JP2004534506A - Secreted protein - Google Patents

Abstract

The present invention provides human secreted proteins (SECPs) and polynucleotides that identify and encode SCCP. The present invention also provides expression vectors, host cells, antibodies, agonists and antagonists. The present invention also provides a method for diagnosing, treating or preventing a disease associated with abnormal expression of SSCP.

Description

【０１２５】
場合によっては、最終コンセンサスポリヌクレオチド配列を確認するために、列５に示すような配列カバレッジと重複するＩｎｃｙｔｅ ｃＤＮＡカバレッジが得られたが、該当するＩｎｃｙｔｅ ｃＤＮＡ識別番号は示さなかった。
【０１２６】
表５は、Ｉｎｃｙｔｅ ｃＤＮＡ配列を用いて構築された完全長ポリヌクレオチド配列のための代表的なｃＤＮＡライブラリを示している。代表的なｃＤＮＡライブラリとはＩｎｃｙｔｅ ｃＤＮＡライブラリであり、これは、最も頻繁にはＩｎｃｙｔｅ ｃＤＮＡ配列群によって代表されるが、これらは、上記のポリヌクレオチド配列を構築および確認するために用いられた。ｃＤＮＡライブラリを作製するために用いた組織およびベクターを表５に示し、表６で説明している。
【０１２７】
本発明はまた、ＳＥＣＰの変異配列をも含む。好適なＳＥＣＰ変異配列は、ＳＥＣＰの機能的あるいは構造的特徴の少なくとも１つを有し、且つＳＥＣＰアミノ酸配列に対して少なくとも約８０％のアミノ酸配列同一性、あるいは少なくとも約９０％のアミノ酸配列同一性、更には少なくとも約９５％のアミノ酸配列同一性を有する。
【０１２８】
本発明はまた、ＳＥＣＰをコードするポリヌクレオチド群を提供する。特定の実施態様において、本発明は、ＳＥＣＰをコードする、ＳＥＱ ＩＤ ＮＯ：６４−１２６からなる一群から選択された１配列を持つポリヌクレオチド配列を提供する。ＳＥＱ ＩＤ ＮＯ：６４−１２６のポリヌクレオチド配列には、配列表に示したように等価ＲＮＡ配列をも含むが、そこでは窒素塩基チミンの出現はウラシルに置換され、糖のバックボーンはデオキシリボースではなくリボースで構成される。
【０１２９】
本発明はまた、ＳＥＣＰをコードするポリヌクレオチド配列の変異配列を含む。詳細には、このような変異体ポリヌクレオチド配列は、ＳＥＣＰをコードするポリヌクレオチド配列との、少なくとも約７０％のポリヌクレオチド配列同一性、あるいは少なくとも約８５％のポリヌクレオチド配列同一性、更には少なくとも約９５％ものポリヌクレオチド配列同一性を持つこととなる。本発明の或る特定の実施態様では、ＳＥＱ ＩＤ ＮＯ：６４−１２６からなる群から選択された或る配列を持つポリヌクレオチド配列の変異配列を含む。この変異配列は、ＳＥＱ ＩＤ ＮＯ：６４−１２６からなる群から選択された或る核酸配列との、少なくとも約７０％、あるいは少なくとも約８５％、または少なくとも約９５％ものポリヌクレオチド配列同一性を有する。前記の任意のポリヌクレオチド変異配列は、ＳＥＣＰの機能的若しくは構造的特徴を少なくとも１つ有するアミノ酸配列をコードできる。
【０１３０】
更に、別の例では、本発明の或るポリヌクレオチド変異配列は、ＳＥＣＰをコードする１ポリヌクレオチド配列のスプライス変異配列である。或るスプライス変異配列はＳＥＣＰをコードするポリヌクレオチド配列との顕著な配列同一性を持つ部分複数を有し得るが、ｍＲＮＡプロセッシング時のエキソン群の選択的スプライシングによって生ずる、配列の数ブロックの付加または欠失により、通常、より多数またはより少数のポリヌクレオチドを持つことになる。或るスプライス変異配列は、ＳＥＣＰをコードするポリヌクレオチド配列の全長との間には、約７０％未満、または約６０％未満、あるいは約５０％未満のポリヌクレオチド配列同一性を持ち得るが、このスプライス変異配列のいくつかの部分は、ＳＥＣＰをコードするポリヌクレオチド配列の各部との、少なくとも約７０％、あるいは少なくとも約８５％、または少なくとも約９５％、なおまたは１００％の、ポリヌクレオチド配列同一性を有するであろう。上記したスプライス変異配列は何れも、ＳＥＣＰの機能的或いは構造的特徴の少なくとも１つを有する或るアミノ酸配列をコードし得る。
【０１３１】
遺伝暗号の縮重により、ＳＥＣＰをコードする種々のポリヌクレオチド配列が作り出され、中には、既知のいかなる天然遺伝子のポリヌクレオチド配列群とも最小の類似性しか有しない配列もあることは、当業者には理解されよう。したがって本発明には、可能コドン選択に基づく組合せの選択によって産出し得るあらゆる可能なポリヌクレオチド配列のバリエーションを網羅し得る。これらの組み合わせは、天然ＳＥＣＰのポリヌクレオチド配列に適用される標準的なトリプレット遺伝暗号を基に成される。このような全ての変異は明確に開示されていると考慮されたい。
【０１３２】
ＳＥＣＰとその変異配列群とをコードするヌクレオチド配列群は一般に、好適に選択されたストリンジェンシー条件下で天然ＳＥＣＰのヌクレオチド配列にハイブリダイズ可能であるが、非天然コドンを含むなど実質的に異なるコドン使用を有する、ＳＥＣＰあるいはその誘導体をコードするヌクレオチド配列群を産生することは有益であり得る。宿主が特定コドンを利用する頻度に基づいて、特定の真核宿主または原核宿主に発生するペプチドの発現率を高めるようにコドンを選択し得る。コードされるアミノ酸配列群を改変せずに、ＳＥＣＰとその誘導体群とをコードするヌクレオチド配列を実質的に改変する別の理由の例として、例えば天然配列から作られる転写物より長い半減期など、より好ましい特性を持つＲＮＡ転写物群の産生がある。
【０１３３】
本発明にはまた、ＳＥＣＰとその誘導体とをコードするＤＮＡ配列またはそれらの断片の、完全に合成化学による作製も含む。作製後、当分野で公知の試薬を用いて、この合成配列を任意の様々な入手可能な発現ベクターおよび細胞系中に挿入し得る。更に、合成化学を用いて、ＳＥＣＰまたはその任意の断片をコードする１配列に突然変異を誘導し得る。
【０１３４】
更に本発明には、種々のストリンジェンシー条件下で、請求項に記載されたポリヌクレオチド配列、特にＳＥＱ ＩＤ ＮＯ：６４−１２６、およびそれらの断片へのハイブリダイズが可能なポリヌクレオチド配列を含む（例えば、Ｗａｈｌ， Ｇ．Ｍ．およびＳ．Ｌ． Ｂｅｒｇｅｒ （１９８７） Ｍｅｔｈｏｄｓ Ｅｎｚｙｍｏｌ． １５２：３９９−４０７； Ｋｉｍｍｅｌ． Ａ．Ｒ． （１９８７） Ｍｅｔｈｏｄｓ Ｅｎｚｙｍｏｌ． １５２：５０７−５１１を参照）。アニーリングおよび洗浄条件を含むハイブリダイゼーションの条件は、「定義」に記載した。
【０１３５】
ＤＮＡシークエンシングの方法は当分野では公知であり、本発明のいずれの実施例も、ＤＮＡシークエンシング方法を用い得る。ＤＮＡシークエンシング方法には酵素を用い得る。例えばＤＮＡポリメラーゼＩのクレノウ断片、ＳＥＱＵＥＮＡＳＥ（ＵＳ Ｂｉｏｃｈｅｍｉｃａｌ， Ｃｌｅｖｅｌａｎｄ ＯＨ）、Ｔａｑポリメラーゼ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）、熱安定性Ｔ７ポリメラーゼ（Ａｍｅｒｓｈａｍ， Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ， Ｐｉｓｃａｔａｗａｙ ＮＪ）を用い得る。あるいは、例えばＥＬＯＮＧＡＳＥ増幅システム（Ｌｉｆｅ Ｔｅｃｈｎｏｌｏｇｉｅｓ， Ｇａｉｔｈｅｒｓｂｕｒｇ ＭＤ）において見られるように、ポリメラーゼと校正エキソヌクレアーゼとを併用し得る。好適には、ＭＩＣＲＯＬＡＢ２２００液体転移システム（Ｈａｍｉｌｔｏｎ， Ｒｅｎｏ， ＮＶ）、ＰＴＣ２００サーマルサイクラー（ＭＪ Ｒｅｓｅａｒｃｈ， Ｗａｔｅｒｔｏｗｎ ＭＡ）およびＡＢＩ ＣＡＴＡＬＹＳＴ ８００サーマルサイクラー（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）などの装置を用いて配列の準備を自動化する。次に、ＡＢＩ ３７３ あるいは ３７７ ＤＮＡシークエンシングシステム（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）、ＭＥＧＡＢＡＣＥ １０００ ＤＮＡシークエンシングシステム（Ｍｏｌｅｃｕｌａｒ Ｄｙｎａｍｉｃｓ， Ｓｕｎｎｙｖａｌｅ ＣＡ）または当分野で公知の他のシステムを用いてシークエンシングを行う。結果として得た配列を、当分野で周知の種々のアルゴリズムを用いて分析する（例えば、Ａｕｓｕｂｅｌ， Ｆ．Ｍ． （１９９７） Ｓｈｏｒｔ Ｐｒｏｔｏｃｏｌｓ ｉｎ Ｍｏｌｅｃｕｌａｒ Ｂｉｏｌｏｇｙ， Ｊｏｈｎ Ｗｉｌｅｙ ＆ Ｓｏｎｓ， Ｎｅｗ Ｙｏｒｋ ＮＹ， ７．７ユニット、Ｍｅｙｅｒｓ， Ｒ．Ａ． （１９９５） Ｍｏｌｅｃｕｌａｒ Ｂｉｏｌｏｇｙ ａｎｄ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ， Ｗｉｌｅｙ ＶＣＨ， Ｎｅｗ Ｙｏｒｋ ＮＹ， ８５６−８５３ページを参照）。
【０１３６】
部分的なヌクレオチド配列を利用し、また、ＰＣＲに基づく当分野で公知の種々の方法で、ＳＥＣＰをコードする核酸配列群を伸長し、プロモーターや調節エレメントなどの、上流にある配列群を検出し得る。例えば、使用し得る方法の１つである制限部位ＰＣＲ法は、ユニバーサルプライマーおよびネステッドプライマーを用いてクローニングベクター内のゲノムＤＮＡからの未知の配列を増幅する方法である（例えば、Ｓａｒｋａｒ， Ｇ． （１９９３） ＰＣＲ Ｍｅｔｈｏｄｓ Ａｐｐｌｉｃ． ２：３１８−３２２を参照）。別の方法に逆ＰＣＲ法があり、これは多岐の方向に伸長するプライマー群を用いて、環状化した鋳型から未知の配列を増幅する方法である。鋳型は、或る既知のゲノム遺伝子座およびその周辺の配列群からなる制限酵素断片群から得る（例えば、Ｔｒｉｇｌｉａ， Ｔ． 他 （１９８８） Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｒｅｓ． １６：８１８６を参照）。第３の方法としてキャプチャＰＣＲ法があり、これにはヒトおよび酵母人工染色体ＤＮＡの既知の配列群に隣接するＤＮＡ断片群をＰＣＲ増幅する方法を含む（例えばＬａｇｅｒｓｔｒｏｍ， Ｍ．他（１９９１） ＰＣＲ Ｍｅｔｈｏｄｓ Ａｐｐｌｉｃ １：１１１−１１９を参照）。この方法では、ＰＣＲを行う前に、複数の制限酵素の消化およびライゲーション反応を用い、未知の配列領域内に組換え二本鎖配列を挿入し得る。未知の配列群を検索するために用い得る他の複数の方法も当分野で公知である（例えばＰａｒｋｅｒ， Ｊ．Ｄ．他 （１９９１） Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｒｅｓ． １９：３０５５−３０６０を参照）。更に、ＰＣＲ、ネステッドプライマー類、およびＰＲＯＭＯＴＥＲＦＩＮＤＥＲライブラリ類（Ｃｌｏｎｔｅｃｈ， Ｐａｌｏ Ａｌｔｏ ＣＡ）を用いてゲノムＤＮＡをウォーキングし得る。この手順は、ライブラリ類をスクリーニングする必要がなく、イントロン／エキソン接合部の発見に有用である。全てのＰＣＲベースの方法では、市販ソフトウェア、例えばＯＬＩＧＯ ４．０６プライマー分析ソフトウェア（Ｎａｔｉｏｎａｌ Ｂｉｏｓｃｉｅｎｃｅｓ， Ｐｌｙｍｏｕｔｈ ＭＮ）或いは別の好適なプログラムを用いて、長さが約２２〜３０ヌクレオチド、ＧＣ含有率が約５０％以上で、温度約６８℃〜７２℃で鋳型に対してアニーリングするように、プライマー群を設計し得る。
【０１３７】
完全長ｃＤＮＡ群をスクリーニングする際は、より大きなｃＤＮＡ群を含むようにサイズ選択されたライブラリ群を用いるのが好ましい。更に、ランダムプライマーのライブラリ群は、しばしば遺伝子群の５’領域を有する配列を含み、オリゴｄ（Ｔ）ライブラリが完全長ｃＤＮＡを作製できない状況に対して好適である。ゲノムライブラリ群は、５’非転写調節領域への、配列の伸長に有用であろう。
【０１３８】
市販のキャピラリー電気泳動システム群を用いて、シークエンシングまたはＰＣＲ産物のサイズを分析し、またはそのヌクレオチド配列を確認し得る。具体的には、キャピラリーシークエンシングは、電気泳動による分離のための流動性ポリマーと、４つの異なるヌクレオチドに特異的であるような、レーザで活性化される蛍光色素と、発光された波長の検出に利用するＣＣＤカメラとを有し得る。出力／光の強度は、適切なソフトウェア（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社のＧＥＮＯＴＹＰＥＲ、ＳＥＱＵＥＮＣＥ ＮＡＶＩＧＡＴＯＲなど）を用いて電気信号に変換し得る。サンプルのロードからコンピュータ分析および電子データ表示までの全プロセスがコンピュータ制御可能である。キャピラリー電気泳動法は、特定のサンプルに少量しか存在しないようなＤＮＡ小断片のシークエンシングに特に適している。
【０１３９】
本発明の別の実施態様では、ＳＥＣＰをコードするポリヌクレオチド配列またはその断片を、ＳＥＣＰ、その断片または機能的等価物を適切な宿主細胞内に発現させる組換えＤＮＡ分子にクローニングし得る。遺伝暗号固有の縮重により、実質的に同じあるいは機能的に等価のアミノ酸配列をコードする別のＤＮＡ配列を産生可能であり、これらの配列をＳＥＣＰの発現に利用可能である。
【０１４０】
種々の目的で、ＳＥＣＰをコードする配列群を改変するために、当分野で一般的に既知の複数の方法を用いて、本発明のヌクレオチド配列群を組換えることができる。この組換えの多様な目的には、遺伝子産物のクローン化の、あるいはプロセッシングおよび／または発現のモディフィケーションが含まれるが、これらに限定されるものではない。遺伝子断片と合成オリゴヌクレオチドとの、ランダムなフラグメンテーションおよびＰＣＲ再アセンブリによるＤＮＡシャッフリングを用い、ヌクレオチド配列を組み換え得る。例えば、オリゴヌクレオチドを介した部位特異的変異誘導を利用して、新規な制限部位の作製、グリコシル化パターンの改変、コドン優先の変更、スプライス変異配列の生成などを起こす突然変異を導入し得る。
【０１４１】
本発明のヌクレオチドは、ＭＯＬＥＣＵＬＡＲＢＲＥＥＤＩＮＧ （Ｍａｘｙｇｅｎ Ｉｎｃ．， Ｓａｎｔａ Ｃｌａｒａ ＣＡ； 米国特許第５，８３７，４５８号； Ｃｈａｎｇ， Ｃ．−Ｃ． 他 （１９９９） Ｎａｔ． Ｂｉｏｔｅｃｈｎｏｌ． １７：７９３−７９７； Ｃｈｒｉｓｔｉａｎｓ， Ｆ．Ｃ． 他 （１９９９） Ｎａｔ． Ｂｉｏｔｅｃｈｎｏｌ． １７：２５９−２６４； Ｃｒａｍｅｒｉ， Ａ． 他 （１９９６） Ｎａｔ． Ｂｉｏｔｅｃｈｎｏｌ． １４：３１５−３１９）などのＤＮＡシャッフリング技術の対象となり得る。これにより、生物活性または酵素活性、あるいは他の分子や化合物と結合する能力など、ＳＥＣＰの生物学的特性を改変あるいは改良し得る。ＤＮＡシャッフリングは、ＰＣＲを介する遺伝子断片組換えを用いて遺伝子変異配列のライブラリを作製するプロセスである。ライブラリはその後、所望の特性を持つ遺伝子変異配列群を同定する、選択またはスクリーニングの手順を経る。続いて、これら好適な変異配列をプールし、更に反復してＤＮＡシャッフリングおよび選択／スクリーニングを行い得る。かくして、「人工的な」育種および急速な分子の進化によって、多様な遺伝子が作られる。例えば、ランダムポイント突然変異を持つ単一の遺伝子の断片を組み換えて、スクリーニングし、その後所望の特性が最適化されるまでシャッフリングし得る。あるいは、所定の遺伝子の断片群を同種または異種のいずれかから得た同一遺伝子ファミリーの相同遺伝子の断片と組み換え、それによって天然に存在する複数の遺伝子の遺伝多様性を、方向付けした制御可能な方法で最大化させることができる。
【０１４２】
別の実施態様によれば、ＳＥＣＰをコードする配列は、当分野で周知の化学的方法を用いて、全体あるいは一部が合成可能である（例えば、Ｃａｒｕｔｈｅｒｓ． Ｍ．Ｈ．他（１９８０）Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｓｙｍｐ． Ｓｅｒ． ７：２１５−２２３； およびＨｏｒｎ， Ｔ．他（１９８０）Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｓｙｍｐ． Ｓｅｒ． ７：２２５−２３２を参照）。別法として、化学的方法を用いて、ＳＥＣＰ自体またはその断片を合成し得る。例えば、種々の液相または固相技術を用いてペプチド合成を行い得る（例えばＣｒｅｉｇｈｔｏｎ， Ｔ．（１９８４）Ｐｒｏｔｅｉｎｓ， Ｓｔｒｕｃｔｕｒｅｓ ａｎｄ Ｍｏｌｅｃｕｌａｒ Ｐｒｏｐｅｒｔｉｅｓ， ＷＨ Ｆｒｅｅｍａｎ， Ｎｅｗ Ｙｏｒｋ ＮＹ， ５５−６０ページ、Ｒｏｂｅｒｇｅ， Ｊ．Ｙ．他（１９９５）Ｓｃｉｅｎｃｅ ２６９：２０２−２０４を参照）。自動合成はＡＢＩ ４３１Ａペプチドシンセサイザ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）を用いて達成し得る。更にＳＥＣＰのアミノ酸配列または任意のその一部は、直接的な合成の際の改変により、および／または他のタンパク質群または任意のその一部からの配列群との組み合わせにより、変異配列ポリペプチドを作製、または、天然ポリペプチドの配列を有するポリペプチドを作製することが可能である。
【０１４３】
このペプチドは、分離用高速液体クロマトグラフィーを用いて実質的に精製し得る（例えばＣｈｉｅｚ， Ｒ．Ｍ．およびＦ．Ｚ． Ｒｅｇｎｉｅｒ （１９９０） Ｍｅｔｈｏｄｓ Ｅｎｚｙｍｏｌ． １８２：３９２−４２１を参照）。この合成ペプチドの組成は、アミノ酸分析またはシークエンシングによって確認できる（例えば前出のＣｒｅｉｇｈｔｏｎ， ２８−５３ページを参照）。
【０１４４】
生物学的に活性なＳＥＣＰを発現させるために、ＳＥＣＰをコードするヌクレオチド配列またはその誘導体を好適な発現ベクターに挿入し得る。好適な発現ベクターとは、好適な宿主に挿入されたコーディング配列の転写および翻訳の制御に必要なエレメント群を持つベクターである。必要なエレメントとしては、ベクター内およびＳＥＣＰをコードするポリヌクレオチド配列における調節配列（例えばエンハンサー、構成型および発現誘導型のプロモーター、５’および３’の非翻訳領域など）が含まれる。必要なエレメント群は、特長および特異性が様々である。特定の開始シグナルによって、ＳＥＣＰをコードする配列の、より効果的な翻訳を達成することも可能である。開始シグナルの例には、ＡＴＧ開始コドンと、コザック配列など近傍の配列とが含まれる。ＳＥＣＰをコードする配列、およびその開始コドンや、上流の調節配列が好適な発現ベクターに挿入された場合は、更なる転写制御シグナルや翻訳制御シグナルは必要ないこともある。しかしながら、コーディング配列あるいはその断片のみが挿入された場合は、インフレームＡＴＧ開始コドンなど外来性の翻訳制御シグナルが発現ベクターに含まれるようにすべきである。外来性の翻訳エレメント群および開始コドン群は、様々な天然物および合成物を起源とし得る。用いられる特定の宿主細胞系に好適なエンハンサーを含めることで発現の効率を高めることが可能である（例えばＳｃｈａｒｆ， Ｄ． 他（１９９４）Ｒｅｓｕｌｔｓ Ｐｒｏｂｌ． Ｃｅｌｌ Ｄｉｆｆｅｒ． ２０：１２５−１６２を参照）。
【０１４５】
当業者に周知の方法を用いて、ＳＥＣＰをコードする配列と、好適な転写および翻訳制御エレメントとを持つ発現ベクターを作製し得る。これらの方法としては、ｉｎ ｖｉｔｒｏ組換えＤＮＡ技術、合成技術、およびｉｎ ｖｉｖｏ遺伝子組換え技術がある（例えば、Ｓａｍｂｒｏｏｋ， Ｊ． 他．（１９８９）Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ， Ａ Ｌａｂｏｒａｔｏｒｙ Ｍａｎｕａｌ， Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｐｒｅｓｓ， Ｐｌａｉｎｖｉｅｗ ＮＹ， ４章および８章， および１６−１７章； および Ａｕｓｕｂｅｌ， Ｆ．Ｍ． 他．（１９９５）Ｃｕｒｒｅｎｔ Ｐｒｏｔｏｃｏｌｓ ｉｎ Ｍｏｌｅｃｕｌａｒ Ｂｉｏｌｏｇｙ， Ｊｏｈｎ Ｗｉｌｅｙ ＆ Ｓｏｎｓ， Ｎｅｗ Ｙｏｒｋ ＮＹ， ９章、１３章および１６章を参照）。
【０１４６】
種々の発現ベクター／宿主系を利用して、ＳＥＣＰをコードする配列の保持および発現が可能である。限定するものではないがこのような発現ベクター／宿主系には、組換えバクテリオファージ、プラスミドまたはコスミドＤＮＡ発現ベクターで形質転換させた細菌や、酵母菌発現ベクターで形質転換させた酵母菌など微生物や、ウイルス発現ベクター（例えばバキュロウイルス）に感染した昆虫細胞系や、ウイルス発現ベクター（例えばカリフラワーモザイクウイルス、ＣａＭＶまたはタバコモザイクウイルス、ＴＭＶ）または細菌発現ベクター（例えばＴｉプラスミドまたはｐＢＲ３２２プラスミド）で形質転換させた植物細胞系、動物細胞系がある（例えば前出のＳａｍｂｒｏｏｋ、前出のＡｕｓｕｂｅｌ、Ｖａｎ Ｈｅｅｋｅ， Ｇ．およびＳ．Ｍ． Ｓｃｈｕｓｔｅｒ（１９８９）Ｊ． Ｂｉｏｌ． Ｃｈｅｍ． ２６４：５５０３−５５０９、Ｅｎｇｅｌｈａｒｄ、Ｅ．Ｋ． 他（１９９４）Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ ９１：３２２４−３２２７、Ｓａｎｄｉｇ， Ｖ． 他（１９９６）Ｈｕｍ． Ｇｅｎｅ Ｔｈｅｒ． ７：１９３７−１９４５、Ｔａｋａｍａｔｓｕ， Ｎ．（１９８７）ＥＭＢＯ Ｊ． ６：３０７−３１１、『マグローヒル科学技術年鑑』（Ｔｈｅ ＭｃＧｒａｗ Ｈｉｌｌ Ｙｅａｒｂｏｏｋ ｏｆ Ｓｃｉｅｎｃｅ ａｎｄ Ｔｅｃｈｎｏｌｏｇｙ）（１９９２）ＭｃＧｒａｗ Ｈｉｌｌ Ｎｅｗ Ｙｏｒｋ ＮＹ， １９１−１９６ページ、Ｌｏｇａｎ， Ｊ． ａｎｄ Ｔ． Ｓｈｅｎｋ（１９８４）Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ ８１：３６５５−３６５９、Ｈａｒｒｉｎｇｔｏｎ， Ｊ．Ｊ． 他（１９９７）Ｎａｔ． Ｇｅｎｅｔ． １５：３４５−３５５を参照）。レトロウイルス、アデノウイルス、ヘルペスウイルスまたはワクシニアウイルス由来の発現ベクター、または種々の細菌性プラスミド由来の発現ベクターを用いて、ヌクレオチド配列を標的器官、組織または細胞集団へ送達することができる（例えばＤｉ Ｎｉｃｏｌａ， Ｍ． 他（１９９８）Ｃａｎｃｅｒ Ｇｅｎ． Ｔｈｅｒ． ５（６）：３５０−３５６、Ｙｕ， Ｍ． 他（１９９３）Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ ９０（１３）：６３４０−６３４４、Ｂｕｌｌｅｒ， Ｒ．Ｍ． 他（１９８５）Ｎａｔｕｒｅ ３１７（６０４０）：８１３−８１５； ＭｃＧｒｅｇｏｒ， Ｄ．Ｐ． 他（１９９４）Ｍｏｌ． Ｉｍｍｕｎｏｌ． ３１（３）：２１９−２２６、Ｖｅｒｍａ， Ｉ．Ｍ． ａｎｄ Ｎ． Ｓｏｍｉａ（１９９７）Ｎａｔｕｒｅ ３８９：２３９−２４２を参照）。本発明は使用される宿主細胞によって限定されるものではない。
【０１４７】
細菌系では、多数のクローニングベクターおよび発現ベクターが、ＳＥＣＰをコードするポリヌクレオチド配列の使用目的に応じて選択可能である。例えば、ＳＥＣＰをコードするポリヌクレオチド配列の慣例的なクローニング、サブクローニング、増殖には、ＰＢＬＵＥＳＣＲＩＰＴ（Ｓｔｒａｔａｇｅｎｅ， Ｌａ Ｊｏｌｌａ ＣＡ）またはＰＳＰＯＲＴ１プラスミド（Ｌｉｆｅ Ｔｅｃｈｎｏｌｏｇｉｅｓ）などの多機能の大腸菌ベクターを用いることができる。ＳＥＣＰをコードする配列をこのベクターのマルチクローニング部位にライゲーションするとｌａｃＺ遺伝子が破壊され、組換え分子を持つ形質転換された細菌の同定のための比色スクリーニング法が可能となる。更にこれらのベクターは、クローニングされた配列におけるｉｎ ｖｉｔｒｏ転写、ジデオキシのシークエンシング、ヘルパーファージによる一本鎖のレスキュー、入れ子状態の欠失の生成にも有用であろう（例えば、Ｖａｎ Ｈｅｅｋｅ， Ｇ．およびＳ．Ｍ． Ｓｃｈｕｓｔｅｒ（１９８９）Ｊ． Ｂｉｏｌ． Ｃｈｅｍ． ２６４：５５０３５５０９を参照）。例えば抗体の産生のためなどに多量のＳＥＣＰが必要な場合は、ＳＥＣＰの発現をハイレベルで誘導するベクターが使用できる。例えば、強力な誘導ＳＰ６バクテリオファージプロモーターまたは誘導Ｔ７バクテリオファージプロモーターを持つベクターが使用できる。
【０１４８】
酵母の発現系を使用してＳＥＣＰを産出することもできる。α因子、アルコールオキシダーゼ、ＰＧＨプロモーターなど、構成型あるいは誘導型のプロモーターを持つ多数のベクターが、出芽酵母菌（Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ）またはピキア酵母（Ｐｉｃｈｉａ ｐａｓｔｏｒｉｓ）内で使用可能である。更に、このようなベクターは、発現したタンパク質の分泌か細胞内での保持かのどちらかを指示し、安定した増殖のために宿主ゲノムの中に外来配列を組み込むことを可能にする（例えば、Ａｕｓｕｂｅｌ， １９９５，前出、Ｂｉｔｔｅｒ， Ｇ．Ａ． 他（１９８７）Ｍｅｔｈｏｄｓ Ｅｎｚｙｍｏｌ．１５３：５１６−５４４、およびＳｃｏｒｅｒ． Ｃ． Ａ． 他（１９９４）Ｂｉｏ／Ｔｅｃｈｎｏｌｏｇｙ １２：１８１−１８４を参照）。
【０１４９】
植物系を使用してＳＥＣＰを発現することも可能である。ＳＥＣＰをコードする配列の転写は、ウイルスプロモーター、例えば単独であるいはＴＭＶ由来のオメガリーダー配列と組み合わせて用いられるようなＣａＭＶ由来の３５Ｓおよび１９Ｓプロモーターによって促進し得る（Ｔａｋａｍａｔｓｕ， Ｎ．（１９８７）ＥＭＢＯ Ｊ ６：３０７−３１１）。あるいは、ＲｕＢｉｓＣＯの小サブユニットなどの植物プロモーター、または熱ショックプロモーターを用い得る（例えば、Ｃｏｒｕｚｚｉ， Ｇ． 他．（１９８４）ＥＭＢＯ Ｊ． ３：１６７１−１６８０； Ｂｒｏｇｌｉｅ， Ｒ． 他（１９８４）Ｓｃｉｅｎｃｅ ２２４：８３８−８４３； および Ｗｉｎｔｅｒ， Ｊ． 他（１９９１）Ｒｅｓｕｌｔｓ Ｐｒｏｂｌ． Ｃｅｌｌ Ｄｉｆｆｅｒ． １７：８５−１０５を参照）。これらの構成物は、直接ＤＮＡ形質転換により、または病原体を媒介とする形質移入により、植物細胞内に導入し得る（例えば『マグローヒル科学技術年鑑』（Ｔｈｅ ＭｃＧｒａｗ Ｈｉｌｌ Ｙｅａｒｂｏｏｋ ｏｆ Ｓｃｉｅｎｃｅ ａｎｄ Ｔｅｃｈｎｏｌｏｇｙ） （１９９２） ＭｃＧｒａｗ Ｈｉｌｌ Ｎｅｗ Ｙｏｒｋ ＮＹ，１９１−１９６ページを参照）。
【０１５０】
哺乳類細胞においては、多数のウイルスベースの発現系を利用し得る。アデノウイルスを発現ベクターとして用いる場合、ＳＥＣＰをコードする配列を、後期プロモーターと３連リーダー配列とからなるアデノウイルス転写／翻訳複合体に連結し得る。アデノウイルスゲノムの非必須のＥ１またはＥ３領域へ挿入することにより、宿主細胞内でＳＥＣＰを発現する感染ウイルスを得ることができる（例えばＬｏｇａｎ， Ｊ．およびＴ． Ｓｈｅｎｋ（１９８４）Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ ８１：３６５５３６５９を参照）。更に、ラウス肉腫ウイルス（ＲＳＶ）エンハンサーなどの転写エンハンサーを用いて、哺乳動物宿主細胞における発現を増大させ得る。ＳＶ４０またはＥＢＶをベースにしたベクターを用いてタンパク質を高レベルで発現させることもできる。
【０１５１】
また、ヒト人工染色体（ＨＡＣ）類を用いて、或るプラスミドに含まれ得る断片やプラスミドから発現し得る断片より大きなＤＮＡの断片群を送達し得る。治療のために約６ｋｂ〜１０ＭｂのＨＡＣｓが作製され、従来の送達方法（リポソーム、ポリカチオンアミノポリマー、またはベシクル）で送達されている（例えばＨａｒｒｉｎｇｔｏｎ． Ｊ．Ｊ． 他（１９９７）Ｎａｔ Ｇｅｎｅｔ． １５：３４５−３５５を参照）。
【０１５２】
哺乳類系の組換えタンパク質の長期にわたる産生のためには、細胞株におけるＳＥＣＰの安定した発現が望ましい。例えば、ＳＥＣＰをコードする配列を細胞株に形質転換するために、発現ベクター類と、同じ或いは別のベクター上の選択可能マーカー遺伝子とを用い得る。用いる発現ベクターは、複製のウイルス起源、及び／または内在性の発現エレメントを持ち得る。ベクターの導入後、選択培地に移す前に強化培地で約１〜２日間、細胞を増殖させ得る。選択可能マーカーの目的は選択剤への抵抗性を与えることであり、選択可能マーカーの存在により、導入した配列をうまく発現するような細胞の成長および回収が可能となる。安定的に形質転換された細胞の耐性クローンは、その細胞型に適した組織培養技術を用いて増殖可能である。
【０１５３】
任意の数の選択系を用いて、形質転換細胞株を回収できる。限定するものではないがこのような選択系には、ｔｋ⁻細胞のために用いられる単純ヘルペスウイルスのチミジンキナーゼ遺伝子と、ａｐｒ⁻細胞のために用いられる単純ヘルペスウイルスのアデニンホスホリボシルトランスフェラーゼ遺伝子がある（例えばＷｉｇｌｅｒ， Ｍ．他 （１９７７） Ｃｅｌｌ １１：２２３−２３２、Ｌｏｗｙ， Ｉ．他（１９８０） Ｃｅｌｌ ２２：８１７−８２３を参照）。また、選択の基礎として代謝拮抗物質、抗生物質あるいは除草剤への耐性を用いることができる。例えばｄｈｆｒはメトトレキセートに対する耐性を与え、ｎｅｏはアミノグリコシッドネオマイシンおよびＧ−４１８に対する耐性を与え、ａｌｓはクロルスルフロンに対する耐性を、ｐａｔはホスフィノトリシンアセチルトランスフェラーゼに対する耐性を各々与える（例えばＷｉｇｌｅｒ， Ｍ．他 （１９８０） Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ ７７：３５６７−３５７０、Ｃｏｌｂｅｒｅ−Ｇａｒａｐｉｎ， Ｆ． 他 （１９８１） Ｊ． Ｍｏｌ． Ｂｉｏｌ． １５０：１−１４を参照）。この他の選択可能な遺伝子、例えば、代謝のための、細胞の必要条件を改変するｔｒｐＢおよびｈｉｓＤも、文献に記載がある（例えばＨａｒｔｍａｎ， Ｓ．Ｃ．およびＲ．Ｃ． Ｍｕｌｌｉｇａｎ （１９８８） Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ ８５：８０４７８０５１を参照）。可視マーカー類、例えばアントシアニンや、緑色蛍光タンパク質（ＧＦＰ；Ｃｌｏｎｔｅｃｈ）、βグルクロニダーゼおよびその基質であるβグルクロニド、またはルシフェラーゼおよびその基質であるルシフェリンを用い得る。これらのマーカーを用いて、トランスフォーマントを同定するだけでなく、特定のベクター系に起因する一過性あるいは安定したタンパク質発現を定量し得る（例えばＲｈｏｄｅｓ， Ｃ．Ａ． （１９９５） Ｍｅｔｈｏｄｓ Ｍｏｌ． Ｂｉｏｌ． ５５：１２１１３１を参照）。
【０１５４】
マーカー遺伝子発現の有無によって目的の遺伝子の存在が示唆されても、その遺伝子の存在および発現の確認が必要な場合もある。例えば、ＳＥＣＰをコードする配列がマーカー遺伝子配列の中に挿入された場合、ＳＥＣＰをコードする配列を持つ形質転換された細胞は、マーカー遺伝子機能の欠落により同定可能である。または、或るマーカー遺伝子を、ＳＥＣＰをコードする配列とタンデムに、１つのプロモーターの制御下に配置し得る。誘導または選択に応答したマーカー遺伝子の発現は通常、タンデム遺伝子の発現も示す。
【０１５５】
一般に、ＳＥＣＰをコードする核酸配列を持ち、ＳＥＣＰを発現する宿主細胞は、当業者に周知の種々の方法を用いて同定し得る。これらの方法には、ＤＮＡ−ＤＮＡあるいはＤＮＡ−ＲＮＡハイブリダイゼーションや、ＰＣＲ法、核酸配列あるいはタンパク質配列の検出および／または数量化のための膜系、溶液ベース、あるいはチップベースの技術を含むタンパク質生物学的試験法または免疫学的アッセイが含まれるが、これらに限定されるものではない。
【０１５６】
特異的ポリクローナル抗体または特異的モノクローナル抗体を用いてＳＥＣＰの発現の検出および計測を行うための免疫学的方法は、当分野で公知である。このような技術の例としては、酵素に結合した免疫吸着剤検定法（ＥＬＩＳＡ）、ラジオイムノアッセイ（ＲＩＡ）、蛍光活性化セルソーティング（ＦＡＣＳ、フローサイトメトリー）などが挙げられる。ＳＥＣＰ上の２つの非干渉エピトープに反応するモノクローナル抗体を用いた、２部位のモノクローナルベースイムノアッセイ（ｔｗｏ−ｓｉｔｅ， ｍｏｎｏｃｌｏｎａｌ−ｂａｓｅｄ ｉｍｍｕｎｏａｓｓａｙ）が好ましいが、競合結合試験を用いることもできる。これらのアッセイおよびこれ以外のアッセイは、当分野で公知である（例えばＨａｍｐｔｏｎ， Ｒ． 他（１９９０）Ｓｅｒｏｌｏｇｉｃａｌ Ｍｅｔｈｏｄｓ， ａ Ｌａｂｏｒａｔｏｒｙ Ｍａｎｕａｌ， ＡＰＳ Ｐｒｅｓｓ． Ｓｔ． Ｐａｕｌ． ＭＮ， Ｓｅｃｔ． ＩＶ、Ｃｏｌｉｇａｎ， Ｊ． Ｅ． 他（１９９７）Ｃｕｒｒｅｎｔ Ｐｒｏｔｏｃｏｌｓ ｉｎ Ｉｍｍｕｎｏｌｏｇｙ， Ｇｒｅｅｎｅ Ｐｕｂ． Ａｓｓｏｃｉａｔｅｓ ａｎｄ Ｗｉｌｅｙ−Ｉｎｔｅｒｓｃｉｅｎｃｅ， Ｎｅｗ Ｙｏｒｋ ＮＹ、Ｐｏｕｎｄ， Ｊ．Ｄ．（１９９８）Ｉｍｍｕｎｏｃｈｅｍｉｃａｌ Ｐｒｏｔｏｃｏｌｓ， Ｈｕｍａｎａ Ｐｒｅｓｓ， Ｔｏｔｏｗａ ＮＪを参照）。
【０１５７】
多岐にわたる標識方法および抱合方法が当業者に知られており、様々な核酸アッセイおよびアミノ酸アッセイにこれらの方法を用い得る。 ＳＥＣＰをコードするポリヌクレオチドに関連する配列を検出するための、標識されたハイブリダイゼーションプローブあるいはＰＣＲプローブを生成する方法には、オリゴ標識化、ニックトランスレーション、末端標識化（エンドラベリング）、または標識されたヌクレオチドを用いるＰＣＲ増幅が含まれる。別法として、ＳＥＣＰをコードする配列、またはその任意の断片を、ｍＲＮＡプローブを生成するためのベクターにクローニングすることも可能である。このようなベクターは、当分野において知られており、市販もされており、Ｔ７、Ｔ３またはＳＰ６などの好適なＲＮＡポリメラーゼおよび標識されたヌクレオチドを加えて、ｉｎ ｖｉｔｒｏでＲＮＡプローブの合成に用いることができる。このような方法は、例えばＡｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ、Ｐｒｏｍｅｇａ（Ｍａｄｉｓｏｎ ＷＩ）、Ｕ．Ｓ． Ｂｉｏｃｈｅｍｉｃａｌなどから市販されている種々のキットを用いて実行することができる。検出を容易にするために用い得る好適なレポーター分子あるいは標識には、基質、補助因子、インヒビター、磁気粒子のほか、放射性核種、酵素、蛍光剤、化学発光剤、発色剤などがある。
【０１５８】
ＳＥＣＰをコードするヌクレオチド配列で形質転換された宿主細胞は、細胞培地でのこのタンパク質の発現および回収に好適な条件下で培養される。形質転換細胞から製造されたタンパク質が分泌されるか細胞内に留まるかは、使用される配列、ベクター、あるいはその両者に依存する。ＳＥＣＰをコードするポリヌクレオチドを持つ発現ベクターは、原核細胞膜または真核細胞膜を透過するＳＥＣＰの分泌を誘導するシグナル配列を含むように設計できることは、当業者には理解されよう。
【０１５９】
更に、宿主細胞株の選択は、挿入した配列の発現をモジュレートする能力、または発現したタンパク質を所望の形に処理する能力によって行い得る。限定するものではないがこのようなポリペプチドの修飾には、アセチル化、カルボキシル化、グリコシル化、リン酸化、脂質化およびアシル化がある。タンパク質の「プレプロ」または「プロ」形を切断する翻訳後のプロセシングを利用して、タンパク質ターゲッティング、折りたたみ、および／または活性を特定することが可能である。翻訳後の活性のための、特定の細胞装置および特徴的な機序を持つ、種々の宿主細胞（例えばＣＨＯ、ＨｅＬａ、ＭＤＣＫ、ＭＥＫ２９３、ＷＩ３８など）は、Ａｍｅｒｉｃａｎ Ｔｙｐｅ Ｃｕｌｔｕｒｅ Ｃｏｌｌｅｃｔｉｏｎ（ＡＴＣＣ， Ｍａｎａｓｓａｓ， ＶＡ）から入手可能であり、外来タンパク質の正しい修飾および処理を確実にするように選択し得る。
【０１６０】
本発明の別の実施態様では、ＳＥＣＰをコードする、自然のあるいは修飾された、または組換えの核酸配列を、或る異種配列に結合させることにより、上記した任意の宿主系内で、１融合タンパク質の翻訳を生じ得る。例えば、市販の抗体によって認識できる異種部分を含むキメラＳＥＣＰタンパク質は、ＳＥＣＰの活性のインヒビターに対するペプチドライブラリのスクリーニングを促進し得る。また、異種タンパク質部分および異種ペプチド部分も、市販されている親和性マトリックスを用いて融合タンパク質の精製を促進し得る。限定されるものではないがこのような部分には、グルタチオンＳトランスフェラーゼ（ＧＳＴ）、マルトース結合タンパク質（ＭＢＰ）、チオレドキシン（Ｔｒｘ）、カルモジュリン結合ペプチド（ＣＢＰ）、６−Ｈｉｓ、ＦＬＡＧ、ｃ−ｍｙｃ、赤血球凝集素（ＨＡ）がある。ＧＳＴは固定化グルタチオン上で、ＭＢＰはマルトース上で、Ｔｒｘはフェニルアルシンオキシド上で、ＣＢＰはカルモジュリン上で、そして６−Ｈｉｓは金属キレート樹脂上で、同族の融合タンパク質の精製を可能にする。ＦＬＡＧ、ｃ−ｍｙｃおよび赤血球凝集素（ＨＡ）は、これらのエピトープ標識を特異的に認識する市販されているモノクローナル抗体およびポリクローナル抗体を用いた、融合タンパク質の免疫親和性精製を可能にする。また、融合タンパク質が、ＳＥＣＰをコードする配列と異種タンパク質配列との間にタンパク質分解切断部位を持つように遺伝子操作すると、精製後にＳＥＣＰが異種部分から切断され得る。融合タンパク質の発現および精製方法は、前出のＡｕｓｕｂｅｌ（１９９５）１０章に記載されている。市販されている種々のキットを用いて融合タンパク質の発現および精製を促進することもできる。
【０１６１】
本発明の別の実施例では、ＴＮＴウサギ網状赤血球可溶化液またはコムギ胚芽抽出系（Ｐｒｏｍｅｇａ）を用いてｉｎ ｖｉｔｒｏで、放射能標識したＳＥＣＰの合成が可能である。これらの系は、Ｔ７、Ｔ３またはＳＰ６プロモーターと機能的に連結したタンパク質コード配列の、転写と翻訳とを結合する。翻訳は、例えば^３５Ｓメチオニンのような放射能標識したアミノ酸前駆体の存在下で起こる。
【０１６２】
本発明のＳＥＣＰまたはその断片を用いて、ＳＥＣＰに特異結合する化合物をスクリーニングすることができる。少なくとも１つまたは複数の試験化合物を用いて、ＳＥＣＰへの特異的な結合をスクリーニングすることが可能である。試験化合物としては、抗体、オリゴヌクレオチド、タンパク質（例えば受容体）または小分子が挙げられる。
【０１６３】
或る実施態様では、このように同定された化合物は、ＳＥＣＰの天然リガンドに密接に関連し、例えばリガンドやその断片であり、または天然基質や、構造的または機能的な擬態物質（ｍｉｍｅｔｉｃ）あるいは自然結合パートナーである（例えばＣｏｌｉｇａｎ， Ｊ．Ｅ． 他（１９９１）Ｃｕｒｒｅｎｔ Ｐｒｏｔｏｃｏｌｓ ｉｎ Ｉｍｍｕｎｏｌｏｇｙ １（２）の５章を参照）。同様に、この化合物は、ＳＥＣＰが結合する天然受容体に、あるいは例えばリガンド結合部位など少なくともこの受容体の或る断片に密接に関連し得る。何れの場合も、既知の技術を用いてこの化合物を合理的に設計することができる。或る実施例では、これら化合物のためのスクリーニングには、分泌タンパク質として、あるいは細胞膜上でＳＥＣＰを発現する、好適な細胞群の作製が含まれる。好適な細胞には、哺乳類、酵母、ショウジョウバエ、または大腸菌からの細胞が含まれる。ＳＥＣＰを発現する細胞、またはＳＥＣＰを含有する細胞膜分画を試験化合物と接触させて、ＳＥＣＰまたは化合物のどちらかの結合、刺激、または活性の阻害を分析する。
【０１６４】
或るアッセイは、単に試験化合物をポリペプチドに実験的に結合させ、蛍光色素、放射性同位体、酵素抱合体またはその他の検出可能な標識によりその結合を検出することができる。例えば、このアッセイは、少なくとも１つの試験化合物を、溶液中の、あるいは固体支持物に固定されたＳＥＣＰと混合するステップと、ＳＥＣＰとこの化合物との結合を検出するステップを含み得る。別法では、標識された競合物の存在下での試験化合物の結合の検出および測定を行うことができる。更にこのアッセイは、無細胞再構成標本、化学ライブラリ、または、天然産物の混合物を用いて実施でき、試験化合物（群）は、溶液中で遊離させるか固体支持体に固定する。
【０１６５】
本発明のＳＥＣＰまたはその断片を用いて、ＳＥＣＰの活性をモジュレートする化合物をスクリーニングすることが可能である。このような化合物には、アゴニスト、アンタゴニスト、或るいは部分的または逆アゴニストなどが含まれる。或る実施例では、ＳＥＣＰが少なくとも１つの試験化合物と結合する、ＳＥＣＰの活性が許容される条件下でアッセイを実施し、試験化合物の存在下でのＳＥＣＰの活性と、試験化合物不在下でのＳＥＣＰの活性とを比較する。試験化合物の存在下でのＳＥＣＰの活性の変化は、ＳＥＣＰの活性をモジュレートする化合物の存在を示唆する。 別法では、試験化合物を、ＳＥＣＰの活性に適した条件下で、ＳＥＣＰを含むｉｎ ｖｉｔｒｏまたは細胞遊離系と結合させてアッセイを実施する。これらアッセイのいずれにおいても、ＳＥＣＰの活性をモジュレートする試験化合物は間接的にモジュレートする場合があり、試験化合物と直接接触する必要がない場合がある。少なくとも１つ、または複数の試験化合物をスクリーニングすることができる。
【０１６６】
別の実施態様では、胚性幹細胞（ＥＳ細胞）における相同組換えを用いて動物モデル系内で、ＳＥＣＰまたはその哺乳類相同体をコードするポリヌクレオチドを「ノックアウト」する。このような技術は当技術分野において周知であり、ヒト疾患動物モデルの作製に有用である（米国特許第５，１７５，３８３号および第５，７６７，３３７号などを参照）。例えば１２９／ＳｖＪ細胞系などのマウスＥＳ細胞は初期のマウス胚に由来し、培地で増殖させることができる。このＥＳ細胞は、ネオマイシンホスホトランスフェラーゼ遺伝子（ｎｅｏ： Ｃａｐｅｃｃｈｉ， Ｍ．Ｒ．（１９８９）Ｓｃｉｅｎｃｅ ２４４：１２８８−１２９２）などのマーカー遺伝子で破壊した、目的の遺伝子を含むベクターで形質転換する。このベクターは、相同組換えにより、宿主ゲノムの対応する領域に組み込まれる。別法では、Ｃｒｅ−ｌｏｘＰ系を用いて相同組換えを行い、組織特異的または発生段階特異的に目的遺伝子をノックアウトする（Ｍａｒｔｈ， Ｊ．Ｄ． （１９９６） Ｃｌｉｎ． Ｉｎｖｅｓｔ． ９７：１９９９−２００２； Ｗａｇｎｅｒ， Ｋ．Ｕ． 他 （１９９７） Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｒｅｓ． ２５：４３２３−４３３０）。形質転換したＥＳ細胞を同定し、例えばＣ５７ＢＬ／６マウス株などから採取したマウス細胞胚盤胞に微量注入する。これらの胚盤胞を偽妊娠メスに外科的に導入し、得られるキメラ子孫の遺伝形質を特定し、これらを交配させてヘテロ接合性系またはホモ接合性系を作製する。このようにして作製した遺伝子組換え動物は、潜在的治療薬や毒性薬剤で検査することができる。
【０１６７】
また、ＳＥＣＰをコードするポリヌクレオチド類を、ｉｎ ｖｉｔｒｏで、ヒト胚盤胞由来のＥＳ細胞において操作し得る。ヒトＥＳ細胞は、内胚葉、中胚葉および外胚葉の細胞タイプを含む、少なくとも８つの別々の細胞系統に分化する可能性を有する。これらの細胞系統は、例えば神経細胞、造血系統および心筋細胞に分化する（Ｔｈｏｍｓｏｎ， Ｊ．Ａ． 他 （１９９８） Ｓｃｉｅｎｃｅ ２８２：１１４５−１１４７）。
【０１６８】
ＳＥＣＰをコードするポリヌクレオチドを用いて、ヒト疾患をモデルとした「ノックイン」ヒト化動物（ブタ）または遺伝子組換え動物（マウスまたはラット）を作製することもできる。ノックイン技術を用いて、ＳＥＣＰをコードするポリヌクレオチドの或る領域を動物ＥＳ細胞に注入し、注入した配列を動物細胞ゲノムに組み込ませる。形質転換細胞を胞胚に注入し、胞胚を上記のように移植する。遺伝子組換え子孫または近交系について試験し、潜在的医薬品を用いて処理し、ヒトの疾患の治療に関する情報を得る。別法では、例えばＳＥＣＰを乳汁内に分泌するなどＳＥＣＰを過剰に発現する哺乳類近交系は、便利なタンパク質源となり得る（Ｊａｎｎｅ， Ｊ． 他（１９９８）Ｂｉｏｔｅｃｈｎｏｌ． Ａｎｎｕ． Ｒｅｖ． ４：５５−７４）。
【０１６９】
（治療）
ＳＥＣＰのいくつかの領域と分泌タンパク質群との間には、例えば配列およびモチーフの文脈における、化学的および構造的類似性が存在する。更に、ＳＥＣＰの発現は、健常な、または腫瘍性の、肺、心臓、脳、皮膚、結腸上皮、および心血管の各組織に密接に関連し、また、神経組織、泌尿組織、生殖組織、消化系組織、免疫組織、罹患組織、および腫瘍組織に密接に関連する。従って、ＳＥＣＰは、細胞増殖異常、自己免疫／炎症性疾患、心血管障害、神経障害、および発達障害において、或る役割を果たすと考えられる。ＳＥＣＰの発現若しくは活性の増大に関連する疾患の治療においては、ＳＥＣＰの発現または活性を低下させることが望ましい。また、ＳＥＣＰの発現または活性の低下に関連する疾患の治療においては、ＳＥＣＰの発現または活性を増大させることが望ましい。
【０１７０】
