 20 ng / ml IL-11 and 10% fetal bovine serum and 100 μl of purified rOCIF (E) were placed in a 96-well microplate. Mouse neonatal skull-derived preadipocyte cell line MC3T3-G2 / PA6 cells (RIKEN Cell Bank-RCB1127) 5 × 10^Three1 x 10 ddy mouse spleen cells about 8 weeks old^FiveAre suspended in α-MEM medium containing 100 μl of 10% fetal calf serum and seeded with 5% CO₂The cells were cultured at 37 ° C. and 100% humidity for 5 days. After 5 days of culturing, the cells were washed with phosphate buffered saline, fixed with ethanol / acetone (1: 1) solution for 1 minute at room temperature, and osteoclast formation was measured for acid phosphatase activity measurement kit (Acid Phosphatase, Leucocyte , Catalog No.387-A, Sigma). The number of acid phosphatase activity positive cells in the presence of tartaric acid was counted, and the decrease was regarded as OCIF activity. The results are shown in Table 9.
Both nOCIF and rOCIF (E) showed activity to suppress osteoclast formation induced by IL-11 in a dose-dependent manner at a concentration of 2 ng / ml or more.
[0075]
[Table 9]

[0076]
Thus, in the test system for osteoclast formation using various target cells, OCIF is vitamin D._ThreeIt was revealed that osteoclast formation by osteoclast formation-inducing factors such as PTH, IL-11 and the like was suppressed at almost the same concentration. Therefore, it was suggested that OCIF could be effectively used to treat different types of osteopenia induced by such various bone resorption promoting substances.
[0077]
[Example 17]
Monomer type and dimer type OCIF Sample preparation
A reverse phase column equilibrated with 30% acetonitrile containing 0.1% TFA after adding 1/100 volume of 25% TFA (trifluoroacetic acid) to a sample containing 100 μg each of rOCIF (E) and rOCIF (C) Elution was performed with a linear gradient of 55% acetonitrile over 50 minutes and a flow rate of 0.2 ml / min to separate each OCIF peak. Monomeric OCIF and dimer OCIF were obtained by freeze-drying the obtained peak fraction.
[0078]
Example 18
Recombinant OCIF Molecular weight measurement
A sample containing monomer type and dimer type nOCIF purified using a reverse phase column by the method of Example 3-vi) and about 1 μg of 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 by the method of Example 4. The results of electrophoresis under non-reducing conditions and reducing conditions are shown in FIGS. 6 and 7, respectively.
[0079]
As a result, a 60 kD protein band was detected in any monomer type sample under non-reducing conditions, and a 120 kD protein band was detected in any dimer type sample. In addition, only about 60 kD protein band was detected in any sample under reducing conditions. Therefore, it was shown that the molecular weights of the monomer type and dimer type of IMR-90 cell-derived nOCIF, 293 / EBNA cell-derived recombinant OCIF, and CHO cell-derived recombinant OCIF were almost the same.
[0080]
Example 19
IMR- 90 Cell-derived natural type OCIF And recombination OCIF Of N-linked sugar chain and molecular weight measurement
A sample containing about 5 μg each of the monomer type and dimer type nOCIF purified by using the reverse phase column in the method of Example 3-vi) and the monomer type and dimer type rOCIF purified by the method of Example 17 was concentrated under reduced pressure. . To these samples, add 50 mM phosphate buffer solution with 100 mM 2-mercaptoethanol, pH 8.6, 9.5 μl to dissolve, and add 0.5 μl of 250 U / ml N-glycanase solution (Seikagaku Corporation) to 37 ° C. Left for a day. To these samples, 20 mM Tris-HCl, pH 8.0, 10 μl containing 2 mM MEDTA, 5% SDS, and 0.02% bromophenol blue was added and heated at 100 ° C. for 5 minutes. 1 μl of these samples were subjected to SDS-polyacrylamide electrophoresis by the method of Example 4 and then stained with silver. The results are shown in FIG.
[0081]
As a result, it was shown that the molecular weight under the reducing condition of the OCIF protein from which the N-linked sugar chain was removed by N-glycanase treatment was about 40 kD. Since the molecular weights under the reducing conditions of IOC-90 cell-derived nOCIF, 293 / EBNA cell-derived rOCIF, and CHO cell-derived rOCIF, which have not been subjected to sugar chain removal, are about 60 kD, these OCIF Was revealed to be a glycoprotein containing an N-linked sugar chain in the molecule.
[0082]
Example 20
OCIF Analogue (variant) cDNA Cloning and nucleotide sequence determination
As shown in Examples 10 and 11, a transformant having a plasmid pBKOCIF in which OCIF cDNA was inserted into pBKCMV (Stratagene) was obtained from one of several purified positive phages. A transformant having a plasmid in which inserts having different lengths were inserted was obtained from several positive phages. Transformants having these plasmids were grown and the plasmids were purified by conventional methods. The base sequences of these insert DNAs were determined using a tack dideoxy terminator cycle sequencing kit (Perkin Elmer). The primers used were T3 and T7 primers (Stratagene) and synthetic primers designed based on the base sequence of OCIF cDNA. In addition to the original OCIF, there were a total of four OCIF variants (OCIF2, 3, 4, 5). The determined base sequence of 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 determined nucleotide sequence of 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 determined nucleotide sequence of 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 determined nucleotide sequence of 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 265th guanine to the 285th guanine in the base sequence of OCIF cDNA (SEQ ID NO: 6). In the amino acid sequence, the 68th glutamic acid (Glu) in the amino acid sequence of OCIF (SEQ ID NO: 5) There is a deletion of 7 amino acids from to 74th glutamine (Gln).
[0084]
OCIF 3
The 9th cytidine in the base sequence of OCIF cDNA (SEQ ID NO: 6) has been converted to guanine. In the amino acid sequence, the -19th asparagine (Asn) in the amino acid sequence of OCIF (SEQ ID NO: 5) is lysine (Lys). It 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 117 bp deletion from the 872nd guanine to the 988th guanine in the base sequence of OCIF cDNA (SEQ ID NO: 6). In the amino acid sequence, the 270th threonine (Thr) in the amino acid sequence of OCIF (SEQ ID NO: 5) There is a 39 amino acid deletion from 308 to leucine (Leu).
[0085]
OCIF 4
The 9th cytidine in the base sequence of OCIF cDNA (SEQ ID NO: 6) has been converted to guanine. In the amino acid sequence, the -19th asparagine (Asn) in the amino acid sequence of OCIF (SEQ ID NO: 5) is lysine (Lys). It has changed to. Further, the 22nd guanine is converted to thymidine, and in the amino acid sequence, the -14th alanine (Ala) in the amino acid sequence of OCIF (SEQ ID NO: 5) is 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 intron 2 between the 400th and 401st positions of the base sequence of OCIF cDNA (SEQ ID NO: 6), and the open reeling frame stops in it. In the amino acid sequence, a novel amino acid sequence consisting of 21 amino acids is added after the 112th alanine (Ala) in the amino acid sequence of OCIF (SEQ ID NO: 5 in the sequence listing).
[0086]
OCIF 5
The 9th cytidine in the base sequence of OCIF cDNA (SEQ ID NO: 6) has been converted to guanine. In the amino acid sequence, the -19th asparagine (Asn) in the amino acid sequence of OCIF (SEQ ID NO: 5) is lysine (Lys). It 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 base sequence of OCIF cDNA (SEQ ID NO: 6), and the open reeling frame stops there. In the amino acid sequence, a new amino acid sequence consisting of 12 amino acids is added after the 112th alanine (Ala) in the amino acid sequence of OCIF (SEQ ID NO: 5 in the sequence listing).
[0087]
Example 21
OCIF Production of analogs (variants)
i) OCIF variant cDNA Of an expression plasmid
Among the OCIF variant cDNAs obtained in Example 20, plasmids pBKOCIF2 and pBKOCIF3 into which OCIF 2 and 3 cDNAs were inserted were digested with restriction enzymes XhoI and BamHI (Takara Shuzo), respectively, and OCIF 2 and 3 cDNAs were respectively obtained. After excision and separation by agarose electrophoresis, purification was performed using a QIAEX gel extraction kit (Qiagen). Insertion of these OCIF 2 and 3 cDNAs into expression plasmid pCEP4 (Invitrogen) previously digested with restriction enzymes XhoI and BamHI (Takara Shuzo) using Ligation Kit Ver.2 (Takara Shuzo) Then, E. coli DH5α (Gibco BRL) was transformed.
Also, of the OCIF variant cDNA obtained in Example 20, the 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 QIAEX gel extraction. Purification was performed using a kit (Qiagen). This OCIF4 cDNA was inserted into the expression plasmid pCEP4 (Invitrogen) previously digested with restriction enzymes NheI and XhoI (Takara Shuzo) using Ligation Kit Ver.2 (Takara Shuzo), and Escherichia coli DH5α (Gibco BRL) was transformed.
[0088]
Further, among the OCIF variant cDNAs obtained in Example 20, the plasmid pBKOCIF5 into which the OCIF5 cDNA was inserted was digested with the restriction enzyme Hind III (Takara Shuzo), and the 5 ′ region of the OCIF5 cDNA coding region was excised. After separation by electrophoresis, purification was performed using a QIAEX gel extraction kit (Qiagen). The OCIF expression plasmid pCEPOCIF obtained in Example 13-i) was digested with the restriction enzyme Hind III (Takara Shuzo), the 5 ′ region of the OCIF cDNA coding region was removed, and the DNA containing the pCEP plasmid and the OCIF cDNA 3 ′ region Fragment pCEPOCIF-3 ′ was separated by agarose electrophoresis and then purified using a QIAEX gel extraction kit (Qiagen). The Hind III fragment of this OCIF5 cDNA was inserted into pCEPOCIF-3 ′ using Ligation Kit Ver. 2 (Takara Shuzo), and E. coli DH5α (Gibco BRL) was transformed.
The obtained transformant was grown and the expression plasmid pCEPOCIF 2, 3, 4, 5 in which the cDNA of OCIF2, 3, 4, 5 was inserted was purified using a Qiagen column (Qiagen). The OCIF variant expression plasmid was precipitated with ethanol, dissolved in sterile distilled water, and used for the following operations.
[0089]
ii) OCIF variant cDNA Of transient expression and activity
Using the OCIF variant expression plasmid pCEPOCIF 2, 3, 4, 5 obtained in Example 21-i), the OCIF variant was expressed transiently by the method described in Example 13-ii), and their activity was determined. Examined. As a result, weak activity was observed in these OCIF variants.
[0090]
[Example 22]
OCIF Creation of mutants
i) OCIF Mutant cDNA Preparation 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 cleaved DNA was subjected to preparative agarose gel electrophoresis.
A DNA fragment of about 1.6 kilobase pairs (kb) including the full-length OCIF cDNA was isolated and purified with a 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 cleaved with restriction enzymes BamHI and XhoI (Takara Shuzo). The cleaved DNA was subjected to preparative agarose gel electrophoresis. A DNA fragment of about 3.0 kb was isolated and purified with a 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, 5 μl of DNA ligation kit ver.2 I solution (Takara Shuzo) was added and mixed, and then incubated at 16 ° C. for 30 minutes to carry out a ligation reaction. In addition, all the following ligation reactions were performed under a heat-retaining condition at 16 ° C. for 30 minutes.
[0091]
Using this ligation reaction solution, E. coli was transformed under the following conditions. Hereafter, E. coli was transformed under the following conditions. 5 μl of this ligation reaction solution and 100 μl of E. coli DH5α competent cells (Gibco BRL) were mixed in a 15 ml sterilization tube (Iwaki Glass Co., Ltd.) and allowed to stand in ice water for 30 minutes. After incubation at 42 ° C. for 45 seconds, 250 μl of L medium (1% tryptone, 0.5% yeast extract, 1% NaCl) was added and 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. The cells were cultured overnight at 37 ° C., and the 6 colonies grown were further cultured overnight in 2 ml of L ampicillin liquid medium, and the structure of the plasmid possessed by each strain was examined. pBluescript IISK⁺A plasmid with a structure in which an approximately 1.6 kb DNA fragment containing the full-length OCIF cDNA is inserted into the BamHI XhoI cleavage site (hereinafter referred to as pSK)⁺-Called OCIF).
[0092]
ii) Creation of mutants with Cys replaced with Ser
(1) Mutation introduction
In the amino acid sequence set forth in SEQ ID NO: 4 in the sequence listing, a mutant was prepared by substituting Cys residues Nos. 174, 181, 256, 298 and 379 with Ser residues. Mutants with 174Cys replaced with Ser are OCIF-C19S, mutants with 181Cys replaced with Ser are OCIF-C20S, mutants with 256Cys replaced with Ser are OCIF-C21S, and mutants with 298Cys replaced with Ser are OCIF- Mutants obtained by substituting C22S and 379 Cys with Ser were named OCIF-C23S2. First, the base sequence encoding each Cys residue was replaced with a base sequence encoding a Ser residue in order to produce a mutant. Mutagenesis was performed by two-stage PCR (polymerase chain reaction). Hereinafter, it is called a two-step PCR reaction. The first stage consists of two PCR reactions (PCR1 and PCR2).
[0093]
PCR1 Reaction liquid
10X Ex Taq buffer (Takara Shuzo) 10 μl
2.5 mM dNTP solution 8 μl
2 μl of the plasmid vector described in Example 11 (8 ng / ml)
Sterile distilled water 73.5μl
20 μM Primer 1 5 μl
100 μM Primer 2 (for mutagenesis) 1 μl
Ex Taq (Takara Shuzo) 0.5μl
[0094]
PCR2 Reaction liquid
10X Ex Taq buffer (Takara Shuzo) 10 μl
2.5 mM dNTP solution 8 μl
2 μl of the plasmid vector described in Example 11 (8 ng / ml)
Sterile distilled water 73.5μl
20 μM Primer 3 5 μl
100 μM Primer 4 (for mutagenesis) 1 μl
Ex Taq (Takara Shuzo) 0.5μl
[0095]
At the time of introducing each mutation, 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 30-40. The PCR1 reaction solution and the PCR2 reaction solution were mixed in separate microcentrifuge tubes, 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 then 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 the desired length was synthesized. After the completion of the first step PCR reaction, the primer is removed from the reaction solution with Amicon Microcon (Amicon), the final solution volume is adjusted to 50 μl with sterilized distilled water, and the resulting DNA fragment is used for further second step PCR reaction (PCR3) was performed.
[0096]
PCR3 Reaction liquid
10X Ex Taq buffer (Takara Shuzo) 10 μl
2.5 mM dNTP solution 8 μl
DNA fragment obtained by PCR1 5 μl
DNA fragment obtained by PCR2 5 μl
Sterile distilled water 61.5 μl
20 μM primer 1 5 μl
20 μM primer 3 5 μl
Ex Taq (Takara Shuzo) 0.5 μl
[0097]
[Table 10]

[0098]
The above solution was placed in a microcentrifuge tube and mixed, and then 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, and it was confirmed that a DNA fragment of the desired length was synthesized. DNA obtained by PCR was precipitated with ethanol, dried in vacuum, and dissolved in 40 μl of sterile distilled water. A solution containing a C19S mutant DNA fragment is solution A, a solution containing a C20S mutant DNA fragment is solution B, a solution containing a C21S mutant DNA fragment is solution C, a solution containing a C22S mutant DNA fragment is solution D, and a solution containing a C23S mutant DNA fragment is contained. The solution was named Solution E.
[0099]
The DNA fragment in 20 μl of solution A was cleaved 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 cleaved with restriction enzymes NdeI and SphI (Takara Shuzo), and 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 5 μl of DNA ligation kit ver.2 I solution was added to carry out a ligation reaction. Escherichia coli DH5α was transformed with 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 analyzing the DNA structure. The DNA structure was analyzed by measuring the length of the fragment obtained by restriction enzyme cleavage and determining the base sequence. The obtained plasmid DNA of interest was named pSK-OCIF-C19S.
[0100]
The C20S mutant DNA fragment in 20 μl of solution B was cleaved 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 DNA solution 5 and 3 μl of DNA solution 4 were mixed, and 5 μl of DNA ligation kit ver.2 I solution was added to carry out a ligation reaction. Escherichia coli DH5α was transformed with 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 analyzing the DNA structure. The DNA structure was analyzed by measuring the length of the fragment obtained by restriction enzyme cleavage and determining the base sequence. The obtained plasmid DNA of interest was named pSK-OCIF-C20S.
[0101]
The DNA fragment in 20 μl of solution C was cleaved 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 5 μl of DNA ligation kit ver.2 I solution was added to carry out a ligation reaction. Escherichia coli DH5α was transformed with 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 analyzing the DNA structure. The DNA structure was analyzed by measuring the length of the fragment obtained by restriction enzyme cleavage and determining the base sequence. The obtained plasmid DNA of interest was named pSK-OCIF-C21S.
[0102]
The DNA fragment in 20 μl of solution D was cleaved 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 pSK⁺-OCIF was cleaved 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 DNA solution 7 and 3 μl of DNA solution 8 were mixed, and 5 μl of DNA ligation kit ver.2 I solution was added to carry out a ligation reaction. Escherichia coli DH5α was transformed with 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 analyzing the DNA structure. The DNA structure was analyzed by measuring the length of the fragment obtained by restriction enzyme cleavage and determining the base sequence. The obtained plasmid DNA of interest was named pSK-OCIF-C22S.
[0103]
The DNA fragment in 20 μl of solution E was cleaved 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 cleaved with restriction enzymes BstEII and EcoRV (Takara Shuzo), and a DNA fragment of about 4.5 kb was separated and purified by preparative electrophoresis, and dissolved in 20 μl of distilled water (DNA solution 10). 2 μl of DNA solution 9 and 3 μl of DNA solution 10 were mixed, and 5 μl of DNA ligation kit ver.2 I solution was further added to carry out a ligation reaction. Escherichia coli DH5α was transformed with 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 analyzing the DNA structure. The DNA structure was analyzed by measuring the length of the fragment obtained by restriction enzyme cleavage and determining the base sequence. The obtained plasmid DNA of interest was named pSK-OCIF-C23S.
[0104]
(2) Construction of mutant expression vector
The obtained plasmid DNA (pSK-OCIF-C19S, pSK-OCIF-C20S, pSK-OCIF-C21S, pSK-OCIF-C22S, pSK-OCIF-C23S) is digested with restriction enzymes BamHI and XhoI (Takara Shuzo). Then, an approximately 1.6 kb DNA fragment (including the target mutation) containing the entire OCIF cDNA was isolated and purified, and dissolved in 20 μl of sterile distilled water. They were named C19SDNA solution, C20SDNA solution, C21SDNA solution, C22SDNA solution, and C23SDNA solution, respectively. Next, 5 μg of the expression vector pCEP4 (Invitrogen) was cleaved 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 pCEP4DNA solution and 6 μl each of C19SDNA solution, C20SDNA solution, C21SDNA solution, C22SDNA solution, and C23SDNA solution were mixed separately, and 7 μl of DNA Ligation Kit Ver.2 I solution was added to each mixture, and ligation reaction was performed. . After the reaction was completed, 7 ml of the reaction solution was used to transform 100 ml of E. coli DH5α competent cell solution. From the obtained ampicillin resistant transformed cells, a total of 5 strains having plasmid DNAs 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- They were named C19S, pCEP4-OCIF-C20S, pCEP4-OCIF-C21S, pCEP4-OCIF-C22S, and pCEP4-OCIF-C23S.
[0105]
ii) Generation of domain deletion mutants
(1) Introduction of domain deletion mutation
Among amino acids listed in SEQ ID NO: 4, from Tyr 2 to 42 Ala, 43 Pro to 84 Cys, 85 Glu to 122 Lys, 123 Arg to 164 To Cys, mutants were deleted from Asp at 177 to Gln at 251 and from Ile at 253 to His at 326, respectively. Mutants deleted from Thr of No. 2 to Ala of No. 42 are OCIF-DCR1, mutants deleted of Pro from No. 43 to Cys of No. 84 are OCIF-DCR2, 122 from Glu of No. 85 OCIF-DCR3 is a mutant deleted from Lys of No. 1, OCIF-DCR4 is a mutant deleted from Arg of No. 123 to Cys of No. 164, and from Asp of No. 177 to Gln of No. 251 The lost mutant was named OCIF-DDD1, and the mutant lacking from 253 Ile to His 326 was named OCIF-DDD2. Domain deletion mutations were also introduced by the two-step PCR method described in Example 22-ii). The primers used in each mutagenesis reaction are shown in Table 11, and their sequences are shown in Sequence Listing SEQ ID NOs: 19, 25, 40 to 53, and 54.
[0106]
[Table 11]

[0107]
DNA obtained by PCR was precipitated with ethanol, dried in vacuum, and dissolved in 40 μl of sterile distilled water. Solution containing DCR1 mutant DNA fragment is solution F, solution containing DCR2 mutant DNA fragment is solution G, solution containing DCR3 mutant DNA fragment is solution H, solution containing DCR4 mutant DNA fragment is solution I, and containing DDD1 mutant DNA fragment The solution was named Solution J, and the solution containing the DDD2 mutant DNA fragment was named Solution K.
