JPWO2002088349A1 - Method for testing allergic disease using alternative splicing form of cathepsin C as index - Google Patents

Abstract

Airway epithelial cells were stimulated with IL-4 or IL-13, and an alternative splicing form of cathepsin C (cathepsin-Alternative splicing form) was obtained as an allergy-related gene whose expression was significantly changed in a plurality of cells. The expression of this gene is enhanced in airway epithelial cells treated with IL-4 or IL-13. The present invention provides a method for testing an allergic disease and a method for screening a compound useful for treatment, using the expression level of this gene in a biological sample as an index.

Description

次に、Ｅｘｐｒｅｓｓｉｏｎ Ａｎａｌｙｓｉｓ Ｔｅｃｈｎｉｃａｌ Ｍａｎｕａｌに従い、ＤＮＡ Ｌｉｇａｓｅ，ＤＮＡ ｐｏｌｙｍｅｒａｓｅ Ｉ及びＲＮａｓｅ Ｈを加え、２本鎖ｃＤＮＡを合成した。ｃＤＮＡをフェノール・クロロホルム抽出後、Ｐｈａｓｅ Ｌｏｃｋ Ｇｅｌｓに通し、エタノール沈澱し精製した。
さらに、ＢｉｏＡｒｒａｙ Ｈｉｇｈ Ｙｉｅｌｄ ＲＮＡ Ｔｒａｎｓｃｒｉｐｔｉｏｎ Ｌａｂｅｌｉｎｇ Ｋｉｔを用い、ビオチンラベルしたｃＲＮＡを合成した。ＲＮｅａｓｙ Ｓｐｉｎ ｃｏｌｕｍｎ（ＱＩＡＧＥＮ）を用いてｃＲＮＡを精製し、熱処理により断片化した。
そのうち１２．５μｇのｃＲＮＡをＥｘｐｒｅｓｓｉｏｎ Ａｎａｌｙｓｉｓ Ｔｅｃｈｎｉｃａｌ Ｍａｎｕａｌに従いＨｙｂｒｉｄｉｚａｔｉｏｎ Ｃｏｃｋｔａｉｌに加えた。これをアレイＨｕ３５Ｋ ＧｅｎｅＣｈｉｐ（Ａｆｆｙｍｅｔｒｉｘ）に入れ、４５℃１６時間ハイブリダイゼーションした。
アレイを洗浄した後、Ｓｔｒｅｐｔａｖｉｄｉｎ Ｐｈｙｃｏｅｒｙｔｈｒｉｎを加え染色した。洗浄後、ｎｏｒｍａｌヤギＩｇＧとビオチン化ヤギＩｇＧの抗体混合液をアレイに加えた。さらに、蛍光強度を増強する目的で、再度Ｓｔｒｅｐｔａｖｉｄｉｎ Ｐｈｙｃｏｅｒｙｔｈｒｉｎを加え染色した。洗浄後、スキャナーにセットし、ＧｅｎｅＣｈｉｐ Ｓｏｆｔｗａｒｅにて解析した。
５．ＧｅｎｅＣｈｉｐ解析
ＧｅｎｅＣｈｉｐ解析ソフトであるＳｕｉｔｅを用いてデータ解析を行った。Ａｂｓｏｌｕｔｅ ａｎａｌｙｓｉｓで、Ａｖｅｒａｇｅ Ｉｎｔｅｎｓｉｔｙ（１）とＢａｃｋｇｒｏｕｎｄ Ａｖｅｒａｇｅ（２）を調べ、（１）から（２）を引いた、未刺激、ＩＬ−４刺激、またはＩＬ−１３刺激の３つの平均を補正値（Ｓｃａｌｅ Ｆａｃｔｏｒ）としてＣｏｍｐａｒｉｓｏｎ Ａｎａｌｙｓｉｓを行った。
まず、Ａｂｓｏｌｕｔｅ Ａｎａｌｙｓｉｓを行い１個のチップデータの解析をした。プローブセットのパーフェクトマッチとミスマッチの蛍光強度を比較して、ｐｏｓｉｔｉｖｅとｎｅｇａｔｉｖｅを決定した。Ｐｏｓ Ｆｒａｃｔｉｏｎ，Ｌｏｇ Ａｖｇ，Ｐｏｓ／Ｎｅｇの値から判定されるＡｂｓｏｌｕｔｅ ＣａｌｌであるＰ（ｐｒｅｓｅｎｔ）、Ａ（ａｂｓｅｎｔ）、およびＭ（ｍａｒｇｉｎａｌ）の３区分の判定をした。
Ｐｏｓ Ｆｒａｃｔｉｏｎ；Ｐｏｓｉｔｉｖｅなペアの割合。
Ｌｏｇ Ａｖｇ；パーフェクトマッチとミスマッチのプローブセルの蛍光強度比の対数の平均
Ｐｏｓ／Ｎｅｇ；Ｐｏｓｉｔｉｖｅペア数とＮｅｇａｔｉｖｅペア数の比
また、それぞれの遺伝子において、パーフェクトマッチとミスマッチのプローブセルの蛍光強度の差の平均値であるＡｖｅｒａｇｅ Ｄｉｆｆｅｒｅｎｃｅ（Ａｖｇ Ｄｉｆｆ）も計算した。
次に２つのデータを比較解析（Ｃｏｍｐａｒｉｓｏｎ Ａｎａｌｙｓｉｓ）した。未刺激とＩＬ−４刺激あるいは未刺激とＩＬ−１３刺激で比較し、発現レベルの差を以下のようにランキングした。Ｉｎｃ／Ｄｅｃ，Ｉｎｃ Ｒａｔｉｏ，Ｄｐｏｓ−Ｄｎｅｇ Ｒａｔｉｏ，Ｌｏｇ Ａｖｇ Ｒａｔｉｏ Ｃｈａｎｇｅの値から判定されるＤｉｆｆｅｒｅｎｃｅ ＣａｌｌであるＩ，Ｄ，ＭＩ，ＭＤ，ＮＣの５区分の判定をした。
Ｉｎｃ：ＩＬ−４刺激あるいはＩＬ−１３刺激と未刺激の対応するプローブペアについてＩＬ−４刺激あるいはＩＬ−１３刺激の方が増加していると判定されたペア数。
Ｄｅｃ：ＩＬ−４刺激あるいはＩＬ−１３刺激の方が減少していると判定されたペア数。
Ｉｎｃ／Ｄｅｃ：Ｉｎｃと判定されたペア数とＤｅｃと判定されたペア数の比。
Ｉｎｃ Ｒａｔｉｏ：Ｉｎｃと判定されたペア数／実際に使用されたペア数。
Ｄｐｏｓ／Ｄｎｅｇ Ｒａｔｉｏ：Ｐｏｓ ＣｈａｎｇｅからＮｅｇ Ｃｈａｎｇｅを引いた数と実際に使用されたペア数の比
Ｐｏｓ Ｃｈａｎｇｅ：ＩＬ−４刺激あるいはＩＬ−１３刺激のＡｂｓｏｌｕｔｅ ＡｎａｌｙｓｉｓでＰｏｓｉｔｉｖｅなペア数と未刺激のＡｂｓｏｌｕｔｅ ＡｎａｌｙｓｉｓでＰｏｓｉｔｉｖｅなペア数の差。
Ｎｅｇ Ｃｈａｎｇｅ：ＩＬ−４刺激あるいはＩＬ−１３刺激のＡｂｓｏｌｕｔｅ ＡｎａｌｙｓｉｓでＮｅｇａｔｉｖｅなペア数と未刺激のＡｂｓｏｌｕｔｅ ＡｎａｌｙｓｉｓでＮｅｇａｔｉｖｅなペア数の差
Ｌｏｇ Ａｖｇ Ｒａｔｉｏ Ｃｈａｎｇｅ：ＩＬ−４刺激あるいはＩＬ−１３刺激と未刺激のＡｂｓｏｌｕｔｅ ＡｎａｌｙｓｉｓでのＬｏｇ Ａｖｇの差。
増加：Ｉ（Ｉｎｃｒｅａｓｅｄ）、
減少：Ｄ（Ｄｅｃｒｅａｓｅｄ）、
わずかに増加：ＭＩ（Ｍａｒｇｉｎａｌｌｙ Ｉｎｃｒｅａｓｅｄ）、
わずかに減少：ＭＤ（Ｍａｒｇｉｎａｌｌｙ Ｄｅｃｒｅａｓｅｄ）、および
変化無し：ＮＣ（ｎｏ ｃｈａｎｇｅ）
また、未刺激とＩＬ−４刺激あるいは未刺激とＩＬ−１３刺激のＡｂｓｏｌｕｔｅ ＡｎａｌｙｓｉｓでのＡｖｇ Ｄｉｆｆの比であるＦｏｌｄ Ｃｈａｎｇｅの値でＩＬ−４刺激、またはＩＬ−１３刺激によって発現変動（増強あるいは減少）が認められる遺伝子として選抜した。気道上皮細胞のＬｏｔ別の発現遺伝子の絞込みの結果を表１に示した。

表中、「［Ｄｉｆｆ Ｃａｌｌ］で増加或は減少」とは、ＩＬ−４刺激、またはＩＬ−１３刺激によって発現レベルに差が見られた遺伝子の数を示す。発現レベルに差が見られた遺伝子には、前記ランキングにおいて、わずかに増加（またはわずかに減少）と判定された遺伝子を含む。各ロットごとに、ＩＬ−４とＩＬ−１３の両方に共通して差が見られた遺伝子の数を、「変動共通遺伝子」として示した。更に、「［Ｆｏｌｄ Ｃｈａｎｇｅ］」として、２倍以上の増減（＞２または＜−２）、および３倍以上の増減（＞３または＜−３）が見られた遺伝子の数をそれぞれ示した。
その結果、２７遺伝子がＩＬ−４及びＩＬ−１３刺激によって共通してＦｏｌｄ Ｃｈａｎｇｅが３倍以上、または１／３倍以下に変動する遺伝子であった。その中で、ＩＬ−４及びＩＬ−１３刺激によって共通でも最も発現上昇の見られる配列としてＨｕ３５Ｋ ｓｕｂｓｅｔ Ｂのチップ上にＥＳＴとして報告されているａａ０１１３０５配列が選抜された。
６．遺伝子の解析
ａａ０１１３０５の配列内にＡＢＩ７７００による定量用のプライマーセット（ａａＦ／配列番号：４とａａＲ／配列番号：５）およびＴａｑＭａｎＰｒｏｂｅを設計した。ＴａｑＭａｎプローブの５’末端はＦＡＭ（６−ｃａｒｂｏｘｙ−ｆｌｕｏｒｅｓｃｅｉｎ）で、また３’末端はＴＡＭＲＡ（６−ｃａｒｂｏｘｙ−Ｎ，Ｎ，Ｎ’，Ｎ’−ｔｅｔｒａｍｅｔｈｙｌｒｈｏｄａｍｉｎｅ）で標識されている。
前述の方法で抽出した全ＲＮＡをＤＮａｓｅ（ニッポンジーン）処理した。その後、ｒａｎｄａｍ ｈｅｘａｍｅｒ（ＧＩＢＣＯ ＢＲＬ）をプライマーとして逆転写したｃＤＮＡを鋳型とした。コピー数を算出する標準曲線のために両プライマーで増幅される塩基配列領域を含むプラスミドクローンを各々の遺伝子について準備し、その段階希釈を鋳型として反応を行った。ＰＣＲ増幅のモニタリングのための反応液の組成は表２に示した。

また、試料中のｃＤＮＡ濃度の差を補正するため、補正用内部標準としてβ−アクチン（β−ａｃｔｉｎ）遺伝子、およびグリセルアルデヒド３リン酸脱水素酵素（ＧＡＰＤＨ）遺伝子について同様の定量解析を行い、それら遺伝子のコピー数を基に補正して、目的遺伝子のコピー数を算出した。
βアクチン、あるいはＧＡＰＤＨ測定用のプライマーとプローブは、ＴａｑＭａｎ β−ａｃｔｉｎ Ｃｏｎｔｒｏｌ Ｒｅａｇｅｎｔｓ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）に添付のものを用いて行った。塩基配列は以下の通りである。βアクチンにより補正した各遺伝子の発現量（ｃｏｐｙ／５ｎｇ全ＲＮＡ）の経時的な変化を図１に、細胞ロット別の発現量の変化を図２に示す。３ロットの気道上皮細胞を用いたＩＬ−４及びＩＬ−１３刺激実験での発現変動を確認したところ、やはりはっきりとした発現の上昇が観察された。
定量的ＰＣＲの結果、実施例１で選択した遺伝子の気道上皮細胞における発現レベルは、いずれもＩＬ−４あるいはＩＬ−１３刺激によって、３つの異なる気道上皮細胞において２倍以上に上昇した。これらの結果に基づいて、気道上皮細胞では、ＩＬ−４やＩＬ−１３に応答して、これらの指標遺伝子の発現レベルが上昇することが予測された。
アレルギー反応との密接な関連が知られているＩＬ−４やＩＬ−１３の刺激によって、本発明の指標遺伝子は、異なるロットの気管支上皮細胞で共通の挙動を示す。したがって、本発明の指標遺伝子は、アレルギー反応の進行を制御する重要な遺伝子であると考えられる。