したがって、一実施態様において、ＳＥＣＰの発現または活性の低下に関連した疾患の治療または予防のために、患者にＳＥＣＰまたはその断片や誘導体を投与することが可能である。限定するものではないが、このような疾患には細胞増殖異常、自己免疫／炎症疾患、心血管障害、神経障害、および発達障害が含まれ、細胞増殖異常の中には日光角化症、動脈硬化、アテローム性動脈硬化、滑液包炎、硬変、肝炎、混合性結合組織病（ＭＣＴＤ）、骨髄線維症、発作性夜間ヘモグロビン尿症、真性多血症、乾癬、原発性血小板血症、並びに腺癌および白血病、リンパ腫、黒色腫、骨髄腫、肉腫、および奇形癌、具体的には、副腎、膀胱、骨、骨髄、脳、乳房、頚部、胆嚢、神経節、消化管、心臓、腎臓、肝臓、肺、筋肉、卵巣、膵臓、副甲状腺、陰茎、前立腺、唾液腺、皮膚、脾臓、精巣、胸腺、甲状腺、および子宮の癌が含まれ、自己免疫／炎症性の疾患の中には、後天性免疫不全症候群（ＡＩＤＳ）、アジソン病（慢性原発性副腎機能不全）、成人呼吸窮迫症候群、アレルギー、強直性脊椎炎、アミロイド症、貧血、喘息、アテローム性動脈硬化、自己免疫性溶血性貧血、自己免疫性甲状腺炎、自己免疫性多腺性内分泌カンジダ性外胚葉ジストロフィー（ＡＰＥＣＥＤ）、気管支炎、胆嚢炎、接触皮膚炎、クローン病、アトピー性皮膚炎、皮膚筋炎、糖尿病、肺気腫、リンパ球傷害因子性偶発性リンパ球減少症、新生児溶血性疾患（胎児赤芽球症）、結節性紅斑、萎縮性胃炎、糸球体腎炎、グッドパスチャー症候群、痛風、グレーブス病、橋本甲状腺炎、過好酸球増加症、過敏性腸症候群、多発性硬化症、重症筋無力症、心筋または心膜炎症、骨関節炎、骨粗しょう症、膵炎、多発性筋炎、乾癬、ライター症候群、リウマチ様関節炎、強皮症、シェーグレン症候群、全身性アナフィラキシー、全身性エリテマトーデス、全身性硬化症、血小板減少性紫斑病、潰瘍性大腸炎、ぶどう膜炎、ウェルナー症候群、癌合併症、血液透析、体外循環、ウイルス感染症、細菌感染症、真菌感染症、寄生虫感染症、原虫感染症、蠕虫感染症、外傷が含まれ、心血管障害の中には、鬱血性心不全、虚血性心疾患、狭心症、心筋梗塞、高血圧性心疾患、変性弁膜性心疾患、石灰化大動脈弁狭窄症、先天性２尖大動脈弁、僧帽弁輪部石灰化（ｍｉｔｒａｌ ａｎｎｕｌａｒ ｃａｌｃｉｆｉｃａｔｉｏｎ）、僧帽弁逸脱、リウマチ熱、リウマチ性心疾患、感染性心内膜炎、非細菌性血栓性心内膜炎、全身性エリテマトーデスの心内膜炎、カルチノイド心疾患、心筋症、心筋炎、心膜炎、腫瘍性心疾患、先天性心臓疾患、心移植の合併症、動静脈瘻、アテローム硬化、高血圧、脈管炎、レイノー病、動脈瘤、動脈解離、静脈瘤、血栓静脈炎および静脈血栓、血管系腫瘍、血栓溶解の合併症、バルーン血管形成術、血管置換術（ｖａｓｃｕｌａｒ ｒｅｐｌａｃｅｍｅｎｔ）、冠動脈バイパス移植術が含まれ、神経の疾患の中には、癲癇、虚血性脳血管障害、脳卒中、脳腫瘍、アルツハイマー病、ピック病、ハンチントン病、痴呆、パーキンソン病およびその他の錐体外路障害、筋萎縮性側索硬化およびその他の運動ニューロン障害、進行性神経性筋萎縮症、網膜色素変性症（色素性網膜炎）、遺伝性運動失調、多発性硬化症および他の脱髄疾患、細菌性およびウイルス性髄膜炎、脳膿瘍、硬膜下膿瘍、硬膜外膿瘍、化膿性頭蓋内血栓性静脈炎、脊髄炎および神経根炎、ウイルス性中枢神経系疾患と、プリオン病（クールー、クロイツフェルト‐ヤコブ病、およびＧｅｒｓｔｍａｎｎ−Ｓｔｒａｕｓｓｌｅｒ−Ｓｃｈｅｉｎｋｅｒ症候群を含む）、致死性家族性不眠症、神経系性栄養病および代謝病、神経線維腫症、結節硬化症、小脳網膜血管腫症（ｃｅｒｅｂｅｌｌｏｒｅｔｉｎａｌ ｈｅｍａｎｇｉｏｂｌａｓｔｏｍａｔｏｓｉｓ）、脳３叉神経血管症候群、ダウン症を含む中枢神経系性精神遅滞および他の発達障害、脳性麻痺、神経骨格異常症、自律神経系障害、脳神経障害、脊髄疾患、筋ジストロフィー他の神経筋障害、末梢神経疾患、皮膚筋炎および多発性筋炎と、遺伝性、代謝性、内分泌性、および中毒性ミオパシーと、重症筋無力症、周期性四肢麻痺、精神障害（気分性、不安性の障害、統合失調症／分裂病）、季節性感情障害（ＳＡＤ）と、静座不能、健忘症、緊張病、糖尿病性ニューロパシー、遅発性ジスキネジア、ジストニー、パラノイド精神病、帯状疱疹後神経痛、およびトゥーレット病と、進行性核上麻痺、大脳皮質基底核変性（ｃｏｒｔｉｃｏｂａｓａｌ ｄｅｇｅｎｅｒａｔｉｏｎ）、および家族性前頭側頭型痴呆とが含まれ、また発達障害も含まれ、その中には尿細管性アシドーシス、貧血、クッシング症候群、軟骨形成不全性小人症、デュシェンヌ／ベッカー型筋ジストロフィー、癲癇、性腺形成異常、ＷＡＧＲ症候群（ウィルムス腫瘍、無虹彩症、尿生殖器異常、精神遅滞）、スミス‐マジェニス症候群（Ｓｍｉｔｈ− Ｍａｇｅｎｉｓ ｓｙｎｄｒｏｍｅ）、骨髄異形成症候群、遺伝性粘膜上皮異形成、遺伝性角皮症、シャルコーマリーツース病および神経線維腫症などの遺伝性神経病、甲状腺機能低下症、水頭症、Ｓｙｎｄｅｎｈａｍ舞踏病（Ｓｙｎｄｅｎｈａｍ’ｓ ｃｈｏｒｅａ）および脳性小児麻痺などの発作障害、二分脊椎、無脳症、頭蓋脊椎披裂、先天性緑内障、白内障、感音難聴が含まれる。
【０１７１】
別の実施態様では、限定するものではないが上記した疾患を含む、ＳＥＣＰの発現または活性の低下に関連した疾患の治療または予防のために、ＳＥＣＰまたはその断片や誘導体を発現し得るベクターを患者に投与することも可能である。
【０１７２】
更に別の実施態様では、限定するものではないが上記した疾患を含む、ＳＥＣＰの発現または活性の低下に関連した疾患の治療または予防のために、実質的に精製されたＳＥＣＰを含む組成物を好適な医薬用キャリアと共に患者に投与することも可能である。
【０１７３】
更に別の実施態様では、限定するものではないが上に列記した疾患を含む、ＳＥＣＰの発現または活性の低下に関連した疾患の治療または予防のために、ＳＥＣＰの活性をモジュレートするアゴニストを患者に投与することも可能である。
【０１７４】
更なる実施例では、ＳＥＣＰの発現または活性の増大に関連した疾患の治療または予防のために、患者にＳＥＣＰのアンタゴニストを投与することが可能である。限定するものではないが、このような疾患としては、上記した細胞増殖異常、自己免疫／炎症疾患、心血管障害、神経障害、および発達障害が含まれる。一実施態様では、ＳＥＣＰと特異的に結合する抗体を、直接にアンタゴニストとして、あるいはＳＥＣＰを発現する細胞または組織に薬剤を運ぶターゲッティングあるいは送達機構として間接的に用い得る。
【０１７５】
別の実施態様では、ＳＥＣＰをコードするポリヌクレオチドの相補配列を発現するベクターを患者に投与して、限定するものではないが上記した疾患を含む、ＳＥＣＰの発現または活性の増大に関連した疾患の治療または予防も可能である。
【０１７６】
別の実施態様では、本発明の任意のタンパク質、アンタゴニスト、抗体、アゴニスト、相補配列、またはベクターを、別の好適な治療薬と組み合わせて投与することもできる。併用療法で用いる好適な治療薬は、当業者が従来の医薬原理に従って選択し得る。治療薬と組み合わせることにより、上記した種々の疾患の治療または予防に相乗効果をもたらし得る。この方法により、少量の各薬剤で医薬効果をあげることが可能となり、それによって副作用の可能性を低減し得る。
【０１７７】
ＳＥＣＰのアンタゴニストは、当分野で一般的な方法を用いて製造し得る。詳しくは、精製されたＳＥＣＰを用いて抗体を作ったり、治療薬のライブラリをスクリーニングしてＳＥＣＰと特異的に結合するものの同定が可能である。ＳＥＣＰへの抗体群も、当分野で一般的な複数の方法を用いて製造することが可能である。このような抗体の例として、ポリクローナル抗体、モノクローナル抗体、キメラ抗体、一本鎖抗体、Ｆａｂ断片、およびＦａｂ発現ライブラリによって作られた断片が挙げられる。但し、これらに限定されるものではない。中和抗体（すなわち二量体の形成を阻害する抗体）は通常、治療用に好適である。
【０１７８】
抗体の産生のためには、ヤギ、ウサギ、ラット、マウス、ヒトなどを含む種々の宿主が、ＳＥＣＰ、若しくは免疫原性の特性を備えるその任意の断片またはオリゴペプチドの注入によって免疫化され得る。宿主の種に応じて、種々のアジュバントを用いて免疫応答を高めることもできる。限定するものではないがこのようなアジュバントには、フロイントアジュバントと、水酸化アルミニウムなどのミネラルゲルアジュバントと、リゾレシチン、プルロニックポリオル、ポリアニオン、ペプチド、油性乳剤、スカシガイのヘモシアニン（ＫＬＨ）、ジニトロフェノールなどの界面活性剤とがある。ヒトに用いられるアジュバントの中では、ＢＣＧ（カルメット‐ゲラン杆菌）およびコリネバクテリウム‐パルヴム（Ｃｏｒｙｎｅｂａｃｔｅｒｉｕｍ ｐａｒｖｕｍ）が特に好ましい。
【０１７９】
ＳＥＣＰに対する抗体を誘導するために用いるオリゴペプチド、ペプチド、または断片は、少なくとも約５個のアミノ酸からなるアミノ酸配列を持つものが好ましく、一般的には約１０個以上のアミノ酸からなるものとなる。これらのオリゴペプチド、ペプチドまたは断片は、天然タンパク質のアミノ酸配列の一部と同一であることが望ましい。ＳＥＣＰアミノ酸の短いストレッチは、ＫＬＨなど別のタンパク質の配列と融合し得、キメラ分子に対する抗体が産生され得る。
【０１８０】
ＳＥＣＰに対するモノクローナル抗体は、培地内の連続した細胞株によって抗体分子を産生する、任意の技術を用いて作製することが可能である。限定するものではないがこのような技術には、ハイブリドーマ技術、ヒトＢ細胞ハイブリドーマ技術、およびＥＢＶ−ハイブリドーマ技術がある（例えばＫｏｈｌｅｒ， Ｇ． 他．（１９７５）Ｎａｔｕｒｅ ２５６：４９５−４９７、Ｋｏｚｂｏｒ， Ｄ． 他（１９８５）Ｊ． Ｉｍｍｕｎｏｌ． Ｍｅｔｈｏｄｓ ８１：３１−４２、Ｃｏｔｅ， Ｒ．Ｊ． 他（１９８３）Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ ８０：２０２６−２０３０、Ｃｏｌｅ， Ｓ．Ｐ． 他（１９８４）Ｍｏｌ． Ｃｅｌｌ Ｂｉｏｌ． ６２：１０９−１２０を参照）。
【０１８１】
更に、「キメラ抗体」作製のために発達した、ヒト抗体遺伝子にマウス抗体遺伝子をスプライシングするなどの技術が、好適な抗原特異性および生物学的活性を備える分子を得るために用いられる（例えばＭｏｒｒｉｓｏｎ， Ｓ．Ｌ．他．（１９８４）Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ ８１：６８５１６８５５、Ｎｅｕｂｅｒｇｅｒ、Ｍ．Ｓ．他．（１９８４）Ｎａｔｕｒｅ ３１２：６０４−６０８、Ｔａｋｅｄａ， Ｓ．他（１９８５）Ｎａｔｕｒｅ ３１４：４５２−４５４を参照）。別法では、当分野で周知の方法を用い、一本鎖抗体の産生のための記載された技術を適用して、ＳＥＣＰ特異的一本鎖抗体を生成する。関連した特異性を有するがイディオタイプ組成が異なるような抗体を、ランダムな組合せの免疫グロブリンライブラリ類からチェーンシャッフリングによって産生することもできる（例えばＢｕｒｔｏｎ Ｄ．Ｒ．（１９９１）Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ ８８：１０１３４−１０１３７を参照）。
【０１８２】
抗体類の産生は、リンパ球集団におけるｉｎ ｖｉｖｏ産生の誘導によって、あるいは免疫グロブリンライブラリ類のスクリーニングまたは文献に開示されているような、高度に特異的な結合試薬のパネル類のスクリーニングによっても行い得る（例えばＯｒｌａｎｄｉ， Ｒ． 他（１９８９）Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ ８６： ３８３３−３８３７、Ｗｉｎｔｅｒ， Ｇ． 他（１９９１）Ｎａｔｕｒｅ ３４９：２９３−２９９を参照）。
【０１８３】
ＳＥＣＰに対する特異的な結合部位を持つ抗体断片群を生成することもできる。例えば、限定するものではないが、このような断片には、抗体分子のペプシン消化によって作製されるＦ（ａｂ’）_２ 断片と、Ｆ（ａｂ’）_２ 断片のジスルフィド架橋を還元することによって作製されるＦａｂ断片とがある。あるいは、Ｆａｂ発現ライブラリを作製することによって、所望の特異性を持つモノクローナルＦａｂ断片を迅速且つ容易に同定することが可能となる（例えばＨｕｓｅ， Ｗ．Ｄ． 他 （１９８９） Ｓｃｉｅｎｃｅ ２４６：１２７５−１２８１を参照）。
【０１８４】
種々の免疫学的検定（イムノアッセイ）を用いてスクリーニングし、所望の特異性を有する抗体を同定することができる。確立された特異性を有するポリクローナル抗体またはモノクローナル抗体のいずれかを用いる競合的な結合、または免疫放射線アッセイのための数々のプロトコルが、当分野では周知である。通常このような免疫学的検定には、ＳＥＣＰとその特異性抗体との間の複合体形成の測定が含まれる。２つの非干渉性ＳＥＣＰエピトープに対して反応性を持つモノクローナル抗体を用いる、２部位モノクローナルベースのイムノアッセイが一般に利用されるが、競合的結合アッセイも利用することができる（Ｐｏｕｎｄ、前出）。
【０１８５】
ラジオイムノアッセイ技術と共にＳｃａｔｃｈａｒｄ分析などの様々な方法を用いて、ＳＥＣＰに対する抗体類の親和性を算定し得る。親和性を結合定数Ｋａで表すが、このＫａは、平衡状態下の、ＳＥＣＰ抗体複合体のモル濃度を、遊離抗体と遊離抗原とのモル濃度で除して得られる値である。多数のＳＥＣＰエピトープに対して親和性が不均一なポリクローナル抗体類の、試薬のＫａは、ＳＥＣＰに対する抗体の、平均親和性または結合活性を表す。特定のＳＥＣＰエピトープに単一特異的なモノクローナル抗体類の、試薬のＫａは、親和性の真の測定値を表す。Ｋａ値が１０^９〜１０^１２ｌｉｔｅｒ／ｍｏｌの高親和性抗体試薬は、ＳＥＣＰ抗体複合体が過酷な処理に耐えなければならないイムノアッセイに用いるのが好ましい。Ｋａ値が１０^６〜１０^７ｌｉｔｅｒ／ｍｏｌの低親和性抗体試薬は、ＳＥＣＰが抗体から最終的に（好ましくは活性化状態で）解離する必要がある免疫精製（ｉｍｍｕｎｏｐｕｒｉｆｉｃａｔｉｏｎ）および類似の処理に用いるのが好ましい（Ｃａｔｔｙ， Ｄ．（１９８８）Ａｎｔｉｂｏｄｉｅｓ， Ｖｏｌｕｍｅ Ｉ： Ａ Ｐｒａｃｔｉｃａｌ Ａｐｐｒｏａｃｈ， ＩＲＬ Ｐｒｅｓｓ， Ｗａｓｈｉｎｇｔｏｎ， ＤＣ； Ｌｉｄｄｅｌｌ， Ｊ． Ｅ． および Ｃｒｙｅｒ， Ａ．（１９９１）Ａ Ｐｒａｃｔｉｃａｌ Ｇｕｉｄｅ ｔｏ Ｍｏｎｏｃｌｏｎａｌ Ａｎｔｉｂｏｄｉｅｓ， Ｊｏｈｎ Ｗｉｌｅｙ ＆ Ｓｏｎｓ， Ｎｅｗ Ｙｏｒｋ ＮＹ）。
【０１８６】
ポリクローナル抗体試薬の抗体価および結合活性を更に評価して、後に使う或る適用例に対するこのような試薬の品質および適性を決定することができる。例えば、少なくとも１〜２ｍｇ／ｍｌの特異的な抗体、好ましくは５〜１０ｍｇ／ｍｌの特異的な抗体を含むポリクローナル抗体医薬は一般に、ＳＥＣＰ抗体複合体を沈殿させなければならない処理に用いられる。抗体の特異性、抗体価、結合活性、様々な適用例における抗体の品質や使用に対する指針については、一般に入手可能である。（前出のＣａｔｔｙの文献、同Ｃｏｌｉｇａｎ 他の文献などを参照）。
【０１８７】
本発明の別の実施例では、ＳＥＣＰをコードするポリヌクレオチド、またはその任意の断片や相補配列を、治療目的で使用できる。或る実施例では、ＳＥＣＰをコードする遺伝子のコーディング領域や調節領域に相補的な配列やアンチセンス分子（ＤＮＡ、ＲＮＡ、ＰＮＡ、または修飾したオリゴヌクレオチド）を設計して遺伝子発現をモディフィケーションし得る。このような技術は当分野では周知であり、アンチセンスオリゴヌクレオチドまたは大きな断片を、ＳＥＣＰをコードする配列の制御領域、またはコード領域に沿ったさまざまな位置から設計可能である（例えばＡｇｒａｗａｌ， Ｓ．，編集（１９９６）Ａｎｔｉｓｅｎｓｅ Ｔｈｅｒａｐｅｕｔｉｃｓ， Ｈｕｍａｎａ Ｐｒｅｓｓ Ｉｎｃ．， Ｔｏｔａｗａ ＮＪを参照）。
【０１８８】
治療に用いる場合、アンチセンス配列を適切な標的細胞に導入するのに好適な、任意の遺伝子送達系を用いることができる。アンチセンス配列は、転写時に標的タンパク質をコードする細胞配列の少なくとも一部に相補的な配列を作製する発現プラスミドの形で、細胞内に送達し得る（例えばＳｌａｔｅｒ， Ｊ．Ｅ． 他（１９９８）Ｊ． Ａｌｌｅｒｇｙ Ｃｌｉｎ． Ｉｍｍｕｎｏｌ． １０２（３）：４６９−４７５ および Ｓｃａｎｌｏｎ， Ｋ．Ｊ． 他（１９９５）９（１３）：１２８８−１２９６を参照）。アンチセンス配列はまた、例えばレトロウイルスやアデノ随伴ウイルスベクターなどのウイルスベクターを用いて細胞内に導入することもできる（例えばＭｉｌｌｅｒ， Ａ．Ｄ．（１９９０）Ｂｌｏｏｄ ７６：２７１、前出のＡｕｓｕｂｅｌ、Ｕｃｋｅｒｔ， Ｗ．およびＷ． Ｗａｌｔｈｅｒ（１９９４）Ｐｈａｒｍａｃｏｌ． Ｔｈｅｒ． ６３（３）：３２３−３４７を参照）。その他の遺伝子送達機構には、リポソーム系、人工的なウイルスエンベロープおよび当分野で公知のその他のシステムが含まれる（Ｒｏｓｓｉ， Ｊ．Ｊ．（１９９５）Ｂｒ． Ｍｅｄ． Ｂｕｌｌ． ５１（１）：２１７−２２５； Ｂｏａｄｏ、Ｒ．Ｊ．他（１９９８）Ｊ． Ｐｈａｒｍ． Ｓｃｉ． ８７（１１）：１３０８−１３１５、Ｍｏｒｒｉｓ， Ｍ．Ｃ． 他（１９９７）Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｒｅｓ． ２５（１４）：２７３０−２７３６などを参照）。
【０１８９】
本発明の別の実施態様では、ＳＥＣＰをコードするポリヌクレオチドを、体細胞若しくは生殖細胞の遺伝子治療に用いることが可能である。遺伝子治療により、（ｉ）遺伝子欠損症を治療し（例えばＸ染色体連鎖遺伝（Ｃａｖａｚｚａｎａ−Ｃａｌｖｏ， Ｍ．他 （２０００） Ｓｃｉｅｎｃｅ ２８８：６６９−６７２）により特徴付けられる重症複合型免疫不全（ＳＣＩＤ）−Ｘ１病の場合）、先天性アデノシンデアミナーゼ（ＡＤＡ）欠損症に関連する重症複合型免疫不全症候群（Ｂｌａｅｓｅ， Ｒ．Ｍ． 他 （１９９５） Ｓｃｉｅｎｃｅ ２７０：４７５−４８０、Ｂｏｒｄｉｇｎｏｎ， Ｃ．他 （１９９５） Ｓｃｉｅｎｃｅ ２７０：４７０−４７５）、嚢胞性繊維症（Ｚａｂｎｅｒ， Ｊ．他 （１９９３） Ｃｅｌｌ ７５：２０７−２１６： Ｃｒｙｓｔａｌ、Ｒ．Ｇ．他 （１９９５） Ｈｕｍ． Ｇｅｎｅ Ｔｈｅｒａｐｙ ６：６４３−６６６、Ｃｒｙｓｔａｌ， Ｒ．Ｇ．他． （１９９５） Ｈｕｍ． Ｇｅｎｅ Ｔｈｅｒａｐｙ ６：６６７−７０３）、サラセミア（ｔｈａｌａｓｓａｍｉａ）、家族性高コレステロール血症や、第ＶＩＩＩ因子若しくは第ＩＸ因子欠損に起因する血友病（Ｃｒｙｓｔａｌ， Ｒ．Ｇ． （１９９５） Ｓｃｉｅｎｃｅ ２７０：４０４−４１０、Ｖｅｒｍａ， Ｉ．Ｍ． および Ｎ． Ｓｏｍｉａ （１９９７） Ｎａｔｕｒｅ ３８９：２３９−２４２）、（ｉｉ）条件的致死性遺伝子産物を発現させ（例えば制御不能な細胞増殖に起因する癌の場合）、（ｉｉｉ）細胞内の寄生生物、例えばヒト免疫不全ウイルス（ＨＩＶ）（Ｂａｌｔｉｍｏｒｅ， Ｄ． （１９８８） Ｎａｔｕｒｅ ３３５：３９５−３９６、Ｐｏｅｓｃｈｌａ， Ｅ．他 （１９９６） Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ． ９３：１１３９５−１１３９９）などヒトレトロウイルス、Ｂ型若しくはＣ型肝炎ウイルス（ＨＢＶ、ＨＣＶ）、Ｃａｎｄｉｄａ ａｌｂｉｃａｎｓおよびＰａｒａｃｏｃｃｉｄｉｏｉｄｅｓ ｂｒａｓｉｌｉｅｎｓｉｓ等の寄生真菌、並びに熱帯熱マラリア原虫およびクルーズトリパノソーマ等の寄生原虫に対する防御機能を有するタンパク質を発現させることができる。ＳＥＣＰの発現若しくは調節に必要な遺伝子の欠損が疾患を引き起こす場合、導入した細胞の好適な集団からＳＥＣＰを発現させて、遺伝子欠損によって起こる症状の発現を緩和することが可能である。
【０１９０】
本発明の更なる実施例では、ＳＥＣＰの欠損による疾患や異常症を、ＳＥＣＰをコードする哺乳類発現ベクターを作製して、これらのベクターを機械的手段によってＳＥＣＰ欠損細胞に導入することによって治療する。ｉｎ ｖｉｖｏあるいはｅｘ ｖｉｔｒｏの細胞に用いる機械的導入技術には、（ｉ）個々の細胞内への直接的なＤＮＡ微量注射法、（ｉｉ）遺伝子銃、（ｉｉｉ）リポソームを介した形質移入、（ｉｖ）受容体を介した遺伝子導入、および（ｖ）ＤＮＡトランスポゾンの使用がある（Ｍｏｒｇａｎ， Ｒ．Ａ． および Ｗ．Ｆ． Ａｎｄｅｒｓｏｎ（１９９３）Ａｎｎｕ． Ｒｅｖ． Ｂｉｏｃｈｅｍ． ６２：１９１−２１７、Ｉｖｉｃｓ， Ｚ．（１９９７）Ｃｅｌｌ ９１：５０１−５１０； Ｂｏｕｌａｙ， Ｊ−Ｌ． およびＨ． Ｒｅｃｉｐｏｎ（１９９８）Ｃｕｒｒ． Ｏｐｉｎ． Ｂｉｏｔｅｃｈｎｏｌ． ９：４４５−４５０）。
【０１９１】
ＳＥＣＰの発現に有効であり得る発現ベクターの例として、限定するものではないがｐｃＤＮＡ ３．１、ＥｐｉＴａｇ、ｐＲｃＣＭＶ２、ｐＲＥＰ、ｐＶＡＸ、ｐＣＲＩＩ−ＴＯＰＯＴＡベクター（Ｉｎｖｉｔｒｏｇｅｎ， Ｃａｒｌｓｂａｄ ＣＡ）、ｐＣＭＶ−Ｓｃｒｉｐｔ、ｐＣＭＶ−Ｔａｇ、ＰＥＧＳＨ／ＰＥＲＶ（Ｓｔｒａｔａｇｅｎｅ， Ｌａ Ｊｏｌｌａ ＣＡ）およびＰＴＥＴ−ＯＦＦ、ＰＴＥＴ−ＯＮ、ＰＴＲＥ２、ＰＴＲＥ２−ＬＵＣ、ＰＴＫ−ＨＹＧ（Ｃｌｏｎｔｅｃｈ， Ｐａｌｏ Ａｌｔｏ ＣＡ）が挙げられる。ＳＥＣＰを発現させるには、（ｉ）構成的に活性なプロモーター（その由来は例えば、サイトメガロウイルス（ＣＭＶ）、ラウス肉腫ウイルス（ＲＳＶ）、ＳＶ４０ウイルス、チミジンキナーゼ（ＴＫ）、若しくはβ−アクチンの遺伝子）、（ｉｉ）誘導性プロモーター（例えば、市販のＴ−ＲＥＸプラスミド（Ｉｎｖｉｔｒｏｇｅｎ）に含まれるテトラサイクリン調節性プロモーター（Ｇｏｓｓｅｎ， Ｍ． および Ｈ． Ｂｕｊａｒｄ （１９９２） Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． Ｕ．Ｓ．Ａ． ８９：５５４７−５５５１； Ｇｏｓｓｅｎ， Ｍ． 他 （１９９５） Ｓｃｉｅｎｃｅ ２６８：１７６６−１７６９； Ｒｏｓｓｉ， Ｆ．Ｍ．Ｖ． および Ｈ．Ｍ． Ｂｌａｕ （１９９８） Ｃｕｒｒ． Ｏｐｉｎ． Ｂｉｏｔｅｃｈｎｏｌ． ９：４５１−４５６））、エクジソン誘導性プロモーター（市販のプラスミドＰＶＧＲＸＲおよびＰＩＮＤに含まれる：Ｉｎｖｉｔｒｏｇｅｎ）、ＦＫ５０６／ラパマイシン誘導性プロモーター、またはＲＵ４８６／ミフェプリストーン誘導性プロモーター（Ｒｏｓｓｉ， Ｆ．Ｍ．Ｖ． および Ｈ．Ｍ． Ｂｌａｕ， 前出）、または（ｉｉｉ）正常な個体に由来する、ＳＥＣＰをコードする内在性遺伝子の天然プロモーター、若しくは組織特異的プロモーターを用い得る。
【０１９２】
市販のリポソーム形質転換キット（例えばＩｎｖｉｔｒｏｇｅｎ社のＰｅｒＦｅｃｔ Ｌｉｐｉｄ Ｔｒａｎｓｆｅｃｔｉｏｎ Ｋｉｔ）を用いれば、当業者は実験の各パラメータを最適化する努力をさほど要さず、ポリヌクレオチド群を、培養中の標的細胞群に送達し得る。別法では、リン酸カルシウム法（Ｇｒａｈａｍ． Ｆ．Ｌ． および Ａ．Ｊ． Ｅｂ（１９７３）Ｖｉｒｏｌｏｇｙ ５２：４５６−４６７）若しくは電気穿孔法（Ｎｅｕｍａｎｎ， Ｂ． 他（１９８２）ＥＭＢＯ Ｊ． １：８４１−８４５）を用いて形質転換を行う。初代培養細胞にＤＮＡを導入するためには、標準化された哺乳類の形質移入プロトコルの修正が必要である。
【０１９３】
本発明の別の実施態様では、ＳＥＣＰの発現に関連する種々の遺伝子欠損によって起こる疾患や障害を、（ｉ）レトロウイルス長末端反復配列（ＬＴＲ）プロモーター若しくは或る独立プロモーターのコントロール下でＳＥＣＰをコードするポリヌクレオチドと、（ｉｉ）好適なＲＮＡパッケージングシグナル群と、（ｉｉｉ）効率的なベクター増殖に必要なコーディング配列群と追加のレトロウイルス・シス作用性ＲＮＡ配列群とを伴うＲｅｖ応答性エレメント（ＲＲＥ）と、からなるレトロウイルスベクターを作製して治療し得る。レトロウイルスベクター（例えばＰＦＢおよびＰＦＢＮＥＯ）はＳｔｒａｔａｇｅｎｅ社から市販されており、刊行データ（Ｒｉｖｉｅｒｅ， Ｉ． 他（１９９５）Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ ９２：６７３３−６７３７）に基づく。上記データを引用することを以て本明細書の一部とする。このベクターは、好適なベクター産生細胞株（ＶＰＣＬ）において増殖される。ＶＰＣＬは、各標的細胞上の受容体への親和性を持つエンベロープ遺伝子を、またはＶＳＶｇなど汎親和性エンベロープタンパク質を発現する（Ａｒｍｅｎｔａｎｏ， Ｄ． 他 （１９８７） Ｊ． Ｖｉｒｏｌ． ６１：１６４７−１６５０、Ｂｅｎｄｅｒ， Ｍ．Ａ． 他 （１９８７） Ｊ． Ｖｉｒｏｌ． ６１：１６３９−１６４６、Ａｄａｍ， Ｍ．Ａ． および Ａ．Ｄ． Ｍｉｌｌｅｒ （１９８８） Ｊ． Ｖｉｒｏｌ． ６２：３８０２−３８０６、Ｄｕｌｌ， Ｔ． 他 （１９９８） Ｊ． Ｖｉｒｏｌ． ７２：８４６３−８４７１、Ｚｕｆｆｅｒｅｙ， Ｒ． 他 （１９９８） Ｊ． Ｖｉｒｏｌ． ７２：９８７３−９８８０）。Ｒｉｇｇに付与された米国特許第５，９１０，４３４号（「Ｍｅｔｈｏｄ ｆｏｒ ｏｂｔａｉｎｉｎｇ ｒｅｔｒｏｖｉｒｕｓ ｐａｃｋａｇｉｎｇ ｃｅｌｌ ｌｉｎｅｓ ｐｒｏｄｕｃｉｎｇ ｈｉｇｈ ｔｒａｎｓｄｕｃｉｎｇ ｅｆｆｉｃｉｅｎｃｙ ｒｅｔｒｏｖｉｒａｌ ｓｕｐｅｒｎａｔａｎｔ」）において、レトロウイルスパッケージング細胞株群を得る方法が開示されており、引用を以て本明細書の一部とする。レトロウイルスベクター類の増殖や、細胞集団（例えばＣＤ４^＋Ｔ細胞群）の形質導入、および形質導入した細胞群の患者への戻しは、遺伝子治療の分野では当業者に周知の手法であり、多数の文献に記載がある（Ｒａｎｇａ， Ｕ． 他（１９９７）Ｊ． Ｖｉｒｏｌ． ７１：７０２０−７０２９、Ｂａｕｅｒ， Ｇ． 他（１９９７）Ｂｌｏｏｄ ８９：２２５９−２２６７、Ｂｏｎｙｈａｄｉ， Ｍ．Ｌ．（１９９７）Ｊ． Ｖｉｒｏｌ． ７１：４７０７−４７１６、Ｒａｎｇａ， Ｕ． 他（１９９８）Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． Ｕ．Ｓ．Ａ． ９５：１２０１−１２０６、Ｓｕ， Ｌ．（１９９７）Ｂｌｏｏｄ ８９：２２８３−２２９０）。
【０１９４】
別法では、アデノウイルス系遺伝子治療の或る送達系を用いて、ＳＥＣＰの発現に関連する１つ以上の遺伝子異常を有する細胞群に、ＳＥＣＰをコードするポリヌクレオチド群を送達する。アデノウイルス系ベクター類の作製およびパッケージングについては、当業者に周知である。複製欠損型アデノウイルスベクター類は、種々の免疫調節タンパク質をコードする遺伝子群を、無損傷の膵島内にインポートする目的で多様に利用し得ることが証明された（Ｃｓｅｔｅ， Ｍ．Ｅ． 他 （１９９５） Ｔｒａｎｓｐｌａｎｔａｔｉｏｎ ２７：２６３−２６８）。潜在的に有用なアデノウイルスベクター類はＡｒｍｅｎｔａｎｏに付与された米国特許第５，７０７，６１８号（「Ａｄｅｎｏｖｉｒｕｓ ｖｅｃｔｏｒｓ ｆｏｒ ｇｅｎｅ ｔｈｅｒａｐｙ」）に記載されており、引用することを以て本明細書の一部とする。アデノウイルスベクター類については、Ａｎｔｉｎｏｚｚｉ， Ｐ．Ａ． 他 （１９９９） Ａｎｎｕ． Ｒｅｖ． Ｎｕｔｒ． １９：５１１−５４４ 並びに、Ｖｅｒｍａ， Ｉ．Ｍ． および Ｎ． Ｓｏｍｉａ （１９９７） Ｎａｔｕｒｅ １８：３８９：２３９−２４２も参照されたい。両文献は、引用を以て本明細書の一部とする。
【０１９５】
別法では、ヘルペス系遺伝子治療の或る送達系を用いて、ＳＥＣＰの発現に関連する１つ以上の遺伝子異常を持つ標的細胞群に、ＳＥＣＰをコードするポリヌクレオチド群を送達する。単純ヘルペスウイルス（ＨＳＶ）系ベクター類は、ＨＳＶが親和性を持つ中枢神経細胞にＳＥＣＰを導入する際に、特に有益に思える。ヘルペス系ベクター類の作製およびパッケージングは、当業者に公知である。或る複製適格性単純ヘルペスウイルス（ＨＳＶ）Ｉ型系のベクターが、或るレポーター遺伝子の、霊長類の眼への送達に用いられている（Ｌｉｕ， Ｘ． 他 （１９９９） Ｅｘｐ． Ｅｙｅ Ｒｅｓ．１６９：３８５−３９５）。ＨＳＶ−１ウイルスベクターの作製についても、ＤｅＬｕｃａに付与された米国特許第５，８０４，４１３号（「Ｈｅｒｐｅｓ ｓｉｍｐｌｅｘ ｖｉｒｕｓ ｓｔｒａｉｎｓ ｆｏｒ ｇｅｎｅ ｔｒａｎｓｆｅｒ」）に詳細に開示されており、該特許の引用を以て本明細書の一部とする。米国特許第５，８０４，４１３号は、ヒト遺伝子治療などの目的で、好適なプロモーターの制御下において細胞に導入される少なくとも１つの外来遺伝子を有するゲノムを持つ、組換えＨＳＶ ｄ９２の利用について記載する。上記特許はまた、ＩＣＰ４、ＩＣＰ２７、およびＩＣＰ２２を欠失した組換えＨＳＶ系統の、作製および使用について開示している。ＨＳＶベクター類については、Ｇｏｉｎｓ， Ｗ．Ｆ． 他 （１９９９） Ｊ． Ｖｉｒｏｌ． ７３：５１９−５３２ および Ｘｕ， Ｈ． 他 （１９９４） Ｄｅｖ． Ｂｉｏｌ． １６３：１５２−１６１も参照されたい。両文献は、引用を以て本明細書の一部とする。クローン化したヘルペスウイルス配列群の操作、大ヘルペスウイルスゲノム（ｌａｒｇｅ ｈｅｒｐｅｓｖｉｒｕｓ ｇｅｎｏｍｅｓ）の様々なセグメントを持つ複数のプラスミドを形質移入した後の組換えウイルスの産生、ヘルペスウイルスの成長および増殖、並びに細胞群へのヘルペスウイルスの感染は、当業者に公知の技術である。
【０１９６】
別法では、或るαウイルス（正の一本鎖ＲＮＡウイルス）ベクターを用いて、ＳＥＣＰをコードするポリヌクレオチド群を標的細胞群に送達する。プロトタイプのαウイルスであるセムリキ森林熱ウイルス（ＳＦＶ）の生物学的研究が広範に行われており、遺伝子導入ベクター類は、ＳＦＶゲノムに基づく（Ｇａｒｏｆｆ， Ｈ． および Ｋ．−Ｊ． Ｌｉ （１９９８） Ｃｕｒｒ． Ｏｐｉｎ． Ｂｉｏｔｅｃｈｎｏｌ． ９：４６４−４６９）。αウイルスＲＮＡの複製中に、ウイルスのキャプシッドタンパク質群を通常はコードする、サブゲノムＲＮＡが産生される。このサブゲノムＲＮＡは、完全長ゲノムＲＮＡよりも高レベルに複製するので、酵素活性（例えばプロテアーゼおよびポリメラーゼ）を持つウイルスタンパク質に比べてキャプシッドタンパク質群が過剰産生される。同様に、ＳＥＣＰをコードする配列をαウイルスゲノムの、キャプシッドをコードする領域に挿入することにより、ベクター導入細胞において、ＳＥＣＰをコードするＲＮＡが多数、産生され、高レベルにＳＥＣＰが合成される。通常、αウイルスの感染は、数日以内の細胞溶解を伴う。一方、シンドビスウイルス（ＳＩＮ）の或る変異体を有するハムスター正常腎臓細胞（ＢＨＫ−２１）群が持続的な感染を確立する能力は、αウイルス類の溶解複製を、遺伝子治療に応用し得るように好適に改変可能であることを示唆する（Ｄｒｙｇａ， Ｓ．Ａ． 他 （１９９７） Ｖｉｒｏｌｏｇｙ ２２８ ：７４−８３）。様々な宿主にαウイルスを導入できるので、様々なタイプの細胞にＳＥＣＰを導入することができる。或る集団における或るサブセットの細胞群の特異的形質導入は、形質導入前に細胞の選別を必要とし得る。αウイルスの感染性ｃＤＮＡクローンの処置方法、αウイルスのｃＤＮＡおよびＲＮＡの形質移入方法およびαウイルスの感染方法は、当業者に公知である。
【０１９７】
転写開始部位由来のオリゴヌクレオチドを用いて遺伝子発現を阻害することも可能である。転写開始部位とは、例えば開始部位から数えて約−１０と約＋１０の間である。同様に、三重らせん塩基対の形成方法を用いて阻害が可能となる。三重らせん塩基対形成は、ポリメラーゼ、転写因子または調節分子の結合のために十分に開く、二重らせんの能力を阻害するので有用である。三重らせんＤＮＡを用いる最近の治療の進歩については文献に記載がある（例えばＧｅｅ， Ｊ．Ｅ． 他（１９９４）ｉｎ Ｈｕｂｅｒ， Ｂ．Ｅ．およびＢ．Ｉ． Ｃａｒｒ， Ｍｏｌｅｃｕｌａｒ ａｎｄ Ｉｍｍｕｎｏｌｏｇｉｃ Ａｐｐｒｏａｃｈｅｓ， Ｆｕｔｕｒａ Ｐｕｂｌｉｓｈｉｎｇ， Ｍｔ． Ｋｉｓｃｏ ＮＹ， １６３−１７７ページを参照）。また、相補配列またはアンチセンス分子を設計し、転写物がリボソームに結合するのを阻止することによってｍＲＮＡの翻訳をブロックし得る。
【０１９８】
リボザイム類は、酵素性ＲＮＡ分子である。ＲＮＡの特異的切断を触媒するためにリボザイムを用い得る。リボザイム作用のメカニズムは、相補的標的ＲＮＡへのリボザイム分子の配列特異性ハイブリダイゼーションと、その後に起こる内ヌクレオチド鎖切断に関与している。例えば、人工的に作製されたハンマーヘッド型リボザイム分子が、ＳＥＣＰをコードする配列の、内ヌクレオチド鎖分解性の切断を特異的且つ効果的に触媒できる可能性がある。
【０１９９】
任意の潜在的ＲＮＡ標的内の特異的リボザイム切断部位を先ず同定するため、ＧＵＡ、ＧＵＵ、ＧＵＣ配列などリボザイム切断部位に対して標的分子をスキャンする。一度同定されると、切断部位を持つ標的遺伝子の領域に対応する１５〜２０リボヌクレオチドの短いＲＮＡ配列が、そのオリゴヌクレオチドを機能不全にするような２次構造の特徴をもっていないかを評価することが可能になる。候補標的の適合性の評価も、リボヌクレアーゼ保護アッセイを用いて相補的オリゴヌクレオチド群とのハイブリダイゼーションへのアクセス可能性をテストすることによって行い得る。
【０２００】
本発明の相補リボ核酸分子およびリボザイムは、核酸分子合成のために当分野でよく知られている任意の方法を用いて作製し得る。作製方法には、固相ホスホラミダイト化学合成など、オリゴヌクレオチドを化学的に合成する方法がある。あるいは、ＳＥＣＰをコードするＤＮＡ配列のｉｎ ｖｉｔｒｏおよびｉｎ ｖｉｖｏ転写によってＲＮＡ分子を作成し得る。このようなＤＮＡ配列は、Ｔ７やＳＰ６などの好適なＲＮＡポリメラーゼプロモーターを用いて多様なベクター内に取り込むことが可能である。あるいは、相補的ＲＮＡを構成的あるいは誘導的に合成するこれらのｃＤＮＡ構成物を、細胞株、細胞または組織内に導入することができる。
【０２０１】
細胞内の安定性を高め、半減期を長くするためにＲＮＡ分子を修飾し得る。限定するものではないが可能な修飾としては、分子の５’末端、３’末端、あるいはその両方において隣接配列群を追加することや、分子の主鎖内においてホスホジエステラーゼ結合ではなくホスホロチオエートまたは２’ Ｏ−メチルを使用することが含まれる。この概念は、本来はＰＮＡ群の産出におけるものであるが、これら全ての分子に拡大することができる。そのためには、内在性エンドヌクレアーゼによって容易には認識されない、アデニン、シチジン、グアニン、チミン、およびウリジンにアセチル−、メチル−、チオ−および同様の修飾をしたものや、非従来型塩基、例えばイノシン、クエオシン（ｑｕｅｏｓｉｎｅ）、ワイブトシン（ｗｙｂｕｔｏｓｉｎｅ）を加える。
【０２０２】
本発明の更なる実施例は、ＳＥＣＰをコードするポリヌクレオチドの発現の改変に有効な化合物をスクリーニングする方法を含む。限定するものではないが特異ポリヌクレオチドの発現改変を起こすのに有効であり得る化合物には、オリゴヌクレオチド、アンチセンスオリゴヌクレオチド、三重らせん形成オリゴヌクレオチド、転写因子などのポリペプチド転写制御因子、および特異ポリヌクレオチド配列と相互作用し得る非高分子化学的実体がある。有効な化合物は、ポリヌクレオチド発現のインヒビターまたはエンハンサーのいずれかとして作用することによりポリヌクレオチド発現を改変し得る。したがって、ＳＥＣＰの発現または活性の増加に関連する疾患の治療においては、ＳＥＣＰをコードするポリヌクレオチドの発現を特異的に阻害する化合物が治療上有用であり、ＳＥＣＰの発現または活性の低下に関連する疾患の治療においては、ＳＥＣＰをコードするポリヌクレオチドの発現を特異的に促進する化合物が治療上有用であり得る。
【０２０３】
或る特定ポリヌクレオチドの発現を改変する際の有効性に対して、少なくとも１個または複数個の試験化合物をスクリーニングし得る。試験化合物を得るには、当分野で公知の任意の方法を用い得る。取得方法としては、以下の場合に有効な既知化合物の化学修飾がある。ポリヌクレオチドの発現を改変する場合と、既存の、商用または専用の、天然または非天然の化合物のライブラリから選択する場合と、標的ポリヌクレオチドの化学的および／または構造的特性に基づく化合物を合理的にデザインする場合と、組合せ的にまたは無作為に生成した化合物のライブラリから選択する場合とである。ＳＥＣＰをコードするポリヌクレオチドを持つサンプルを、このようにして得た試験化合物の少なくとも１つに曝露する。サンプルは例えば、無傷細胞、透過化処理した細胞、ｉｎ ｖｉｔｒｏ無細胞系すなわち再構成生化学系があり得る。ＳＥＣＰをコードするポリヌクレオチドの発現の改変は、当分野で公知の任意の方法でアッセイする。通常、或る特定ヌクレオチドの発現を検出するには、ＳＥＣＰをコードするポリヌクレオチドの配列に相補的なヌクレオチド配列を持つプローブとのハイブリダイゼーションを用いる。ハイブリダイゼーション量を定量することにより、１つ以上の試験化合物に曝露される、または曝露されないポリヌクレオチドの発現の比較のための基礎を形成し得る。試験化合物に曝露されるポリヌクレオチドの発現における変化の検出は、ポリヌクレオチドの発現を改変する際に試験化合物が有効であることを示す。或る特定ポリヌクレオチドの改変発現に有効な化合物のためのスクリーニングを実行でき、例えば分裂酵母（Ｓｃｈｉｚｏｓａｃｃｈａｒｏｍｙｃｅｓ ｐｏｍｂｅ）遺伝子発現系（Ａｔｋｉｎｓ， Ｄ． 他 （１９９９） 米国特許第５，９３２，４３５号、Ａｒｎｄｔ， Ｇ．Ｍ． 他 （２０００） Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｒｅｓ． ２８：Ｅ１５）またはＨｅＬａ細胞等のヒト細胞株（Ｃｌａｒｋｅ， Ｍ．Ｌ． 他 （２０００） Ｂｉｏｃｈｅｍ． Ｂｉｏｐｈｙｓ． Ｒｅｓ． Ｃｏｍｍｕｎ． ２６８：８−１３）を用いる。本発明の或る特定の実施例は、或る特定ポリヌクレオチド配列に対するアンチセンス活性について、オリゴヌクレオチド（デオキシリボヌクレオチド、リボヌクレオチド、ペプチド核酸、修飾したオリゴヌクレオチド）の組み合わせライブラリをスクリーニングすることに関する（Ｂｒｕｉｃｅ， Ｔ．Ｗ． 他 （１９９７） 米国特許第５，６８６，２４２号、Ｂｒｕｉｃｅ， Ｔ．Ｗ． 他 （２０００） 米国特許第６，０２２，６９１号）。
【０２０４】
ベクターを細胞または組織に導入する多数の方法が利用可能であり、ｉｎ ｖｉｖｏ、ｉｎ ｖｉｔｒｏおよびｅｘ ｖｉｖｏの使用に対して同程度に適している。ｅｘ ｖｉｖｏ治療の場合、ベクターを、患者から採取した幹細胞内に導入し、クローニング増殖して同患者に自家移植で戻すことができる。トランスフェクションによる、またはリボソーム注入やポリカチオンアミノポリマーによる送達は、当分野でよく知られている方法を用いて実行することができる（Ｇｏｌｄｍａｎ， Ｃ．Ｋ． 他（１９９７）Ｎａｔ． Ｂｉｏｔｅｃｈｎｏｌ． １５：４６２−４６６などを参照）。
【０２０５】
上記の治療方法はいずれも、例えば、ヒト、イヌ、ネコ、ウシ、ウマ、ウサギ、サルなどの哺乳類を含めて治療が必要な全ての対象に適用できる。
【０２０６】
本発明の或る更なる実施例は、薬剤として許容できる賦形剤と共に処方される活性成分を一般に持つ、或る組成物の投与に関する。賦形剤には例えば、糖、でんぷん、セルロース、ゴムおよびタンパク質がある。様々な処方が広く知られており、詳細はＲｅｍｉｎｇｔｏｎ’ｓ Ｐｈａｒｍａｃｅｕｔｉｃａｌ Ｓｃｉｅｎｃｅｓ（Ｍａａｃｋ Ｐｕｂｌｉｓｈｉｎｇ， Ｅａｓｔｏｎ ＰＡ）の最新版に記載されている。このような組成物は、ＳＥＣＰ、ＳＥＣＰの抗体、擬態物質、アゴニスト、アンタゴニスト、またはＳＥＣＰのインヒビターなどからなる。
【０２０７】
本発明に用いる組成物は、任意の数の経路によって投与することができ、限定するものではないが経路には、経口、静脈内、筋肉内、動脈内、骨髄内、クモ膜下腔内、心室内、肺、経皮、皮下、腹腔内、鼻腔内、腸内、局所、舌下または直腸がある。
【０２０８】
肺から投与する組成物は、液状または乾燥粉末状で調製し得る。このような組成物は通常、患者が吸入する直前にエアロゾル化する。小分子（例えば伝統的な低分子量有機薬）の場合には、速効製剤のエアロゾル送達は当分野で公知である。高分子（例えばより大きなペプチドおよびタンパク質）の場合には、当該分野において肺の肺胞領域を介しての肺送達が最近向上したことにより、インスリンなどの薬剤を実質的に血液循環へ輸送することを可能にした（Ｐａｔｔｏｎ， Ｊ．Ｓ． 他， 米国特許第５，９９７，８４８号などを参照）。肺送達は、針注射なしに投与する点で優れており、有毒な可能性のある浸透エンハンサーの必要性をなくす。
【０２０９】
本発明での使用に適した組成物には、所定の目的を達成するために必要なだけの量の活性成分を含有する組成物が含まれる。有効投与量の決定は、当業者の能力の範囲内で行う。
【０２１０】
ＳＥＣＰまたはその断片を含む高分子を直接に細胞内に送達すべく、特殊な形態の組成物群を調製し得る。例えば、細胞不透過性高分子を含むリポソーム製剤は、その高分子の細胞融合と細胞内送達とを促進し得る。別法では、ＳＥＣＰまたはその断片を、ＨＩＶ Ｔａｔ−１タンパク質の陽イオンＮ末端部に結合することもできる。このようにして生成された融合タンパク質類は、或るマウスモデル系の、脳を含む全ての組織の細胞群に形質導入することがわかっている（Ｓｃｈｗａｒｚｅ， Ｓ．Ｒ． 他 （１９９９） Ｓｃｉｅｎｃｅ ２８５：１５６９−１５７２）。
【０２１１】
任意の化合物に対して、先ず細胞培養アッセイ、例えば新生物性細胞の細胞培養アッセイにおいて、あるいは、動物モデル、例えばマウス、ウサギ、イヌまたはブタなどにおいて、治療の有効投与量を推定することができる。動物モデルはまた、好適な濃度範囲および投与経路を決定するためにも用い得る。このような情報を用いて、次にヒトに対する有益な投与量および投与経路を決定することができる。
【０２１２】
医薬的有効量とは、活性処方成分、たとえばＳＥＣＰまたはその断片、ＳＥＣＰの抗体、ＳＥＣＰのアゴニストまたはアンタゴニスト、インヒビターなどの、症状や容態を回復させる量を指す。薬用有効度および毒性は、細胞培養または動物実験における標準的な薬学手法によって、例えばＥＤ_５０（集団の５０％の医薬的有効量）またはＬＤ_５０（集団の５０％の致死量）統計を計算するなどして判定できる。毒性効果の、治療効果に対する投与量比が治療指数であり、ＬＤ_５０／ＥＤ_５０比として表すことができる。高い治療指数を示すような組成物が望ましい。細胞培養アッセイと動物実験とから得られたデータは、ヒトに用いる投与量の範囲での調剤に用いられる。このような組成物に含まれる投与量は、毒性を殆どあるいは全く持たず、ＥＤ_５０を含むような血中濃度の範囲にあることが好ましい。用いられる投与形態、患者の感受性および投与の経路によって、投与量はこの範囲内で様々に変わる。
【０２１３】
正確な投与量は、治療が必要な被験者に関する要素を考慮して、現場の医者が決定することになる。充分なレベルの活性成分を与え、あるいは所望の効果を維持すべく、用法および用量を調整する。被験者に関する要素としては、疾患の重症度、患者の一般的な健康状態、患者の年齢、体重および性別、投与の時間および頻度、薬剤の配合、反応感受性および治療に対する応答などを考慮し得る。作用期間が長い組成物は、特定の製剤の半減期およびクリアランス率によって３〜４日毎に１度、１週間に１度、あるいは２週間に１度の間隔で投与し得る。
【０２１４】
通常の投与量は、投与の経路にもよるが約０．１〜１００，０００μｇであり、合計で約１ｇまでとする。特定の投与量および送達方法に関するガイダンスは文献に記載されており、現場の医者は通常それを利用することができる。当業者は、タンパク質またはそれらのインヒビター類についての処方とは異なる、ヌクレオチドについての処方を利用することになる。同様に、ポリヌクレオチドまたはポリペプチドの送達は、特定の細胞、症状、部位などに特異的なものとなる。
【０２１５】
（診断）
別の実施例では、ＳＥＣＰに特異的に結合する抗体を、ＳＥＣＰの発現によって特徴付けられる疾患の診断に、またはＳＥＣＰやＳＥＣＰのアゴニストまたはアンタゴニスト、インヒビターで治療を受けている患者をモニターするためのアッセイに用いる。診断目的に有用な抗体は、上記の治療の箇所で記載した方法と同じ方法で調合される。ＳＥＣＰの診断アッセイには、ヒトの体液から、あるいは細胞や組織の抽出物から、抗体および標識を用いてＳＥＣＰを検出する方法が含まれる。この抗体は修飾されたものもされていないものも可能であり、レポーター分子との共有結合または非共有結合で標識化できる。レポーター分子としては広くさまざまな種類が本分野で知られており、また使用可能であるが、そのうちのいくつかは上記で説明されている。
【０２１６】
ＳＥＣＰを測定するための種々のプロトコル、例えばＥＬＩＳＡ，ＲＩＡ，およびＦＡＣＳなどが当分野では周知であり、変化した、あるいは異常なレベルのＳＥＣＰの発現を診断するための基盤を提供する。正常あるいは標準的なＳＥＣＰの発現の値を確定するため、正常な哺乳類、例えばヒトなどの被験者から採取した体液または細胞抽出物と、ＳＥＣＰに対する抗体類とを、複合体の形成に適した条件下で混合する。標準複合体形成量は、種々の方法、例えば測光法で定量できる。被験者での、または対照での、または生検組織群からの疾患サンプルでのＳＥＣＰ発現の量が、基準値と比較される。標準値と被験者との偏差が、疾患を診断するパラメータを確定する。
【０２１７】
本発明の別の実施態様では、ＳＥＣＰをコードするポリヌクレオチドを診断に用いることもできる。用い得るポリヌクレオチドには、オリゴヌクレオチド配列、相補的ＲＮＡおよびＤＮＡ分子、そしてＰＮＡが含まれる。これらのポリヌクレオチドを用いて、ＳＥＣＰの発現が疾患と相関し得る生検組織での遺伝子発現を、検出し定量し得る。この診断アッセイを用いて、ＳＥＣＰの存在の有無、更には過剰な発現を調べ、治療中のＳＥＣＰ値の調節を監視する。
【０２１８】
一実施形態では、ＳＥＣＰをコードまたは近縁の分子をコードするポリヌクレオチド配列（例えばゲノム配列）を検出可能なＰＣＲプローブ類とのハイブリダイゼーションによって、ＳＥＣＰをコードする核酸配列を同定できる。例えば５’調節領域など高度に特異的な領域から作られた、または例えば保存されたモチーフなどやや特異性の低い領域から作られたというプローブの特異性は、またハイブリダイゼーションのまたは増幅のストリンジェンシーは、プローブが、ＳＥＣＰをコードする天然配列のみを同定するかどうか、あるいは対立遺伝子変異配列や関連配列を同定するかどうかを決めることになる。
【０２１９】
プローブ類はまた、関連配列の検出に利用され得る他、ＳＥＣＰをコードする任意の配列との少なくとも５０％の配列同一性を持ち得る。本発明のハイブリダイゼーションプローブ類はＤＮＡまたはＲＮＡであることがあり、ＳＥＱ ＩＤ ＮＯ：６４−１２６の配列に由来、或いはゲノム配列群、例えばＳＥＣＰ遺伝子のプロモーター、エンハンサー、イントロンなどに由来し得る。
【０２２０】
ＳＥＣＰをコードするＤＮＡ群に対して特異的なハイブリダイゼーションプローブの作製方法として、ＳＥＣＰまたはＳＥＣＰ誘導体をコードするポリヌクレオチド配列を、ｍＲＮＡプローブ作製用ベクターにクローニングする方法がある。ｍＲＮＡプローブ作製用ベクター類は、当業者に知られており、市販されており、好適なＲＮＡポリメラーゼと好適な標識されたヌクレオチドとを加えることによって、ｉｎ ｖｉｔｒｏでＲＮＡプローブを合成するために用い得る。ハイブリダイゼーションプローブ類は、種々のレポーターの集団によって標識され得る。レポーター集団の例としては、^３２Ｐまたは^３５Ｓなどの放射性核種が、あるいはアビジン／ビオチン結合系を介してプローブに結合されたアルカリホスファターゼなど酵素標識が挙げられる。
【０２２１】