[0108]
The DNA fragment in 20 μl of solution F was cleaved 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 cleaved with restriction enzymes NdeI and XhoI (Takara Shuzo), and 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 DNA solution 11 and 3 μl of DNA solution 12 were mixed, and 5 μl of DNA ligation kit ver.2 I solution was further added to carry out a ligation reaction. Escherichia coli DH5α was transformed with 5 μl of the ligation solution after the reaction. From the resulting ampicillin-resistant transformed cells, strains having plasmid DNA in which the desired mutation was introduced into OCIF cDNA were selected by analyzing the DNA structure. The DNA structure was analyzed by measuring the length of the fragment obtained by restriction enzyme cleavage and determining the base sequence. The obtained plasmid DNA of interest was named pSK-OCIF-DCR1.
[0109]
The DNA fragment in 20 μl of solution G was cleaved 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 DNA solution 13 and 3 μl of DNA solution 12 were mixed, and 5 μl of DNA ligation kit ver.2 I solution was further added to carry out a ligation reaction. Escherichia coli DH5α was transformed with 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 analyzing the DNA structure. The DNA structure was analyzed by measuring the length of the fragment obtained by restriction enzyme cleavage and determining the base sequence. The obtained plasmid DNA of interest was named pSK-OCIF-DCR2.
[0110]
The DNA fragment in 20 μl of solution H was cleaved 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 DNA solution 14 and 3 μl of DNA solution 12 were mixed, and 5 μl of DNA ligation kit ver.2 I solution was further added to carry out a ligation reaction. Escherichia coli DH5α was transformed with 5 μl of the ligation solution after the reaction. From the resulting ampicillin-resistant transformed cells, strains having plasmid DNA in which the desired mutation was introduced into OCIF cDNA were selected by analyzing the DNA structure. The DNA structure was analyzed by measuring the length of the fragment obtained by restriction enzyme cleavage and determining the base sequence. The obtained plasmid DNA of interest was named pSK-OCIF-DCR3.
[0111]
The DNA fragment in 20 μl of solution I was cleaved 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 cleaved with restriction enzymes XhoI and SphI (Takara Shuzo), and 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 DNA solution 15 and 3 μl of DNA solution 16 were mixed, and 5 μl of DNA ligation kit ver.2 I solution was further added to carry out a ligation reaction. Escherichia coli DH5α was transformed with 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 analyzing the DNA structure. The DNA structure was analyzed by measuring the length of the fragment obtained by restriction enzyme cleavage and determining the base sequence. The obtained plasmid DNA of interest was named pSK-OCIF-DCR4.
[0112]
The DNA fragment in 20 μl of the solution J was cleaved 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 DNA solution 17 and 3 μl of DNA solution 8 were mixed, and 5 μl of DNA ligation kit ver.2 I solution was further added to carry out a ligation reaction. Escherichia coli DH5α was transformed with 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 analyzing the DNA structure. The DNA structure was analyzed by measuring the length of the fragment obtained by restriction enzyme cleavage and determining the base sequence. The obtained plasmid DNA of interest was named pSK-OCIF-DDD1.
[0113]
The DNA fragment in 20 μl of the solution K was cleaved 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 DNA solution 18 and 3 μl of DNA solution 8 were mixed, and 5 μl of DNA ligation kit ver.2 I solution was further added to carry out a ligation reaction. Escherichia coli DH5α was transformed with 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 analyzing the DNA structure. The DNA structure was analyzed by measuring the length of the fragment obtained by restriction enzyme cleavage and determining the base sequence. The obtained plasmid DNA of interest was named pSK-OCIF-DDD2.
[0114]
(2) Construction of mutant expression vector
The obtained plasmid DNAs (pSK-OCIF-DCR1, pSK-OCIF-DCR2, pSK-OCIF-XR3, pSK-OCIF-DCR4, pSK-OCIF-DDD1, pSK-OCIF-DDD2) are used as restriction enzymes BamHI and XhoI. A DNA fragment (including the target mutation) of about 1.4-1.5 kb containing the entire OCIF cDNA was isolated and purified by cutting with Takara Shuzo, and dissolved in 20 μl of sterile distilled water. These were named DCR1DNA solution, DCR2DNA solution, DCR3DNA solution, DCR4DNA solution, DDD1DNA solution, and DDD2DNA solution, respectively. 1 μl of pCEP4 DNA solution described in Example 22-ii) and 6 μl each of DCR1DNA solution, DCR2DNA solution, DCR3DNA solution, DCR4DNA solution, DDD1DNA solution, DDD2DNA solution are mixed separately, and 7 μl of DNA ligation buffer is added to each mixture. The ligation reaction was carried out. After completion of the reaction, E. coli DH5α was transformed with 7 μl of the reaction solution. From the obtained ampicillin resistant transformed cells, a total of 6 strains having plasmid DNAs having a structure in which each 1.4-1.5 kb fragment was inserted into the pCEP4BamHI XhoI site were selected. The plasmids having the target 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
Of the amino acids listed in SEQ ID NO: 4, Cys at 379 and Leu at 380, Ser from 331 to Leu at 380, Asp from 252 to Leu at 380, Asp from 177 to 380 Leu Thus, mutants in which 123 from Arg to 380 Leu and 86 from Cys to 380 Leu were deleted, respectively. Mutants lacking 379 Cys and 380 Leu are OCIF-CL, mutants deleted from Ser 331 to 380 Leu are OCIF-CC, 252 Asp to 380 Mutants deleted from Leu to OCIF-CDD2 and mutants deleted from Asp to 380 to Leu at 380 are deleted from OCIF-CDD1, Arg from 123 to Leu at 380 The resulting mutant was named OCIF-CCR4, and the mutant lacking Cys from 86 to 380 Leu was named OCIF-CCR3.
[0116]
Mutagenesis for 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. DNA obtained by PCR was precipitated with ethanol, dried in vacuum, and dissolved in 40 μl of sterile distilled water (solution L).
[0117]
The DNA fragment in 20 μl of solution L was cleaved 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 DNA solution 9 and 3 μl of DNA solution 10 described in Example 22-ii) were mixed, and 5 μl of DNA ligation kit ver.2 I solution was further added to carry out a ligation reaction. Escherichia coli DH5α was transformed with 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 analyzing the DNA structure. The DNA structure was analyzed by measuring the length of the fragment obtained by restriction enzyme cleavage and determining the base sequence. The obtained plasmid DNA of interest was named pSK-OCIF-CL. Mutant OCIF-CC, mutant OCIF-CDD2, mutant OCIF-CDD1, mutant OCIF-CCR4 and mutant OCIF-CCR3 were introduced using a one-step PCR method. The reaction conditions are shown below.
[0118]
For C-terminal domain deletion mutation introduction PCR Reaction liquid
10X Ex Taq buffer (Takara Shuzo) 10 μl
2.5 mM dNTP solution 8 μl
2 μl of the plasmid vector described in Example 11 (8 ng / ml)
Sterile distilled water 73.5μl
20μM Primer OCIF Xho F 5 μl
100 μM mutagenesis primer 1 μl
Ex Taq (Takara Shuzo) 0.5 μl
[0119]
[Table 12]

[0120]
At the time of introducing each mutation, only the type of primer was changed, and the other reaction compositions were the same.
The mutagenesis primers for each reaction are shown in Table 13, and their sequences are shown in SEQ ID NOs: 57 to 61 in the sequence listing. The PCR reaction solution was placed in a microcentrifuge tube and mixed, and then PCR was performed under the following conditions. After 3 minutes of treatment at 97 ° C., a three-step reaction of 95 ° C. for 30 seconds, 50 ° C. for 30 seconds and 70 ° C. for 3 minutes was repeated 25 times, and then 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 the desired length was synthesized. Primers were removed from the reaction solution with Amicon Microcon, 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 cleaved with restriction enzymes XhoI and BamHI. After the enzymatic cleavage, 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, respectively.
[0121]
[Table 13]

[0122]
(2) Construction of mutant expression vector
pSK-OCIF-CL was cleaved with restriction enzymes BamHI and XhoI (Takara Shuzo), and an approximately 1.5 kb DNA fragment (including the target mutation) containing OCIF cDNA was isolated and purified, and dissolved in 20 μl of sterile distilled water (CLDNA). solution). 1 μl of pCEP4 DNA solution described in Example 22-ii) and 6 μl each of CLDNA solution, CCDNA solution, CDD2DNA solution, CDD1DNA solution, CCR4DNA solution, CCR3DNA solution are mixed separately, and 7 μl of DNA Ligation Kit Ver.2 I solution Was added to perform a ligation reaction. After completion of the reaction, E. coli DH5α was transformed with 7 μl of the reaction solution. From the obtained ampicillin resistant transformed cells, a total of 6 strains having plasmid DNAs having a structure in which the OCIF cDNA fragment having the target mutation was inserted into the XhoI-BamHI site of pCEP4 were selected. The plasmids having the target structure were named pCEP4-OCIF-CL, pCEP4-OCIF-CC, pCEP4-OCIF-CDD2, pCEP4-OCIF-CDD1, pCEP4-OCIF-CCR4, and pCEP4-OCIF-CCR3, respectively.
[0123]
iv) Creation of C-terminal deletion mutant
(1) Introduction of C-terminal deletion mutation
Among the amino acids listed in SEQ ID NO: 4, a mutant (OCIF-CBst) in which 371 Gln to 380 Leu are deleted and 2 Leu-Val residues are added, 298 Cys to 380 Leu is deleted Mutants with added Ser-Leu-Asp residues (OCIF-CSph), mutants deleted from No. 167 Asn to No. 380 (OCIF-CBsp), No. 62 Cys to No. 380 Leu A mutant (OCIF-CPst) with deletion and addition of two Leu-Val residues was prepared. 2 μg each of pSK⁺-OCIF was cleaved with restriction enzymes BstPI, SphI, PstI (Takara Shuzo) and BspEI (New England Biolab), and the DNA was purified by phenol treatment and ethanol precipitation, and dissolved in 10 µl of sterile distilled water. Each 2 μl of the solution was used to smooth the ends of each DNA with a DNA branding kit (Takara Shuzo) (final volume 5 μl). To this reaction solution, 1 μg (1 μl) of XbaI linker (5′-CTAGTCTAGACTAG-3 ′) containing an amber codon and 6 μl of DNA ligation kit ver.2 I solution were added to carry out a ligation reaction. Escherichia coli DH5α was transformed with 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 analyzing the DNA structure. The DNA structure was analyzed by measuring the length of the fragment obtained by restriction enzyme cleavage and determining the base sequence. The obtained plasmid DNAs of interest were named pSK-OCIF-CBst, pSK-OCIF-CSph, pSK-OCIF-CBsp and pSK-OCIF-CPst.
[0124]
(2) Construction of mutant expression vector
The obtained plasmid DNA (pSK-OCIF-CBst, pSK-OCIF-CSph, pSK-OCIF-CBsp, pSK-OCIF-CPst) was cleaved with restriction enzymes BamHI and XhoI (Takara Shuzo Co., Ltd.) to obtain about 1.5 including OCIF cDNA full length. A kilobase pair (kb) DNA fragment (including the target mutation) was isolated and purified, and dissolved in 20 μl of sterile distilled water (named CBstDNA solution, CSphDNA solution, CBspDNA solution, and CPstDNA solution, respectively).
1 μl of pCEP4 DNA solution described in Example 22-ii) and 6 μl each of CBstDNA solution, CSphDNA solution, CBspDNA solution and CPstDNA solution are mixed separately, and 7 μl of DNA Ligation Kit Ver.2 I solution is added to each mixture. Then, a ligation reaction was performed. After completion of the reaction, E. coli DH5α was transformed with 7 μl of the reaction solution. From the obtained ampicillin-resistant transformed cells, a total of 5 strains having plasmid DNA having a structure in which the OCIF cDNA fragment having the target mutation was inserted between the XhoI BamHI sites of pCEP4 were selected. The plasmids having the target 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 mutant expression vectors was grown, and various mutant expression vectors were purified using Qiagen columns (Qiagen). Each expression vector was precipitated with ethanol, dissolved in sterilized distilled water, and used for the following operations.
[0126]
vi ) Mutant cDNA Of transient expression and activity
Using the various OCIF variant expression plasmids purified in Example 22-v), the OCIF variant 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 × 10^Five293 / EBNA cells were implanted in each well using IMDM medium containing 10% fetal bovine serum. The amounts of mutant expression vector and lipofectamine used for DNA introduction were 1 μg and 4 μl, respectively. Diluted with OPTI-MEM medium (Gibco BRL) to a final volume of 0.5 ml. Add the mixture of mutant expression vector and lipofectamine to the cells and mix with CO for 24 hours at 37 ° C.₂After culturing in the incubator, remove the mixture, add 0.5 ml of Ex-cell 301 medium (JSR), and then add CO for 48 hours at 37 ° C.₂Cultured in an incubator. The medium was collected and used as a sample for measuring mutant activity. The nucleotide sequences of the obtained mutants are shown in SEQ ID NO: 83 to 103, and the amino acid sequences deduced from the sequences are shown in SEQ ID NO: 62 to 82, respectively. The activity measurement of OCIF was according to Example 13. Further, the amount of OCIF antigen was quantified by the EIA method described in Example 24. Table 14 shows the activity per antigen amount compared to unmodified OCIF.
[0127]
[Table 14]

[0128]
vi) Western blotting analysis
10 μl of the sample used for activity measurement was subjected to Western blot analysis. 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)) to 10 µl of sample, boil at 100 ° C for 3 minutes, non-reducing state At 10% SDS polyacrylamide electrophoresis. After completion of the electrophoresis, the protein was blotted onto a PVDF membrane (ProBlott®, Perkin Elmer) using a semi-drive blotting device (Bio-Rad). After blocking the membrane, the membrane was incubated with a horseradish peroxidase-labeled anti-OCIF antibody for EIA described in Example 24 at 37 ° C. for 2 hours.
After washing, proteins bound to the anti-OCIF antibody were detected by ECL system (Amersham). OCIF detected bands of approximately 120 kilodaltons (kD) and 60 kD. On the other hand, in OCIF-C23S, OCIF-CL, and OCIF-CC, only the 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 the main bands, respectively. From the above results, in OCIF, the 379th Cys residue in the amino acid sequence of Sequence Listing SEQ ID NO: 4 is involved in dimer formation, the activity is retained even in the monomer, and the number 177 It was revealed that the activity was retained even when residues from Asp to 380 Leu were deleted.
[0129]
Example 23
Human OCIF genome DNA Separation
I) Human genome DNA Library screening
A genomic library prepared using human placenta chromosomal DNA and the λFIX II vector was purchased from Stratagene and screened using OCIF cDNA as a probe. Screening was basically performed according to the protocol attached to the genomic library, but general methods for handling phage, E. coli, and DNA were performed according to Molecular Cloning: A Laboratory Manual.
1 × 10 after testing the titer of the purchased genomic DNA library⁶ pfu phages were infected with 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 Hybond-N nylon membrane (Amersham) was placed on the agar plate to transfer the phage. The nylon membrane with the transferred phage is placed on a filter paper moistened with 1.5M NaCl / 0.5M NaOH solution for 1 minute, and then 1M Tris-HCl (pH 7.5) and 1.5M NaCl / 0.5M Tris-HCl (pH 7.5). ), Each was treated for 1 minute and neutralized, and finally transferred onto filter paper moistened with 2XSSC. Thereafter, the nylon membrane was irradiated with 1200 microjoules of UV using a stratalinker (Stratagene) to immobilize the phage DNA on the membrane. Next, this nylon membrane was immersed in a rapid hybridization buffer (Amersham) to perform prehybridization. After 1 hour prehybridization,³²P-labeled OCIF cDNA was added and hybridization was performed overnight at 65 ° C. This cDNA probe was prepared by cleaving the plasmid pBKOCIF having the 1.6 kb OCIF cDNA obtained in Example 11 using restriction enzymes BamHI and XhoI, isolating the OCIF cDNA by agarose gel electrophoresis, and then subjecting the OCIF cDNA to megaprime. Using 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 buffer^FiveA cpm probe was used. After hybridization, the nylon membrane was washed with 2XSSC for 5 minutes at room temperature, and then washed 4 times with 0.5XSSC / 0.1% SDS at 65 ° C. for 20 minutes each. After the fourth washing, the nylon membrane was dried, and autoradiography was performed at −80 ° C. using Fujifilm X-ray film, Super HR-H and intensifying screen. Since 6 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 SM buffer to which 1% chloroform was added. Left overnight to extract the phage. Each phage extract was diluted 1000-fold with SM buffer, and 1 μl and 20 μl were taken out of it, and again infected with the E. coli, and spread on agar plates together with top agarose by the above method. After transferring the phage to a nylon membrane, pre-hybridization, hybridization, washing, drying, and autoradiography were performed by the above-described method. This phage purification procedure was performed for all six signals initially detected by autoradiography and repeated until all phage plaques on the agar plate hybridized with the cDNA probe. Purified phage plaques were excised and 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, and λOIF17, respectively.
[0131]
II) Human by restriction enzyme digestion and Southern blot hybridization OCIF genome DNA Analysis of clones
The purified 6 kinds of phage DNA were purified by plate lysis according to the method described in Molecular Cloning: A Laboratory Manual. These DNAs were digested with restriction enzymes, and the resulting fragments were separated by agarose electrophoresis. The fragments separated on the agarose gel were transferred to a nylon membrane by a general method, and then Southern blot hybridization was performed using OCIF cDNA as a probe. As a result of these analyses, it was found that the six types of purified phages were different clones. Among DNA fragments obtained by restriction enzyme digestion, those that hybridize with OCIF cDNA were subcloned into a plasmid vector, and then analyzed for nucleotide sequences by the following method.
[0132]
iii) genome DNA Obtained from a clone by restriction enzyme digestion DNA Subcloning of fragments into plasmid vectors and determination of nucleotide sequences
λOIF8 DNA was digested with restriction enzymes EcoRI and NotI and the resulting fragment was donated to a 0.7% agarose gel and separated. A 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 the pBluescriptII SK + vector (Stratagene) previously cut with EcoRI and NotI and Ready-To-Go T4Ligase (Pharmacia) according to the attached protocol. The obtained recombinant plasmid was introduced into competent DH5α E. coli (Amersham) and then spread on an agarose plate containing 50 μg / ml ampicillin to select E. coli having the plasmid. 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 generated by digesting pBSG8-5.8 with the restriction enzyme HindIII was separated on an agarose gel, extracted according to the above method, and then pBluescriptII SK- (Stratagene previously cleaved with HindIII. And cloned according to the method described above. This recombinant plasmid having the 0.9 kb HindIII fragment was designated pBS8H0.9. On the other hand, 6 kb, 3.6 kb, and 2.6 kb fragments generated by digesting λOIF11 DNA with EcoRI were isolated and then inserted into the pBluescriptII SK + vector and cloned in the same manner as described above. The recombinant plasmids having 6 kb, 3.6 kb, and 2.6 kb EcoRI fragments thus prepared were named pBSG11-6, pBSG11-3.6, and pBSG11-2.6, respectively.
[0133]
Furthermore, three fragments of 2.2 kb, 1.1 kb and 1.05 kb generated by digesting pBSG11-6 with the restriction enzyme HindIII were separated by agarose gel electrophoresis and cloned by inserting them into the HindIII site of pBluescriptII SK-, respectively. did. These recombinant plasmids having HindIII fragments of 2.2 kb, 1.1 kb and 1.05 kb were named pBS6H2.2, pBS6H1.1 and pBS6H1.05, respectively. ABI Dyedeoxy Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer) and 373 DNA Sequencing System (Applied Biosystems) were used for the analysis of the genomic DNA base sequence. Molecular Cloning: Prepare 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 used as a casting mold. The nucleotide sequences of human OCIF genomic DNA are shown in SEQ ID NOs: 104 and 105 in Sequence Listing. The base sequence intervening between exon 1 and exon 2 is not completely determined, and it is confirmed that about 17 kb nucleotide intervenes between the base sequences shown in SEQ ID NOs: 104 and 105 in the sequence listing. Has been.
[0134]
Example 24
EIA by OCIF Quantification of
i) Rabbit anti OCIF Antibody preparation
Three male white rabbits (weighing 2.5-3.0 kg, obtained from Kitayama Labes Co., Ltd.) were mixed subcutaneously with 1 ml of rOCIF 200 μg / ml mixed with Freund's complete adjuvant (DIFCO) in an equal volume. I was immunized. Immunization was performed 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 antiserum diluted 2-fold with PBS to a final concentration of 40 w / v% and left at 4 ° C. for 1 hour, followed by centrifugation 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 loaded onto 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 neutralized with 1.5 M Tris hydrochloride buffer (pH 8.7). After dialyzing the eluted protein fraction against PBS, the absorbance at 280 nm was measured to determine its concentration (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 purified antibody and reacted at room temperature for 30 minutes. After 5 mg of hydroxylamine was added to this for deacetylation, 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 Quantification of
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 overnight at 4 ° C. Then, the antibody was immobilized. Add 300 μl of 25% Block Ace (Snow Brand Milk Products Co., Ltd.) prepared in PBS to each well, block at 37 ° C. for 1 hour, block, then add sample (100 μl / well) and react at room temperature for 2 hours. It was. After washing three times with PBS containing 0.05% Tween 20 (PBST), 100 μl of horseradish peroxidase-labeled anti-OCIF antibody diluted 10,000 times was added and incubated at room temperature for 2 hours. After washing 3 times with PBST, 100 μl of enzyme substrate solution (TMB, ScyTek) was added to develop color at room temperature, and then the reaction was stopped. Absorbance at 450 nm was measured using a microplate reader (Immunoleader NJ2000, Nippon Intermed), and the OCIF concentration of the specimen was quantified from a calibration curve using purified recombinant OCIF as a standard. The calibration curve for OCIF is shown in FIG.