次に、発現レベルの上昇が認められた配列であるａａ０１１３０５配列をＨｕｍａｎ Ｇｅｎｏｍｅ Ｂｌａｓｔ検索したところ、カテプシンＣ遺伝子の第２イントロンに一致することが分かった。ゲノムを増幅してしまった可能性が考えられたので、ＬｏｎｇＰＣＲによる検証を行った。すなわち、ａａ０１１３０５配列内に設計したプライマー（ａａＦおよびａａＲ）と、ａａ０１１３０５配列の含まれる第２イントロンに近接するエキソン（５’側は第２エキソン，３’側は第３エキソン）内に設計したＰＣＲプライマー（３Ｆ／配列番号：１３および３Ｒ／配列番号：１４）を用い、増幅領域が各エキソンとａａ０１１３０５配列を結ぶようなｌｏｎｇ−ＰＣＲを行った。操作は次のとおりである。
すなわち、２．５ｕｎｉｔ／μｌ ＫＯＤ Ｄａｓｈ（ＴＯＹＯＢＯ）、０．２μｌ、１０ｎｇ／μｌ気道上皮細胞（ＩＬ−４刺激）由来ｃＤＮＡ ２μｌ、１０μＭ ｐｒｉｍｅｒ各２μｌ、２ｍＭ ｄＮＴＰｓ １μｌ、ｄＨ_２Ｏ １０．８μｌを混合し、ｔｏｔａｌ ２０μｌとした。ＡＢＩ９７００を用いて９４℃ ２ｍｉｎの後９４℃ ３０ｓｅｃ、６０℃ ２ｓｅｃ、７４℃ １ｍｉｎ×３５ｃｙｃｌｅの増幅反応を行った。用いたプライマーの塩基配列を以下に示す。

プライマーａａＦと３Ｆを用いた５’側の増幅では、プライマー同士のゲノム配列上の距離は約８ｋｂあるのにもかかわらず、２００ｂｐ程度の増幅産物が得られた。この断片をｐＣＲ２．１ｖｅｃｔｏｒ（ｉｎｖｉｔｒｏｇｅｎ）へ通常の方法によって組み込み、ＴＯＰ１０Ｆ’（ｉｎｖｉｔｒｏｇｅｎ）へ形質転換し、その塩基配列を解析した。その結果、ａａ０１１３０５配列よりも５’側に６９ｂｐ伸長した所で第２エキソンとつながる新規の配列が得られた（配列番号：２３として示した配列の１−２０６）。この配列は第２エキソンから既知のカテプシンＣとは異なる３１アミノ酸および終止コドンを含む新規のＯＲＦをコードしており、また、伸長した６９ｂｐをａａ０１１３０５配列に付加してＨｕｍａｎ Ｇｅｎｏｍｅ Ｂｌａｓｔ検索したところ、カテプシンＣ遺伝子の第２イントロン内で２つの領域に分断されていた。図３に本発明のオータナティブスプライシングフォームのエキソン、カテプシンＣのエキソン、並びにＤＮＡチップ上のＥＳＴであるａａ０１１３０５のゲノムにおける位置関係を模式的に示した。また本発明のオータナティブスプライシングフォームのイントロン１およびイントロン２の、エキソンとイントロンの間の塩基配列と、ＧＴ−ＡＧルールの関係を図４に示した。見出された２つの新規エキソンと隣接するイントロンの間のＧＴ−ＡＧ規則はかなりよく保存されており、ｃａｔｈｅｐｓｉｎ−Ｃ遺伝子の新規なオータナティブスプライシングフォームであると考えられた。
ａａ０１１３０５配列上のプライマーａａＲと第３エキソン上に設計したプライマー３Ｒを用いたＰＣＲでは増幅は認められなかった。また、第２エキソン・第３エキソン上のプライマー（３Ｆおよび３Ｒ）を用いた場合、および第１エキソン・第７（最終）エキソン上のプライマー（２Ｆおよび２Ｒ）を用いたＰＣＲではカテプシンＣとして既知の配列以外は得られなかった。
しかし、第１エキソンの開始コドン近傍に設計したプライマー１Ｆとａａ０１１３０５配列上のプライマーａａＦでのＰＣＲでは第１エキソンから第２エキソンを経て今回見出された新規エキソンに切り替わる配列が得られた。ａａ０１１３０５配列中に終止コドンが存在することと併せて考慮すると、今回得られたオータナティブスプライシングフォームにはａａ０１１３０５配列以降のエキソンはなく、少なくとも第１エキソンの途中から第２エキソンまでは既知の配列と同一で、第２エキソンの終わりから新規の３１アミノ酸を付加したところで終結する短いペプチドをコードしていることが示唆される。
さらに、このオータナティブスプライシングフォームの５’末端と３’末端を決定するために、Ｍａｒａｔｈｏｎ ｃＤＮＡ Ａｍｐｌｉｆｉｃａｔｉｏｎ Ｋｉｔ（ＣＬＯＮＴＥＣＨ）により、ＨＬ６０由来のｍＲＮＡを用いて作成されたＭａｒａｔｈｏｎ用ライブラリーを鋳型として３’ＲＡＣＥおよび５’ＲＡＣＥを行った。
５’ＲＡＣＥでは、キット付属のＡＰ１プライマーと５’ＲＡＣＥ１（配列番号：１８）でＰＣＲを行った。さらに、この増幅断片を鋳型にアダプター内の配列［ＡＰ２］と５’ＲＡＣＥ２（配列番号：１９）を用いてｎｅｓｔｅｄ ＰＣＲを行った。一方、３’ＲＡＣＥではキット付属のＡＰ１プライマーとプライマー３’ＲＡＣＥ１（配列番号：２０）でＰＣＲを行った。さらに、この増幅断片を鋳型にアダプター内の配列［ＡＰ２］とプライマー３’ＲＡＣＥ２（配列番号：２１）を用いてｎｅｓｔｅｄ ＰＣＲを行った。

この結果、ａａ０１１３０５として報告されている配列の５’端がこのオータナティブスプライシングフォームの３’端であり、３’末端から１９ｂｐに存在するｐｏｌｙＡ付加シグナルを認識しているらしいことが分かった。以上の実験で得られた新規オータナティブスプライシングフォームの全長の塩基配列を配列番号：１に、このｃＤＮＡによってコードされるアミノ酸配列を配列番号：２に示す。
［実施例２］ノーザンハイブリダイゼーション
カテプシン遺伝子及びオータナティブスプライシングフォームがｍＲＮＡとして発現していることを確認するためにノーザンハイブリダイゼーションを行った。次のプライマーセットによる増幅産物をＲＥＣＯ Ｃｈｉｐ（ＴＡＫＡＲＡ）を用いて精製し、Ｒａｎｄｏｍ Ｐｒｉｍｅｒ Ｌａｂｅｌｉｎｇ Ｋｉｔ（ＴＡＫＡＲＡ）により^３２Ｐを用いて標識しプローブとした。鋳型には、Ｌｏｎｇ ＰＣＲ実験で得た２Ｆ／２Ｒ間の塩基配列をインサートとして持つプラスミドを用いた。
既知配列のみを認識するプローブ５５２ｂｐ

既知配列及びオータナティブスプライシングフォーム双方を認識するプローブ１３０１ｂｐ

メンブレンは、ＣＬＯＮＴＥＣＨ社のＨｕｍａｎ Ｉｍｍｕｎｅ Ｓｙｓｔｅｍ ＭＴＮ Ｂｌｏｔ ＩＩおよびＨｕｍａｎ ＭＴＮ Ｂｌｏｔを用いた。Ｅｘｐｒｅｓｓ Ｈｙｂｒｉｄｉｚａｔｉｏｎ Ｓｏｌｕｔｉｏｎ（ＣＬＯＮＴＥＣＨ）を用いて添付使用書通りにノーザンハイブリダイゼーションおよび洗浄を行った。洗浄は２ｘＳＳＣ−０．１％ＳＤＳを用いて室温で約４０分、０．１×ＳＳＣ−０．１％ＳＤＳを用いて５６℃で１０分行った。一晩イメージングプレートに暴露し画像を取得した。画像はＭｏｌｅｃｕｌａｒ Ｉｍａｇｅｒ Ｓｙｓｔｅｍ（ＢＩＯ−ＲＡＤ）によって取得した。
既知カテプシンＣ遺伝子の第６エキソン・第７エキソン上の配列を持ち、既知カテプシンＣ遺伝子のみを認識することが期待されるプローブ（ｘ８７ｆ／２Ｒ）と、第１エキソンから第７エキソンまでの配列を持ち、既知カテプシンＣ遺伝子及び新規オータナティブスプライシングフォーム双方を認識するプローブ（２Ｆ／２Ｒ）を用いた場合は約２ｋｂのバンドが検出された。このサイズは、報告されている既知遺伝子の１．８ｋｂとほぼ一致した。また、検出されたバンドの強度は肺・腎臓・胎盤で強く、脳で弱いという過去の報告（Ｊ．Ｂｉｏｌ．Ｃｈｅｍ，２７２，１０２６０−１０２６５，１９９７）と同一の結果を示した。さらに、既知カテプシンＣ遺伝子および新規オータナティブスプライシングフォーム双方を認識するプローブを用いた場合にのみ、非常に弱いシグナルではあるが１ｋｂ付近にバンドが認められ、これが前述のオータナティブスプライシングフォームであることが予測された。
オータナティブスプライシングフォームのみを検出するために、本実験で新規に得られた領域（配列番号：２３）をもとにｒｉｂｏｐｒｏｂｅによるノーザンハイブリダイゼーションを行った。プローブは次のようにして調製した。まず、プライマーａａＦ（配列番号：４）とプライマー３’ＲＡＣＥ１（配列番号：２０）を用いて増幅される領域をｐＧＥＭ−Ｔ Ｅａｓｙ ｖｅｃｔｏｒ（ｐｒｏｍｅｇａ）に組み込んだ。鋳型には、Ｌｏｎｇ ＰＣＲ実験で得た１Ｆ／ａａＦ間の塩基配列をインサートとして持つプラスミドを用いた。次に、Ｔ７ ｐｒｏｍｏｔｅｒから標的配列のａｎｔｉｓｅｎｓｅ鎖が読まれるクローンを選択し、プラスミドを精製した。得られたプラスミドを制限酵素ＮｄｅＩを用いて直鎖状とし、これを鋳型として^３２Ｐ−ｒＣＴＰで標識されたａｎｔｉｓｅｎｓｅ ＲＮＡ鎖をＴ７ ＲＮＡ ｐｏｌｙｍｅｒａｓｅ（ｐｒｏｍｅｇａ）を用いて合成し、プローブとして用いた。一連の操作は、ｒｉｂｏｐｒｏｂｅ Ｉｎ Ｖｉｔｒｏ Ｔｒａｎｓｃｒｉｐｔｉｏｎ Ｓｙｓｔｅｍｓ（ｐｒｏｍｅｇａ）の指示書にしたがった。
同種のメンブランを用い、５ｘＳＳＰＥ，５０％ホルムアミド、５ｘデンハルト、０．５％ＳＤＳのハイブリソリューション中、５５℃で一晩ハイブリダイゼーションを行った。洗浄は２ｘＳＳＣ−０．１％ＳＤＳを用いて室温で約４０分、０．１×ＳＳＣ−０．１％ＳＤＳを用いて５６℃で１時間、６０℃で１時間、７０度で３０分行った。一晩イメージングプレートに暴露し画像を取得した。画像はＭｏｌｅｃｕｌａｒ Ｉｍａｇｅｒ Ｓｙｓｔｅｍ（ＢＩＯ−ＲＡＤ）によって取得した。
この結果、１ｋｂ付近にシグナルが見出され、このオータナティブスプライシングフォームが実際にｍＲＮＡとして存在していることが示された。また、腎臓・胎盤・リンパ節で発現が高いことが分かった（図５）。
［実施例３］指標遺伝子の発現レベルの比較
培養気道上皮細胞（Ｃｌｏｎｅｔｉｃｓ社）におけるカテプシンＣのオータナティブスプライシングフォームの発現レベルを定量的ＰＣＲにより測定し、カテプシンＣと比較した。培養細胞としては、８Ｆ１７５６、８Ｆ１５４８、および８Ｆ１８０５の３つのロットを用いた。測定方法は、６に準じた。オータナティブスプライシングフォームのためのプライマーおよびＴａｑＭａｎＰｒｏｂｅは、［実施例１］の６で用いたａａＦ（配列番号：４）およびａａＲ（配列番号：５）、およびＴａｑＭａｎＰｒｏｂｅ（配列番号：６）である。カテプシンＣ用のプライマーとＴａｑＭａｎＰｒｏｂｅの塩基配列は以下に示す。