ＳＥＣＰをコードするポリヌクレオチド配列を用いて、ＳＥＣＰの発現に関連する疾患を診断することが可能である。限定するものではないが、このような疾患には細胞増殖異常、自己免疫／炎症疾患、心血管障害、神経障害、および発達障害が含まれ、細胞増殖異常の中には日光角化症、動脈硬化、アテローム硬化、滑液包炎、硬変、肝炎、混合性結合組織病（ＭＣＴＤ）、骨髄線維症、発作性夜間ヘモグロビン尿症、真性多血症、乾癬、原発性血小板血症、並びに、腺癌、白血病、リンパ腫、黒色腫、骨髄腫、肉腫、および奇形癌など癌、具体的には、副腎、膀胱、骨、骨髄、脳、乳房、頚部、胆嚢、神経節、消化管、心臓、腎臓、肝臓、肺、筋肉、卵巣、膵臓、副甲状腺、陰茎、前立腺、唾液腺、皮膚、脾臓、精巣、胸腺、甲状腺、および子宮の癌が含まれ、自己免疫／炎症性の疾患の中には、後天性免疫不全症候群（ＡＩＤＳ）、アジソン病（慢性原発性副腎機能不全）、成人呼吸窮迫症候群、アレルギー、強直性脊椎炎、アミロイド症、貧血、喘息、アテローム性動脈硬化、自己免疫性溶血性貧血、自己免疫性甲状腺炎、自己免疫性多腺性内分泌カンジダ性外胚葉ジストロフィー（ＡＰＥＣＥＤ）、気管支炎、胆嚢炎、接触皮膚炎、クローン病、アトピー性皮膚炎、皮膚筋炎、糖尿病、肺気腫、リンパ球傷害因子性偶発性リンパ球減少症、新生児溶血性疾患（胎児赤芽球症）、結節性紅斑、萎縮性胃炎、糸球体腎炎、グッドパスチャー症候群、痛風、グレーブス病、橋本甲状腺炎、過好酸球増加症、過敏性腸症候群、多発性硬化症、重症筋無力症、心筋または心膜炎症、骨関節炎、骨粗しょう症、膵炎、多発性筋炎、乾癬、ライター症候群、リウマチ様関節炎、強皮症、シェーグレン症候群、全身性アナフィラキシー、全身性エリテマトーデス、全身性硬化症、血小板減少性紫斑病、潰瘍性大腸炎、ウェルナー症候群、癌合併症、血液透析、体外循環、ウイルス感染症、細菌感染症、真菌感染症、寄生虫感染症、原虫感染症、蠕虫感染症、外傷が含まれ、心血管障害の中には、鬱血性心不全、虚血性心疾患、狭心症、心筋梗塞、高血圧性心疾患、変性弁膜性心疾患、石灰化大動脈弁狭窄症、先天性２尖大動脈弁、僧帽弁輪部石灰化（ｍｉｔｒａｌ ａｎｎｕｌａｒ ｃａｌｃｉｆｉｃａｔｉｏｎ）、僧帽弁逸脱、リウマチ熱、リウマチ性心疾患、感染性心内膜炎、非細菌性血栓性心内膜炎、全身性エリテマトーデスの心内膜炎、カルチノイド心疾患、心筋症、心筋炎、心膜炎、腫瘍性心疾患、先天性心臓疾患、心移植の合併症、動静脈瘻、アテローム硬化、高血圧、脈管炎、レイノー病、動脈瘤、動脈解離、静脈瘤、血栓静脈炎および静脈血栓、血管系腫瘍、血栓溶解の合併症、バルーン血管形成術、血管置換術（ｖａｓｃｕｌａｒ ｒｅｐｌａｃｅｍｅｎｔ）、冠動脈バイパス移植術が含まれ、神経障害の中には、癲癇、虚血性脳血管障害、脳卒中、脳腫瘍、アルツハイマー病、ピック病、ハンチントン病、痴呆、パーキンソン病およびその他の錐体外路障害、筋萎縮性側索硬化およびその他の運動ニューロン障害、進行性神経性筋萎縮症、網膜色素変性症（色素性網膜炎）、遺伝性運動失調、多発性硬化症および他の脱髄疾患、細菌性およびウイルス性髄膜炎、脳膿瘍、硬膜下膿瘍、硬膜外膿瘍、化膿性頭蓋内血栓性静脈炎、脊髄炎および神経根炎、ウイルス性中枢神経系疾患と、プリオン病（クールー、クロイツフェルト‐ヤコブ病、およびＧｅｒｓｔｍａｎｎ−Ｓｔｒａｕｓｓｌｅｒ−Ｓｃｈｅｉｎｋｅｒ症候群を含む）、致死性家族性不眠症、神経系性栄養病および代謝病、神経線維腫症、結節硬化症、小脳網膜血管腫症（ｃｅｒｅｂｅｌｌｏｒｅｔｉｎａｌ ｈｅｍａｎｇｉｏｂｌａｓｔｏｍａｔｏｓｉｓ）、脳３叉神経血管症候群、ダウン症を含む中枢神経系性精神遅滞および他の発達障害、脳性麻痺、神経骨格異常症、自律神経系障害、脳神経障害、脊髄疾患、筋ジストロフィー他の神経筋障害、末梢神経疾患、皮膚筋炎および多発性筋炎と、遺伝性、代謝性、内分泌性、および中毒性ミオパシーと、重症筋無力症、周期性四肢麻痺、精神障害（気分性、不安性の障害、統合失調症／分裂病）、季節性感情障害（ＳＡＤ）、静座不能、健忘症、緊張病、糖尿病性ニューロパシー、遅発性ジスキネジア、ジストニー、パラノイド精神病、帯状疱疹後神経痛、トゥーレット病、進行性核上麻痺、大脳皮質基底核変性（ｃｏｒｔｉｃｏｂａｓａｌ ｄｅｇｅｎｅｒａｔｉｏｎ）、および家族性前頭側頭型痴呆とが含まれ、また発達障害も含まれ、その中には尿細管性アシドーシス、貧血、クッシング症候群、軟骨形成不全性小人症、デュシェンヌ／ベッカー型筋ジストロフィー、癲癇、性腺形成異常、ＷＡＧＲ症候群（ウィルムス腫瘍、無虹彩症、尿生殖器異常、精神遅滞）、スミス‐マジェニス症候群（Ｓｍｉｔｈ− Ｍａｇｅｎｉｓ ｓｙｎｄｒｏｍｅ）、骨髄異形成症候群、遺伝性粘膜上皮異形成、遺伝性角皮症や、シャルコーマリーツース病および神経線維腫症などの遺伝性神経病、甲状腺機能低下症、水頭症や、Ｓｙｎｄｅｎｈａｍ舞踏病（Ｓｙｎｄｅｎｈａｍ’ｓ ｃｈｏｒｅａ）および脳性小児麻痺などの発作障害、二分脊椎、無脳症、頭蓋脊椎披裂、先天性緑内障、白内障、感音難聴が含まれる。ＳＥＣＰをコードするポリヌクレオチド配列は、変容したＳＥＣＰ発現を検出するために患者から採取した体液あるいは組織を利用する、サザーン法やノーザン法、ドットブロット法、あるいはその他の膜系の技術、ＰＣＲ法、ディップスティック（ｄｉｐｓｔｉｃｋ）、ピン（ｐｉｎ）、およびマルチフォーマットのＥＬＩＳＡ式アッセイ、およびマイクロアレイに使用可能である。このような定性方法または定量方法は、当分野で公知である。
【０２２２】
ある実施態様では、ＳＥＣＰをコードするヌクレオチド配列は、関連する疾患、特に上記した疾患を検出するアッセイにおいて有用であろう。ＳＥＣＰをコードするヌクレオチド配列は、標準的な方法で標識化され、ハイブリダイゼーション複合体の形成に好適な条件下で、患者から採取した体液あるいは組織のサンプルに加えることができるであろう。好適なインキュベーション期間が経過したらサンプルを洗浄し、シグナルを定量して標準値と比較する。患者のサンプルのシグナルの量が対照サンプルに比較して著しく変化している場合は、そのサンプル内のＳＥＣＰをコードするヌクレオチド配列群のレベル変化の存在により、関連する疾患の存在が明らかになる。このようなアッセイは、動物実験、臨床試験における特定の治療効果を推定するため、あるいは個々の患者の治療をモニターするために用いることもできる。
【０２２３】
ＳＥＣＰの発現に関連する疾患の診断の基礎を提供するために、発現の正常すなわち標準的なプロフィールが確立される。これは、ハイブリダイゼーションあるいは増幅に好適な条件下で、動物あるいはヒトのいずれかの正常な被験者から採取した体液あるいは細胞抽出物と、ＳＥＣＰをコードする配列あるいはその断片とを混合することにより達成され得る。実質的に精製されたポリヌクレオチドを既知量で用いて行った実験から得た値を正常な対象から得た値と比較することにより、標準ハイブリダイゼーションを定量することができる。このようにして得た標準値は、疾患の徴候を示す患者から得たサンプルから得た値と比較することができる。標準値からの偏差を用いて疾患の存在を確定する。
【０２２４】
疾患の存在が確定されて治療プロトコルが開始されると、患者の発現レベルが正常な被検者に観察されるレベルに近づき始めたかどうかを測定するため、ハイブリダイゼーションアッセイを定期的に繰り返し得る。連続アッセイから得られた結果を用いて、数日から数ヶ月の期間にわたる治療の効果を示し得る。
【０２２５】
癌に関しては、個体からの生体組織における異常な量の転写物（過少発現または過剰発現）の存在は、疾患の発生素質を示したり、実際に臨床的症状が現れる前に疾患を検出する方法を提供し得る。この種のより明確な診断により、医療の専門家が予防方法または積極的な治療法を早くから利用し、それによって癌の発生または更なる進行を防止することが可能となる。
【０２２６】
ＳＥＣＰをコードする配列群から設計したオリゴヌクレオチド群の更なる診断的利用には、ＰＣＲの利用を含み得る。これらのオリゴマーは、化学的に合成するか、酵素により生産するか、あるいはｉｎ ｖｉｔｒｏで産出し得る。オリゴマーは、好ましくはＳＥＣＰをコードするポリヌクレオチドの断片、あるいはＳＥＣＰをコードするポリヌクレオチドと相補的なポリヌクレオチドの断片を含み、最適化した条件下で、特定の遺伝子や条件を識別するために利用される。また、オリゴマーは、やや緩いストリンジェンシー条件下で、近縁のＤＮＡあるいはＲＮＡ配列の検出、定量、あるいはその両方のため用いることが可能である。
【０２２７】
或る実施態様においては、ＳＥＣＰをコードするポリヌクレオチド配列群に由来するオリゴヌクレオチドプライマー類を用いて、一塩基多型（ＳＮＰ）を検出し得る。ＳＮＰは、多くの場合にヒトの先天性または後天性遺伝病の原因となるような置換、挿入および欠失である。限定するものではないがＳＮＰの検出方法には、ＳＳＣＰ（ｓｉｎｇｌｅ−ｓｔｒａｎｄｅｄ ｃｏｎｆｏｒｍａｔｉｏｎ ｐｏｌｙｍｏｒｐｈｉｓｍ）および蛍光ＳＳＣＰ（ｆＳＳＣＰ）がある。ＳＳＣＰでは、ＳＥＣＰをコードするポリヌクレオチド配列に由来するオリゴヌクレオチドプライマーを用いたポリメラーゼ連鎖反応（ＰＣＲ）でＤＮＡを増幅する。ＤＮＡは例えば、病変組織または正常組織、生検サンプル、体液などに由来し得る。ＤＮＡ内のＳＮＰは、一本鎖形のＰＣＲ生成物の２次および３次構造に差異を生じさせる。差異は非変性ゲル中でのゲル電気泳動法を用いて検出可能である。ｆＳＣＣＰでは、オリゴヌクレオチドプライマーを蛍光性に標識する。それによってＤＮＡシークエンシング機などの高処理機器でアンプリマー（ａｍｐｌｉｍｅｒ）の検出が可能になる。更に、インシリコＳＮＰ（ｉｎ ｓｉｌｉｃｏ ＳＮＰ， ｉｓＳＮＰ）と呼ばれる配列データベース分析法は、一般的なコンセンサス配列に配列されるような個々のオーバーラップするＤＮＡ断片群の配列を比較することにより、多型性を同定し得る。これらのコンピュータベースの方法は、ＤＮＡの実験室での調製に、または統計モデルとＤＮＡ配列クロマトグラムの自動分析とを用いたシークエンシングのエラーに起因する配列変異を、フィルタリングして除去する。別の態様では、例えば高処理ＭＡＳＳＡＲＲＡＹシステム（Ｓｅｑｕｅｎｏｍ， Ｉｎｃ．， Ｓａｎ Ｄｉｅｇｏ ＣＡ）を用いた質量分析によりＳＮＰを検出し、特徴付ける。
【０２２８】
ＳＥＣＰの発現を定量するために用い得る別の方法の例としては、ヌクレオチド群の放射標識またはビオチン標識、対照核酸の共増幅（ｃｏａｍｐｌｉｆｉｃａｔｉｏｎ）、および、標準曲線から得た結果の補間もある（例えば、Ｍｅｌｂｙ， Ｐ．Ｃ．他（１９９３）Ｊ． Ｉｍｍｕｎｏｌ． Ｍｅｔｈｏｄｓ， １５９：２３５−２４４； Ｄｕｐｌａａ， Ｃ．他（１９９３）Ａｎａｌ． Ｂｉｏｃｈｅｍ． ２１２：２２９−２３６を参照）。複数サンプルの定量速度を加速するには、目的のオリゴマーまたはポリヌクレオチドを種々の希釈液中に置き、分光光度法または比色反応によって定量が迅速になるような高処理フォーマットでのアッセイを行い得る。
【０２２９】
更に別の実施例では、本明細書で記載した任意のポリヌクレオチド配列に由来するオリゴヌクレオチドまたはより長い断片を、マイクロアレイにおけるエレメントとして用いることができる。多数の遺伝子の相対発現レベルを同時にモニターする転写イメージング技術にマイクロアレイを用いることが可能である。これについては、以下に記載する。マイクロアレイはまた、遺伝変異体、突然変異および多型性の同定に用いることができる。この情報を用いることで、遺伝子機能を決定し、疾患の遺伝的根拠を理解し、疾患を診断し、遺伝子発現の機能としての疾病の進行／後退をモニターし、疾病治療における薬剤の活性を開発およびモニターすることができる。特に、患者にとって最もふさわしく、有効な治療法を選択するために、この情報を用いて患者の薬理ゲノムプロフィールを開発することができる。例えば、患者の薬理ゲノムプロフィールに基づき、患者に対して高度に効果的で副作用を殆ど示さない治療薬を選択することができる。
【０２３０】
別の実施例では、ＳＥＣＰ、ＳＥＣＰの断片、ＳＥＣＰに特異的な抗体を、マイクロアレイ上のエレメントとして用いることができる。マイクロアレイを用いて、上記のようなタンパク質−タンパク質相互作用、薬剤−標的相互作用および遺伝子発現プロフィールをモニターまたは測定することが可能である。
【０２３１】
或る実施態様は、或る組織または細胞タイプの転写イメージを作製する、本発明のポリヌクレオチドの使用に関連する。転写イメージは、特定の組織または細胞タイプによる、遺伝子発現の包括的パターンを表す。包括的遺伝子発現パターンは、所与の条件下で所与の時間に発現した遺伝子の数および相対存在量を定量することにより分析し得る（Ｓｅｉｌｈａｍｅｒ 他の米国特許第５，８４０，４８４号 「Ｃｏｍｐａｒａｔｉｖｅ Ｇｅｎｅ Ｔｒａｎｓｃｒｉｐｔ Ａｎａｌｙｓｉｓ」を参照。該特許は特に引用することを以って本明細書の一部となす）。したがって、特定の組織または細胞タイプの転写物または逆転写物全体に、本発明のポリヌクレオチドまたはそれらの相補体をハイブリダイズすることにより、転写イメージを作製し得る。或る実施態様では、本発明のポリヌクレオチドまたはそれらの相補体が１マイクロアレイ上に複数エレメントの１サブセットを持つような高処理フォーマットでハイブリダイゼーションを発生させる。結果として得られる転写イメージは、遺伝子活性のプロフィールを提供し得る。
【０２３２】
転写イメージは、組織、細胞株、生検またはその生物学的サンプルから単離した転写物を用いて作製し得る。転写イメージはしたがって、組織または生検サンプルの場合にはｉｎ ｖｉｖｏ、細胞株の場合にはｉｎ ｖｉｔｒｏでの遺伝子発現を反映する。
【０２３３】
本発明のポリヌクレオチドの発現のプロフィールを作製する転写イメージはまた、ｉｎ ｖｉｔｒｏモデル系および薬剤の前臨床評価に、あるいは工業的または天然の環境化合物の毒性試験に関連して使用し得る。全ての化合物は、作用および毒性のメカニズムを標示し、しばしば分子フィンガープリントまたは毒性シグネチャと称される、特徴的な遺伝子発現パターンを惹起する（Ｎｕｗａｙｓｉｒ， Ｅ．Ｆ． 他（１９９９）Ｍｏｌ． Ｃａｒｃｉｎｏｇ． ２４：１５３−１５９、Ｓｔｅｉｎｅｒ， Ｓ． および Ｎ．Ｌ． Ａｎｄｅｒｓｏｎ（２０００）Ｔｏｘｉｃｏｌ． Ｌｅｔｔ． １１２−１１３：４６７−４７１、該文献は特に引用を以って本明細書の一部となす）。試験化合物が、既知毒性を有する化合物のシグネチャと同一のシグネチャを有する場合には、毒性特性を共有している可能性がある。フィンガープリントまたはシグネチャは、多数の遺伝子および遺伝子ファミリーからの発現情報を含んでいる場合に、最も有用且つ正確である。理想的には、ゲノム全域にわたる発現の測定が、最高品質のシグネチャを提供する。たとえ、発現が任意の試験された化合物によって変容しない遺伝子があったとしても、それらの発現レベルを残りの発現データを標準化することに使用できるため、それらの遺伝子は重要である。正規化手順は、種々の化合物で処理した後の発現データの比較に有用である。或る毒性シグネチャのエレメント群に遺伝子機能を割り当てることは毒性機序の解釈に役立つが、毒性の予測につながる、シグネチャ群の統計的マッチングには、遺伝子機能の知識は必要でない（例えば２０００年２月２９日に米国国立環境健康科学研究所（Ｎａｔｉｏｎａｌ Ｉｎｓｔｉｔｕｔｅ ｏｆ Ｅｎｖｉｒｏｎｍｅｎｔａｌ Ｈｅａｌｔｈ Ｓｃｉｅｎｃｅｓ）より発行されたＰｒｅｓｓ Ｒｅｌｅａｓｅ ００−０２を参照されたい。これについてはｈｔｔｐ：／／ｗｗｗ．ｎｉｅｈｓ．ｎｉｈ．ｇｏｖ／ｏｃ／ｎｅｗｓ／ｔｏｘｃｈｉｐ．ｈｔｍで入手可能である）。したがって、毒性シグネチャを用いる中毒学的スクリーニングの際に、全ての発現した遺伝子配列を含めることは、重要且つ望ましい。
【０２３４】
或る実施例では、核酸を有する生物学的サンプルを試験化合物で処理することにより、この試験化合物の毒性を算定する。処理した生物学的サンプル中で発現した核酸は、本発明のポリヌクレオチドに特異的な１つ以上のプローブでハイブリダイズし、それによって本発明のポリヌクレオチドに対応する転写レベルを定量し得る。処理した生物学的サンプル中の転写レベルを、未処理生物学的サンプル中のレベルと比較する。両サンプルの転写レベルの差は、処理済サンプル中で試験化合物が引き起こす毒性反応を標示する。
【０２３５】
別の実施態様は、本発明のポリペプチド配列群を用いて或る組織または細胞タイプのプロテオームを分析することに関する。プロテオームの語は、特定の組織または細胞タイプでのタンパク質発現の包括的パターンを指す。プロテオームの各タンパク質成分は、個々に更なる分析の対象とすることができる。プロテオーム発現パターンすなわちプロフィールは、所与の条件下で所与の時間に発現したタンパク質の数および相対存在量を定量することにより分析し得る。したがって、或る細胞のプロテオームのプロフィールは、特定の組織または細胞タイプのポリペプチドを分離および分析することにより作成し得る。或る実施例では、１次元等電点電気泳動によりサンプルからタンパク質を分離し、２次元ドデシル硫酸ナトリウムスラブゲル電気泳動により分子量に応じて分離するような２次元ゲル電気泳動により、分離が達成される（前出のＳｔｅｉｎｅｒ および Ａｎｄｅｒｓｏｎ）。タンパク質は、通常はクーマシーブルー、あるいは銀染色液または蛍光染色液などの物質を用いてゲルを染色することにより、分散した、独自の位置にある点としてゲル中で可視化される。各タンパク質スポットの光学密度は、通常、サンプル中のタンパク質レベルに比例する。異なるサンプル、例えば試験化合物または治療薬で処理または未処理のいずれかの生物学的サンプルからの、同等に位置したタンパク質スポットの光学密度を比較し、処理に関連するタンパク質スポット密度の変化を同定する。スポット内のタンパク質は、例えば化学的または酵素的切断とそれに続く質量分析を用いる標準的な方法を用いて部分的にシークエンシングする。或るスポット内のタンパク質の同一性は、その部分配列を、好適には少なくとも５個の連続するアミノ酸残基を、本発明のポリペプチド配列と比較することにより決定し得る。場合によっては、決定的なタンパク質同定のための更なる配列データが得られる。
【０２３６】
プロテオームのプロフィールは、ＳＥＣＰに特異的な抗体を用いてＳＥＣＰ発現レベルを定量することによっても作成可能である。或る実施態様では、マイクロアレイ上のエレメントとしてこれら抗体を用い、マイクロアレイをサンプルに曝して各アレイエレメントへのタンパク質結合レベルを検出することにより、タンパク質発現レベルを定量する（Ｌｕｅｋｉｎｇ， Ａ． 他 （１９９９） Ａｎａｌ． Ｂｉｏｃｈｅｍ． ２７０：１０３−１１１、Ｍｅｎｄｏｚｅ， Ｌ．Ｇ． 他 （１９９９） Ｂｉｏｔｅｃｈｎｉｑｕｅｓ ２７：７７８−７８８）。検出は当分野で既知の様々な方法で行うことができ、例えば、チオール反応性またはアミノ反応性蛍光化合物とサンプル中のタンパク質を反応させ、各アレイエレメントにおける蛍光結合の量を検出し得る。
【０２３７】
プロテオームレベルでの毒性シグネチャも中毒学的スクリーニングに有用であり、転写レベルでの毒性シグネチャと並行に分析するべきである。いくつかの組織のいくつかのタンパク質については、転写物の存在量とタンパク質の存在量との相関が乏しいので（Ａｎｄｅｒｓｏｎ， Ｎ．Ｌ． および Ｊ． Ｓｅｉｌｈａｍｅｒ（１９９７）Ｅｌｅｃｔｒｏｐｈｏｒｅｓｉｓ １８：５３３−５３７）、転写イメージにはそれ程影響しないがプロテオームのプロフィールを改変するような化合物の分析において、プロテオーム毒性シグネチャは有用たり得る。更に、体液中の転写物の分析はｍＲＮＡの急速な分解のために困難なので、プロテオームのプロフィール作成はこのような場合により信頼し得、情報価値があり得る。
【０２３８】
別の実施例では、タンパク質を含有する生物学的サンプルを試験化合物で処理することにより試験化合物の毒性を算定する。処理された生物学的サンプル中で発現したタンパク質は、各タンパク質の量を定量し得るように分離する。各タンパク質の量を、未処理生物学的サンプル中の対応するタンパク質の量と比較する。両サンプルのタンパク質の量の差は、処理サンプル中の試験化合物に対する毒性反応を標示する。個々のタンパク質は、個々のタンパク質のアミノ酸残基をシークエンシングし、これら部分配列を本発明のポリペプチドと比較することにより同定する。
【０２３９】
別の実施例では、タンパク質を含有する生物学的サンプルを試験化合物で処理することにより試験化合物の毒性を算定する。生物学的サンプルから得たタンパク質は、本発明のポリペプチドに特異的な抗体を用いてインキュベートする。抗体により認識されたタンパク質の量を定量する。処理された生物学的サンプル中のタンパク質の量を、未処理生物学的サンプル中のタンパク質の量と比較する。両サンプルのタンパク質の量の差は、処理サンプル中の試験化合物に対する毒性反応を標示する。
【０２４０】
マイクロアレイは、本技術分野で既知の方法で調製し、使用し、分析する（例えばＢｒｅｎｎａｎ， Ｔ．Ｍ． 他（１９９５）米国特許第５，４７４，７９６号、Ｓｃｈｅｎａ， Ｍ． 他（１９９６）Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ ９３：１０６１４−１０６１９、Ｂａｌｄｅｓｃｈｗｅｉｌｅｒ 他（１９９５）ＰＣＴ出願第ＷＯ９５／２５１１１６号、Ｓｈａｌｏｎ， Ｄ．他（１９９５）ＰＣＴ出願第ＷＯ９５／３５５０５号、Ｈｅｌｌｅｒ， Ｒ．Ａ． 他（１９９７）Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ ９４：２１５０−２１５５、Ｈｅｌｌｅｒ， Ｍ．Ｊ． 他（１９９７）米国特許第５，６０５，６６２号を参照）。様々なタイプのマイクロアレイが公知であり、詳細については、ＤＮＡ Ｍｉｃｒｏａｒｒａｙｓ： Ａ Ｐｒａｃｔｉｃａｌ Ａｐｐｒｏａｃｈ， Ｍ． Ｓｃｈｅｎａ， 編集 （１９９９） Ｏｘｆｏｒｄ Ｕｎｉｖｅｒｓｉｔｙ Ｐｒｅｓｓ， Ｌｏｎｄｏｎに記載がある。該文献は、特に引用を以って本明細書の一部となす。
【０２４１】
本発明の別の実施例ではまた、ＳＥＣＰをコードする核酸配列を用いて、天然のゲノム配列をマッピングするのに有用なハイブリダイゼーションプローブを作製することが可能である。コード配列または非コード配列のいずれかを用いることができ、いくつかの例では、コード配列より非コード配列が好ましい。例えば、多重遺伝子族のメンバー内でのコード配列の保存により、染色体マッピング中に望ましくないクロスハイブリダイゼーションが生じる可能性がある。配列は、或る特定の染色体に、または或る染色体の或る特定領域に、または人為形成の染色体、例えば、ヒト人工染色体（ＨＡＣ）、酵母人工染色体（ＹＡＣ）、細菌人工染色体（ＢＡＣ）、細菌Ｐ１産物、あるいは単一染色体ｃＤＮＡライブラリ群に対してマッピングされる（例えばＨａｒｒｉｎｇｔｏｎ， Ｊ．Ｊ． 他（１９９７）Ｎａｔ． Ｇｅｎｅｔ． １５：３４５−３５５； Ｐｒｉｃｅ， Ｃ．Ｍ．（１９９３）Ｂｌｏｏｄ Ｒｅｖ． ７：１２７−１３４； Ｔｒａｓｋ， Ｂ．Ｊ．（１９９１）Ｔｒｅｎｄｓ Ｇｅｎｅｔ． ７：１４９−１５４を参照）。一度マッピングすると、本発明の核酸配列群を用いて、例えば或る病状の遺伝を特定染色体領域の遺伝とまたは制限酵素断片長多型（ＲＦＬＰ）と相関させるような遺伝子連鎖地図を開発し得る（例えば、Ｌａｎｄｅｒ， Ｅ．Ｓ．およびＤ． Ｂｏｔｓｔｅｉｎ（１９８６）Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ ８３：７３５３−７３５７を参照）。
【０２４２】
蛍光原位置ハイブリッド形成法（ＦＩＳＨ）は、他の物理的および遺伝的地図データと相関し得る（例えば前出のＨｅｉｎｚ−Ｕｌｒｉｃｈ， 他（１９９５）ｉｎ Ｍｅｙｅｒｓ， ９６５−９６８ページを参照）。遺伝地図データの例は、種々の科学雑誌あるいはＯｎｌｉｎｅ Ｍｅｎｄｅｌｉａｎ Ｉｎｈｅｒｉｔａｎｃｅ ｉｎ Ｍａｎ（ＯＭＩＭ）のウェブサイトに見られる。物理的地図上の、ＳＥＣＰをコードする遺伝子の位置と、特定の疾患との相関性、あるいは特定の疾患に対する素因との相関性は、この疾患と関連するＤＮＡの領域の決定に役立ち得るため、位置を決定するクローニングの作業を促進し得る。
【０２４３】
確定した染色体マーカー類を用いた連鎖分析などの物理的マッピング技術、および染色体標本の原位置ハイブリッド形成法を用いて、遺伝地図を拡張することができる。例えばマウスなど別の哺乳類の染色体上に遺伝子を配置することにより、正確な染色体上の遺伝子座が未知でも、関連するマーカー類をしばしば明らかにし得る。この情報は、位置クローニングなどの遺伝子発見技術を用いて疾患遺伝子を探る研究者にとって価値がある。いったん疾患または症候群に関与する遺伝子（群）が、血管拡張性失調症の１１ｑ２２−２３領域など、特定のゲノム領域への遺伝的連鎖によって、大まかに位置決めがなされると、該領域にマップされるいかなる配列も、更なる調査のための関連遺伝子あるいは調節遺伝子を表し得る（例えばＧａｔｔｉ， Ｒ．Ａ．他（１９８８）Ｎａｔｕｒｅ ３３６：５７７−５８０を参照）。転座、反転などに起因する、健常者、保有者、罹病者の三者間における染色体位置の相違を検出する場合にも、本発明のヌクレオチド配列を用い得る。
【０２４４】
本発明の別の実施態様では、ＳＥＣＰ、その触媒作用断片あるいは免疫原性断片またはそのオリゴペプチドを、種々の任意の薬剤スクリーニング技術における、化合物のライブラリ類のスクリーニングに用い得る。薬剤スクリーニングに用いる断片は、溶液中に遊離しているか、固体支持物に固定されるか、細胞表面上に保持されるか、細胞内に位置することができる。ＳＥＣＰと検査する薬剤との結合による複合体の形成を測定してもよい。
【０２４５】
別の薬剤スクリーニング方法は、目的のタンパク質に対して好適な結合親和性を有する化合物を高い処理能力でスクリーニングするために用いられる（例えばＧｅｙｓｅｎ他（１９８４）ＰＣＴ出願番号 ＷＯ８４／０３５６４を参照）。この方法においては、多数の様々な低分子の試験用化合物を固体基板上で合成する。試験用化合物は、ＳＥＣＰ、あるいはその断片と反応してから洗浄される。次に、結合したＳＥＣＰを本技術分野で周知の方法で検出する。精製したＳＥＣＰはまた、上記した薬剤スクリーニング技術に用いるプレート上に直接コーティングできる。別法では、非中和抗体を用いてペプチドを捕捉し、ペプチドを固体支持物に固定することもできる。
【０２４６】
別の実施態様では、競合的薬剤スクリーニングアッセイを用い、ＳＥＣＰと特異結合可能な中和抗体をＳＥＣＰとの結合について試験化合物と競合させ得る。この方法では、抗体を用いて、１つ以上の抗原決定基をＳＥＣＰと共有するどのペプチドの存在をも検出できる。
【０２４７】
別の実施態様では、ＳＥＣＰをコードするヌクレオチド配列を、将来に開発される分子生物学技術であり、現在知られているヌクレオチド配列の特性（限定はされないが、例えばトリプレット遺伝コード、特異的な塩基対相互作用等）に依存する新技術に用い得る。
【０２４８】
更に詳細説明をしなくとも、当業者であれば以上の説明を以って本発明を最大限に利用できるであろう。したがって、これ以下に記載する実施例は単なる例示目的にすぎず、いかようにも本発明を限定するものではない。
【０２４９】
本明細書において開示した全ての特許、特許出願および刊行物、特に米国特許出願第６０／２４７，６４２号、第６０／２４９，８２４号、第６０／２５２，８２４号、第６０／２４７，５０５号、第６０／２５４，３０５号および第６０／２５６，４４８号は、言及することを以て本明細書の一部となす。
【０２５０】
（実施例）
１ ｃＤＮＡ ライブラリの作製
Ｉｎｃｙｔｅ ｃＤＮＡ群の由来は、ＬＩＦＥＳＥＱ ＧＯＬＤデータベース（Ｉｎｃｙｔｅ Ｇｅｎｏｍｉｃｓ， Ｐａｌｏ Ａｌｔｏ ＣＡ）に記載されたｃＤＮＡライブラリ群であり、表４の列５に列記した。幾つかの組織はホモジナイズしてグアニジニウムイソチオシアネート溶液に溶解し、他の組織はホモジナイズしてフェノールにまたは変性剤群の好適な混合液に溶解した。混合液の１例であるＴＲＩＺＯＬ（Ｌｉｆｅ Ｔｅｃｈｎｏｌｏｇｉｅｓ）は、フェノールとグアニジンイソチオシアネートとの単相溶液である。結果として得た溶解物は、塩化セシウムのクッション液の上に重層して遠心分離するか、クロロホルムで抽出した。イソプロパノールか、酢酸ナトリウムとエタノールか、いずれか一方、あるいは別の慣例的方法で、溶解物からＲＮＡを沈殿させた。
【０２５１】
ＲＮＡの純度を高めるため、フェノールによるＲＮＡの抽出および沈殿を、必要な回数繰り返した。場合によっては、ＤＮアーゼでＲＮＡを処理した。殆どのライブラリでは、オリゴｄ（Ｔ）連結常磁性粒子（Ｐｒｏｍｅｇａ）、ＯＬＩＧＯＴＥＸラテックス粒子（ＱＩＡＧＥＮ， Ｃｈａｔｓｗｏｒｔｈ ＣＡ）またはＯＬＩＧＯＴＥＸ ｍＲＮＡ精製キット（ＱＩＡＧＥＮ）を用いて、ポリ（Ａ）＋ＲＮＡを単離した。別法では、別のＲＮＡ単離キット、例えばＰＯＬＹ（Ａ）ＰＵＲＥ ｍＲＮＡ精製キット（Ａｍｂｉｏｎ， Ａｕｓｔｉｎ ＴＸ）を用いて、組織溶解物からＲＮＡを直接単離した。
【０２５２】
場合によってはＳｔｒａｔａｇｅｎｅ社にＲＮＡを提供し、対応するｃＤＮＡライブラリ群をＳｔｒａｔａｇｅｎｅ社が作製した。そうでない場合は、ＵＮＩＺＡＰベクターシステム（Ｓｔｒａｔａｇｅｎｅ）またはＳＵＰＥＲＳＣＲＩＰＴプラスミドシステム（Ｌｉｆｅ Ｔｅｃｈｎｏｌｏｇｉｅｓ）を用いて本技術分野で既知の推奨方法または類似の方法でｃＤＮＡを合成し、ｃＤＮＡライブラリを作製した（前出のＡｕｓｕｂｅｌ， １９９７， ５．１−６．６ユニットなどを参照）。逆転写は、オリゴｄ（Ｔ）またはランダムプライマーを用いて開始した。合成オリゴヌクレオチドアダプターを二本鎖ｃＤＮＡに連結反応させ、好適な制限酵素または酵素群でｃＤＮＡを消化した。殆どのライブラリに対して、ｃＤＮＡのサイズ選択（３００〜１０００ｂｐ）は、ＳＥＰＨＡＣＲＹＬ Ｓ１０００、ＳＥＰＨＡＲＯＳＥ ＣＬ２ＢまたはＳＥＰＨＡＲＯＳＥ ＣＬ４Ｂカラムクロマトグラフィー（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ）、あるいは調製用アガロースゲル電気泳動法を用いて行った。ｃＤＮＡは好適なプラスミドのポリリンカーの、適合する制限酵素部位にライゲーションされた。好適なプラスミドは、例えばＰＢＬＵＥＳＣＲＩＰＴプラスミド（Ｓｔｒａｔａｇｅｎｅ）、ＰＳＰＯＲＴ１プラスミド（Ｌｉｆｅ Ｔｅｃｈｎｏｌｏｇｉｅｓ）ＰＣＤＮＡ２．１プラスミド（Ｉｎｖｉｔｒｏｇｅｎ， Ｃａｒｌｓｂａｄ ＣＡ）、ＰＢＫ−ＣＭＶプラスミド（Ｓｔｒａｔａｇｅｎｅ）、ＰＣＲ２−ＴＯＰＯＴＡプラスミド（Ｉｎｖｉｔｒｏｇｅｎ）、ＰＣＭＶ−ＩＣＩＳプラスミド（Ｓｔｒａｔａｇｅｎｅ）、ｐＩＧＥＮ（Ｉｎｃｙｔｅ Ｇｅｎｏｍｉｃｓ， Ｐａｌｏ Ａｌｔｏ ＣＡ）またはｐｌＮＣＹ（Ｉｎｃｙｔｅ Ｇｅｎｏｍｉｃｓ）、またはこれらの誘導体である。組換えプラスミドは、Ｓｔｒａｔａｇｅｎｅ社のＸＬ１−Ｂｌｕｅ、ＸＬ１−ＢＩｕｅＭＲＦまたはＳＯＬＲ、あるいはＬｉｆｅ Ｔｅｃｈｎｏｌｏｇｉｅｓ社のＤＨ５α、ＤＨ１０ＢまたはＥｌｅｃｔｒｏＭＡＸ ＤＨ１０Ｂなど適格な大腸菌細胞に形質転換した。
【０２５３】
２ ｃＤＮＡ クローンの単離
実施例１のようにして得たプラスミドの、宿主細胞からの回収は、ＵＮＩＺＡＰベクターシステム（Ｓｔｒａｔａｇｅｎｅ）を用いたｉｎ ｖｉｖｏ切除によって、あるいは細胞溶解によって行った。プラスミド群の精製には、次の内、少なくとも１つを用いた。ＭａｇｉｃまたはＷＩＺＡＲＤ Ｍｉｎｉｐｒｅｐｓ ＤＮＡ精製システム（Ｐｒｏｍｅｇａ）、ＡＧＴＣ Ｍｉｎｉｐｒｅｐ精製キット（Ｅｄｇｅ Ｂｉｏｓｙｓｔｅｍｓ， Ｇａｉｔｈｅｒｓｂｕｒｇ ＭＤ）、ＱＩＡＧＥＮ社のＱＩＡＷＥＬＬ ８ Ｐｌａｓｍｉｄ、ＱＩＡＷＥＬＬ ８ Ｐｌｕｓ ＰｌａｓｍｉｄおよびＱＩＡＷＥＬＬ ８ Ｕｌｔｒａ Ｐｌａｓｍｉｄ 精製システムまたはＲ．Ｅ．Ａ．Ｌ． Ｐｒｅｐ ９６プラスミド精製キットである。沈殿させた後、プラスミドを０．１ｍｌの蒸留水に再懸濁して、凍結乾燥して或いは凍結乾燥せずに、４℃で保管した。
【０２５４】
別法では、高処理フォーマットで直接結合ＰＣＲ法を用い、宿主細胞溶解物からプラスミドＤＮＡを増幅した（Ｒａｏ， Ｖ．Ｂ．（１９９４）Ａｎａｌ． Ｂｉｏｃｈｅｍ． ２１６：１−１４）。宿主細胞の溶解および熱サイクリング過程は、単一反応混合液中で行った。サンプルを加工し、それを３８４穴プレート内で保管し、増幅したプラスミドＤＮＡの濃度をＰＩＣＯＧＲＥＥＮ色素（Ｍｏｌｅｃｕｌａｒ Ｐｒｏｂｅｓ， Ｅｕｇｅｎｅ ＯＲ）およびＦＬＵＯＲＯＳＫＡＮＩＩ蛍光スキャナ（Ｌａｂｓｙｓｔｅｍｓ Ｏｙ， Ｈｅｌｓｉｎｋｉ， Ｆｉｎｌａｎｄ）を用いて蛍光分析的に定量した。
【０２５５】
３ シークエンシングおよび分析
実施例２に記載したようにプラスミドから回収したＩｎｃｙｔｅ ｃＤＮＡを、以下に示すようにシークエンシングした。ｃＤＮＡのシークエンス反応は、標準的方法あるいは高処理装置、例えばＡＢＩ ＣＡＴＡＬＹＳＴ ８００ サーマルサイクラー（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）またはＰＴＣ−２００ サーマルサイクラー（ＭＪ Ｒｅｓｅａｒｃｈ）を、ＨＹＤＲＡマイクロディスペンサー（Ｒｏｂｂｉｎｓ Ｓｃｉｅｎｔｉｆｉｃ）またはＭＩＣＲＯＬＡＢ ２２００（Ｈａｍｉｌｔｏｎ）液体転移システムと併用して処理した。ｃＤＮＡのシークエンス反応は、Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ社が提供する試薬、またはＡＢＩシークエンシングキット、例えばＡＢＩ ＰＲＩＳＭ ＢＩＧＤＹＥ Ｔｅｒｍｉｎａｔｏｒ ｃｙｃｌｅ ｓｅｑｕｅｎｃｉｎｇ ｒｅａｄｙ ｒｅａｃｔｉｏｎ ｋｉｔ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）の試薬を用いて準備した。ｃＤＮＡのシークエンス反応の電気泳動的分離には、また、標識したポリヌクレオチドの検出には、ＭＥＧＡＢＡＣＥ １０００ ＤＮＡシークエンシングシステム（Ｍｏｌｅｃｕｌａｒ Ｄｙｎａｍｉｃｓ）か、標準ＡＢＩプロトコルと塩基対呼び出しソフトウェアとを用いるＡＢＩ ＰＲＩＳＭ ３７３または３７７シークエンシングシステム（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）か、あるいはその他の本技術分野で既知の配列解析システムを用いた。ｃＤＮＡ配列内のリーディングフレームは、標準的方法（前出のＡｕｓｕｂｅｌ， １９９７， ７．７ユニットに概説）を用いて同定した。いくつかのｃＤＮＡ配列を選択し、実施例８に開示する技術で配列を伸長させた。
【０２５６】
Ｉｎｃｙｔｅ ｃＤＮＡに由来するポリヌクレオチド配列は、ベクター、リンカーおよびポリ（Ａ）配列を除去し、あいまいな塩基をマスクすることによって有効性を確認した。その際、ＢＬＡＳＴと、動的プログラミングと、隣接ジヌクレオチド頻度分析とに基づく、アルゴリズムとプログラムとを用いた。次に、Ｉｎｃｙｔｅ ｃＤＮＡ配列またはそれらの翻訳の問い合わせを、以下のデータベース群に対して行った。すなわち、選抜した公共のデータベース群（例えばＧｅｎＢａｎｋの霊長類、げっ歯類、哺乳類、脊椎動物、真核生物のデータベースと、ＢＬＯＣＫＳ、ＰＲＩＮＴＳ、ＤＯＭＯ、ＰＲＯＤＯＭ）と、ヒト、ラット、マウス、線虫（Ｃａｅｎｏｒｈａｂｄｉｔｉｓ ｅｌｅｇａｎｓ）、出芽酵母（Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ）、分裂酵母（Ｓｃｈｉｚｏｓａｃｃｈａｒｏｍｙｃｅｓ ｐｏｍｂｅ）およびＣａｎｄｉｄａ ａｌｂｉｃａｎｓからの配列群を持つＰＲＯＴＥＯＭＥデータベース群（Ｉｎｃｙｔｅ Ｇｅｎｏｍｉｃｓ， Ｐａｌｏ Ａｌｔｏ ＣＡ）、および、隠れマルコフモデル（ＨＭＭ）ベースのタンパク質ファミリーデータベース群（ＰＦＡＭ等）である（ＨＭＭは、遺伝子ファミリーのコンセンサス１次構造を分析する確率的アプローチである。例えば、Ｅｄｄｙ， Ｓ．Ｒ． （１９９６） Ｃｕｒｒ． Ｏｐｉｎ． Ｓｔｒｕｃｔ． Ｂｉｏｌ． ６：３６１−３６５を参照）。問合せは、ＢＬＡＳＴ、ＦＡＳＴＡ、ＢＬＩＭＰＳ、およびＨＭＭＥＲに基づくプログラムを用いて行った。Ｉｎｃｙｔｅ ｃＤＮＡ配列を構築し、完全長のポリヌクレオチド配列を産出した。あるいは、ＧｅｎＢａｎｋ ｃＤＮＡ群、ＧｅｎＢａｎｋ ＥＳＴ群、スティッチされた配列群、ストレッチされた配列群、またはＧｅｎｓｃａｎ予測コード配列群（実施例４および５を参照）を用い、Ｉｎｃｙｔｅ ｃＤＮＡの集団を完全長まで伸長させた。ＰｈｒｅｄとＰｈｒａｐとＣｏｎｓｅｄとに基づくプログラムを用いて構築し、ＧｅｎＭａｒｋとＢＬＡＳＴとＦＡＳＴＡとに基づくプログラムを用いて、ｃＤＮＡの集団を、オープンリーディングフレームについてスクリーニングした。完全長ポリヌクレオチド配列を翻訳し、対応する完全長ポリペプチド配列を誘導した。あるいは、本発明のポリペプチドは、完全長翻訳ポリペプチドの任意のメチオニン残基で開始し得る。完全長ポリペプチド配列群の続いての分析としての問い合わせを、ＧｅｎＢａｎｋタンパク質データベース群（ｇｅｎｐｅｐｔ）、ＳｗｉｓｓＰｒｏｔ、ＰＲＯＴＥＯＭＥデータベース群、ＢＬＯＣＫＳ、ＰＲＩＮＴＳ、ＤＯＭＯ、ＰＲＯＤＯＭおよびＰｒｏｓｉｔｅ等のデータベースや、ＰＦＡＭ等の隠れマルコフモデル（ＨＭＭ）ベースのタンパク質ファミリーデータベース群に対し行った。完全長ポリヌクレオチド配列はまた、ＭＡＣＤＮＡＳＩＳ ＰＲＯソフトウェア（日立ソフトウェアエンジニアリング， Ｓｏｕｔｈ Ｓａｎ Ｆｒａｎｃｉｓｃｏ ＣＡ）およびＬＡＳＥＲＧＥＮＥソフトウェア（ＤＮＡＳＴＡＲ）を用いて分析した。ポリヌクレオチドとポリペプチドとの配列アラインメントは、ＭＥＧＡＬＩＧＮマルチシークエンスアラインメントプログラム（ＤＮＡＳＴＡＲ）に組み込まれたＣＬＵＳＴＡＬアルゴリズムが指定する、デフォルトパラメータを用いて作製する。これは、アラインメントした配列間の一致率も計算する。
【０２５７】
表７は、Ｉｎｃｙｔｅ ｃＤＮＡと完全長配列との分析と構築とに利用したツールとプログラムとアルゴリズムとの概略と、適用可能な説明、参照文献、閾値パラメータを示す。用いたツール、プログラムおよびアルゴリズムを表７の列１に、それらの簡単な説明を列２に示す。列３は好適な参照文献であり、全ての文献は引用を以って全文を本明細書の一部となす。適用可能な場合には、列４は、２つの配列が一致する強さを評価するために用いた、スコア、確率値などのパラメータを示す（スコアが高いほど、または確率値が低いほど、２配列間の同一性が高い）。
【０２５８】
完全長ポリヌクレオチド配列群とポリペプチド配列群との構築と分析とに用いた上記プログラム群は、ＳＥＱ ＩＤ ＮＯ：６４−１２６のポリヌクレオチド配列断片群の同定にも利用した。ハイブリダイゼーション技術と増幅技術とに有用な約２０〜約４０００ヌクレオチドの断片群を、表４の列４に示した。
【０２５９】
４ ゲノム ＤＮＡ からのコード配列の同定および編集
推定上の分泌タンパク質類の同定には、先ずＧｅｎｓｃａｎ遺伝子同定プログラムを、公共のゲノム配列データベース（例えば、ｇｂｐｒｉやｇｂｈｔｇ）に対して実行した。Ｇｅｎｓｃａｎは汎用遺伝子同定プログラムであり、様々な生物からのゲノムＤＮＡ配列を分析する（Ｂｕｒｇｅ， Ｃ． および Ｓ． Ｋａｒｌｉｎ（１９９７）Ｊ． Ｍｏｌ． Ｂｉｏｌ． ２６８：７８−９４、Ｂｕｒｇｅ， Ｃ． および Ｓ． Ｋａｒｌｉｎ（１９９８）Ｃｕｒｒ． Ｏｐｉｎ． Ｓｔｒｕｃｔ． Ｂｉｏｌ． ８：３４６−３５４参照）。このプログラムは予測されたエキソン群を連結し、メチオニンから終止コドンに及ぶ、構築されたｃＤＮＡ配列を形成する。Ｇｅｎｓｃａｎの出力は、ポリヌクレオチドおよびポリペプチド配列のＦＡＳＴＡデータベースである。Ｇｅｎｓｃａｎが一度に分析する配列の最大範囲は、３０ｋｂに設定した。これらのＧｅｎｓｃａｎ予測ｃＤＮＡ配列の内、どの配列が分泌タンパク質をコードするかを決定するために、コードされるポリペプチドをＰＦＡＭモデル群に対し分泌タンパク質について問合せて分析した。潜在的な分泌タンパク質はまた、既に分泌タンパク質として注釈が付けられたＩｎｃｙｔｅ ｃＤＮＡ配列群に対する相同性により、同定した。これら選択したＧｅｎｓｃａｎ予測配列を、次にＢＬＡＳＴ分析により、公共データベースｇｅｎｐｅｐｔおよびｇｂｐｒｉと比較した。必要であれば、ｇｅｎｐｅｐｔからのトップＢＬＡＳＴヒットと比較することによりＧｅｎｓｃａｎ予測配列を編集し、余分なまたは省略されたエキソンなど、Ｇｅｎｓｃａｎが予測した配列におけるエラーを補正した。ＢＬＡＳＴ分析はまた、Ｇｅｎｓｃａｎ予測配列の、いかなるＩｎｃｙｔｅ ｃＤＮＡまたは公共ｃＤＮＡカバレッジ（ｃｏｖｅｒａｇｅ）の発見にも用いられ、したがって転写の証拠を提供した。Ｉｎｃｙｔｅ ｃＤＮＡカバレッジが利用できた場合には、この情報を用いてＧｅｎｓｃａｎ予測配列を補正または確認した。完全長ポリヌクレオチド配列は、実施例３に記載した構築プロセスを用いて、Ｉｎｃｙｔｅ ｃＤＮＡ配列および／または公共ｃＤＮＡ配列でＧｅｎｓｃａｎ予測コード配列を構築して得た。あるいは、完全長ポリヌクレオチド配列は、編集後または非編集のＧｅｎｓｃａｎ予測コード配列に、完全に由来する。
【０２６０】
５ ｃＤＮＡ 配列データを使ったゲノム配列データの構築
スティッチ配列（ Ｓｔｉｃｈｅｄ Ｓｅｑｕｅｎｃｅ ）
部分ｃＤＮＡ配列群を伸長させるため、実施例４に記載のＧｅｎｓｃａｎ遺伝子同定プログラムが予測したエキソン群を用いた。実施例３に記載したように構築した部分ｃＤＮＡ群を、ゲノムＤＮＡにマッピングし、また、関連するｃＤＮＡ群と、１つ以上のゲノム配列からＧｅｎｓｃａｎ予測されたエキソン群とを有するクラスター群に分解した。ｃＤＮＡとゲノムとの情報を統合すべく、グラフ理論および動的プログラミングに基づく或るアルゴリズムを用いて各クラスターを分析し、潜在的スプライス変異配列群を生成した。配列群を続いて確認、編集または伸長し、完全長配列を創出した。区間の全長が、２つ以上の配列に在るような配列区間群をクラスター内で同定し、そのように同定された区間群は、推移性により、等しいと考えた。例えば、或る区間が、１つのｃＤＮＡと２つのゲノム配列とに在る場合、３つの区間は全て等しいと考えた。このプロセスにより、無関係だが連続したゲノム配列群を、ｃＤＮＡ配列で結び合わせて架橋し得る。このようにして同定した区間を、それらの親配列（ｐａｒｅｎｔ ｓｅｑｕｅｎｃｅｓ）に沿って現われる順にスティッチアルゴリズムで「縫い合わせ」、可能な限り最長の配列と変異配列群とを生成した。１種類の親配列に沿って続く区間間の連鎖（ｃＤＮＡ−ｃＤＮＡまたはゲノム配列−ゲノム配列）は、親の種類を変える連鎖（ｃＤＮＡ−ゲノム配列）より優先した。結果として得たスティッチ配列群を翻訳し、ＢＬＡＳＴ分析で公共データベースｇｅｎｐｅｐｔおよびｇｂｐｒｉと比較した。Ｇｅｎｓｃａｎが予測した不正確なエキソン群は、ｇｅｎｐｅｐｔからのトップＢＬＡＳＴヒットとの比較により補正した。必要な場合、追加ｃＤＮＡ配列群を用いるかゲノムＤＮＡの検査により、配列群を更に伸長させた。
【０２６１】
ストレッチ配列（ Ｓｔｒｅｔｃｈｅｄ Ｓｅｑｕｅｎｃｅ ）
部分ＤＮＡ配列群を、ＢＬＡＳＴ分析に基づく１アルゴリズムにより完全長まで伸長した。先ず、ＢＬＡＳＴプログラムを用いて、ＧｅｎＢａｎｋの霊長類、げっ歯類、哺乳類、脊椎動物および真核生物のデータベースなどの公共データベースに対し、実施例３に記載されたように構築された部分ｃＤＮＡを問い合わせた。次に、最も近いＧｅｎＢａｎｋタンパク質相同体を、ＢＬＡＳＴ分析により、Ｉｎｃｙｔｅ ｃＤＮＡ配列または実施例４に記載のＧｅｎＳｃａｎエキソン予測配列のいずれかと比較した。結果として得られる高スコアリングセグメント対（ＨＳＰ）を用いてキメラタンパク質を産出し、翻訳した配列をＧｅｎＢａｎｋタンパク質相同体上にマッピングした。元のＧｅｎＢａｎｋタンパク質相同体に対し、キメラタンパク質内では挿入または欠失が起こり得る。ＧｅｎＢａｎｋタンパク質相同体、キメラタンパク質またはその両方をプローブとして用い、公共のヒトゲノムデータベースから相同ゲノム配列を検索した。このようにして、部分的なＤＮＡ配列を、相同ゲノム配列の付加によりストレッチすなわち伸長した。結果として得られるストレッチ配列を検査し、完全遺伝子を含んでいるか否かを決定した。
【０２６２】
６ ＳＥＣＰをコードするポリヌクレオチドの染色体マッピング
ＳＥＱ ＩＤ ＮＯ：６４−１２６を構築するために用いた配列群を、ＢＬＡＳＴ他のＳｍｉｔｈ−Ｗａｔｅｒｍａｎアルゴリズムの実装群（ｉｍｐｌｅｍｅｎｔａｔｉｏｎｓ）を用いて、Ｉｎｃｙｔｅ ＬＩＦＥＳＥＱデータベースおよび公共ドメインデータベース群の配列群と比較した。これらのデータベースからの配列群の内、ＳＥＱ ＩＤ ＮＯ：６４−１２６と一致した配列を、Ｐｈｒａｐなどの構築アルゴリズム（表７）を使用して、連続しオーバーラップした配列群のクラスター群に組み入れた。スタンフォード・ヒトゲノムセンター（ＳＨＧＣ）、ホワイトヘッド・ゲノム研究所（ＷＩＧＲ）、Ｇｅｎｅｔｈｏｎなどの公的な情報源から入手可能な放射線ハイブリッドおよび遺伝地図データを用いて、いずれかのクラスター化された配列が既にマッピングされているかを判定した。マッピングされた配列が或るクラスターに含まれている場合、そのクラスターの全配列が、個々の配列番号と共に、地図上の位置に割り当てられた。
【０２６３】
地図上の位置は、ヒト染色体の範囲または間隔として表される。センチモルガン間隔の地図上の位置は、染色体のｐアームの末端に対して測定する（センチモルガン（ｃＭ）は、染色体マーカー間の組換え頻度に基づく計測単位である。平均して、１ｃＭは、ヒトＤＮＡの１メガベース（Ｍｂ）にほぼ等しい。尤も、この値は、組換えのホットスポットおよびコールドスポットに起因して広範囲に変化する）。ｃＭ距離は、各クラスタ内に配列が含まれる放射線ハイブリッドマーカー類に対して境界を提供するＧｅｎｅｔｈｏｎによってマッピングされた遺伝マーカー群に基づく。ＮＣＢＩ「ＧｅｎｅＭａｐ’９９」（ｈｔｔｐ：／／ｗｗｗ．ｎｃｂｉ．ｎｌｍ．ｎｉｈ．ｇｏｖ／ｇｅｎｅｍａｐ／）など一般個人が入手可能なヒトゲノム地図などの資源を用いて、既に同定された疾患遺伝子類が、上記の区間内若しくは近傍にマップされているかを判定できる。
【０２６４】
７ ポリヌクレオチド発現の分析
ノーザン分析は、遺伝子の転写物の存在を検出するために用いられる実験技術であり、特定の細胞種または組織からのＲＮＡが結合している膜への、標識されたヌクレオチド配列のハイブリダイゼーションに関与する（例えば前出のＳａｍｂｒｏｏｋ， ７章、同Ａｕｓｕｂｅｌ （１９９５） ４章および１６章を参照）。
【０２６５】
ＢＬＡＳＴを応用した類似のコンピュータ技術を用いて、ＧｅｎＢａｎｋやＬＩＦＥＳＥＱ（Ｉｎｃｙｔｅ Ｇｅｎｏｍｉｃｓ）などのｃＤＮＡデータベースにおいて、同一または関連分子を検索した。ノーザン分析は、いくつかの膜系ハイブリダイゼーションよりも非常に速い。更に、任意の特定の一致を厳密なあるいは相同的なものとして分類するか否かを決定するため、コンピュータ検索の感度を修正することができる。検索の基準は積スコアであり、次式で定義される。
【０２６６】
【数１】