[0136]
Example 25
Anti OCIF Monoclonal antibody
i) Human OCIF Preparation of antibody-producing hybridoma
Human fibroblast IMR-90 was cultured, and OCIF was purified from the culture solution by the method described in Example 11. Purified OCIF was dissolved in PBS to a concentration of 10 μg / 100 μl, and this solution was intraperitoneally administered to BALB / c mice every two weeks for immunization. In the first and second immunizations, an equal volume of Freund's complete adjuvant mixture was administered. On the third day from the final immunization, the spleen was removed, B lymphocytes were isolated, and cell fusion was performed with mouse myeloma cells P3x63-AG8.653 by the commonly used polyethylene glycol method. Subsequently, hybridoma cells were selected by culturing in HAT medium in order to select fused cells. Next, to confirm whether the selected cells are producing OCIF-specific antibodies, 100 μl of OCIF solution (10 μg / ml) dissolved in 0.1 M sodium bicarbonate solution was added to a 96-well microplate (Nunc) In addition to the above, OCIF-specific antibodies in the hybridoma culture were measured using a solid phase ELISA prepared. The hybridoma in which antibody production was recognized was cloned 3-5 times by the limiting dilution method, and the amount of antibody production was checked by the above-mentioned ELISA each time. Clones with high antibody production were selected from the obtained antibody-producing strains.
[0137]
ii) Production of monoclonal antibodies
The antibody producing strains obtained in Example 25-i) were each 1 × 10⁶Was transplanted intraperitoneally into BALB / c mice previously inoculated with pristane (Aldrich Chemical Co.). Two weeks after the transplantation, the accumulated ascites was collected to obtain ascites containing the monoclonal antibody of the present invention. From this ascites, a purified antibody was obtained by affinity chromatography using Affigel Protein A Sepharose (manufactured by Bio-Rad). That is, ascites was diluted with an equal amount of binding buffer (Bio-Rad), loaded onto a protein A column, and then washed with a sufficient amount of the same buffer. The elution of IgG was performed using an elution buffer (Bio-Rad). The obtained eluate was dialyzed with water and then freeze-dried. When the purified antibody thus obtained was subjected to a purity test by SDS-PAGE, a uniform band was observed at a molecular weight of about 150,000.
[0138]
iii) OCIF Of monoclonal antibodies with high affinity for
The antibody obtained in Example 25-ii) was dissolved in PBS, and protein quantification was performed by the Raleigh method. Subsequently, each antibody was dissolved in PBS so that the protein concentration was constant, and this solution was diluted by a serial dilution method. Using the solid phase ELISA described in Example 25-ii), monoclonal antibodies that react with OCIF up to the high dilution step were selected. As a result, three types of antibodies, A1G5, E3H8, and D2F4, were obtained.
[0139]
iv) Antibody subclass assay
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). The assay was performed according to the protocol specified in the kit. The results are shown in Table 15. E3H8, A1G5, and D2F4 are IgG₁, IgG_2aAnd IgG_2b Met.
[0140]
[Table 15]

[0141]
v) OCIF of ELISA Measurement method
The three monoclonal antibodies A1G5, E3H8, and D2F4 obtained in Example 25-iv) were used as a solid phase antibody and a labeled antibody, respectively. A sandwich ELISA was constructed for each combination. The antibody was labeled 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 100 μl per well of a 96-well immunoplate (Nunc), and allowed to stand overnight at room temperature. Each plate was then blocked with 1/2 concentration of Block Ace (Snow Brand Milk Products) and washed 3 times with PBS containing 0.1% Tween20 (wash buffer). Each concentration of OCIF was prepared in the primary reaction buffer (0.2 M Tris-HCl buffer, pH 7.4 containing 1 / 2.5 concentration of Block Ace and 0.1% Tween20).
[0142]
100 μl of the prepared OCIF solution of each concentration was added to each well, allowed to stand at 37 ° C. for 3 hours, and then washed 3 times with a washing buffer. For dilution of the labeled antibody, a secondary reaction buffer (0.1 M Tris-HCl buffer, pH 7.4 containing 1/4 concentration of Block Ace and 0.1% Tween 20) was used. Each labeled antibody was diluted 400 times with the secondary reaction buffer, and 100 μl of each was added to each well. Each plate was allowed to stand at 37 ° C. for 2 hours and then washed 3 times before the substrate solution (0.1 M citrate-phosphate buffer containing 0.4 mg / ml orthophenylenediamine hydrochloride, 0.006% hydrogen peroxide, pH 4.5). 100 μl was added to each well. After leaving in a dark room 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 using an immunoleader (NJ2000, Nihon Intermed). Good results were obtained with any combination of the three antibodies, each of which was a solid phase antibody or a labeled antibody, and the three antibodies were recognized to recognize different epitopes of OCIF. As a representative example, a calibration curve when A1G5 is a solid phase antibody and E3H8 is a labeled antibody is shown in FIG.
[0143]
vi) In human serum OCIF Measurement
OCIF in the serum of 5 healthy individuals was measured by the ELISA system of FIG. 14 in Example 25- (v). That is, A1G5 was immobilized on an immunoplate in the same manner as in Example 25- (v), 50 μl of the primary reaction buffer was added to each well, and then 50 μl of each human serum was added and left at 37 ° C. for 3 hours. After washing 3 times with the washing buffer, 100 μl of labeled antibody of E3H8 diluted 400 times with the secondary reaction buffer was added to each well and left at 37 ° C. for 2 hours. After washing the plate 3 times with the washing buffer, 100 μl of the above substrate solution was added to each well and allowed to react 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 first reaction buffer containing a known amount of OCIF was similarly operated 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. The results are shown in Table 16.
[0144]
[Table 16]

[0145]
Example 26
Therapeutic effect on osteoporosis
We confirmed the therapeutic effect of OCIF on immobility bone atrophy model by nerve resection.
A Fischer male rat was used to excise the left brachial plexus at 6 weeks of age (body weight approximately 120 g), thereby causing immobilization of the left forelimb and creating a bone atrophy model. OCIF was adjusted with PBS (-) containing 0.01% Tween 80 and from the next day, it was intravenously administered twice a day for 2 weeks every 12 hours at a dose of 5 μg / kg and 50 μg / kg. A sham operation was performed on the normal group, and PBS (-) containing 0.01% Tween80 was similarly administered to the control group. After the administration, the left upper arm was removed 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 compared with the normal group, but an improvement was observed in the OCIF 50 μg / kg administration group.
[0146]
【The invention's effect】
  According to the present invention, a novel protein having osteoclast formation inhibitory activity and its efficient
A manufacturing method is provided. The protein of the present invention has osteoclast formation inhibitory activity, and osteoporosis
As a therapeutic agent for various bone loss diseases such as infectious diseases or immunological diagnosis of these diseases
It can be used as an antigen for the purpose.
[Sequence Listing]
SEQUENCELISTING
<110> Sankyo Co., Ltd
<120> Novel protein and methods for theproduction of the same
<130> 2003-119SW
<140>
<141>
<150> JP H07-54977
<151> 1995-2-20
<150> JP H07-207508
<151> 1995-7-21
<150> PCT / JP96 / 00374
<151> 1996-2-20
<160> 105
<170> PatentIn version 3.1.2
<210> 1
<211> 6
<212> PRT
<213> human OCIF
<220>
<221> Xaa
<222> 1
<223> Inventor: Goto, Masaaki
      Inventor: Tsuda, Eisuke
      Inventor: Mochizuki, Shin'ichi
      Inventor: Yano, Kazuki
      Inventor: Kobayashi, Fumie
      Inventor: Shima, Nobuyuki
      Inventor: Yasuda, Hisataka
      Inventor: Nakagawa, Nobuaki
      Inventor: Morinaga, Tomonori
      Inventor: Ueda, Masatsugu
      Inventor: Higashio, Kanji
<400> 1
Xaa Tyr His Phe Pro Lys
  1 5
<210> 2
<211> 14
<212> PRT
<213> human OCIF
<220>
<221> Xaa
<222> 1
<400> 2
Xaa Gln His Ser Xaa Gln Glu Gln Thr Phe Gln Leu Xaa Lys
  1 5 10
<210> 3
<211> 12
<212> PRT
<213> human OCIF
<220>
<221> Xaa
<222> 1
<400> 3
Xaa Ile Arg Phe Leu His Ser Phe Thr Met Tyr Lys
1 5 10
<210> 4
<211> 380
<212> PRT
<213> human OCIF
<400> 4
  Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr Asp Glu Glu Thr Ser
   1 5 10 15
  His Gln Leu Leu Cys Asp Lys Cys Pro Pro Gly Thr Tyr Leu Lys
                  20 25 30
  Gln His Cys Thr Ala Lys Trp Lys Thr Val Cys Ala Pro Cys Pro
                  35 40 45
  Asp His Tyr Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Cys Leu
                  50 55 60
  Tyr Cys Ser Pro Val Cys Lys Glu Leu Gln Tyr Val Lys Gln Glu
                  65 70 75
  Cys Asn Arg Thr His Asn Arg Val Cys Glu Cys Lys Glu Gly Arg
                  80 85 90
  Tyr Leu Glu Ile Glu Phe Cys Leu Lys His Arg Ser Cys Pro Pro
                  95 100 105
  Gly Phe Gly Val Val Gln Ala Gly Thr Pro Glu Arg Asn Thr Val
                  110 115 120
  Cys Lys Arg Cys Pro Asp Gly Phe Phe Ser Asn Glu Thr Ser Ser
                  125 130 135
  Lys Ala Pro Cys Arg Lys His Thr Asn Cys Ser Val Phe Gly Leu
                  140 145 150
  Leu Leu Thr Gln Lys Gly Asn Ala Thr His Asp Asn Ile Cys Ser
                  155 160 165
  Gly Asn Ser Glu Ser Thr Gln Lys Cys Gly Ile Asp Val Thr Leu
                  170 175 180
  Cys Glu Glu Ala Phe Phe Arg Phe Ala Val Pro Thr Lys Phe Thr
                  185 190 195
  Pro Asn Trp Leu Ser Val Leu Val Asp Asn Leu Pro Gly Thr Lys
                  200 205 210
  Val Asn Ala Glu Ser Val Glu Arg Ile Lys Arg Gln His Ser Ser
                  215 220 225
  Gln Glu Gln Thr Phe Gln Leu Leu Lys Leu Trp Lys His Gln Asn
                  230 235 240
  Lys Asp Gln Asp Ile Val Lys Lys Ile Ile Gln Asp Ile Asp Leu
                  245 250 255
  Cys Glu Asn Ser Val Gln Arg His Ile Gly His Ala Asn Leu Thr
                  260 265 270
  Phe Glu Gln Leu Arg Ser Leu Met Glu Ser Leu Pro Gly Lys Lys
                  275 280 285
  Val Gly Ala Glu Asp Ile Glu Lys Thr Ile Lys Ala Cys Lys Pro
                  290 295 300
  Ser Asp Gln Ile Leu Lys Leu Leu Ser Leu Trp Arg Ile Lys Asn
                  305 310 315
  Gly Asp Gln Asp Thr Leu Lys Gly Leu Met His Ala Leu Lys His
                  320 325 330
  Ser Lys Thr Tyr His Phe Pro Lys Thr Val Thr Gln Ser Leu Lys
                  335 340 345
  Lys Thr Ile Arg Phe Leu His Ser Phe Thr Met Tyr Lys Leu Tyr
                  350 355 360
  Gln Lys Leu Phe Leu Glu Met Ile Gly Asn Gln Val Gln Ser Val
                  365 370 375
  Lys Ile Ser Cys Leu
                  380
<210> 5
<211> 401
<212> PRT
<213> human OCIF
<400> 5
  Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
      -20 -15 -10
  Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
      -5 -1 1 5
  Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
  10 15 20
  Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
  25 30 35
  Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
  40 45 50
  Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
  55 60 65
  Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys
  70 75 80
  Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys
  85 90 95
  His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly Thr
  100 105 110
  Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe Phe
  115 120 125
  Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn
  130 135 140
  Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys Gly Asn Ala Thr
  145 150 155
  His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr Gln Lys Cys
  160 165 170
  Gly Ile Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg Phe Ala
  175 180 185
  Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val Asp
  190 195 200
  Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg Ile
  205 210 215
  Lys Arg Gln His Ser Ser Gln Glu Gln Thr Phe Gln Leu Leu Lys
  220 225 230
  Leu Trp Lys His Gln Asn Lys Asp Gln Asp Ile Val Lys Lys Ile
  235 240 245
  Ile Gln Asp Ile Asp Leu Cys Glu Asn Ser Val Gln Arg His Ile
  250 255 260
  Gly His Ala Asn Leu Thr Phe Glu Gln Leu Arg Ser Leu Met Glu
  265 270 275
  Ser Leu Pro Gly Lys Lys Val Gly Ala Glu Asp Ile Glu Lys Thr
  280 285 290
  Ile Lys Ala Cys Lys Pro Ser Asp Gln Ile Leu Lys Leu Leu Ser
  295 300 305
  Leu Trp Arg Ile Lys Asn Gly Asp Gln Asp Thr Leu Lys Gly Leu
  310 315 320
  Met His Ala Leu Lys His Ser Lys Thr Tyr His Phe Pro Lys Thr
  325 330 335
  Val Thr Gln Ser Leu Lys Lys Thr Ile Arg Phe Leu His Ser Phe
  340 345 350
  Thr Met Tyr Lys Leu Tyr Gln Lys Leu Phe Leu Glu Met Ile Gly
  355 360 365
  Asn Gln Val Gln Ser Val Lys Ile Ser Cys Leu
  370 375 380
<210> 6
<211> 1206
<212> DNA
<213> human OCIF
<400> 6
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gaaccccaga gcgaaataca 420
gtttgcaaaa gatgtccaga tgggttcttc tcaaatgaga cgtcatctaa agcaccctgt 480
agaaaacaca caaattgcag tgtctttggt ctcctgctaa ctcagaaagg aaatgcaaca 540
cacgacaaca tatgttccgg aaacagtgaa tcaactcaaa aatgtggaat agatgttacc 600
ctgtgtgagg aggcattctt caggtttgct gttcctacaa agtttacgcc taactggctt 660
agtgtcttgg tagacaattt gcctggcacc aaagtaaacg cagagagtgt agagaggata 720
aaacggcaac acagctcaca agaacagact ttccagctgc tgaagttatg gaaacatcaa 780
aacaaagacc aagatatagt caagaagatc atccaagata ttgacctctg tgaaaacagc 840
gtgcagcggc acattggaca tgctaacctc accttcgagc agcttcgtag cttgatggaa 900
agcttaccgg gaaagaaagt gggagcagaa gacattgaaa aaacaataaa ggcatgcaaa 960
cccagtgacc agatcctgaa gctgctcagt ttgtggcgaa taaaaaatgg cgaccaagac 1020
accttgaagg gcctaatgca cgcactaaag cactcaaaga cgtaccactt tcccaaaact 1080
gtcactcaga gtctaaagaa gaccatcagg ttccttcaca gcttcacaat gtacaaattg 1140
tatcagaagt tatttttaga aatgataggt aaccaggtcc aatcagtaaa aataagctgc 1200
ttataa 1206
<210> 7
<211> 15
<212> PRT
<213> human OCIF
<400> 7
Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr Asp Glu Glu Thr Ser
1 5 10 15
<210> 8
<211> 1185
<212> DNA
<213> OCIF2
<400> 8
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagtgc aatcgcaccc acaaccgcgt gtgcgaatgc 300
aaggaagggc gctaccttga gatagagttc tgcttgaaac ataggagctg ccctcctgga 360
tttggagtgg tgcaagctgg aaccccagag cgaaatacag tttgcaaaag atgtccagat 420
gggttcttct caaatgagac gtcatctaaa gcaccctgta gaaaacacac aaattgcagt 480
gtctttggtc tcctgctaac tcagaaagga aatgcaacac acgacaacat atgttccgga 540
aacagtgaat caactcaaaa atgtggaata gatgttaccc tgtgtgagga ggcattcttc 600
aggtttgctg ttcctacaaa gtttacgcct aactggctta gtgtcttggt agacaatttg 660
cctggcacca aagtaaacgc agagagtgta gagaggataa aacggcaaca cagctcacaa 720
gaacagactt tccagctgct gaagttatgg aaacatcaaa acaaagacca agatatagtc 780
aagaagatca tccaagatat tgacctctgt gaaaacagcg tgcagcggca cattggacat 840
gctaacctca ccttcgagca gcttcgtagc ttgatggaaa gcttaccggg aaagaaagtg 900
ggagcagaag acattgaaaa aacaataaag gcatgcaaac ccagtgacca gatcctgaag 960
ctgctcagtt tgtggcgaat aaaaaatggc gaccaagaca ccttgaaggg cctaatgcac 1020
gcactaaagc actcaaagac gtaccacttt cccaaaactg tcactcagag tctaaagaag 1080
accatcaggt tccttcacag cttcacaatg tacaaattgt atcagaagtt atttttagaa 1140
atgataggta accaggtcca atcagtaaaa ataagctgct tataa 1185
<210> 9
<211> 394
<212> PRT
<213> OCIF2
<400> 9
  Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
      -20 -15 -10
  Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
      -5 1 5
  Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
  10 15 20
  Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
  25 30 35
  Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
  40 45 50
  Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Cys
  55 60 65
  Asn Arg Thr His Asn Arg Val Cys Glu Cys Lys Glu Gly Arg Tyr
  70 75 80
  Leu Glu Ile Glu Phe Cys Leu Lys His Arg Ser Cys Pro Pro Gly
  85 90 95
  Phe Gly Val Val Gln Ala Gly Thr Pro Glu Arg Asn Thr Val Cys
  100 105 110
  Lys Arg Cys Pro Asp Gly Phe Phe Ser Asn Glu Thr Ser Ser Lys
  115 120 125
  Ala Pro Cys Arg Lys His Thr Asn Cys Ser Val Phe Gly Leu Leu
  130 135 140
  Leu Thr Gln Lys Gly Asn Ala Thr His Asp Asn Ile Cys Ser Gly
  145 150 155
  Asn Ser Glu Ser Thr Gln Lys Cys Gly Ile Asp Val Thr Leu Cys
  160 165 170
  Glu Glu Ala Phe Phe Arg Phe Ala Val Pro Thr Lys Phe Thr Pro
  175 180 185
  Asn Trp Leu Ser Val Leu Val Asp Asn Leu Pro Gly Thr Lys Val
  190 195 200
  Asn Ala Glu Ser Val Glu Arg Ile Lys Arg Gln His Ser Ser Gln
  205 210 215
  Glu Gln Thr Phe Gln Leu Leu Lys Leu Trp Lys His Gln Asn Lys
  220 225 230
  Asp Gln Asp Ile Val Lys Lys Ile Ile Gln Asp Ile Asp Leu Cys
  235 240 245
  Glu Asn Ser Val Gln Arg His Ile Gly His Ala Asn Leu Thr Phe
  250 255 260
  Glu Gln Leu Arg Ser Leu Met Glu Ser Leu Pro Gly Lys Lys Val
  265 270 275
  Gly Ala Glu Asp Ile Glu Lys Thr Ile Lys Ala Cys Lys Pro Ser
  280 285 290
  Asp Gln Ile Leu Lys Leu Leu Ser Leu Trp Arg Ile Lys Asn Gly
  295 300 305
  Asp Gln Asp Thr Leu Lys Gly Leu Met His Ala Leu Lys His Ser
  310 315 320
  Lys Thr Tyr His Phe Pro Lys Thr Val Thr Gln Ser Leu Lys Lys
  325 330 335
  Thr Ile Arg Phe Leu His Ser Phe Thr Met Tyr Lys Leu Tyr Gln
  340 345 350
  Lys Leu Phe Leu Glu Met Ile Gly Asn Gln Val Gln Ser Val Lys
  355 360 365
  Ile Ser Cys Leu
  370
<210> 10
<211> 1089
<212> DNA
<213> OCIF3
<400> 10
atgaacaagt tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gaaccccaga gcgaaataca 420
gtttgcaaaa gatgtccaga tgggttcttc tcaaatgaga cgtcatctaa agcaccctgt 480
agaaaacaca caaattgcag tgtctttggt ctcctgctaa ctcagaaagg aaatgcaaca 540
cacgacaaca tatgttccgg aaacagtgaa tcaactcaaa aatgtggaat agatgttacc 600
ctgtgtgagg aggcattctt caggtttgct gttcctacaa agtttacgcc taactggctt 660
agtgtcttgg tagacaattt gcctggcacc aaagtaaacg cagagagtgt agagaggata 720
aaacggcaac acagctcaca agaacagact ttccagctgc tgaagttatg gaaacatcaa 780
aacaaagacc aagatatagt caagaagatc atccaagata ttgacctctg tgaaaacagc 840
gtgcagcggc acattggaca tgctaacctc agtttgtggc gaataaaaaa tggcgaccaa 900
gacaccttga agggcctaat gcacgcacta aagcactcaa agacgtacca ctttcccaaa 960
actgtcactc agagtctaaa gaagaccatc aggttccttc acagcttcac aatgtacaaa 1020
ttgtatcaga agttattttt agaaatgata ggtaaccagg tccaatcagt aaaaataagc 1080
tgcttataa 1089
<210> 11
<211> 362
<212> PRT
<213> OCIF3
<400> 11
  Met Asn Lys Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
      -20 -15 -10
  Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
      -5 1 5
  Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
  10 15 20
  Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
  25 30 35
  Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
  40 45 50
  Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
  55 60 65
  Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys
  70 75 80
  Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys
  85 90 95
  His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly Thr
  100 105 110
  Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe Phe
  115 120 125
  Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn
  130 135 140
  Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys Gly Asn Ala Thr
  145 150 155
  His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr Gln Lys Cys
  160 165 170
  Gly Ile Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg Phe Ala
  175 180 185
  Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val Asp
  190 195 200
  Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg Ile
  205 210 215
  Lys Arg Gln His Ser Ser Gln Glu Gln Thr Phe Gln Leu Leu Lys
  220 225 230
  Leu Trp Lys His Gln Asn Lys Asp Gln Asp Ile Val Lys Lys Ile
  235 240 245
  Ile Gln Asp Ile Asp Leu Cys Glu Asn Ser Val Gln Arg His Ile
  250 255 260
  Gly His Ala Asn Leu Ser Leu Trp Arg Ile Lys Asn Gly Asp Gln
  265 270 275
  Asp Thr Leu Lys Gly Leu Met His Ala Leu Lys His Ser Lys Thr
  280 285 290
  Tyr His Phe Pro Lys Thr Val Thr Gln Ser Leu Lys Lys Thr Ile
  295 300 305
  Arg Phe Leu His Ser Phe Thr Met Tyr Lys Leu Tyr Gln Lys Leu
  310 315 320
  Phe Leu Glu Met Ile Gly Asn Gln Val Gln Ser Val Lys Ile Ser
  325 330 335
  Cys Leu
  340
<210> 12
<211> 465
<212> DNA
<213> OCIF4
<400> 12
atgaacaagt tgctgtgctg ctcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gtacgtgtca atgtgcagca 420
aaattaatta ggatcatgca aagtcagata gttgtgacag tttag 465
<210> 13
<211> 154
<212> PRT
<213> OCIF4
<400> 13
  Met Asn Lys Leu Leu Cys Cys Ser Leu Val Phe Leu Asp Ile Ser
      -20 -15 -10
  Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
      -5 1 5
  Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
  10 15 20
  Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
  25 30 35
  Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
  40 45 50
  Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
  55 60 65
  Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys
  70 75 80
  Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys
  85 90 95
  His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly Thr
  100 105 110
  Cys Gln Cys Ala Ala Lys Leu Ile Arg Ile Met Gln Ser Gln Ile
  115 120 125
  Val Val Thr Val
  130
<210> 14
<211> 438
<212> DNA
<213> OCIF5
<400> 14
atgaacaagt tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gatgcaggag aagacccaag 420
ccacagatat gtatctga 438
<210> 15
<211> 145
<212> PRT
<213> OCIF5
<400> 15
  Met Asn Lys Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
      -20 -15 -10
  Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
      -5 1 5
  Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
  10 15 20
  Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
  25 30 35
  Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
  40 45 50
  Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
  55 60 65
  Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys
  70 75 80
  Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys
  85 90 95
  His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly Cys
  100 105 110
  Arg Arg Arg Pro Lys Pro Gln Ile Cys Ile
  115 120 125
<210> 16
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer T3
<400> 16
aattaaccct cactaaaggg 20
<210> 17
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer T7
<400> 17
gtaatacgac tcactatagg gc 22
<210> 18
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer IF1
<400> 18
acatcaaaac aaagaccaag 20
<210> 19
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer IF2
<400> 19
tcttggtctt tgttttgatg 20
<210> 20
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer IF3
<400> 20
ttattcgcca caaactgagc 20
<210> 21
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer IF4
<400> 21
ttgtgaagct gtgaaggaac 20
<210> 22
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer IF5
<400> 22
gctcagtttg tggcgaataa 20
<210> 23
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer IF6
<400> 23
gtgggagcag aagacattga 20
<210> 24
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer IF7
<400> 24
aatgaacaac ttgctgtgct 20
<210> 25
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer IF8
<400> 25
tgacaaatgt cctcctggta 20
<210> 26
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer IF9
<400> 26
aggtaggtac caggaggaca 20
<210> 27
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer IF10
<400> 27
gagctgccct cctggatttg 20
<210> 28
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer IF11
<400> 28
caaactgtat ttcgctctgg 20
<210> 29
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer IF12
<400> 29
gtgtgaggag gcattcttca 20
<210> 30
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer C19SF
<400> 30
gaatcaactc aaaaaagtgg aatagatgtt ac 32
<210> 31
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer C19SR
<400> 31
gtaacatcta ttccactttt ttgagttgat tc 32
<210> 32
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer C20SF
<400> 32
atagatgtta ccctgagtga ggaggcattc 30
<210> 33
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer C20SR
<400> 33
gaatgcctcc tcactcaggg taacatctat 30
<210> 34
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer C21SF
<400> 34
caagatattg acctcagtga aaacagcgtg c 31
<210> 35
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer C21SR
<400> 35
gcacgctgtt ttcactgagg tcaatatctt g 31
<210> 36
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer C22SF
<400> 36
aaaacaataa aggcaagcaa acccagtgac c 31
<210> 37
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer C22SR
<400> 37
ggtcactggg tttgcttgcc tttattgttt t 31
<210> 38
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer C23SF
<400> 38
tcagtaaaaa taagcagctt ataactggcc a 31
<210> 39
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer C23SR
<400> 39
tggccagtta taagctgctt atttttactg a 31
<210> 40
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer IF 14
<400> 40
ttggggttta ttggaggaga tg 22
<210> 41
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer DCR1F
<400> 41
accacccagg aaccttgccc tgaccactac tacaca 36
<210> 42
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer DCR1R
<400> 42
gtcagggcaa ggttcctggg tggtccactt aatgga 36
<210> 43
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer DCR2F
<400> 43
accgtgtgcg ccgaatgcaa ggaagggcgc tacctt 36
<210> 44
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer DCR2R
<400> 44
ttccttgcat tcggcgcaca cggtcttcca ctttgc 36
<210> 45
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer DCR3F
<400> 45
aaccgcgtgt gcagatgtcc agatgggttc ttctca 36
<210> 46
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer DCR3R
<400> 46
atctggacat ctgcacacgc ggttgtgggt gcgatt 36
<210> 47
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer DCR4F
<400> 47
acagtttgca aatccggaaa cagtgaatca actcaa 36
<210> 48
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer DCR4R
<400> 48
actgtttccg gatttgcaaa ctgtatttcg ctctgg 36
<210> 49
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer DDD1F
<400> 49
aatgtggaat agatattgac ctctgtgaaa acagcg 36
<210> 50
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer DDD1R
<400> 50
agaggtcaat atctattcca catttttgag ttgatt 36
<210> 51
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer DDD2F
<400> 51
agatcatcca agacgcacta aagcactcaa agacgt 36
<210> 52
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer DDD2R
<400> 52
gctttagtgc gtcttggatg atcttcttga ctatat 36
<210> 53
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer XhoI F
<400> 53
ggctcgagcg cccagccgcc gcctccaag 29
<210> 54
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer IF 16
<400> 54
tttgagtgct ttagtgcgtg 20
<210> 55
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer CL F
<400> 55
tcagtaaaaa taagctaact ggaaatggcc 30
<210> 56
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer CL R
<400> 56
ggccatttcc agttagctta tttttactga 30
<210> 57
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer CC R
<400> 57
ccggatcctc agtgctttag tgcgtgcat 29
<210> 58
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer CCD2 R
<400> 58
ccggatcctc attggatgat cttcttgac 29
<210> 59
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer CCD1 R
<400> 59
ccggatcctc atattccaca tttttgagt 29
<210> 60
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer CCR4 R
<400> 60
ccggatcctc atttgcaaac tgtatttcg 29
<210> 61
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCRprimer CCR3 R
<400> 61
ccggatcctc attcgcacac gcggttgtg 29
<210> 62
<211> 401
<212> PRT
<213> OCIF-C19S
<400> 62
  Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
      -20 -15 -10
  Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
      -5 -1 1 5
  Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
  10 15 20
  Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
  25 30 35
  Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
  40 45 50
  Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
  55 60 65
  Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys
  70 75 80
  Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys
  85 90 95
  His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly Thr
  100 105 110
  Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe Phe
  115 120 125
  Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn
  130 135 140
  Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys Gly Asn Ala Thr
  145 150 155
  His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr Gln Lys Ser
  160 165 170
  Gly Ile Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg Phe Ala
  175 180 185
  Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val Asp
  190 195 200
  Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg Ile
  205 210 215
  Lys Arg Gln His Ser Ser Gln Glu Gln Thr Phe Gln Leu Leu Lys
  220 225 230
  Leu Trp Lys His Gln Asn Lys Asp Gln Asp Ile Val Lys Lys Ile
  235 240 245
  Ile Gln Asp Ile Asp Leu Cys Glu Asn Ser Val Gln Arg His Ile
  250 255 260
  Gly His Ala Asn Leu Thr Phe Glu Gln Leu Arg Ser Leu Met Glu
  265 270 275
  Ser Leu Pro Gly Lys Lys Val Gly Ala Glu Asp Ile Glu Lys Thr
  280 285 290
  Ile Lys Ala Cys Lys Pro Ser Asp Gln Ile Leu Lys Leu Leu Ser
  295 300 305
  Leu Trp Arg Ile Lys Asn Gly Asp Gln Asp Thr Leu Lys Gly Leu
  310 315 320
  Met His Ala Leu Lys His Ser Lys Thr Tyr His Phe Pro Lys Thr
  325 330 335
  Val Thr Gln Ser Leu Lys Lys Thr Ile Arg Phe Leu His Ser Phe
  340 345 350
  Thr Met Tyr Lys Leu Tyr Gln Lys Leu Phe Leu Glu Met Ile Gly
  355 360 365
  Asn Gln Val Gln Ser Val Lys Ile Ser Cys Leu
  370 375 380
<210> 63
<211> 401
<212> PRT
<213> OCIF-C20S
<400> 63
  Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
      -20 -15 -10
  Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
      -5 -1 1 5
  Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
  10 15 20
  Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
  25 30 35
  Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
  40 45 50
  Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
  55 60 65
  Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys
  70 75 80
  Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys
  85 90 95
  His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly Thr
  100 105 110
  Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe Phe
  115 120 125
  Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn
  130 135 140
  Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys Gly Asn Ala Thr
  145 150 155
  His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr Gln Lys Cys
  160 165 170
  Gly Ile Asp Val Thr Leu Ser Glu Glu Ala Phe Phe Arg Phe Ala
  175 180 185
  Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val Asp
  190 195 200
  Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg Ile
  205 210 215
  Lys Arg Gln His Ser Ser Gln Glu Gln Thr Phe Gln Leu Leu Lys
  220 225 230
  Leu Trp Lys His Gln Asn Lys Asp Gln Asp Ile Val Lys Lys Ile
  235 240 245
  Ile Gln Asp Ile Asp Leu Cys Glu Asn Ser Val Gln Arg His Ile
  250 255 260
  Gly His Ala Asn Leu Thr Phe Glu Gln Leu Arg Ser Leu Met Glu
  265 270 275
  Ser Leu Pro Gly Lys Lys Val Gly Ala Glu Asp Ile Glu Lys Thr
  280 285 290
  Ile Lys Ala Cys Lys Pro Ser Asp Gln Ile Leu Lys Leu Leu Ser
  295 300 305
  Leu Trp Arg Ile Lys Asn Gly Asp Gln Asp Thr Leu Lys Gly Leu
  310 315 320
  Met His Ala Leu Lys His Ser Lys Thr Tyr His Phe Pro Lys Thr
  325 330 335
  Val Thr Gln Ser Leu Lys Lys Thr Ile Arg Phe Leu His Ser Phe
  340 345 350
  Thr Met Tyr Lys Leu Tyr Gln Lys Leu Phe Leu Glu Met Ile Gly
  355 360 365
  Asn Gln Val Gln Ser Val Lys Ile Ser Cys Leu
  370 375 380
<210> 64
<211> 401
<212> PRT
<213> OCIF-C21S
<400> 64
  Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
      -20 -15 -10
  Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
      -5 -1 1 5
  Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
  10 15 20
  Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
  25 30 35
  Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
  40 45 50
  Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
  55 60 65
  Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys
  70 75 80
  Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys
  85 90 95
  His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly Thr
  100 105 110
  Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe Phe
  115 120 125
  Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn
  130 135 140
  Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys Gly Asn Ala Thr
  145 150 155
  His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr Gln Lys Cys
  160 165 170
  Gly Ile Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg Phe Ala
  175 180 185
  Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val Asp
  190 195 200
  Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg Ile
  205 210 215
  Lys Arg Gln His Ser Ser Gln Glu Gln Thr Phe Gln Leu Leu Lys
  220 225 230
  Leu Trp Lys His Gln Asn Lys Asp Gln Asp Ile Val Lys Lys Ile
  235 240 245
  Ile Gln Asp Ile Asp Leu Ser Glu Asn Ser Val Gln Arg His Ile
  250 255 260
  Gly His Ala Asn Leu Thr Phe Glu Gln Leu Arg Ser Leu Met Glu
  265 270 275
  Ser Leu Pro Gly Lys Lys Val Gly Ala Glu Asp Ile Glu Lys Thr
  280 285 290
  Ile Lys Ala Cys Lys Pro Ser Asp Gln Ile Leu Lys Leu Leu Ser
  295 300 305
  Leu Trp Arg Ile Lys Asn Gly Asp Gln Asp Thr Leu Lys Gly Leu
  310 315 320
  Met His Ala Leu Lys His Ser Lys Thr Tyr His Phe Pro Lys Thr
  325 330 335
  Val Thr Gln Ser Leu Lys Lys Thr Ile Arg Phe Leu His Ser Phe
  340 345 350
  Thr Met Tyr Lys Leu Tyr Gln Lys Leu Phe Leu Glu Met Ile Gly
  355 360 365
  Asn Gln Val Gln Ser Val Lys Ile Ser Cys Leu
  370 375 380
<210> 65
<211> 401
<212> PRT
<213> OCIF-C22S
<400> 65
  Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
      -20 -15 -10
  Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
      -5 -1 1 5
  Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
  10 15 20
  Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
  25 30 35
  Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
  40 45 50
  Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
  55 60 65
  Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys
  70 75 80
  Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys
  85 90 95
  His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly Thr
  100 105 110
  Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe Phe
  115 120 125
  Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn
  130 135 140
  Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys Gly Asn Ala Thr
  145 150 155
  His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr Gln Lys Cys
  160 165 170
  Gly Ile Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg Phe Ala
  175 180 185
  Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val Asp
  190 195 200
  Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg Ile
  205 210 215
  Lys Arg Gln His Ser Ser Gln Glu Gln Thr Phe Gln Leu Leu Lys
  220 225 230
  Leu Trp Lys His Gln Asn Lys Asp Gln Asp Ile Val Lys Lys Ile
  235 240 245
  Ile Gln Asp Ile Asp Leu Cys Glu Asn Ser Val Gln Arg His Ile
  250 255 260
  Gly His Ala Asn Leu Thr Phe Glu Gln Leu Arg Ser Leu Met Glu
  265 270 275
  Ser Leu Pro Gly Lys Lys Val Gly Ala Glu Asp Ile Glu Lys Thr
  280 285 290
  Ile Lys Ala Ser Lys Pro Ser Asp Gln Ile Leu Lys Leu Leu Ser
  295 300 305
  Leu Trp Arg Ile Lys Asn Gly Asp Gln Asp Thr Leu Lys Gly Leu
  310 315 320
  Met His Ala Leu Lys His Ser Lys Thr Tyr His Phe Pro Lys Thr
  325 330 335
  Val Thr Gln Ser Leu Lys Lys Thr Ile Arg Phe Leu His Ser Phe
  340 345 350
  Thr Met Tyr Lys Leu Tyr Gln Lys Leu Phe Leu Glu Met Ile Gly
  355 360 365
  Asn Gln Val Gln Ser Val Lys Ile Ser Cys Leu
  370 375 380
<210> 66
<211> 401
<212> PRT
<213> OCIF-C23S
<400> 66
  Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
      -20 -15 -10
  Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
      -5 -1 1 5
  Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
  10 15 20
  Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
  25 30 35
  Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
  40 45 50
  Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
  55 60 65
  Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys
  70 75 80
  Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys
  85 90 95
  His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly Thr
  100 105 110
  Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe Phe
  115 120 125
  Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn
  130 135 140
  Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys Gly Asn Ala Thr
  145 150 155
  His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr Gln Lys Cys
  160 165 170
  Gly Ile Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg Phe Ala
  175 180 185
  Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val Asp
  190 195 200
  Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg Ile
  205 210 215
  Lys Arg Gln His Ser Ser Gln Glu Gln Thr Phe Gln Leu Leu Lys
  220 225 230
  Leu Trp Lys His Gln Asn Lys Asp Gln Asp Ile Val Lys Lys Ile
  235 240 245
  Ile Gln Asp Ile Asp Leu Cys Glu Asn Ser Val Gln Arg His Ile
  250 255 260
  Gly His Ala Asn Leu Thr Phe Glu Gln Leu Arg Ser Leu Met Glu
  265 270 275
  Ser Leu Pro Gly Lys Lys Val Gly Ala Glu Asp Ile Glu Lys Thr
  280 285 290
  Ile Lys Ala Cys Lys Pro Ser Asp Gln Ile Leu Lys Leu Leu Ser
  295 300 305
  Leu Trp Arg Ile Lys Asn Gly Asp Gln Asp Thr Leu Lys Gly Leu
  310 315 320
  Met His Ala Leu Lys His Ser Lys Thr Tyr His Phe Pro Lys Thr
  325 330 335
  Val Thr Gln Ser Leu Lys Lys Thr Ile Arg Phe Leu His Ser Phe
  340 345 350
  Thr Met Tyr Lys Leu Tyr Gln Lys Leu Phe Leu Glu Met Ile Gly
  355 360 365
  Asn Gln Val Gln Ser Val Lys Ile Ser Ser Leu
  370 375 380
<210> 67
<211> 360
<212> PRT
<213> OCIF-DCR1
<400> 67
  Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
      -20 -15 -10
  Ile Lys Trp Thr Thr Gln Glu Pro Cys Pro Asp His Tyr Tyr Thr