前述の方法で抽出した全ＲＮＡをＤＮａｓｅ（ニッポンジーン）処理した。その後、ｒａｎｄａｍ ｈｅｘａｍｅｒ（ＧＩＢＣＯ ＢＲＬ）をプライマーとして逆転写したｃＤＮＡを鋳型とした。コピー数を算出する標準曲線のために両プライマーで増幅される塩基配列領域を含むプラスミドクローンを各々の遺伝子について準備し、その段階希釈を鋳型として反応を行った。ＰＣＲ増幅のモニタリングのための反応液の組成も［実施例１］の６で表２に示したものと同じである。
また、試料中のｃＤＮＡ濃度の差を補正するため、補正用内部標準としてβ−アクチン（β−ａｃｔｉｎ）遺伝子、およびグリセルアルデヒド３リン酸脱水素酵素（ＧＡＰＤＨ）遺伝子について同様の定量解析を行い、それら遺伝子のコピー数を基に補正して、目的遺伝子のコピー数を算出した。
βアクチン、あるいはＧＡＰＤＨ測定用のプライマーとプローブは、ＴａｑＭａｎ β−ａｃｔｉｎ Ｃｏｎｔｒｏｌ Ｒｅａｇｅｎｔｓ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）に添付のもの（６で用いたのと同じもの）を用いて行った。ＧＡＰＤＨにより補正した各遺伝子の発現量（ｃｏｐｙ／５ｎｇ全ＲＮＡ）を図６に示す。
定量的ＰＣＲの結果、カテプシンＣとそのオータナティブスプライシングフォームの発現レベルは、いずれもＩＬ−４あるいはＩＬ−１３刺激によって、３つの異なる気道上皮細胞において２倍以上に上昇していた。気道上皮細胞では、ＩＬ−４やＩＬ−１３に応答して、これらの指標遺伝子の発現レベルが上昇することが予測された。しかし、両者の発現レベルは大きく相違していた。すなわち、オータナティブスプライシングフォームの発現レベルは、カテプシンＣの約２０倍に達することが明らかとなった。
アレルギー反応との密接な関連が知られているＩＬ−４やＩＬ−１３の刺激によって、本発明の指標遺伝子は、異なるロットの気管支上皮細胞で共通の挙動を示す。したがって、本発明の指標遺伝子は、アレルギー反応の進行を制御する重要な遺伝子であると考えられる。またカテプシンＣと比べてはるかに高い発現レベルを示すオータナティブスプライシングフォームは、アレルギーに関連する遺伝子の中でも、特に高感度な測定を行うことができる指標として有用である。
［実施例４］カテプシンＣオータナティブスプライシングフォームのマウスカウンターパートのクローニング
まず、既知のマウスカテプシンＣ配列（ｕ８９２６９）の第２エキソン（ＪＢＣ，ｖｏｌ．２７２，Ｎｏ．１６，１０６９５−１０７０３，１９９７より）上に５’側のプライマーを、また３’側にはヒトのオータナティブスプライシングフォームの配列に基づいて縮重プライマー（表３）を作成し、マウスｃＤＮＡライブラリー（肺）を鋳型としたＰＣＲを行った。

得られた断片の配列を決定したところ、ヒトの場合と同位置（エキソン２の終了点）から新規配列に切り替わる配列の存在が確認された。このことからヒトと類似のオータナティブスプライシングフォームがマウスにも存在している可能性が高いと考えられたため、前述のＰＣＲで得たオータナティブスプライシングフォームの一部と思われる配列上にＲＡＣＥ用のプライマー（表３）を設計し、Ｍａｒａｔｈｏｎ ｃＤＮＡ ａｍｐｌｉｆｉｃａｔｉｏｎ Ｋｉｔ（ＣＬＯＮＴＥＣＨ）を用いてマウスｃＤＮＡライブラリー（ｌｕｎｇ）をｔｅｍｐｌａｔｅとした５’ＲＡＣＥおよび３’ＲＡＣＥを行った。
この結果、開始コドンから第２エキソンまではマウスで報告されているカテプシンＣと同一だが、そこから未報告の３６２ｂｐの新規配列を付加して終結するｍＲＮＡの存在が確認された。マウスカウンターパートの塩基配列を配列番号：２６に（図７）、該塩基配列によってコードされるタンパク質のアミノ酸配列を配列番号：２７に示す。このマウスカウンターパートはヒト遺伝子の塩基配列と高い相同性を持ち（図８）、ヒトと同様に３１アミノ酸の新規ペプチド（配列番号：２８／ＤＶＴＤＦＩＳＱＬＦＭＱＬＧＴＶＧＭＹＤＬＰＨＬＲＮＫＬＶＩＫ／ヒトと異なるアミノ酸を下線で示す）を付加する。クローニングした領域中（９３ｂｐ＋ＴＡＧ）には、３つの塩基置換が見られ、一つは同義置換、残りはＨｉｓ（ヒト）→Ｇｌｎ、Ｉｌｅ（ヒト）→Ｍｅｔの非同義置換であった。また３’ＵＴＲは、ヒトの３２４ｂｐに対してマウスでは２４６ｂｐとなっていた。
産業上の利用の可能性
本発明により、ＩＬ−４あるいはＩＬ−１３で刺激した気道上皮細胞において発現が増加する遺伝子が見出された。ＩＬ−４あるいはＩＬ−１３で刺激した気道上皮細胞において発現が高まる遺伝子は、気管支喘息におけるアレルギー症状の本質的な原因となっている可能性が高い。したがって本発明によって提供された指標遺伝子は、気管支喘息がアレルギー症状によってもたらされたものかどうかを確実に知ることができる有用な指標となる。アレルギーによってもたらされた気管支喘息を確実に診断できることにより、的確な治療方法を早期に選択することができる。
ＩＬ−４あるいはＩＬ−１３は、アレルギー反応を増強する重要な因子である。したがって、これらの因子の刺激に伴って発現が増加する遺伝子は、アレルギー症状の病態形成において重要な役割を果たしていると考えられる。しかも本発明によって提供された指標遺伝子は、いずれも複数の気道上皮細胞で、明確な発現の増強が見られた。ＩＬ−４あるいはＩＬ−１３の刺激に伴う遺伝子の発現レベルの変動に着目した研究は初めてではない。しかし、本発明によって提供される遺伝子は、いずれも、ここで述べたような厳しいクライテリアによって見出されたアレルギー関連遺伝子である。したがって、本発明の指標遺伝子は、類似のアプローチによって得られた公知のアレルギー関連遺伝子に比べて、アレルギー反応における重要な役割を果たしている遺伝子であると考えられる。なぜなら、本発明の指標遺伝子は、異なる細胞において、常にＩＬ−４あるいはＩＬ−１３の刺激に鋭敏に応答している。このことは、アレルギー反応に本発明の指標遺伝子が欠かせない存在であることを裏付けている。
このように、本発明の指標遺伝子は、いずれもその発現亢進が病態と結びついていることから、それを抑えることがアレルギー性疾患の治療戦略のターゲットとなるとともに、そのような新しい治療法におけるモニタリングのための新しい臨床診断指標としての有用性が期待できる。特に本発明の指標遺伝子は、健常者とアレルギー性疾患の患者との間で発現レベルの違いが大きい。たとえば本発明者らは、ＩＬ−４やＩＬ−１３で処理した気道上皮細胞において、カテプシンＣの発現レベルが有意に高いことを確認している。しかし本発明の指標遺伝子の発現レベルは、カテプシンＣの発現レベルに比較して約２０倍高い。したがって、本発明の指標遺伝子は、容易に高感度な測定が可能である。このように、本発明の指標遺伝子は、診断指標としての有用性が高い。
本発明によって提供された指標遺伝子は、アレルゲンの種類に関わらず、簡便にその発現レベルを知ることができる。したがって、アレルギー反応の病態を総合的に把握することができる。
また本発明によるアレルギーの検査方法は、生体試料を試料としてその発現レベルを解析することができるので、患者に対する侵襲性が低い。しかも遺伝子発現解析に関しては、微量サンプルによる高感度な測定が可能である。遺伝子解析技術は、年々ハイスループット化、低価格化が進行している。したがって本発明によるアレルギーの検査方法は、近い将来、ベッドサイドにおける重要な診断方法となることが期待される。この意味でこれらの病態関連遺伝子の診断的価値は高い。
【配列表】