積スコアは、２つの配列間の類似度と、配列が一致する長さとの両方を考慮している。積スコアは、０〜１００の正規化された値であり、次のようにして求める。ＢＬＡＳＴスコアにヌクレオチドの配列一致率を乗じ、その積を２つの配列の短い方の長さの５倍で除する。ＢＬＡＳＴスコアを計算するには、或る高スコアリングセグメント対（ＨＳＰ）内の一致する各塩基に＋５のスコアを割り当て、各不一致塩基に−４を割り当てる。２つの配列は、２以上のＨＳＰを共有し得る（ギャップにより隔離される）。２以上のＨＳＰがある場合には、最高ＢＬＡＳＴスコアのセグメント対を用いて積スコアを計算する。積スコアは、断片的オーバーラップとＢＬＡＳＴアラインメントの質とのバランスを表す。例えば積スコア１００は、比較した２つの配列の短い方の長さ全体にわたって１００％一致する場合のみ得られる。積スコア７０は、一端が１００％一致し、７０％オーバーラップしているか、他端が８８％一致し、１００％オーバーラップしているかのいずれかの場合に得られる。積スコア５０は、一端が１００％一致し、５０％オーバーラップしているか、７９％一致し、１００％オーバーラップしているかのいずれかの場合に得られる。
【０２６７】
あるいは、ＳＥＣＰをコードするポリヌクレオチド配列は、由来する組織源について分析する。例えば幾つかの完全長配列は、少なくとも一部は、オーバーラップするＩｎｃｙｔｅ ｃＤＮＡ配列群を用いて構築される（実施例３を参照）。各ｃＤＮＡ配列は、ヒト組織から作製されたｃＤＮＡライブラリに由来する。各ヒト組織は、以下の臓器／組織カテゴリーの１つに分類される。すなわち心血管系、結合組織、消化器系、胎芽構造、内分泌系、外分泌腺、女性生殖器、男性生殖器、生殖細胞、血液および免疫系、肝、筋骨格系、神経系、膵臓、呼吸器系、感覚器、皮膚、顎口腔系、非分類性／混合性または尿路である。各カテゴリーのライブラリ数を数えて、全カテゴリーの総ライブラリ数で除する。同様に、各ヒト組織は、以下の疾患／病状カテゴリーすなわち癌、細胞株、発達、炎症、神経性、外傷、心血管、プール、その他の１つに分類される。各カテゴリーのライブラリ数を数えて、全カテゴリーの総ライブラリ数で除する。得られるパーセンテージは、ＳＥＣＰをコードするｃＤＮＡの、組織特異的または疾患特異的な発現を反映する。ｃＤＮＡ配列およびｃＤＮＡライブラリ／組織の情報は、ＬＩＦＥＳＥＱ ＧＯＬＤ データベース（Ｉｎｃｙｔｅ Ｇｅｎｏｍｉｃｓ， Ｐａｌｏ Ａｌｔｏ ＣＡ）から得ることができる。
【０２６８】
８ ＳＥＣＰをコードするポリヌクレオチドの伸長
完全長のポリヌクレオチド配列はまた、完全長分子の適切な断片から設計したオリゴヌクレオチドプライマー群を用いて該断片を伸長させて産生した。或るプライマーは既知の断片の５’伸長を開始するべく合成し、別のプライマーは既知の断片の３’伸長を開始するべく合成した。開始プライマー群の設計にはＯＬＩＧＯ ４．０６ソフトウェア（Ｎａｔｉｏｎａｌ Ｂｉｏｓｃｉｅｎｃｅｓ）或いは別の適切なプログラムを用い、長さが約２２〜３０ヌクレオチド、ＧＣ含有率が約５０％以上となり、約６８〜約７２℃の温度で標的配列にアニーリングするようにした。ヘアピン構造およびプライマー−プライマー二量体を生ずるようなヌクレオチド群の伸長は、全て回避した。
【０２６９】
選択したヒトｃＤＮＡライブラリ群を用い、配列を伸長した。２段階以上の伸長が必要または望ましい場合には、付加的プライマーあるいはプライマーのネステッドセットを設計した。
【０２７０】
高忠実度の増幅が、当業者によく知られている方法を利用したＰＣＲ法によって得られた。ＰＣＲは、ＰＴＣ−２００ サーマルサイクラー（ＭＪ Ｒｅｓｅａｒｃｈ， Ｉｎｃ．）を用いて９６穴プレート内で行った。反応混合液は、鋳型ＤＮＡを有し、また、２００ ｎｍｏｌの各プライマーを有する。また、Ｍｇ^{２ ＋}と（ＮＨ_４）_２ＳＯ_４と２−メルカプトエタノールとを含有する反応バッファーと、Ｔａｑ ＤＮＡポリメラーゼ（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ）と、ＥＬＯＮＧＡＳＥ酵素（Ｌｉｆｅ Ｔｅｃｈｎｏｌｏｇｉｅｓ）と、Ｐｆｕ ＤＮＡポリメラーゼ（Ｓｔｒａｔａｇｅｎｅ）とを含有する。プライマー対であるＰＣＩ ＡとＰＣＩ Ｂとに対し、以下のパラメータで増幅した。
ステップ１ ９４℃で３分間
ステップ２ ９４℃で１５秒間
ステップ３ ６０℃で１分間
ステップ４ ６８℃で２分間
ステップ５ ステップ２、３、及び４を２０回繰り返す
ステップ６ ６８℃で５分間
ステップ７ ４℃で保存
別法では、プライマー対であるＴ７とＳＫ＋に対し、以下のパラメータで増幅した。
ステップ１ ９４℃で３分間
ステップ２ ９４℃で１５秒間
ステップ３ ５７℃で１分間
ステップ４ ６８℃で２分間
ステップ５ ステップ２、３、及び４を２０回繰り返す
ステップ６ ６８℃で５分間
ステップ７ ４℃で保存
各ウェルのＤＮＡ濃度は、１× ＴＥおよび０．５μｌの希釈していないＰＣＲ産物に溶解した１００μｌのＰＩＣＯＧＲＥＥＮ定量試薬（０．２５％（ｖ／ｖ） ＰＩＣＯＧＲＥＥＮ； Ｍｏｌｅｃｕｌａｒ Ｐｒｏｂｅｓ， Ｅｕｇｅｎｅ ＯＲ）を不透明な蛍光光度計プレート（Ｃｏｒｎｉｎｇ Ｃｏｓｔａｒ， Ａｃｔｏｎ ＭＡ）の各ウェルに分配し、ＤＮＡが試薬と結合できるようにして測定した。サンプルの蛍光を計測してＤＮＡの濃度を定量すべく、プレートをＦｌｕｏｒｏｓｋａｎ ＩＩ（Ｌａｂｓｙｓｔｅｍｓ Ｏｙ， Ｈｅｌｓｉｎｋｉ， Ｆｉｎｌａｎｄ）でスキャンした。反応混合物のアリコット５〜１０μｌを１％アガロースゲル上で電気泳動法によって解析し、どの反応が配列の伸長に成功したかを判定した。
【０２７１】
伸長したヌクレオチドは、脱塩および濃縮して３８４穴プレートに移し、ＣｖｉＪＩコレラウイルスエンドヌクレアーゼ（Ｍｏｌｅｃｕｌａｒ Ｂｉｏｌｏｇｙ Ｒｅｓｅａｒｃｈ， Ｍａｄｉｓｏｎ ＷＩ）を用いて消化し、音波処理またはせん断し、ｐＵＣ １８ベクター（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ）への再連結を行った。ショットガン・シークエンシングのために、消化したヌクレオチドを低濃度（０．６〜０．８％）のアガロースゲル上で分離し、断片を切除し、寒天をＡｇａｒ ＡＣＥ（Ｐｒｏｍｅｇａ）で消化した。伸長させたクローンをＴ４リガーゼ（Ｎｅｗ Ｅｎｇｌａｎｄ Ｂｉｏｌａｂｓ， Ｂｅｖｅｒｌｙ ＭＡ）を用いてｐＵＣ １８ベクター（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ）に再連結し、Ｐｆｕ ＤＮＡポリメラーゼ（Ｓｔｒａｔａｇｅｎｅ）で処理して制限部位のオーバーハングを満たし、適格な大腸菌細胞に形質移入した。形質移入した細胞群の選択を抗生物質を含む培地で行い、それぞれのコロニーを採取し、ＬＢ／２Ｘカルベニシリン培養液中の３８４ウェルプレート群に３７℃で一晩培養した。
【０２７２】
細胞を溶解し、Ｔａｑ ＤＮＡポリメラーゼ（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ）およびＰｆｕ ＤＮＡポリメラーゼ（Ｓｔｒａｔａｇｅｎｅ）を用いて以下の手順でＤＮＡをＰＣＲ増幅した。
ステップ１ ９４℃で３分間
ステップ２ ９４℃で１５秒間
ステップ３ ６０℃で１分間
ステップ４ ７２℃で２分間
ステップ５ ステップ２、３、及び４を２９回繰り返す
ステップ６ ７２℃で５分間
ステップ７ ４℃で保存
ＤＮＡの定量化には、上記したようにＰＩＣＯＧＲＥＥＮ試薬（Ｍｏｌｅｃｕｌａｒ Ｐｒｏｂｅｓ）を用いた。ＤＮＡの回収率が低いサンプルは、上記と同一の条件を用いて再増幅した。サンプルは２０％ジメチルスルホキシド（１：２， ｖ／ｖ）で希釈し、ＤＹＥＮＡＭＩＣ エネルギートランスファー シークエンシングプライマー、およびＤＹＥＮＡＭＩＣ ＤＩＲＥＣＴ ｋｉｔ（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ）またはＡＢＩ ＰＲＩＳＭ ＢＩＧＤＹＥ ターミネーターサイクル シークエンシングレディ反応キット（Ｔｅｒｍｉｎａｔｏｒ ｃｙｃｌｅ ｓｅｑｕｅｎｃｉｎｇ ｒｅａｄｙ ｒｅａｃｔｉｏｎ ｋｉｔ）（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）を用いてシークエンシングした。
【０２７３】
同様に、上記手順を用いて完全長ポリヌクレオチド配列を検証した。あるいは、完全長ポリヌクレオチドを用い、上記手順で、そのような伸長のために設計したオリゴヌクレオチド類と、或る適切なゲノムライブラリとを用いて５’調節配列を得た。
【０２７４】
９ 個々のハイブリダイゼーションプローブの標識及び使用
ＳＥＱ ＩＤ ＮＯ：６４−１２６由来のハイブリダイゼーションプローブ群を用いて、ｃＤＮＡ、ｍＲＮＡ、またはゲノムＤＮＡをスクリーニングする。約２０塩基対からなるオリゴヌクレオチドの標識について特に記載するが、より大きなヌクレオチド断片に対しても本質的に同一の手順が用いられる。オリゴヌクレオチドは、ＯＬＩＧＯ ４．０６ソフトウェア（Ｎａｔｉｏｎａｌ Ｂｉｏｓｃｉｅｎｃｅｓ）等の最新ソフトウェアを用いて設計し、各オリゴマー５０ｐｍｏｌと、［γ−^３２Ｐ］アデノシン３リン酸（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ）２５０μＣｉと、Ｔ４ポリヌクレオチドキナーゼ（ＤｕＰｏｎｔ ＮＥＮ， Ｂｏｓｔｏｎ ＭＡ）とを混合することにより標識する。標識したオリゴヌクレオチドは、ＳＥＰＨＡＤＥＸ Ｇ−２５超細繊分子サイズ排除デキストラン ビーズカラム（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ）を用いて実質的に精製する。Ａｓｅ Ｉ、Ｂｇｌ ＩＩ、Ｅｃｏ ＲＩ、Ｐｓｔ Ｉ、ＸｂａＩまたはＰｖｕ ＩＩ（ＤｕＰｏｎｔ ＮＥＮ）のいずれか１つのエンドヌクレアーゼで消化されたヒトゲノムＤＮＡの、典型的な膜ベースのハイブリダイゼーション解析において、毎分１０^７カウントの標識されたプローブを含むアリコットを用いる。
【０２７５】
各消化物から得たＤＮＡは、０．７％アガロースゲル上で分画してナイロン膜（Ｎｙｔｒａｎ Ｐｌｕｓ， Ｓｃｈｌｅｉｃｈｅｒ ＆ Ｓｃｈｕｅｌｌ， Ｄｕｒｈａｍ ＮＨ）に移す。ハイブリダイゼーションは、４０℃で１６時間行う。非特異的シグナル群を除去するため、最大で例えば０．１×クエン酸ナトリウム食塩水および０．５％ドデシル硫酸ナトリウムの条件下で、ブロット群を室温で順次洗浄する。オートラジオグラフィーまたはそれに代わるイメージング手段を用いてハイブリダイゼーションパターンを視覚化し、比較する。
【０２７６】
１０ マイクロアレイ
或るマイクロアレイ上でのアレイエレメント群の結合または合成は、フォトリソグラフィ、圧電印刷（インクジェット印刷、前出のＢａｌｄｅｓｃｈｗｅｉｌｅｒなどを参照）、機械的マイクロスポッティング技術およびこれらから派生した技術を用いて達成し得る。上記各技術において基板は、均一な、非多孔性の表面を持つ固体とすべきである（Ｓｃｈｅｎａ（１９９９）前出）。推奨する基板には、シリコン、シリカ、スライドガラス、ガラスチップおよびシリコンウエハがある。あるいは、ドットブロット法またはスロットブロット法に類似した手順を利用し、熱的、紫外線的、化学的または機械的結合手順を用いて基板の表面にエレメントを配置および結合させてもよい。通常のアレイは、利用可能な、当業者に公知の方法と機械とを用いて作製でき、任意の適正数のエレメントを有し得る（例えばＳｃｈｅｎａ， Ｍ． 他（１９９５）Ｓｃｉｅｎｃｅ ２７０：４６７−４７０、Ｓｈａｌｏｎ． Ｄ． 他（１９９６）Ｇｅｎｏｍｅ Ｒｅｓ． ６：６３９−６４５、Ｍａｒｓｈａｌｌ， Ａ． および Ｊ． Ｈｏｄｇｓｏｎ（１９９８）Ｎａｔ． Ｂｉｏｔｅｃｈｎｏｌ． １６：２７−３１を参照）。
【０２７７】
完全長ｃＤＮＡ、発現配列タグ（ＥＳＴ）、またはそれらの断片またはオリゴマーが、マイクロアレイのエレメントと成り得る。ハイブリダイゼーションに好適な断片またはオリゴマーを、ＬＡＳＥＲＧＥＮＥソフトウェア（ＤＮＡＳＴＡＲ）など本技術分野で公知のソフトウェアを用いて選択することが可能である。アレイエレメント群を、生物学的サンプル中のポリヌクレオチド群とハイブリダイズする。生物学的サンプル中のポリヌクレオチドは、検出を容易にするために蛍光標識などの分子タグに抱合させる。ハイブリダイゼーション後、生物学的サンプルからのハイブリダイズされていないヌクレオチドを除去し、蛍光スキャナを用いて各アレイエレメントでのハイブリダイゼーションを検出する。あるいは、レーザ脱離および質量スペクトロメトリを用いてもハイブリダイゼーションを検出し得る。マイクロアレイ上の或るエレメントにハイブリダイズする各ポリヌクレオチドの、相補性の度合と相対存在度とを算定し得る。 一実施態様におけるマイクロアレイの調整および使用について、以下に詳述する。
【０２７８】
組織または細胞サンプルの調製
全ＲＮＡを組織サンプル群から単離するためグアニジニウム チオシアネート法を用い、ポリ（Ａ）^＋ＲＮＡを精製するためオリゴ（ｄＴ）セルロース法を用いる。各ポリ（Ａ）^＋ＲＮＡサンプルを逆転写するため、ＭＭＬＶ逆転写酵素を用い、また、０．０５ｐｇ／μｌのオリゴ（ｄＴ）プライマー（２１ｍｅｒ）、１×第一鎖バッファー、０．０３ｕｎｉｔ／μｌのＲＮアーゼ阻害因子、５００μＭのｄＡＴＰ、５００μＭのｄＧＴＰ、５００μＭのｄＴＴＰ、４０μＭのｄＣＴＰ、４０μＭのｄＣＴＰ−Ｃｙ３（ＢＤＳ）またはｄＣＴＰ−Ｃｙ５（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ）を用いる。逆転写反応は、ＧＥＭＢＲＩＧＨＴキット（Ｉｎｃｙｔｅ）を用い、２００ｎｇのポリ（Ａ）^＋ＲＮＡを含有する体積２５ｍｌで行う。特異的対照ポリ（Ａ）^＋ＲＮＡ群は、非コード酵母ゲノムＤＮＡからｉｎ ｖｉｔｒｏ転写により合成する。３７℃で２時間インキュベートした後、各反応サンプル（１つはＣｙ３、もう１つはＣｙ５標識）は、２．５ｍｌの０．５Ｍ水酸化ナトリウムで処理し、８５℃で２０分間インキュベートし、反応を停止させてＲＮＡを分解させる。サンプル群の精製には、２つの連続するＣＨＲＯＭＡ ＳＰＩＮ ３０ゲル濾過スピンカラム（ＣＬＯＮＴＥＣＨ Ｌａｂｏｒａｔｏｒｉｅｓ， Ｉｎｃ． （ＣＬＯＮＴＥＣＨ）， Ｐａｌｏ Ａｌｔｏ ＣＡ）を用いる。混合後、２つの反応サンプルのエタノール沈殿を、１ｍｌのグリコーゲン（１ｍｇ／ｍｌ）、６０ｍｌの酢酸ナトリウム、および３００ｍｌの１００％エタノールで行う。サンプルは次にＳｐｅｅｄＶＡＣ（Ｓａｖａｎｔ Ｉｎｓｔｒｕｍｅｎｔｓ Ｉｎｃ．， Ｈｏｌｂｒｏｏｋ ＮＹ）を用いて完全に乾燥させ、１４μｌの５×ＳＳＣ／０．２％ＳＤＳ中で再懸濁する。
【０２７９】
マイクロアレイの調製
本発明の配列を用いて、アレイエレメントを作製する。各アレイエレメントは、クローン化したｃＤＮＡインサート群により、ベクター含有細菌細胞群から増幅する。ＰＣＲ増幅は、ｃＤＮＡインサートの側面に位置するベクター配列群に相補的なプライマー類を用いる。３０サイクルのＰＣＲによって、１〜２ｎｇの初期量から５μｇを超える最終量まで、アレイエレメントを増幅する。増幅したアレイエレメントは、ＳＥＰＨＡＣＲＹＬ−４００（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ）を用いて精製する。
【０２８０】
精製したアレイエレメントは、ポリマーコートされたスライドグラス上に固定する。顕微鏡スライドグラス（Ｃｏｒｎｉｎｇ）は、０．１％のＳＤＳおよびアセトン中で超音波洗浄し、処理中および処理後に多量の蒸留水で洗浄する。スライドグラスは、４％フッ化水素酸（ＶＷＲ Ｓｃｉｅｎｔｉｆｉｃ Ｐｒｏｄｕｃｔｓ Ｃｏｒｐｏｒａｔｉｏｎ（ＶＷＲ）， Ｗｅｓｔ Ｃｈｅｓｔｅｒ ＰＡ）中でエッチングし、多量の蒸留水中で洗浄し、９５％エタノール中で０．０５％アミノプロピルシラン（Ｓｉｇｍａ）を用いてコーティングする。コーティングしたスライドは、１１０℃のオーブンで硬化させる。
【０２８１】
コーティングしたガラス基板にアレイエレメント群を付加する手順は、米国特許第５，８０７，５２２号に記載されている。この特許は、引用を以って本明細書の一部とする。平均濃度１００ｎｇ／μｌのアレイエレメントＤＮＡ１μｌを、高速ロボット装置（ｒｏｂｏｔｉｃ ａｐｐａｒａｔｕｓ）により、開放型キャピラリープリンティングエレメント（ｏｐｅｎ ｃａｐｉｌｌａｒｙ ｐｒｉｎｔｉｎｇ ｅｌｅｍｅｎｔ）に充填する。装置はここで、スライド毎に約５ｎｌのアレイエレメントサンプルを置く。
【０２８２】
マイクロアレイをＵＶ架橋するため、ＳＴＲＡＴＡＬＩＮＫＥＲ ＵＶ架橋剤（Ｓｔｒａｔａｇｅｎｅ）を用いる。マイクロアレイの洗浄を、室温において、０．２％ＳＤＳで１回、蒸留水で３回行う。非特異結合部位をブロックするため、マイクロアレイのインキュベートをリン酸緩衝生理食塩水（ＰＢＳ）（Ｔｒｏｐｉｘ， Ｉｎｃ．， Ｂｅｄｆｏｒｄ ＭＡ）中の０．２％カゼイン中において６０℃で３０分間行った後、前に行ったように０．２％ＳＤＳおよび蒸留水で洗浄する。
【０２８３】
ハイブリダイゼーション
ハイブリダイゼーション反応に用いる９μｌのサンプル混合体には、Ｃｙ３またはＣｙ５で標識したｃＤＮＡ合成産物群の各０．２μｇを、５×ＳＳＣ，０．２％ＳＤＳハイブリダイゼーション緩衝液中に含む。サンプル混合体は、６５℃まで５分間加熱し、マイクロアレイ表面上で等分して１．８ｃｍ^２のカバーガラスで覆う。アレイを、顕微鏡用スライドよりわずかに大きい空洞を有する防水チェンバーに移す。チェンバー内を湿度１００に保持するため、１４０μｌの５×ＳＳＣをチェンバーの或るコーナーに加える。アレイを含むチェンバーは、６０℃で約６．５時間インキュベートする。アレイは、第１洗浄緩衝液中（１×ＳＳＣ，０．１％ＳＤＳ）において４５℃で１０分間洗浄し、第２洗浄緩衝液中（０．１×ＳＳＣ）において４５℃で１０分間ずつ３回洗浄して乾燥させる。
【０２８４】
検出
レポーター標識したハイブリダイゼーション複合体を検出するには、Ｃｙ３の励起のために４８８ｎｍ、Ｃｙ５の励起のために６３２ｎｍでスペクトル線を発生し得るＩｎｎｏｖａ７０混合ガス１０Ｗレーザ（Ｃｏｈｅｒｅｎｔ， Ｉｎｃ．， Ｓａｎｔａ Ｃｌａｒａ ＣＡ）を備えた顕微鏡を用いる。励起レーザ光の焦点をアレイ上に置くため、２０×顕微鏡対物レンズ（Ｎｉｋｏｎ， Ｉｎｃ．， Ｍｅｌｖｉｌｌｅ ＮＹ）を用いる。アレイを含むスライドを、顕微鏡の、コンピュータ制御のＸ−Ｙステージに置き、対物レンズを通してラスタースキャンする。本実施例で用いる１．８ｃｍ×１．８ｃｍのアレイは、解像度２０μｍでスキャンする。
【０２８５】
異なる２回のスキャンで、混合ガスマルチラインレーザは２つの蛍光色素を連続的に励起する。発光された光は、波長に基づき分離され、２つの蛍光色素に対応する２つの光電子増倍管検出器（ＰＭＴ Ｒ１４７７， Ｈａｍａｍａｔｓｕ Ｐｈｏｔｏｎｉｃｓ Ｓｙｓｔｅｍｓ， Ｂｒｉｄｇｅｗａｔｅｒ ＮＪ）に送られる。好適なフィルター群をアレイと光電子増倍管との間に設置して、シグナルをフィルターする。用いる蛍光色素の最大発光の波長は、Ｃｙ３では５６５ｎｍ、Ｃｙ５では６５０ｎｍである。装置は両方の蛍光色素からのスペクトルを同時に記録し得るが、レーザ源において好適なフィルターを用いて、蛍光色素１つにつき１度スキャンし、各アレイを通常２度スキャンする。
【０２８６】
通常、各スキャンの感度を較正するため、ｃＤＮＡ対照種を或る既知濃度でサンプル混合体に添加し、対照種が発生するシグナル強度を用いる。アレイ上の或る特定の位置には或る相補的ＤＮＡ配列が含まれ、その位置におけるシグナルの強度をハイブリダイゼーション種の重量比１：１００，０００で相関させる。異なる源泉（例えば代表的な試験細胞および対照細胞など）からの２つのサンプルを、各々異なる蛍光色素で標識し、異なる発現をする遺伝子群を同定するために単一のアレイにハイブリダイズする場合には、較正するｃＤＮＡのサンプルを２種の蛍光色素で標識し、ハイブリダイゼーション混合液に各々等量を加えることによって較正を行う。
【０２８７】
光電子増倍管の出力をディジタル化するため、ＩＢＭコンパチブルＰＣコンピュータにインストールした１２ビットＲＴＩ−８３５Ｈアナログディジタル（Ａ／Ｄ）変換ボード（Ａｎａｌｏｇ Ｄｅｖｉｃｅｓ， Ｉｎｃ．， Ｎｏｒｗｏｏｄ ＭＡ）を用いる。ディジタル化したデータは、青色（低シグナル）から赤色（高シグナル）までの擬似カラースケールへのリニア２０色変換を用いて、シグナル強度がマッピングされたイメージとして表示される。データは、定量的にも分析される。２つの異なる蛍光色素を同時に励起および測定する場合には、各蛍光体の発光スペクトルを用いて、データは先ず蛍光色素間の光学的クロストーク（発光スペクトルの重なりに起因する）を補正される。
【０２８８】
グリッドが蛍光シグナルイメージ上に重ねられ、それによって各スポットからのシグナルはグリッドの各エレメントに集められる。各エレメント内の蛍光シグナルは統合され、シグナルの平均強度に応じた数値が得られる。シグナル分析に用いるソフトウェアは、ＧＥＭＴＯＯＬＳ遺伝子発現分析プログラム（Ｉｎｃｙｔｅ）である。
【０２８９】
１１ 相補的ポリヌクレオチド
ＳＥＣＰをコードする配列あるいはその任意の一部に対して相補的な配列は、天然ＳＥＣＰの発現を検出、低減または阻害するために用いられる。約１５〜３０塩基対を含むオリゴヌクレオチドの使用について記すが、これより小さなあるいは大きな配列の断片の場合でも、本質的に同じ手順を用いる。適切なオリゴヌクレオチドを設計するため、Ｏｌｉｇｏ ４．０６ソフトウェア（Ｎａｔｉｏｎａｌ Ｂｉｏｓｃｉｅｎｃｅｓ）およびＳＥＣＰのコーディング配列を用いる。転写を阻害するためには、最も独特な５’配列から相補的オリゴヌクレオチドを設計し、これを用いて、プロモーターがコーディング配列に結合するのを防止する。翻訳を阻害するには、相補的なオリゴヌクレオチドを設計して、リボソームがＳＥＣＰをコードする転写物に結合するのを防ぐ。
【０２９０】
１２ ＳＥＣＰの発現
ＳＥＣＰの発現および精製は、細菌若しくはウイルスを基にした発現系を用いて行うことができる。細菌内でＳＥＣＰを発現させるためには、抗生物質耐性遺伝子およびｃＤＮＡの転写レベルを高める誘導性のプロモーターを含む好適なベクターにｃＤＮＡをサブクローニングする。このようなプロモーターとしては、ｌａｃオペレーター調節エレメントに関連するＴ５またはＴ７バクテリオファージプロモーターおよびｔｒｐ−ｌａｃ（ｔａｃ）ハイブリッドプロモーターが含まれるが、これらに限定するものではない。組換えベクターを、ＢＬ２１（ＤＥ３）などの好適な細菌宿主に形質転換する。抗生物質耐性細菌は、イソプロピルβ−Ｄチオガラクトピラノシド（ＩＰＴＧ）で誘発されるとＳＥＣＰを発現する。真核細胞でのＳＥＣＰの発現は、昆虫細胞株または哺乳類細胞株に、一般にバキュロウイルスとして知られているＡｕ ｔｏｇｒａｐｈｉｃａ ｃａｌｉｆｏｒｎｉｃａ核多角体病ウイルス（ＡｃＭＮＰＶ）の組換え型を感染させて行う。バキュロウイルスの非必須ポリヘドリン遺伝子を、相同組換えによって、あるいは転移プラスミドの媒介を伴う細菌の媒介による遺伝子転移によって、ＳＥＣＰをコードするｃＤＮＡと置換する。ウイルスの感染力は維持され、強力な多角体プロモーターによって高レベルのｃＤＮＡ転写が行われる。組換えバキュロウイルスは、多くの場合は夜蛾の１種Ｓｐｏｄｏｐｔｅｒａ ｆｒｕｇｉｐｅｒｄａ（Ｓｆ９）昆虫細胞への感染に用いるが、ヒト肝細胞への感染に用いることもある。後者の感染の場合は、バキュロウイルスへの更なる遺伝的修飾が必要になる（Ｅｎｇｅｌｈａｒｄ． Ｅ． Ｋ．他（１９９４）Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ ９１：３２２４−３２２７、Ｓａｎｄｉｇ， Ｖ． 他（１９９６）Ｈｕｍ． Ｇｅｎｅ Ｔｈｅｒ． ７：１９３７−１９４５を参照）。
【０２９１】
殆どの発現系では、ＳＥＣＰが、例えばグルタチオンＳトランスフェラーゼ（ＧＳＴ）で、またはＦＬＡＧや６−Ｈｉｓなどのペプチドエピトープ標識で合成された融合タンパク質となるため、未精製の細胞溶解物からの組換え融合タンパク質の、親和性ベースの精製を迅速に１回で行うことができる。ＧＳＴは日本住血吸虫からの２６ｋＤａの酵素であり、タンパク質の活性および抗原性を維持した状態で、固定化したグルタチオン上での融合タンパク質の精製を可能とする（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ）。精製の後、ＧＳＴ部分を、特異的に操作した各部位でＳＥＣＰからタンパク質分解的に切断できる。ＦＬＡＧは８アミノ酸のペプチドであり、市販されているモノクローナルおよびポリクローナル抗ＦＬＡＧ抗体を用いて、免疫親和性精製を可能にする（Ｅａｓｔｍａｎ Ｋｏｄａｋ）。６ヒスチジン残基が連続して伸長した６−Ｈｉｓは、金属キレート樹脂上での精製を可能にする（ＱＩＡＧＥＮ）。タンパク質の発現および精製の方法は、前出のＡｕｓｕｂｅｌ（１９９５）１０章、１６章に記載されている。これらの方法で精製したＳＥＣＰを直接用いて以下の実施例１６、１７、および１８の適用可能なアッセイを行うことができる。
【０２９２】
１３ 機能的アッセイ
ＳＥＣＰの機能は、哺乳類細胞培養系において生理学的に高められたレベルでのＳＥＣＰをコードする配列の発現によって算定する。ｃＤＮＡを高いレベルで発現する強いプロモーターを含む哺乳類発現ベクターにｃＤＮＡをサブクローニングする。選択されるベクターとしては、ｐＣＭＶ ＳＰＯＲＴ（Ｌｉｆｅ Ｔｅｃｈｎｏｌｏｇｉｅｓ）およびｐＣＲ ３．１（Ｉｎｖｉｔｒｏｇｅｎ， Ｃａｒｌｓｂａｄ ＣＡ）があり、どちらもサイトメガロウイルスプロモーターを持つ。リポソーム製剤あるいは電気穿孔法を用いて、５〜１０μｇの組換えベクターをヒト細胞株、例えば内皮由来または造血由来の細胞株に、一過的に形質移入する。更に、標識タンパク質をコードする配列を含む１〜２μｇのプラスミドを同時に形質移入する。標識タンパク質の発現により、形質移入細胞と非形質移入細胞を区別する手段が与えられる。また、標識タンパク質の発現によって、組換えベクターからのｃＤＮＡ発現を正確に予想できる。標識タンパク質は、例えば緑色蛍光タンパク質（ＧＦＰ；Ｃｌｏｎｔｅｃｈ）、ＣＤ６４またはＣＤ６４−ＧＦＰ融合タンパク質から選択できる。自動化された、レーザー光学に基づく技術であるフローサイトメトリー（ＦＣＭ）を用いて、ＧＦＰまたはＣＤ６４−ＧＦＰを発現する形質移入された細胞を同定し、それらの細胞のアポトーシス状態や他の細胞特性を評価する。ＦＣＭは、細胞死に先行するかあるいは同時に発生する現象を診断する蛍光分子の取込を検出して計量する。このような現象として挙げられるのは、プロピジウムヨウ化物によるＤＮＡ染色によって計測される核ＤＮＡ内容物の変化、前方散乱光と９０°側方散乱光によって計測される細胞サイズと顆粒性の変化、ブロモデオキシウリジンの取込量の低下によって計測されるＤＮＡ合成の下方調節、特異抗体との反応性によって計測される細胞表面および細胞内におけるタンパク質の発現の変容、およびフルオレセイン抱合したアネキシンＶタンパク質の細胞表面への結合によって計測される原形質膜組成の変容がある。フローサイトメトリー法については、Ｏｒｍｅｒｏｄ， Ｍ． Ｇ．（１９９４）Ｆｌｏｗ Ｃｙｔｏｍｅｔｒｙ， Ｏｘｆｏｒｄ， Ｎｅｗ Ｙｏｒｋ， ＮＹに記述がある。
【０２９３】
遺伝子発現におけるＳＥＣＰの影響は、ＳＥＣＰをコードする配列とＣＤ６４またはＣＤ６４−ＧＦＰのどちらかが形質移入された高度に精製された細胞集団を用いて算定することができる。ＣＤ６４またはＣＤ６４−ＧＦＰは、形質転換された細胞表面で発現し、ヒト免疫グロブリンＧ（ＩｇＧ）の保存された領域と結合する。形質転換された細胞と形質転換されない細胞とは、ヒトＩｇＧかＣＤ６４に対する抗体のどちらかで被覆された磁気ビーズを用いて効率的に分離することができる（ＤＹＮＡＬ， Ｌａｋｅ Ｓｕｃｃｅｓｓ ＮＹ）。ｍＲＮＡは、当業者に周知の方法で細胞から精製することができる。ＳＥＣＰと、目的とする他の遺伝子とをコードするｍＲＮＡの発現は、ノーザン分析やマイクロアレイ技術で分析することができる。
【０２９４】
１４ ＳＥＣＰに特異的な抗体の作製
実質的に精製されたＳＥＣＰを、ポリアクリルアミドゲル電気泳動法（ＰＡＧＥ；例えば、Ｈａｒｒｉｎｇｔｏｎ， Ｍ．Ｇ．（１９９０）Ｍｅｔｈｏｄｓ Ｅｎｚｙｍｏｌ． １８２：４８８−４９５を参照）または他の精製技術で作製し、これを用いて標準的なプロトコルでウサギを免疫化して抗体を作り出す。
【０２９５】
あるいは、ＬＡＳＥＲＧＥＮＥソフトウェア（ＤＮＡＳＴＡＲ）を用いてＳＥＣＰアミノ酸配列を解析し、免疫原性の高い領域を決定する。そして対応するオリゴペプチドを合成し、このオリゴペプチドを用いて当業者によく知られている方法で抗体を生成する。Ｃ末端付近あるいは親水性領域などの、適切なエピトープの選択方法については、当分野で公知である（前出のＡｕｓｕｂｅｌ， １９９５， １１章などを参照）。
【０２９６】
通常は、長さ約１５残基のオリゴペプチドを、ＦＭＯＣケミストリを用いるＡＢＩ ４３１Ａ ペプチドシンセサイザ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）を用いて合成し、Ｎ−マレイミドベンゾイル−Ｎ−ヒドロキシスクシンイミドエステル（ＭＢＳ）を用いた反応によってＫＬＨ（Ｓｉｇｍａ−Ａｌｄｒｉｃｈ， Ｓｔ． Ｌｏｕｉｓ ＭＯ）に結合させて、免疫原性を高める（例えば前出のＡｕｓｕｂｅｌ， １９９５を参照）。完全フロイントアジュバントにおいて、オリゴペプチド−ＫＬＨ複合体を用いてウサギを免疫化する。得られた抗血清の抗ペプチド活性および抗ＳＥＣＰ活性を検査するため、例えば、ペプチドまたはＳＥＣＰを或る基板に結合し、１％ＢＳＡを用いてブロッキング処理し、ウサギ抗血清と反応させて洗浄し、更に、放射性ヨウ素標識したヤギ抗ウサギＩｇＧと反応させる。
【０２９７】
１５ 特異的抗体を用いる天然ＳＥＣＰの精製
天然ＳＥＣＰあるいは組換えＳＥＣＰを実質的に精製するため、ＳＥＣＰに特異的な抗体群を用いるイムノアフィニティークロマトグラフィを行う。イムノアフィニティーカラムは、ＣＮＢｒ−活性化ＳＥＰＨＡＲＯＳＥ（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ）などの、或る活性化したクロマトグラフィー用レジンと抗ＳＥＣＰ抗体とを共有結合させることにより構成する。結合後に、製造者の使用説明書に従ってこのレジンをブロックし、洗浄する。
【０２９８】
ＳＥＣＰを含む培養液をイムノアフィニティーカラムに通し、ＳＥＣＰを優先的に吸着できる条件で（例えば、界面活性剤の存在下において高イオン強度のバッファー類で）そのカラムを洗浄する。そのカラムを、抗体とＳＥＣＰとの結合を切るような条件で（例えば、或るｐＨ２〜３のバッファー、あるいは高濃度の、例えば尿素またはチオシアン酸イオンなどのカオトロープで）溶出させ、ＳＥＣＰを回収する。
【０２９９】
１６ ＳＥＣＰと相互作用する分子の同定
ＳＥＣＰまたは生物学的に活性なその断片を、^１２５Ｉボルトンハンター試薬で標識する（例えば Ｂｏｌｔｏｎ， Ａ．Ｅ．およびＷ．Ｍ． Ｈｕｎｔｅｒ（１９７３）Ｂｉｏｃｈｅｍ． Ｊ． １３３：５２９−５３９を参照）。マルチウェルプレートの各ウェルに予め配列しておいた候補の分子群を、標識したＳＥＣＰと共にインキュベートし、洗浄して、標識したＳＥＣＰ複合体を有する全てのウェルをアッセイする。様々なＳＥＣＰ濃度で得られたデータを用いて、候補分子と結合したＳＥＣＰの数量および親和性、会合についての値を計算する。
【０３００】
別法では、ＳＥＣＰと相互作用する分子を、Ｆｉｅｌｄｓ， Ｓ．およびＯ． Ｓｏｎｇ（１９８９， Ｎａｔｕｒｅ ３４０：２４５−２４６）に記載の酵母２−ハイブリッドシステム（ｙｅａｓｔ ｔｗｏ−ｈｙｂｒｉｄ ｓｙｓｔｅｍ）や、ＭＡＴＣＨＭＡＫＥＲシステム（Ｃｌｏｎｔｅｃｈ）などの２−ハイブリッドシステムに基づいた市販のキットを用いて分析する。
【０３０１】
ＳＥＣＰはまた、ハイスループット型の酵母２ハイブリッドシステムを使用するＰＡＴＨＣＡＬＬＩＮＧプロセス（ＣｕｒａＧｅｎ Ｃｏｒｐ．， Ｎｅｗ Ｈａｖｅｎ ＣＴ）に用いて、遺伝子の２つの大きなライブラリによってコードされるタンパク質間の全ての相互作用を決定することができる（Ｎａｎｄａｂａｌａｎ， Ｋ． 他 （２０００） 米国特許第６，０５７，１０１号）。
【０３０２】
１７ ＳＥＣＰ活性の実証
ＳＥＣＰのペルオキシダーゼ活性の測定には、分光光度アッセイ（例えばＪｅｏｎｇ， Ｍ． 他 （２０００） Ｊ． Ｂｉｏｌ． Ｃｈｅｍ． ２７５：２９２４−２９３０を参照）を用いる。または、アッセイキット、例えばＡＭＰＬＥＸ Ｒｅｄ Ｐｅｒｏｘｉｄａｓｅ Ａｓｓａｙ Ｋｉｔ（Ｍｏｌｅｃｕｌａｒ Ｐｒｏｂｅｓ）を用い、蛍光マイクロプレートリーダーまたは蛍光光度計を併用する。
【０３０３】
ＳＥＣＰの成長刺激活性若しくは成長阻害活性の為のアッセイでは、スイスマウス３Ｔ３細胞におけるＤＮＡ合成の量を測定する（ＭｃＫａｙ， Ｉ． および Ｌｅｉｇｈ， Ｉ．， ｅｄｓ．（１９９３）Ｇｒｏｗｔｈ Ｆａｃｔｏｒｓ ： Ａ Ｐｒａｃｔｉｃａｌ Ａｐｐｒｏａｃｈ， Ｏｘｆｏｒｄ Ｕｎｉｖｅｒｓｉｔｙ Ｐｒｅｓｓ， Ｎｅｗ Ｙｏｒｋ， ＮＹ）。このアッセイにおいては、可変量のＳＥＣＰを、静止状態３Ｔ３培養細胞に、放射性ＤＮＡ前駆物質である［^３Ｈ］チミジンの存在下で加える。このアッセイのためのＳＥＣＰを得る手段は、組み替えでも良く、生化学的な標本より得ても良い。酸−沈殿可能ＤＮＡへの［^３Ｈ］チミジンの混合は適切な時間間隔で測定し、混合量は、新規に合成されたＤＮＡの量に直接比例する。少なくとも１００倍のＳＥＣＰ濃度レンジにわたる一次の用量‐応答曲線は、成長モジュレート活性を示す。ミリリットルあたりの活性の１ユニットは、５０％の応答レベルを産生するＳＥＣＰの濃度として定義される。ここで、１００％の応答レベルとは、酸−沈殿可能ＤＮＡへの、［^３Ｈ］チミジンの最大の混合を表す。
【０３０４】
別法では、ＴＧＦ−β活性を、上皮成長因子２．５ ｎｇ／ｍｌの存在中で、非腫瘍性正常ラット腎臓線維芽細胞群を足場非依存性増殖に誘導することによって測定する。この方法の記載はＡｓｓｏｉａｎ， Ｒ．Ｋ． 他 （１９８３） Ｊ． Ｂｉｏｌ． Ｃｈｅｍ． ２５８：７１５５７１６０にある。
【０３０５】
別法では、ＳＥＣＰ活性のためのアッセイで、培養細胞中の神経伝達の、刺激作用若しくは抑制を測定する。培養ＣＨＯ繊維芽細胞をＳＥＣＰに曝す。エンドサイトーシスによるＳＥＣＰ取込後に、これらの細胞を新鮮培養液で洗浄し、細胞全膜電位固定したアフリカツメガエル筋細胞を、ＳＥＣＰを含まない媒質中の線維芽細胞の１つと接触させる。膜電流を、この筋細胞から記録する。対照値に対し増加若しくは減少した電流は、ＳＥＣＰの神経調節効果を示す（Ｍｏｒｉｍｏｔｏ， Ｔ．他（１９９５）Ｎｅｕｒｏｎ １５：６８９−６９６）。
【０３０６】
別法では、ＳＥＣＰ活性のアッセイで、分泌性の膜結合細胞小器官におけるＳＥＣＰの量を測定する。上述したように形質移入された細胞を採取し、溶解する。ライセートは、当業者に既知の、ショ糖密度勾配超遠心法などの方法で分画する。そのような方法は、ゴルジ体、ＥＲ、小膜結合小胞、およびその他の分泌細胞小器官のような、細胞内区画の単離を可能とする。分画した細胞ライセートおよび全体の細胞ライセートよりの免疫沈降は、ＳＥＣＰ特異抗体を用いて実行され、また免疫沈降サンプルは、ＳＤＳ−ＰＡＧＥおよび免疫ブロット技術を用いて解析される。全細胞ライセート中ＳＥＣＰに対する分泌オルガネラ中ＳＥＣＰ濃度は、分泌経路を通るＳＥＣＰの量に比例する。
【０３０７】
別法では、或るアッセイでＳＥＣＰのプロテインキナーゼ活性を測定するが、これは、γ標識した^３２Ｐ−ＡＴＰの存在下での、ＳＥＣＰによるタンパク質基質のリン酸化を定量化することで行う。ＳＥＣＰを、タンパク質基質、^３２Ｐ−ＡＴＰ、および或る適切なキナーゼバッファと共にインキュベートする。基質に組み込まれた^３２Ｐは、電気泳動法で遊離^３２Ｐ−ＡＴＰより分離され、組み込まれた^３２Ｐをラジオアイソトープカウンターでカウントする。組み込まれた^３２Ｐの量は、ＳＥＣＰの活性に比例する。リン酸化した特異的アミノ酸残基の判定を、加水分解したタンパク質のホスホアミノ酸解析で行う。
【０３０８】
別法では、ＡＭＰ結合活性の測定を、ＳＥＣＰと^３２Ｐ標識したＡＭＰとを混合して行う。この反応溶液を３７℃でインキュベートし、反応はトリクロロ酢酸の添加で終了する。酸抽出物を中和し、ゲル電気泳動にかけて非結合標識を除去する。ゲル内に留まる放射活性が、ＳＥＣＰの活性に比例する。
【０３０９】
１８ 免疫グロブリン活性の実証
ＳＥＣＰ活性の或るアッセイでは、ＳＥＣＰが血清からの抗原類を認識し沈殿させる能力を測定する。この活性の測定は、定量沈降反応で成し得る（Ｇｏｌｕｂ， Ｅ． Ｓ． 他（１９８７）Ｉｍｍｕｎｏｌｏｇｙ： Ａ Ｓｙｎｔｈｅｓｉｓ， Ｓｉｎａｕｅｒ Ａｓｓｏｃｉａｔｅｓ， Ｓｕｎｄｅｒｌａｎｄ， ＭＡ， １１３−１１５ページ）。ＳＥＣＰを、当分野で既知の方法で同位体標識する。一定量の標識ＳＥＣＰに種々の血清濃度を加える。ＳＥＣＰ−抗原複合体を溶液から沈殿させ、遠心分離で収集する。沈殿性ＳＥＣＰ−抗原複合体の量は、沈殿物中に検出される放射性同位元素の量に比例する。沈殿性ＳＥＣＰ−抗原複合体の量を、血清濃度に対してプロットする。多様な血清中濃度に対しての特徴的沈降素曲線が得られ、沈殿性ＳＥＣＰ−抗原複合体の量は、初め血清中濃度の増加に比例して増加し、当量点を頂点とし、その後は血清中濃度の増加に比例して減少する。このように、沈澱性ＳＥＣＰ−抗原複合体の量は、抗原の制限量と過剰量に対する感受性を特徴とするＳＥＣＰ活性の測定方法となる。
【０３１０】
別法として、ＳＥＣＰ活性の或るアッセイは、細胞表面におけるＳＥＣＰの発現を測定する。 ＳＥＣＰをコードするｃＤＮＡを、非白血球細胞株に形質移入する。細胞表面タンパク質類をビオチンで標識する（ｄｅ ｌａ Ｆｕｅｎｔｅ， Ｍ．Ａ． 他（１９９７）Ｂｌｏｏｄ ９０：２３９８−２４０５）。ＳＥＣＰ特異的抗体群を用いて免疫沈降を行い、免疫沈降したサンプルをＳＤＳ−ＰＡＧＥと免疫ブロット法で分析する。標識した免疫沈降剤と未標識免疫沈降剤の比は、細胞表面に発現したＳＥＣＰの量に比例する。
【０３１１】
別法で、ＳＥＣＰ活性の或るアッセイは、ＳＥＣＰの過剰発現が誘発する、細胞凝集の量を測定する。このアッセイにおいては、ＮＩＨ３Ｔ３などの培養細胞にＳＥＣＰをコードするｃＤＮＡで形質移入する。このｃＤＮＡは、或る強力なプロモーターの制御下にある或る適切な哺乳類発現ベクター内に含まれるようにする。緑色蛍光タンパク質（ＣＬＯＮＴＥＣＨ）などの蛍光標識タンパク質をコードするｃＤＮＡとの共形質移入を行うと、安定な形質移入体を同定するのに役立つ。形質移入された細胞と形質移入されない細胞で、細胞の凝集（塊化）量を比較する。細胞の凝集量により、ＳＥＣＰの活性を直接に測定できる。
【０３１２】
当業者には、本発明の要旨および精神から逸脱しない範囲での、記載した本発明の方法およびシステムの、種々の修正および変更の手段は自明であろう。本発明について説明するにあたり幾つかの実施例に関連して説明を行ったが、本発明の請求の範囲が、そのような特定の実施例に不当に制限されるべきではないことを理解されたい。分子生物学または関連分野の専門家には明らかな、本明細書に記載する本発明の実施方法の様々な修正は、明確に特許請求の範囲内にあるものとする。
【０３１３】
（表の簡単な説明）
表１は、本発明の完全長ポリヌクレオチド配列およびポリペプチド配列の命名法の概略を示す。
【０３１４】
表２は、本発明のポリペプチド群のＧｅｎＢａｎｋ識別番号と、最も近いＧｅｎＢａｎｋ相同体の注釈（ａｎｎｏｔａｔｉｏｎ）とを示す。また、各ポリペプチドとその相同体（１つ以上）が一致する確率スコアも併せて示す。
【０３１５】
表３は、予測されるモチーフおよびドメインなど、本発明のポリヌクレオチド配列の構造的特徴を、ポリペプチドの分析に用いる方法、アルゴリズムおよび検索可能なデータベースと共に示す。
【０３１６】
表４は、本発明のポリヌクレオチド配列を構築するために用いたｃＤＮＡ断片やゲノムＤＮＡ断片を、ポリヌクレオチド配列の選択した断片と共に示す。
【０３１７】
表５は、本発明のポリヌクレオチドの代表的なｃＤＮＡライブラリを示す。
【０３１８】
表６は、表５に示したｃＤＮＡライブラリの作製に用いた組織およびベクターを説明する付表である。
【０３１９】
表７は、本発明のポリヌクレオチドとポリペプチドの分析に用いたツール、プログラム、アルゴリズムを、適用可能な説明、引用文献および閾値パラメータと共に示す。
【表１】