      -5 -1 1 5
  Asp Ser Trp His Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val
  10 15 20
  Cys Lys Glu Leu Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His
  25 30 35
  Asn Arg Val Cys Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu
  40 45 50
  Phe Cys Leu Lys His Arg Ser Cys Pro Pro Gly Phe Gly Val Val
  55 60 65
  Gln Ala Gly Thr Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro
  70 75 80
  Asp Gly Phe Phe Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg
  85 90 95
  Lys His Thr Asn Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys
  100 105 110
  Gly Asn Ala Thr His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser
  115 120 125
  Thr Gln Lys Cys Gly Ile Asp Val Thr Leu Cys Glu Glu Ala Phe
  130 135 140
  Phe Arg Phe Ala Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser
  145 150 155
  Val Leu Val Asp Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser
  160 165 170
  Val Glu Arg Ile Lys Arg Gln His Ser Ser Gln Glu Gln Thr Phe
  175 180 185
  Gln Leu Leu Lys Leu Trp Lys His Gln Asn Lys Asp Gln Asp Ile
  190 195 200
  Val Lys Lys Ile Ile Gln Asp Ile Asp Leu Cys Glu Asn Ser Val
  205 210 215
  Gln Arg His Ile Gly His Ala Asn Leu Thr Phe Glu Gln Leu Arg
  220 225 230
  Ser Leu Met Glu Ser Leu Pro Gly Lys Lys Val Gly Ala Glu Asp
  235 240 245
  Ile Glu Lys Thr Ile Lys Ala Cys Lys Pro Ser Asp Gln Ile Leu
  250 255 260
  Lys Leu Leu Ser Leu Trp Arg Ile Lys Asn Gly Asp Gln Asp Thr
  265 270 275
  Leu Lys Gly Leu Met His Ala Leu Lys His Ser Lys Thr Tyr His
  280 285 290
  Phe Pro Lys Thr Val Thr Gln Ser Leu Lys Lys Thr Ile Arg Phe
  295 300 305
  Leu His Ser Phe Thr Met Tyr Lys Leu Tyr Gln Lys Leu Phe Leu
  310 315 320
  Glu Met Ile Gly Asn Gln Val Gln Ser Val Lys Ile Ser Cys Leu
  325 330 335
<210> 68
<211> 359
<212> PRT
<213> OCIF-DCR2
<400> 68
  Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
      -20 -15 -10
  Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
      -5 -1 1 5
  Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
  10 15 20
  Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
  25 30 35
  Val Cys Ala Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe
  40 45 50
  Cys Leu Lys His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln
  55 60 65
  Ala Gly Thr Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp
  70 75 80
  Gly Phe Phe Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys
  85 90 95
  His Thr Asn Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys Gly
  100 105 110
  Asn Ala Thr His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr
  115 120 125
  Gln Lys Cys Gly Ile Asp Val Thr Leu Cys Glu Glu Ala Phe Phe
  130 135 140
  Arg Phe Ala Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val
  145 150 155
  Leu Val Asp Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val
  160 165 170
  Glu Arg Ile Lys Arg Gln His Ser Ser Gln Glu Gln Thr Phe Gln
  175 180 185
  Leu Leu Lys Leu Trp Lys His Gln Asn Lys Asp Gln Asp Ile Val
  190 195 200
  Lys Lys Ile Ile Gln Asp Ile Asp Leu Cys Glu Asn Ser Val Gln
  205 210 215
  Arg His Ile Gly His Ala Asn Leu Thr Phe Glu Gln Leu Arg Ser
  220 225 230
  Leu Met Glu Ser Leu Pro Gly Lys Lys Val Gly Ala Glu Asp Ile
  235 240 245
  Glu Lys Thr Ile Lys Ala Cys Lys Pro Ser Asp Gln Ile Leu Lys
  250 255 260
  Leu Leu Ser Leu Trp Arg Ile Lys Asn Gly Asp Gln Asp Thr Leu
  265 270 275
  Lys Gly Leu Met His Ala Leu Lys His Ser Lys Thr Tyr His Phe
  280 285 290
  Pro Lys Thr Val Thr Gln Ser Leu Lys Lys Thr Ile Arg Phe Leu
  295 300 305
  His Ser Phe Thr Met Tyr Lys Leu Tyr Gln Lys Leu Phe Leu Glu
  310 315 320
  Met Ile Gly Asn Gln Val Gln Ser Val Lys Ile Ser Cys Leu
  325 330 335
<210> 69
<211> 363
<212> PRT
<213> OCIF-DCR3
<400> 69
  Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
      -20 -15 -10
  Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
      -5 -1 1 5
  Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
  10 15 20
  Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
  25 30 35
  Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
  40 45 50
  Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
  55 60 65
  Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys
  70 75 80
  Arg Cys Pro Asp Gly Phe Phe Ser Asn Glu Thr Ser Ser Lys Ala
  85 90 95
  Pro Cys Arg Lys His Thr Asn Cys Ser Val Phe Gly Leu Leu Leu
  100 105 110
  Thr Gln Lys Gly Asn Ala Thr His Asp Asn Ile Cys Ser Gly Asn
  115 120 125
  Ser Glu Ser Thr Gln Lys Cys Gly Ile Asp Val Thr Leu Cys Glu
  130 135 140
  Glu Ala Phe Phe Arg Phe Ala Val Pro Thr Lys Phe Thr Pro Asn
  145 150 155
  Trp Leu Ser Val Leu Val Asp Asn Leu Pro Gly Thr Lys Val Asn
  160 165 170
  Ala Glu Ser Val Glu Arg Ile Lys Arg Gln His Ser Ser Gln Glu
  175 180 185
  Gln Thr Phe Gln Leu Leu Lys Leu Trp Lys His Gln Asn Lys Asp
  190 195 200
  Gln Asp Ile Val Lys Lys Ile Ile Gln Asp Ile Asp Leu Cys Glu
  205 210 215
  Asn Ser Val Gln Arg His Ile Gly His Ala Asn Leu Thr Phe Glu
  220 225 230
  Gln Leu Arg Ser Leu Met Glu Ser Leu Pro Gly Lys Lys Val Gly
  235 240 245
  Ala Glu Asp Ile Glu Lys Thr Ile Lys Ala Cys Lys Pro Ser Asp
  250 255 260
  Gln Ile Leu Lys Leu Leu Ser Leu Trp Arg Ile Lys Asn Gly Asp
  265 270 275
  Gln Asp Thr Leu Lys Gly Leu Met His Ala Leu Lys His Ser Lys
  280 285 290
  Thr Tyr His Phe Pro Lys Thr Val Thr Gln Ser Leu Lys Lys Thr
  295 300 305
  Ile Arg Phe Leu His Ser Phe Thr Met Tyr Lys Leu Tyr Gln Lys
  310 315 320
  Leu Phe Leu Glu Met Ile Gly Asn Gln Val Gln Ser Val Lys Ile
  325 330 335
  Ser Cys Leu
  340
<210> 70
<211> 359
<212> PRT
<213> OCIF-DCR4
<400> 70
  Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
      -20 -15 -10
  Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
      -5 -1 1 5
  Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
  10 15 20
  Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
  25 30 35
  Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
  40 45 50
  Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
  55 60 65
  Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys
  70 75 80
  Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys
  85 90 95
  His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly Thr
  100 105 110
  Pro Glu Arg Asn Thr Val Cys Lys Ser Gly Asn Ser Glu Ser Thr
  115 120 125
  Gln Lys Cys Gly Ile Asp Val Thr Leu Cys Glu Glu Ala Phe Phe
  130 135 140
  Arg Phe Ala Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val
  145 150 155
  Leu Val Asp Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val
  160 165 170
  Glu Arg Ile Lys Arg Gln His Ser Ser Gln Glu Gln Thr Phe Gln
  175 180 185
  Leu Leu Lys Leu Trp Lys His Gln Asn Lys Asp Gln Asp Ile Val
  190 195 200
  Lys Lys Ile Ile Gln Asp Ile Asp Leu Cys Glu Asn Ser Val Gln
  205 210 215
  Arg His Ile Gly His Ala Asn Leu Thr Phe Glu Gln Leu Arg Ser
  220 225 230
  Leu Met Glu Ser Leu Pro Gly Lys Lys Val Gly Ala Glu Asp Ile
  235 240 245
  Glu Lys Thr Ile Lys Ala Cys Lys Pro Ser Asp Gln Ile Leu Lys
  250 255 260
  Leu Leu Ser Leu Trp Arg Ile Lys Asn Gly Asp Gln Asp Thr Leu
  265 270 275
  Lys Gly Leu Met His Ala Leu Lys His Ser Lys Thr Tyr His Phe
  280 285 290
  Pro Lys Thr Val Thr Gln Ser Leu Lys Lys Thr Ile Arg Phe Leu
  295 300 305
  His Ser Phe Thr Met Tyr Lys Leu Tyr Gln Lys Leu Phe Leu Glu
  310 315 320
  Met Ile Gly Asn Gln Val Gln Ser Val Lys Ile Ser Cys Leu
  325 330 335
<210> 71
<211> 326
<212> PRT
<213> OCIF-DDD1
<400> 71
  Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
      -20 -15 -10
  Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
      -5 -1 1 5
  Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
  10 15 20
  Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
  25 30 35
  Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
  40 45 50
  Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
  55 60 65
  Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys
  70 75 80
  Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys
  85 90 95
  His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly Thr
  100 105 110
  Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe Phe
  115 120 125
  Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn
  130 135 140
  Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys Gly Asn Ala Thr
  145 150 155
  His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr Gln Lys Cys
  160 165 170
  Gly Ile Asp Ile Asp Leu Cys Glu Asn Ser Val Gln Arg His Ile
  175 180 185
  Gly His Ala Asn Leu Thr Phe Glu Gln Leu Arg Ser Leu Met Glu
  190 195 200
  Ser Leu Pro Gly Lys Lys Val Gly Ala Glu Asp Ile Glu Lys Thr
  205 210 215
  Ile Lys Ala Cys Lys Pro Ser Asp Gln Ile Leu Lys Leu Leu Ser
  220 225 230
  Leu Trp Arg Ile Lys Asn Gly Asp Gln Asp Thr Leu Lys Gly Leu
  235 240 245
  Met His Ala Leu Lys His Ser Lys Thr Tyr His Phe Pro Lys Thr
  250 255 260
  Val Thr Gln Ser Leu Lys Lys Thr Ile Arg Phe Leu His Ser Phe
  265 270 275
  Thr Met Tyr Lys Leu Tyr Gln Lys Leu Phe Leu Glu Met Ile Gly
  280 285 290
  Asn Gln Val Gln Ser Val Lys Ile Ser Cys Leu
  295 300 305
<210> 72
<211> 327
<212> PRT
<213> OCIF-DDD2
<400> 72
  Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
      -20 -15 -10
  Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
      -5 -1 1 5
  Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
  10 15 20
  Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
  25 30 35
  Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
  40 45 50
  Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
  55 60 65
  Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys
  70 75 80
  Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys
  85 90 95
  His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly Thr
  100 105 110
  Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe Phe
  115 120 125
  Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn
  130 135 140
  Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys Gly Asn Ala Thr
  145 150 155
  His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr Gln Lys Cys
  160 165 170
  Gly Ile Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg Phe Ala
  175 180 185
  Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val Asp
  190 195 200
  Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg Ile
  205 210 215
  Lys Arg Gln His Ser Ser Gln Glu Gln Thr Phe Gln Leu Leu Lys
  220 225 230
  Leu Trp Lys His Gln Asn Lys Asp Gln Asp Ile Val Lys Lys Ile
  235 240 245
  Ile Gln Asp Ala Leu Lys His Ser Lys Thr Tyr His Phe Pro Lys
  250 255 260
  Thr Val Thr Gln Ser Leu Lys Lys Thr Ile Arg Phe Leu His Ser
  265 270 275
  Phe Thr Met Tyr Lys Leu Tyr Gln Lys Leu Phe Leu Glu Met Ile
  280 285 290
  Gly Asn Gln Val Gln Ser Val Lys Ile Ser Cys Leu
  295 300 305
<210> 73
<211> 399
<212> PRT
<213> OCIF-CL
<400> 73
  Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
      -20 -15 -10
  Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
      -5 -1 1 5
  Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
  10 15 20
  Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
  25 30 35
  Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
  40 45 50
  Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
  55 60 65
  Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys
  70 75 80
  Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys
  85 90 95
  His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly Thr
  100 105 110
  Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe Phe
  115 120 125
  Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn
  130 135 140
  Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys Gly Asn Ala Thr
  145 150 155
  His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr Gln Lys Cys
  160 165 170
  Gly Ile Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg Phe Ala
  175 180 185
  Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val Asp
  190 195 200
  Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg Ile
  205 210 215
  Lys Arg Gln His Ser Ser Gln Glu Gln Thr Phe Gln Leu Leu Lys
  220 225 230
  Leu Trp Lys His Gln Asn Lys Asp Gln Asp Ile Val Lys Lys Ile
  235 240 245
  Ile Gln Asp Ile Asp Leu Cys Glu Asn Ser Val Gln Arg His Ile
  250 255 260
  Gly His Ala Asn Leu Thr Phe Glu Gln Leu Arg Ser Leu Met Glu
  265 270 275
  Ser Leu Pro Gly Lys Lys Val Gly Ala Glu Asp Ile Glu Lys Thr
  280 285 290
  Ile Lys Ala Cys Lys Pro Ser Asp Gln Ile Leu Lys Leu Leu Ser
  295 300 305
  Leu Trp Arg Ile Lys Asn Gly Asp Gln Asp Thr Leu Lys Gly Leu
  310 315 320
  Met His Ala Leu Lys His Ser Lys Thr Tyr His Phe Pro Lys Thr
  325 330 335
  Val Thr Gln Ser Leu Lys Lys Thr Ile Arg Phe Leu His Ser Phe
  340 345 350
  Thr Met Tyr Lys Leu Tyr Gln Lys Leu Phe Leu Glu Met Ile Gly
  355 360 365
  Asn Gln Val Gln Ser Val Lys Ile Ser
  370 375
<210> 74
<211> 351
<212> PRT
<213> OCIF-CC
<400> 74
  Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
      -20 -15 -10
  Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
      -5 -1 1 5
  Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
  10 15 20
  Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
  25 30 35
  Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
  40 45 50
  Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
  55 60 65
  Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys
  70 75 80
  Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys
  85 90 95
  His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly Thr
  100 105 110
  Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe Phe
  115 120 125
  Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn
  130 135 140
  Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys Gly Asn Ala Thr
  145 150 155
  His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr Gln Lys Cys
  160 165 170
  Gly Ile Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg Phe Ala
  175 180 185
  Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val Asp
  190 195 200
  Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg Ile
  205 210 215
  Lys Arg Gln His Ser Ser Gln Glu Gln Thr Phe Gln Leu Leu Lys
  220 225 230
  Leu Trp Lys His Gln Asn Lys Asp Gln Asp Ile Val Lys Lys Ile
  235 240 245
  Ile Gln Asp Ile Asp Leu Cys Glu Asn Ser Val Gln Arg His Ile
  250 255 260
  Gly His Ala Asn Leu Thr Phe Glu Gln Leu Arg Ser Leu Met Glu
  265 270 275
  Ser Leu Pro Gly Lys Lys Val Gly Ala Glu Asp Ile Glu Lys Thr
  280 285 290
  Ile Lys Ala Cys Lys Pro Ser Asp Gln Ile Leu Lys Leu Leu Ser
  295 300 305
  Leu Trp Arg Ile Lys Asn Gly Asp Gln Asp Thr Leu Lys Gly Leu
  310 315 320
  Met His Ala Leu Lys His
  325 330
<210> 75
<211> 272
<212> PRT
<213> OCIF-CDD2
<400> 75
  Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
      -20 -15 -10
  Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
      -5 -1 1 5
  Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
  10 15 20
  Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
  25 30 35
  Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
  40 45 50
  Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
  55 60 65
  Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys
  70 75 80
  Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys
  85 90 95
  His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly Thr
  100 105 110
  Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe Phe
  115 120 125
  Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn
  130 135 140
  Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys Gly Asn Ala Thr
  145 150 155
  His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr Gln Lys Cys
  160 165 170
  Gly Ile Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg Phe Ala
  175 180 185
  Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val Asp
  190 195 200
  Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg Ile
  205 210 215
  Lys Arg Gln His Ser Ser Gln Glu Gln Thr Phe Gln Leu Leu Lys
  220 225 230
  Leu Trp Lys His Gln Asn Lys Asp Gln Asp Ile Val Lys Lys Ile
  235 240 245
  Ile Gln
  250
<210> 76
<211> 197
<212> PRT
<213> OCIF-CDD1
<400> 76
  Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
      -20 -15 -10
  Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
      -5 -1 1 5
  Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
  10 15 20
  Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
  25 30 35
  Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
  40 45 50
  Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
  55 60 65
  Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys
  70 75 80
  Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys
  85 90 95
  His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly Thr
  100 105 110
  Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe Phe
  115 120 125
  Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn
  130 135 140
  Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys Gly Asn Ala Thr
  145 150 155
  His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr Gln Lys Cys
  160 165 170
  Gly Ile
  175
<210> 77
<211> 143
<212> PRT
<213> OCIF-CCR4
<400> 77
  Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
      -20 -15 -10
  Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
      -5 -1 1 5
  Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
  10 15 20
  Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
  25 30 35
  Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
  40 45 50
  Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
  55 60 65
  Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys
  70 75 80
  Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys
  85 90 95
  His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly Thr
  100 105 110
  Pro Glu Arg Asn Thr Val Cys Lys
  115 120
<210> 78
<211> 106
<212> PRT
<213> OCIF-CCR3
<400> 78
  Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
      -20 -15 -10
  Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
      -5 -1 1 5
  Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
  10 15 20
  Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
  25 30 35
  Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
  40 45 50
  Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
  55 60 65
  Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys
  70 75 80
  Glu
  85
<210> 79
<211> 393
<212> PRT
<213> OCIF-CBst
<400> 79
  Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
      -20 -15 -10
  Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
      -5 -1 1 5
  Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
  10 15 20
  Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
  25 30 35
  Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
  40 45 50
  Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
  55 60 65
  Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys
  70 75 80
  Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys
  85 90 95
  His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly Thr