【図面の簡単な説明】
図１は、ＩＬ−４およびＩＬ−１３で刺激した培養気管支上皮細胞における指標遺伝子の処理後から０、６、１２、２４、および４８時間後の発現レベルの経時的な変化を示したグラフである。縦軸は遺伝子の発現量（ｃｏｐｙ／５ｎｇ全ＲＮＡ）を、横軸は処理したサイトカインの種類と処理ごの経過時間を示す。Ｂ２−２４−ｃ〜Ｂ２−２４−ＩＬ９で示したカラムは、サイトカインとして、ＴＮＦα、ＩＬ−１β、ＩＬ−５、ＩＬ−６、およびＩＬ−９を用いた場合の、処理２４時間後の測定結果である。カラムは左から順に、生データ、β−アクチン補正値、およびＧＡＰＤＨ補正値を示している。
図２は、ＩＬ−４およびＩＬ−１３で刺激した培養気管支上皮細胞における指標遺伝子の発現レベルを細胞ロットごとに測定した結果を示すグラフ。縦軸は遺伝子の発現量（ｃｏｐｙ／５ｎｇ全ＲＮＡ）を、横軸は細胞のロットと処理したサイトカインの種類を示す。カラムは左から順に、生データ、β−アクチン補正値、およびＧＡＰＤＨ補正値を示している。
図３は、本発明のオータナティブスプライシングフォームのエキソン、カテプシンＣのエキソン、並びにＤＮＡチップ上のＥＳＴであるａａ０１１３０５のゲノムにおける位置関係を模式的に示した図。
図４は、本発明のオータナティブスプライシングフォームのイントロン１およびイントロン２の、エキソンとイントロンの間の塩基配列と、ＧＴ−ＡＧルールの関係を示ず図。
図５は、ｒｉｂｏｐｒｏｂｅによるノーザンハイブリダイゼーションの結果を示す写真。
図６は、カテプシンＣ、およびそのオータナティブスプライシングフォームの、培養細胞における発現レベル（ＧＡＰＤＨ補正値）の測定結果を示すグラフ。
図７は、カテプシンＣオータナティブスプライシングフォームのマウスカウンターパート全長配列を表す図である。開始コドン、終始コドン、ｐｏｌｙＡ付加シグナルを下線で表した。＊はエキソン２との境界を示す。
図８は、ヒトとマウスのカテプシンＣオータナティブスプライシングフォームの塩基配列を整列化した図である。ギャップは「−」で表した。▼は既知のスプライシング部位を表し、▽は新規なスプライシング部位を表し、下線はヒトのＡＡ０１１３０５のＥＳＴ配列を表す。
図９は、ヒトとマウスのアミノ酸のアライメントを表す図である。Ｂｏｘで囲った領域は、ホモロジーのある領域を表す。 Technical field
The present invention relates to a method for testing an allergic disease.
Background art
Bronchial asthma is considered to be a multifactorial disease. Thus, bronchial asthma results from the interaction of the expression of many different genes, and the expression of these individual genes is affected by multiple environmental factors. For this reason, it has been very difficult to elucidate the specific genes that cause bronchial asthma.
However, bronchial asthma is currently positioned as a chronic inflammatory disease in the airways. It has been pointed out that allergic reactions in airway mucosa and bronchial smooth muscle are closely involved in the pathogenesis of bronchial asthma. Therefore, in diagnosing bronchial asthma, it is important to understand the state of allergic reactions in these tissues. In the treatment of bronchial asthma, control of an allergic reaction becomes an issue.
On the other hand, allergic diseases are considered to involve the expression of genes having mutations or deficiencies, or overexpression or reduction of the expression level of specific genes. To understand the role of gene expression in disease, it is necessary to understand how genes are involved in the pathogenesis and how external stimuli such as drugs alter gene expression.
By the way, many patients with bronchial asthma have an atopic predisposition accompanied by enhanced production of IgE antibodies. Although there are a variety of causes for bronchial asthma, there is no doubt that atopic predisposition causes hypersensitivity in many patients. As a mechanism of airway obstruction due to asthma attack, contraction of bronchial smooth muscle, edema of airway mucosa, and enhancement of airway endocrine secretion are expected. In these changes in the airways, a type I allergic reaction in the airways due to exposure to etiological allergens plays an important role.
Recently, it has been suggested that IL-4 and IL-13 have an important role in the development of bronchial asthma. Therefore, for example, genes whose expression levels are changed by IL-4 or IL-13 in airway epithelial cells or bronchial smooth muscle are considered to be related to bronchial asthma. However, there is no report on the isolation of a gene whose expression level is specifically changed by IL-4 or IL-13 based on such a concept.
Now, in the diagnosis of allergic diseases, interviews, family histories, and confirmation of the patient's history are important factors in general. In order to diagnose allergy based on more objective information, a test method using blood as a sample and a method of observing a patient's immunological response to an allergen have been implemented. Examples of the former include an allergen-specific IgE measurement, a leukocyte histamine release test, and a lymphocyte blastogenesis test. The presence of allergen-specific IgE is evidence of an allergic reaction to the allergen. However, in some patients, it is not always possible to detect allergen-specific IgE. In addition, due to its measurement principle, tests must be performed for all allergens required for diagnosis. The leukocyte histamine release test and lymphocyte blastogenesis test are methods for observing the response of the immune system to allergens in vitro. These methods are complicated in operation.
On the other hand, a method of using an immune response observed when a patient is actually contacted with an allergen to diagnose allergy (the latter) is also known. Such tests include brick tests, scratch tests, patch tests, intradermal reactions, or provocation tests. While these test methods can directly diagnose a patient's allergic reaction, they are tests involving invasiveness that actually exposes a subject to an allergen.
In addition, test methods for proving the involvement of allergic reactions regardless of allergens have been attempted. For example, when the serum IgE level is high, it can be estimated that the patient has an allergic reaction. The serum IgE value is information corresponding to the total amount of allergen-specific IgE. Although it is easy to determine the total amount of IgE regardless of the type of allergen, IgE may be low in patients with diseases such as non-atopic bronchitis.
Therefore, it would be useful to provide a marker for an allergic disease, which has a low risk to the patient and which can easily obtain information necessary for diagnosis. Since such a marker is considered to be deeply involved in the development of allergic diseases, it may be an important target not only in diagnosis but also in control of allergic symptoms.
Disclosure of the invention
An object of the present invention is to provide an index that enables a test for an allergic disease. Another object of the present invention is to provide a method for testing an allergic disease and a method for screening a candidate compound for a therapeutic drug for an allergic disease based on the index.
Several reports have suggested that IL-4 and IL-13 are deeply involved in allergic reactions. For example, IL-4 (Yssel, H and Groux, H: Int. Arch. Allergy Immunol., 121; 10-18, 2000) and STAT6 (Akimoto, T. et al .: J. Exp. Med., 187, 1537-1542, 1998), the airway hyperresponsiveness disappears in mice knocked out. In model mice, IL-13 is involved in the formation of asthma-like pathologies irrespective of IgE production or Th2 type (Wills-Karp, M. et al .: Science, 282, 2258-2261, 1998; Grunig, G. et al .: Science, 282, 2261-2263, 1998; Zhu, Z. et al .: J. Clin. Invest., 103, 779-788, 1999).
In addition, IL-4 receptor and IL-13 receptor are highly expressed in human airway epithelial cells and bronchial smooth muscle (Heinzmann, A. et al .: Hum. Mol. Genet., 9: 549-559, 2000). This suggests that these tissues are target cells for IL-4 and IL-13. On the other hand, it has been shown that SNPs present in IL-4 receptor α and IL-13 are one of the genetic factors of allergic diseases (Mitsuyasu, H., et al .: Nature Genet., 19, Mitsuyasu, H., et al .: J. Immunol., 162: 1227-1231, 1999; Kruse, S., et al .: Immunol., 96, 365-371, 1999; Heinzmann, A. et al .: Hum.Mol.Genet., 9: 549-559, 2000). Furthermore, it has been shown that inhibiting the action of IL-4 or IL-13 by soluble IL-4 receptor α is effective as a treatment for bronchial asthma (Borish, LC et al .: Am. J. Respir. Crit. Care Med., 160: 912-922, 1999).
As described above, it has been suggested that IL-4 and IL-13 have a deep relationship with allergic reactions, particularly respiratory symptoms. In other words, genes constituting a signal transduction pathway by IL-4 and IL-13 can be said to be genes closely related to allergic reactions. Therefore, by isolating these genes and elucidating their relevance to allergic reactions, new targets for the treatment of allergic diseases can be found.
Based on such a concept, the present inventors will search for a gene showing a change in the expression level when human bronchial epithelial cells are treated with IL-4 and IL-13. We thought that it would be possible to isolate a gene that would According to a similar approach, there have been reports of attempts to isolate a gene whose expression level changes by treatment with IL-4 or IL-13 (Wang et al., Immunology 2000, Seattle, May 12-16, 2000). However, in the known search method, since the number of lots of cells used for analysis is small and the range of change in the expression level is not clear, specificity for IL-4 or IL-13 stimulation cannot be expected.
In order to isolate a gene that responds more specifically to IL-4 or IL-13 stimulation, the present inventors increased the number of lots of cells to be analyzed and further changed the expression level. Was selected twice or more times. Next, it was confirmed that the expression level of the gene thus selected significantly increased in airway epithelial cells stimulated with IL-4 or IL-13. Based on the above findings, the present inventors have clarified that the alternative splicing form of cathepsin C is closely related to allergic diseases.
Based on the above findings, the present inventors have found that it is possible to test for allergic diseases by using the alternative splicing form of cathepsin C and the protein encoded by the nucleotide sequence as an index. Completed the invention. In addition, the present inventors have found that a therapeutic agent for an allergic disease can be screened by using the expression levels of these genes or the activities of the proteins encoded by these genes as indicators, and completed the present invention.
That is, the present invention relates to the following inspection methods and screening methods.
[1] A method for testing an allergic disease, comprising the following steps:
a) a step of measuring the expression level of a gene containing the nucleotide sequence of SEQ ID NO: 1 in a biological sample of a subject
b) comparing with the expression level of the gene in a biological sample of a healthy subject
[2] The test method according to [1], wherein the allergic disease is bronchial asthma.
[3] The test method according to [1], wherein the expression level of the gene is measured by cDNA PCR.
[4] The test method according to [1], wherein the expression level of the gene is measured by detecting a protein encoded by the gene.
[5] a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 23, or a polynucleotide comprising a nucleotide sequence complementary to the complementary strand thereof and comprising the nucleotide sequence of SEQ ID NO: 1, or a complementary strand thereof; A reagent for testing an allergic disease, comprising an oligonucleotide having a length of at least 15 bases selected from a complementary base sequence.
[6] recognizes a peptide consisting of at least 7 consecutive amino acid residues selected from the amino acid sequence described in SEQ ID NO: 2, and containing an amino acid sequence selected from the amino acid sequence at positions 107 to 137 from the N-terminus An allergic disease test reagent consisting of an antibody.
[7] A method for screening a therapeutic drug for an allergic disease, comprising the following steps.
(1) a step of contacting a candidate compound with a cell that expresses a gene containing the nucleotide sequence of SEQ ID NO: 1 and / or any gene functionally equivalent to these genes
(2) measuring the expression level of the gene;
(3) selecting a compound that reduces the expression level of the gene as compared to a control that is not contacted with the candidate compound
[8] The method of [7], wherein the cells are established airway epithelial cells.
[9] A method for screening a therapeutic drug for an allergic disease, comprising the following steps.
(1) administering a candidate compound to a test animal,
(2) measuring the expression intensity of a gene containing the nucleotide sequence of SEQ ID NO: 1 and / or a gene functionally equivalent to the gene in a biological sample of a test animal; and
(3) selecting a compound that reduces the expression level of the gene as compared to a control to which no candidate compound is administered
[10] A method for screening a therapeutic drug for an allergic disease, comprising the following steps.
(1) contacting a candidate substance with a protein encoded by a gene comprising the nucleotide sequence of SEQ ID NO: 1 and / or a protein functionally equivalent to the protein;
(2) measuring the activity of the protein, and
(3) selecting a compound that reduces the activity of the protein as compared to a control that is not contacted with a candidate compound
[11] A therapeutic agent for an allergic disease, comprising as an active ingredient a compound obtainable by the screening method according to any one of [7], [9], and [10].
[12] A therapeutic agent for an allergic disease, comprising a gene containing the nucleotide sequence of SEQ ID NO: 23 or a part thereof as an antisense DNA as a main component.
[13] a polynucleotide comprising a nucleotide sequence of a gene comprising the nucleotide sequence of SEQ ID NO: 1, or an oligonucleotide having a nucleotide sequence complementary to a complementary strand thereof and having a length of at least 15 nucleotides; A kit for screening a therapeutic drug candidate compound for an allergic disease, comprising a cell that expresses a gene containing the nucleotide sequence according to 1.
[14] an antibody that recognizes a peptide containing the amino acid sequence of the protein encoded by the gene containing the nucleotide sequence of SEQ ID NO: 1, and a cell that expresses the gene containing the nucleotide sequence of SEQ ID NO: 1 And a kit for screening candidate compounds for the treatment of allergic diseases.
[15] Allergic disease of a transgenic non-human vertebrate in which the expression intensity of a gene comprising the nucleotide sequence of SEQ ID NO: 1 and / or a gene functionally equivalent to this gene in airway epithelial cells is increased. Use as a model animal.
The present invention also relates to a method for treating an allergic disease, comprising a step of administering a compound obtainable by the screening method according to any one of [7], [9], and [10]. Alternatively, the present invention relates to the use of a compound obtainable by the screening method according to any one of [7], [9], and [10] in the manufacture of a pharmaceutical composition for treating an allergic disease. Furthermore, the present invention relates to a method for treating an allergic disease, which comprises a step of administering the component (a) or (b) described below. Alternatively, the present invention relates to the use of component (a) or (b) described below in the manufacture of a pharmaceutical composition for treating an allergic disease.
(A) a gene comprising the nucleotide sequence of SEQ ID NO: 23, or a partial antisense DNA thereof
(B) an antibody that recognizes a peptide containing an amino acid sequence of a protein encoded by a gene containing the nucleotide sequence of SEQ ID NO: 1
In the present invention, an allergic disease is a general term for diseases in which an allergic reaction is involved. More specifically, it can be defined as identifying an allergen, demonstrating a deep link between allergen exposure and the development of a lesion, and demonstrating an immunological mechanism for the lesion. Here, the immunological mechanism means that white blood cells show an immune response by allergen stimulation. Examples of the allergen include a mite antigen and a pollen antigen.
Representative allergic diseases can include bronchial asthma, allergic rhinitis, hay fever, or insect allergy. An allergic diathesis is a genetic factor transmitted from a parent having an allergic disease to a child. An allergic disease that develops familially is also called atopic disease, and a genetically transmitted factor that causes it is atopic predisposition. Asthma is a general term given to atopic diseases, particularly those associated with respiratory symptoms.
The method for testing an allergic disease of the present invention includes the step of measuring the expression level of an alternative splicing form of cathepsin C in a biological sample of a subject and comparing the measured level with the measurement value of a healthy subject. As a result of comparison between the two, when the expression is higher than that in a healthy subject, the subject is determined to have an allergic disease. For comparison of the expression level, a standard value is usually set based on, for example, the expression level of the indicator gene in a healthy person. Based on this standard value, for example, ± 2S. D. Is regarded as an allowable range. Techniques for setting a standard value and an allowable range based on a measured value of an indicator gene are known. If the expression level of the indicator gene in the subject is higher than the allowable range, the subject is determined to have an allergic disease. If the expression level is within the allowable range, the possibility of having an allergic disease is expected to be low.
In the present invention, an alternative splicing form of cathepsin C that can be used as an indicator of an allergic disease may be simply referred to as an indicator gene.
It is already known that a gene having a nucleotide sequence corresponding to the alternative splicing form of cathepsin C is expressed in human tissues. For example, a gene having a nucleotide sequence that matches the alternative splicing form of cathepsin C has been selected as a cancer-related gene (WO99 / 38972). In addition, it has been shown that a cathepsin C homolog is useful as a diagnostic or therapeutic agent for monocyte and macrophage diseases. Alternatively, use of type II collagen as a therapeutic or diagnostic agent has been reported. However, it is not known that the alternative splicing form of cathepsin C enhances expression in airway epithelial cells in response to IL-4 or IL-13.
Furthermore, according to the findings of the present inventors, the expression level of cathepsin C (GenBank Acc. No. X87212) is significantly increased in airway epithelial cells treated with IL-4 or IL-13. Although the presence of cathepsin C is already known, there is no report on its association with allergic diseases. On the other hand, the alternative splicing form of cathepsin C selected as an indicator gene in the present invention shows an expression level about 20 times higher than that of cathepsin C. Therefore, the indicator gene of the present invention can be expected to have higher sensitivity than, for example, cathepsin C. Alternatively, various measurement techniques can be applied since the expression level itself of the gene serving as an index is high.
In the method for testing an allergy according to the present invention, the indicator gene for which the expression level or activity is to be measured is an alternative splicing form of cathepsin C. In the method for testing an allergy according to the present invention, a gene other than the indicator gene of the present invention, which can be used as an index for an allergic disease, can also be combined. For example, the present applicant has reported the following genes that can be used in a method for testing allergy.
Hay fever-related gene 373 (WO 00/65046) hay fever-related gene 627 (WO 00/65051) hay fever-related gene 419 (WO 00/65045)
Hay fever-related gene 441 (WO 00/73435) Hay fever-related gene 513 (WO 00/65049) Hay fever-related gene 465 (WO 00/73439)
Hay fever-related gene 581 (WO 00/65048) Hay fever-related gene 787 (WO 00/73440) Hay fever-related gene 795 (WO 00/65050)
Any of these genes can be combined with the test method of the present invention. Alternatively, other genes that can be used as indicators of allergic diseases can also be used. By using a plurality of genes as indices, test accuracy can be improved. Since bronchial asthma patients are heterogeneous (heterogeneous) populations, more reliable diagnosis can be made by using a plurality of genes as indices.
In the present invention, the expression level of the indicator gene includes transcription of the gene into mRNA and translation into a protein. Therefore, the method for testing for an allergic disease according to the present invention is performed based on a comparison of the expression intensity of mRNA corresponding to the gene or the expression level of a protein encoded by the gene.
The measurement of the expression level of the indicator gene in the test for an allergic disease according to the present invention can be performed according to a known gene analysis method. Specifically, for example, a hybridization technique using a nucleic acid that hybridizes to the gene as a probe or a gene amplification technique using a DNA that hybridizes to the gene of the present invention as a primer can be used.
The probe or primer used for the test of the present invention can be set based on the base sequence of the indicator gene. The nucleotide sequence of the indicator gene revealed by the present inventors is shown in SEQ ID NO: 1. As mentioned earlier, the existence of alternative splicing forms of cathepsin C has been reported. However, the known base sequence of the alternative splicing form of cathepsin C disclosed in WO 99/38972 is incomplete, including some undetermined bases. On the other hand, the present inventors have analyzed the nucleotide sequence of this gene in a more complete manner.
In general, higher animal genes are frequently associated with polymorphism. Also, there are many molecules that generate isoforms composed of mutually different amino acid sequences in the process of splicing. Even if the gene has a mutation in the nucleotide sequence due to polymorphism or alternative splicing, any gene that has the same activity as the indicator gene and is involved in allergy is included in the indicator gene of the present invention.
The primer or the probe includes a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 23, or a polynucleotide comprising a nucleotide sequence complementary to the complementary strand thereof and comprising the nucleotide sequence of SEQ ID NO: 1, or a polynucleotide thereof. Oligonucleotides having a length of at least 15 bases having a base sequence complementary to the complementary strand can be used. Here, the “complementary strand” refers to one strand of a double-stranded DNA consisting of A: T (U for RNA) and G: C base pairs with respect to the other strand. The term "complementary" is not limited to a case where the sequence is completely complementary to at least 15 contiguous nucleotide regions, but is at least 70%, preferably at least 80%, more preferably 90%, and still more preferably 95%. It is only necessary to have the above homology on the base sequence. The homology of the nucleotide sequences can be determined by an algorithm such as BLAST.
For specifically recognizing the indicator gene of the present invention, a polynucleotide that recognizes a base sequence specifically present in the indicator gene is important. SEQ ID NO: 23 shows a nucleotide sequence unique to the indicator gene. In the present invention, in the indicator gene represented by SEQ ID NO: 1, the region consisting of the base sequence represented by SEQ ID NO: 23 and its complementary strand may be referred to as “specific region”. In the present invention, the following polynucleotides are included in the polynucleotide recognizing the region consisting of the nucleotide sequence shown in SEQ ID NO: 23, which constitutes the indicator gene.
First, a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 23, or a complementary strand thereof, that is, a polynucleotide containing at least 15 nucleotides complementary to the specific region can be used.
Next, a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 23, or a polynucleotide comprising a nucleotide sequence complementary to the complementary strand thereof and comprising the nucleotide sequence of SEQ ID NO: 1, or a complementary strand thereof A polynucleotide having a base sequence selected from a complementary base sequence is exemplified. Such a polynucleotide can hybridize to a region consisting of a base sequence unique to the indicator gene of the present invention and a boundary region consisting of a region consisting of a base sequence shared with cathepsin C. On the other hand, in the polynucleotide encoding cathepsin C, such a polynucleotide can be used for hybridization only in a region shared with the alternative splicing form. Therefore, at least under stringent conditions, after all, it can hybridize only to the indicator gene, even if it is a polynucleotide containing a nucleotide sequence complementary to the shared region, it is used for detection of the indicator gene It is possible. In particular, when used as a primer, a primer specific to an indicator gene can be obtained by positioning at least one base at the 3 'end of the primer in a specific region.
When the probe or the primer contains a polynucleotide containing a base sequence shared with cathepsin C or a base sequence complementary to a complementary strand thereof, it is desirable to make the number of constituent bases less than 15 bases. . More specifically, it is set to 14 or less, usually 10 or less, preferably 8 or less, more preferably 7 or less, and still more preferably 5 or less.
Such a polynucleotide can be used as a probe for detecting the indicator gene and as a primer for amplifying the indicator gene. When used as a primer, it usually has a chain length of 15 bp to 100 bp, preferably 15 bp to 35 bp. When used as a probe, a DNA having at least a part or all of the sequence of the indicator gene (or its complementary strand) and a chain length of at least 15 bp is used. When used as a primer, the region on the 3 'side must be complementary, but a restriction enzyme recognition sequence, a tag, or the like can be added to the 5' side.
The “polynucleotide” in the present invention can be DNA or RNA. These polynucleotides may be synthetic or natural. The probe DNA used for hybridization is usually labeled. As the labeling method, for example, the following method can be shown. The term oligonucleotide means a polynucleotide having a relatively low degree of polymerization among polynucleotides. Oligonucleotides are included in polynucleotides.
Labeling by nick translation using DNA polymerase I
・ End labeling using polynucleotide kinase
-Fill-in end labeling with Klenow fragment (Berger SL, Kimmel AR. (1987) Guide to Molecular Cloning Technologies, Methods in Enzymology, Academic Press; Hames BD. J, Fritsch EF, Maniatis T. (1989) Molecular Cloning: a Laboratory Manual, 2nd Edn. Cold Spring Harbor Laboratory Press.
Labeling by transcription using RNA polymerase (Melton DA, Krieg, PA, Rebagkiati MR, Maniatis T, Zinn K, Green MR. (1984) Nucleic Acid Res., 12, 7035-7056)
A method of incorporating a modified nucleotide without using a radioisotope into DNA (Kricka LJ. (1992) Nonisotopic DNA Probing Technologies. Academic Press)
Testing for allergic diseases using hybridization techniques can be performed using, for example, Northern hybridization, dot blotting, a method using a DNA microarray, and the like. Further, a gene amplification technique such as an RT-PCR method can be used. In the RT-PCR method, more quantitative analysis can be performed on the expression of the gene of the present invention by using the PCR amplification monitoring method in the gene amplification process.
In the PCR gene amplification monitoring method, a probe whose both ends are labeled with different fluorescent dyes that cancel each other's fluorescence is hybridized to a detection target (reverse transcript of DNA or RNA). When the PCR proceeds and the probe is decomposed by the 5'-3 'exonuclease activity of Taq polymerase, the two fluorescent dyes are separated and the fluorescence is detected. This fluorescence is detected in real time. The number of copies of the detection target in the target sample is determined based on the number of linear cycles of the PCR amplification by simultaneously measuring a standard sample whose copy number is apparent for the detection target (Holland, PM et al., Natl. Acad. Sci. USA 88: 7276-7280; Livak, KJ et al., 1995, PCR Methods and Applications 4 (6): 357-362; Heid, CA et al. , Genome Research 6: 986-994; Gibson, EMU et al., 1996, Genome Research 6: 995-1001). In the PCR amplification monitoring method, for example, ABI PRISM7700 (Applied Biosystems) can be used.
Further, the method for testing an allergic disease of the present invention can also be performed by detecting a protein encoded by the indicator gene. Hereinafter, in this specification, a protein encoded by the indicator gene is referred to as an indicator protein. As such a test method, for example, a Western blotting method, an immunoprecipitation method, an ELISA method, or the like using an antibody that binds to these indicator proteins can be used.
Antibodies that bind to the indicator protein used for this detection can be obtained using techniques well known to those skilled in the art. In particular, an antibody recognizing a peptide consisting of at least 7 consecutive amino acid residues selected from the amino acid sequence of SEQ ID NO: 2 and containing an amino acid sequence selected from the amino acid sequence at positions 107 to 137 from the N-terminus Is useful as an antibody that enables specific detection of an indicator protein. In the amino acid sequence shown in SEQ ID NO: 2, the region at positions 107 to 137 from the N-terminus corresponds to the amino acid sequence encoded by the nucleotide sequence containing the specific region. That is, it can be said that the amino acid sequence is specific to the indicator protein.
The antibody used in the present invention can be a polyclonal antibody or a monoclonal antibody (Milstein C, et al., 1983, Nature 305 (5934): 537-40). For example, a polyclonal antibody against an indicator protein is obtained by extracting blood of a mammal sensitized with an antigen and separating serum from the blood by a known method. Serum containing the polyclonal antibody can be used as the polyclonal antibody. Alternatively, if necessary, a fraction containing a polyclonal antibody can be further isolated from the serum. In addition, to obtain a monoclonal antibody, immune cells are removed from a mammal sensitized with the above antigen, and are fused with myeloma cells or the like. The hybridoma thus obtained is cloned, and the antibody is recovered from the culture to obtain a monoclonal antibody.
These antibodies may be appropriately labeled and used for detection of the indicator protein. Further, without labeling the antibody, a substance that specifically binds to the antibody, for example, protein A or protein G can be labeled and detected indirectly. As a specific detection method, for example, an ELISA method can be mentioned.
A protein or a partial peptide thereof used as an antigen can be prepared, for example, by incorporating the gene or a part thereof into an expression vector, introducing this into an appropriate host cell, preparing a transformant, culturing the transformant, and performing recombination. It can be obtained by expressing a protein and purifying the expressed recombinant protein from a culture or a culture supernatant. Alternatively, an oligopeptide consisting of an amino acid sequence encoded by these genes or a partial amino acid sequence of an amino acid sequence encoded by a full-length cDNA can be chemically synthesized and used as an immunogen.
Furthermore, in the present invention, not only the expression level of the indicator gene but also the activity of the indicator protein in the biological sample can be used as an indicator for testing for allergic diseases. The activity of the indicator protein refers to the biological activity of each protein. The activity of the indicator protein can be detected based on a known method.
In the test method of the present invention, usually, a biological sample of a subject is used as a sample. As a biological sample, airway epithelial cells and the like can be used. Methods for obtaining airway epithelial cells are known. That is, under a bronchoscope, it is possible to physically exfoliate and collect using a rod. The obtained sample can be prepared by freezing, fixing in formalin, or culturing in a medium. In addition, blood, sputum, nasal mucosa secretion, bronchoalveolar lavage fluid, lung scraping cells, and the like can also be used as the biological sample in the present invention. Methods for collecting these biological samples are also known.
When the biological sample is cells such as airway epithelial cells, a sample for immunological measurement of the protein can be prepared by preparing a lysate. Alternatively, if mRNA is extracted from this lysate, it can be used as a sample for measuring mRNA corresponding to the gene. It is convenient to use a commercially available kit for extracting a lysate or mRNA from a biological sample. If the indicator protein is secreted into the blood, the expression level of the gene encoding it can be compared by measuring the amount of the target protein contained in a body fluid sample such as blood or serum of the subject. It is possible. Liquid biological samples such as blood, nasal mucosal secretions, and bronchoalveolar lavage fluid can be diluted with a buffer or the like, if necessary, to prepare a sample for protein or gene measurement.
When measuring mRNA, the measured value of the expression level of the indicator gene in airway epithelial cells can be corrected by a known method. With the correction, changes in the expression level of the gene in the cells can be compared. Correction of the measured value is based on the measured value of the expression level of a gene (housekeeping gene) which is expressed in airway epithelial cells and whose expression level does not fluctuate greatly irrespective of the state of the cell. Is performed by correcting the measured value of the expression level. Examples of the gene whose expression level does not fluctuate greatly include β-actin, GAPDH and the like.
The indicator gene of the present invention showed an increased expression level in a plurality of airway epithelial cell lines stimulated with IL-4 or IL-13. Therefore, allergic diseases such as bronchial asthma can be tested using the expression level of the indicator gene as an indicator.
The test for allergic disease in the present invention includes, for example, the following tests. Even if a patient cannot show a symptom of bronchial asthma and cannot be determined to be an allergic disease by a general test, it can be easily determined to be a patient with an allergic disease by performing the test according to the present invention. More specifically, an increase in the expression of an indicator gene in a patient exhibiting a symptom suspected of having an allergic disease indicates that the cause of the symptom is likely to be an allergic disease. Some bronchial asthma is caused by an allergic reaction, while others are not. Diagnosing which of these causes the symptoms of bronchial asthma is a very important therapeutic step, since the treatment methods are quite different. The test method of the present invention can provide extremely important information in identifying the cause of bronchial asthma.
Alternatively, a test can be performed to determine whether the allergic symptoms are improving. The indicator gene of the present invention showed an increased expression level in airway epithelial cells stimulated with IL-4 or IL-13. Airway epithelium is a tissue that shows significant lesions in bronchial asthma. Therefore, a gene whose expression fluctuates in airway epithelial cells stimulated with IL-4 or IL-13, which is a cytokine that strongly induces an allergic reaction, is useful for determining the therapeutic effect. More specifically, an increase in the expression of an indicator gene in a patient diagnosed with an allergic disease indicates that there is a high possibility that allergic symptoms are progressing.
The present invention also relates to the use of a transgenic non-human animal having an increased expression level of an indicator gene in airway epithelial cells as a model animal for an allergic disease. Allergic disease model animals are useful for elucidating in vivo changes in bronchial asthma. Furthermore, the allergic disease model animal of the present invention is useful for evaluating a therapeutic drug for allergic bronchial asthma.
According to the present invention, it has been revealed that the expression level of the indicator gene in airway epithelial cells is increased by stimulation of IL-4 or IL-13. Therefore, animals in which expression levels of these genes or genes functionally equivalent to these genes are artificially enhanced in airway epithelial cells can be used as model animals for allergic diseases. The increase in the expression level in airway epithelial cells includes an increase in the expression level of the target gene in the whole airway tissue. That is, the expression level of the gene is increased not only in the airway epithelium but also in the entire airway tissue or when the expression level of the gene is increased systemically.
In the present invention, a functionally equivalent gene is a gene that encodes a protein having the same activity as that of the protein encoded by each indicator gene. Representative examples of functionally equivalent genes include a counterpart of the indicator gene in the animal species that the test animal originally has. For example, the present inventors have succeeded in isolating a counterpart in a mouse of the gene of the present invention. This gene has the nucleotide sequence of SEQ ID NO: 26 and encodes the amino acid sequence of SEQ ID NO: 27. The human and mouse nucleotide sequences showed about 73% homology (FIG. 8). In addition, the amino acid sequences of human and mouse showed about 78% homology (FIG. 9). Therefore, a gene consisting of a base sequence having 73% or more identity with the base sequence described in SEQ ID NO: 1 is desirable as a functionally equivalent gene in the present invention. Alternatively, a gene consisting of a base sequence encoding an amino acid sequence having 78% or more homology with the amino acid sequence described in SEQ ID NO: 2 is desirable as a functionally equivalent gene in the present invention.
Genes whose expression is increased by stimulation of IL-4 or IL-13 can be said to be genes on the pathway of signal transduction by these cytokines. In other words, it is considered that the stimulation of IL-4 or IL-13 appears as an allergic symptom through the enhancement of the expression of these genes. In other words, a gene whose expression is increased by stimulation of IL-4 or IL-13 can be said to be a gene that plays an important role in the formation of allergic pathological conditions in the respiratory epithelium. Therefore, a drug that suppresses the expression of this gene or inhibits its activity is expected to not only improve the allergic symptoms but also eliminate the essential cause of allergic pathogenesis in the treatment of allergy.
As described above, genes whose expression increases in airway epithelial cells stimulated with IL-4 or IL-13 have important significance. Therefore, it is of great significance to use transgenic animals obtained by increasing the expression level of this gene as model animals for allergic diseases and to evaluate the role of the gene and drugs targeting the gene. .
Further, the allergic disease model animal according to the present invention can be used not only for screening pharmaceuticals for treating or preventing allergic diseases described later, but also for elucidating the mechanism of allergic diseases and further testing the safety of the screened compounds. Useful.
For example, if the allergic disease model animal according to the present invention develops bronchial asthma or shows a change in a measured value related to any allergic disease, a screening system for searching for a compound having an action to restore it can be constructed.
In the present invention, the expression level increase refers to a state in which the target gene is forcibly expressed by being introduced as a foreign gene, or a state in which transcription and translation of a gene provided in a host are enhanced, and a translation product. Which means that the degradation of the protein is suppressed. The expression level of the gene can be confirmed, for example, by quantitative PCR as described in Examples. Further, the activity of the protein as a translation product can be confirmed by comparing with the normal state.
A typical transgenic animal is an animal into which a target gene has been introduced and forcedly expressed. Other transgenic animals include, for example, animals in which a mutation has been introduced into the coding region of a gene to enhance its activity or have been modified to an amino acid sequence that is hardly degraded. Mutations in the amino acid sequence can indicate substitutions, deletions, insertions, or additions. In addition, the expression itself of the gene of the present invention can be regulated by introducing a mutation into the transcription regulatory region of the gene.
Methods for obtaining a transgenic animal for a specific gene are known. That is, a method in which a gene and an egg are mixed and treated with calcium phosphate, a method in which a gene is directly introduced into a nucleus of a pronuclear stage egg with a micropipette under a phase-contrast microscope (microinjection method, US Pat. A transgenic animal can be obtained by a method using embryonic stem cells (ES cells) or the like. In addition, a method of inserting a gene into a retrovirus vector and infecting an egg, a method of introducing a gene into an egg via sperm, and a sperm vector method have also been developed. The sperm vector method is a genetic recombination method in which a foreign gene is introduced into sperm cells by a method such as attachment or electroporation to a sperm and then fertilized by an egg to introduce the foreign gene (M. Lavitranoet et al., Cell, 57, 717, 1989).
The transgenic animal used as the allergic disease model animal of the present invention can be prepared using any vertebrate other than human. Specifically, transgenic animals in which various genes have been introduced and expression levels of which have been modified in vertebrates such as mice, rats, rabbits, minipigs, goats, sheep, and cattle have been created.
Furthermore, the present invention relates to a method for screening a candidate compound for a therapeutic drug for an allergic disease. In the present invention, the expression levels of the indicator genes are significantly increased in airway epithelial cells stimulated with IL-4 or IL-13. Therefore, a therapeutic agent for an allergic disease can be obtained by selecting a compound that can reduce the expression levels of these genes. In the present invention, the compound that reduces the expression level of a gene is a compound that acts to inhibit any of the steps of gene transcription, translation, and protein activity expression.
The method for screening a candidate compound for treating an allergic disease of the present invention can be performed in vivo or in vitro. This screening can be performed, for example, according to the following steps. The indicator gene in the screening method of the present invention includes, in addition to those listed above as indicator genes, any gene functionally equivalent to those genes.
(1) administering a candidate compound to a test animal;
(2) measuring the expression level of the indicator gene in a biological sample of a test animal;
(3) selecting a compound that reduces the expression level of the indicator gene as compared to a control to which no candidate compound is administered
Allergic disease model animals can be used as test animals in the screening method of the present invention. Allergic disease model animals are known. For example, as a model similar to human asthma, an ovalbumin (hereinafter OVA) antigen-exposed asthma model using Balb / c mice has been reported. The mouse is immunized intraperitoneally twice on Day 0 and Day 10 with OVA and aluminum hydroxide as an adjuvant, and after 10 days, the mouse is inhaled with OVA three times every four days using a nebulizer. Increased airway hyperresponsiveness in mice after inhalation of the final antigen can induce symptoms very similar to asthma. The screening according to the present invention can be performed by administering a candidate compound to such a model animal and tracking the change in the expression level of the indicator gene of the present invention.
Thus, by administering the drug candidate compound to the test animal and monitoring the effect of the compound on the expression of the indicator gene in the biological sample of the test animal, detecting the effect of the drug candidate compound on the expression level of the indicator gene Can be. Fluctuations in the expression level of the indicator gene in the biological sample of the test animal can be monitored by the same method as the above-described test method of the present invention. Further, by selecting a drug candidate compound that reduces the expression level of the indicator gene based on the result of this detection, the drug candidate compound can be screened.
More specifically, the screening according to the present invention can be performed by collecting a biological sample from a test animal and comparing the expression level of the indicator gene with a control to which no candidate compound is administered. As a biological sample, smooth muscle cells, keratinocytes, nasal mucosal epithelial cells, intestinal epithelial cells, lymphocytes, mast cells, eosinophils, basophils, neutrophils, and the like can be used. Methods for collecting and preparing these biological samples are known.
By such a screening, it is possible to select drugs that are involved in various ways in the expression of the indicator gene. Specifically, for example, drug candidate compounds having the following actions can be found.
Suppression of signaling pathways leading to expression of indicator genes,
Decrease in transcription activity of the indicator gene,
Promotion of degradation of transcripts of indicator genes, etc.
In addition, in the in vitro screening, for example, a method of bringing a candidate compound into contact with cells expressing the indicator gene and selecting a compound that reduces the expression level of these genes can be mentioned. This screening can be performed, for example, according to the following steps.
(1) A step of bringing a candidate compound into contact with cells expressing the indicator gene
(2) measuring the expression level of the indicator gene;
(3) selecting a compound that reduces the expression level of the indicator gene as compared to a control that does not contact the candidate compound
In the present invention, cells that express the indicator gene can be obtained by inserting the indicator gene into an appropriate expression vector and introducing the vector into an appropriate host cell. Usable vectors and host cells may be any as long as they can express the gene of the present invention. Examples of host cells in the host-vector system include Escherichia coli, yeast, insect cells, animal cells, and the like, and any available vector can be appropriately selected.
Examples of a method for introducing a vector into a host include a biological method, a physical method, and a chemical method. Examples of biological methods include a method using a viral vector, a method using a specific receptor, a cell fusion method (HVJ (Sendai virus), polyethylene glycol (PEG), an electric cell fusion method, micronucleus fusion). Method (chromosome transfer)). Examples of the physical method include a microinjection method, an electroporation method, and a method using a gene particle gun. Examples of the chemical method include a calcium phosphate precipitation method, a liposome method, a DEAE dextran method, a protoplast method, an erythrocyte ghost method, an erythrocyte membrane ghost method, and a microcapsule method.
As a cell that expresses the indicator gene, a cell line established from human respiratory tissue can be used. For example, human lung cancer cell line A549 and human bronchial epithelial cell line BEAS-2B are suitable for the screening method of the present invention. These cells can be purchased from the ATCC.
In the screening method of the present invention, first, a candidate compound is added to the cell line. Thereafter, the expression level of the indicator gene in the cell line is measured, and a compound that reduces the expression level of the gene is selected.
In the screening method of the present invention, the expression levels of the indicator genes can be compared not only by the expression levels of the proteins encoded by these genes but also by detecting the corresponding mRNA. In order to compare the expression levels using mRNA, the above-described mRNA sample preparation step is performed instead of the protein sample preparation step. Detection of mRNA and protein can be performed by the known methods as described above.
As an in vitro screening method according to the present invention, a screening method based on the activity of an indicator protein can also be used. That is, the present invention provides a method for screening a candidate compound for treating an allergic disease, comprising the following steps, wherein the indicator gene is functionally equivalent to an alternative splicing form of cathepsin C or an alternative splicing form of cathepsin C. A method that is a gene.
(1) contacting a protein encoded by the indicator gene with a candidate compound;
(2) measuring the activity of the protein, and
(3) selecting a compound that reduces the activity of the protein compared to a control
The activity of the alternative splicing form of cathepsin C as the indicator protein in the present invention has already been described. Using this activity as an index, compounds having an activity to promote the activity can be screened. The compound thus obtained suppresses the action of the alternative splicing form of cathepsin C. As a result, allergic diseases can be controlled through the inhibition of the activity of the indicator protein whose expression is induced in airway epithelial cells.
The test candidate compounds used in these screenings include compound preparations synthesized by existing chemical methods such as steroid derivatives, compound preparations synthesized by combinatorial chemistry, extracts of animal and plant tissues, or cultures of microorganisms. And a mixture containing a plurality of compounds, and a sample purified therefrom.
Polynucleotides, antibodies, cell lines, or model animals required for various screening methods according to the present invention can be combined in advance to form a kit. These kits must be packaged with the substrate compound used to detect the label, culture media and containers for culturing cells, positive and negative standard samples, and instructions describing how to use the kit. You can also.
The compound selected by the screening method of the present invention is useful as a therapeutic drug for allergic diseases. Alternatively, antisense DNA, which can suppress the expression of a gene containing the nucleotide sequence of SEQ ID NO: 1, is also useful as a therapeutic drug for allergic diseases. Further, an antibody that recognizes a protein encoded by the nucleotide sequence of SEQ ID NO: 1 is useful as a therapeutic drug for allergic diseases.
The gene containing the nucleotide sequence of SEQ ID NO: 1 is a gene whose expression level is significantly increased in airway epithelial cells stimulated with IL-4 or IL-13. Therefore, by suppressing the expression of these genes or suppressing the functions of the proteins encoded by these genes, a therapeutic effect for allergic diseases can be expected.
The remedy for allergic diseases of the present invention can be produced by mixing the compound selected by the screening method as an active ingredient and mixing with a physiologically acceptable carrier, excipient, or diluent. it can. The therapeutic agent for an allergic disease of the present invention can be administered orally or parenterally for the purpose of improving allergic symptoms.
As oral preparations, dosage forms such as granules, powders, tablets, capsules, solvents, emulsions, and suspensions can be selected. Injections include subcutaneous injections, intramuscular injections, and intraperitoneal injections.
When the compound to be administered consists of a protein, a therapeutic effect can be achieved by introducing a gene encoding the protein into a living body using a gene therapy technique. Techniques for treating a disease by introducing a gene encoding a protein having a therapeutic effect into a living body and expressing the gene in a living body are known.
Alternatively, antisense DNA can be incorporated downstream of an appropriate promoter sequence and administered as an antisense RNA expression vector. When this expression vector is introduced into T cells of an allergic disease patient, the antisense of these genes is expressed, and a therapeutic effect on allergy can be achieved by reducing the expression level of the genes. As a method for introducing an expression vector into a T cell, a method in which the expression vector is performed in vivo or ex vivo is known. .
The dose varies depending on the age, sex, weight and condition of the patient, therapeutic effect, administration method, treatment time, or the type of active ingredient contained in the pharmaceutical composition. It can be administered in the range 0.1 mg to 500 mg, preferably in the range 0.5 mg to 20 mg. However, since the dose varies depending on various conditions, a dose smaller than the above dose may be sufficient, and a dose exceeding the above range may be required in some cases.
Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to these Examples.
All prior art documents cited in this specification are incorporated herein by reference.
BEST MODE FOR CARRYING OUT THE INVENTION
[Example 1] Selection of candidate genes using DNA microarray
1. Culture of normal human bronchial epithelial cells and stimulation with IL-4 or IL-13
Three lots of normal human bronchial epithelial cells sold by Clonetics were purchased (8F1756, 8F1548, 8F1805). 5x10 in one vial⁵The cells were divided into three equal parts (1.67 × 10 6) for unstimulated, IL-4 stimulated, and IL-13 stimulated.⁵/ 75cm²flash) and a SABM medium (Clonetics) while changing the medium for about 8-10 days. At that time, BPE (bovine pituitary extract), Hydrocortisone, hEGF, Epinephrine, Transferrin, Insulin, Retinoic Acid, BSA-FAF, Triodothyronine, and GA-1000 (Gentamicin / Ampho-attached with Genomicin) were added to the medium. did.
The cells were washed with PBS before cytokine stimulation, and then replaced with SABM from which additional factors had been removed. IL-4 (10 ng / ml) and IL-13 (50 ng / ml) were added thereto (both are manufactured by Peprotech) and cultured for 24 hours. Observation of changes over time (0, 6, 12, 24, 48 hours) was performed in the same manner.
2. Other cytokine stimulation of normal human bronchial epithelial cells
The cells of lot 8F1548 were cultured in the same manner as in 1. Instead of IL-4 and IL-13, 50 ng / ml of TNFα, IL-1β, IL-5, IL-6, and IL-9 (all manufactured by Peprotech) were added, and the cells were cultured for 24 hours.
3. Preparation of RNA for GeneChip
The airway epithelial cells treated as described above were dissolved in Isogen (Nippon Gene; Wako Pure Chemical Industries), and RNA was isolated from this solution according to the protocol attached to Isogen. Chloroform was added, and the mixture was centrifuged with stirring to collect an aqueous layer. Next, isopropanol was added, and the mixture was centrifuged with stirring to collect the total RNA in the precipitate.
4. CDNA synthesis for GeneChip
T7- (dT) was prepared from 5 μg of total RNA prepared from cells of lot 8F1756.₂₄(Amersham Pharmacia) as a primer and according to the method of Affymetrix's Expression Analysis Technical Manual, a Superscript II Reverse Transcriptase (a reverse transcript of the cDNA obtained by reverse cloning of a cDNA obtained by reverse technology from Lifescripts) was used. T7- (dT)₂₄The primer has d (T) in the base sequence of the T7 promoter as follows.₂₄Consists of a base sequence to which is added.