【表２】

【表３】

【表４】

【表５】

【表６】

【表７】

【表８】

【表９】

【表１０】

【表１１】

【表１２】

【表１３】

【表１４】

【表１５】

【表１６】

【表１７】

【表１８】

【表１９】

【表２０】

【表２１】

【表２２】

【表２３】

【表２４】

【表２５】

【表２６】

【表２７】

【表２８】

【表２９】

【表３０】

【表３１】

【表３２】

【表３３】

【表３４】

【表３５】

【表３６】

【表３７】

【表３８】

【表３９】

【表４０】

【表４１】

【表４２】

【表４３】

[0001]
(Technical field)
The present invention relates to nucleic acid and amino acid sequences of secreted proteins. The present invention also relates to the diagnosis, treatment, and prevention of abnormal cell proliferation, autoimmune / inflammatory disorders, cardiovascular disorders, neurological disorders, and developmental disorders using these sequences. The invention further relates to calculating the effect of a foreign compound on the expression of nucleic acid and amino acid sequences of secreted proteins.
[0002]
(Background of the Invention)
Protein transport and secretion are essential for cellular function. Protein transport is mediated by a signal peptide at the amino terminus of the protein being transported or secreted. The signal peptide is composed of about 10 to 20 hydrophobic amino acids and directs nascent proteins from ribosomes to specific compartments surrounded by membranes, such as the endoplasmic reticulum (ER). Proteins that target the ER include those that go through the secretory pathway and those that remain in any secretory cell organ (eg, the ER, Golgi apparatus, or lysosome). Proteins that travel the secretory pathway are either secreted into the extracellular space or remain in the plasma membrane. Proteins retained in the plasma membrane have one or more transmembrane domains, each domain consisting of about 20 hydrophobic amino acid residues. Secreted proteins are usually synthesized as inactive precursors and are activated by post-translational processing events as they progress through the secretory pathway. Such events include glycosylation, proteolysis, and removal of signal peptides by signal peptidases. Other events that can occur during the transport of a protein include the chaperone-dependent unfolding and folding of a nascent protein and the interaction of the protein with a receptor or pore complex. Examples of secreted proteins having an amino-terminal signal peptide are described below, but some proteins have important roles in intercellular signaling. Such proteins include transmembrane receptors and cell surface markers, extracellular matrix molecules, cytokines, hormones, growth and differentiation factors, enzymes, neuropeptides, vasomediators, cell surface markers, and antigen recognition There are molecules (Alberts, B. et al. (1994)Molecular Biology of The Cell, Garland Publishing, New York, NY, 557-560, 582, 582-592).
[0003]
Cell surface markers include cell surface antigens identified on white blood cells of the immune system. To identify these antigens, a systematic, monoclonal antibody (mAb) based "shot gun" technique is utilized. With these techniques, hundreds of mAbs have been produced against unknown cell surface leukocyte antigens. Those antigens have been grouped into "clusters of differentiation". Classification is based on immunocytochemical localization patterns common to a variety of differentiated and undifferentiated leukocyte cell types. Antigens within a cluster are assumed to identify a single cell surface protein and have been designated a "differentiation cluster" or "CD (cluster of difference)" name. Several genes encoding proteins identified by CD antigens have been cloned and validated by standard molecular biology techniques. CD antigens are characterized by being transmembrane proteins and cell surface proteins that are anchored to the plasma membrane via covalent adhesion to fatty acid-containing glycolipids such as glycosylphosphatidylinositol (GPI). (Barclay, A. N. et al. (1995)The Leucocyte Antigen Facts Book, Academic Press, San Diego, CA, review on pages 17-20).
[0004]
Matrix proteins (MPs) are transmembrane and extracellular proteins that function in the formation, growth, remodeling, and maintenance of each tissue and serve as important mediators and regulators of the inflammatory response. Causes of biochemical changes that can disrupt the expression and balance of MPs include congenital, epigenetic, or infectious diseases. MPs also affect leukocyte migration, proliferation, differentiation, and activation in the immune response.
[0005]
Often, MPs are characterized by the presence of one or more domains, such domains can include collagen-like domains, EGF-like domains, immunoglobulin-like domains, and fibronectin-like domains. In addition, MPs can be heavily glycosylated. It may also have an arginine-glycine-aspartic acid (RGD) tripeptide motif, which may play a role in adhesive interactions. As MP, extracellular proteins include fibronectin, collagen, galectin, vitronectin and its proteolytic derivative somatomedin B, and cell adhesion receptors include cell adhesion molecules (CAM), cadherin, and integrins ( For a review, see Ayad, S. et al. (1994).The Extracellular Matrix Facts Book, Academic Press, San Diego, CA, pp. 2-16; Ruoslahti, E. et al. (1997) Kidney Int. 51: 1413-1417; Sjaastad, M .; D. And Nelson, W.C. J. (1997) BioEssays 19: 47-55). Peroxidasin is a Drosophila protein that has both a peroxidase motif and an extracellular matrix motif. The 1512 amino acid peroxidase protein has a peroxidase domain that is homologous to human myeloperoxidase and eosinophil peroxidase, six leucine-rich repeats, and four immunoglobulin domains. There are certain regions of thrombospondin / procollagen homology. The secretion of peroxidasin is performed by blood cells. These transmissions are throughout the developing Drosophila embryo. It is thought that this protein functions in extracellular matrix consolidation, phagocytosis, and defense (Nelson, RE (1994) EMBO J. 13: 3438-3347). Certain human homologs of the Drosophila peroxidasin gene have recently been discovered to be up-regulated in certain colon cancer cell lines that undergo p53 tumor suppressor-dependent apoptosis, and thus have a role. May play a variety of mechanisms in p53-dependent apoptosis (Horikoshi, N. et al. (1999) Biochem. Biophy. Res. Commun. 261: 864-869). Mucins are highly glycosylated glycoproteins and are the major structural components of mucus gels.
The physiological functions of mucins are cell protection, mechanical protection, retention of secretion viscosity, and cell recognition. MUC6 is a human gastric juice mucin, but has also been found in the gallbladder, pancreas, seminal vesicle, and female reproductive tract (Toribara, NW, et al. (1997) J. Biol. Chem. 272: 16398-16403). The MUC6 gene has been mapped to human chromosome 11 (Toribara, NW, et al. (1993) J. Biol. Chem. 268: 5879-5885). Hemomucin is a novel Drosophila surface mucin and may be involved in the induction of antimicrobial effector molecules (Theopold, U. et al. (1996) J. Biol. Chem. 217: 12708-12715).
[0006]
Tuftelins are one of the four enamel matrix proteins identified to date. The other three known enamel matrix proteins are amelogenin, enamelin and ameloblastin. Construction of an enamel extracellular matrix from these component proteins is believed to be important in producing a matrix that is eligible for mineral replacement (Paine CT et al. (1998) Connect Tissue Res. .38: 257-267). Tufterin mRNA has been found to be expressed in human non-mineralized odontogenic tumor, human ameloblastoma (Deutsch D. et al. (1998) Connect. Tissue Res. 39: 177-184).
[0007]
Olfactomedin-related proteins are secreted glycoproteins of the extracellular matrix and have a conserved group of C-terminal motifs. These proteins are nematodes (Caenorhabditis elegans) To humans in a wide variety of species ranging from humans. Olfactomedin-related proteins include a gene family having at least five family members in humans. One of the five, TIGR / myocillin protein, is expressed in the eye and is associated with the pathogenesis of glaucoma (Kulkarni, NH et al. (2000) Genet. Res. 76: 41-50. ). (1996) found that in a neuroblastoma cell line cDNA library, AMY, a 135 amino acid protein, had 96% sequence identity to the olfactomedin-related ER-localized protein in rat neurons. Was found, suggesting an important role for AMY in neural tissue (Yokoyama, M. et al. (1996) DNA Res. 3: 311-320). Nerve cell-specific olfactomedin-related glycoproteins isolated from rat brain cDNA libraries show strong sequence similarity with olfactomedin. This similarity suggests a matrix-related function of these glycoproteins in neurons and neurosecretory cells (Danielson, PE et al. (1994) J. Neurosci. Res. 38: 468-478). .
[0008]
The Mac-2 binding protein is a 90 kDa serum protein (90K), a secreted glycoprotein isolated from both human breast cancer cell line SK-BR-3 and human breast milk. 90K specifically binds to Mac-2, a human macrophage-related lectin. Structurally, the mature protein is 567 amino acids long, preceded by an 18 amino acid leader. There are 16 cysteines and 7 potential N-linked glycosylation sites. The first 106 amino acids represent a domain that is very similar to an older protein superfamily defined by a macrophage scavenger receptor cysteine-rich domain (Koths, K. et al. (1993) J. Biol. Chem. 268). : 14245-14249). 90K is elevated in the serum of a subpopulation of AIDS patients and is expressed at varying levels in primary tumor samples and tumor cell lines. (1994) demonstrated that 90K can stimulate multiple host defense systems and induce secretion of interleukin-2. The cause of this immune stimulation has been proposed to be oncogenic transformation, viral infection or pathogenic invasion (Ullrich, A., et al. (1994) J. Biol. Chem. 269: 18401-18407).
[0009]
Semaphorins are a large group of axon guidance molecules with at least 30 members, and have been found in vertebrates, invertebrates, and even several viruses. All semaphorins have a sema domain of about 500 amino acids in length. Neuropilin, a semaphorin receptor,in in vitroPromotes the formation of neurites. The extracellular region of neuropilin consists of three domains: CUB, discoidin, and the MAM domain. The CUB motif and MAM motif of neuropilin have been suggested to play several roles in protein-protein interactions and may be involved in the binding of semaphorins via the sema and C-terminal domains ( Raper, JA (2000) Curr. Opin. Neurobiol. 10: 8894).
Plexins are neuronal cell surface molecules that mediate cell adhesion by a homophilic binding mechanism in the presence of calcium ions. Plexins have been shown to be expressed in receptors of certain sensory systems and in neurons (Ohta, K. et al. (1995) Cell 14: 1189-1199). Evidence suggests that some plexins function to control motor and central axon guidance in the developing nervous system. Plexins themselves have a complete semaphorin domain and may be the ancestors of both classic semaphorins and binding partners of semaphorins (Winberg, ML et al. (1998)). Cell 95: 903-916).
[0010]
Human pregnancy-specific β1 glycoprotein (PSG) is a family of closely related glycoproteins with molecular weights of 72 KDa, 64 KDa, 62 KDa and 54 KDa. PSG, together with oncofetal antigens, constitutes a subfamily within the immunoglobulin superfamily (Plouzek CA and Chou JY (1991) Endocrinology 129: 950958). Various subpopulations of PSG have been found to be produced by vegetative germ layers of the human placenta and amniotic membrane and serosa (Plouzek CA et al. (1993) Placenta 14: 277285).
[0011]
Autocrine prokinetic factor (AMF) is one of the motility cytokines that regulates tumor cell migration, and thus the identification of the associated signaling pathways is critical. Autocrine motility factor receptor (AMFR) expression has been found to be associated with tumor progression in thymoma (Ohta Y. et al. (2000) Int. J. Oncol. 17: 259264). AMFR is a cell surface glycoprotein with a molecular weight of 78 KDa.
[0012]
Hormones are secreted molecules that are carried by the blood circulation and bind to specific receptors on or inside target cells. Hormones have diverse biochemical compositions and mechanisms of action, but can be divided into two categories. The first category is small lipophilic hormones, which diffuse through the plasma membrane of target cells, bind to cytosolic or nuclear receptors, form complexes and alter gene expression. These molecules include retinoic acid and thyroxine, and also cholesterol-derived steroid hormones such as progesterone, estrogen, testosterone, cortisol, and aldosterone. The second category of hydrophilic hormones functions by binding to cell surface receptors that transmit signals inside the plasma membrane. Examples of such hormones include catecholamines (epinephrine, norepinephrine) and histamine as amino acid derivatives, and glucagon, insulin, gastrin, secretin, cholecystokinin, adrenocorticotropic hormone, follicle stimulating hormone, luteinizing hormone as peptide hormones. There are hormones, thyroid-stimulating hormone, and vasopressin (Lodish et al. (1995)Molecular Cell Biology, Scientific American Books Inc. , New York, NY, pp. 856-864).
[0013]
Proopiomelanocortin (POMC) is a precursor polypeptide of corticotropin (ACTH), a hormone synthesized by the anterior pituitary gland, and functions in stimulating the adrenal cortex. POMC is also a precursor polypeptide of β-lipotropin hormone (β-LPH). Each hormone has a smaller group of peptides with unique biological activities. α-melanocyte stimulating hormone (α-MSH) and adrenocorticotropic hormone-like middle lobe peptide (CLIP) are formed from ACTH, and γ-lipotropin (γ-LPH) and β-endorphin are peptide components of β-LPH. , Β-MSH is contained in γ-LPH. Adrenal insufficiency due to ACTH deficiency, due to genetic mutations in exons 2 and 3 of POMC, results in endocrine disorders characterized by early onset obesity, adrenal insufficiency and red hair pigmentation (Chretien, M. et al (1979) 57: 1111-11121, Krude, H. et al. (1998) Nature Genet. 19: 155-157, Online Mendelian Inheritance in Man (OMIM) 176830.
[0014]
Growth and differentiation factors are secreted proteins that function in intercellular communication. The activity of some factors requires oligomerization or association with membrane proteins. Complex interactions between these factors and their receptors trigger intracellular signaling pathways that stimulate or suppress cell division, cell differentiation, cell signaling and cell motility. Most growth and differentiation factors act on cells in their local environment (paracrine signaling). There are three main classes of growth and differentiation factors. The first class includes large polypeptide growth factors, such as epidermal growth factor, fibroblast growth factor, transforming growth factor, insulin-like growth factor, and platelet-derived growth factor. The second class includes hematopoietic growth factors such as colony stimulating factor (CSF). Hematopoietic growth factors stimulate the proliferation and differentiation of blood cells, such as B lymphocytes, T lymphocytes, erythrocytes, platelets, eosinophils, basophils, neutrophils, macrophages, and their stem cell precursors . The third class includes small peptide factors, such as bombesin, vasopressin, oxytocin, endothelin, transferrin, angiotensin II, vasoactive intestinal peptide, and bradykinin, which function as hormones that regulate cellular functions other than proliferation. .
[0015]
Growth and differentiation factorsin in vitroNeoplastic transformation of cells atin VivoPlays several crucial roles in tumor progression. Improper expression of growth factors by tumor cells can contribute to tumor angiogenesis and metastasis. Anemia, leukemia, and lymphoma can result from dysregulation of growth factors during hematopoiesis. Some growth factors, such as interferon,in in vitroandin VivoAre cytotoxic to a group of tumor cells. In addition, some growth factors and growth factor receptors are structurally and functionally related to oncoproteins. In addition, growth factors influence the transcriptional regulation of both proto-oncogenes and tumor suppressor genes (Pimmentel, E. (1994)).Handbook of Growth Factors, CRC Press, Ann Arbor, MI, review on page 1-9).
[0016]
The Slit protein, first identified in Drosophila, is important in central nervous system midline formation and possibly in nervous tissue histogenesis and axon guidance. Itoh et al. ((1998) Brain Res. Mol. Brain Res. 62: 175-186) identified mammalian homologues of the slit gene (human Slit-1, Slit-2, Slit-3 and rat Slit-1). . The encoded proteins are putative secretory proteins, with EFG-like motifs and leucine-rich repeats, both of which are conserved protein-protein interaction domains. Slit-1 mRNA, Slit-2 mRNA and Slit-3 mRNA are expressed in the brain, spinal cord and thyroid gland, respectively (Itoh, A. et al., Supra). The Slit family of proteins has been shown to be a functional ligand for glypican-1 in neural tissue, suggesting that their interactions may be important at several stages during central nervous system tissue development. (Liang, Y. et al. (1999) J. Biol. Chem. 274: 17885-17892).
[0017]
Neuropeptides and vascular mediators (NP / VM) make up a large family of endogenous signaling molecules. The molecules included in the NP / VM family include, as neuropeptides and neuropeptide hormones, bombesin, neuropeptide Y, neurotensin, neuromedin N, melanocortins, opioids, galanin, somatostatin, tachykinins, urotensin II and smooth muscle. There are related peptides for stimulation, vasopressin, and vasoactive intestinal peptides. In addition, signaling molecules carried to the circulatory system include angiotensin, complement, calcitonin, endothelins, formylmethionyl peptides, glucagon, cholecystokinin, and gastrin. NP / VM can transmit signals directly, modulate the activity or release of other neurotransmitters and hormones, or function as a catalytic enzyme in a cascade. The effects of NP / VM range from very short to long lasting (Martin, CR et al. (1985)).Endocrine Physiology, Oxford University Press, New York, NY, page 57-62).
[0018]
NP / VM is involved in many neurological and cardiovascular disorders. For example, neuropeptide Y is involved in hypertension, congestive heart failure, affective disorders, and appetite regulation. Somatostatin inhibits secretion of growth hormone and prolactin in the anterior pituitary gland, and also inhibits secretion in intestine, pancreatic acinar cells, and pancreatic β cells. Decreased somatostatin levels have been reported in Alzheimer's disease and Parkinson's disease. Vasopressin increases water and sodium absorption in the kidney and, at high concentrations, stimulates vascular smooth muscle contraction, platelet activation, and glycogenolysis in the liver. Vasopressin and its analogs are used clinically for the treatment of diabetes insipidus. Endothelin and angiotensin are involved in hypertension, and drugs such as captopril, which reduce plasma levels of angiotensin, are used to lower blood pressure (Watson, S. and S. Arkinstall (1994)).The G-protein Linked Receptor Facts Book, Academic Press, San Diego CA, 194, 252, 284, 55, and 111).
[0019]
Neuropeptides have also been shown to have some role in nociception (pain sensation). Vasoactive intestinal peptides appear to play an important role in chronic neuropathic pain. Nociceptin, an endogenous ligand for the opioid receptor-like 1 receptor, is thought to have mainly antinociceptive effects and has analgesic properties in various animal models of persistent pain or chronic pain. (Dickinson, T. and Fleetwood-Walker, SM (1998) Trends Pharmacol. Sci. 19: 346-348).
[0020]
Other proteins having a signal peptide include secreted proteins having enzymatic activity. Enzyme activities include, for example, oxidoreductase / dehydrogenase activity, transferase activity, hydrolase activity, lyase activity, isomerase activity, or ligase activity. For example, matrix metalloproteinases are secretory hydrolases that degrade extracellular matrix and therefore play a key role in tumor metastasis, tissue morphogenesis, and arthritis (Reponen, P. et al. (1995) Dev). Firen, GS (1992) Curr. Opin. Rheumatol. 4: 348-354; Ray, JM and Steller-Stevenson, WG (1994) Eur., Dyn. 202: 388-396; Respir. J. 7: 2062-2072; Mignatti, P. and Rifkin, DB (1993) Physiol. Rev. 73: 161-195). Another example is acetyl-CoA synthase, which activates acetic acid for lipid synthesis or for energy production (Long, A. et al. (2000) J. Biol. Chem. 275: 26458-26466). When acetyl-CoA synthase is activated, acetyl-CoA is formed from acetic acid and CoA. Acetyl-CoA synthase shares certain regions of sequence similarity identified as AMP binding domain signatures. Acetyl-CoA synthase has been shown to be associated with hypertension (H. Toh (1991) Protein Seq. Data Anal. 4: 111-117 and Iwai, N. et al. (1994) Hypertension 23: 375-380).
[0021]
Many isomerases catalyze protein folding, light transmission, and steps in various protein assimilation and catabolic pathways. One class of isomerases is known as peptidylprolyl cis-trans isomerase (PPIase). PPIases catalyze the cis-to-trans isomerization of certain prolinimide bonds in proteins. Two families of PPIases are FK506 binding protein (FKBP) and cyclophilin (CyP). The binding of FKBPs to FK506 and rapamycin, both of which are potent immunosuppressive drugs, suppresses multiple signaling pathways in T cells. In particular, the FKBP PPIase activity is inhibited by the binding of FK506 or rapamycin. Five members of the FKBP family (FKBP12, FKBP13, FKBP25, FKBP52 and FKBP65), named according to the calculated molecular weight, are located in various regions of the cell, which associate with various protein complexes (Coss, M. et al.). (1995) J. Biol. Chem. 270: 29336-29341; Schreiber, SL (1991) Science 251: 283-287).
[0022]
The peptidylprolyl isomerase activity of CyP may be part of a signaling pathway leading to T cell activation. CyP isomerase activity is involved in protein folding and protein transport, and may also be involved in the assembly or disassembly of protein complexes and regulation of protein activity. For example, in Drosophila CyP NinaA is required for accurate localization of rhodopsin, and mammalian CyP (Cyp40) is part of the Hsp90 / Hsc70 complex that binds to steroid receptors. Mammalian CypA binds to the gag protein from human immunodeficiency virus 1 (HIV-1), but it has been shown that this interaction can be suppressed by cyclosporin. Because cyclosporine has potent anti-HIV-1 activity, CypA may play an important function in HIV-1 replication. Finally, Cyp40 binds to and inactivates the transcription factor c-Myb, but this effect has been shown to be reversed by cyclosporine. This effect implies the involvement of Cyps in the regulation of transcription, transformation and differentiation (Bergsma, DJ et al. (1991) J. Biol. Chem. 266: 23204-23214; Hunter, T. (1998). ) Cell 92: 141-143; Leverson, JD and Ness, SA (1998) Mol. Cell. 1: 203-211).
[0023]
Proline-rich γ-carboxyglutamic acid (Gla) proteins (PRGPs) are members of the vitamin K-dependent single-transmembrane integral membrane protein family. The PRGP protein is characterized by a Gla-rich, extracellular amino-terminal domain of approximately 45 amino acids. The intracellular carboxyl-terminal region has one or two copies of the sequence PPXY, which is present on a variety of proteins involved in various functions of the cell, such as signal transduction, cell cycle progression, and protein turnover. (Kulman, JD, et al., (2001) Proc. Natl. Acad. Sci. USA 98: 1370-1375). The process of post-translational modification of glutamic acid residues forming Gla is vitamin K-dependent carboxylation. As a protein containing Gla, there is a plasma protein involved in blood coagulation. These proteins are prothrombin, proteins C, S and Z, and blood coagulation factors VII, IX and X. Osteocalcin (bone-Gla protein, BGP) and matrix Gla protein (MGP) also include Gla (Friedman, PA and CT Przysiecki (1987) Int. J. Biochem. 19: 1-7; C. Vermeer (1990) Biochem. J. 266: 625-636).
[0024]
A feature of the Drosophila gene crossveinless 2 is that it has a putative signal sequence or transmembrane sequence. It also has a partial Von Willebrand factor D domain, which is similar to a group of domains known to regulate the formation of intramolecular and intermolecular bonds. It also has five cysteine-rich domains, which are said to bind BMP (bone morphogenetic protein) -like ligands. These features indicate that crossveinless 2 acts to enable ligand signaling directly extracellularly or in the secretory pathway, and thus plays a role in vein specification. (Conley, CA et al. (2000) Development 127: 3947-3959). Dorsal-central patterning in vertebrate and Drosophila embryos requires a conserved system of extracellular proteins to generate a positional informational gradient.
[0025]
The discovery of new secreted proteins and polynucleotides encoding them provides a new set of compositions that can meet the needs of the art. These novel compositions are useful in the diagnosis, treatment, and prevention of abnormal cell proliferation, autoimmune / inflammatory diseases, cardiovascular disorders, neurological disorders, and developmental disorders, and have the nucleic acid and amino acid sequences of secreted proteins. It is also useful for calculating the effect of a foreign compound group on expression.
[0026]
(Summary of the Invention)
The present invention is generally referred to as "SECP", and individually referred to as "SECP-1", "SECP-2", "SECP-3", "SECP-4", "SECP-5", and "SECP-6", respectively. , "SECP-7", "SCCP-8", "SCCP-9", "SCCP-10", "SCCP-11", "SCCP-12", "SSCP-13", "SCCP-14", " "SECP-15", "SECP-16", "SECP-17", "SECP-18", "SECP-19", "SECP-20", "SECP-21", "SECP-22", "SECP- 23 "," SCCP-24 "," SCCP-25 "," SCCP-26 "," SCCP-27 "," SCCP-28 "," SSCP-29 "," SSCP-30 "," SCCP-31 " , "SECP-32 , "SECP-33", "SCCP-34", "SECT-35", "SECT-36", "SECT-37", "SECT-38", "SECT-39", "SECT-40", " "SECP-41", "SECP-42", "SECP-43", "SECP-44", "SECP-45", "SECP-46", "SECP-47", "SECP-48", "SECP- 49 "," SECP-50 "," SECP-51 "," SECP-52 "," SECP-53 "," SECP-54 "," SECP-55 "," SECP-56 "," SECP-57 " , "SECP-58", "SECP-59", "SECP-60", "SECP-61", "SECP-62", "SECP-63" To provide the tides. In certain embodiments, the present invention provides a polypeptide having an amino acid sequence selected from the group consisting of (a) SEQ ID NO: 1-63; and (b) a polypeptide selected from the group consisting of SEQ ID NO: 1-63. A polypeptide having a natural amino acid sequence having at least 90% identity to an amino acid sequence; (c) a biological sequence of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-63. An isolated fragment selected from the group consisting of: an active fragment; and (d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-63. . In one embodiment, the invention provides an isolated polypeptide having the amino acid sequence of SEQ ID NO: 1-63.
[0027]
The present invention also relates to (a) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-63, and (b) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-63. A polypeptide having at least 90% identity with a native amino acid sequence, (c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-63. Or (d) an immunogenic fragment of a polynucleotide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-63, which encodes a polypeptide selected from the group consisting of A polynucleotide is provided. In one embodiment, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NOs: 1-63. In another embodiment, the polynucleotide is selected from the group consisting of SEQ ID NOs: 64-126.
[0028]
The present invention further provides (a) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-63, and (b) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-63. A polypeptide having a certain amino acid sequence having at least 90% identity with (c) a biologically active fragment of a polypeptide having a certain amino acid sequence selected from the group consisting of SEQ ID NOs: 1-63 And (d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-63, and a polynucleotide encoding a polypeptide selected from the group consisting of The present invention provides a recombinant polynucleotide having a promoter sequence linked to In one embodiment, the invention provides a cell transformed with the recombinant polynucleotide. In another embodiment, the present invention provides a genetic transformant having the recombinant polynucleotide.
[0029]
Further, the present invention provides (a) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-63, and (b) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-63. A polypeptide having a certain natural amino acid sequence having at least 90% or more identity with the sequence; and (c) a biology of a polypeptide having a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-63. A method for producing a polypeptide selected from the group consisting of: an active fragment; and (d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-63. . The production method comprises the steps of (a) culturing a cell under conditions suitable for expression of the polypeptide and transforming the cell with a recombinant polynucleotide; It consists of recovering the peptide. The recombinant polynucleotide has a promoter sequence operably linked to the polynucleotide encoding the polypeptide.
[0030]
The present invention further provides (a) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-63, and (b) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-63. A polypeptide having at least 90% identity with a natural amino acid sequence, (c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-63, and (D) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-63, an isolated antibody that specifically binds to a polypeptide selected from the group consisting of I will provide a.
[0031]
The invention further relates to (a) a polynucleotide having a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 64-126, and (b) a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 64-126. A polynucleotide having at least 90% identity with a natural polynucleotide sequence, a polynucleotide complementary to (c) (a), a polynucleotide complementary to (d) (b), and (e) C) providing an isolated polynucleotide selected from the group consisting of: RNA equivalents of (a)-(d). In one embodiment, the polynucleotide consists of at least 60 contiguous nucleotides.
[0032]
The present invention further provides one method for detecting a target polynucleotide in a sample. Here, the target polynucleotide was selected from the group consisting of (a) a polynucleotide having a certain polynucleotide sequence selected from the group consisting of SEQ ID NOs: 64-126, and (b) the polynucleotide having a certain polynucleotide sequence selected from the group consisting of SEQ ID NOs: 64-126. A polynucleotide having a natural polynucleotide sequence having at least 90% identity to a polynucleotide sequence, a polynucleotide complementary to the polynucleotide of (c) (a), or a polynucleotide of (d) (b). Having the sequence of a polynucleotide selected from the group consisting of complementary polynucleotides and (e) RNA equivalents of (a)-(d). The detection method comprises: (a) hybridizing the sample with a probe consisting of at least 20 contiguous nucleotides consisting of a sequence complementary to the target polynucleotide in the sample; (b) Detecting the presence or absence of the hybridization complex and optionally detecting the amount of the complex, if any. The probe hybridizes specifically to the target polynucleotide under conditions such that a hybridization complex is formed between the probe and the target polynucleotide or a fragment thereof. In one embodiment, the probe consists of at least 60 contiguous nucleotides.
[0033]
The present invention also provides one method for detecting a target polynucleotide in a sample. Here, the target polynucleotide was selected from the group consisting of (a) a polynucleotide having a certain polynucleotide sequence selected from the group consisting of SEQ ID NOs: 64-126, and (b) the polynucleotide having a certain polynucleotide sequence selected from the group consisting of SEQ ID NOs: 64-126. A polynucleotide having a natural polynucleotide sequence having at least 90% identity to a polynucleotide sequence, a polynucleotide complementary to the polynucleotide of (c) (a), or a polynucleotide of (d) (b). Having the sequence of a polynucleotide selected from the group consisting of complementary polynucleotides and (e) RNA equivalents of (a)-(d). The detection method includes (a) a process of amplifying a target polynucleotide or a fragment thereof using polymerase chain reaction amplification, and (b) detecting the presence or absence of the amplified target polynucleotide or a fragment thereof, and If present, the process comprises optionally detecting its amount.
[0034]
The present invention further provides certain compositions comprising an effective amount of the polypeptide and certain pharmaceutically acceptable excipients. An effective amount of a polypeptide is (a) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-63, and (b) a polypeptide selected from the group consisting of SEQ ID NOs: 1-63. A polypeptide comprising a native amino acid sequence having at least 90% identity to the amino acid sequence; (c) a biological activity of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-63. And (d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-63. In one embodiment, the composition has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-63. Further, the present invention provides a method of administering the above composition to a patient in need of treatment for a disease or condition accompanied by a decrease in the expression of functional SSCP.
[0035]
The present invention also provides (a) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-63, and (b) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-63. A polypeptide having a natural amino acid sequence having at least 90% identity to (c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-63; A) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-63, in order to confirm the effectiveness of a polypeptide selected from the group consisting of Methods for screening compounds are provided. The screening method comprises (a) exposing a sample containing the polypeptide to a certain compound, and (b) detecting agonist activity in the sample. Alternatively, the invention provides a composition comprising an agonist compound identified in this way and an acceptable pharmaceutical excipient. In yet another alternative, the invention provides a method of administering the composition to a patient in need of treatment for a disease or condition associated with reduced expression of functional SSCP.
[0036]
The present invention further provides (a) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-63, and (b) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-63. A polypeptide having a natural amino acid sequence having at least 90% identity to (c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-63; A) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-63, and a method of screening a compound for the antagonistic efficacy of a polypeptide selected from the group consisting of provide. The screening method comprises (a) exposing a sample containing the polypeptide to a certain compound, and (b) detecting antagonist activity in the sample. In another embodiment, the invention provides a composition comprising the antagonist compound identified in this way and a pharmaceutically acceptable excipient. In yet another alternative, the invention provides a method of administering the composition to a patient in need of treatment for a disease or condition involving overexpression of functional SSCP.
[0037]
The present invention further provides (a) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-63, and (b) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-63. A polypeptide having at least 90% identity with a natural amino acid sequence, (c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-63, or (D) a method of screening for a compound that specifically binds to a polypeptide selected from the group consisting of: an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-63. provide. The screening method comprises: (a) mixing the polypeptide with at least one test compound under appropriate conditions; and (b) detecting the binding of the test compound to the polypeptide, thereby providing the polypeptide with It consists of the process of identifying compounds that specifically bind.
[0038]
The present invention further relates to (a) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-63, and (b) an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-63. A polypeptide having a certain amino acid sequence having at least 90% identity; (c) a biologically active fragment of a polypeptide having a certain amino acid sequence selected from the group consisting of SEQ ID NOs: 1-63; (D) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-63, a compound modulating the activity of a polypeptide selected from the group consisting of A method for screening is provided. The screening method comprises: (a) mixing the polypeptide with at least one test compound under conditions acceptable for the activity of the polypeptide; and (b) determining the activity of the polypeptide in the presence of the test compound. And (c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound. An alteration in the activity of the polypeptide at indicates a compound that modulates the activity of the polypeptide.
[0039]
The invention further provides a method of screening a compound for the effect of altering the expression of a target polynucleotide having a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 64-126. The method comprises: (a) exposing a sample having the target polynucleotide to a compound; and (b) detecting a modification in the expression of the target polynucleotide.
[0040]
The present invention further provides a method for determining the toxicity of a test compound. This method includes the following steps. (A) treating a biological sample having a nucleic acid group with a test compound; (B) A step of hybridizing the nucleic acid group of the treated biological sample. In this process, the following probes are used. (I) a polynucleotide having a polynucleotide sequence selected from the group consisting of SEQ ID NO: 64-126, and (ii) a polynucleotide having a polynucleotide sequence selected from the group consisting of SEQ ID NO: 64-126. A polynucleotide having a 90% identity natural polynucleotide sequence, a polynucleotide having a sequence complementary to (iii) (i), a polynucleotide complementary to the polynucleotide of (iv) (ii), (v A) a probe consisting of at least 20 contiguous nucleotides of a polynucleotide selected from the group consisting of the RNA equivalents of (i)-(iv). Hybridization occurs under conditions such that a specific hybridization complex is formed between the probe and a target polynucleotide in a biological sample. The target polynucleotide is (i) a polynucleotide having a polynucleotide sequence selected from the group consisting of SEQ ID NO: 64-126, and (ii) a polynucleotide selected from the group consisting of SEQ ID NO: 64-126. A polynucleotide having a native polynucleotide sequence having at least 90% identity to a polynucleotide sequence, (iii) a polynucleotide complementary to the polynucleotide of (i), a polynucleotide complementary to the polynucleotide of (iv) (ii). And (v) RNA equivalents of (i) to (iv). Alternatively, the target polynucleotide has a fragment of a polynucleotide sequence selected from the group consisting of (i) to (v) above. The toxicity calculation method further includes the following process. (C) quantifying the amount of hybridization complex; and (d) determining the amount of hybridization complex in the treated biological sample by the amount of hybridization complex in the untreated biological sample. This is the process of comparison. Differences in the amount of hybridization complex in the treated biological sample indicate the toxicity of the test compound.
[0041]
(About the description of the present invention)
Before describing the proteins, nucleotide sequences and methods of the present invention, it is to be understood that the invention is not limited to the particular devices, materials and methods described, as such may be modified. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention, which is limited only by the claims. Please understand at the same time.
[0042]
As used in the claims and the specification, the singular forms “a” and “the” may refer to the plural unless the context clearly dictates otherwise. Note that Thus, for example, when referring to "a certain host cell", there may be a plurality of such host cells, and when "a certain antibody" is described, one or more antibodies, and Reference is also made to equivalents of antibodies known to those skilled in the art.
[0043]
All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art, unless otherwise defined. Although any apparatus, materials, and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred devices, materials, and methods are now described. All publications mentioned in the present invention are cited for the purpose of describing and disclosing the cells, protocols, reagents and vectors that are reported in the publications and that may be of interest to the present invention. . Nothing herein is an admission that the present invention is not entitled to antedate such disclosure by virtue of prior art.
[0044]
(Definition)
The term "SECP" refers to substantially purified SCCP obtained from all species, particularly natural, synthetic, semi-synthetic or recombinant, including mammals, including cows, sheep, pigs, rats, horses and humans. Of amino acid sequences.
[0045]
The term "agonist" refers to a molecule that enhances or mimics the biological activity of SSCP. Agonists include proteins, nucleic acids, carbohydrates, small molecules, or any other protein that interacts directly with SCCP or acts on a component of a biological pathway involving SSCP to modulate the activity of SSCP. There can be compounds and compositions.
[0046]
The term "allelic variant" is another form of the gene encoding SSCP. Allelic variants can result from at least one mutation in a nucleic acid sequence, as well as altered mRNAs. Also, while it may give rise to polypeptides, the structure or function of those polypeptides may or may not be altered. A gene may have no, or more than one, of its natural allelic variant. The usual mutagenic changes that give rise to allelic variant sequences are generally due to natural deletions, additions or substitutions of nucleotides. These various changes, alone or with other changes, can occur more than once in an arrangement.
[0047]
An "altered / altered" nucleic acid sequence that encodes SSCP includes a polypeptide that has the same polypeptide as SSCP, or that has at least one of the functional properties of SSCP, with various nucleotide deletions, insertions, or substitutions. Some sequences result in peptides. This definition includes improper or unexpected hybridization to the allelic variant at a position other than the normal chromosomal locus for the polynucleotide sequence encoding SSCP, and also includes the polynucleotide encoding SSCP. Or polymorphisms that are easily detectable or difficult to detect using certain oligonucleotide probes. The encoded protein may also be "altered / altered" and may have deletions, insertions, or substitutions of amino acid residues that produce a silent change and result in a functionally equivalent SSCP. . Intentional amino acid substitutions may be directed to the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the residues as long as the biological or immunological activity of the SSCP is maintained. , Based on similarity. For example, negatively charged amino acids include aspartic acid and glutamic acid, and positively charged amino acids include lysine and arginine. Amino acids having uncharged polar side chains with similar hydrophilicity values include asparagine and glutamine, and serine and threonine. Amino acids having uncharged side chains with similar hydrophilicity values include leucine and isoleucine and valine, glycine and alanine, and phenylalanine and tyrosine.
[0048]
The terms "amino acid" and "amino acid sequence" refer to oligopeptides, peptides, polypeptides, protein sequences, or fragments thereof, and refer to natural or synthetic molecules. When "amino acid sequence" refers to the sequence of a native protein molecule, "amino acid sequence" and similar terms are not limited to the complete, intact amino acid sequence associated with the protein molecule described.
[0049]
The term "amplification" relates to the act of making additional copies of a nucleic acid sequence. Amplification generally employs the polymerase chain reaction (PCR) technique known to those skilled in the art.
[0050]
The term "antagonist" refers to a molecule that inhibits or attenuates the biological activity of SSCP. As antagonists, proteins, such as antibodies, nucleic acids, carbohydrates, small molecules, or antibodies that modulate the activity of SSCP by interacting directly with SCCP or by acting on components of the biological pathways involving SSCP There can be any other compounds or compositions.
[0051]
The term "antibody" refers to intact immunoglobulin molecules or fragments thereof, e.g., Fab, F (ab '), which are capable of binding epitope determinants.₂ , And Fv fragments. For the production of antibodies that bind to the SSEC polypeptides, intact polypeptides can be used as the immunizing antigen, or fragments having the relevant small peptides can be used. The origin of the polypeptides or oligopeptides used to immunize animals such as mice, rats, rabbits and the like may be in the case of translation of RNA or by chemical synthesis. Also, they can be conjugated to a carrier protein, if desired. Examples of commonly used carriers that chemically bond to the peptide include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KLH). The conjugated peptide is used to immunize the animal.
[0052]
The term "antigenic determinant" refers to a region of a molecule (ie, an epitope) that contacts a particular antibody. When immunizing a host animal with a protein or protein fragment, a number of regions of the protein can trigger the production of antibodies that specifically bind to an antigenic determinant (a particular region or three-dimensional structure of the protein). Certain antigenic determinants can compete with the intact antigen (ie, the immunogen used to elicit the immune response) for binding to certain antibodies.
[0053]
The term "aptamer" refers to a nucleic acid or oligonucleotide molecule that binds to a specific molecular target. Aptamers arein in vitro(Eg, SELEX, short for Systematic Evolution of Ligands by Exponential Enrichment, in vitro selection method), described in US Pat. No. 5,270,163. This is the process of selecting target-specific aptamer sequences from a large set of combinatorial libraries. Aptamer configurations can be double-stranded or single-stranded and can include deoxyribonucleotides, ribonucleotides, nucleotide derivatives or other nucleotide-like molecules. Each nucleotide component of the aptamer may have a modified sugar group (eg, the 2'-OH group of a ribonucleotide is 2'-F or 2'-NH₂Which may improve desired properties, such as resistance to nucleases or longer lifespan in the blood. Conjugation of the aptamer with other molecules (eg, high molecular weight carriers) can slow the clearance of this aptamer from the circulation. Aptamers can be specifically cross-linked with their cognate ligand group, for example, by photoactivation of certain cross-linkers (eg, Brody, EN and L. Gold (2000) J. Biotechnol. 74: 5). -13).
[0054]
The term "intramer"in VivoRefers to the aptamer expressed in For example, certain RNA aptamers are expressed at high levels in the cytoplasm of leukocytes using an RNA expression system based on vaccinia virus (Blind, M. et al. (1999) Proc. Natl Acad. Sci. USA 96). : 3606-3610).
[0055]
The term "spiegelmer" refers to a levorotatory nucleotide derivative such as L-DNA, L-RNA or a levorotatory nucleotide-like molecule among aptamers. Aptamers with levorotatory nucleotides are resistant to degradation by natural enzymes, which normally act on substrates with dextrorotatory nucleotides.
[0056]
The term "antisense" refers to any composition capable of forming base pairs with the "sense" (coding) strand of a particular nucleic acid sequence. Antisense compositions include DNA, RNA, peptide nucleic acids (PNA), oligonucleotides having modified backbone linkages such as phosphorothioic acid, methylphosphonic acid or benzylphosphonic acid, and modified sugar groups such as 2 ′ Oligonucleotides having -methoxyethyl sugar or 2'-methoxyethoxy sugar, and oligonucleotides having a modified base such as 5-methylcytosine, 2-deoxyuracil or 7-deaza-2'-deoxyguanosine. obtain. Antisense molecules can be produced by any method, such as chemical synthesis or transcription. When introduced into a cell, the complementary antisense molecule will base pair with the natural nucleic acid sequence produced by the cell, forming a duplex and preventing transcription or translation. The expression "negative" or "minus" may refer to the antisense strand of a reference DNA molecule, and the expression "positive" or "plus" may refer to the sense strand of a reference DNA molecule.
[0057]
The term "biologically active" refers to a protein that has structural, regulatory, or biochemical functions of a natural molecule. Similarly, the term "immunologically active" or "immunogenic" refers to the natural or recombinant SSCP, synthetic SSCP, or any oligopeptide thereof, for the specific immunization of a suitable animal or cell. Refers to the ability to elicit a response and bind to a group of specific antibodies.
[0058]
The term "complementary" refers to the relationship between two single-stranded nucleic acid sequences that anneal by base pairing. For example, "5'-AGT-3 '" pairs with its complementary sequence "3'-TCA-5'".
[0059]
“Composition comprising (having) a polynucleotide sequence of” or “composition comprising (having) an amino acid sequence of” in a broad sense refers to any composition having a specified polynucleotide sequence or amino acid sequence. Point. The composition may include a dry formulation or an aqueous solution. Compositions having a polynucleotide sequence that encodes SCCP, or a fragment of SSCP, can be used as a hybridization probe. This probe can be stored in a lyophilized state, and can be bound to a stabilizer such as a carbohydrate. In hybridization, a probe is dispersed in an aqueous solution containing a salt (eg, NaCl), a surfactant (eg, sodium dodecyl sulfate; SDS), and other components (eg, denhardt's solution, milk powder, DNA of salmon sperm, etc.). Can be.
[0060]
The “consensus sequence” is repeatedly subjected to DNA sequence analysis to remove unnecessary bases, extended in the 5 ′ and / or 3 ′ direction using an XL-PCR kit (Applied Biosystems, Foster City CA), and sequenced again. One or more overlapping cDNAs or ESTs using a computer program for constructing fragmented nucleic acid sequences or fragment constructs such as the GELVIEW fragment construction system (GCG, Madison, Wis.) Or Phrap (University of Washington, Seattle WA). , Or a nucleic acid sequence constructed from genomic DNA fragments. Some sequences both extend and assemble to produce a consensus sequence.
[0061]
The term "conservative amino acid substitution" refers to a substitution that is expected to change little the properties of the original protein. That is, the structure of the protein, especially its function, is conserved and does not change significantly by substitution. The following table shows the amino acids that can be substituted for the original amino acid in the protein and that are recognized as conservative amino acid substitutions.
Original residue Conservative substitution
Ala Gly, Ser
Arg His, Lys
Asn Asp, Gln, His
Asp Asn, Glu
Cys Ala, Ser
Gln Asn, Glu, His
Glu Asp, Gln, His
Gly Ala
His Asn, Arg, Gln, Glu
Ile Leu, Val
Leu Ile, Val
Lys Arg, Gln, Glu
Met Leu, Ile
Phe His, Met, Leu, Trp, Tyr
Ser Cys, Thr
Thr Ser, Val
Trp Phe, Tyr
Tyr His, Phe, Trp
Val Ile. Leu, Thr
Conservative amino acid substitutions typically involve (a) the backbone structure of the polypeptide in the substitution region, such as a β-sheet or α-helical structure, (b) the charge or hydrophobicity of the molecule at the substitution site, and / or (c) the side chain For the most part, hold.
[0062]
The term "deletion" refers to a change in the amino acid sequence that lacks one or more amino acid residues, or a change in the nucleotide sequence that lacks one or more nucleotides.
[0063]
The term “derivative” refers to a chemically modified polynucleotide or polypeptide. For example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or amino group can be included in chemical modification of a polynucleotide. A polynucleotide derivative encodes a polypeptide that retains at least one of the biological or immunological functions of the natural molecule. A polypeptide derivative is a polypeptide that has been modified by the following process. That is, glycosylation, polyethylene glycolation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it is derived.
[0064]
"Detectable label" refers to a reporter molecule or enzyme that is capable of generating a measurable signal and that is covalently or non-covalently linked to a polynucleotide or polypeptide.
[0065]
"Differential expression" refers to increased (up-regulation) or decreased (down-regulation) or defective gene or protein expression as determined by comparing at least two samples. Such comparisons can be made, for example, between a treated sample and an untreated sample, or between a disease state sample and a healthy sample.
[0066]
"Exon shuffling" refers to the recombination of different coding regions (exons). Certain exons can represent one structural or functional domain of the encoding protein, so that new combinations of stable substructures can be used to construct new proteins and new protein functions Can promote the evolution of
[0067]
The term "fragment" refers to a unique portion of a SSCP or a polynucleotide encoding a SSCP that is identical in sequence to its parent sequence, but shorter in length than the parent sequence. A fragment may have a length that is less than the defined length of the sequence minus one nucleotide / amino acid residue. For example, a fragment may have from 5 to 1000 contiguous nucleotide or amino acid residues. Certain fragments used as probes, primers, antigens, therapeutic molecules or for other purposes may be at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 75, 100, It can be 150, 250 or 500 contiguous nucleotides or amino acid residues. Fragments can be preferentially selected from particular regions of a molecule. For example, a polypeptide fragment may be a certain length selected from the first 250 or 500 amino acids (or the first 25% or 50%) of a polypeptide as found in a defined sequence. Of consecutive amino acids. These lengths are clearly given by way of example and, in embodiments of the present invention, may be any length supported herein, including the sequence listing, tables and figures.
[0068]
Certain fragments of SEQ ID NOs: 64-126 have certain unique polynucleotide sequence regions. This region specifically identifies SEQ ID NO: 64-126, for example, and differs from any other sequence in the genome from which this fragment was obtained. Certain fragments of SEQ ID NOs: 64-126 are useful, for example, in hybridization and amplification techniques, or similar methods to distinguish SEQ ID NOs: 64-126 from related polynucleotide sequences. The exact length of a fragment of SEQ ID NO: 64-126, and the region of SEQ ID NO: 64-126 corresponding to that fragment, are routinely determined by one of ordinary skill in the art based on the purpose intended for the fragment. Can decide.
[0069]
Certain fragments of SEQ ID NOs: 1-63 are encoded by certain fragments of SEQ ID NOs: 64-126. Certain fragments of SEQ ID NOs: 1-63 have regions of unique amino acid sequences that specifically identify SEQ ID NOs: 1-63. For example, certain fragments of SEQ ID NO: 1-63 are useful as immunogenic peptides in the development of antibodies that specifically recognize SEQ ID NO: 1-63. The exact length of a fragment of SEQ ID NO: 1-63, and the region of SEQ ID NO: 1-63 corresponding to this fragment, will be determined by those of ordinary skill in the art based on the intended purpose for the fragment. Can be determined.
[0070]
A “full-length” polynucleotide sequence is a sequence that has at least one translation initiation codon (eg, methionine) followed by one open reading frame and a translation stop codon. A "full-length" polynucleotide sequence encodes a "full-length" polypeptide sequence.
[0071]
The term "homology" refers to sequence similarity, interchangeability, or sequence identity between two or more polynucleotide sequences or two or more polypeptide sequences.
[0072]
The term "percent match" or "% match" as applied to polynucleotide sequences refers to the percentage of residues that match between two or more polynucleotide sequences, aligned using a standardized algorithm. The standardization algorithm can insert gaps in the two sequences to be compared in a standardized and reproducible manner to optimize the alignment between the two sequences, so that the two sequences can be compared more significantly.
[0073]
The default parameter group of the CLUSTAL V algorithm incorporated in the MEGALIGN version 3.12e sequence alignment program or the like can be used to determine the percent identity between polynucleotide sequences. This program is part of the LASERGENE software package (a set of molecular biological analysis programs) (DNASTAR, Madison WI). This CLUSTAL V is available from Higgins, D.W. G. FIG. And P. M. Sharp (1989) CABIOS 5: 151-153; Higgins, D .; G. FIG. (1992) CABIOS 8: 189-191. Default parameters for pairwise alignment of polynucleotide sequences are set as Ktuple = 2, gap penalty = 5, window = 4, and “diagonals saved” = 4. The "weighted" residue weight table is selected by default. Reports of percent identity by CLUSTAL V are reported as "percent similarity" between the aligned polynucleotide sequences.
[0074]
Alternatively, the Basic Local Alignment Search Tool (BLAST) of the National Center for Biotechnology Information (NCBI) provides a set of commonly used and freely available sequence comparison algorithms (Altschul, SF). (1990) J. Mol. Biol. 215: 403-410). The BLAST algorithm is available from several sources and is also available from NCBI in Bethesda, MD and the Internet (http://www.ncbi.nlm.nih.gov/BLAST/). The BLAST software suite includes a variety of sequence analysis programs, including "blastn" used to align known polynucleotide sequences with other polynucleotide sequences in various databases. A tool is also available called "BLAST 2 Sequences", which is used to compare two nucleotide sequences directly pairwise. “BLAST 2 Sequences” is available at http: // www. ncbi. nlm. nih. gov / gorf / bl2. html and can be used interactively. The "BLAST 2 Sequences" tool can be used for both blastn and blastp (described below). The BLAST program generally uses parameters such as gaps set to default settings. For example, to compare two nucleotide sequences, blastn can be performed using the "BLAST 2 Sequences" tool Version 2.0.12 (April 21, 2000) set to default parameters. An example of setting default parameters is shown below.
[0075]
Matrix: BLOSUM62
Reward for match: 1
Penalty for Mismatch: -2
Open Gap: 5 and Extension Gap: 2 penalties
Gap x drop-off: 50
Expect: 10
Word Size: 11
Filter: on
The percent identity can be measured over the entire length of a defined sequence (eg, the sequence defined by a particular SEQ ID number). Alternatively, percentage identity of shorter lengths, eg, fragments from defined, larger sequences (eg, fragments of at least 20, 30, 40, 50, 70, 100 or at least 200 contiguous nucleotides) Can also be measured. The lengths listed here are merely exemplary, and any fragment length supported by the sequences described herein, including tables, figures and sequence listings, may be used to determine percent identity. It should be understood that certain lengths can be described.
[0076]
Nucleic acid sequences that do not exhibit a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is to be understood that this degeneracy can be used to effect changes in a nucleic acid sequence to produce a large number of nucleic acid sequences such that all nucleic acid sequences encode substantially the same protein.
[0077]
The term “percent identity” or “percent identity” as used for polypeptide sequences refers to the percentage of matching residues between two or more polypeptide sequences that are aligned using a standardized algorithm. Methods for polypeptide sequence alignment are known. There are also alignment methods that take into account conservative amino acid substitutions. Such conservative substitutions, as detailed above, typically conserve the charge and hydrophobicity of the substitution site, thus preserving the structure (and therefore function) of the polypeptide.
[0078]
The percent identity between polypeptide sequences can be determined using the default parameter set of the CLUSTAL V algorithm, such as that incorporated in the MEGALIGN version 3.12e sequence alignment program (described above with references). Default parameters for pairwise alignment of polypeptide sequences using CLUSTAL V are set to Ktuple = 1, gap penalty = 3, window = 5, and “diagonals saved” = 5. The PAM250 matrix is selected as the default residue weight table. Similar to polynucleotide alignments, the percent identity of aligned polypeptide sequence pairs is reported by CLUSTAL V as "percent similarity".
[0079]
Alternatively, the NCBI BLAST software suite may be used. For example, when comparing two polypeptide sequences on a pair-wise basis, blastp of the “BLAST 2 Sequences” tool Version 2.0.12 (April 21, 2000) can be used with default parameters set.
An example of setting default parameters is shown below.
[0080]
Matrix: BLOSUM62
Open Gap: 11 and Extension Gap: 1 penalties
Gap x drop-off: 50
Expect: 10
Word Size: 3
Filter: on
Identity can be measured over the entire length of a defined polypeptide sequence (eg, a sequence defined by a particular SEQ ID number). Alternatively, shorter lengths, eg, fragments obtained from a defined, larger polypeptide sequence (eg, a fragment of at least 15, 20, 30, 40, 50, 70, or at least 150 contiguous residues) Can also be measured. The lengths listed here are merely exemplary, and any fragment length supported by the sequences described herein, including tables, figures and sequence listings, may be used to determine percent identity. It should be understood that certain lengths can be described.
[0081]
“Human artificial chromosomes (HACs)” are linear microchromosomes that can have a DNA sequence of about 6 kb to 10 Mb in size. It also has all the elements necessary for chromosome replication, segregation and maintenance.
[0082]
The term "humanized antibody" refers to an antibody molecule in which the amino acid sequence of the non-antigen binding region has been modified to resemble a human antibody while retaining its original binding ability.
[0083]
"Hybridization" is the process of annealing in which a single-stranded polynucleotide base pairs with a complementary single-strand under predetermined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share high complementarity. Specific hybridization complexes are formed under acceptable annealing conditions and remain hybridized after one or more "wash" steps. The washing step is particularly important in determining the stringency of the hybridization process; under more stringent conditions, non-specific binding (ie, binding between pairs of nucleic acid strands that do not perfectly match) is reduced. The permissive conditions for annealing a nucleic acid sequence can be routinely determined by those skilled in the art. The annealing conditions can be constant for any hybridization experiment, but the washing conditions can be varied from experiment to experiment to obtain the desired stringency, and thus hybridization specificity. Acceptable annealing conditions include, for example, at a temperature of 68 ° C. in the presence of about 6 × SSC, about 1% (w / v) SDS, and about 100 μg / ml of shear denatured salmon sperm DNA. is there.
[0084]
In general, the stringency of hybridization can be expressed, in part, in reference to the temperature at which the washing step is performed. Such washing temperatures are usually selected to be about 5-20 ° C. below the melting point (Tm) of the particular sequence at a given ionic strength and pH. This Tm is the temperature at which 50% of the target sequence hybridizes to a perfectly matched probe under the conditions of a predetermined ionic strength and pH. The formula for calculating Tm and hybridization conditions for nucleic acids are well known and are described in Sambrook, J .; Others (1989)Molecular Cloning: A Laboratory Manual, Second Edition, Volumes 1-3, Cold Spring Harbor Press, Plainview NY, particularly see Volume 9, Chapter 9.
[0085]
Hybridization under high stringency conditions between polynucleotides of the invention includes washing conditions at 68 ° C. for 1 hour in the presence of about 0.2 × SSC and about 0.1% SDS. Alternatively, temperatures of about 65 ° C, 60 ° C, 55 ° C, or 42 ° C may be used. The SSC concentration can vary from about 0.1 to 2 × SSC in the presence of about 0.1% SDS. Usually, blocking agents are used to block non-specific hybridization. An example of such a blocking agent is sheared denatured salmon sperm DNA at about 100-200 μg / ml. Under certain conditions, for example, for hybridization of RNA and DNA, an organic solvent such as formamide at a concentration of about 35-50% v / v can also be used. Useful variations of washing conditions will be apparent to those skilled in the art. Hybridization can indicate evolutionary similarity between nucleotides, especially under high stringency conditions. Evolutionary similarity strongly suggests a similar role for the nucleotides and for the polypeptides they encode.
[0086]
The term "hybridization complex" refers to a complex of two nucleic acid sequences formed by the formation of hydrogen bonds between complementary bases. Hybridization complexes can be formed in solution (C₀t or R₀t analysis). Alternatively, one nucleic acid sequence is present in solution and the other nucleic acid sequence is a solid support (eg, paper, membrane, filter, chip, pin or glass slide, or other suitable substrate, such as a cell or its nucleic acid). May be formed between two nucleic acid sequences, such as those immobilized on a substrate on which is immobilized.
[0087]
The term "insertion" or "addition" refers to a change in an amino acid or nucleic acid sequence where one or more amino acid residues or nucleotides are added, respectively.
[0088]
"Immune response" can refer to a condition associated with inflammation, trauma, immune disorders, infectious or genetic diseases, and the like. These conditions can be characterized by the expression of various factors that can affect cells and the system of defense, such as cytokines, chemokines and other signaling molecules.
[0089]
The term “immunogenic fragment” refers to a polypeptide or oligopeptide fragment of SSCP that can elicit an immune response when introduced into an organism (eg, a mammal). The term "immunogenic fragment" also refers to any polypeptide or oligopeptide fragment of SSCP that is useful herein for any of the antibody production methods disclosed herein or known in the art.
[0090]
The term "microarray" refers to an arrangement of a plurality of polynucleotides, polypeptides or other compounds on a substrate.
[0091]
The term "element" or "array element" refers to a polynucleotide, polypeptide or other compound that has a unique, designated location on a microarray.
[0092]
The terms "modulate" or "modulate activity" refer to altering the activity of a SCCP. For example, modulation can cause an increase or decrease in the protein activity of SSCP, or a change in binding or other biological, functional or immunological properties.
[0093]
The terms "nucleic acid" and "nucleic acid sequence" refer to nucleotides, oligonucleotides, polynucleotides or fragments thereof. The terms “nucleic acid” and “nucleic acid sequence” also refer to DNA or RNA of genomic or synthetic origin that can be single-stranded or double-stranded or represent the sense or antisense strand. Or peptide nucleic acid (PNA) or any DNA-like or RNA-like substance.
[0094]
"Operably linked" refers to the state in which a first nucleic acid sequence and a second nucleic acid sequence are in a functional relationship. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. The operably linked DNA sequences can be very close or contiguous, and can be in the same reading frame, if necessary to join the two protein-coding regions.
[0095]
"Peptide nucleic acid (PNA)" refers to an antisense molecule or an antigenic agent having an oligonucleotide at least about 5 nucleotides in length attached to the peptide backbone at amino acid residues terminating in lysine. Terminal lysine confers solubility to this composition. PNAs preferentially bind to complementary single-stranded DNA or RNA and stop transcription elongation. Polyethylene glycolation can extend the life of PNA in cells.
[0096]
“Post-translational modifications” of a SCCP may include lipidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage and other modifications known in the art. These processes can occur synthetically or biochemically. Biochemical modification depends on the enzymatic environment of SCCP and will vary from cell type to cell type.
[0097]
The term "probe" refers to a sequence that encodes SSCP, its complementary sequence, or a fragment thereof among nucleic acid sequences and is used for detection of an identical sequence, an allelic nucleic acid sequence, or a related nucleic acid sequence. A probe is an isolated oligonucleotide or polynucleotide, a sequence attached to a detectable label or reporter molecule. Typical labels include radioisotopes, ligands, chemiluminescent reagents and enzymes. "Primers" are short nucleic acids, usually DNA oligonucleotides, that can form complementary base pairs and anneal to a target polynucleotide. The primer can then be extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification (and identification) of nucleic acid sequences, for example, by the polymerase chain reaction (PCR).
[0098]
Probes and primers for use in the invention usually consist of at least 15 contiguous nucleotides of known sequence. To increase specificity, longer probes and primers, such as those consisting of at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or at least 150 contiguous nucleotides of the disclosed nucleic acid sequences And primers can also be used. Some probes and primers are considerably longer. It is to be understood that any length of nucleotides supported herein, including tables, figures and sequence listings, may be used.
[0099]
For methods of preparing and using probes and primers, see Sambrook, J. et al. Others (1989)Molecular Cloning: A Laboratory Manual, Second Edition, Volumes 1-3, Cold Spring Harbor Press, Plainview NY, Ausubel, F.C. M. Other (1987)Current Protocols in Molecular Biology, Green Publ. Assoc. & Wiley-Intersciences, New York NY, Innis, M .; Others (1990)PCR Protocols, A Guide to Methods and Applications, Academic Press, San Diego CA, and the like. To obtain a PCR primer pair from a known sequence, for example, a computer program therefor, such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge MA) can be used.
[0100]
The selection of oligonucleotides to use as primers is made using software well known in the art for such purpose. For example, OLIGO 4.06 software is useful for selecting PCR primer pairs of up to 100 nucleotides each, from oligonucleotides and input polynucleotide sequences of up to 5,000 larger polynucleotides up to 32 kilobases. It is also useful for analyzing the obtained sequence. Similar primer selection programs incorporate additional features for expansion capabilities. For example, the PrimOU Primer Selection Program (available publicly from the Genome Center at the University of Texas Southwestern Medical Center in Dallas, Texas) is capable of selecting specific primers from megabase sequences and thus spans the entire genome. Useful for designing primers. Using the Primer3 primer selection program (available from the Whitehead Institute / MIT Center for Genome Research (Cambridge, Mass.)), A "non-priming library" that can specify the sequence to be avoided as a primer binding site can be input. Primer 3 is particularly useful for selecting oligonucleotides for microarrays (the source code of the latter two primer selection programs can be obtained from their respective sources and modified to meet user-specific needs). The PrimerGen program (publicly available from the UK Human Genome Mapping Project-Resource Center in Cambridge, UK) designs primers based on a number of sequence alignments, thereby maximally conserving or minimally conserving aligned nucleic acid sequences Allows selection of primers that hybridize to any of the regions. Thus, this program, whether unique or stored, is useful for identifying oligonucleotides and polynucleotide fragments. Oligonucleotides and polynucleotide fragments identified by any of the above selection methods identify fully or partially complementary polynucleotides in hybridization techniques, for example, as PCR or sequencing primers, as microarray elements, or in nucleic acid samples. Useful as a specific probe. The method for selecting the oligonucleotide is not limited to the above method.
[0101]
A "recombinant nucleic acid" is a sequence that is not a native sequence, but rather an artificial combination of two or more separate segments of the sequence. This artificial combination is often achieved by chemical synthesis, but more generally by artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques as described in Sambrook, supra. I do. The term recombinant nucleic acid also includes a nucleic acid in which a part of the nucleic acid is simply modified by addition, substitution or deletion. Often, a recombinant nucleic acid includes a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid can form part of a vector used, for example, to transform a cell.
[0102]
Alternatively, such a recombinant nucleic acid can be part of a viral vector, which can be based, for example, on vaccinia virus. Such a vector can be inoculated into a mammal and the recombinant nucleic acid expressed and used to induce a protective immune response in the mammal.
[0103]
A "regulatory element" is a nucleic acid sequence usually derived from the untranslated region of a gene, including enhancers, promoters, introns, and 5 'and 3' untranslated regions (UTRs). Regulatory elements interact with host or viral proteins that control transcription, translation or RNA stability.
[0104]
A "reporter molecule" is a chemical or biochemical component used to label nucleic acids, amino acids or antibodies. Reporter molecules include radionuclides, enzymes, fluorescent agents, chemiluminescent agents, chromogenic agents, substrates, cofactors, inhibitors, magnetic particles and other components known in the art.
[0105]
An "RNA equivalent" for a DNA sequence consists of the same linear nucleotide sequence as the reference DNA sequence, but all the resulting nitrogenous bases, thymine, are replaced with uracil and the backbone of the sugar chain is deoxyribose. But not ribose.
[0106]
The term "sample" is used in its broadest sense. Examples of a sample presumed to contain SSCP, a nucleic acid group encoding SCCP, or a fragment group thereof include body fluids, chromosomes isolated from cells and cells, organelles and extracts from membranes, There may be cells, genomic DNA, RNA, cDNA present in solution or immobilized on a substrate, tissues, tissue prints, and the like.
[0107]
The terms "specific binding" and "specifically bind" refer to the interaction between a protein or peptide, an agonist, antibody, antagonist, small molecule, or any natural or synthetic binding composition. This interaction depends on the presence or absence of a particular structure of the protein (eg, an antigenic determinant or epitope) that the binding molecule recognizes. For example, if an antibody is specific for epitope "A", then in a reaction involving unbound labeled "A" and the antibody, a polypeptide having epitope A, or The presence of unlabeled "A" reduces the amount of labeled A that binds to the antibody.
[0108]
The term "substantially purified" refers to an isolated or isolated nucleic acid or amino acid sequence that has been removed from its natural environment and has at least about 60% of its naturally associated composition removed. Preferably at least about 75% removed, most preferably at least 90% removed.
[0109]
"Substitution" refers to the replacement of one or more amino acid residues or nucleotides with another amino acid residue or nucleotide, respectively.
[0110]
The term "substrate" refers to any suitable solid or semi-solid support, including membranes and filters, chips, slides, wafers, fibers, magnetic or non-magnetic beads, gels, tubes, plates, polymers, microparticles, capillaries. Is included. The substrate may have various surface morphologies such as wells, grooves, pins, channels, holes, etc., and polynucleotides and polypeptides are bound to the substrate surface.
[0111]
“Transcription image” or “expression profile” refers to the collective pattern of gene expression by a particular cell type or tissue at a given time under a given condition.
[0112]
"Transformation" refers to the process by which exogenous DNA is introduced into a recipient cell. Transformation can occur under natural or artificial conditions according to a variety of methods known in the art, and include any known method for inserting an exogenous nucleic acid sequence into a prokaryotic or eukaryotic host cell. Method. The method of transformation is selected depending on the type of host cell to be transformed. Transformation methods include, but are not limited to, viral infection, electroporation, heat shock, lipofection, and microprojectile bombardment. "Transformed cells" include stably transformed cells in which the introduced DNA is replicable, either as an autonomously replicating plasmid or as part of the host chromosome. Furthermore, cells that transiently express the introduced DNA or introduced RNA for a limited period of time are also included.
[0113]
As used herein, a "genetically transformant" is any organism, including but not limited to animals and plants, wherein one or more cells of the organism are transformed by human intervention, for example, as is known in the art. It has a heterologous nucleic acid introduced by a transformation technique. The introduction of the nucleic acid into the cell is performed by directly or indirectly introducing into the precursor of the cell. This is done by deliberate genetic manipulation, for example by microinjection or by introduction of a recombinant virus. The term genetic manipulation refers to classical cross breeding orin in vitroIt does not refer to fertilization, but refers to the introduction of a recombinant DNA molecule. Genetic transformants contemplated according to the present invention include bacteria, cyanobacteria, fungi and plants and animals. The isolated DNA of the present invention can be introduced into a host by methods known in the art, for example, infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are known and described in references such as Sambrook et al. (1989), supra.
[0114]
A “variant / variant sequence” of a particular nucleic acid sequence is a sequence determined as a nucleic acid sequence having at least 40% sequence identity to the particular nucleic acid sequence over a length of one nucleic acid sequence. is there. For the determination, blastn is executed by using the “BLAST 2 Sequences” tool Version 2.0.9 (May 7, 1999) set as the default parameter. Such a nucleic acid pair can be, for example, at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, It may exhibit 96%, 97%, 98%, 99% or more sequence identity. Variant sequences can be described, for example, as "allelic" variant sequences (described above) or "splice" variant sequences, "species" variant sequences, "polymorphic" variant sequences. Although splice variant sequences may have significant identity to a reference molecule, alternative splicing of groups of exons during mRNA processing usually results in having more or fewer polynucleotides. The corresponding polypeptide may have additional functional domains or may lack the domains present in the reference molecule. Species variant sequences are polynucleotide sequences that vary from species to species. The resulting polypeptides usually have significant amino acid identity to one another. A polymorphic variant sequence is a variation within the polynucleotide sequence of a particular gene between certain individuals. Polymorphic variant sequences may also include "single nucleotide polymorphisms" (SNPs) in which one nucleotide base of the polynucleotide sequence differs. The presence of a SNP may be indicative of, for example, a particular population, condition or propensity for a condition.
[0115]
A “variant sequence” of a particular polypeptide sequence is a sequence determined as a polypeptide sequence having at least 40% sequence identity to the particular polypeptide sequence over a certain length of one polypeptide sequence It is. For the determination, blastp is executed by using a “BLAST 2 Sequences” tool Version 2.0.9 (May 7, 1999) set as a default parameter. Such a polypeptide pair can be, for example, at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, for one predetermined length of the polypeptide. , 95%, 96%, 97%, 98%, 99% or more sequence identity.
[0116]
(invention)
The present invention is based on the discovery of a new family of human secreted proteins (SECPs) and polynucleotides encoding SCCP. Also based on the development of the diagnosis, treatment and prevention of abnormal cell proliferation, autoimmune / inflammatory disorders, cardiovascular disorders, neurological disorders and developmental disorders using these compositions.
[0117]
Table 1 is a summary of the nomenclature of the full length polynucleotide and polypeptide sequences of the present invention. Each polynucleotide and its corresponding polypeptide correlates to one Incyte project identification number (Incyte project ID). Each polypeptide sequence was designated by a polypeptide sequence identification number (polypeptide SEQ ID NO :) and an Incyte polypeptide sequence number (Incyte polypeptide ID). Each polynucleotide sequence was designated by a polynucleotide sequence identification number (polynucleotide SEQ ID NO :) and an Incyte polynucleotide consensus sequence number (Incyte polynucleotide ID).
[0118]
Table 2 shows sequences homologous to the polypeptides of the invention, identified by BLAST analysis against the GenBank protein (genpept) database. Columns 1 and 2 show the polypeptide sequence identification number (polypeptide SEQ ID NO :) and the corresponding Incyte polypeptide sequence number (Incyte polypeptide ID) for each polypeptide of the invention. Column 3 shows the GenBank identification number (GenBank ID NO :) of the closest GenBank homolog. Column 4 shows the probability score for a match between each polypeptide and one or more of its homologs. Column 5 shows appropriate citations where applicable and indicates one or more annotations of the GenBank homologs, all of which are incorporated herein by reference.
[0119]
Table 3 shows various structural features of the polypeptides of the invention. Columns 1 and 2 show the polypeptide sequence identification number (SEQ ID NO :) and the corresponding Incyte polypeptide sequence number (Incyte polypeptide ID) for each polypeptide of the invention. Column 3 shows the number of amino acid residues in each polypeptide. Rows 4 and 5 show potential phosphorylation and glycosylation sites, respectively, as determined by the MOTIFS program (Genetics Computer Group, Madison WI) of the GCG sequence analysis software package. Column 6 shows the amino acid residues that contain the signature sequence, domain, and motif. Column 7 shows the analysis method for the analysis of the structure / function of the protein, and in the corresponding part, a searchable database used for the analysis method.
[0120]
Tables 2 and 3 together summarize the properties of each of the polypeptides of the present invention, which properties establish that the claimed polypeptides are secreted proteins. For example, SEQ ID NO: 1 has been shown by the Basic Local Alignment Search Tool (BLAST) to have 34% identity with the human epileptic seizure-related gene 6 (mouse) -like protein, isoform 1 (GenBank ID g69416112). Was. The BLAST probability score is 8.5e-34, indicating the probability that the observed polypeptide sequence alignment will be obtained by chance. SEQ ID NO: 1 also has two CUB domains and one sushi domain (SCR repeat), which are based on the hidden Markov model (HMM) in the PFAM database of conserved protein family domains: It was determined by searching for a statistically significant match (see Table 3). As another example, SEQ ID NO: 2 was shown by Basic Local Alignment Search Tool (BLAST) to have 40% identity with Drosophila melanogaster peroxidasin precursor (GenBank ID g53385) (BLAST). See Table 2). The BLAST probability score is 7.8e-266, indicating the probability that the observed polypeptide sequence alignment would be obtained by chance. SEQ ID NO: 2 also has one peroxidase domain, four immunoglobulin domains, six leucine-rich repeats, one leucine-rich repeat C-terminal domain, and one von Willebrand factor type C domain, which And were determined by searching for statistically significant matches in the PFAM database of conserved protein family domains based on the Hidden Markov Model (HMM) (see Table 3). Data from BLIMPS and MOTIFS analyzes provide evidence further confirming that SEQ ID NO: 2 is a peroxidasin homolog. As another example, SEQ ID NO: 4 has been shown by Basic Local Alignment Search Tool (BLAST) to have 98% identity to rat neulexophilin (GenBank ID g508574) (Table 2). reference). The BLAST probability score is 4.7e-148, indicating the probability that the observed polypeptide sequence alignment would be obtained by chance. Data from SPSCAN analysis and BLAST_PRODOM analysis provide more conclusive evidence that SEQ ID NO: 4 is a secreted neulexophilin. In another example, SEQ ID NO: 6 has 68% identity with porcine preprosecretin (GenBank ID g164671), as identified by Basic Local Alignment Search Tool (BLAST) (see Table 2). . The BLAST probability score is 2.3e-36, indicating the probability that the observed polypeptide sequence alignment will be obtained by chance. SEQ ID NO: 6 was predicted by HMMER and SPSCAN to have a signal peptide. SEQ ID NO: 6 also has one polypeptide hormone domain, which makes a statistically significant match in the PFAM database of conserved protein family domains based on the Hidden Markov Model (HMM). It was determined by searching (see Table 3). The presence of this domain is confirmed by BLIMPS analysis and MOTIFS analysis, providing further empirical evidence that SEQ ID NO: 6 is a secreted hormone. In another example, SEQ ID NO: 28 has 78% identity with the mouse nodal TGF-β-like gene (GenBank ID g296605), as identified by Basic Local Alignment Search Tool (BLAST) (Table 1). 2). The BLAST probability score is 7.5e-148, indicating the probability that the observed polypeptide sequence alignment will be obtained by chance. SEQ ID NO: 28 also has one TGF-β-like domain, which is a statistically significant match in the PFAM database of conserved protein family domains based on the Hidden Markov Model (HMM) (See Table 3). Data from BLIMPS, MOTIFS, and PROFILESCAN analyzes provide further empirical evidence that SEQ ID NO: 28 is a TGF-β-like protein. In another example, SEQ ID NO: 63 has 86% identity to rat late pregnancy lung protein 1 (GenBank ID g4324682), as identified by Basic Local Alignment Search Tool (BLAST) (see Table 2). The BLAST probability score is 3.4e-97, indicating the probability that the observed polypeptide sequence alignment will be obtained by chance. SEQ ID NO: 63 also has one SCP (sperm-coated glycoprotein) -like extracellular protein domain, which is stored in the PFAM database of conserved protein family domains based on a hidden Markov model (HMM). Was determined by searching for a statistically significant match (see Table 3). Data from BLIMPS analysis and MOTIFS analysis provide evidence further confirming that SEQ ID NO: 63 is a protease inhibitor-like protein. SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7-27, and SEQ ID NO: 29-62 were analyzed and annotated in a similar manner. Table 7 describes the algorithm and parameters for the analysis of SEQ ID NOs: 1-63.
[0121]
As shown in Table 4, the full-length polynucleotide sequence of the present invention was constructed using a cDNA sequence or a coding (exon) sequence derived from genomic DNA, or by arbitrarily combining these two types of sequences. Columns 1 and 2 show the polynucleotide sequence identification number (polynucleotide SEQ ID NO :) and the corresponding Incyte polynucleotide consensus sequence number (Incyte polynucleotide ID) for each polynucleotide of the invention. Column 3 shows the length of each polynucleotide sequence in base pairs. Column 4 is useful for hybridization or amplification techniques, for example, to identify SEQ ID NO: 64-126, or to distinguish SEQ ID NO: 64-126 from related polynucleotide sequences. 3 shows fragments of polynucleotide sequences. Column 5 shows the identification number corresponding to the cDNA sequence, or the coding sequence (exon) predicted from genomic DNA, and / or the identification number corresponding to the sequence set having both cDNA and genomic DNA. These sequences were used to construct the full length polynucleotide sequences of the present invention. Columns 6 and 7 of Table 4 indicate the starting nucleotide (5 ') and ending nucleotide (3') positions of the cDNA and / or genomic sequences of column 5, respectively, related to their full length sequences.
[0122]
The identification numbers in column 5 of Table 4 may particularly refer to, for example, Incyte cDNAs and their corresponding cDNA libraries. For example, 27199959T6 is the identification number of a certain Incyte cDNA sequence, and LUNGTUT10 is the cDNA library from which it was derived.
[0123]
Incyte cDNAs for which no cDNA library is indicated (eg, 56002879J1) are from pooled cDNA libraries. Alternatively, the identification number in row 5 may indicate a GenBank cDNA or EST that contributed to the construction of the full-length polynucleotide sequence family (eg, g147765). Further, the identification number in column 5 may indicate a sequence from the ENSEMBL (The Sanger Center, Cambridge, UK) database (ie, a sequence containing the designation "ENST"). Alternatively, the identification number in column 5 may be from the NCBI RefSeq Nucleotide Sequence Records database (ie, a sequence that includes the designation "NM" or "NT"), or may be from the NCBI RefSeq Protein Sequences (if it is from NCBI RefSeq Protein Sequence Records). That is, a sequence containing the designation "NP"). Or the identification number in column 5 may refer to the group consisting of both the cDNA and the Genscan predicted exon, linked by an "exon-stitching" algorithm. For example, FL_XXXXXX_N₁_N₂_YYYYY_N₃_N₄ Is the "stitched" sequence, where XXXXXX is the identification number of the cluster of sequences to which the algorithm applies, YYYYY is the number of predictions the algorithm makes, and N_{1,2,3. . .}If present, represents a particular group of exons that were manually edited during the analysis (Example 5reference). Alternatively, the identification number in column 5 may refer to a set of exons connected by an “exon-stretching” algorithm. For example, FLXXXXXX_gAAAAAA_gBBBBBB_1_N is the identification number of the “stretch” sequence. Where XXXXXX is the Incyte project identification number, gAAAAAA is the GenBank identification number of the human genome sequence to which the “exon stretching” algorithm has been applied, gBBBBBB is the GenBank identification number of the closest GenBank protein homolog, or NCBI RefSeq identification number, and N is the identification number. Is an exon (Example 5See). If a RefSeq sequence was used as a protein homolog for the “exon stretching” algorithm, the RefSeq identifier represented by “NM”, “NP”, or “NT” would be the GenBank identifier (ie, gBBBBBB). ) Can be used instead.
[0124]
Alternatively, the prefix identifies a manually edited constituent sequence, a constituent sequence predicted from a genomic DNA sequence, or a constituent sequence derived from a combined sequence analysis method. The following table lists the prefixes of the constituent sequences and examples of sequence analysis methods corresponding to the prefixes (Examples 4 and 5See).