  100 105 110
  Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe Phe
  115 120 125
  Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn
  130 135 140
  Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys Gly Asn Ala Thr
  145 150 155
  His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr Gln Lys Cys
  160 165 170
  Gly Ile Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg Phe Ala
  175 180 185
  Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val Asp
  190 195 200
  Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg Ile
  205 210 215
  Lys Arg Gln His Ser Ser Gln Glu Gln Thr Phe Gln Leu Leu Lys
  220 225 230
  Leu Trp Lys His Gln Asn Lys Asp Gln Asp Ile Val Lys Lys Ile
  235 240 245
  Ile Gln Asp Ile Asp Leu Cys Glu Asn Ser Val Gln Arg His Ile
  250 255 260
  Gly His Ala Asn Leu Thr Phe Glu Gln Leu Arg Ser Leu Met Glu
  265 270 275
  Ser Leu Pro Gly Lys Lys Val Gly Ala Glu Asp Ile Glu Lys Thr
  280 285 290
  Ile Lys Ala Cys Lys Pro Ser Asp Gln Ile Leu Lys Leu Leu Ser
  295 300 305
  Leu Trp Arg Ile Lys Asn Gly Asp Gln Asp Thr Leu Lys Gly Leu
  310 315 320
  Met His Ala Leu Lys His Ser Lys Thr Tyr His Phe Pro Lys Thr
  325 330 335
  Val Thr Gln Ser Leu Lys Lys Thr Ile Arg Phe Leu His Ser Phe
  340 345 350
  Thr Met Tyr Lys Leu Tyr Gln Lys Leu Phe Leu Glu Met Ile Gly
  355 360 365
  Asn Leu Val
  370
<210> 80
<211> 321
<212> PRT
<213> OCIF-CSph
<400> 80
  Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
      -20 -15 -10
  Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
      -5 -1 1 5
  Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
  10 15 20
  Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
  25 30 35
  Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
  40 45 50
  Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
  55 60 65
  Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys
  70 75 80
  Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys
  85 90 95
  His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly Thr
  100 105 110
  Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe Phe
  115 120 125
  Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn
  130 135 140
  Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys Gly Asn Ala Thr
  145 150 155
  His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr Gln Lys Cys
  160 165 170
  Gly Ile Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg Phe Ala
  175 180 185
  Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val Asp
  190 195 200
  Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg Ile
  205 210 215
  Lys Arg Gln His Ser Ser Gln Glu Gln Thr Phe Gln Leu Leu Lys
  220 225 230
  Leu Trp Lys His Gln Asn Lys Asp Gln Asp Ile Val Lys Lys Ile
  235 240 245
  Ile Gln Asp Ile Asp Leu Cys Glu Asn Ser Val Gln Arg His Ile
  250 255 260
  Gly His Ala Asn Leu Thr Phe Glu Gln Leu Arg Ser Leu Met Glu
  265 270 275
  Ser Leu Pro Gly Lys Lys Val Gly Ala Glu Asp Ile Glu Lys Thr
  280 285 290
  Ile Lys Ala Ser Leu Asp
  295 300
<210> 81
<211> 187
<212> PRT
<213> OCIF-CBsp
<400> 81
  Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
      -20 -15 -10
  Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
      -5 -1 1 5
  Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
  10 15 20
  Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
  25 30 35
  Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
  40 45 50
  Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
  55 60 65
  Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys
  70 75 80
  Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys
  85 90 95
  His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly Thr
  100 105 110
  Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe Phe
  115 120 125
  Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn
  130 135 140
  Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys Gly Asn Ala Thr
  145 150 155
  His Asp Asn Ile Cys Ser Gly
  160 165
<210> 82
<211> 84
<212> PRT
<213> OCIF-CPst
<400> 82
  Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser
      -20 -15 -10
  Ile Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
      -5 -1 1 5
  Tyr Asp Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
  10 15 20
  Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
  25 30 35
  Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
  40 45 50
  Thr Ser Asp Glu Cys Leu Tyr Leu Val
  55 60
<210> 83
<211> 1206
<212> DNA
<213> OCIF-C19S
<400> 83
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gaaccccaga gcgaaataca 420
gtttgcaaaa gatgtccaga tgggttcttc tcaaatgaga cgtcatctaa agcaccctgt 480
agaaaacaca caaattgcag tgtctttggt ctcctgctaa ctcagaaagg aaatgcaaca 540
cacgacaaca tatgttccgg aaacagtgaa tcaactcaaa aaagtggaat agatgttacc 600
ctgtgtgagg aggcattctt caggtttgct gttcctacaa agtttacgcc taactggctt 660
agtgtcttgg tagacaattt gcctggcacc aaagtaaacg cagagagtgt agagaggata 720
aaacggcaac acagctcaca agaacagact ttccagctgc tgaagttatg gaaacatcaa 780
aacaaagacc aagatatagt caagaagatc atccaagata ttgacctctg tgaaaacagc 840
gtgcagcggc acattggaca tgctaacctc accttcgagc agcttcgtag cttgatggaa 900
agcttaccgg gaaagaaagt gggagcagaa gacattgaaa aaacaataaa ggcatgcaaa 960
cccagtgacc agatcctgaa gctgctcagt ttgtggcgaa taaaaaatgg cgaccaagac 1020
accttgaagg gcctaatgca cgcactaaag cactcaaaga cgtaccactt tcccaaaact 1080
gtcactcaga gtctaaagaa gaccatcagg ttccttcaca gcttcacaat gtacaaattg 1140
tatcagaagt tatttttaga aatgataggt aaccaggtcc aatcagtaaa aataagctgc 1200
ttataa 1206
<210> 84
<211> 1206
<212> DNA
<213> OCIF-C20S
<400> 84
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gaaccccaga gcgaaataca 420
gtttgcaaaa gatgtccaga tgggttcttc tcaaatgaga cgtcatctaa agcaccctgt 480
agaaaacaca caaattgcag tgtctttggt ctcctgctaa ctcagaaagg aaatgcaaca 540
cacgacaaca tatgttccgg aaacagtgaa tcaactcaaa aatgtggaat agatgttacc 600
ctgagtgagg aggcattctt caggtttgct gttcctacaa agtttacgcc taactggctt 660
agtgtcttgg tagacaattt gcctggcacc aaagtaaacg cagagagtgt agagaggata 720
aaacggcaac acagctcaca agaacagact ttccagctgc tgaagttatg gaaacatcaa 780
aacaaagacc aagatatagt caagaagatc atccaagata ttgacctctg tgaaaacagc 840
gtgcagcggc acattggaca tgctaacctc accttcgagc agcttcgtag cttgatggaa 900
agcttaccgg gaaagaaagt gggagcagaa gacattgaaa aaacaataaa ggcatgcaaa 960
cccagtgacc agatcctgaa gctgctcagt ttgtggcgaa taaaaaatgg cgaccaagac 1020
accttgaagg gcctaatgca cgcactaaag cactcaaaga cgtaccactt tcccaaaact 1080
gtcactcaga gtctaaagaa gaccatcagg ttccttcaca gcttcacaat gtacaaattg 1140
tatcagaagt tatttttaga aatgataggt aaccaggtcc aatcagtaaa aataagctgc 1200
ttataa 1206
<210> 85
<211> 1206
<212> DNA
<213> OCIF-C21S
<400> 85
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gaaccccaga gcgaaataca 420
gtttgcaaaa gatgtccaga tgggttcttc tcaaatgaga cgtcatctaa agcaccctgt 480
agaaaacaca caaattgcag tgtctttggt ctcctgctaa ctcagaaagg aaatgcaaca 540
cacgacaaca tatgttccgg aaacagtgaa tcaactcaaa aatgtggaat agatgttacc 600
ctgtgtgagg aggcattctt caggtttgct gttcctacaa agtttacgcc taactggctt 660
agtgtcttgg tagacaattt gcctggcacc aaagtaaacg cagagagtgt agagaggata 720
aaacggcaac acagctcaca agaacagact ttccagctgc tgaagttatg gaaacatcaa 780
aacaaagacc aagatatagt caagaagatc atccaagata ttgacctcag tgaaaacagc 840
gtgcagcggc acattggaca tgctaacctc accttcgagc agcttcgtag cttgatggaa 900
agcttaccgg gaaagaaagt gggagcagaa gacattgaaa aaacaataaa ggcatgcaaa 960
cccagtgacc agatcctgaa gctgctcagt ttgtggcgaa taaaaaatgg cgaccaagac 1020
accttgaagg gcctaatgca cgcactaaag cactcaaaga cgtaccactt tcccaaaact 1080
gtcactcaga gtctaaagaa gaccatcagg ttccttcaca gcttcacaat gtacaaattg 1140
tatcagaagt tatttttaga aatgataggt aaccaggtcc aatcagtaaa aataagctgc 1200
ttataa 1206
<210> 86
<211> 1206
<212> DNA
<213> OCIF-C22S
<400> 86
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gaaccccaga gcgaaataca 420
gtttgcaaaa gatgtccaga tgggttcttc tcaaatgaga cgtcatctaa agcaccctgt 480
agaaaacaca caaattgcag tgtctttggt ctcctgctaa ctcagaaagg aaatgcaaca 540
cacgacaaca tatgttccgg aaacagtgaa tcaactcaaa aatgtggaat agatgttacc 600
ctgtgtgagg aggcattctt caggtttgct gttcctacaa agtttacgcc taactggctt 660
agtgtcttgg tagacaattt gcctggcacc aaagtaaacg cagagagtgt agagaggata 720
aaacggcaac acagctcaca agaacagact ttccagctgc tgaagttatg gaaacatcaa 780
aacaaagacc aagatatagt caagaagatc atccaagata ttgacctctg tgaaaacagc 840
gtgcagcggc acattggaca tgctaacctc accttcgagc agcttcgtag cttgatggaa 900
agcttaccgg gaaagaaagt gggagcagaa gacattgaaa aaacaataaa ggcaagcaaa 960
cccagtgacc agatcctgaa gctgctcagt ttgtggcgaa taaaaaatgg cgaccaagac 1020
accttgaagg gcctaatgca cgcactaaag cactcaaaga cgtaccactt tcccaaaact 1080
gtcactcaga gtctaaagaa gaccatcagg ttccttcaca gcttcacaat gtacaaattg 1140
tatcagaagt tatttttaga aatgataggt aaccaggtcc aatcagtaaa aataagctgc 1200
ttataa 1206
<210> 87
<211> 1206
<212> DNA
<213> OCIF-C23S
<400> 87
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gaaccccaga gcgaaataca 420
gtttgcaaaa gatgtccaga tgggttcttc tcaaatgaga cgtcatctaa agcaccctgt 480
agaaaacaca caaattgcag tgtctttggt ctcctgctaa ctcagaaagg aaatgcaaca 540
cacgacaaca tatgttccgg aaacagtgaa tcaactcaaa aatgtggaat agatgttacc 600
ctgtgtgagg aggcattctt caggtttgct gttcctacaa agtttacgcc taactggctt 660
agtgtcttgg tagacaattt gcctggcacc aaagtaaacg cagagagtgt agagaggata 720
aaacggcaac acagctcaca agaacagact ttccagctgc tgaagttatg gaaacatcaa 780
aacaaagacc aagatatagt caagaagatc atccaagata ttgacctctg tgaaaacagc 840
gtgcagcggc acattggaca tgctaacctc accttcgagc agcttcgtag cttgatggaa 900
agcttaccgg gaaagaaagt gggagcagaa gacattgaaa aaacaataaa ggcatgcaaa 960
cccagtgacc agatcctgaa gctgctcagt ttgtggcgaa taaaaaatgg cgaccaagac 1020
accttgaagg gcctaatgca cgcactaaag cactcaaaga cgtaccactt tcccaaaact 1080
gtcactcaga gtctaaagaa gaccatcagg ttccttcaca gcttcacaat gtacaaattg 1140
tatcagaagt tatttttaga aatgataggt aaccaggtcc aatcagtaaa aataagcagc 1200
ttataa 1206
<210> 88
<211> 1083
<212> DNA
<213> OCIF-DCR1
<400> 88
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaacctt gccctgacca ctactacaca gacagctggc acaccagtga cgagtgtcta 120
tactgcagcc ccgtgtgcaa ggagctgcag tacgtcaagc aggagtgcaa tcgcacccac 180
aaccgcgtgt gcgaatgcaa ggaagggcgc taccttgaga tagagttctg cttgaaacat 240
aggagctgcc ctcctggatt tggagtggtg caagctggaa ccccagagcg aaatacagtt 300
tgcaaaagat gtccagatgg gttcttctca aatgagacgt catctaaagc accctgtaga 360
aaacacacaa attgcagtgt ctttggtctc ctgctaactc agaaaggaaa tgcaacacac 420
gacaacatat gttccggaaa cagtgaatca actcaaaaat gtggaataga tgttaccctg 480
tgtgaggagg cattcttcag gtttgctgtt cctacaaagt ttacgcctaa ctggcttagt 540
gtcttggtag acaatttgcc tggcaccaaa gtaaacgcag agagtgtaga gaggataaaa 600
cggcaacaca gctcacaaga acagactttc cagctgctga agttatggaa acatcaaaac 660
aaagaccaag atatagtcaa gaagatcatc caagatattg acctctgtga aaacagcgtg 720
cagcggcaca ttggacatgc taacctcacc ttcgagcagc ttcgtagctt gatggaaagc 780
ttaccgggaa agaaagtggg agcagaagac attgaaaaaa caataaaggc atgcaaaccc 840
agtgaccaga tcctgaagct gctcagtttg tggcgaataa aaaatggcga ccaagacacc 900
ttgaagggcc taatgcacgc actaaagcac tcaaagacgt accactttcc caaaactgtc 960
actcagagtc taaagaagac catcaggttc cttcacagct tcacaatgta caaattgtat 1020
cagaagttat ttttagaaat gataggtaac caggtccaat cagtaaaaat aagctgctta 1080
taa 1083
<210> 89
<211> 1080
<212> DNA
<213> OCIF-DCR2
<400> 89
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccg aatgcaagga agggcgctac cttgagatag agttctgctt gaaacatagg 240
agctgccctc ctggatttgg agtggtgcaa gctggaaccc cagagcgaaa tacagtttgc 300
aaaagatgtc cagatgggtt cttctcaaat gagacgtcat ctaaagcacc ctgtagaaaa 360
cacacaaatt gcagtgtctt tggtctcctg ctaactcaga aaggaaatgc aacacacgac 420
aacatatgtt ccggaaacag tgaatcaact caaaaatgtg gaatagatgt taccctgtgt 480
gaggaggcat tcttcaggtt tgctgttcct acaaagttta cgcctaactg gcttagtgtc 540
ttggtagaca atttgcctgg caccaaagta aacgcagaga gtgtagagag gataaaacgg 600
caacacagct cacaagaaca gactttccag ctgctgaagt tatggaaaca tcaaaacaaa 660
gaccaagata tagtcaagaa gatcatccaa gatattgacc tctgtgaaaa cagcgtgcag 720
cggcacattg gacatgctaa cctcaccttc gagcagcttc gtagcttgat ggaaagctta 780
ccgggaaaga aagtgggagc agaagacatt gaaaaaacaa taaaggcatg caaacccagt 840
gaccagatcc tgaagctgct cagtttgtgg cgaataaaaa atggcgacca agacaccttg 900
aagggcctaa tgcacgcact aaagcactca aagacgtacc actttcccaa aactgtcact 960
cagagtctaa agaagaccat caggttcctt cacagcttca caatgtacaa attgtatcag 1020
aagttatttt tagaaatgat aggtaaccag gtccaatcag taaaaataag ctgcttataa 1080
<210> 90
<211> 1092
<212> DNA
<213> OCIF-DCR3
<400> 90
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcagatg tccagatggg ttcttctcaa atgagacgtc atctaaagca 360
ccctgtagaa aacacacaaa ttgcagtgtc tttggtctcc tgctaactca gaaaggaaat 420
gcaacacacg acaacatatg ttccggaaac agtgaatcaa ctcaaaaatg tggaatagat 480
gttaccctgt gtgaggaggc attcttcagg tttgctgttc ctacaaagtt tacgcctaac 540
tggcttagtg tcttggtaga caatttgcct ggcaccaaag taaacgcaga gagtgtagag 600
aggataaaac ggcaacacag ctcacaagaa cagactttcc agctgctgaa gttatggaaa 660
catcaaaaca aagaccaaga tatagtcaag aagatcatcc aagatattga cctctgtgaa 720
aacagcgtgc agcggcacat tggacatgct aacctcacct tcgagcagct tcgtagcttg 780
atggaaagct taccgggaaa gaaagtggga gcagaagaca ttgaaaaaac aataaaggca 840
tgcaaaccca gtgaccagat cctgaagctg ctcagtttgt ggcgaataaa aaatggcgac 900
caagacacct tgaagggcct aatgcacgca ctaaagcact caaagacgta ccactttccc 960
aaaactgtca ctcagagtct aaagaagacc atcaggttcc ttcacagctt cacaatgtac 1020
aaattgtatc agaagttatt tttagaaatg ataggtaacc aggtccaatc agtaaaaata 1080
agctgcttat aa 1092
<210> 91
<211> 1080
<212> DNA
<213> OCIF-DCR4
<400> 91
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gaaccccaga gcgaaataca 420
gtttgcaaat ccggaaacag tgaatcaact caaaaatgtg gaatagatgt taccctgtgt 480
gaggaggcat tcttcaggtt tgctgttcct acaaagttta cgcctaactg gcttagtgtc 540
ttggtagaca atttgcctgg caccaaagta aacgcagaga gtgtagagag gataaaacgg 600
caacacagct cacaagaaca gactttccag ctgctgaagt tatggaaaca tcaaaacaaa 660
gaccaagata tagtcaagaa gatcatccaa gatattgacc tctgtgaaaa cagcgtgcag 720
cggcacattg gacatgctaa cctcaccttc gagcagcttc gtagcttgat ggaaagctta 780
ccgggaaaga aagtgggagc agaagacatt gaaaaaacaa taaaggcatg caaacccagt 840
gaccagatcc tgaagctgct cagtttgtgg cgaataaaaa atggcgacca agacaccttg 900
aagggcctaa tgcacgcact aaagcactca aagacgtacc actttcccaa aactgtcact 960
cagagtctaa agaagaccat caggttcctt cacagcttca caatgtacaa attgtatcag 1020
aagttatttt tagaaatgat aggtaaccag gtccaatcag taaaaataag ctgcttataa 1080
<210> 92
<211> 981
<212> DNA
<213> OCIF-DDD1
<400> 92
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gaaccccaga gcgaaataca 420
gtttgcaaaa gatgtccaga tgggttcttc tcaaatgaga cgtcatctaa agcaccctgt 480
agaaaacaca caaattgcag tgtctttggt ctcctgctaa ctcagaaagg aaatgcaaca 540
cacgacaaca tatgttccgg aaacagtgaa tcaactcaaa aatgtggaat agatattgac 600
ctctgtgaaa acagcgtgca gcggcacatt ggacatgcta acctcacctt cgagcagctt 660
cgtagcttga tggaaagctt accgggaaag aaagtgggag cagaagacat tgaaaaaaca 720
ataaaggcat gcaaacccag tgaccagatc ctgaagctgc tcagtttgtg gcgaataaaa 780
aatggcgacc aagacacctt gaagggccta atgcacgcac taaagcactc aaagacgtac 840
cactttccca aaactgtcac tcagagtcta aagaagacca tcaggttcct tcacagcttc 900
acaatgtaca aattgtatca gaagttattt ttagaaatga taggtaacca ggtccaatca 960
gtaaaaataa gctgcttata a 981
<210> 93
<211> 984
<212> DNA
<213> OCIF-DDD2
<400> 93
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gaaccccaga gcgaaataca 420
gtttgcaaaa gatgtccaga tgggttcttc tcaaatgaga cgtcatctaa agcaccctgt 480
agaaaacaca caaattgcag tgtctttggt ctcctgctaa ctcagaaagg aaatgcaaca 540
cacgacaaca tatgttccgg aaacagtgaa tcaactcaaa aatgtggaat agatgttacc 600
ctgtgtgagg aggcattctt caggtttgct gttcctacaa agtttacgcc taactggctt 660
agtgtcttgg tagacaattt gcctggcacc aaagtaaacg cagagagtgt agagaggata 720
aaacggcaac acagctcaca agaacagact ttccagctgc tgaagttatg gaaacatcaa 780
aacaaagacc aagatatagt caagaagatc atccaagacg cactaaagca ctcaaagacg 840
taccactttc ccaaaactgt cactcagagt ctaaagaaga ccatcaggtt ccttcacagc 900
ttcacaatgt acaaattgta tcagaagtta tttttagaaa tgataggtaa ccaggtccaa 960
tcagtaaaaa taagctgctt ataa 984
<210> 94
<211> 1200
<212> DNA
<213> OCIF-CL
<400> 94
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gaaccccaga gcgaaataca 420
gtttgcaaaa gatgtccaga tgggttcttc tcaaatgaga cgtcatctaa agcaccctgt 480
agaaaacaca caaattgcag tgtctttggt ctcctgctaa ctcagaaagg aaatgcaaca 540
cacgacaaca tatgttccgg aaacagtgaa tcaactcaaa aatgtggaat agatgttacc 600
ctgtgtgagg aggcattctt caggtttgct gttcctacaa agtttacgcc taactggctt 660
agtgtcttgg tagacaattt gcctggcacc aaagtaaacg cagagagtgt agagaggata 720
aaacggcaac acagctcaca agaacagact ttccagctgc tgaagttatg gaaacatcaa 780
aacaaagacc aagatatagt caagaagatc atccaagata ttgacctctg tgaaaacagc 840
gtgcagcggc acattggaca tgctaacctc accttcgagc agcttcgtag cttgatggaa 900
agcttaccgg gaaagaaagt gggagcagaa gacattgaaa aaacaataaa ggcatgcaaa 960
cccagtgacc agatcctgaa gctgctcagt ttgtggcgaa taaaaaatgg cgaccaagac 1020
accttgaagg gcctaatgca cgcactaaag cactcaaaga cgtaccactt tcccaaaact 1080
gtcactcaga gtctaaagaa gaccatcagg ttccttcaca gcttcacaat gtacaaattg 1140
tatcagaagt tatttttaga aatgataggt aaccaggtcc aatcagtaaa aataagctaa 1200
<210> 95
<211> 1056
<212> DNA
<213> OCIF-CC
<400> 95
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gaaccccaga gcgaaataca 420
gtttgcaaaa gatgtccaga tgggttcttc tcaaatgaga cgtcatctaa agcaccctgt 480
agaaaacaca caaattgcag tgtctttggt ctcctgctaa ctcagaaagg aaatgcaaca 540
cacgacaaca tatgttccgg aaacagtgaa tcaactcaaa aatgtggaat agatgttacc 600
ctgtgtgagg aggcattctt caggtttgct gttcctacaa agtttacgcc taactggctt 660
agtgtcttgg tagacaattt gcctggcacc aaagtaaacg cagagagtgt agagaggata 720
aaacggcaac acagctcaca agaacagact ttccagctgc tgaagttatg gaaacatcaa 780
aacaaagacc aagatatagt caagaagatc atccaagata ttgacctctg tgaaaacagc 840
gtgcagcggc acattggaca tgctaacctc accttcgagc agcttcgtag cttgatggaa 900
agcttaccgg gaaagaaagt gggagcagaa gacattgaaa aaacaataaa ggcatgcaaa 960
cccagtgacc agatcctgaa gctgctcagt ttgtggcgaa taaaaaatgg cgaccaagac 1020
accttgaagg gcctaatgca cgcactaaag cactga 1056
<210> 96
<211> 819
<212> DNA
<213> OCIF-CDD2
<400> 96
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gaaccccaga gcgaaataca 420
gtttgcaaaa gatgtccaga tgggttcttc tcaaatgaga cgtcatctaa agcaccctgt 480
agaaaacaca caaattgcag tgtctttggt ctcctgctaa ctcagaaagg aaatgcaaca 540
cacgacaaca tatgttccgg aaacagtgaa tcaactcaaa aatgtggaat agatgttacc 600
ctgtgtgagg aggcattctt caggtttgct gttcctacaa agtttacgcc taactggctt 660
agtgtcttgg tagacaattt gcctggcacc aaagtaaacg cagagagtgt agagaggata 720
aaacggcaac acagctcaca agaacagact ttccagctgc tgaagttatg gaaacatcaa 780
aacaaagacc aagatatagt caagaagatc atccaatga 819
<210> 97
<211> 594
<212> DNA
<213> OCIF-CDD1
<400> 97
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gaaccccaga gcgaaataca 420
gtttgcaaaa gatgtccaga tgggttcttc tcaaatgaga cgtcatctaa agcaccctgt 480
agaaaacaca caaattgcag tgtctttggt ctcctgctaa ctcagaaagg aaatgcaaca 540
cacgacaaca tatgttccgg aaacagtgaa tcaactcaaa aatgtggaat atga 594
<210> 98
<211> 432
<212> DNA
<213> OCIF-CCR4
<400> 98
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gaaccccaga gcgaaataca 420
gtttgcaaat ga 432
<210> 99
<211> 321
<212> DNA
<213> OCIF-CCR3
<400> 99
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg a 321
<210> 100
<211> 1182
<212> DNA
<213> OCIF-CBst
<400> 100
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gaaccccaga gcgaaataca 420