Next, DNA Ligase, DNA polymerase I and RNase H were added according to Expression Analysis Technical Manual to synthesize a double-stranded cDNA. After cDNA was extracted with phenol / chloroform, the cDNA was passed through Phase Lock Gels, precipitated with ethanol, and purified.
Furthermore, using the BioArray High Yield RNA Transcription Labeling Kit, biotin-labeled cRNA was synthesized. The cRNA was purified using RNeasy Spin column (QIAGEN) and fragmented by heat treatment.
12.5 μg of the cRNA was added to the Hybridization Cocktail according to the Expression Analysis Technical Manual. This was put into an array Hu35K GeneChip (Affymetrix) and hybridized at 45 ° C. for 16 hours.
After washing the array, Streptavidin Phycoerythrin was added for staining. After washing, an antibody mixture of normal goat IgG and biotinylated goat IgG was added to the array. Further, for the purpose of enhancing the fluorescence intensity, Streptavidin Phycoerythrin was added again and stained. After washing, it was set on a scanner and analyzed by GeneChip Software.
5. GeneChip analysis
Data analysis was performed using Suite, which is GeneChip analysis software. In Absolute analysis, examine Average Intensity (1) and Background Average (2), and subtract (2) from (1), and average the three averages of unstimulated, IL-4 stimulated, or IL-13 stimulated correction values ( Comparison Analysis was performed as a Scale Factor).
First, Absolute Analysis was performed to analyze one chip data. The positive and negative fluorescent intensities of the probe set were compared to determine positive and negative. Three classifications of P (present), A (absent), and M (marginal), which are Absolute Calls determined from the values of Pos Fraction, Log Avg, and Pos / Neg, were determined.
Pos Fraction; Positive pair ratio.
Log Avg; average of logarithm of fluorescence intensity ratio of perfect match and mismatch probe cells
Pos / Neg; ratio of the number of positive pairs to the number of negative pairs
In addition, Average Difference (Avg Diff), which is the average value of the difference between the fluorescence intensities of the perfect match and mismatch probe cells, was calculated for each gene.
Next, the two data were compared and analyzed (Comparison Analysis). Comparison was made between unstimulated and IL-4 stimulated or unstimulated and IL-13 stimulated, and differences in expression levels were ranked as follows. Five classifications of I, D, MI, MD, and NC, which are Difference Calls determined from the values of Inc / Dec, Inc Ratio, Dpos-Dneg Ratio, and Log Avg Ratio Change, were performed.
Inc: The number of pairs for which it has been determined that IL-4 stimulation or IL-13 stimulation has increased relative to the corresponding probe pairs that have not been stimulated with IL-4 stimulation or IL-13 stimulation.
Dec: The number of pairs determined to be decreased in IL-4 stimulation or IL-13 stimulation.
Inc / Dec: The ratio of the number of pairs determined to be Inc to the number of pairs determined to be Dec.
Inc Ratio: The number of pairs determined to be Inc / the number of pairs actually used.
Dpos / Dneg Ratio: Ratio of Pos Change minus Neg Change to the number of pairs actually used
Pos Change: The difference between the number of positive pairs in Absolute Analysis with IL-4 stimulation or IL-13 stimulation and the number of positive pairs in unstimulated Absolute Analysis.
Neg Change: Difference between the number of negative pairs in Absolute Analysis with IL-4 stimulation or IL-13 stimulation and the number of negative pairs in non-stimulated Absolute Analysis.
Log Avg Ratio Change: Difference between Log Avg between IL-4 stimulation or IL-13 stimulation and unstimulated Absolute Analysis.
Increase: I (Increased),
Decrease: D (Decreed),
Slight increase: MI (Marginally Increased),
Slightly reduced: MD (Marginally Decreased), and
No change: NC (no change)
In addition, the expression change (enhancement or decrease) is caused by IL-4 stimulation or IL-13 stimulation at the value of Fold Change, which is the ratio of Avg Diff in unstimulated and IL-4 stimulation or in Absolute Analysis of unstimulated and IL-13 stimulation ) Was selected as a gene in which) was observed. Table 1 shows the results of narrowing down the expressed genes of the airway epithelial cells by Lot.