[0125]
In some cases, to confirm the final consensus polynucleotide sequence, Incyte cDNA coverage overlapping sequence coverage as shown in column 5 was obtained, but without the corresponding Incyte cDNA identification number.
[0126]
Table 5 shows a representative cDNA library for full-length polynucleotide sequences constructed using Incyte cDNA sequences. Representative cDNA libraries are Incyte cDNA libraries, which are most frequently represented by the Incyte cDNA sequence family, which were used to construct and confirm the polynucleotide sequences described above. The tissues and vectors used to generate the cDNA library are shown in Table 5 and described in Table 6.
[0127]
The present invention also includes mutant sequences of SSCP. Preferred SSCP variant sequences have at least one of the functional or structural characteristics of SSCP and have at least about 80% amino acid sequence identity to the SSCP amino acid sequence, or at least about 90% amino acid sequence identity. And even at least about 95% amino acid sequence identity.
[0128]
The present invention also provides a group of polynucleotides encoding SSCP. In certain embodiments, the present invention provides a polynucleotide sequence having one sequence selected from the group consisting of SEQ ID NOs: 64-126, which encodes SSCP. The polynucleotide sequence of SEQ ID NOs: 64-126 also includes equivalent RNA sequences as shown in the Sequence Listing, wherein the appearance of the nitrogenous base thymine is replaced by uracil and the sugar backbone is not deoxyribose. Consists of ribose.
[0129]
The invention also includes mutant sequences of the polynucleotide sequence encoding SSCP. In particular, such variant polynucleotide sequences will have at least about 70% polynucleotide sequence identity, or at least about 85% polynucleotide sequence identity, or even at least about 85% polynucleotide sequence identity with the polynucleotide sequence encoding SSCP. It will have as much as about 95% polynucleotide sequence identity. Certain embodiments of the present invention include a variant of the polynucleotide sequence having a sequence selected from the group consisting of SEQ ID NOs: 64-126. The variant sequence has at least about 70%, or at least about 85%, or even at least about 95%, polynucleotide sequence identity with a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 64-126. . Any of the foregoing polynucleotide variant sequences can encode an amino acid sequence having at least one functional or structural characteristic of SSCP.
[0130]
In yet another example, certain polynucleotide variants of the present invention are splice variants of one polynucleotide sequence encoding SSCP. Certain splice variant sequences may have a plurality of portions with significant sequence identity to the polynucleotide sequence encoding SSCP, but may add several blocks of sequence or may result from alternative splicing of exons during mRNA processing. Deletions usually result in having more or fewer polynucleotides. Certain splice variant sequences may have less than about 70%, or less than about 60%, or even less than about 50% polynucleotide sequence identity to the full length of the polynucleotide sequence encoding SSCP, although Some portions of the splice variant sequence have at least about 70%, or at least about 85%, or at least about 95%, or even 100%, polynucleotide sequence identity with each portion of the polynucleotide sequence encoding SSCP. Will have. Any of the above splice variant sequences may encode a certain amino acid sequence having at least one of the functional or structural features of SSCP.
[0131]
The degeneracy of the genetic code creates various polynucleotide sequences that encode SSCP, some of which have minimal similarity to any of the known natural gene polynucleotide sequences. Will understand. Thus, the present invention may cover all possible polynucleotide sequence variations that can be generated by selecting combinations based on possible codon choices. These combinations are based on the standard triplet genetic code as applied to the polynucleotide sequence of native SSCP. All such variations are to be considered explicitly disclosed.
[0132]
Nucleotide sequences encoding SSCP and variants thereof are generally capable of hybridizing to a nucleotide sequence of native SSCP under suitably selected stringency conditions, but are substantially different in codons, including unnatural codons. It may be advantageous to produce nucleotide sequences encoding SSCP or derivatives thereof that have use. Based on the frequency with which a host utilizes a particular codon, codons can be selected to increase the expression of a peptide that occurs in a particular eukaryotic or prokaryotic host. Other reasons for substantially altering the nucleotide sequences encoding SSCP and its derivatives without altering the encoded amino acid sequences include, for example, longer half-lives than transcripts made from the native sequences. There is the production of a group of RNA transcripts with more favorable properties.
[0133]
The invention also includes the production of DNA sequences encoding SSCP and its derivatives, or fragments thereof, entirely by synthetic chemistry. After production, the synthetic sequence may be inserted into any of the various available expression vectors and cell lines using reagents known in the art. In addition, synthetic chemistry can be used to induce mutations in one sequence encoding SSCP or any fragment thereof.
[0134]
The invention further includes polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, under various stringency conditions, particularly SEQ ID NOs: 64-126, and fragments thereof ( See, for example, Wahl, GM and SL Berger (1987) Methods Enzymol. 152: 399-407; Kimmel.AR (1987) Methods Enzymol.152: 507-511). Hybridization conditions, including annealing and washing conditions, are described in “Definitions”.
[0135]
Methods for DNA sequencing are known in the art, and any of the embodiments of the present invention may employ a DNA sequencing method. Enzymes can be used in DNA sequencing methods. For example, Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (Applied Biosystems), thermostable T7 polymerase (Amersham, Pharmacia Biotech, Piscataway, NY). Alternatively, a polymerase and a proofreading exonuclease may be used in combination, for example, as found in the ELONGASE amplification system (Life Technologies, Gaithersburg MD). Preferably, an apparatus such as a MICROLAB2200 liquid transfer system (Hamilton, Reno, NV), a PTC200 thermal cycler (MJ Research, Watertown MA) and an ABI CATARYST 800 thermal cycler (Applied Biosystems) are used to automate the arrangement. Next, sequencing is performed using an ABI 373 or 377 DNA sequencing system (Applied Biosystems), a MEGABACE 1000 DNA sequencing system (Molecular Dynamics, Sunnyvale CA) or other systems known in the art. The resulting sequence is analyzed using various algorithms well known in the art (eg, Ausubel, FM (1997)).Short Protocols in Molecular Biology, John Wiley & Sons, New York NY, 7.7 units, Meyers, R.E. A. (1995)Molecular Biology and Biotechnology, Wiley VCH, New York NY, pages 856-853).
[0136]
Utilizing partial nucleotide sequences and extending PCR-based nucleic acid sequences using various methods known in the art based on PCR to detect upstream sequences such as promoters and regulatory elements. obtain. For example, one of the methods that can be used, restriction site PCR, is a method of amplifying an unknown sequence from genomic DNA in a cloning vector using universal primers and nested primers (see, for example, Sarkar, G. et al. 1993) PCR Methods Applic. 2: 318-322). Another method is the inverse PCR method, in which an unknown sequence is amplified from a circularized template using primers extending in various directions. The template is obtained from a restriction enzyme fragment group consisting of a sequence group of a known genomic locus and surrounding sequences (see, for example, Triglia, T. et al. (1988) Nucleic Acids Res. 16: 8186). A third method is the capture PCR method, which includes a method of PCR-amplifying DNA fragments adjacent to a known sequence of human and yeast artificial chromosomal DNA (eg, Lagerstrom, M. et al. (1991) PCR Methods). Appl. 1: 111-119). In this method, a recombinant double-stranded sequence can be inserted within an unknown sequence region using digestion and ligation reactions of multiple restriction enzymes before performing PCR. Other methods that can be used to search for unknown sequences are also known in the art (see, eg, Parker, JD, et al. (1991) Nucleic Acids Res. 19: 3055-3060). In addition, PCR, nested primers, and PROMOTERFINDER libraries (Clontech, Palo Alto CA) can be used to walk genomic DNA. This procedure eliminates the need to screen libraries and is useful for finding intron / exon junctions. All PCR-based methods use commercially available software, such as OLIGO 4.06 Primer Analysis Software (National Biosciences, Plymouth MN) or another suitable program, and have a GC content of about 22-30 nucleotides. Primers can be designed to anneal to the template at a temperature of about 68 ° C. to 72 ° C. at 50% or more.
[0137]
When screening full-length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. In addition, libraries of random primers often include sequences having the 5 'region of the genes, and are suitable for situations where an oligo d (T) library cannot produce a full-length cDNA. Genomic libraries may be useful for extension of sequence into 5 'non-transcribed regulatory regions.
[0138]
Commercially available capillary electrophoresis systems can be used to analyze the size of the sequencing or PCR product or to confirm its nucleotide sequence. Specifically, capillary sequencing consists of a flowable polymer for electrophoretic separation, a laser-activated fluorescent dye that is specific for four different nucleotides, and detection of the emitted wavelength. And a CCD camera used for The output / light intensity can be converted to an electrical signal using appropriate software (such as GENOTYPER, SEQUENCE NAVIGATOR from Applied Biosystems). The entire process from sample loading to computer analysis and electronic data display is computer controllable. Capillary electrophoresis is particularly suitable for sequencing small DNA fragments that are present in small amounts in a particular sample.
[0139]
In another embodiment of the invention, the polynucleotide sequence encoding SSCP, or a fragment thereof, may be cloned into a recombinant DNA molecule that expresses SSCP, a fragment thereof, or a functional equivalent in a suitable host cell. Due to the inherent degeneracy of the genetic code, other DNA sequences encoding substantially the same or a functionally equivalent amino acid sequence can be produced, and these sequences can be used for the expression of SSCP.
[0140]
For various purposes, the nucleotide sequences of the present invention can be recombined using a number of methods generally known in the art to modify the sequences encoding SSCP. The various purposes of this recombination include, but are not limited to, the cloning of gene products or the modification of processing and / or expression. Nucleotide sequences can be recombined using random fragmentation of gene fragments and synthetic oligonucleotides and DNA shuffling by PCR reassembly. For example, site-directed mutagenesis via oligonucleotides can be used to introduce mutations that create new restriction sites, alter glycosylation patterns, alter codon preferences, generate splice variant sequences, and the like.
[0141]
The nucleotides of the present invention may be obtained from MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; U.S. Pat. No. 5,837,458; Chang, C.-C. et al. (1999) Nat. C. et al. (1999) Nat. Biotechnol. 17: 259-264; Crameri, A. et al. (1996) Nat. Biotechnol. 14: 315-319). This can modify or improve the biological properties of SSCP, such as biological or enzymatic activity, or the ability to bind to other molecules or compounds. DNA shuffling is a process of preparing a library of gene mutation sequences using gene fragment recombination via PCR. The library then goes through a selection or screening procedure that identifies gene variants with the desired properties. Subsequently, these suitable mutated sequences can be pooled and subjected to repeated DNA shuffling and selection / screening. Thus, by "artificial" breeding and rapid molecular evolution, diverse genes are created. For example, fragments of a single gene with random point mutations can be recombined, screened, and then shuffled until the desired properties are optimized. Alternatively, a group of fragments of a given gene can be recombined with a fragment of a homologous gene of the same gene family obtained from either the same species or a different species, thereby controlling the genetic diversity of a plurality of naturally occurring genes. Can be maximized in a way.
[0142]
According to another embodiment, the sequence encoding SSCP can be synthesized in whole or in part using chemical methods well known in the art (eg, Caruthers. MH. Et al. (1980) Nucleic). Acids Symp. Ser. 7: 215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7: 225-232). Alternatively, SCCP itself or a fragment thereof may be synthesized using chemical methods. For example, peptide synthesis can be performed using various liquid or solid phase techniques (eg, Creighton, T. (1984)).Proteins, Structures and Molecular Properties, WH Freeman, New York NY, pp. 55-60, Robert, J. et al. Y. (1995) Science 269: 202-204). Automated synthesis can be achieved using an ABI 431A peptide synthesizer (Applied Biosystems). Furthermore, the amino acid sequence of SSCP, or any portion thereof, may be modified by modification during direct synthesis and / or in combination with sequences from other proteins or any portion thereof to form the mutant sequence polypeptide. It is possible to produce or to produce a polypeptide having the sequence of the native polypeptide.
[0143]
The peptide can be substantially purified using preparative high performance liquid chromatography (see, for example, Chiez, RM and FZ Regnier (1990) Methods Enzymol. 182: 392-421). The composition of the synthetic peptide can be confirmed by amino acid analysis or sequencing (see, eg, Creighton, supra, pages 28-53).
[0144]
To express a biologically active SSCP, the nucleotide sequence encoding SSCP or a derivative thereof can be inserted into a suitable expression vector. A suitable expression vector is a vector having a group of elements necessary for controlling transcription and translation of a coding sequence inserted into a suitable host. Necessary elements include regulatory sequences (e.g., enhancers, constitutive and inducible promoters, 5 'and 3' untranslated regions, etc.) in the vector and in the polynucleotide sequence encoding SSCP. The required elements vary in their characteristics and specificities. With a specific initiation signal, it is also possible to achieve a more efficient translation of the sequence encoding SSCP. Examples of initiation signals include the ATG initiation codon and nearby sequences such as the Kozak sequence. If the sequence encoding SSCP, its initiation codon, and upstream regulatory sequences have been inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be required. However, when only the coding sequence or a fragment thereof is inserted, exogenous translational control signals, such as an in-frame ATG start codon, should be included in the expression vector. Exogenous translation elements and initiation codons can be of various natural and synthetic origin. Increasing the efficiency of expression can be achieved by including an enhancer suitable for the particular host cell line used (see, eg, Scharf, D. et al. (1994) Results Probl. Cell Differ. 20: 125-162).
[0145]
Using well known methods to those skilled in the art, an expression vector having a sequence encoding SSCP and suitable transcription and translation control elements can be generated. These methods include:in in vitroRecombinant DNA technology, synthetic technology, andin VivoThere is a gene recombination technique (for example, Sambrook, J. et al. (1989)).Molecular Cloning, A Laboratory ManualAusubel, F., Cold Spring Harbor Press, Plainview NY, Chapters 4 and 8, and 16-17; M. other. (1995)Current Protocols in Molecular Biology, John Wiley & Sons, New York NY, Chapters 9, 13, and 16).
[0146]
A variety of expression vector / host systems can be utilized to retain and express the sequence encoding SSCP. Such expression vector / host systems include, but are not limited to, bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors, and microorganisms such as yeast transformed with yeast expression vectors. Insect cell lines infected with a viral expression vector (eg, baculovirus), or transformed with a viral expression vector (eg, cauliflower mosaic virus, CaMV or tobacco mosaic virus, TMV) or a bacterial expression vector (eg, Ti plasmid or pBR322 plasmid). (Eg, Sambrook, supra, Ausubel, Van Heke, G. and SM Schuster (1989) J. Biol. Chem. 264: 5503-550). Natl. Acad. Sci. USA 91: 3224-3227, Sandig, V. et al. (1996) Hum. Gene Ther. 7: 1937-1945, Takamatsu, N., Engelhard, EK, et al., (1994) Proc. 1987) EMBO J. 6: 307-311, "The McGraw Hill Yearbook of Science and Technology" (1992) McGraw Hill New York, NY, Jan. 1-96, 1968. Natl. Acad. Sci. USA 81: 3655-3659, Harrington, JJ et al. (1997) Nat. Genet. 5: see 345-355). Nucleotide sequences can be delivered to target organs, tissues or cell populations using expression vectors derived from retroviruses, adenoviruses, herpesviruses or vaccinia viruses, or expression vectors derived from various bacterial plasmids (eg, Di Nicola). 5 (6): 350-356, Yu, M. et al. (1993) Proc. Natl. Acad. Sci. USA 90 (13): 6340-6344, Buller, R., M. et al., (1998) Cancer Gen. Ther. (1985) Nature 317 (6040): 813-815; McGregor, DP, et al. (1994) Mol. Immunol. 31 (3): 219-226, Verma, IM and N. Somia. (1997) Na cure 389: 239-242). The present invention is not limited by the host cell used.
[0147]
In bacterial systems, a number of cloning and expression vectors are available depending upon the use for which the polynucleotide sequence encoding SSCP will be used. For example, multifunctional E. coli vectors such as PBLUESCRIPT (Stratagene, La Jolla CA) or PSPORT1 plasmid (Life Technologies) can be used for routine cloning, subcloning, and propagation of a polynucleotide sequence encoding SSCP. Ligation of the sequence encoding SCCP into the multicloning site of this vector disrupts the lacZ gene, allowing a colorimetric screening method to identify transformed bacteria harboring the recombinant molecule. In addition, these vectors are used in the cloned sequence.in in vitroIt may also be useful for transcription, dideoxy sequencing, single-stranded rescue by helper phage, generation of nested deletions (see, eg, Van Heake, G. and SM Schuster (1989) J. Biol. Chem. 264: 55035509). When a large amount of SSCP is required, for example, for the production of an antibody, a vector that induces a high level of SSCP expression can be used. For example, vectors having a strong inducible SP6 or T7 bacteriophage promoter can be used.
[0148]
Yeast expression systems can also be used to produce SSCP. Numerous vectors having constitutive or inducible promoters, such as α-factor, alcohol oxidase, PGH promoter, etc.Saccharomyces cerevisiae) Or Pichia yeast (Pichia pastoris) Can be used. In addition, such vectors direct either the secretion of the expressed protein or its retention in cells, and allow the integration of foreign sequences into the host genome for stable growth (eg, Ausubel, 1995, supra, Bitter, GA, et al. (1987) Methods Enzymol. 153: 516-544, and Scorer.CA, et al. (1994) Bio / Technology 12: 181-184).
[0149]
It is also possible to express SSCP using a plant system. Transcription of the sequence encoding SSCP can be promoted by a viral promoter, such as the 35S and 19S promoters from CaMV as used alone or in combination with an omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6: 307-311). Alternatively, a plant promoter, such as the small subunit of RuBisCO, or a heat shock promoter can be used (eg, Coruzzi, G. et al. (1984) EMBO J. 3: 1671-1680; Broglie, R. et al. (1984) Science 224). : 838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ. 17: 85-105). These constructs can be introduced into plant cells by direct DNA transformation or by pathogen-mediated transfection (eg, the McGraw-Hill Science and Technology Yearbook).The McGraw Hill Yearbook of Science and Technology(1992) McGraw Hill New York NY, pp. 191-196).
[0150]
In mammalian cells, a number of viral-based expression systems may be utilized. When an adenovirus is used as an expression vector, the sequence encoding SSCP can be ligated to an adenovirus transcription / translation complex consisting of the late promoter and tripartite leader sequence. Insertion into a non-essential E1 or E3 region of the adenovirus genome can result in an infectious virus expressing SSCP in host cells (eg, Logan, J. and T. Shenk (1984) Proc. Natl. Acad. ScL USA 81: 36553659). In addition, transcription enhancers such as the Rous sarcoma virus (RSV) enhancer may be used to increase expression in mammalian host cells. High levels of protein expression can also be achieved using vectors based on SV40 or EBV.
[0151]
In addition, human artificial chromosomes (HACs) can be used to deliver DNA fragments larger than fragments that can be contained in or expressed from a certain plasmid. Approximately 6 kb to 10 Mb of HACs have been made for treatment and delivered using conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) (eg, Harrington. JJ et al. (1997) Nat Genet. 15). : 345-355).
[0152]
For long-term production of recombinant proteins of mammalian origin, stable expression of SSCP in cell lines is desirable. For example, expression vectors and a selectable marker gene on the same or a different vector may be used to transform a sequence encoding SSCP into a cell line. The expression vector used may have a viral origin of replication and / or endogenous expression elements. After the introduction of the vector, the cells may be allowed to grow for about 1-2 days in an enriched medium before being transferred to a selective medium. The purpose of the selectable marker is to confer resistance to the selection agent, and the presence of the selectable marker allows for the growth and recovery of cells that successfully express the introduced sequence. Resistant clones of stably transformed cells can be propagated using tissue culture techniques appropriate for the cell type.
[0153]
The transformed cell line can be recovered using any number of selection systems. Such selection systems include, but are not limited to, tk⁻A herpes simplex virus thymidine kinase gene used for cells, and apr⁻There are herpes simplex virus adenine phosphoribosyltransferase genes used for cells (see, eg, Wigler, M. et al. (1977) Cell 11: 223-232; Lowy, I. et al. (1980) Cell 22: 817-823. ). Also, resistance to antimetabolites, antibiotics or herbicides can be used as the basis for selection. For example, dhfr confers resistance to methotrexate, neo confers resistance to aminoglycosid neomycin and G-418, als confers resistance to chlorsulfuron, and pat confers resistance to phosphinothricin acetyltransferase (eg, Wigler, M. Natl. Acad. Sci. USA 77: 3567-3570, Colbere-Garapin, F. et al. (1981) J. Mol. Biol. 150: 1-14). Other selectable genes, such as trpB and hisD, which alter cellular requirements for metabolism, are also described in the literature (eg, Hartman, SC and RC Mulligan (1988) Proc). Natl.Acad.Sci.USA 85: 80478051). Visible markers such as anthocyanins, green fluorescent protein (GFP; Clontech), β-glucuronidase and its substrate β-glucuronide, or luciferase and its substrate luciferin can be used. These markers can be used to not only identify transformants, but also quantify transient or stable protein expression due to a particular vector system (eg, Rhodes, CA (1995) Methods Mol. Biol). 55: 121131).
[0154]
Even if the presence or absence of marker gene expression indicates the presence of the gene of interest, it may be necessary to confirm the presence and expression of that gene. For example, if the sequence encoding SSCP is inserted into a marker gene sequence, transformed cells having the sequence encoding SSCP can be identified by a lack of marker gene function. Alternatively, a marker gene can be placed in tandem with the sequence encoding SSCP under the control of one promoter. Expression of the marker gene in response to induction or selection usually also indicates expression of the tandem gene.
[0155]
In general, host cells that have a nucleic acid sequence that encodes a SSCP and that express a SCCP can be identified using a variety of methods well known to those of skill in the art. These methods include DNA-DNA or DNA-RNA hybridization, PCR techniques, and protein organisms including membrane-based, solution-based, or chip-based techniques for detecting and / or quantifying nucleic acid or protein sequences. Including but not limited to biological testing or immunological assays.
[0156]
Immunological methods for detecting and measuring SCCP expression using specific polyclonal or specific monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence activated cell sorting (FACS, flow cytometry), and the like. A two-site, monoclonal-based immunoassay using monoclonal antibodies reactive to two non-interfering epitopes on SCCP is preferred, but a competitive binding test can also be used. These and other assays are known in the art (eg, Hampton, R. et al. (1990)).Serological Methods, a Laboratory ManualAPS Press. St. Paul. MN, Sect. IV, Coligan, J.M. E. FIG. Others (1997)Current Protocols in Immunology, Green Pub. Associates and Wiley-Interscience, New York NY, Pound, J .; D. (1998)Immunochemical Protocols, Humana Press, Totowa NJ).
[0157]
A wide variety of methods for labeling and conjugation are known to those skilled in the art and may be used in various nucleic acid and amino acid assays. Methods for generating labeled hybridization or PCR probes for detecting sequences related to a polynucleotide encoding SSCP include oligo labeling, nick translation, end labeling (end labeling), or labeling. PCR amplification using the nucleotides provided. Alternatively, the sequence encoding SSCP, or any fragment thereof, can be cloned into a vector for generating an mRNA probe. Such vectors are known in the art and are commercially available, with the addition of a suitable RNA polymerase such as T7, T3 or SP6 and labeled nucleotides,in in vitroCan be used for the synthesis of RNA probes. Such methods are described, for example, in Amersham Pharmacia Biotech, Promega (Madison WI), U.S.A. S. It can be performed using various kits commercially available from Biochemical and the like. Suitable reporter molecules or labels that can be used to facilitate detection include substrates, cofactors, inhibitors, magnetic particles, as well as radionuclides, enzymes, fluorescent agents, chemiluminescent agents, chromogenic agents, and the like.
[0158]
Host cells transformed with the nucleotide sequence encoding SSCP are cultured under conditions suitable for the expression and recovery of the protein from cell culture. Whether a protein produced from a transformed cell is secreted or remains intracellular depends on the sequence used, the vector, or both. One of skill in the art will appreciate that expression vectors carrying a polynucleotide encoding SSCP can be designed to include a signal sequence that directs secretion of SSCP across a prokaryotic or eukaryotic cell membrane.
[0159]
In addition, selection of a host cell strain may be made by its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing that cleaves the "prepro" or "pro" form of the protein can be used to identify protein targeting, folding, and / or activity. Various host cells (eg, CHO, HeLa, MDCK, MEK293, WI38, etc.) with specific cellular machinery and characteristic mechanisms for post-translational activity are available from the American Type Culture Collection (ATCC, Manassas, VA). ) And can be selected to ensure correct modification and processing of the foreign protein.
[0160]
In another embodiment of the invention, the fusion of a native or modified or recombinant nucleic acid sequence encoding SSCP to a heterologous sequence in any of the host systems described above This can result in translation of the protein. For example, a chimeric SSCP protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate screening of a peptide library for inhibitors of SSCP activity. Heterologous protein moieties and heterologous peptide moieties can also facilitate purification of the fusion protein using commercially available affinity matrices. Such portions include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, There is hemagglutinin (HA). GST on immobilized glutathione, MBP on maltose, Trx on phenylarsine oxide, CBP on calmodulin, and 6-His on metal chelating resin allow purification of homologous fusion proteins. FLAG, c-myc and hemagglutinin (HA) allow immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags. Alternatively, if the fusion protein is genetically engineered to have a proteolytic cleavage site between the sequence encoding the SSCP and the heterologous protein sequence, the SSCP can be cleaved from the heterologous portion after purification. Methods for expression and purification of the fusion protein are described in Ausubel (1995), Chapter 10, supra. Various commercially available kits can also be used to facilitate expression and purification of the fusion protein.
[0161]
In another embodiment of the invention, a TNT rabbit reticulocyte lysate or a wheat germ extract system (Promega) is used.in in vitroThus, synthesis of radiolabeled SSCP is possible. These systems couple the transcription and translation of a protein coding sequence operably linked to a T7, T3 or SP6 promoter. Translation, for example³⁵It occurs in the presence of a radiolabeled amino acid precursor such as S-methionine.
[0162]
A compound that specifically binds to SCCP can be screened using the SSCP of the present invention or a fragment thereof. At least one or more test compounds can be used to screen for specific binding to SCCP. Test compounds include antibodies, oligonucleotides, proteins (eg, receptors) or small molecules.
[0163]
In certain embodiments, the compound so identified is closely related to the natural ligand of SCCP, for example, a ligand or a fragment thereof, or a natural substrate, a structural or functional mimetic or A natural binding partner (eg, Coligan, JE et al. (1991)Current Protocols in Immunology See Chapter 5 of 1 (2)). Similarly, the compound may be closely related to the natural receptor to which the SSCP binds, or at least to some fragment of the receptor, for example, a ligand binding site. In each case, the compound can be rationally designed using known techniques. In some embodiments, screening for these compounds involves the generation of suitable populations of cells that express SSCP as a secreted protein or on the cell membrane. Suitable cells include cells from mammals, yeast, Drosophila, or E. coli. Cells expressing SCCP, or cell membrane fractions containing SSCP, are contacted with a test compound and analyzed for inhibition of binding, stimulation, or activity of either SCCP or the compound.
[0164]
Some assays simply allow the test compound to be conjugated experimentally to the polypeptide, and the binding detected by a fluorescent dye, radioisotope, enzyme conjugate or other detectable label. For example, the assay may include mixing at least one test compound with SSCP in solution or immobilized on a solid support, and detecting binding of the compound to SSCP. Alternatively, the detection and measurement of binding of a test compound in the presence of a labeled competitor can be performed. In addition, the assay can be performed using cell-free reconstituted specimens, chemical libraries, or mixtures of natural products, where the test compound (s) are released in solution or immobilized on a solid support.
[0165]
Using the SSCP of the present invention or a fragment thereof, it is possible to screen for a compound that modulates the activity of SSCP. Such compounds include agonists, antagonists, or partial or inverse agonists, and the like. In certain embodiments, the assay is performed under conditions where the activity of the SSCP binds to the at least one test compound, and the activity of the SSCP in the presence of the test compound and the activity of the SSCP in the absence of the test compound. Compare with the activity of SECP. A change in the activity of SSCP in the presence of the test compound indicates the presence of a compound that modulates the activity of SSCP. Alternatively, the test compound comprises SSCP under conditions suitable for the activity of SSCP.in in vitroAlternatively, the assay is performed in combination with a cell release system. In any of these assays, the test compound that modulates the activity of SSCP may modulate indirectly and may not require direct contact with the test compound. At least one or more test compounds can be screened.
[0166]
In another embodiment, a polynucleotide encoding SSCP or a mammalian homolog thereof is "knocked out" in an animal model system using homologous recombination in embryonic stem cells (ES cells). Such techniques are well known in the art and are useful for generating animal models of human disease (see, eg, US Pat. Nos. 5,175,383 and 5,767,337). Mouse ES cells, such as the 129 / SvJ cell line, are derived from early mouse embryos and can be grown in culture. The ES cells are transformed with a vector containing the gene of interest disrupted with a marker gene such as the neomycin phosphotransferase gene (neo: Capecchi, MR (1989) Science 244: 1288-1292). This vector is integrated into the corresponding region of the host genome by homologous recombination. In another method, homologous recombination is performed using the Cre-loxP system to knock out the target gene in a tissue-specific or developmental stage-specific manner (Marth, JD (1996) Clin. Invest. 97: 1999-2002). Wagner, KU, et al. (1997) Nucleic Acids Res. 25: 4323-4330). The transformed ES cells are identified and microinjected into mouse cell blastocysts collected from, for example, the C57BL / 6 mouse strain. These blastocysts are surgically introduced into pseudopregnant females, the genetic traits of the resulting chimeric progeny are identified, and they are bred to produce heterozygous or homozygous lines. The transgenic animals thus produced can be tested with potential therapeutic or toxic agents.
[0167]
Further, polynucleotides encoding SSCP arein in vitroCan operate on ES cells derived from human blastocysts. Human ES cells have the potential to differentiate into at least eight distinct cell lineages, including endoderm, mesoderm and ectoderm cell types. These cell lines differentiate into, for example, neurons, hematopoietic lineages and cardiomyocytes (Thomson, JA, et al. (1998) Science 282: 1145-1147).
[0168]
Polynucleotides that encode SSCP can also be used to create "knock-in" humanized animals (pigs) or transgenic animals (mouse or rat) modeled on human disease. Using the knock-in technique, a region of the polynucleotide encoding SSCP is injected into animal ES cells and the injected sequence is integrated into the animal cell genome. The transformed cells are injected into a blastula and the blastula is implanted as described above. Tested on transgenic progeny or inbreds, treated with potential medicines to obtain information on treatment of human disease. Alternatively, inbred mammalian strains that overexpress SSCP, eg, secrete SSCP into milk, can be a convenient source of proteins (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4: 55-). 74).
[0169]
(Treatment)
There are chemical and structural similarities between some regions of the SCCP and the secreted proteins, for example, in the context of sequences and motifs. In addition, SSCP expression is closely associated with healthy or neoplastic lung, heart, brain, skin, colon epithelium, and cardiovascular tissues, as well as in nervous, urinary, reproductive, and digestive tissues. Closely related to lineage, immune, diseased, and tumor tissues. Thus, SSCP is thought to play a role in abnormal cell proliferation, autoimmune / inflammatory diseases, cardiovascular disorders, neurological disorders, and developmental disorders. In the treatment of diseases associated with increased expression or activity of SSCP, it is desirable to reduce the expression or activity of SSCP. In the treatment of a disease associated with decreased expression or activity of SSCP, it is desirable to increase the expression or activity of SSCP.
[0170]
Thus, in one embodiment, it is possible to administer SSCP or a fragment or derivative thereof to a patient for the treatment or prevention of a disease associated with reduced expression or activity of SSCP. Such disorders include, but are not limited to, abnormal cell proliferation, autoimmune / inflammatory diseases, cardiovascular disorders, neurological disorders, and developmental disorders, including actinic keratosis, arterial disorders, Sclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia, psoriasis, primary thrombocythemia, And adenocarcinoma and leukemia, lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma, specifically, adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gallbladder, ganglion, digestive tract, heart, kidney Including cancer of the liver, lungs, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary gland, skin, spleen, testis, thymus, thyroid, and uterus; among autoimmune / inflammatory diseases, Acquired immunodeficiency syndrome (AIDS), Addison's disease (chronic Adult adrenal insufficiency syndrome), adult respiratory distress syndrome, allergy, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyglandularity Endocrine Candida ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes, pulmonary emphysema, lymphocytic factor accidental lymphopenia, neonatal hemolysis Disease (erythroblastosis fetalis), erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis , Myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's disease Group, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, cancer complications, hemodialysis, extracorporeal circulation, viral infection, bacterial infection Includes fungal infections, parasitic infections, protozoan infections, helminth infections, trauma, cardiovascular disorders including congestive heart failure, ischemic heart disease, angina pectoris, myocardial infarction, hypertensive heart Disease, degenerative valvular heart disease, calcified aortic stenosis, congenital bicuspid aortic valve, mitral annular calcification, mitral valve prolapse, rheumatic fever, rheumatic heart disease, infectious Endocarditis, non-bacterial thrombotic endocarditis, systemic lupus erythematosus endocarditis, carcinoid heart disease, cardiomyopathy, myocarditis, pericarditis, neoplastic heart disease, congenital heart disease, heart transplantation Complications, arteriovenous fistula, atherosclerosis, hypertension, vasculitis, Raynaud's disease, aneurysm, arterial dissection, varicose veins, thrombophlebitis and venous thrombosis, vascular tumors, thrombolytic complications, balloon angioplasty, It includes vascular replacement, coronary artery bypass graft surgery, and among neurological disorders, epilepsy, ischemic cerebrovascular disease, stroke, brain tumor, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and Other extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neuromuscular atrophy, retinitis pigmentosa (retinitis pigmentosa), hereditary ataxia, multiple sclerosis and others Demyelinating disease, bacterial and viral meningitis, brain abscess, subdural abscess, epidural abscess, purulent intracranial thrombophlebitis, myelitis and radiculitis, c Ruth central nervous system diseases and prion diseases (including Kuru, Creutzfeldt-Jakob disease and Gerstmann-Straussler-Scheinker syndrome), fatal familial insomnia, nervous nutritional and metabolic diseases, neurofibromatosis Central nervous system mental retardation and other developmental disorders, including tuberous sclerosis, cerebellar retinal hemangioblastomatosis, cerebral trigeminal neurovascular syndrome, Down syndrome, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, Cranial nerve disorders, spinal cord disorders, muscular dystrophy and other neuromuscular disorders, peripheral nerve disorders, dermatomyositis and polymyositis, and hereditary, metabolic, endocrine, and toxic myopathy, and myasthenia gravis, periodic quadriplegia, Mental disorders (mood disorders, anxiety disorders, schizophrenia) Schizophrenia), seasonal affective disorder (SAD), restlessness, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonia, paranoid psychosis, postherpetic neuralgia, and Tourette's disease, and progressive nucleus Includes epilepsy, corticobasal degeneration, and familial frontotemporal dementia, and also includes developmental disorders, including tubular acidosis, anemia, Cushing's syndrome, chondrodysplasia Dwarfism, Duchenne / Becker muscular dystrophy, epilepsy, gonadal dysplasia, WAGR syndrome (Wilms tumor, aniridia, genitourinary disorders, mental retardation), Smith-Magenis syndrome, myelodysplastic syndrome , Hereditary mucosal epithelial dysplasia, hereditary keratoderma, Hereditary neuropathies such as Jarco-Marie-Tooth disease and neurofibromatosis, seizure disorders such as hypothyroidism, hydrocephalus, Syndenham's chorea and cerebral palsy, spina bifida, anencephaly, skull Includes spina bifida, congenital glaucoma, cataract, and sensorineural hearing loss.
[0171]
In another embodiment, a vector capable of expressing SSCP or a fragment or derivative thereof for the treatment or prevention of a disease associated with decreased expression or activity of SSCP, including but not limited to the diseases described above, is administered to a patient. Can also be administered.
[0172]
In yet another embodiment, a composition comprising substantially purified SSCP for the treatment or prevention of a disease associated with reduced expression or activity of SSCP, including, but not limited to, the diseases described above. It can also be administered to a patient together with a suitable pharmaceutical carrier.
[0173]
In yet another embodiment, an agonist that modulates the activity of SSCP is administered to a patient for the treatment or prevention of a disease associated with reduced expression or activity of SSCP, including but not limited to the diseases listed above. Can also be administered.
[0174]
In a further example, a patient can be administered an antagonist of SSCP for the treatment or prevention of a disease associated with increased expression or activity of SSCP. Such disorders include, but are not limited to, the abnormal cell proliferation described above, autoimmune / inflammatory disorders, cardiovascular disorders, neurological disorders, and developmental disorders. In one embodiment, an antibody that specifically binds to SSCP can be used directly as an antagonist or indirectly as a targeting or delivery mechanism to deliver the agent to cells or tissues that express SSCP.
[0175]
In another embodiment, a vector expressing the complement of the polynucleotide encoding SSCP is administered to the patient to treat a disease associated with increased expression or activity of SSCP, including, but not limited to, the diseases described above. Treatment or prevention is also possible.
[0176]
In another embodiment, any of the proteins, antagonists, antibodies, agonists, complementary sequences, or vectors of the invention may be administered in combination with another suitable therapeutic agent. Suitable therapeutic agents for use in combination therapy may be selected by one skilled in the art according to conventional pharmaceutical principles. Combination with a therapeutic agent can have a synergistic effect in the treatment or prevention of the various diseases described above. By this method, a small amount of each drug can achieve a medicinal effect, thereby reducing the possibility of side effects.
[0177]
Antagonists of SCCP may be prepared using methods common in the art. Specifically, antibodies can be produced using purified SSCP, or a therapeutic drug library can be screened to identify those that specifically bind to SSCP. Antibodies to SSCP can also be produced using several methods common in the art. Examples of such antibodies include polyclonal, monoclonal, chimeric, single chain, Fab fragments, and fragments produced by Fab expression libraries. However, it is not limited to these. Neutralizing antibodies (ie, antibodies that inhibit dimer formation) are generally suitable for therapeutic use.
[0178]
For the production of antibodies, various hosts, including goats, rabbits, rats, mice, humans, etc., can be immunized by injection of SSCP, or any fragment or oligopeptide thereof with immunogenic properties. Depending on the host species, various adjuvants can be used to enhance the immune response. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gel adjuvants such as aluminum hydroxide, lysolecithin, pluronic polyols, polyanions, peptides, oily emulsions, keyhole limpet hemocyanin (KLH), dinitrophenol, etc. Surfactants. Among the adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum (Corynebacterium parvumIs particularly preferred.
[0179]
The oligopeptide, peptide, or fragment used to induce an antibody against SCCP preferably has an amino acid sequence of at least about 5 amino acids, and generally has at least about 10 amino acids. Desirably, these oligopeptides, peptides or fragments are identical to a portion of the amino acid sequence of the native protein. Short stretches of SCCP amino acids can be fused to sequences of another protein, such as KLH, and antibodies to the chimeric molecule can be produced.
[0180]
Monoclonal antibodies to SSCP can be made using any technique that produces antibody molecules by continuous cell lines in culture. Such techniques include, but are not limited to, hybridoma technology, human B cell hybridoma technology, and EBV-hybridoma technology (eg, Kohler, G. et al. (1975) Nature 256: 495-497, Kozbor, D.). (1985) J. Immunol. Methods 81: 31-42, Cote, RJ, et al. (1983) Proc. Natl. Acad. Sci. USA 80: 2026-2030, Cole, SP, et al. ) Mol. Cell Biol. 62: 109-120).
[0181]
In addition, techniques developed for the production of "chimeric antibodies", such as splicing mouse antibody genes to human antibody genes, are used to obtain molecules with suitable antigen specificity and biological activity (eg, Morrison (1984) Proc. Natl. Acad. Sci. USA 81: 68516855, Neuberger, MS. Et al. (1984) Nature 312: 604-608, Takeda, S. et al. (1985) Nature. 314: 452-454). Alternatively, SCCP-specific single-chain antibodies are generated using techniques well known in the art and applying the described techniques for the production of single-chain antibodies. Antibodies with related specificities but differing idiotypic compositions can also be produced by chain shuffling from random combinations of immunoglobulin libraries (eg, Burton DR (1991) Proc. Natl. Acad. ScL USA 88: 10134-10137).
[0182]
The production of antibodies in lymphocyte populationsin VivoIt can also be done by inducing production or by screening immunoglobulin libraries or a panel of highly specific binding reagents as disclosed in the literature (eg, Orlandi, R. et al. (1989) Proc. Natl.Acad.Sci.USA 86: 3833-3837, Winter, G. et al. (1991) Nature 349: 293-299).
[0183]
Antibody fragments having a specific binding site for SCCP can also be generated. For example, but not by way of limitation, such fragments may include F (ab ') 2 produced by pepsin digestion of the antibody molecule.₂ Fragment and F (ab ')₂ Some Fab fragments are made by reducing the disulfide bridges of the fragment. Alternatively, by creating a Fab expression library, monoclonal Fab fragments with the desired specificity can be quickly and easily identified (eg, Huse, WD, et al. (1989) Science 246: 1275-1281). See).
[0184]
Screening can be performed using various immunoassays (immunoassays) to identify antibodies with the desired specificity. Numerous protocols for competitive binding, using either polyclonal or monoclonal antibodies with established specificity, or immunoradiation assays, are well known in the art. Usually such immunoassays involve the measurement of complex formation between SSCP and its specific antibody. A two-site, monoclonal-based immunoassay using monoclonal antibodies reactive to two non-interfering SSCP epitopes is commonly used, but competitive binding assays can also be used (Pound, supra).
[0185]
Various methods, such as Scatchard analysis, can be used with radioimmunoassay techniques to calculate the affinity of antibodies for SSCP. The affinity is represented by a binding constant Ka, which is a value obtained by dividing the molar concentration of the SSCP antibody complex under the equilibrium state by the molar concentration of the free antibody and the free antigen. The Ka of the reagent, for polyclonal antibodies with heterogeneous affinity for a number of SSCP epitopes, represents the average affinity or avidity of the antibody for SSCP. For monoclonal antibodies monospecific for a particular SCCP epitope, the Ka of the reagent represents a true measure of affinity. Ka value is 10⁹-10¹²The liter / mol high affinity antibody reagent is preferably used in immunoassays where the SCCP antibody conjugate must withstand harsh treatments. Ka value is 10⁶-10⁷liter / mol of low affinity antibody reagents is preferably used for immunopurification and similar treatments in which SCCP must ultimately (preferably in the activated state) dissociate from the antibody (Catty, D.C.). (1988)Antibodies, Volume I: A Practical ApproachJ., IRL Press, Washington, DC; E. FIG. And Cryer, A .; (1991)A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York NY).
[0186]
The titer and avidity of the polyclonal antibody reagents can be further evaluated to determine the quality and suitability of such reagents for certain subsequent applications. For example, polyclonal antibody medicaments containing at least 1-2 mg / ml of specific antibody, preferably 5-10 mg / ml of specific antibody, are generally used in processes where the SSCP antibody complex must be precipitated. Information on antibody specificity, titer, avidity, and guidelines for antibody quality and use in various applications is generally available. (Refer to the above-mentioned Catty reference, the same Colligan et al. Reference).
[0187]
In another embodiment of the invention, a polynucleotide encoding SSCP, or any fragment or complement thereof, can be used for therapeutic purposes. In one embodiment, sequences that are complementary to the coding and regulatory regions of the gene encoding SSCP and antisense molecules (DNA, RNA, PNA, or modified oligonucleotides) are designed to modify gene expression. obtain. Such techniques are well known in the art, and antisense oligonucleotides or large fragments can be designed from the control region of the sequence encoding SSCP, or from various locations along the coding region (see, eg, Agrawal, S. et al. , Editing (1996)Antisense Therapeutics, Humana Press Inc. , Totawa NJ).
[0188]
For use in therapy, any gene delivery system suitable to introduce the antisense sequence into appropriate target cells can be used. The antisense sequence can be delivered intracellularly in the form of an expression plasmid that upon transcription creates a sequence complementary to at least a portion of the cell sequence encoding the target protein (eg, Slater, JE et al. (1998). J. Allergy Clin. Immunol. 102 (3): 469-475 and Scanlon, KJ. Et al. (1995) 9 (13): 1288-1296). Antisense sequences can also be introduced into cells using viral vectors, such as, for example, retroviruses and adeno-associated virus vectors (eg, Miller, AD (1990) Blood 76: 271, Ausubel, supra). Uckert, W. and W. Walther (1994) Pharmacol. Ther. 63 (3): 323-347). Other gene delivery mechanisms include liposome systems, artificial virus envelopes, and other systems known in the art (Rossi, JJ (1995) Br. Med. Bull. 51 (1): 217). -225; Boado, RJ, et al. (1998) J. Pharm. Sci. 87 (11): 1308-1315, Morris, MC, et al. (1997) Nucleic Acids Res. 25 (14): 2730-2736. Etc.).
[0189]
In another embodiment of the present invention, polynucleotides encoding SSCP can be used for somatic or germ cell gene therapy. Severe combined immunodeficiency (SCID) characterized by (i) treating gene deficiency by gene therapy (e.g., characterized by X chromosomal linkage inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288: 669-672)). X1 disease), severe combined immunodeficiency syndrome associated with congenital adenosine deaminase (ADA) deficiency (Blaese, RM, et al. (1995) Science 270: 475-480, Bordignon, C. et al. (1995) Science 270: 470-475), cystic fibrosis (Zabner, J. et al. (1993) Cell 75: 207-216: Crystal, RG et al. (1995) Hum. Gene Therapy 6: 643-666, Crystal, R. G. et al. (1995) Hum. Gene Therapy 6: 667-703), thalassemia, familial hypercholesterolemia, and hemophilia due to factor VIII or factor IX deficiency (Crystal, RG (1995)). ) Science 270: 404-410, Verma, IM and N. Somia (1997) Nature 389: 239-242), and (ii) expressing a conditional lethal gene product (eg, due to uncontrolled cell growth). (Iii) parasites within cells, such as the human immunodeficiency virus (HIV) (Baltimore, D. (1988) Nature 335: 395-396, Poeschla, E. et al. (1996) Proc. Natl. Acad.Sci. USA 93: 11395-11399), hepatitis B or C virus (HBV, HCV),Candida albicansandParacoccidioides brasiliensisAnd proteins having a protective function against parasites such as Plasmodium falciparum and Trypanosoma cruzi. If a deficiency in the gene required for SCCP expression or regulation causes the disease, SSCP can be expressed from a suitable population of the introduced cells to mitigate the onset of the symptoms caused by the deficiency.
[0190]
In a further embodiment of the present invention, diseases and disorders due to SSCP deficiency are treated by creating mammalian expression vectors encoding SSCP and introducing these vectors into SSCP-deficient cells by mechanical means.in VivoOrex in vitroThe mechanical transfection techniques used for cells of (i) include (i) direct DNA microinjection into individual cells, (ii) gene guns, (iii) transfection via liposomes, and (iv) receptors. Mediated gene transfer, and (v) the use of DNA transposons (Morgan, RA and WF Anderson (1993) Annu. Rev. Biochem. 62: 191-217, Ivics, Z. (1997)). Cell 91: 501-510; Boulay, JL and H. Recipon (1998) Curr. Opin. Biotechnol. 9: 445-450).
[0191]
Examples of expression vectors that may be effective for SSCP expression include, but are not limited to, pcDNA 3.1, EpiTag, pRcCMV2, pREP, pVAX, pCRII-TOPOTA vector (Invitrogen, Carlsbad CA), pCMV-Script, pCMV- Tag, PEGSH / PERV (Stratagene, La Jolla CA) and PTT-OFF, PETTE-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA). Expression of SCCP requires the expression of (i) a constitutively active promoter (derived from, for example, cytomegalovirus (CMV), rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or β-actin. Gene), (ii) an inducible promoter (eg, a tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl. Acad. Sci. U.S.A.) contained in a commercially available T-REX plasmid (Invitrogen). SA 89: 5547-5551; Gossen, M. et al. (1995) Science 268: 1766-1769; Rossi, FMV and HM Blaau (1998) Curr. Opin. Biotechnol. 9: 451-456)), an ecdysone inducible promoter (included in commercially available plasmids PVGRXR and PIND: Invitrogen), an FK506 / rapamycin inducible promoter, or a RU486 / mifepristone inducible promoter (Rossi, FM. V. and HM Blau, supra), or (iii) the native or tissue-specific promoter of the endogenous gene encoding SSCP from a normal individual.
[0192]
Using a commercially available liposome transformation kit (eg, PerFect Lipid Transfection Kit from Invitrogen), a person skilled in the art does not need to make much effort to optimize each parameter of the experiment, and can transfer the polynucleotide group to the target cell group in culture. Can be delivered. Alternative methods include the calcium phosphate method (Graham. FL and AJ Eb (1973) Virology 52: 456-467) or the electroporation method (Neumann, B. et al. (1982) EMBO J. 1: 841-845). ). The introduction of DNA into primary cells requires modification of standardized mammalian transfection protocols.
[0193]
In another embodiment of the present invention, a disease or disorder caused by various gene deletions associated with the expression of SSCP can be used to: (i) control SCP under the control of a retroviral long terminal repeat (LTR) promoter or an independent promoter; Rev responsiveness with the encoding polynucleotide, (ii) suitable RNA packaging signals, (iii) the coding sequences required for efficient vector propagation and additional retroviral cis-acting RNA sequences A retroviral vector consisting of an element (RRE) can be made and treated. Retroviral vectors (eg, PFB and PFBNEO) are commercially available from Stratagene and are based on published data (Riviere, I. et al. (1995) Proc. Natl. Acad. Sci. USA 92: 6733-6737). The above data is incorporated herein by reference. This vector is propagated in a suitable vector producing cell line (VPCL). VPCL expresses an envelope gene with affinity for the receptor on each target cell or a pan-affinity envelope protein such as VSVg (Armentano, D. et al. (1987) J. Virol. 61: 1647-1650; Bender, MA, et al. (1987) J. Virol. 61: 1639-1646, Adam, MA and AD Miller (1988) J. Virol. 62: 3802-3806, Dull, T. et al. (1998) J. Virol. 72: 8463-8471, Zufferey, R. et al. (1998) J. Virol. 72: 9873-9880). U.S. Pat. No. 5,910,434 to Rigg ("Method for obtaining retroviral packaging cell lines producing high transducing strains"; "Retrospectroscopy"; Is a part of the present specification. Propagation of retroviral vectors, cell populations (eg, CD4⁺Transduction of the T cell group) and return of the transduced cell group to the patient are procedures well known to those skilled in the art of gene therapy and are described in a large number of documents (Ranga, U. et al. (1997). ) J. Virol. 71: 7020-7029, Bauer, G. et al. (1997) Blood 89: 2259-2267, Bonyhadi, ML (1997) J. Virol. 71: 4707-4716, Ranga, U. et al. (1998) Proc. Natl. Acad. Sci. USA 95: 1201-1206, Su, L. (1997) Blood 89: 2283- 2290).
[0194]
Alternatively, certain delivery systems of adenovirus-based gene therapy are used to deliver a group of polynucleotides encoding SSCP to a group of cells having one or more genetic abnormalities associated with the expression of SSCP. The production and packaging of adenovirus-based vectors is well known to those skilled in the art. It has been demonstrated that replication-defective adenovirus vectors can be used in a variety of ways to import genes encoding various immunoregulatory proteins into intact pancreatic islets (Csete, ME et al. 1995) Transplantation 27: 263-268). Potentially useful adenoviral vectors are described in U.S. Patent No. 5,707,618 to Armentano ("Adenovirus vectors for gene therapy"), which is hereby incorporated herein by reference. I do. For adenovirus vectors, see Antinozzi, P. et al. A. Et al. (1999) Annu. Rev .. Nutr. 19: 511-544 and Verma, I .; M. And N.I. See also Somia (1997) Nature 18: 389: 239-242. Both documents are hereby incorporated by reference.
[0195]
Alternatively, certain delivery systems of herpes-based gene therapy are used to deliver a group of polynucleotides encoding SSCP to a group of target cells having one or more genetic abnormalities associated with the expression of SSCP. Herpes simplex virus (HSV) -based vectors appear to be particularly beneficial in introducing SSCP into central nervous cells to which HSV has affinity. The production and packaging of herpes-based vectors is known to those skilled in the art. Certain replication-competent herpes simplex virus (HSV) type I vectors have been used to deliver certain reporter genes to primate eyes (Liu, X. et al. (1999) Exp. Eye Res. 169: 385-395). The construction of the HSV-1 viral vector is also disclosed in detail in U.S. Patent No. 5,804,413 to DeLuca ("Herpes simplex strains for gene transfer"), which is hereby incorporated by reference. Book. US Patent No. 5,804,413 describes the use of recombinant HSV d92 having a genome with at least one foreign gene introduced into a cell under the control of a suitable promoter, for purposes such as human gene therapy. I do. The patent also discloses the generation and use of a recombinant HSV line lacking ICP4, ICP27, and ICP22. For HSV vectors, see Goins, W. et al. F. Et al. (1999) J. Am. Virol. 73: 519-532 and Xu, H .; Et al. (1994) Dev. Biol. 163: 152-161. Both documents are hereby incorporated by reference. Manipulation of Cloned Herpesvirus Sequences, Production of Recombinant Virus After Transfection with Multiple Plasmids Having Various Segments of Large Herpesvirus Genomes, Herpesvirus Growth and Propagation, and Cell Groups Infection of the herpes virus into the is a technique known to those skilled in the art.
[0196]
Alternatively, certain alphavirus (positive single-stranded RNA virus) vectors are used to deliver a group of polynucleotides encoding SSCP to a group of target cells. Biological studies of the prototype alphavirus, Semliki Forest Fever Virus (SFV), have been extensively performed, and gene transfer vectors are based on the SFV genome (Garoff, H. and K.-J. Li (1998). ) Curr. Opin. Biotechnol. 9: 464-469). During replication of alpha viral RNA, subgenomic RNA is produced, usually encoding the capsid proteins of the virus. This subgenomic RNA replicates at a higher level than full-length genomic RNA, resulting in overproduction of capsid proteins compared to viral proteins having enzymatic activity (eg, proteases and polymerases). Similarly, by inserting the sequence encoding SSCP into the region encoding the capsid of the α virus genome, a large number of RNAs encoding SCCP are produced in the vector-transfected cells, and SSCP is synthesized at a high level. Usually, infection with the alpha virus involves cell lysis within a few days. On the other hand, the ability of hamster normal kidney cell (BHK-21) populations with certain mutants of Sindbis virus (SIN) to establish persistent infection can apply the lytic replication of alpha viruses to gene therapy. (Dryga, SA et al. (1997) Virology 228: 74-83). The ability to introduce α-virus into various hosts allows the introduction of SSCP into various types of cells. Specific transduction of a subset of cells in a population may require sorting of cells prior to transduction. Methods for treating α-virus infectious cDNA clones, transfecting α-virus cDNA and RNA, and infecting α-virus are known to those skilled in the art.
[0197]
It is also possible to inhibit gene expression using an oligonucleotide derived from the transcription start site. The transcription start site is, for example, between about -10 and about +10 counted from the start site. Similarly, inhibition can be achieved using triple helix base pairing methods. Triple helix base pairing is useful because it inhibits the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors or regulatory molecules. Recent therapeutic advances using triple helical DNA have been described in the literature (eg, Gee, JE et al. (1994) in Huber, BE and BI Carr,Molecular and Immunological Approaches, Futura Publishing, Mt. Kisco NY, see pages 163-177). Also, complementary sequences or antisense molecules can be designed to block translation of mRNA by preventing transcripts from binding to ribosomes.
[0198]
Ribozymes are enzymatic RNA molecules. Ribozymes can be used to catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. For example, artificially produced hammerhead ribozyme molecules may be able to specifically and effectively catalyze endonucleolytic cleavage of the sequence encoding SSCP.
[0199]
To first identify a specific ribozyme cleavage site within any potential RNA target, the target molecule is scanned for ribozyme cleavage sites, such as GUA, GUU, GUC sequences. Once identified, assess whether the short RNA sequence of 15-20 ribonucleotides corresponding to the region of the target gene having the cleavage site has secondary structural features that render the oligonucleotide dysfunctional. Becomes possible. An assessment of the suitability of a candidate target can also be made by testing accessibility to hybridization with a group of complementary oligonucleotides using a ribonuclease protection assay.
[0200]
The complementary ribonucleic acid molecules and ribozymes of the invention can be made using any method well known in the art for nucleic acid molecule synthesis. As a production method, there is a method of chemically synthesizing an oligonucleotide, such as solid phase phosphoramidite chemical synthesis. Alternatively, the DNA sequence encoding SSCPin in vitroandin VivoAn RNA molecule may be created by transcription. Such a DNA sequence can be incorporated into a variety of vectors using a suitable RNA polymerase promoter such as T7 or SP6. Alternatively, these cDNA constructs that synthesize complementary RNA constitutively or inducibly can be introduced into cell lines, cells or tissues.
[0201]
RNA molecules can be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of contiguous sequences at the 5 'end, 3' end, or both, of the molecule, and phosphorothioate or 2'O instead of phosphodiesterase linkages within the backbone of the molecule. -The use of methyl. This concept is originally in the production of the PNA group, but can be extended to all these molecules. To do so, adenine, cytidine, guanine, thymine, and uridine, which are not readily recognized by endogenous endonucleases, have acetyl-, methyl-, thio-, and similar modifications, and unconventional bases such as inosine , Queosine, wybutosine.
[0202]
A further embodiment of the invention includes a method of screening for a compound that is effective in altering the expression of a polynucleotide encoding SSCP. Compounds that may be effective in causing expression modification of a specific polynucleotide include, but are not limited to, oligonucleotides, antisense oligonucleotides, triple helix-forming oligonucleotides, polypeptide transcription regulators such as transcription factors, and specific There are non-polymeric chemical entities that can interact with the polynucleotide sequence. Effective compounds can alter polynucleotide expression by acting as either inhibitors or enhancers of polynucleotide expression. Therefore, in the treatment of diseases associated with increased expression or activity of SSCP, compounds that specifically inhibit the expression of a polynucleotide encoding SSCP are therapeutically useful, and are associated with decreased expression or activity of SSCP. In treating disease, compounds that specifically enhance the expression of a polynucleotide encoding SSCP may be therapeutically useful.
[0203]
At least one or more test compounds may be screened for effectiveness in altering the expression of a particular polynucleotide. Any method known in the art can be used to obtain a test compound. As an acquisition method, there is a chemical modification of a known compound that is effective in the following cases. Whether modifying the expression of a polynucleotide, selecting from a library of existing, commercial or dedicated, natural or unnatural compounds, and rationalizing compounds based on the chemical and / or structural properties of the target polynucleotide And selecting from a library of compounds generated in combination or randomly. A sample carrying a polynucleotide encoding SSEC is exposed to at least one of the test compounds thus obtained. Samples include, for example, intact cells, permeabilized cells,in in vitroThere can be a cell-free or reconstituted biochemical system. Modification of the expression of a polynucleotide encoding SSCP is assayed by any method known in the art. Usually, to detect the expression of a specific nucleotide, hybridization with a probe having a nucleotide sequence complementary to the sequence of the polynucleotide encoding SSCP is used. Quantifying the amount of hybridization can form the basis for comparison of the expression of polynucleotides exposed or unexposed to one or more test compounds. Detection of a change in the expression of the polynucleotide exposed to the test compound indicates that the test compound is effective in altering the expression of the polynucleotide. Screening for compounds that are effective for the modified expression of certain polynucleotides can be performed, for example, by using fission yeast (Schizosaccharomyces pombe) Gene expression system (Atkins, D. et al. (1999) US Patent No. 5,932,435, Arndt, GM et al. (2000) Nucleic Acids Res. 28: E15) or human cell lines such as HeLa cells ( Clarke, ML, et al. (2000) Biochem. Biophys. Res. Commun. 268: 8-13). Certain embodiments of the present invention relate to screening combinatorial libraries of oligonucleotides (deoxyribonucleotides, ribonucleotides, peptide nucleic acids, modified oligonucleotides) for antisense activity against certain polynucleotide sequences (Bruice). (1997) U.S. Patent No. 5,686,242, Bruice, TW et al. (2000) U.S. Patent No. 6,022,691).
[0204]
Numerous methods for introducing vectors into cells or tissues are available,in Vivo,in in vitroandex VivoEqually suitable for the use ofex VivoFor treatment, the vector can be introduced into stem cells taken from a patient, cloned, propagated and returned to the patient by autotransplant. Delivery by transfection or by ribosome injection or polycationic amino polymer can be performed using methods well known in the art (Goldman, CK et al. (1997) Nat. Biotechnol. 15: 462-466).
[0205]
Any of the above treatment methods can be applied to all subjects in need of treatment, including, for example, mammals such as humans, dogs, cats, cows, horses, rabbits, monkeys, and the like.
[0206]
Certain further embodiments of the present invention relate to the administration of certain compositions that generally have the active ingredient formulated with a pharmaceutically acceptable excipient. Excipients include, for example, sugars, starches, celluloses, gums and proteins. Various prescriptions are widely known,Remington's Pharmaceutical Sciences(Maack Publishing, Easton PA). Such compositions comprise SSCP, antibodies to SSCP, mimetics, agonists, antagonists, or inhibitors of SSCP, and the like.
[0207]
The compositions used in the present invention can be administered by any number of routes, including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intrathecal, Intraventricular, lung, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or rectal.
[0208]
Compositions for pulmonary administration may be prepared in liquid or dry powder form. Such compositions typically aerosolize shortly before inhalation by the patient. For small molecules (eg, traditional low molecular weight organic drugs), aerosol delivery of fast-acting formulations is known in the art. In the case of macromolecules (e.g., larger peptides and proteins), the recent improvement in the art of pulmonary delivery through the alveolar region of the lung will substantially transport drugs such as insulin into the blood circulation. (See Patton, JS, et al., US Pat. No. 5,997,848, etc.). Pulmonary delivery is advantageous in that it is administered without needle injection, eliminating the need for potentially toxic penetration enhancers.
[0209]
Compositions suitable for use in the present invention include compositions that contain as much active ingredient as necessary to achieve the intended purpose. Determination of the effective dosage is within the ability of those skilled in the art.
[0210]
Special forms of compositions can be prepared to deliver macromolecules, including SSCP or fragments thereof, directly into cells. For example, a liposome formulation containing a cell-impermeable polymer can facilitate cell fusion and intracellular delivery of the polymer. Alternatively, SSCP or a fragment thereof can be attached to the cationic N-terminus of the HIV Tat-1 protein. The fusion proteins thus produced have been shown to transduce cells of all tissues, including the brain, of certain mouse model systems (Schwarze, SR, et al. (1999) Science 285). : 1569-1572).
[0211]
For any compound, an effective dosage of treatment can be estimated initially in cell culture assays, such as those for neoplastic cells, or in animal models such as mice, rabbits, dogs or pigs. . Animal models can also be used to determine suitable concentration ranges and routes of administration. Such information can then be used to determine useful doses and routes for administration in humans.
[0212]
A pharmaceutically effective amount refers to an amount that ameliorates a symptom or condition, such as an active ingredient, eg, SSCP or a fragment thereof, an antibody to SSCP, an agonist or antagonist of SSCP, or an inhibitor. Medicinal efficacy and toxicity can be determined by standard pharmaceutical techniques in cell culture or animal studies, for example,₅₀(Pharmaceutically effective amount of 50% of the population) or LD₅₀(The lethal dose of 50% of the population) can be determined by calculating statistics or the like. The dose ratio of toxic to therapeutic effects is the therapeutic index and the LD₅₀/ ED₅₀It can be expressed as a ratio. Compositions that exhibit high therapeutic indices are desirable. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for use in humans. The dosage contained in such compositions will have little or no toxicity,₅₀It is preferable that the concentration be in the blood concentration range containing Depending on the dosage form employed, the sensitivity of the patient and the route of administration, the dosage will vary within this range.
[0213]
The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. Factors relating to the subject can take into account the severity of the disease, the general health of the patient, the age, weight and sex of the patient, the time and frequency of administration, drug incorporation, response sensitivity and response to treatment, and the like. Compositions with a longer duration of action may be administered once every 3 to 4 days, once a week, or once every two weeks depending on the half-life and clearance rate of the particular formulation.
[0214]
Normal dosage amounts range from about 0.1 to 100,000 μg, depending on the route of administration, up to a total of about 1 g. Guidance as to specific dosages and methods of delivery is provided in the literature and is readily available to practitioners in the field. One skilled in the art will utilize formulations for nucleotides that are different from those for proteins or their inhibitors. Similarly, delivery of a polynucleotide or polypeptide will be specific to a particular cell, condition, site, etc.
[0215]
(Diagnosis)
In another embodiment, an antibody that specifically binds to SCCP is used to diagnose a disease characterized by the expression of SCCP or to monitor a patient being treated with SSCP or an agonist or antagonist or inhibitor of SSCP. Used for assay. Antibodies useful for diagnostic purposes are prepared in the same manner as described above for treatment. Diagnostic assays for SCCP include methods for detecting SSCP using antibodies and labels from human body fluids or from extracts of cells and tissues. The antibody can be modified or unmodified and can be labeled covalently or non-covalently with a reporter molecule. A wide variety of reporter molecules are known in the art and can be used, some of which are described above.
[0216]
Various protocols for measuring SSCP, such as ELISA, RIA, and FACS, are well known in the art and provide the basis for diagnosing altered or abnormal levels of SSCP expression. To determine the value of normal or standard SSCP expression, a body fluid or cell extract obtained from a subject such as a normal mammal, for example, a human, and antibodies to SSCP are combined under conditions suitable for complex formation. Mix with. The amount of the standard complex formed can be determined by various methods, for example, by photometry. The amount of SSCP expression in a subject or in a control or in a disease sample from a group of biopsy tissues is compared to a reference value. The deviation between the standard value and the subject determines the parameters for diagnosing the disease.
[0217]
In another embodiment of the present invention, polynucleotides encoding SSCP may be used for diagnosis. Polynucleotides that can be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNA. These polynucleotides can be used to detect and quantify gene expression in biopsy tissues where SSCP expression may correlate with disease. This diagnostic assay is used to check for the presence or even overexpression of SSCP and to monitor the regulation of SSCP values during treatment.
[0218]
In one embodiment, the nucleic acid sequence encoding SSCP can be identified by hybridization with PCR probes capable of detecting a polynucleotide sequence (eg, a genomic sequence) encoding SCCP or a closely related molecule. The specificity of a probe, eg, made from a highly specific region, such as the 5 'regulatory region, or from a less specific region, such as a conserved motif, may also be attributed to the stringency of hybridization or amplification. Will determine whether the probe identifies only the native sequence that encodes SSCP, or whether it identifies an allelic variant or related sequence.
[0219]
Probes can also be used for the detection of related sequences and can have at least 50% sequence identity with any sequence encoding SSCP. The hybridization probes of the present invention may be DNA or RNA and may be derived from the sequence of SEQ ID NO: 64-126, or may be derived from a group of genomic sequences, such as the promoter, enhancer, intron, etc. of the SCCP gene.
[0220]
As a method for preparing a hybridization probe specific to a DNA group encoding SSCP, there is a method of cloning a polynucleotide sequence encoding SSCP or a SSCP derivative into a vector for producing an mRNA probe. Vectors for making mRNA probes are known to those of skill in the art, and are commercially available. By adding a suitable RNA polymerase and a suitable labeled nucleotide,in in vitroCan be used to synthesize RNA probes. Hybridization probes can be labeled with a population of different reporters. Examples of reporter populations include:³²P or³⁵A radionuclide such as S, or an enzyme label such as alkaline phosphatase bound to a probe via an avidin / biotin binding system may be used.
[0221]
A polynucleotide sequence encoding SCCP can be used to diagnose a disease associated with SSCP expression. Such disorders include, but are not limited to, abnormal cell proliferation, autoimmune / inflammatory diseases, cardiovascular disorders, neurological disorders, and developmental disorders, including actinic keratosis, arterial disorders, Sclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and Cancers such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma, specifically, adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gallbladder, ganglion, digestive tract, heart, Includes cancers of the kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary gland, skin, spleen, testis, thymus, thyroid, and uterus, and some autoimmune / inflammatory diseases , Acquired immunodeficiency syndrome (AIDS), Addison's disease (chronic Adult renal dysfunction), adult respiratory distress syndrome, allergy, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyglandular endocrine Candida ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes, pulmonary emphysema, lymphocytic factor accidental lymphopenia, neonatal hemolytic disease (Erythroblastosis fetalis), erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, Myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome , Systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, Werner syndrome, cancer complications, hemodialysis, extracorporeal circulation, viral infections, bacterial infections, fungal infections, Includes parasitic infections, protozoan infections, helminth infections, trauma, and cardiovascular disorders include congestive heart failure, ischemic heart disease, angina pectoris, myocardial infarction, hypertensive heart disease, degenerative valvular disease Heart disease, calcified aortic stenosis, congenital bicuspid aortic valve, mitral annular calcification, mitral valve prolapse, rheumatic fever, rheumatic heart disease, infective endocarditis, Nonbacterial thrombotic endocarditis, systemic lupus erythematosus endocarditis, carcinoid heart disease, cardiomyopathy, myocarditis, pericarditis, neoplastic heart disease, congenital heart disease, complications of heart transplantation, movement Fistula, atherosclerosis, hypertension, vasculitis, Raynaud's disease, aneurysm, arterial dissection, varicose vein, thrombophlebitis and venous thrombosis, vascular tumors, thrombolytic complications, balloon angioplasty, vascular replacement replacement, coronary artery bypass graft surgery, and among neurological disorders are epilepsy, ischemic cerebrovascular disease, stroke, brain tumor, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders , Amyotrophic lateral sclerosis and other motor neuron disorders, progressive neuromuscular atrophy, retinitis pigmentosa (retinitis pigmentosa), hereditary ataxia, multiple sclerosis and other demyelinating diseases, bacteria And viral meningitis, brain abscess, subdural abscess, epidural abscess, purulent intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system Diseases include prion disease (including Kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome), fatal familial insomnia, neurotrophic and metabolic diseases, neurofibromatosis, tuberous sclerosis, Central nervous system mental retardation and other developmental disorders including cerebellar retinal hemangioblastomatosis, trigeminal neurovascular syndrome, Down syndrome, cerebral palsy, neuroskeletal abnormalities, autonomic nervous system disorders, cranial nerve disorders, spinal cord disorders , Muscular dystrophy and other neuromuscular disorders, peripheral neuropathy, dermatomyositis and polymyositis, hereditary, metabolic, endocrine, and toxic myopathy, myasthenia gravis, periodic limb paralysis, mental disorders (mood , Anxiety disorders, schizophrenia / schizophrenia), season Affective disorder (SAD), restlessness, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonia, paranoid psychosis, postherpetic neuralgia, Tourette's disease, progressive supranuclear palsy, basal ganglia degeneration ( corticobasal generation, and familial frontotemporal dementia, and also includes developmental disorders, among which are tubular acidosis, anemia, Cushing's syndrome, chondrodysplastic dwarfism, Duchenne / Becker type Muscular dystrophy, epilepsy, gonadal dysplasia, WAGR syndrome (Wilms tumor, aniridia, genitourinary dysfunction, mental retardation), Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucosal epithelial dysplasia, inheritance Keratoderma and Charcot-Marie-Tooth disease And seizure disorders, such as hereditary neuropathy such as neurofibromatosis, hypothyroidism, hydrocephalus, and Syndenham's chorea and cerebral palsy, spina bifida, anencephaly, cranial spine avulsion, congenital Includes glaucoma, cataracts, and sensorineural hearing loss. The polynucleotide sequence encoding SSCP can be obtained by using a bodily fluid or tissue collected from a patient to detect altered SSCP expression, using Southern, Northern, dot blot, or other membrane-based techniques, PCR, It can be used for dipstick, pin, and multi-format ELISA-based assays, and microarrays. Such qualitative or quantitative methods are known in the art.
[0222]
In certain embodiments, the nucleotide sequence encoding SSCP will be useful in assays that detect related diseases, particularly those described above. The nucleotide sequence encoding SSCP could be labeled by standard methods and added to a sample of body fluid or tissue taken from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantified and compared to a standard value. If the amount of signal in the patient's sample is significantly altered relative to the control sample, the presence of a change in the level of the nucleotide sequence encoding SSCP in that sample will reveal the presence of the associated disease. Such assays can also be used to estimate the effect of a particular treatment in animal studies, clinical trials, or to monitor the treatment of individual patients.
[0223]
A normal or standard profile of expression is established to provide a basis for the diagnosis of a disease associated with the expression of SCCP. This is accomplished by mixing a body fluid or cell extract from a normal subject, either animal or human, with a sequence encoding SSCP or a fragment thereof under conditions suitable for hybridization or amplification. obtain. Standard hybridization can be quantified by comparing values obtained from experiments performed with known amounts of substantially purified polynucleotide to values obtained from normal subjects. The standard values thus obtained can be compared to values obtained from samples obtained from patients showing signs of the disease. Deviation from standard values is used to determine the presence of disease.
[0224]
Once the presence of the disease has been determined and the treatment protocol has begun, the hybridization assay may be repeated periodically to determine whether the patient's expression levels have begun to approach those observed in normal subjects. The results from serial assays can be used to show the effect of treatment over a period of days to months.
[0225]
With respect to cancer, the presence of abnormal amounts of transcripts (under- or over-expression) in living tissue from an individual indicates the predisposition of the disease or a method of detecting the disease before actual clinical symptoms appear. Can provide. This type of more definitive diagnosis allows medical professionals to use preventative or aggressive treatment early on, thereby preventing the development or further progression of cancer.
[0226]
Further diagnostic uses of oligonucleotides designed from the sequences encoding SSCP may include the use of PCR. These oligomers can be chemically synthesized, produced enzymatically, orin in vitroCan be produced in Oligomers, preferably including fragments of a polynucleotide encoding SSCP or fragments of a polynucleotide complementary to a polynucleotide encoding SSCP, are used under optimized conditions to identify a particular gene or condition. Is done. Oligomers can also be used under relatively mild stringency conditions to detect and / or quantify closely related DNA or RNA sequences.
[0227]
In certain embodiments, single nucleotide polymorphisms (SNPs) can be detected using oligonucleotide primers derived from a set of polynucleotide sequences encoding SSCP. SNPs are substitutions, insertions and deletions that often cause a congenital or acquired genetic disease in humans. Non-limiting methods for detecting SNPs include single-stranded conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP). In SSCP, DNA is amplified by the polymerase chain reaction (PCR) using oligonucleotide primers derived from the polynucleotide sequence encoding SSCP. DNA can be derived, for example, from diseased or normal tissue, biopsy samples, body fluids, and the like. SNPs in the DNA cause differences in the secondary and tertiary structure of the single-stranded form of the PCR product. Differences can be detected using gel electrophoresis in a non-denaturing gel. In fSCCP, oligonucleotide primers are fluorescently labeled. This allows the detection of amplimers on high throughput equipment such as DNA sequencing machines. Furthermore, a sequence database analysis method called in silico SNP (isSNP) is used to compare polymorphisms by comparing the sequences of individual overlapping DNA fragments as arranged in a general consensus sequence. Can be identified. These computer-based methods filter out sequence variations due to errors in the laboratory preparation of DNA or sequencing errors using statistical models and automated analysis of DNA sequence chromatograms. In another embodiment, SNPs are detected and characterized by mass spectrometry, for example, using a high-throughput MASSARRAY system (Sequenom, Inc., San Diego CA).
[0228]
Examples of other methods that can be used to quantify SSCP expression include radiolabeling or biotin labeling of nucleotides, co-amplification of control nucleic acids, and interpolation of results from a standard curve (eg, (Melby, PC, et al. (1993) J. Immunol. Methods, 159: 235-244; Duplaa, C. et al. (1993) Anal. Biochem. 212: 229-236). To accelerate the rate of quantification of multiple samples, the oligomer or polynucleotide of interest can be placed in various diluents and assayed in a high-throughput format where quantitation is rapid by spectrophotometric or colorimetric reactions. .
[0229]
In yet another embodiment, oligonucleotides or longer fragments from any of the polynucleotide sequences described herein can be used as elements in a microarray. Microarrays can be used in transcription imaging techniques to simultaneously monitor the relative expression levels of many genes. This is described below. Microarrays can also be used to identify genetic variants, mutations and polymorphisms. This information can be used to determine gene function, understand the genetic basis of disease, diagnose disease, monitor disease progression / regression as a function of gene expression, and develop drug activity in disease treatment And can be monitored. In particular, this information can be used to develop a pharmacological genomic profile for the patient in order to select the most appropriate and effective treatment for the patient. For example, based on a patient's pharmacogenomic profile, a therapeutic agent that is highly effective and has few side effects for the patient can be selected.
[0230]
In another embodiment, SSCP, fragments of SSCP, antibodies specific to SSCP can be used as elements on the microarray. Microarrays can be used to monitor or measure protein-protein interactions, drug-target interactions and gene expression profiles as described above.
[0231]
Certain embodiments relate to the use of a polynucleotide of the invention to produce a transcribed image of a tissue or cell type. The transcribed image represents the global pattern of gene expression by a particular tissue or cell type. Global gene expression patterns can be analyzed by quantifying the number and relative abundance of genes expressed at a given time under given conditions (Seilhamer et al., US Pat. No. 5,840,484, “Comparative”). See Gene Transcript Analysis, which is hereby incorporated by reference. Thus, a transcribed image can be generated by hybridizing a polynucleotide of the invention or its complement to an entire transcript or reverse transcript of a particular tissue or cell type. In some embodiments, hybridization occurs in a high-throughput format such that the polynucleotides of the invention or their complement have a subset of multiple elements on a microarray. The resulting transcribed image can provide a profile of gene activity.
[0232]
Transcript images can be created using transcripts isolated from tissues, cell lines, biopsies, or biological samples thereof. The transcribed image is therefore in the case of a tissue or biopsy samplein Vivo, In the case of cell linesin in vitroReflects gene expression at
[0233]
Transcribed images that create a profile of expression of a polynucleotide of the invention may also includein in vitroIt can be used for preclinical evaluation of model systems and drugs, or in connection with toxicity testing of industrial or natural environmental compounds. All compounds exhibit mechanisms of action and toxicity, and elicit a characteristic pattern of gene expression, often referred to as molecular fingerprints or toxic signatures (Nuwaysir, EF et al. (1999) Mol. Carcinog. 24: 153-159, Steiner, S. and NL Anderson (2000) Toxicol. Lett. 112-113: 467-471, which are hereby specifically incorporated by reference. If the test compounds have the same signature as the compound with known toxicity, they may share toxic properties. Fingerprints or signatures are most useful and accurate when they contain expression information from many genes and gene families. Ideally, measuring expression across the genome will provide the highest quality signature. Even if there are genes whose expression is not altered by any of the tested compounds, their genes are important because their expression levels can be used to normalize the remaining expression data. The normalization procedure is useful for comparing expression data after treatment with various compounds. Assigning a gene function to a group of elements of a certain toxic signature is helpful in interpreting the toxic mechanism, but knowledge of the gene function is not required for statistical matching of the group of signatures, which leads to prediction of toxicity (eg, 2000 See Press Release 00-02, published by the National Institute of Environmental Health Sciences on the 29th of the month, which is available at http://www.niehs.nih.gov/oc/. news / toxchip.htm). Therefore, it is important and desirable to include all expressed gene sequences during toxicological screening using toxic signatures.
[0234]
In certain embodiments, the toxicity of a test compound is calculated by treating a biological sample having nucleic acids with the test compound. Nucleic acids expressed in the treated biological sample can be hybridized with one or more probes specific for a polynucleotide of the invention, thereby quantifying the level of transcription corresponding to the polynucleotide of the invention. The level of transcription in the treated biological sample is compared to the level in an untreated biological sample. The difference in transcript levels between the two samples is indicative of a toxic response caused by the test compound in the treated sample.
[0235]
Another embodiment relates to analyzing the proteome of a tissue or cell type using the polypeptide sequences of the invention. The term proteome refers to the global pattern of protein expression in a particular tissue or cell type. Each protein component of the proteome can be individually subjected to further analysis. Proteome expression patterns or profiles can be analyzed by quantifying the number and relative abundance of proteins expressed at a given time under given conditions. Thus, a proteomic profile of a cell can be generated by separating and analyzing polypeptides of a particular tissue or cell type. In some embodiments, separation is achieved by two-dimensional gel electrophoresis, such as separating proteins from a sample by one-dimensional isoelectric focusing and separating according to molecular weight by two-dimensional sodium dodecyl sulfate slab gel electrophoresis. (Steiner and Anderson, supra). Proteins are visualized in the gel as dispersed, uniquely located points, usually by staining the gel with a substance such as Coomassie blue or silver or fluorescent stains. The optical density of each protein spot is usually proportional to the protein level in the sample. Compare the optical densities of similarly located protein spots from different samples, for example, biological samples, either treated or untreated with a test compound or therapeutic agent, to identify changes in protein spot density associated with treatment . The proteins in the spots are partially sequenced using standard methods using, for example, chemical or enzymatic cleavage followed by mass spectrometry. The identity of a protein within a spot can be determined by comparing its subsequence, preferably at least 5 contiguous amino acid residues, to a polypeptide sequence of the present invention. In some cases, additional sequence data is obtained for definitive protein identification.
[0236]
Proteome profiles can also be generated by quantifying SSCP expression levels using an antibody specific for SCCP. In one embodiment, protein expression levels are quantified using these antibodies as elements on the microarray and exposing the microarray to a sample to detect the level of protein binding to each array element (Lueking, A. et al. (1999). ) Anal. Biochem. 270: 103-111, Mendose, LG et al. (1999) Biotechniques 27: 778-788). Detection can be performed by various methods known in the art, for example, by reacting a thiol-reactive or amino-reactive fluorescent compound with a protein in a sample and detecting the amount of fluorescent binding at each array element.
[0237]
Toxic signatures at the proteome level are also useful for toxicological screening and should be analyzed in parallel with toxic signatures at the transcript level. For some proteins in some tissues, the correlation between transcript abundance and protein abundance is poor (Anderson, NL and J. Seilhammer (1997) Electrophoresis 18: 533-537). The proteome toxic signature can be useful in the analysis of compounds that do not significantly affect the transcript image but alter the proteome profile. In addition, proteome profiling may be more reliable and informative in such cases, since the analysis of transcripts in body fluids is difficult due to the rapid degradation of mRNA.
[0238]
In another example, the toxicity of a test compound is calculated by treating a biological sample containing the protein with the test compound. The proteins expressed in the processed biological sample are separated so that the amount of each protein can be quantified. The amount of each protein is compared to the amount of the corresponding protein in the untreated biological sample. The difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample. Individual proteins are identified by sequencing the amino acid residues of the individual proteins and comparing these subsequences to the polypeptides of the invention.
[0239]
In another example, the toxicity of a test compound is calculated by treating a biological sample containing the protein with the test compound. Proteins obtained from a biological sample are incubated with an antibody specific for a polypeptide of the invention. The amount of protein recognized by the antibody is quantified. The amount of protein in the treated biological sample is compared to the amount of protein in the untreated biological sample. The difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.
[0240]
Microarrays are prepared, used, and analyzed by methods known in the art (eg, Brennan, TM et al. (1995) US Patent No. 5,474,796; Schena, M. et al. (1996) Proc. Natl.Acad.Sci.USA 93: 10614-10619, Baldeschweiler et al. (1995) PCT Application No. WO 95/251116, Shalon, D. et al. (1995) PCT Application No. WO 95/35505, Haller, RA. (1997) Proc. Natl. Acad. Sci. USA 94: 2150-2155; Heller, MJ. Et al. (1997) U.S. Patent No. 5,605,662). Various types of microarrays are known, and for details, seeDNA Microarrays: A Practical Approach, M .; Schena, Edit (1999) Oxford University Press, London. This document is specifically incorporated herein by reference.
[0241]
In another embodiment of the invention, nucleic acid sequences encoding SSCP can also be used to generate hybridization probes useful for mapping native genomic sequences. Either coding or non-coding sequences can be used, and in some instances, non-coding sequences are preferred over coding sequences. For example, conservation of coding sequences within members of a multigene family may result in unwanted cross-hybridization during chromosome mapping. The sequence may be on a particular chromosome, or on a particular region of a chromosome, or on an artificial chromosome, such as a human artificial chromosome (HAC), a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), Bacterial P1 products or mapped to single chromosome cDNA libraries (eg, Harrington, JJ et al. (1997) Nat. Genet. 15: 345-355; Price, CM (1993) Blood Rev. 7: 127-134; See Trask, BJ (1991) Trends Genet. 7: 149-154). Once mapped, the nucleic acid sequences of the invention can be used to develop a genetic linkage map, for example, that correlates the inheritance of a disease state with the inheritance of a particular chromosomal region or with restriction fragment length polymorphisms (RFLPs) ( See, for example, Lander, ES and D. Botstein (1986) Proc. Natl. Acad. Sci. USA 83: 7353-7357).
[0242]
Fluorescence in situ hybridization (FISH) can be correlated with other physical and genetic map data (see, eg, Heinz-Ulrich, et al., Supra, (1995) in Meyers, 965-968). Examples of genetic map data can be found in various scientific journals or on the website of the Online Mendelian Inheritance in Man (OMIM). Correlation of the location of the gene encoding SSCP on the physical map with a particular disease, or with a predisposition to a particular disease, can help determine regions of DNA associated with the disease, It may facilitate the task of cloning to determine the location.
[0243]
The genetic map can be extended using physical mapping techniques, such as linkage analysis using established chromosomal markers, and in situ hybridization of chromosomal specimens. Placing a gene on the chromosome of another mammal, such as a mouse, can often reveal related markers, even though the exact locus on the chromosome is unknown. This information is valuable to researchers searching for disease genes using gene discovery techniques such as positional cloning. Once the gene (s) involved in the disease or syndrome is loosely mapped by genetic linkage to a particular genomic region, such as the 11q22-23 region of telangiectasia, it is mapped to that region Any sequence may represent a related or regulatory gene for further investigation (see, e.g., Gatti, RA et al. (1988) Nature 336: 577-580). The nucleotide sequence of the present invention can also be used for detecting a difference in chromosomal position among a healthy person, a carrier, and a diseased person due to translocation, inversion, and the like.
[0244]
In another embodiment of the present invention, SSCP, its catalytic or immunogenic fragments or oligopeptides thereof, may be used for screening libraries of compounds in any of a variety of drug screening techniques. Fragments used for drug screening can be free in solution, fixed to a solid support, retained on the cell surface, or located intracellularly. The formation of a complex due to the binding of the SCCP to the agent to be tested may be measured.
[0245]
Another drug screening method is used to screen compounds with suitable binding affinity for the protein of interest with high throughput (see, eg, Geysen et al. (1984) PCT Application No. WO84 / 03564). In this method, a number of different small molecule test compounds are synthesized on a solid substrate. The test compound is washed after reacting with SSCP, or a fragment thereof. Next, the bound SSCP is detected by methods well known in the art. Purified SSCP can also be coated directly onto plates used in the drug screening techniques described above. Alternatively, the peptide can be captured using a non-neutralizing antibody and the peptide immobilized on a solid support.
[0246]
In another embodiment, a competitive drug screening assay may be used to compete with a test compound for a neutralizing antibody capable of specific binding to SSCP for binding to SSCP. In this way, antibodies can be used to detect the presence of any peptide that shares one or more antigenic determinants with SSCP.
[0247]
In another embodiment, the nucleotide sequence encoding SSCP is converted to a molecular biology technology that will be developed in the future, using the characteristics of currently known nucleotide sequences, including but not limited to the triplet genetic code, specific base Pair interaction etc.).
[0248]
Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. Accordingly, the examples described below are for illustrative purposes only and are not intended to limit the invention in any way.
[0249]
All patents, patent applications and publications disclosed herein, particularly US patent applications Ser. Nos. 60 / 247,642, 60 / 249,824, 60 / 252,824, 60 / 247,505. Nos. 60 / 254,305 and 60 / 256,448 are hereby incorporated by reference.
[0250]
(Example)
1 cDNA Creating a library
The origin of the Incyte cDNA group is the cDNA library group described in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA), and listed in column 5 of Table 4. Some tissues were homogenized and dissolved in a guanidinium isothiocyanate solution, while others were homogenized and dissolved in phenol or a suitable mixture of denaturants. TRIZOL (Life Technologies), which is an example of the mixed solution, is a single-phase solution of phenol and guanidine isothiocyanate. The resulting lysate was layered on a cesium chloride cushion solution and centrifuged or extracted with chloroform. RNA was precipitated from the lysate by isopropanol, sodium acetate and ethanol, or one of the other conventional methods.
[0251]
Extraction and precipitation of RNA with phenol were repeated as necessary to increase the purity of the RNA. In some cases, the RNA was treated with DNase. For most libraries, poly (A) + RNA was isolated using oligo d (T) -linked paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworth CA) or OLIGOTEX mRNA purification kit (QIAGEN). Alternatively, RNA was isolated directly from tissue lysates using another RNA isolation kit, such as the POLY (A) PURE mRNA purification kit (Ambion, Austin TX).
[0252]
In some cases, RNA was provided to Stratagene, and a corresponding cDNA library group was created by Stratagene. Otherwise, cDNA was synthesized using the UNIZAP vector system (Stratagene) or the SUPERSCRIPT plasmid system (Life Technologies) according to the recommended method or a similar method known in the art, and a cDNA library was prepared (Ausubel, supra). , 1997, 5.1-6.6 units, etc.). Reverse transcription was started with oligo d (T) or random primer. The synthetic oligonucleotide adapter was ligated to the double-stranded cDNA, and the cDNA was digested with a suitable restriction enzyme or enzymes. For most libraries, cDNA size selection (300-1000 bp) was performed using SEPHACRYL S1000, SEPHAROSE CL2B or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech), or preparative agarose gel electrophoresis. The cDNA was ligated into the appropriate plasmid polylinker at compatible restriction enzyme sites. Suitable plasmids include, for example, PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies) PCDNA2.1 plasmid (Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), PCR2-TOPOTA plasmid (Invitrogen) (Invitrogen). Stratagene), pIGEN (Incyte Genomics, Palo Alto CA) or plNCY (Incyte Genomics), or derivatives thereof. Recombinant plasmids were transformed into competent Escherichia coli cells such as XL1-Blue, XL1-BIueMRF or SOLR from Stratagene, or DH5α, DH10B or ElectroMAX DH10B from Life Technologies.
[0253]
2 cDNA Clone isolation
Example 1The plasmid obtained as described above was recovered from host cells using a UNIZAP vector system (Stratagene).in VivoThis was done by excision or by cell lysis. For purification of the plasmid group, at least one of the following was used. Magic or WIZARD Minipreps DNA Purification System (Promega), AGTC Miniprep Purification Kit (Edge Biosystems, Gaithersburg MD), QIAWELL 8PlaimPlasmid, QIAWELLPlasmid, QIAWELLPlasmid, QIAWELLPlasmid, QIAWELLPlasmid E. FIG. A. L. Prep 96 plasmid purification kit. After precipitation, the plasmid was resuspended in 0.1 ml of distilled water and stored at 4 ° C. with or without lyophilization.
[0254]
Alternatively, plasmid DNA was amplified from host cell lysate using direct binding PCR in a high-throughput format (Rao, VB (1994) Anal. Biochem. 216: 1-14). The lysis and thermal cycling process of the host cells was performed in a single reaction mixture. Samples were processed, stored in 384-well plates, and the concentration of amplified plasmid DNA was determined fluorometrically using a PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKANII fluorescence scanner (Labsystems Oy, Helsinki, Finland). Quantified.
[0255]
3 Sequencing and analysis
Example 2The Incyte cDNA recovered from the plasmid as described in, was sequenced as shown below. The cDNA sequencing reaction can be performed using standard methods or high-throughput equipment such as an ABI CATALYST 800 thermal cycler (Applied Biosystems) or a PTC-200 thermal cycler (MJ Research) and a HYDRA microdispenser (Robins ScientificAMIBlACI200B). Treated in conjunction with a transfer system. The cDNA sequencing reaction was performed using a reagent provided by Amersham Pharmacia Biotech or an ABI sequencing kit, for example, an ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied reagent prepared from Applied reagent kit). For electrophoretic separation of cDNA sequencing reactions, and for detection of labeled polynucleotides, the MEGABACE 1000 DNA sequencing system (Molecular Dynamics) or ABI PRISM 373 using standard ABI protocols and base pairing software or A 377 sequencing system (Applied Biosystems) or other sequence analysis system known in the art was used. The reading frame within the cDNA sequence was identified using standard methods (outlined in Ausubel, 1997, 7.7 Units, supra). Select some cDNA sequences,Example 8The sequence was extended by the technique disclosed in (1).
[0256]
The polynucleotide sequence derived from Incyte cDNA was validated by removing the vector, linker and poly (A) sequences and masking ambiguous bases. An algorithm and a program based on BLAST, dynamic programming and flanking dinucleotide frequency analysis were used. Next, the following database groups were queried for the Incyte cDNA sequences or their translations. That is, selected public databases (for example, GenBank primates, rodents, mammals, vertebrates, eukaryotes, and BLOCKS, PRINTS, DOMO, PRODOM), humans, rats, mice, nematodes (Caenorhabditis elegans), Budding yeast (Saccharomyces cerevisiae), Fission yeast (Schizosaccharomyces pombe)andCandida albicansAnd a protein family database (such as PFAM) based on a hidden Markov model (HMM) (HMM is the primary consensus structure of a gene family), and a PROTEOME database group (Incyte Genomics, Palo Alto CA) having a sequence group from Stochastic approaches to analysis, see, for example, Eddy, SR (1996) Curr. Opin. Struct. Biol. 6: 361-365). Queries were made using programs based on BLAST, FASTA, BLIMPS, and HMMER. An Incyte cDNA sequence was constructed to generate a full-length polynucleotide sequence. Alternatively, GenBank cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan predicted coding sequences (Examples 4 and 5) Was used to extend the Incyte cDNA population to full length. A population of cDNAs was screened for open reading frames using a program based on Phred, Prap and Consed, and a program based on GenMark, BLAST and FASTA. The full length polynucleotide sequence was translated to derive the corresponding full length polypeptide sequence. Alternatively, the polypeptides of the invention may start at any methionine residue of the full length translated polypeptide. Subsequent queries of the full-length polypeptide sequence group as a query were sent to databases such as GenBank protein database group (genpept), SwissProt, PROTEOME database group, BLOCKS, PRINTS, DOMO, PRODOM and Prosite, and hidden Markov models such as PFAM. (HMM) -based protein family databases. Full-length polynucleotide sequences were also analyzed using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE software (DNASTAR). Sequence alignments between polynucleotides and polypeptides are made using default parameters specified by the CLUSTAL algorithm incorporated into the MEGALIGN Multi-Sequence Alignment Program (DNASTAR). It also calculates the percent identity between aligned sequences.
[0257]
Table 7 outlines the tools, programs, and algorithms used for analysis and construction of Incyte cDNA and full-length sequences, along with applicable descriptions, references, and threshold parameters. The tools, programs and algorithms used are shown in column 1 of Table 7 and a brief description of them is shown in column 2. Column 3 is a preferred reference, all references being incorporated by reference in their entirety. If applicable, column 4 shows the parameters, such as score, probability value, used to evaluate the strength of the match between the two sequences (higher score or lower probability value, 2 Sequence identity is high).
[0258]
The programs used for the construction and analysis of full-length polynucleotide sequences and polypeptide sequences were also used to identify the polynucleotide sequence fragments of SEQ ID NO: 64-126. Fragments of about 20 to about 4000 nucleotides useful for hybridization and amplification techniques are shown in Table 4, column 4.
[0259]
4 Genome DNA And editing of coding sequences from
To identify putative secretory proteins, the Genscan gene identification program was first run against public genomic sequence databases (eg, gbpri and gbhtg). Genscan is a universal gene identification program that analyzes genomic DNA sequences from various organisms (Burge, C. and S. Karlin (1997) J. Mol. Biol. 268: 78-94, Burge, C. and S. Karlin (1998) Curr. Opin. Struct. Biol. 8: 346-354). This program ligates the predicted exons to form an assembled cDNA sequence, ranging from methionine to stop codon. The output of Genscan is a FASTA database of polynucleotide and polypeptide sequences. The maximum range of sequences analyzed at one time by Genscan was set at 30 kb. To determine which of these Genscan predicted cDNA sequences encodes a secreted protein, the encoded polypeptides were interrogated against a group of PFAM models for the secreted protein and analyzed. Potential secreted proteins were also identified by homology to the Incyte cDNA sequences already annotated as secreted proteins. These selected Genscan predicted sequences were then compared to public databases genpept and gbpri by BLAST analysis. If necessary, the Genscan predicted sequence was edited by comparing to the top BLAST hits from genpept to correct for errors in the Genscan predicted sequence, such as extra or omitted exons. BLAST analysis was also used to find any Incyte cDNA or public cDNA coverage of the Genscan predicted sequence and thus provided evidence of transcription. If Incyte cDNA coverage was available, this information was used to correct or confirm the Genscan predicted sequence. The full-length polynucleotide sequence isExample 3Genscan predicted coding sequences were constructed from Incyte cDNA sequences and / or public cDNA sequences using the construction process described in. Alternatively, the full length polynucleotide sequence is fully derived from the edited or unedited Genscan predicted coding sequence.
[0260]
5 cDNA Construction of genome sequence data using sequence data
Stitch array ( Stitched Sequence )
To extend the partial cDNA sequence group,Example 4The exon group predicted by the Genscan gene identification program described in (1) was used.Example 3The partial cDNAs constructed as described in (1) were mapped to genomic DNA and also decomposed into clusters having related cDNAs and exons that were Genscan predicted from one or more genomic sequences. To integrate cDNA and genomic information, each cluster was analyzed using some algorithm based on graph theory and dynamic programming to generate a group of potential splice variant sequences. Sequences were subsequently verified, edited or extended to create full length sequences. Sequence section groups in which the total length of the sections was in two or more sequences were identified in the cluster, and the section groups identified in this manner were considered to be equal due to transitivity. For example, if a section is present in one cDNA and two genomic sequences, all three sections were considered equal. This process allows unrelated but contiguous genomic sequences to be linked and cross-linked with cDNA sequences. The sections identified in this way were "stitched" by stitch algorithm in the order in which they appeared along their parent sequences to generate the longest possible sequence and the mutated sequence group. The linkage between sections that follow along one type of parent sequence (cDNA-cDNA or genomic sequence-genomic sequence) has priority over the linkage that changes parent type (cDNA-genomic sequence). The resulting stitch sequences were translated and compared by BLAST analysis to the public databases genpept and gbpri. The incorrect exon groups predicted by Genscan were corrected by comparison to the top BLAST hits from genpept. If necessary, the sequences were further extended using additional cDNA sequences or by examining genomic DNA.
[0261]
Stretch array ( Stretched Sequence )
The partial DNA sequences were extended to full length by one algorithm based on BLAST analysis. First, the BLAST program was used to access public databases such as GenBank primates, rodents, mammals, vertebrates and eukaryotes databases.Example 3Were queried for the partial cDNAs constructed as described in. Next, the closest GenBank protein homolog was identified by BLAST analysis with the Incyte cDNA sequence orExample 4As described in any of the GenScan exon predicted sequences described above. The resulting high scoring segment pair (HSP) was used to generate a chimeric protein and the translated sequence was mapped onto a GenBank protein homolog. Insertions or deletions may occur within the chimeric protein relative to the original GenBank protein homolog. GenBank protein homologs, chimeric proteins or both were used as probes to search homologous genomic sequences from public human genome databases. In this way, the partial DNA sequence was stretched or extended by the addition of a homologous genomic sequence. The resulting stretch sequences were examined to determine if they contained the complete gene.
[0262]
6 Chromosome mapping of polynucleotide encoding SSCP
The sequences used to construct SEQ ID NOs: 64-126 were compared to those in the Incyte LIFESEQ database and the public domain database using BLAST and other Smith-Waterman algorithm implementations. Of the sequences from these databases, those that matched SEQ ID NO: 64-126 were incorporated into a cluster of contiguous and overlapping sequences using a construction algorithm such as Phrap (Table 7). . Using radiation hybrid and genetic map data available from public sources such as the Stanford Human Genome Center (SHGC), Whitehead Genome Institute (WIGR), and Genethon, any clustered sequence is already It was determined whether it was mapped. If the mapped sequence was included in a cluster, the entire sequence of that cluster was assigned to a location on the map, along with the individual SEQ ID NO.
[0263]
Locations on the map are represented as ranges or intervals of human chromosomes. The location on the map of centiMorgan spacing is measured relative to the end of the p-arm of the chromosome (Centimorgan (cM) is a unit of measure based on the recombination frequency between chromosomal markers. On average, 1 cM is Approximately one megabase (Mb) of human DNA, although this value varies widely due to recombination hotspots and cold spots). The cM distance is based on Genethon-mapped genetic markers that provide boundaries for radiation hybrid markers whose sequences are contained within each cluster. Disease genes that have already been identified using the resources such as the NCBI “GeneMap '99” (http://www.ncbi.nlm.nih.gov/genemap/), which are available to the general public, such as the human genome map, It can be determined whether or not it is mapped in the section or in the vicinity.
[0264]
7. Analysis of polynucleotide expression
Northern analysis is an experimental technique used to detect the presence of gene transcripts and involves the hybridization of a labeled nucleotide sequence to membranes bound by RNA from specific cell types or tissues. (See, for example, Sambrook, supra, Chapter 7, and Ausubel (1995), Chapters 4 and 16).
[0265]
The same or related molecules were searched in cDNA databases such as GenBank and LIFESEQ (Incyte Genomics) using a similar computer technique applying BLAST. Northern analysis is much faster than some membrane-based hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether to classify any particular match as exact or homologous. The search criterion is the product score, which is defined by the following equation.
[0266]
(Equation 1)