gtttgcaaaa gatgtccaga tgggttcttc tcaaatgaga cgtcatctaa agcaccctgt 480
agaaaacaca caaattgcag tgtctttggt ctcctgctaa ctcagaaagg aaatgcaaca 540
cacgacaaca tatgttccgg aaacagtgaa tcaactcaaa aatgtggaat agatgttacc 600
ctgtgtgagg aggcattctt caggtttgct gttcctacaa agtttacgcc taactggctt 660
agtgtcttgg tagacaattt gcctggcacc aaagtaaacg cagagagtgt agagaggata 720
aaacggcaac acagctcaca agaacagact ttccagctgc tgaagttatg gaaacatcaa 780
aacaaagacc aagatatagt caagaagatc atccaagata ttgacctctg tgaaaacagc 840
gtgcagcggc acattggaca tgctaacctc accttcgagc agcttcgtag cttgatggaa 900
agcttaccgg gaaagaaagt gggagcagaa gacattgaaa aaacaataaa ggcatgcaaa 960
cccagtgacc agatcctgaa gctgctcagt ttgtggcgaa taaaaaatgg cgaccaagac 1020
accttgaagg gcctaatgca cgcactaaag cactcaaaga cgtaccactt tcccaaaact 1080
gtcactcaga gtctaaagaa gaccatcagg ttccttcaca gcttcacaat gtacaaattg 1140
tatcagaagt tatttttaga aatgataggt aacctagtct ag 1182
<210> 101
<211> 966
<212> DNA
<213> OCIF-CSph
<400> 101
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gaaccccaga gcgaaataca 420
gtttgcaaaa gatgtccaga tgggttcttc tcaaatgaga cgtcatctaa agcaccctgt 480
agaaaacaca caaattgcag tgtctttggt ctcctgctaa ctcagaaagg aaatgcaaca 540
cacgacaaca tatgttccgg aaacagtgaa tcaactcaaa aatgtggaat agatgttacc 600
ctgtgtgagg aggcattctt caggtttgct gttcctacaa agtttacgcc taactggctt 660
agtgtcttgg tagacaattt gcctggcacc aaagtaaacg cagagagtgt agagaggata 720
aaacggcaac acagctcaca agaacagact ttccagctgc tgaagttatg gaaacatcaa 780
aacaaagacc aagatatagt caagaagatc atccaagata ttgacctctg tgaaaacagc 840
gtgcagcggc acattggaca tgctaacctc accttcgagc agcttcgtag cttgatggaa 900
agcttaccgg gaaagaaagt gggagcagaa gacattgaaa aaacaataaa ggctagtcta 960
gactag 966
<210> 102
<211> 564
<212> DNA
<213> OCIF-CBsp
<400> 102
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gaaccccaga gcgaaataca 420
gtttgcaaaa gatgtccaga tgggttcttc tcaaatgaga cgtcatctaa agcaccctgt 480
agaaaacaca caaattgcag tgtctttggt ctcctgctaa ctcagaaagg aaatgcaaca 540
cacgacaaca tatgttccgg ctag 564
<210> 103
<211> 255
<212> DNA
<213> OCIF-CPst
<400> 103
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatacctag tctag 255
<210> 104
<211> 1317
<212> DNA
<213> human OCIF genome
<220>
<221> exon
<222> 1173..1193
<400> 104
ctggagacat ataacttgaa cacttggccc tgatggggaa gcagctctgc agggactttt 60
tcagccatct gtaaacaatt tcagtggcaa cccgcgaact gtaatccatg aatgggacca 120
cactttacaa gtcatcaagt ctaacttcta gaccagggaa ttaatggggg agacagcgaa 180
ccctagagca aagtgccaaa cttctgtcga tagcttgagg ctagtggaaa gacctcgagg 240
aggctactcc agaagttcag cgcgtaggaa gctccgatac caatagccct ttgatgatgg 300
tggggttggt gaagggaaca gtgctccgca aggttatccc tgccccaggc agtccaattt 360
tcactctgca gattctctct ggctctaact accccagata acaaggagtg aatgcagaat 420
agcacgggct ttagggccaa tcagacatta gttagaaaaa ttcctactac atggtttatg 480
taaacttgaa gatgaatgat tgcgaactcc ccgaaaaggg ctcagacaat gccatgcata 540
aagaggggcc ctgtaatttg aggtttcaga acccgaagtg aaggggtcag gcagccgggt 600
acggcggaaa ctcacagctt tcgcccagcg agaggacaaa ggtctgggac acactccaac 660
tgcgtccgga tcttggctgg atcggactct cagggtggag gagacacaag cacagcagct 720
gcccagcgtg tgcccagccc tcccaccgct ggtcccggct gccaggaggc tggccgctgg 780
cgggaagggg ccgggaaacc tcagagcccc gcggagacag cagccgcctt gttcctcagc 840
ccggtggctt ttttttcccc tgctctccca ggggacagac accaccgccc cacccctcac 900
gccccacctc cctgggggat cctttccgcc ccagccctga aagcgttaat cctggagctt 960
tctgcacacc ccccgaccgc tcccgcccaa gcttcctaaa aaagaaaggt gcaaagtttg 1020
gtccaggata gaaaaatgac tgatcaaagg caggcgatac ttcctgttgc cgggacgcta 1080
tatataacgt gatgagcgca cgggctgcgg agacgcaccg gagcgctcgc ccagccgccg 1140
cctccaagcc cctgaggttt ccggggacca ca atg aac aag ttg ctg tgc tgc 1193
                                    Met Asn Lys Leu Leu Cys Cys
                                        -20 -15
gcg ctc gtg gtaagtccct gggccagccg acgggtgccc ggcgcctggg 1242
ala leu val
gaggctgctg ccacctggtc tcccaacctc ccagcggacc ggcggggaaa aaggctccac 1302
tcgctccctc ccaag 1317
<210> 105
<211> 10190
<212> DNA
<213> human OCIF genome
<220>
<221> exon
<222> 131..499
<220>
<221> exon
<222> 4504..4694
<220>
<221> exon
<222> 5043..6939
<220>
<221> exon
<222> 8960..9347
<400> 105
gcttactttg tgccaaatct cattaggctt aaggtaatac aggactttga gtcaaatgat 60
actgttgcac ataagaacaa acctattttc atgctaagat gatgccactg tgttcctttc 120
tccttctag ttt ctg gac atc tcc att aag tgg acc acc cag gaa acg ttt 171
          Phe Leu Asp Ile Ser Ile Lys Trp Thr Thr Gln Glu Thr Phe
              -10 -5 -1 +1
cct cca aag tac ctt cat tat gac gaa gaa acc tct cat cag ctg ttg 219
Pro Pro Lys Tyr Leu His Tyr Asp Glu Glu Thr Ser His Gln Leu Leu
      5 10 15
tgt gac aaa tgt cct cct ggt acc tac cta aaa caa cac tgt aca gca 267
Cys Asp Lys Cys Pro Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala
20 25 30 35
aag tgg aag acc gtg tgc gcc cct tgc cct gac cac tac tac aca gac 315
Lys Trp Lys Thr Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp
                40 45 50
agc tgg cac acc agt gac gag tgt cta tac tgc agc ccc gtg tgc aag 363
Ser Trp His Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys
             55 60 65
gag ctg cag tac gtc aag cag gag tgc aat cgc acc cac aac cgc gtg 411
Glu Leu Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val
         70 75 80
tgc gaa tgc aag gaa ggg cgc tac ctt gag ata gag ttc tgc ttg aaa 459
Cys Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys
     85 90 95
cat agg agc tgc cct cct gga ttt gga gtg gtg caa gct g gtacgtgtca 509
His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala
100 105 110
atgtgcagca aaattaatta ggatcatgca aagtcagata gttgtgacag tttaggagaa 569
cacttttgtt ctgatgacat tataggatag caaattgcaa aggtaatgaa acctgccagg 629
taggtactat gtgtctggag tgcttccaaa ggaccattgc tcagaggaat actttgccac 689
tacagggcaa tttaatgaca aatctcaaat gcagcaaatt attctctcat gagatgcatg 749
atggtttttt tttttttttt taaagaaaca aactcaagtt gcactattga tagttgatct 809
atacctctat atttcacttc agcatggaca ccttcaaact gcagcacttt ttgacaaaca 869
tcagaaatgt taatttatac caagagagta attatgctca tattaatgag actctggagt 929
gctaacaata agcagttata attaattatg taaaaaatga gaatggtgag gggaattgca 989
tttcattatt aaaaacaagg ctagttcttc ctttagcatg ggagctgagt gtttgggagg 1049
gtaaggacta tagcagaatc tcttcaatga gcttattctt tatcttagac aaaacagatt 1109
gtcaagccaa gagcaagcac ttgcctataa accaagtgct ttctcttttg cattttgaac 1169
agcattggtc agggctcatg tgtattgaat cttttaaacc agtaacccac gttttttttc 1229
tgccacattt gcgaagcttc agtgcagcct ataacttttc atagcttgag aaaattaaga 1289
gtatccactt acttagatgg aagaagtaat cagtatagat tctgatgact cagtttgaag 1349
cagtgtttct caactgaagc cctgctgata ttttaagaaa tatctggatt cctaggctgg 1409
actccttttt gtgggcagct gtcctgcgca ttgtagaatt ttggcagcac ccctggactc 1469
tagccactag ataccaatag cagtccttcc cccatgtgac agccaaaaat gtcttcagac 1529
actgtcaaat gtcgccaggt ggcaaaatca ctcctggttg agaacagggt catcaatgct 1589
aagtatctgt aactatttta actctcaaaa cttgtgatat acaaagtcta aattattaga 1649
cgaccaatac tttaggttta aaggcataca aatgaaacat tcaaaaatca aaatctattc 1709
tgtttctcaa atagtgaatc ttataaaatt aatcacagaa gatgcaaatt gcatcagagt 1769
cccttaaaat tcctcttcgt atgagtattt gagggaggaa ttggtgatag ttcctacttt 1829
ctattggatg gtactttgag actcaaaagc taagctaagt tgtgtgtgtg tcagggtgcg 1889
gggtgtggaa tcccatcaga taaaagcaaa tccatgtaat tcattcagta agttgtatat 1949
gtagaaaaat gaaaagtggg ctatgcagct tggaaactag agaattttga aaaataatgg 2009
aaatcacaag gatctttctt aaataagtaa gaaaatctgt ttgtagaatg aagcaagcag 2069
gcagccagaa gactcagaac aaaagtacac attttactct gtgtacactg gcagcacagt 2129
gggatttatt tacctctccc tccctaaaaa cccacacagc ggttcctctt gggaaataag 2189
aggtttccag cccaaagaga aggaaagact atgtggtgtt actctaaaaa gtatttaata 2249
accgttttgt tgttgctgtt gctgttttga aatcagattg tctcctctcc atattttatt 2309
tacttcattc tgttaattcc tgtggaatta cttagagcaa gcatggtgaa ttctcaactg 2369
taaagccaaa tttctccatc attataattt cacattttgc ctggcaggtt ataattttta 2429
tatttccact gatagtaata aggtaaaatc attacttaga tggatagatc tttttcataa 2489
aaagtaccat cagttataga gggaagtcat gttcatgttc aggaaggtca ttagataaag 2549
cttctgaata tattatgaaa cattagttct gtcattctta gattcttttt gttaaataac 2609
tttaaaagct aacttaccta aaagaaatat ctgacacata tgaacttctc attaggatgc 2669
aggagaagac ccaagccaca gatatgtatc tgaagaatga acaagattct taggcccggc 2729
acggtggctc acatctgtaa tctcaagagt ttgagaggtc aaggcgggca gatcacctga 2789
ggtcaggagt tcaagaccag cctggccaac atgatgaaac cctgcctcta ctaaaaatac 2849
aaaaattagc agggcatggt ggtgcatgcc tgcaacccta gctactcagg aggctgagac 2909
aggagaatct cttgaaccct cgaggcggag gttgtggtga gctgagatcc ctctactgca 2969
ctccagcctg ggtgacagag atgagactcc gtccctgccg ccgcccccgc cttccccccc 3029
aaaaagattc ttcttcatgc agaacatacg gcagtcaaca aagggagacc tgggtccagg 3089
tgtccaagtc acttatttcg agtaaattag caatgaaaga atgccatgga atccctgccc 3149
aaatacctct gcttatgata ttgtagaatt tgatatagag ttgtatccca tttaaggagt 3209
aggatgtagt aggaaagtac taaaaacaaa cacacaaaca gaaaaccctc tttgctttgt 3269
aaggtggttc ctaagataat gtcagtgcaa tgctggaaat aatatttaat atgtgaaggt 3329
tttaggctgt gttttcccct cctgttcttt ttttctgcca gccctttgtc atttttgcag 3389
gtcaatgaat catgtagaaa gagacaggag atgaaactag aaccagtcca ttttgcccct 3449
ttttttattt tctggttttg gtaaaagata caatgaggta ggaggttgag atttataaat 3509
gaagtttaat aagtttctgt agctttgatt tttctctttc atatttgtta tcttgcataa 3569
gccagaattg gcctgtaaaa tctacatatg gatattgaag tctaaatctg ttcaactagc 3629
ttacactaga tggagatatt ttcatattca gatacactgg aatgtatgat ctagccatgc 3689
gtaatatagt caagtgtttg aaggtattta tttttaatag cgtctttagt tgtggactgg 3749
ttcaagtttt tctgccaatg atttcttcaa atttatcaaa tatttttcca tcatgaagta 3809
aaatgccctt gcagtcaccc ttcctgaagt ttgaacgact ctgctgtttt aaacagttta 3869
agcaaatggt atatcatctt ccgtttacta tgtagcttaa ctgcaggctt acgcttttga 3929
gtcagcggcc aactttattg ccaccttcaa aagtttatta taatgttgta aatttttact 3989
tctcaaggtt agcatactta ggagttgctt cacaattagg attcaggaaa gaaagaactt 4049
cagtaggaac tgattggaat ttaatgatgc agcattcaat gggtactaat ttcaaagaat 4109
gatattacag cagacacaca gcagttatct tgattttcta ggaataattg tatgaagaat 4169
atggctgaca acacggcctt actgccactc agcggaggct ggactaatga acaccctacc 4229
cttctttcct ttcctctcac atttcatgag cgttttgtag gtaacgagaa aattgacttg 4289
catttgcatt acaaggagga gaaactggca aaggggatga tggtggaagt tttgttctgt 4349
ctaatgaagt gaaaaatgaa aatgctagag ttttgtgcaa cataatagta gcagtaaaaa 4409
ccaagtgaaa agtctttcca aaactgtgtt aagagggcat ctgctgggaa acgatttgag 4469
gagaaggtac taaattgctt ggtattttcc gtag ga acc cca gag cga aat aca 4523
                                     gly thr pro glu arg asn thr
                                             115
gtt tgc aaa aga tgt cca gat ggg ttc ttc tca aat gag acg tca tct 4571
Val Cys Lys Arg Cys Pro Asp Gly Phe Phe Ser Asn Glu Thr Ser Ser
120 125 130 135
aaa gca ccc tgt aga aaa cac aca aat tgc agt gtc ttt ggt ctc ctg 4619
Lys Ala Pro Cys Arg Lys His Thr Asn Cys Ser Val Phe Gly Leu Leu
                140 145 150
cta act cag aaa gga aat gca aca cac gac aac ata tgt tcc gga aac 4667
Leu Thr Gln Lys Gly Asn Ala Thr His Asp Asn Ile Cys Ser Gly Asn
            155 160 165
agt gaa tca act caa aaa tgt gga ata g gtaattacat tccaaaatac 4715
Ser Glu Ser Thr Gln Lys Cys Gly Ile
        170 175
gtctttgtac gattttgtag tatcatctct ctctctgagt tgaacacaag gcctccagcc 4775
acattcttgg tcaaacttac attttccctt tcttgaatct taaccagcta aggctactct 4835
cgatgcatta ctgctaaagc taccactcag aatctctcaa aaactcatct tctcacagat 4895
aacacctcaa agcttgattt tctctccttt cacactgaaa tcaaatcttg cccataggca 4955
aagggcagtg tcaagtttgc cactgagatg aaattaggag agtccaaact gtagaattca 5015
cgttgtgtgt tattactttc acgaatgtct gtattattaa ctaaagtata tattggcaac 5075
taagaagcaa agtgatataa acatgatgac aaattaggcc aggcatggtg gcttactcct 5135
ataatcccaa cattttgggg ggccaaggta ggcagatcac ttgaggtcag gatttcaaga 5195
ccagcctgac caacatggtg aaaccttgtc tctactaaaa atacaaaaat tagctgggca 5255
tggtagcagg cacttctagt accagctact cagggctgag gcaggagaat cgcttgaacc 5315
caggagatgg aggttgcagt gagctgagat tgtaccactg cactccagtc tgggcaacag 5375
agcaagattt catcacacac acacacacac acacacacac acacattaga aatgtgtact 5435
tggctttgtt acctatggta ttagtgcatc tattgcatgg aacttccaag ctactctggt 5495
tgtgttaagc tcttcattgg gtacaggtca ctagtattaa gttcaggtta ttcggatgca 5555
ttccacggta gtgatgacaa ttcatcaggc tagtgtgtgt gttcaccttg tcactcccac 5615
cactagacta atctcagacc ttcactcaaa gacacattac actaaagatg atttgctttt 5675
ttgtgtttaa tcaagcaatg gtataaacca gcttgactct ccccaaacag tttttcgtac 5735
tacaaagaag tttatgaagc agagaaatgt gaattgatat atatatgaga ttctaaccca 5795
gttccagcat tgtttcattg tgtaattgaa atcatagaca agccatttta gcctttgctt 5855
tcttatctaa aaaaaaaaaa aaaaaaatga aggaaggggt attaaaagga gtgatcaaat 5915
tttaacattc tctttaatta attcattttt aattttactt tttttcattt attgtgcact 5975
tactatgtgg tactgtgcta tagaggcttt aacatttata aaaacactgt gaaagttgct 6035
tcagatgaat ataggtagta gaacggcaga actagtattc aaagccaggt ctgatgaatc 6095
caaaaacaaa cacccattac tcccattttc tgggacatac ttactctacc cagatgctct 6155
gggctttgta atgcctatgt aaataacata gttttatgtt tggttatttt cctatgtaat 6215
gtctacttat atatctgtat ctatctcttg ctttgtttcc aaaggtaaac tatgtgtcta 6275
aatgtgggca aaaaataaca cactattcca aattactgtt caaattcctt taagtcagtg 6335
ataattattt gttttgacat taatcatgaa gttccctgtg ggtactaggt aaacctttaa 6395
tagaatgtta atgtttgtat tcattataag aatttttggc tgttacttat ttacaacaat 6455
atttcactct aattagacat ttactaaact ttctcttgaa aacaatgccc aaaaaagaac 6515
attagaagac acgtaagctc agttggtctc tgccactaag accagccaac agaagcttga 6575
ttttattcaa actttgcatt ttagcatatt ttatcttgga aaattcaatt gtgttggttt 6635
tttgtttttg tttgtattga atagactctc agaaatccaa ttgttgagta aatcttctgg 6695
gttttctaac ctttctttag at gtt acc ctg tgt gag gag gca ttc ttc agg 6747
                     Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg
                                 180 185
ttt gct gtt cct aca aag ttt acg cct aac tgg ctt agt gtc ttg gta 6795
Phe Ala Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val
        190 195 200
gac aat ttg cct ggc acc aaa gta aac gca gag agt gta gag agg ata 6843
Asp Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg Ile
    205 210 215
aaa cgg caa cac agc tca caa gaa cag act ttc cag ctg ctg aag tta 6891
Lys Arg Gln His Ser Ser Gln Glu Gln Thr Phe Gln Leu Leu Lys Leu
220 225 230 235
tgg aaa cat caa aac aaa gac caa gat ata gtc aag aag atc atc caa g 6940
Trp Lys His Gln Asn Lys Asp Gln Asp Ile Val Lys Lys Ile Ile Gln
                240 245 250
gtaattacat tccaaaatac gtctttgtac gattttgtag tatcatctct ctctctgagt 7000
tgaacacaag gcctccagcc acattcttgg tcaaacttac attttccctt tcttgaatct 7060
taaccagcta aggctactct cgatgcatta ctgctaaagc taccactcag aatctctcaa 7120
aaactcatct tctcacagat aacacctcaa agcttgattt tctctccttt cacactgaaa 7180
tcaaatcttg cccataggca aagggcagtg tcaagtttgc cactgagatg aaattaggag 7240
agtccaaact gtagaattca cgttgtgtgt tattactttc acgaatgtct gtattattaa 7300
ctaaagtata tattggcaac taagaagcaa agtgatataa acatgatgac aaattaggcc 7360
aggcatggtg gcttactcct ataatcccaa cattttgggg ggccaaggta ggcagatcac 7420
ttgaggtcag gatttcaaga ccagcctgac caacatggtg aaaccttgtc tctactaaaa 7480
atacaaaaat tagctgggca tggtagcagg cacttctagt accagctact cagggctgag 7540
gcaggagaat cgcttgaacc caggagatgg aggttgcagt gagctgagat tgtaccactg 7600
cactccagtc tgggcaacag agcaagattt catcacacac acacacacac acacacacac 7660
acacattaga aatgtgtact tggctttgtt acctatggta ttagtgcatc tattgcatgg 7720
aacttccaag ctactctggt tgtgttaagc tcttcattgg gtacaggtca ctagtattaa 7780
gttcaggtta ttcggatgca ttccacggta gtgatgacaa ttcatcaggc tagtgtgtgt 7840
gttcaccttg tcactcccac cactagacta atctcagacc ttcactcaaa gacacattac 7900
actaaagatg atttgctttt ttgtgtttaa tcaagcaatg gtataaacca gcttgactct 7960
ccccaaacag tttttcgtac tacaaagaag tttatgaagc agagaaatgt gaattgatat 8020
atatatgaga ttctaaccca gttccagcat tgtttcattg tgtaattgaa atcatagaca 8080
agccatttta gcctttgctt tcttatctaa aaaaaaaaaa aaaaaaatga aggaaggggt 8140
attaaaagga gtgatcaaat tttaacattc tctttaatta attcattttt aattttactt 8200
tttttcattt attgtgcact tactatgtgg tactgtgcta tagaggcttt aacatttata 8260
aaaacactgt gaaagttgct tcagatgaat ataggtagta gaacggcaga actagtattc 8320
aaagccaggt ctgatgaatc caaaaacaaa cacccattac tcccattttc tgggacatac 8380
ttactctacc cagatgctct gggctttgta atgcctatgt aaataacata gttttatgtt 8440
tggttatttt cctatgtaat gtctacttat atatctgtat ctatctcttg ctttgtttcc 8500
aaaggtaaac tatgtgtcta aatgtgggca aaaaataaca cactattcca aattactgtt 8560
caaattcctt taagtcagtg ataattattt gttttgacat taatcatgaa gttccctgtg 8620
ggtactaggt aaacctttaa tagaatgtta atgtttgtat tcattataag aatttttggc 8680
tgttacttat ttacaacaat atttcactct aattagacat ttactaaact ttctcttgaa 8740
aacaatgccc aaaaaagaac attagaagac acgtaagctc agttggtctc tgccactaag 8800
accagccaac agaagcttga ttttattcaa actttgcatt ttagcatatt ttatcttgga 8860
aaattcaatt gtgttggttt tttgtttttg tttgtattga atagactctc agaaatccaa 8920
ttgttgagta aatcttctgg gttttctaac ctttctttag at att gac ctc tgt 8974
                                           Asp Ile Asp Leu Cys
                                                       255
gaa aac agc gtg cag cgg cac att gga cat gct aac ctc acc ttc gag 9022
Glu Asn Ser Val Gln Arg His Ile Gly His Ala Asn Leu Thr Phe Glu
            260 265 270
cag ctt cgt agc ttg atg gaa agc tta ccg gga aag aaa gtg gga gca 9070
Gln Leu Arg Ser Leu Met Glu Ser Leu Pro Gly Lys Lys Val Gly Ala
        275 280 285
gaa gac att gaa aaa aca ata aag gca tgc aaa ccc agt gac cag atc 9118
Glu Asp Ile Glu Lys Thr Ile Lys Ala Cys Lys Pro Ser Asp Gln Ile
    290 295 300
ctg aag ctg ctc agt ttg tgg cga ata aaa aat ggc gac caa gac acc 9166
Leu Lys Leu Leu Ser Leu Trp Arg Ile Lys Asn Gly Asp Gln Asp Thr
305 310 315 320
ttg aag ggc cta atg cac gca cta aag cac tca aag acg tac cac ttt 9214
Leu Lys Gly Leu Met His Ala Leu Lys His Ser Lys Thr Tyr His Phe
                325 330 335
ccc aaa act gtc act cag agt cta aag aag acc atc agg ttc ctt cac 9262
Pro Lys Thr Val Thr Gln Ser Leu Lys Lys Thr Ile Arg Phe Leu His
            340 345 350
agc ttc aca atg tac aaa ttg tat cag aag tta ttt tta gaa atg ata 9310
Ser Phe Thr Met Tyr Lys Leu Tyr Gln Lys Leu Phe Leu Glu Met Ile
        355 360 365
ggt aac cag gtc caa tca gta aaa ata agc tgc tta taactggaaa 9356
Gly Asn Gln Val Gln Ser Val Lys Ile Ser Cys Leu
    370 375 380
tggccattga gctgtttcct cacaattggc gagatcccat ggatgagtaa actgtttctc
9416aggcacttga ggctttcagt gatatctttc tcattaccag tgactaattt tgccacaggg
9476tactaaaaga aactatgatg tggagaaagg actaacatct cctccaataa accccaaatg
9536gttaatccaa ctgtcagatc tggatcgtta tctactgact atattttccc ttattactgc
9596ttgcagtaat tcaactggaa attaaaaaaa aaaaactaga ctccactggg ccttactaaa
9656tatgggaatg tctaacttaa atagctttgg gattccagct atgctagagg cttttattag
9716aaagccatat ttttttctgt aaaagttact aatatatctg taacactatt acagtattgc
9776tatttatatt cattcagata taagatttgg acatattatc atcctataaa gaaacggtat
9836gacttaattt tagaaagaaa attatattct gtttattatg acaaatgaaa gagaaaatat
9896atatttttaa tggaaagttt gtagcatttt tctaataggt actgccatat ttttctgtgt
9956ggagtatttt tataatttta tctgtataag ctgtaatatc attttataga aaatgcatta 10
016tttagtcaat tgtttaatgt tggaaaacat atgaaatata aattatctga atattagatg 100
76ctctgagaaa ttgaatgtac cttatttaaa agattttatg gttttataac tatataaatg 1013
6acattattaa agttttcaaa ttatttttta ttgctttctc tgttgctttt attt 1019
0
[Brief description of the drawings]
FIG. 1 shows an elution profile when a HiLoad-Q / FF non-adsorbed fraction crude product (sample 3) is applied to a HiLoad-S / HP column.