In the table, “increase or decrease with [Diff Call]” indicates the number of genes whose expression levels differed by IL-4 stimulation or IL-13 stimulation. Genes having a difference in expression level include genes determined to be slightly increased (or slightly decreased) in the ranking. For each lot, the number of genes that showed a difference in both IL-4 and IL-13 was indicated as “variable common genes”. Furthermore, “[Fold Change]” indicates the number of genes in which a change of 2 times or more (> 2 or <−2) and a change of 3 times or more (> 3 or <−3) were observed, respectively.
As a result, 27 genes were genes whose Fold Change fluctuated to 3 times or more or 1/3 times or less in common by IL-4 and IL-13 stimulation. Among them, the aa011305 sequence reported as EST on the Hu35K subset B chip was selected as the sequence whose expression was most increased even when stimulated by IL-4 and IL-13.
6. Gene analysis
A primer set (aaF / SEQ ID NO: 4 and aaR / SEQ ID NO: 5) for quantification by ABI7700 and TaqManProbe were designed within the sequence of aa011305. The 5 'end of the TaqMan probe is labeled with FAM (6-carboxy-fluorescein), and the 3' end is labeled with TAMRA (6-carboxy-N, N, N ', N'-tetramethylrhodamine).
The total RNA extracted by the method described above was treated with DNase (Nippon Gene). Thereafter, reverse transcribed cDNA was used as a template with random hexammer (GIBCO BRL) as a primer. For a standard curve for calculating the copy number, a plasmid clone containing a nucleotide sequence region amplified by both primers was prepared for each gene, and a reaction was performed using the serial dilution as a template. The composition of the reaction solution for monitoring the PCR amplification is shown in Table 2.