The product score takes into account both the similarity between the two sequences and the length at which the sequences match. The product score is a normalized value of 0 to 100, and is obtained as follows. Multiply the BLAST score by the percent sequence identity of the nucleotides and divide the product by 5 times the shorter length of the two sequences. To calculate the BLAST score, a +5 score is assigned to each matching base in a pair of high scoring segments (HSP) and -4 is assigned to each mismatching base. Two sequences may share more than one HSP (separated by a gap). If there is more than one HSP, the product score is calculated using the segment pair with the highest BLAST score. The product score represents a balance between fragmented overlap and the quality of the BLAST alignment. For example, a product score of 100 is only obtained if there is a 100% match over the entire shorter length of the two sequences compared. The product score 70 is obtained when either end is 100% matched and 70% overlap, or the other end is 88% matched and 100% overlap. A product score of 50 is obtained when either end is 100% consistent and 50% overlapping, or 79% consistent and 100% overlapping.
[0267]
Alternatively, the polynucleotide sequence encoding SSCP is analyzed for tissue sources from which it was derived. For example, some full-length sequences are constructed, at least in part, using overlapping Incyte cDNA sequences (Example 3See). Each cDNA sequence is derived from a cDNA library made from human tissue. Each human tissue is classified into one of the following organ / tissue categories. Cardiovascular system, connective tissue, digestive system, embryonic structure, endocrine system, exocrine glands, female genitalia, male genitalia, germ cells, blood and immune system, liver, musculoskeletal system, nervous system, pancreas, respiratory system, Sensory organs, skin, stomatognathic system, non-classified / mixed or urinary tract. Count the number of libraries in each category and divide by the total number of libraries in all categories. Similarly, each human tissue is classified into one of the following disease / pathology categories: cancer, cell lines, development, inflammation, nervous, trauma, cardiovascular, pool, and others. Count the number of libraries in each category and divide by the total number of libraries in all categories. The percentages obtained reflect tissue-specific or disease-specific expression of the cDNA encoding SSCP. cDNA sequence and cDNA library / tissue information can be obtained from the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA).
[0268]
8. Elongation of polynucleotide encoding SSCP
Full-length polynucleotide sequences were also generated by extending the fragment using oligonucleotide primers designed from the appropriate fragment of the full-length molecule. One primer was synthesized to initiate 5 'extension of a known fragment, and another primer was synthesized to initiate 3' extension of a known fragment. The starting primers were designed using OLIGO 4.06 software (National Biosciences) or another suitable program, with a length of about 22-30 nucleotides, a GC content of about 50% or more, and about 68-about 72 ° C. At the target temperature. All extensions of the nucleotides that would result in hairpin structures and primer-primer dimers were avoided.
[0269]
The sequence was extended using the selected human cDNA library group. If more than one step extension was needed or desired, additional primers or nested sets of primers were designed.
[0270]
High fidelity amplification was obtained by PCR using methods well known to those skilled in the art. PCR was performed in a 96-well plate using a PTC-200 thermal cycler (MJ Research, Inc.). The reaction mixture has the template DNA and 200 nmol of each primer. In addition, Mg^{2 +}And (NH₄)₂SO₄And a reaction buffer containing 2-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene). Amplification was performed on the primer pairs PCI A and PCI B with the following parameters.
Step 1 3 minutes at 94 ° C
Step 2 15 seconds at 94 ° C
Step 3 at 60 ° C for 1 minute
Step 4 2 minutes at 68 ° C
Step 5 Repeat steps 2, 3, and 4 20 times
Step 6 5 minutes at 68 ° C
Step 7 Store at 4 ° C
Alternatively, amplification was performed with the following parameters for primer pair T7 and SK +.
Step 1 3 minutes at 94 ° C
Step 2 15 seconds at 94 ° C
Step 3 1 minute at 57 ° C
Step 4 2 minutes at 68 ° C
Step 5 Repeat steps 2, 3, and 4 20 times
Step 6 5 minutes at 68 ° C
Step 7 Store at 4 ° C
The DNA concentration in each well was opaque with 1 × TE and 100 μl of PICOGREEN quantitative reagent (0.25% (v / v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in 0.5 μl of undiluted PCR product. The solution was distributed to each well of a fluorometer plate (Corning Costar, Acton MA), and the measurement was performed so that the DNA could bind to the reagent. Plates were scanned with a Fluoroskan II (Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and quantify the DNA concentration. An aliquot of 5-10 μl of the reaction mixture was analyzed by electrophoresis on a 1% agarose gel to determine which reaction was successful in extending the sequence.
[0271]
The extended nucleotides were desalted and concentrated, transferred to a 384-well plate, digested with CviJI choleravirus endonuclease (Molecular Biology Research, Madison WI), sonicated or sheared, and pUC18 vector (Amersham PharmaciaBiacia). Was reconnected. For shotgun sequencing, the digested nucleotides were separated on a low concentration (0.6-0.8%) agarose gel, the fragments were excised, and the agar was digested with Agar ACE (Promega). The extended clone was religated to a pUC18 vector (Amersham Pharmacia Biotech) using T4 ligase (New England Biolabs, Beverly MA), treated with Pfu DNA polymerase (Stratagene) and filled in with a restriction site. E. coli cells were transfected. The transfected cell group was selected in a medium containing an antibiotic, and each colony was collected and cultured in a 384-well plate group in a culture medium of LB / 2X carbenicillin at 37 ° C. overnight.
[0272]
The cells were lysed, and the DNA was PCR-amplified using Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) according to the following procedure.
Step 1 3 minutes at 94 ° C
Step 2 15 seconds at 94 ° C
Step 3 at 60 ° C for 1 minute
Step 4 2 minutes at 72 ° C
Step 5 Repeat steps 2, 3, and 4 29 times
Step 6 5 minutes at 72 ° C
Step 7 Store at 4 ° C
For quantification of DNA, PICOGREEN reagent (Molecular Probes) was used as described above. Samples with low DNA recovery were reamplified using the same conditions as above. Samples were diluted with 20% dimethyl sulfoxide (1: 2, v / v), DYENAMIC energy transfer sequencing primer, and DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or ABI PRISM BIGDYE terminator cycle sequencing kit. It was sequenced using a ready reaction kit (Applied Biosystems).
[0273]
Similarly, the full length polynucleotide sequence was verified using the procedure described above. Alternatively, 5 'regulatory sequences were obtained using oligonucleotides designed for such extensions and some suitable genomic libraries using the full length polynucleotides and the procedure described above.
[0274]
9 Labeling and use of individual hybridization probes
A cDNA, mRNA, or genomic DNA is screened using a hybridization probe group derived from SEQ ID NO: 64-126. Although the labeling of oligonucleotides of about 20 base pairs is specifically described, essentially the same procedure is used for larger nucleotide fragments. Oligonucleotides were designed using the latest software such as OLIGO 4.06 software (National Biosciences) and 50 pmol of each oligomer and [γ-³²P] Adenosine triphosphate (Amersham Pharmacia Biotech) 250 μCi is mixed with T4 polynucleotide kinase (DuPont NEN, Boston MA). The labeled oligonucleotide is substantially purified using a SEPHADEX G-25 ultrafine molecular size exclusion dextran bead column (Amersham Pharmacia Biotech). In a typical membrane-based hybridization analysis of human genomic DNA digested with any one of the endonucleases of Ase I, Bgl II, Eco RI, Pst I, Xba I or Pvu II (DuPont NEN), 10 min / min⁷An aliquot containing a count of labeled probes is used.
[0275]
DNA from each digest is fractionated on a 0.7% agarose gel and transferred to a nylon membrane (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is performed at 40 ° C. for 16 hours. To remove non-specific signals, blots are sequentially washed at room temperature under conditions of, for example, 0.1 × sodium citrate saline and 0.5% sodium dodecyl sulfate. Hybridization patterns are visualized and compared using autoradiography or an alternative imaging means.
[0276]
10 microarray
Coupling or synthesizing array elements on a microarray can be achieved using photolithography, piezoelectric printing (see inkjet printing, see Baldschweiler, supra), mechanical microspotting techniques and techniques derived therefrom. . In each of the above techniques, the substrate should be a solid with a uniform, non-porous surface (Schena (1999) supra). Recommended substrates include silicon, silica, glass slides, glass chips and silicon wafers. Alternatively, a procedure similar to dot blot or slot blot may be used to place and attach elements to the surface of the substrate using thermal, ultraviolet, chemical or mechanical binding procedures. Regular arrays can be made using available methods and machines known to those of skill in the art and can have any suitable number of elements (eg, Schena, M. et al. (1995) Science 270: 467-470). (Sharon. D. et al. (1996) Genome Res. 6: 639-645, Marshall, A. and J. Hodgson (1998) Nat. Biotechnol. 16: 27-31).
[0277]
Full-length cDNAs, expressed sequence tags (ESTs), or fragments or oligomers thereof, can be elements of a microarray. Fragments or oligomers suitable for hybridization can be selected using software known in the art, such as LASERGENE software (DNASTAR). The array elements hybridize with the polynucleotide groups in the biological sample. Polynucleotides in a biological sample are conjugated to a molecular tag, such as a fluorescent label, to facilitate detection. After hybridization, unhybridized nucleotides from the biological sample are removed and hybridization at each array element is detected using a fluorescence scanner. Alternatively, laser desorption and mass spectrometry can be used to detect hybridization. The degree of complementarity and relative abundance of each polynucleotide that hybridizes to an element on the microarray can be calculated. The preparation and use of a microarray in one embodiment is described in detail below.
[0278]
Preparation of tissue or cell samples
Using the guanidinium thiocyanate method to isolate total RNA from tissue samples, poly (A)⁺The oligo (dT) cellulose method is used to purify RNA. Each poly (A)⁺MMLV reverse transcriptase was used to reverse transcribe the RNA sample, and 0.05 pg / μl oligo (dT) primer (21 mer), 1 × first strand buffer, 0.03 unit / μl RNase inhibitor, Use 500 μM dATP, 500 μM dGTP, 500 μM dTTP, 40 μM dCTP, 40 μM dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The reverse transcription reaction was performed using a GEMBRIGHT kit (Incyte) using 200 ng of poly (A).⁺Performed in a 25 ml volume containing RNA. Specific control poly (A)⁺RNAs are derived from non-coding yeast genomic DNAin in vitroSynthesized by transcription. After incubation at 37 ° C for 2 hours, each reaction sample (one labeled Cy3 and the other labeled Cy5) was treated with 2.5 ml of 0.5 M sodium hydroxide, incubated at 85 ° C for 20 minutes, and reacted. To degrade the RNA. Two consecutive CHROMA SPIN 30 gel filtration spin columns (CLONTECH Laboratories, Inc. (CLONTECH), Palo Alto CA) are used for sample group purification. After mixing, ethanol precipitation of the two reaction samples is performed with 1 ml glycogen (1 mg / ml), 60 ml sodium acetate, and 300 ml 100% ethanol. The sample is then dried thoroughly using a SpeedVAC (Savant Instruments Inc., Holbrook NY) and resuspended in 14 μl of 5 × SSC / 0.2% SDS.
[0279]
Preparation of microarray
Array elements are made using the sequences of the present invention. Each array element is amplified from a vector-containing bacterial cell population by the cloned cDNA inserts. PCR amplification uses primers complementary to the vector sequences flanking the cDNA insert. The array elements are amplified from an initial amount of 1-2 ng to a final amount of more than 5 μg by 30 cycles of PCR. The amplified array elements are purified using SEPHACRYL-400 (Amersham Pharmacia Biotech).
[0280]
The purified array element is immobilized on a polymer-coated glass slide. Microscope slides (Corning) are ultrasonically washed in 0.1% SDS and acetone, and during and after treatment with copious amounts of distilled water. Slides were etched in 4% hydrofluoric acid (VWR Scientific Products Corporation (VWR), West Chester PA), washed in copious amounts of distilled water, and 0.05% aminopropylsilane (Sigma) in 95% ethanol. ). The coated slides are cured in a 110 ° C. oven.
[0281]
The procedure for adding array elements to a coated glass substrate is described in US Pat. No. 5,807,522. This patent is hereby incorporated by reference. 1 μl of array element DNA having an average concentration of 100 ng / μl is filled into an open capillary printing element by a high-speed robotic apparatus (robotic apparatus). The apparatus now places approximately 5 nl of array element samples per slide.
[0282]
To UV-crosslink the microarray, use the STRATALLINKER UV crosslinker (Stratagene). Wash the microarray once with 0.2% SDS and three times with distilled water at room temperature. To block non-specific binding sites, the microarray was incubated for 30 minutes at 60 ° C. in 0.2% casein in phosphate buffered saline (PBS) (Tropix, Inc., Bedford MA), followed by Wash with 0.2% SDS and distilled water as done in.
[0283]
Hybridization
9 μl of the sample mixture used for the hybridization reaction contains 0.2 μg of each of the cDNA synthesis products labeled with Cy3 or Cy5 in 5 × SSC, 0.2% SDS hybridization buffer. The sample mixture was heated to 65 ° C. for 5 minutes and aliquoted 1.8 cm on the microarray surface.²Cover with cover glass. Transfer the array to a waterproof chamber with a cavity slightly larger than the microscope slide. To keep the chamber at a humidity of 100, add 140 μl of 5 × SSC to a corner of the chamber. The chamber containing the array is incubated at 60 ° C. for about 6.5 hours. The array was washed in a first wash buffer (1 × SSC, 0.1% SDS) at 45 ° C. for 10 minutes, and in a second wash buffer (0.1 × SSC) at 45 ° C. for 10 minutes. Wash and dry twice.
[0284]
detection
To detect reporter-labeled hybridization complexes, an Innova 70 mixed gas 10W laser (Coherent, Inc., Santa Clara CA) capable of generating spectral lines at 488 nm for Cy3 excitation and 632 nm for Cy5 excitation. A microscope equipped with A 20 × microscope objective (Nikon, Inc., Melville NY) is used to focus the excitation laser light on the array. The slide containing the array is placed on a computer controlled XY stage of the microscope and raster scanned through the objective. The 1.8 cm × 1.8 cm array used in this example is scanned at a resolution of 20 μm.
[0285]
In two different scans, the mixed gas multiline laser excites the two fluorescent dyes sequentially. The emitted light is separated based on the wavelength and sent to two photomultiplier detectors (PMT R1277, Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two fluorescent dyes. Suitable filters are placed between the array and the photomultiplier to filter the signal. The maximum emission wavelength of the fluorescent dye used is 565 nm for Cy3 and 650 nm for Cy5. The instrument can record spectra from both fluorochromes simultaneously, but scans once for each fluorochrome using a suitable filter in the laser source, and typically scans each array twice.
[0286]
Usually, to calibrate the sensitivity of each scan, a cDNA control species is added to the sample mixture at some known concentration, and the signal intensity generated by the control species is used. A particular location on the array contains a complementary DNA sequence, and the intensity of the signal at that location is correlated with a 1: 100,000 weight ratio of hybridization species. When two samples from different sources (eg, representative test and control cells) are each labeled with a different fluorescent dye and hybridized to a single array to identify genes that express differently. Performs calibration by labeling a cDNA sample to be calibrated with two types of fluorescent dyes and adding equal amounts to each of the hybridization mixtures.
[0287]
To digitize the output of the photomultiplier, a 12-bit RTI-835H analog-to-digital (A / D) converter board (Analog Devices, Inc., Norwood MA) installed on an IBM compatible PC computer is used. The digitized data is displayed as a signal intensity mapped image using a linear 20-color conversion from a blue (low signal) to a red (high signal) pseudo-color scale. The data is also analyzed quantitatively. If two different fluorochromes are excited and measured simultaneously, the data is first corrected for optical crosstalk between the fluorochromes (due to overlapping emission spectra) using the emission spectra of each fluorophore.
[0288]
A grid is overlaid on the fluorescent signal image, whereby the signal from each spot is collected on each element of the grid. The fluorescent signal in each element is integrated, and a value corresponding to the average intensity of the signal is obtained. The software used for signal analysis is a GEMTOOOLS gene expression analysis program (Incyte).
[0289]
11 Complementary polynucleotide
A sequence that is complementary to the sequence encoding SSCP or any part thereof is used to detect, reduce or inhibit the expression of native SSCP. Although the use of oligonucleotides containing about 15-30 base pairs is described, essentially the same procedure is used for fragments of smaller or larger sequences. Oligo 4.06 software (National Biosciences) and the coding sequence of SSCP are used to design the appropriate oligonucleotides. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5 'sequence and used to prevent the promoter from binding to the coding sequence. To inhibit translation, complementary oligonucleotides are designed to prevent ribosomes from binding to transcripts encoding SSCP.
[0290]
12. Expression of SSCP
Expression and purification of SSCP can be performed using a bacterial or viral based expression system. To express SSCP in bacteria, the cDNA is subcloned into a suitable vector that contains an antibiotic resistance gene and an inducible promoter that increases the level of cDNA transcription. Such promoters include, but are not limited to, the T5 or T7 bacteriophage promoter associated with the lac operator regulatory element and the trp-lac (tac) hybrid promoter. The recombinant vector is transformed into a suitable bacterial host, such as BL21 (DE3). Antibiotic resistant bacteria express SSCP when induced with isopropyl β-D thiogalactopyranoside (IPTG). Expression of SSCP in eukaryotic cells is commonly known as baculovirus in insect or mammalian cell lines.Au tographica californicaIt is performed by infecting a recombinant form of nuclear polyhedrosis virus (AcMNPV). The non-essential polyhedrin gene of the baculovirus is replaced with the cDNA encoding SSCP by homologous recombination or by bacterial-mediated gene transfer with the transfer plasmid mediated. Viral infectivity is maintained and a strong polyhedron promoter drives high levels of cDNA transcription. Recombinant baculoviruses are often a type of night mothSpodoptera frugiperda(Sf9) Used for infection of insect cells, but may also be used for infection of human hepatocytes. In the latter case, further genetic modification to the baculovirus is required (Engelhard. EK et al. (1994) Proc. Natl. Acad. Sci. USA 91: 3224-2227, Sandig, V. et al. (1996) Hum. Gene Ther. 7: 1937-1945).
[0291]
In most expression systems, SSCP is a fusion protein synthesized with, for example, glutathione S-transferase (GST), or with a peptide epitope tag such as FLAG or 6-His, so recombinant fusion from crude cell lysates Affinity-based purification of proteins can be performed quickly and in one go. GST is a 26 kDa enzyme from Schistosoma japonicum, which allows purification of the fusion protein on immobilized glutathione while maintaining protein activity and antigenicity (Amersham Pharmacia Biotech). After purification, the GST moiety can be proteolytically cleaved from the SSCP at each of the specifically engineered sites. FLAG is an 8-amino acid peptide that allows immunoaffinity purification using commercially available monoclonal and polyclonal anti-FLAG antibodies (Eastman Kodak). 6-His with 6 consecutive histidine residues extended allows purification on metal chelating resins (QIAGEN). Methods for protein expression and purification are described in Ausubel (1995) supra, Chapters 10 and 16. Using the SSCP purified by these methods directly,Examples 16, 17, and 18Can be performed.
[0292]
13 Functional Assay
The function of SSCP is assessed by the expression of sequences encoding SSCP at physiologically elevated levels in mammalian cell culture systems. The cDNA is subcloned into a mammalian expression vector containing a strong promoter that expresses the cDNA at high levels. Vectors to be selected include pCMV SPORT (Life Technologies) and pCR 3.1 (Invitrogen, Carlsbad CA), both of which have a cytomegalovirus promoter. Using a liposome formulation or electroporation, 5-10 μg of the recombinant vector is transiently transfected into a human cell line, eg, an endothelial or hematopoietic cell line. In addition, 1-2 μg of plasmid containing the sequence encoding the labeled protein is co-transfected. Expression of the labeled protein provides a means of distinguishing transfected from non-transfected cells. In addition, expression of the cDNA from the recombinant vector can be accurately predicted by expression of the labeled protein. The labeling protein can be selected from, for example, green fluorescent protein (GFP; Clontech), CD64 or a CD64-GFP fusion protein. Using an automated, laser optics-based technique, flow cytometry (FCM), transfected cells expressing GFP or CD64-GFP are identified, and their apoptotic state and other cellular properties are determined. evaluate. FCM detects and quantifies the uptake of fluorescent molecules that diagnose phenomena that precede or coincide with cell death. Such phenomena include changes in nuclear DNA content measured by DNA staining with propidium iodide, changes in cell size and granularity measured by forward scattered light and 90 ° side scattered light, Downregulation of DNA synthesis as measured by reduced uptake of deoxyuridine, altered cell surface and intracellular protein expression as measured by reactivity with specific antibodies, and cell surface of fluorescein-conjugated annexin V protein There is a change in plasma membrane composition as measured by binding to. For flow cytometry, see Ormerod, M .; G. FIG. (1994)Flow Cytometry, Oxford, New York, NY.
[0293]
The effect of SSCP on gene expression can be calculated using a highly purified cell population transfected with the sequence encoding SSCP and either CD64 or CD64-GFP. CD64 or CD64-GFP is expressed on the transformed cell surface and binds to a conserved region of human immunoglobulin G (IgG). Transformed and non-transformed cells can be efficiently separated using magnetic beads coated with either human IgG or an antibody to CD64 (DYNAL, Lake Success NY). mRNA can be purified from cells by methods well known to those skilled in the art. The expression of mRNA encoding SSEC and another gene of interest can be analyzed by Northern analysis or microarray technology.
[0294]
14 Production of Antibodies Specific to SSCP
Substantially purified SSCP is produced by polyacrylamide gel electrophoresis (PAGE; see, eg, Harrington, MG (1990) Methods Enzymol. 182: 488-495) or other purification techniques. Immunize rabbits using standard protocols to produce antibodies.
[0295]
Alternatively, the SCCP amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine a region with high immunogenicity. Then, the corresponding oligopeptide is synthesized, and the oligopeptide is used to generate an antibody by a method well known to those skilled in the art. Methods for selecting appropriate epitopes, such as near the C-terminus or hydrophilic regions, are known in the art (see Ausubel, 1995, Chapter 11, supra).
[0296]
Usually, oligopeptides of about 15 residues in length are synthesized using an ABI 431A peptide synthesizer (Applied Biosystems) using FMOC chemistry and by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS). Conjugated to KLH (Sigma-Aldrich, St. Louis MO) to enhance immunogenicity (see, eg, Ausubel, 1995, supra). Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant. In order to examine the anti-peptide activity and anti-SECP activity of the obtained antiserum, for example, a peptide or SSCP is bound to a certain substrate, blocked with 1% BSA, reacted with rabbit antiserum, and washed. And further reacted with radioactive iodine-labeled goat anti-rabbit IgG.
[0297]
15 Purification of natural SSCP using specific antibodies
In order to substantially purify natural or recombinant SSCP, immunoaffinity chromatography using a group of antibodies specific to SSCP is performed. Immunoaffinity columns are constructed by covalently linking an activated chromatography resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech), to an anti-SECP antibody. After binding, the resin is blocked and washed according to the manufacturer's instructions.
[0298]
The culture solution containing SSCP is passed through an immunoaffinity column, and the column is washed under conditions that allow preferential adsorption of SCCP (eg, with high ionic strength buffers in the presence of a surfactant). The column is eluted under conditions that disrupt the binding of the antibody to the SSCP (eg, with a buffer of some pH 2-3, or with a high concentration of a chaotrope, eg, urea or thiocyanate ions) to recover the SSCP. .
[0299]
16 Identification of molecules interacting with SSEC
SCCP or a biologically active fragment thereof,¹²⁵Label with I Bolton Hunter reagent (see, eg, Bolton, AE and WM Hunter (1973) Biochem. J. 133: 529-539). The candidate molecules pre-arranged in each well of the multiwell plate are incubated with labeled SSCP, washed, and all wells with the labeled SSCP complex are assayed. Using the data obtained at the various SSCP concentrations, values are calculated for the quantity and affinity of SSCP bound to the candidate molecule and for association.
[0300]
Alternatively, molecules that interact with SSCP are identified in Fields, S .; And O. Analysis using a commercially available kit based on a two-hybrid system such as the yeast two-hybrid system described in Song (1989, Nature 340: 245-246) or the MATCHMAKER system (Clontech). .
[0301]
SECP is also used in a PATHHCALLING process (CuraGen Corp., New Haven CT) using a high-throughput yeast two-hybrid system to determine all interactions between proteins encoded by two large libraries of genes. (Nandabalan, K. et al. (2000) U.S. Patent No. 6,057,101).
[0302]
17 Demonstration of SSCP activity
A spectrophotometric assay (see, for example, Jeong, M. et al. (2000) J. Biol. Chem. 275: 2924-2930) is used to measure the peroxidase activity of SSCP. Alternatively, an assay kit, for example, an AMPLEX Red Peroxidase Assay Kit (Molecular Probes) is used in combination with a fluorescent microplate reader or a fluorometer.
[0303]
An assay for the growth-stimulating or growth-inhibiting activity of SECP measures the amount of DNA synthesis in Swiss mouse 3T3 cells (McKay, I. and Leigh, I., eds. (1993) Growth Factors: A Practical Approach, Oxford University Press, New York, NY). In this assay, variable amounts of SSCP are added to quiescent 3T3 cultured cells as a radioactive DNA precursor [³H] in the presence of thymidine. The means for obtaining SSCP for this assay may be recombinant or obtained from biochemical specimens. To acid-precipitable DNA³The mixing of [H] thymidine is measured at appropriate time intervals, and the amount of mixing is directly proportional to the amount of newly synthesized DNA. Primary dose-response curves over at least a 100-fold SSCP concentration range indicate growth-modulating activity. One unit of activity per milliliter is defined as the concentration of SSCP that produces a 50% response level. Here, a response level of 100% refers to [³[H] represents the maximum mixing of thymidine.
[0304]
Alternatively, TGF-β activity is measured by inducing non-neoplastic normal rat kidney fibroblasts to anchorage-independent proliferation in the presence of 2.5 ng / ml of epidermal growth factor. A description of this method can be found in Assian, R .; K. Et al. (1983) Biol. Chem. 258: 71557160.
[0305]
Alternatively, assays for SSCP activity measure the stimulatory effect or inhibition of neurotransmission in cultured cells. The cultured CHO fibroblasts are exposed to SSCP. After SSCP uptake by endocytosis, the cells are washed with fresh culture medium and whole cell voltage-clamped Xenopus myocytes are contacted with one of the fibroblasts in a medium free of SSCP. Membrane currents are recorded from the muscle cells. Increased or decreased currents relative to control values indicate a neuromodulatory effect of SSCP (Morimoto, T. et al. (1995) Neuron 15: 689-696).
[0306]
Alternatively, an assay for SSCP activity measures the amount of SSCP in secreted membrane-associated organelles. The cells transfected as described above are harvested and lysed. The lysate is fractionated by methods known to those skilled in the art, such as sucrose gradient ultracentrifugation. Such methods allow for the isolation of intracellular compartments, such as the Golgi, ER, membrane-associated vesicles, and other secretory organelles. Immunoprecipitation from fractionated and whole cell lysates is performed using SSCP specific antibodies, and immunoprecipitated samples are analyzed using SDS-PAGE and immunoblot techniques. The concentration of SSCP in secreting organelles relative to SSCP in whole cell lysates is proportional to the amount of SSCP through the secretory pathway.
[0307]
Alternatively, an assay measures the protein kinase activity of SSCP, which is gamma-labeled.³²This is done by quantifying the phosphorylation of the protein substrate by SSCP in the presence of P-ATP. SECP is a protein substrate,³²Incubate with P-ATP and some appropriate kinase buffer. Embedded in the substrate³²P is released by electrophoresis³²Separated and incorporated from P-ATP³²P is counted with a radioisotope counter. Incorporated³²The amount of P is proportional to the activity of SSCP. Determination of phosphorylated specific amino acid residues is performed by phosphoamino acid analysis of the hydrolyzed protein.
[0308]
Alternatively, the measurement of AMP binding activity may be³²It is performed by mixing with P-labeled AMP. The reaction solution is incubated at 37 ° C. and the reaction is terminated by the addition of trichloroacetic acid. The acid extract is neutralized and subjected to gel electrophoresis to remove unbound label. The radioactivity that remains in the gel is proportional to the activity of the SSCP.
[0309]
18 Demonstration of immunoglobulin activity
One assay for SCCP activity measures the ability of SCCP to recognize and precipitate antigens from serum. Measurement of this activity can be made by a quantitative precipitation reaction (Golub, ES et al. (1987)).Immunology: A Synthesis, Sinauer Associates, Sunderland, MA, pp. 113-115). SSCP is isotopically labeled by methods known in the art. Various serum concentrations are added to a fixed amount of labeled SSCP. The SSCP-antigen complex precipitates out of solution and is collected by centrifugation. The amount of precipitated SSCP-antigen complex is proportional to the amount of radioisotope detected in the precipitate. The amount of precipitating SSCP-antigen complex is plotted against serum concentration. Characteristic sedimentation curves for various serum concentrations were obtained, and the amount of precipitating SSCP-antigen complex initially increased in proportion to the increase in serum concentration, peaked at the equivalence point, and thereafter It decreases in proportion to the increase in serum concentration. Thus, the amount of precipitating SSCP-antigen complex is a method of measuring SSCP activity characterized by sensitivity to limiting and excess amounts of antigen.
[0310]
Alternatively, some assays for SCCP activity measure the expression of SSCP at the cell surface. The cDNA encoding SCCP is transfected into a non-leukocyte cell line. Cell surface proteins are labeled with biotin (de la Fuente, MA et al. (1997) Blood 90: 2398-2405). Immunoprecipitation is performed using a SCP-specific antibody group, and the immunoprecipitated sample is analyzed by SDS-PAGE and immunoblotting. The ratio of labeled to unlabeled immunoprecipitant is proportional to the amount of SSCP expressed on the cell surface.
[0311]
Alternatively, some assays for SCCP activity measure the amount of cell aggregation that is induced by SCCP overexpression. In this assay, cultured cells such as NIH3T3 are transfected with cDNA encoding SSCP. The cDNA is included in a suitable mammalian expression vector under the control of a strong promoter. Co-transfection with a cDNA encoding a fluorescently labeled protein such as green fluorescent protein (CLONTECH) helps to identify stable transfectants. The amount of cell aggregation (agglomeration) of the transfected and non-transfected cells is compared. The activity of SSCP can be directly measured by the amount of cell aggregation.
[0312]
Various modifications and alterations of the described method and system of the invention will be apparent to those skilled in the art without departing from the spirit and spirit of the invention. While the invention has been described in connection with several embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. . Various modifications of the methods of practicing the invention described herein that are apparent to those skilled in molecular biology or related fields are clearly within the scope of the following claims.
[0313]
(Brief description of the table)
Table 1 outlines the nomenclature of the full length polynucleotide and polypeptide sequences of the present invention.
[0314]
Table 2 shows the GenBank identification numbers of the polypeptides of the invention and the nearest GenBank homolog annotation. The probability score that each polypeptide and its homolog (one or more) match is also shown.
[0315]
Table 3 shows the structural features of the polynucleotide sequences of the present invention, including predicted motifs and domains, along with the methods, algorithms and searchable databases used to analyze the polypeptides.
[0316]
Table 4 shows the cDNA and genomic DNA fragments used to construct the polynucleotide sequences of the present invention, along with selected fragments of the polynucleotide sequence.
[0317]
Table 5 shows a representative cDNA library of the polynucleotide of the present invention.
[0318]
Table 6 is a supplementary table explaining tissues and vectors used for preparing the cDNA library shown in Table 5.
[0319]
Table 7 shows the tools, programs, and algorithms used to analyze the polynucleotides and polypeptides of the present invention, along with applicable descriptions, references, and threshold parameters.
[Table 1]