FIG. 2 shows an elution profile when heparin-5PW crude product (sample 5) is applied to a blue-5PW column.
FIG. 3 shows an elution profile when Blue-5PW elution fractions 49-50 are applied to a reverse phase column.
FIG. 4 shows the results of SDS-PAGE of the final purified product under reducing and non-reducing conditions.
[Explanation of symbols]
Lanes 1 and 4; molecular weight markers
Lanes 2 and 5; peak 6
Lanes 3 and 6; peak 7
FIG. 5 shows an elution profile when peak 7 treated with lysyl endoprotease is applied to a reverse phase column after reductive pyridylethylation.
FIG. 6 shows the results of SDS-PAGE of natural (n) and recombinant (r) OCIF under non-reducing conditions. (E) shows the product produced by 293 / EBNA cells, and (C) shows the product produced by CHO cells.
[Explanation of symbols]
Lane 1; molecular weight marker
Lane 2: Monomer type nOCIF
Lane 3; Dimer type nOCIF
Lane 4: Monomer type rOCIF (E)
Lane 5: Dimer type rOCIF (E)
Lane 6: Monomeric rOCIF (C)
Lane 7: Dimer type rOCIF (C)
FIG. 7 shows the results of SDS-PAGE of natural (n) and recombinant (r) OCIF under reducing conditions. In addition, (E) shows that produced by 293 / EBNA cells, and (C) that produced by CHO cells.
[Explanation of symbols]
Lane 8: molecular weight marker
Lane 9: Monomeric nOCIF
Lane 10: Dimer type nOCIF
Lane 11: Monomeric rOCIF (E)
Lane 12: Dimer type rOCIF (E)
Lane 13: Monomeric rOCIF (C)
Lane 14: Dimer type rOCIF (C)
FIG. 8 shows the results of SDS-PAGE of natural (n) and recombinant (r) OCIF from which N-linked sugar chains have been removed under reducing conditions. (E) shows the product produced by 293 / EBNA cells, and (C) shows the product produced by CHO cells.
[Explanation 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 type rOCIF (E)
Lane 20; monomeric rOCIF (C)
Lane 21: Dimer type rOCIF (C)
FIG. 9 shows comparison of amino acid sequences of OCIF and OCIF2.
FIG. 10 shows comparison of amino acid sequences between OCIF and OCIF3.
FIG. 11 shows comparison of amino acid sequences between OCIF and OCIF4.
FIG. 12 shows a comparison of amino acid sequences between OCIF and OCIF5.
FIG. 13 shows a calibration curve for OCIF when an anti-OCIF polyclonal antibody is used.
FIG. 14 shows a calibration curve of OCIF when an anti-OCIF monoclonal antibody is used.
FIG. 15 shows the therapeutic effect of OCIF on osteoporosis.

Claims (63)

 A cDNA represented by the nucleotide sequence consisting of nucleotide numbers 1 to 1206 or nucleotide numbers 64 to 1206 in SEQ ID NO: 83 in the Sequence Listing.  A protein represented by an amino acid sequence encoded by a nucleotide sequence consisting of nucleotide numbers 1 to 1206 or nucleotide numbers 64 to 1206 in SEQ ID NO: 83 in the Sequence Listing.  A cDNA represented by a nucleotide sequence encoding an amino acid sequence consisting of amino acid Nos. -21 to 380 or Nos. 1 to 380 of SEQ ID NO: 62 in the Sequence Listing. A cDNA represented by the nucleotide sequence consisting of nucleotide numbers 1 to 1206 or nucleotide numbers 64 to 1206 of SEQ ID NO: 86.  A protein represented by an amino acid sequence encoded by a nucleotide sequence consisting of nucleotide numbers 1 to 1206 or nucleotide numbers 64 to 1206 in SEQ ID NO: 86 in the Sequence Listing.  A cDNA represented by a nucleotide sequence encoding an amino acid sequence consisting of amino acid Nos. -21 to 380 or 1 to 380 of SEQ ID NO: 65 in the Sequence Listing.  A cDNA represented by the nucleotide sequence consisting of nucleotide numbers 1 to 1206 or nucleotide numbers 64 to 1206 in SEQ ID NO: 87 in the Sequence Listing.  A protein represented by an amino acid sequence encoded by a nucleotide sequence consisting of nucleotide numbers 1 to 1206 or nucleotide numbers 64 to 1206 in SEQ ID NO: 87 in the Sequence Listing.  A cDNA represented by a nucleotide sequence encoding an amino acid sequence consisting of amino acid Nos. 21 to 380 or Nos. 1 to 380 of SEQ ID NO: 66 in the Sequence Listing.  A cDNA represented by the nucleotide sequence consisting of nucleotide numbers 1 to 981 or nucleotide numbers 64 to 981 in SEQ ID NO: 92 in the Sequence Listing.  A protein represented by an amino acid sequence encoded by a nucleotide sequence consisting of nucleotide numbers 1 to 981 or nucleotide numbers 64 to 981 in SEQ ID NO: 92 in the Sequence Listing.  A cDNA represented by a base sequence encoding an amino acid sequence consisting of amino acid Nos. -21 to 305 or 1 to 305 of SEQ ID NO: 71 in the Sequence Listing.  A cDNA represented by the nucleotide sequence consisting of nucleotide numbers 1 to 1200 or nucleotide numbers 64 to 1200 of SEQ ID NO: 94 in the sequence listing.  A protein represented by an amino acid sequence encoded by a base sequence consisting of base Nos. 1 to 1200 or bases Nos. 64 to 1200 of SEQ ID NO: 94.  A cDNA represented by a nucleotide sequence encoding an amino acid sequence consisting of amino acid Nos. 21 to 378 or Nos. 1 to 378 of SEQ ID NO: 73 in the Sequence Listing.  A cDNA represented by the nucleotide sequence consisting of nucleotide numbers 1 to 1056 or nucleotide numbers 64 to 1056 in SEQ ID NO: 95 of the Sequence Listing.  A protein represented by an amino acid sequence encoded by a nucleotide sequence consisting of nucleotide numbers 1 to 1056 or nucleotide numbers 64 to 1056 in SEQ ID NO: 95 of the Sequence Listing.  A cDNA represented by a base sequence encoding an amino acid sequence consisting of amino acid Nos. -21 to 330 or Nos. 1 to 330 of SEQ ID NO: 74 in the Sequence Listing.  A cDNA represented by the nucleotide sequence consisting of nucleotide numbers 1 to 819 or nucleotide numbers 64 to 819 in SEQ ID NO: 96 in the sequence listing.  A protein represented by an amino acid sequence encoded by a nucleotide sequence consisting of nucleotide numbers 1 to 819 or nucleotide numbers 64 to 819 in SEQ ID NO: 96 in the sequence listing.  A cDNA represented by a nucleotide sequence encoding an amino acid sequence consisting of amino acid Nos. -21 to 251 or 1 to 251 of SEQ ID NO: 75 in the Sequence Listing.   A cDNA represented by the nucleotide sequence consisting of nucleotide numbers 1 to 594 or nucleotide numbers 64 to 594 in SEQ ID NO: 97 in the sequence listing.   A protein represented by an amino acid sequence encoded by a nucleotide sequence consisting of nucleotide numbers 1 to 594 or nucleotide numbers 64 to 594 in SEQ ID NO: 97 in the Sequence Listing.  A cDNA represented by a nucleotide sequence encoding an amino acid sequence consisting of amino acid Nos. -21 to 176 or Nos. 1 to 176 of SEQ ID NO: 76 in the Sequence Listing.   A cDNA represented by the nucleotide sequence consisting of nucleotide numbers 1 to 1182 or nucleotide numbers 64 to 1182 of SEQ ID NO: 100 in the sequence listing.   A protein represented by an amino acid sequence encoded by a nucleotide sequence consisting of nucleotide numbers 1 to 1182 or nucleotide numbers 64 to 1182 of SEQ ID NO: 100 in the sequence listing.  A cDNA represented by a base sequence encoding an amino acid sequence consisting of amino acid Nos. 21 to 372 or Nos. 1 to 372 of SEQ ID NO: 79 in the Sequence Listing.  A cDNA represented by the nucleotide sequence consisting of nucleotide numbers 1 to 966 or nucleotide numbers 64 to 966 of SEQ ID NO: 101 in the sequence listing.   A protein represented by an amino acid sequence encoded by a nucleotide sequence consisting of nucleotide numbers 1 to 966 or nucleotide numbers 64 to 966 in SEQ ID NO: 101 in the sequence listing.  A cDNA represented by a base sequence encoding an amino acid sequence consisting of amino acid Nos. 21 to 300 or Nos. 1 to 300 of SEQ ID NO: 80 in the Sequence Listing.   A protein comprising the amino acid sequence shown in SEQ ID NO: 5 in the sequence listing. A vector into which any one of the following (1) to (4) cDNA is inserted:
(1) cDNA encoding the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing;
(2) cDNA encoding the amino acid sequence shown in SEQ ID NO: 5 in the sequence listing;
(3) cDNA represented by the nucleotide sequence of SEQ ID NO: 6 in the Sequence Listing;
(4) cDNA encoding a protein inserted into a plasmid vector pBKOCIF possessed by transformed E. coli pBK / 01F10 (FERM BP-5267) and having an activity of suppressing osteoclast differentiation and / or maturation.   33. The vector according to claim 32, which is carried by transformed Escherichia coli pBK / 01F10 (FERM BP-5267).   A host cell into which the vector according to claim 32 or 33 has been introduced. A method for producing a protein having the following physicochemical properties (a) to (d) comprising the following steps (I) and (II) and having an activity of inhibiting osteoclast differentiation and / or maturation:
(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; has cation exchanger and heparin affinity.
(c) Heat stability; osteoclast differentiation / maturation inhibitory activity is reduced by heat treatment at 70 ° C for 10 minutes or 56 ° C for 30 minutes, and osteoclast differentiation / maturation by 90 ° C for 10 minutes. Maturation inhibitory activity is lost.
(d) Amino acid sequence; as an internal amino acid sequence, it has the amino acid sequence of SEQ ID NO: 1 to 3 in Sequence Listing.
(I) culturing the host cell of claim 34;
(II) A step of recovering a protein having the physicochemical properties (a) to (d) and having osteoclast differentiation and / or maturation inhibitory activity from the culture solution. The following physicochemical properties (a) obtainable by the method of claim 33:
A protein having an activity of inhibiting osteoclast differentiation and / or maturation, having ~ (d).
(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; has cation exchanger and heparin affinity.
(c) Heat stability; osteoclast differentiation / maturation inhibitory activity is reduced by heat treatment at 70 ° C for 10 minutes or 56 ° C for 30 minutes, and osteoclast differentiation / maturation by 90 ° C for 10 minutes. Maturation inhibitory activity is lost.
(d) Amino acid sequence; as an internal amino acid sequence, it has the amino acid sequence of SEQ ID NO: 1 to 3 in Sequence Listing.   A protein represented by an amino acid sequence consisting of amino acid numbers -21 to 373 or amino acid numbers 1 to 373 in SEQ ID NO: 9 in the sequence listing.   A cDNA represented by a nucleotide sequence consisting of nucleotide numbers 64 to 1185 in SEQ ID NO: 8 in the sequence listing.   A protein represented by an amino acid sequence encoded by a nucleotide sequence consisting of nucleotide numbers 1 to 1185 or 64 to 1185 in SEQ ID NO: 8 in the sequence listing.   A protein represented by an amino acid sequence consisting of amino acid numbers -21 to 341 or amino acid numbers 1 to 341 in SEQ ID NO: 11 in the sequence listing.   A cDNA represented by a nucleotide sequence consisting of nucleotide numbers 64 to 1089 in SEQ ID NO: 10 in the Sequence Listing.   A protein represented by an amino acid sequence encoded by a nucleotide sequence consisting of nucleotide numbers 1 to 1089 or 64 to 1089 in SEQ ID NO: 10 in the Sequence Listing.   A protein represented by amino acids consisting of amino acid Nos. -21 to 133 or amino acids Nos. 1 to 133 in SEQ ID NO: 13 in the Sequence Listing.   A cDNA represented by a nucleotide sequence consisting of nucleotide numbers 64 to 465 of SEQ ID NO: 12 in the Sequence Listing.   A protein represented by an amino acid sequence encoded by a nucleotide sequence consisting of nucleotide numbers 1 to 465 or 64 to 465 in SEQ ID NO: 12 in the sequence listing.   A protein represented by amino acids consisting of amino acid Nos. -21 to 124 or amino acids Nos. 1 to 124 in SEQ ID NO: 15 in the Sequence Listing.   A cDNA represented by the nucleotide sequence consisting of nucleotide numbers 64 to 438 in SEQ ID NO: 14 in the sequence listing.   A protein represented by an amino acid sequence encoded by a nucleotide sequence consisting of nucleotide numbers 1 to 438 or 64 to 438 in SEQ ID NO: 14 in the sequence listing.   A protein represented by an amino acid sequence consisting of amino acid numbers -21 to 380 or amino acid numbers 1 to 380 of SEQ ID NO: 62 in the sequence listing.   A protein represented by an amino acid sequence consisting of amino acid numbers -21 to 380 or amino acid numbers 1 to 380 of SEQ ID NO: 65 in the sequence listing.   A protein represented by an amino acid sequence consisting of amino acid numbers -21 to 380 or amino acid numbers 1 to 380 of SEQ ID NO: 66 in the sequence listing.   A protein represented by an amino acid sequence consisting of amino acid numbers -21 to 305 or amino acid numbers 1 to 305 of SEQ ID NO: 71 in the sequence listing.   A protein represented by an amino acid sequence consisting of amino acid numbers -21 to 378 or amino acid numbers 1 to 378 of SEQ ID NO: 73 in the sequence listing.   A protein represented by an amino acid sequence consisting of amino acid numbers -21 to 330 or amino acid numbers 1 to 330 of SEQ ID NO: 74 in the sequence listing.   A protein represented by an amino acid sequence consisting of amino acid numbers -21 to 251 or amino acid numbers 1 to 251 of SEQ ID NO: 75 in the sequence listing.   A protein represented by an amino acid sequence consisting of amino acid Nos. -21 to 176 or amino acid Nos. 1 to 176 of SEQ ID NO: 76 in the Sequence Listing.     A protein represented by an amino acid sequence consisting of amino acid numbers -21 to 372 or amino acid numbers 1 to 372 of SEQ ID NO: 79 in the sequence listing.   A protein represented by an amino acid sequence consisting of amino acid Nos. -21 to 300 or amino acid Nos. 1 to 300 in SEQ ID NO: 80 in the Sequence Listing. A transformed Escherichia coli pBK / 01F10 (FERM BP-5267) having a plasmid vector pOBKOCIF inserted with a cDNA encoding a protein having an activity of suppressing osteoclast differentiation and / or maturation.   Selected from the group consisting of claims 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 31, 36, 37, 38, 40, 42, 43, 45, 46, and 48-58 A pharmaceutical composition comprising the protein according to claim 1.   Selected from the group consisting of claims 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 31, 36, 37, 38, 40, 42, 43, 45, 46, and 48-58 An agent for preventing or treating osteoporosis, comprising the protein according to claim 1.   Selected from the group consisting of claims 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 31, 36, 37, 38, 40, 42, 43, 45, 46, and 48-58 A preventive or therapeutic agent for osteopenia containing the protein according to claim 1.   Selected from the group consisting of claims 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 31, 36, 37, 38, 40, 42, 43, 45, 46, and 48-58 An agent for preventing or treating bone metabolic disorders, comprising the protein according to claim 1.

Images