In order to correct the difference in cDNA concentration in the sample, the same quantitative analysis was performed on the β-actin (β-actin) gene and the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene as internal standards for correction. Then, the copy number of the target gene was calculated by correcting based on the copy number of those genes.
Primers and probes for β-actin or GAPDH measurement were used as attached to TaqMan β-actin Control Reagents (Applied Biosystems). The base sequence is as follows. FIG. 1 shows the change over time in the expression level (copy / 5 ng total RNA) of each gene corrected by β-actin, and FIG. 2 shows the change in the expression level for each cell lot. When the expression fluctuation was confirmed in IL-4 and IL-13 stimulation experiments using three lots of airway epithelial cells, a clear increase in expression was also observed.
As a result of the quantitative PCR, the expression level of the gene selected in Example 1 in airway epithelial cells was increased at least twice in three different airway epithelial cells by IL-4 or IL-13 stimulation. Based on these results, it was predicted that in airway epithelial cells, the expression levels of these indicator genes would increase in response to IL-4 and IL-13.
By stimulation of IL-4 or IL-13, which is known to be closely related to allergic reactions, the indicator gene of the present invention shows a common behavior in bronchial epithelial cells of different lots. Therefore, the indicator gene of the present invention is considered to be an important gene that controls the progress of an allergic reaction.

Next, a human Genome Blast search was performed on the aa011305 sequence, which was a sequence in which the expression level was increased, and it was found that the sequence matched the second intron of the cathepsin C gene. Since there was a possibility that the genome was amplified, verification by LongPCR was performed. That is, a primer designed in the aa011305 sequence (aaF and aaR) and a PCR designed in an exon adjacent to the second intron containing the aa011305 sequence (the second exon on the 5 ′ side and the third exon on the 3 ′ side) Using the primers (3F / SEQ ID NO: 13 and 3R / SEQ ID NO: 14), long-PCR was performed so that the amplification region would connect each exon to the aa011305 sequence. The operation is as follows.
That is, 2.5 unit / μl KOD Dash (TOYOBO), 0.2 μl, 10 ng / μl cDNA derived from airway epithelial cells (IL-4 stimulated) 2 μl, 10 μM primer 2 μl each, 2 mM dNTPs 1 μl, dH₂10.8 μl of O was mixed to make a total of 20 μl. Using ABI9700, an amplification reaction was performed at 94 ° C. for 2 minutes and then at 94 ° C. for 30 seconds, 60 ° C. for 2 seconds and 74 ° C. for 1 minute × 35 cycles. The base sequence of the primer used is shown below.

In the 5'-side amplification using primers aaF and 3F, an amplification product of about 200 bp was obtained despite the fact that the distance between the primers in the genomic sequence was about 8 kb. This fragment was incorporated into pCR2.1 vector (invitrogen) by an ordinary method, transformed into TOP10F '(invitrogen), and its nucleotide sequence was analyzed. As a result, a novel sequence linked to the second exon was obtained at the position extended 69 bp to the 5 'side of the aa011305 sequence (1-206 of the sequence shown as SEQ ID NO: 23). This sequence encodes a novel ORF containing 31 amino acids different from the known cathepsin C from the second exon and a stop codon, and a human Genome Blast search by adding an extended 69 bp to the aa011305 sequence revealed that cathepsin was found. It was split into two regions within the second intron of the C gene. FIG. 3 schematically shows the positional relationship in the genome of the exon of the alternative splicing form of the present invention, the exon of cathepsin C, and the EST aa011305 on the DNA chip. FIG. 4 shows the relationship between the base sequence between exons and introns of intron 1 and intron 2 of the alternative splicing form of the present invention and the GT-AG rule. The GT-AG rules between the two new exons found and the adjacent introns are fairly well conserved and were considered to be a novel alternative splicing form of the cathepsin-C gene.
No amplification was observed in PCR using the primer aaR on the aa011305 sequence and the primer 3R designed on the third exon. Also known as cathepsin C when using primers (3F and 3R) on the second and third exons and in PCR using primers (2F and 2R) on the first and seventh (final) exons. No sequence other than was obtained.
However, PCR using primer 1F designed near the start codon of the first exon and primer aaF on the aa011305 sequence yielded a sequence that switches from the first exon through the second exon to the novel exon found this time. Considering the presence of a stop codon in the aa011305 sequence, there is no exon after the aa011305 sequence in the alternative splicing form obtained this time, and at least a known sequence from the middle of the first exon to the second exon. And suggests that it encodes a short peptide that terminates at the addition of 31 new amino acids from the end of the second exon.
Furthermore, in order to determine the 5 'end and 3' end of this alternative splicing form, the Marathon cDNA Amplification Kit (CLONTECH) was used as a template with a Marathon library prepared using HL60-derived mRNA as a template. RACE and 5'RACE were performed.
In 5'RACE, PCR was performed using the AP1 primer included in the kit and 5'RACE1 (SEQ ID NO: 18). Further, nested PCR was performed using the amplified fragment as a template and the sequence [AP2] in the adapter and 5'RACE2 (SEQ ID NO: 19). On the other hand, in 3'RACE, PCR was performed with the AP1 primer attached to the kit and the primer 3'RACE1 (SEQ ID NO: 20). Further, nested PCR was performed using the amplified fragment as a template and the sequence [AP2] in the adapter and the primer 3'RACE2 (SEQ ID NO: 21).