[Table 2]

[Table 3]

[Table 4]

[Table 5]

[Table 6]

[Table 7]

[Table 8]

[Table 9]

[Table 10]

[Table 11]

[Table 12]

[Table 13]

[Table 14]

[Table 15]

[Table 16]

[Table 17]

[Table 18]

[Table 19]

[Table 20]

[Table 21]

[Table 22]

[Table 23]

[Table 24]

[Table 25]

[Table 26]

[Table 27]

[Table 28]

[Table 29]

[Table 30]

[Table 31]

[Table 32]

[Table 33]

[Table 34]

[Table 35]

[Table 36]

[Table 37]

[Table 38]

[Table 39]

[Table 40]

[Table 41]

[Table 42]

[Table 43]

Claims (181)

An isolated polypeptide selected from the group consisting of the following (a) to (d):
(A) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-63 (SEQ ID NOs: 1-63); and (b) at least 90 amino acid sequences selected from the group having SEQ ID NOs: 1-63. A polypeptide having an amino acid sequence selected from the group having SEQ ID NO: 1-63; and (d) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group having SEQ ID NO: 1-63. Immunogenic fragments of a polypeptide having an amino acid sequence selected from the group having 1-63 2. The isolated polypeptide of claim 1, having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-63. An isolated polynucleotide encoding the polypeptide of claim 1. An isolated polynucleotide encoding the polypeptide of claim 2. 5. The isolated polynucleotide of claim 4, having a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 64-126. A recombinant polynucleotide comprising a promoter sequence operably linked to the polynucleotide of claim 3. A cell transformed with the recombinant polynucleotide according to claim 6. A genetic transformant comprising the recombinant polynucleotide according to claim 6. A method for producing the polypeptide of claim 1,
(A) culturing cells transformed with a recombinant polynucleotide having a promoter sequence operably linked to the polynucleotide encoding the polypeptide of claim 1 under conditions suitable for expression of said polypeptide. When,
(B) recovering the polypeptide thus expressed, wherein the recombinant polynucleotide has a promoter sequence operably linked to the polynucleotide encoding the polypeptide of claim 1. A method comprising: 10. The method of claim 9, wherein the polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-63. An isolated antibody that specifically binds to the polypeptide of claim 1. An isolated polynucleotide selected from the group consisting of the following (a) to (e):
(A) a polynucleotide having a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 64-126 (b) at least 90% identical to a polynucleotide sequence selected from the group having SEQ ID NOs: 64-126 Polynucleotides (e) (a) to (d) complementary to the polynucleotides (d) and (b) complementary to the polynucleotides of the polynucleotides (c) and (a) having such natural polynucleotide sequences. ) RNA equivalents 13. An isolated polynucleotide having at least 60 contiguous nucleotides of the polynucleotide of claim 12. A method for detecting a target polynucleotide having the polynucleotide sequence according to claim 12 in a sample,
(A) hybridizing the sample with a probe having at least 20 contiguous nucleotides having a sequence complementary to the target polynucleotide in the sample, and combining the probe with the target polynucleotide or a fragment thereof; A process wherein a probe specifically hybridizes to the target polynucleotide under conditions in which a hybridization complex is formed,
(B) detecting the presence or absence of the hybridization complex, and optionally detecting the amount of the complex, if any, if present. The method of claim 14, wherein the probe comprises at least 60 contiguous nucleotides. A method for detecting a target polynucleotide having the polynucleotide sequence according to claim 12 in a sample,
(A) amplifying the target polynucleotide or a fragment thereof using polymerase chain reaction amplification;
(B) detecting the presence or absence of the amplified target polynucleotide or a fragment thereof, and optionally detecting the amount of the target polynucleotide or a fragment thereof when the target polynucleotide or the fragment exists. Method. A composition comprising the polypeptide of claim 1 and a pharmaceutically acceptable excipient. 18. The composition of claim 17, wherein said polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-63. 18. A method of treating a disease or condition associated with reduced expression of a functional SECP, comprising administering the composition of claim 17 to a patient in need of such treatment. A method for screening a compound to confirm the effectiveness of the polypeptide of claim 1 as an agonist,
(A) exposing a sample comprising the polypeptide of claim 1 to a compound;
(B) detecting agonist activity in the sample. A composition comprising the agonist compound identified by the method of claim 20 and also comprising a pharmaceutically acceptable excipient. 22. A method of treating a disease or condition associated with reduced expression of functional SSCP, comprising administering a composition of claim 21 to a patient in need of such treatment. A method for screening a compound for confirming the effectiveness of the polypeptide of claim 1 as an antagonist,
(A) exposing a sample comprising the polypeptide of claim 1 to a compound;
(B) detecting antagonist activity in the sample. A composition comprising the antagonist compound identified by the method of claim 23 and a pharmaceutically acceptable excipient. 25. A method of treating a disease or condition associated with overexpression of a functional SECP, comprising administering a composition of claim 24 to a patient in need of such treatment. A method for screening a compound that specifically binds to the polypeptide of claim 1,
(A) mixing the polypeptide of claim 1 with at least one test compound under suitable conditions;
And (b) detecting the binding of the polypeptide of claim 1 to a test compound, thereby identifying a compound that specifically binds to the polypeptide of claim 1. A method for screening a compound that modulates the activity of the polypeptide according to claim 1,
(A) mixing the polypeptide of claim 1 with at least one test compound under conditions that permit the activity of the polypeptide of claim 1;
(B) calculating the activity of the polypeptide of claim 1 in the presence of a test compound;
(C) comparing the activity of the polypeptide of claim 1 in the presence of the test compound to the activity of the polypeptide of claim 1 in the absence of the test compound, A method wherein the altered activity of the polypeptide of claim 1 indicates a compound that modulates the activity of the polypeptide of claim 1. A method for screening a compound effective for altering expression of a target polynucleotide having the sequence of claim 5, comprising:
(A) exposing a sample containing the target polynucleotide to a compound under conditions suitable for expression of the target polynucleotide;
(B) detecting the altered expression of the target polynucleotide;
(C) comparing the expression of said target polynucleotide in the presence of said compound in a variable amount and in the absence of said compound. A method for calculating the toxicity of a test compound, comprising:
(A) treating a biological sample containing nucleic acids with the test compound;
(B) hybridizing the treated nucleic acid of the biological sample with a probe having at least 20 contiguous nucleotides of the polynucleotide of claim 12, wherein the hybridization comprises the hybridization of the probe and the organism. Under conditions where a specific hybridization complex is formed with a target polynucleotide in a biological sample, said target polynucleotide having the polynucleotide sequence of the polynucleotide of claim 12 or a fragment thereof. The above process, which is a polynucleotide;
(C) quantifying the yield of the hybridization complex;
(D) comparing the amount of the hybridization complex in the treated biological sample with the amount of the hybridization complex in the untreated biological sample. A method wherein a difference in the amount of hybridization complex in a biological sample is indicative of the toxicity of the test compound. A diagnostic test for a condition or disease associated with the expression of SSCP in a biological sample, comprising:
(A) mixing the biological sample with the antibody of claim 11 under conditions suitable for the antibody to bind to the polypeptide and form a complex between the antibody and the polypeptide; ,
(B) detecting the complex, wherein the presence of the complex correlates with the presence of the polypeptide in the biological sample. The antibody of claim 11, wherein
(A) a chimeric antibody (b) a single-chain antibody (c) a Fab fragment (d) an F (ab ') ₂ fragment (e) a humanized antibody. A composition comprising the antibody of claim 11 and an acceptable excipient. 33. A method for diagnosing a condition or disease associated with the expression of SSCP in a subject, comprising administering to the subject an effective amount of the composition of claim 32. 33. The composition of claim 32, wherein said antibody is labeled. 35. A method of diagnosing a condition or disease associated with the expression of SSCP in a subject, the method comprising administering to the subject an effective amount of the composition of claim 34. A method for preparing a polyclonal antibody having the specificity of the antibody of claim 11,
(A) immunizing an animal with a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-63, or having an immunogenic fragment thereof, under conditions which elicit an antibody response; Process
(B) isolating the antibody from the animal;
(C) screening the isolated antibody with the polypeptide, thereby identifying a polyclonal antibody that specifically binds to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-63. Such a method including. A polyclonal antibody produced by the method of claim 36. A composition comprising the polyclonal antibody of claim 37 and a suitable carrier. A method for producing a monoclonal antibody having the specificity of the antibody according to claim 11,
(A) immunizing an animal with a polypeptide comprising an amino acid sequence selected from the group having SEQ ID NO: 1-63 or an immunogenic fragment thereof under conditions for inducing an antibody response;
(B) isolating antibody-producing cells from the animal;
(C) forming a hybridoma cell producing a monoclonal antibody by fusing the antibody-producing cell with the immortalized cell;
(D) culturing the hybridoma cells;
(E) isolating from the cultured monoclonal antibody that specifically binds to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-63. A monoclonal antibody produced by the method of claim 39. A composition comprising the monoclonal antibody of claim 40 and a suitable carrier. The antibody according to claim 11, which is produced by screening a Fab expression library. The antibody according to claim 11, which is produced by screening a recombinant immunoglobulin library. A method for detecting a polypeptide having an amino acid sequence selected from the group having SEQ ID NOs: 1-63 in a sample, comprising:
(A) incubating the antibody according to claim 11 with a sample under conditions permitting specific binding between the antibody and the polypeptide;
(B) detecting specific binding, said specific binding indicating that a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-63 is present in the sample. And how. A method for purifying a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-63 from a sample,
(A) incubating the antibody according to claim 11 with a sample under conditions permitting specific binding between the antibody and the polypeptide;
(B) separating the antibody from the sample to obtain a purified polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-63. A microarray, wherein at least one element of the microarray is the polynucleotide according to claim 13. A method for producing an expression profile of a sample having a polynucleotide, comprising:
(A) labeling a polynucleotide in a sample;
(B) contacting the elements of the microarray of claim 46 with labeled polynucleotides in a sample under conditions suitable for formation of a hybridization complex;
(C) quantifying the expression of the polynucleotide in the sample. An array comprising various nucleotide molecules attached to unique physical locations on a solid substrate, wherein at least one of said nucleotide molecules is specifically hybridizable to at least 30 contiguous nucleotides of a target polynucleotide. An array having an initial oligonucleotide or polynucleotide sequence, wherein said target polynucleotide is a polynucleotide of claim 12. 49. The array of claim 48, wherein the initial sequence of the oligonucleotide or polynucleotide is completely complementary to at least 30 contiguous nucleotides of the target polynucleotide. 49. The array of claim 48, wherein the initial sequence of the oligonucleotide or polynucleotide is completely complementary to at least 60 contiguous nucleotides of the target polynucleotide. 49. The array of claim 48, wherein the initial sequence of the oligonucleotide or polynucleotide is completely complementary to the target polynucleotide. 49. The array of claim 48, wherein the array is a microarray. 49. The array of claim 48, further comprising said target polynucleotide hybridized to a nucleotide molecule having an initial sequence of said oligonucleotide or polynucleotide. 49. The array of claim 48, wherein a linker connects at least one of said nucleotide molecules to said solid substrate. 49. The array of claim 48, wherein each unique physical location on the substrate comprises a plurality of nucleotide molecules, wherein the plurality of nucleotide molecules at any single unique physical location has the same sequence. An array, wherein each unique physical location on the substrate comprises a nucleotide molecule having a sequence that is different from a sequence of nucleotide molecules at another unique physical location on the substrate. 2. The polypeptide according to claim 1, which has the amino acid sequence of SEQ ID NO: 1. 3. The polypeptide according to claim 2, which has the amino acid sequence of SEQ ID NO: 1. The polypeptide according to claim 3, which has the amino acid sequence of SEQ ID NO: 1. The polypeptide according to claim 4, which has the amino acid sequence of SEQ ID NO: 1. The polypeptide according to claim 5, which has the amino acid sequence of SEQ ID NO: 1. The polypeptide according to claim 6, which has the amino acid sequence of SEQ ID NO: 1. The polypeptide according to claim 7, which has the amino acid sequence of SEQ ID NO: 1. 9. The polypeptide according to claim 8, which has the amino acid sequence of SEQ ID NO: 1. The polypeptide according to claim 9, which has the amino acid sequence of SEQ ID NO: 1. The polypeptide according to claim 10, which has the amino acid sequence of SEQ ID NO: 1. The polypeptide according to claim 11, which has the amino acid sequence of SEQ ID NO: 1. 13. The polypeptide according to claim 12, which has the amino acid sequence of SEQ ID NO: 1. 14. The polypeptide according to claim 13, having the amino acid sequence of SEQ ID NO: 1. 15. The polypeptide according to claim 14, having the amino acid sequence of SEQ ID NO: 1. The polypeptide according to claim 15, which has the amino acid sequence of SEQ ID NO: 1. 17. The polypeptide according to claim 16, having the amino acid sequence of SEQ ID NO: 1. 18. The polypeptide according to claim 17, having the amino acid sequence of SEQ ID NO: 1. 19. The polypeptide according to claim 18, having the amino acid sequence of SEQ ID NO: 1. 20. The polypeptide according to claim 19, which has the amino acid sequence of SEQ ID NO: 1. 21. The polypeptide according to claim 20, which has the amino acid sequence of SEQ ID NO: 1. 22. The polypeptide according to claim 21 having the amino acid sequence of SEQ ID NO: 1. 23. The polypeptide according to claim 22, which has the amino acid sequence of SEQ ID NO: 1. 24. The polypeptide of claim 23 having the amino acid sequence of SEQ ID NO: 1. 25. The polypeptide according to claim 24 having the amino acid sequence of SEQ ID NO: 1. 26. The polypeptide of claim 25 having the amino acid sequence of SEQ ID NO: 1. 27. The polypeptide of claim 26 having the amino acid sequence of SEQ ID NO: 1. 28. The polypeptide of claim 27 having the amino acid sequence of SEQ ID NO: 1. 29. The polypeptide of claim 28 having the amino acid sequence of SEQ ID NO: 1. 30. The polypeptide of claim 29 having the amino acid sequence of SEQ ID NO: 1. 31. The polypeptide of claim 30, having the amino acid sequence of SEQ ID NO: 1. 32. The polypeptide of claim 31, having the amino acid sequence of SEQ ID NO: 1. 33. The polypeptide of claim 32 having the amino acid sequence of SEQ ID NO: 1. 34. The polypeptide of claim 33 having the amino acid sequence of SEQ ID NO: 1. 35. The polypeptide of claim 34 having the amino acid sequence of SEQ ID NO: 1. 36. The polypeptide of claim 35 having the amino acid sequence of SEQ ID NO: 1. 37. The polypeptide of claim 36 having the amino acid sequence of SEQ ID NO: 1. 38. The polypeptide of claim 37 having the amino acid sequence of SEQ ID NO: 1. 39. The polypeptide of claim 38 having the amino acid sequence of SEQ ID NO: 1. 40. The polypeptide of claim 39 having the amino acid sequence of SEQ ID NO: 1. 41. The polypeptide of claim 40 having the amino acid sequence of SEQ ID NO: 1. 42. The polypeptide of claim 41 having the amino acid sequence of SEQ ID NO: 1. 43. The polypeptide of claim 42 having the amino acid sequence of SEQ ID NO: 1. 44. The polypeptide of claim 43 having the amino acid sequence of SEQ ID NO: 1. 45. The polypeptide of claim 44 having the amino acid sequence of SEQ ID NO: 1. 46. The polypeptide of claim 45 having the amino acid sequence of SEQ ID NO: 1. 47. The polypeptide of claim 46 having the amino acid sequence of SEQ ID NO: 1. 48. The polypeptide of claim 47 having the amino acid sequence of SEQ ID NO: 1. 49. The polypeptide of claim 48 having the amino acid sequence of SEQ ID NO: 1. 50. The polypeptide of claim 49 having the amino acid sequence of SEQ ID NO: 1. 51. The polypeptide of claim 50 having the amino acid sequence of SEQ ID NO: 1. 52. The polypeptide of claim 51 having the amino acid sequence of SEQ ID NO: 1. 53. The polypeptide of claim 52 having the amino acid sequence of SEQ ID NO: 1. 54. The polypeptide of claim 53 having the amino acid sequence of SEQ ID NO: 1. 55. The polypeptide of claim 54 having the amino acid sequence of SEQ ID NO: 1. 56. The polypeptide of claim 55 having the amino acid sequence of SEQ ID NO: 1. 57. The polypeptide of claim 56 having the amino acid sequence of SEQ ID NO: 1. 58. The polypeptide of claim 57 having the amino acid sequence of SEQ ID NO: 1. 59. The polypeptide of claim 58 having the amino acid sequence of SEQ ID NO: 1. 60. The polypeptide of claim 59 having the amino acid sequence of SEQ ID NO: 1. 61. The polypeptide of claim 60 having the amino acid sequence of SEQ ID NO: 1. 62. The polypeptide of claim 61 having the amino acid sequence of SEQ ID NO: 1. 63. The polypeptide of claim 62 having the amino acid sequence of SEQ ID NO: 1. 64. The polypeptide of claim 63 having the amino acid sequence of SEQ ID NO: 1. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 64. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 65. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 66. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 67. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 68. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 69. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 70. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 71. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 72. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 73. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 74. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 75. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 76. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 77. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 78. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 79. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 80. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 81. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 82. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 83. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 84. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 85. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 86. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 87. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 88. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 89. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 90. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 91. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 92. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 93. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 94. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 95. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 96. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 97. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 98. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 99. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 100. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 101. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 102. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 103. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 104. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 105. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 106. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 107. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 108. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 109. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 110. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 111. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 112. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 113. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 114. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 115. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 116. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 117. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 118. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 119. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 120. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 121. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 122. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 123. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 124. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 125. 13. The polynucleotide of claim 12, having the polynucleotide sequence of SEQ ID NO: 126.