As a result, it was found that the 5 'end of the sequence reported as aa011305 was the 3' end of this alternative splicing form, and seemed to recognize a polyA addition signal existing 19 bp from the 3 'end. The full-length nucleotide sequence of the novel alternative splicing form obtained in the above experiment is shown in SEQ ID NO: 1, and the amino acid sequence encoded by this cDNA is shown in SEQ ID NO: 2.
[Example 2] Northern hybridization
Northern hybridization was performed to confirm that the cathepsin gene and the alternative splicing form were expressed as mRNA. The amplification product of the following primer set was purified using RECO Chip (TAKARA), and was purified using Random Primer Labeling Kit (TAKARA).³²It was labeled with P to be used as a probe. As a template, a plasmid having, as an insert, a base sequence between 2F / 2R obtained by a Long PCR experiment was used.
Probe 552 bp that recognizes only known sequence

Probe 1301 bp recognizing both known sequence and alternative splicing form

As the membrane, Human Immun System MTN Blot II and Human MTN Blot manufactured by CLONTECH were used. Northern Hybridization and washing were performed using Express Hybridization Solution (CLONTECH) as instructed. Washing was performed using 2 × SSC-0.1% SDS at room temperature for about 40 minutes, and using 0.1 × SSC-0.1% SDS at 56 ° C. for 10 minutes. The image was exposed by exposing to the imaging plate overnight. Images were acquired with a Molecular Imager System (BIO-RAD).
A probe (x87f / 2R) having a sequence on the sixth and seventh exons of the known cathepsin C gene and expected to recognize only the known cathepsin C gene, and a sequence from the first exon to the seventh exon When a probe (2F / 2R), which recognizes both the known cathepsin C gene and the novel alternative splicing form, was used, a band of about 2 kb was detected. This size almost coincided with the reported 1.8 kb of the known gene. In addition, the intensity of the detected band was strong in the lung, kidney and placenta, and weak in the brain, showing the same results as in the previous report (J. Biol. Chem, 272, 10260-10265, 1997). Furthermore, only when a probe recognizing both the known cathepsin C gene and the novel alternative splicing form was used, a band was observed at around 1 kb although the signal was very weak, which is the aforementioned alternative splicing form. Was predicted.
In order to detect only the alternative splicing form, northern hybridization using riboprobe was performed based on the region newly obtained in this experiment (SEQ ID NO: 23). The probe was prepared as follows. First, a region to be amplified using the primer aaF (SEQ ID NO: 4) and the primer 3′RACE1 (SEQ ID NO: 20) was incorporated into pGEM-T Easy vector (promega). As a template, a plasmid having, as an insert, a base sequence between 1F / aaF obtained in a Long PCR experiment was used. Next, a clone from which the antisense strand of the target sequence was read was selected from T7 promoter, and the plasmid was purified. The resulting plasmid was linearized using the restriction enzyme NdeI, and this was used as a template.³²Antisense RNA strand labeled with P-rCTP was synthesized using T7 RNA polymerase (promega) and used as a probe. A series of operations followed the instructions of riboprobe In Vitro Transcription Systems (promega).
Using the same kind of membrane, hybridization was carried out at 55 ° C. overnight in a hybrid solution of 5 × SSPE, 50% formamide, 5 × Denhardt, 0.5% SDS. Washing is performed at room temperature for about 40 minutes using 2 × SSC-0.1% SDS, at 56 ° C. for 1 hour, at 60 ° C. for 1 hour, and at 70 ° C. for 30 minutes using 0.1 × SSC-0.1% SDS. Was. The image was exposed by exposing to the imaging plate overnight. Images were acquired with a Molecular Imager System (BIO-RAD).
As a result, a signal was found around 1 kb, indicating that this alternative splicing form actually exists as mRNA. It was also found that the expression was high in kidney, placenta and lymph nodes (FIG. 5).
[Example 3] Comparison of expression levels of indicator genes
The expression level of the alternative splicing form of cathepsin C in cultured airway epithelial cells (Clonetics) was measured by quantitative PCR and compared with cathepsin C. Three lots of 8F1756, 8F1548, and 8F1805 were used as cultured cells. The measuring method conformed to 6. Primers and TaqManProbe for the alternative splicing form are aaF (SEQ ID NO: 4) and aaR (SEQ ID NO: 5) used in 6 of [Example 1], and TaqManProbe (SEQ ID NO: 6). The primers for cathepsin C and the base sequence of TaqManProbe are shown below.

The total RNA extracted by the method described above was treated with DNase (Nippon Gene). Thereafter, reverse transcribed cDNA was used as a template with random hexammer (GIBCO BRL) as a primer. For a standard curve for calculating the copy number, a plasmid clone containing a nucleotide sequence region amplified by both primers was prepared for each gene, and a reaction was performed using the serial dilution as a template. The composition of the reaction solution for monitoring the PCR amplification was the same as that shown in Table 2 in 6 of [Example 1].
In order to correct the difference in cDNA concentration in the sample, the same quantitative analysis was performed on the β-actin (β-actin) gene and the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene as internal standards for correction. Then, the copy number of the target gene was calculated by correcting based on the copy number of those genes.
Primers and probes for β-actin or GAPDH measurement were carried out using those attached to TaqMan β-actin Control Reagents (Applied Biosystems) (the same ones used in 6). FIG. 6 shows the expression level (copy / 5 ng total RNA) of each gene corrected by GAPDH.
As a result of quantitative PCR, the expression levels of cathepsin C and its alternative splicing form were all increased more than 2-fold in three different airway epithelial cells by IL-4 or IL-13 stimulation. In airway epithelial cells, the expression levels of these indicator genes were expected to increase in response to IL-4 and IL-13. However, the expression levels of both were significantly different. That is, it was revealed that the expression level of the alternative splicing form reaches about 20 times that of cathepsin C.
By stimulation of IL-4 or IL-13, which is known to be closely related to allergic reactions, the indicator gene of the present invention shows a common behavior in bronchial epithelial cells of different lots. Therefore, the indicator gene of the present invention is considered to be an important gene that controls the progress of an allergic reaction. Further, an alternative splicing form showing a much higher expression level than that of cathepsin C is useful as an index capable of performing highly sensitive measurement among allergy-related genes.
[Example 4] Cloning of mouse counterpart of cathepsin C alternative splicing form
First, a 5′-side primer was placed on the second exon (from JBC, vol. 272, No. 16, 10695-10703, 1997) of the known mouse cathepsin C sequence (u89269), and a human primer was placed on the 3′-side. A degenerate primer (Table 3) was prepared based on the sequence of the alternative splicing form, and PCR was performed using a mouse cDNA library (lung) as a template.

When the sequence of the obtained fragment was determined, the presence of a sequence that switched to the new sequence at the same position as in humans (end point of exon 2) was confirmed. From this, it was considered that there is a high possibility that an alternative splicing form similar to human is also present in the mouse. Primers (Table 3) were designed, and 5'RACE and 3'RACE using a mouse cDNA library (lung) as a template were performed using Marathon cDNA amplification Kit (CLONTECH).
As a result, from the initiation codon to the second exon, the presence of mRNA which was the same as that of cathepsin C reported in the mouse but was terminated by adding an unreported new 362 bp sequence was confirmed. The nucleotide sequence of the mouse counterpart is shown in SEQ ID NO: 26 (FIG. 7), and the amino acid sequence of the protein encoded by the nucleotide sequence is shown in SEQ ID NO: 27. This mouse counterpart has high homology to the nucleotide sequence of the human gene (FIG. 8), and has a novel peptide of 31 amino acids (SEQ ID NO: 28 / DVTDFIS) as in humans.QLFMQLGTVGMYDLPHLRNKLVK / amino acids different from human are underlined). In the cloned region (93 bp + TAG), three base substitutions were found, one was a synonymous substitution, and the other was a non-synonymous substitution of His (human) → Gln, Ile (human) → Met. The 3'UTR was 324 bp for humans and 246 bp for mice.
Industrial potential
According to the present invention, a gene whose expression is increased in airway epithelial cells stimulated with IL-4 or IL-13 has been found. Genes with increased expression in airway epithelial cells stimulated with IL-4 or IL-13 are likely to be an essential cause of allergic symptoms in bronchial asthma. Therefore, the indicator gene provided by the present invention is a useful indicator that can reliably determine whether bronchial asthma is caused by allergic symptoms. By being able to reliably diagnose bronchial asthma caused by allergy, an appropriate treatment method can be selected at an early stage.
IL-4 or IL-13 is an important factor that enhances allergic reactions. Therefore, genes whose expression increases in response to stimulation of these factors are considered to play an important role in the pathogenesis of allergic symptoms. In addition, all of the indicator genes provided by the present invention clearly showed enhanced expression in a plurality of airway epithelial cells. This is not the first study focusing on the change in the expression level of a gene accompanying the stimulation of IL-4 or IL-13. However, all of the genes provided by the present invention are allergy-related genes found by the strict criteria described herein. Therefore, the indicator gene of the present invention is considered to be a gene that plays an important role in allergic reactions as compared to known allergy-related genes obtained by a similar approach. This is because the indicator gene of the present invention always responds sharply to the stimulation of IL-4 or IL-13 in different cells. This supports that the indicator gene of the present invention is indispensable for allergic reactions.
As described above, since all of the indicator genes of the present invention are associated with the upregulation of their expression, suppressing their expression is a target of therapeutic strategies for allergic diseases and monitoring such new therapeutic methods. Can be expected to be useful as a new clinical diagnostic index. In particular, the expression level of the indicator gene of the present invention is large between healthy subjects and patients with allergic diseases. For example, the present inventors have confirmed that the expression level of cathepsin C is significantly higher in airway epithelial cells treated with IL-4 or IL-13. However, the expression level of the indicator gene of the present invention is about 20 times higher than that of cathepsin C. Therefore, the indicator gene of the present invention can easily be measured with high sensitivity. Thus, the indicator gene of the present invention has high utility as a diagnostic indicator.
The expression level of the indicator gene provided by the present invention can be easily determined regardless of the type of allergen. Therefore, the pathology of the allergic reaction can be comprehensively grasped.
In addition, the method for testing allergy according to the present invention can analyze the expression level of a biological sample as a sample, and thus has low invasiveness to patients. In addition, regarding gene expression analysis, highly sensitive measurement using a small amount of sample is possible. Gene analysis technology has been increasing in throughput and lowering its price year by year. Therefore, the method of testing for allergy according to the present invention is expected to be an important diagnostic method at the bedside in the near future. In this sense, the diagnostic value of these disease-related genes is high.
[Sequence list]

[Brief description of the drawings]
FIG. 1 is a graph showing changes over time in expression levels of 0, 6, 12, 24, and 48 hours after treatment of an indicator gene in cultured bronchial epithelial cells stimulated with IL-4 and IL-13. is there. The vertical axis shows the gene expression level (copy / 5 ng total RNA), and the horizontal axis shows the type of cytokine treated and the elapsed time after each treatment. The columns indicated by B2-24-c to B2-24-IL9 show the measurement 24 hours after treatment when using TNFα, IL-1β, IL-5, IL-6, and IL-9 as cytokines. The result. The columns show, in order from the left, raw data, β-actin correction value, and GAPDH correction value.
FIG. 2 is a graph showing the result of measuring the expression level of an indicator gene in cultured bronchial epithelial cells stimulated with IL-4 and IL-13 for each cell lot. The vertical axis indicates the gene expression level (copy / 5 ng total RNA), and the horizontal axis indicates the cell lot and the type of cytokine processed. The columns show, in order from the left, raw data, β-actin correction value, and GAPDH correction value.
FIG. 3 is a diagram schematically showing the positional relationship in the genome of the exon of the alternative splicing form of the present invention, the exon of cathepsin C, and the EST aa011305 on the DNA chip.
FIG. 4 is a diagram without showing the relationship between the base sequences between exons and introns of intron 1 and intron 2 of the alternative splicing form of the present invention, and the GT-AG rule.
FIG. 5 is a photograph showing the results of Northern hybridization using riboprobe.
FIG. 6 is a graph showing measurement results of expression levels (GAPDH correction values) of cathepsin C and its alternative splicing form in cultured cells.
FIG. 7 is a diagram showing the full-length sequence of the mouse counterpart of the cathepsin C alternative splicing form. The start codon, the stop codon, and the polyA addition signal are underlined. * Indicates a boundary with exon 2.
FIG. 8 is a diagram in which the nucleotide sequences of human and mouse cathepsin C alternative splicing forms are aligned. The gap is represented by "-". Indicates a known splicing site, ▽ indicates a novel splicing site, and the underline indicates the EST sequence of human AA011305.
FIG. 9 is a diagram showing an alignment of amino acids between human and mouse. The region surrounded by Box represents a region having homology.

Claims (15)

A method for testing an allergic disease, comprising the following steps.
a) a step of measuring the expression level of a gene containing the nucleotide sequence of SEQ ID NO: 1 in a biological sample of a subject b) a step of comparing the expression level of the gene with a biological sample of a healthy subject The test method according to claim 1, wherein the allergic disease is bronchial asthma. The test method according to claim 1, wherein the expression level of the gene is measured by cDNA PCR. The test method according to claim 1, wherein the expression level of the gene is measured by detecting a protein encoded by the gene. A polynucleotide comprising the nucleotide sequence of SEQ ID NO: 23, or a polynucleotide comprising a nucleotide sequence complementary to the complementary strand thereof and comprising the nucleotide sequence of SEQ ID NO: 1, or complementary to the complementary strand thereof A reagent for testing an allergic disease, comprising an oligonucleotide having a length of at least 15 bases selected from a base sequence. It consists of an antibody that recognizes a peptide consisting of at least 7 consecutive amino acid residues selected from the amino acid sequence of SEQ ID NO: 2 and containing an amino acid sequence selected from the amino acid sequence at positions 107 to 137 from the N-terminus. , Allergic disease test reagent. A method for screening a therapeutic drug for an allergic disease, comprising the following steps.
(1) a step of bringing a candidate compound into contact with a cell that expresses a gene containing the nucleotide sequence of SEQ ID NO: 1 and / or any gene functionally equivalent to these genes (2) expression of the gene The process of measuring the level,
(3) selecting a compound that reduces the expression level of the gene as compared to a control that is not contacted with the candidate compound The method according to claim 7, wherein the cells are established airway epithelial cells. A method for screening a therapeutic drug for an allergic disease, comprising the following steps.
(1) administering a candidate compound to a test animal,
(2) a step of measuring the expression intensity of a gene containing the nucleotide sequence of SEQ ID NO: 1 and / or a gene functionally equivalent to this gene in a biological sample of a test animal; and (3) administering a candidate compound Selecting a compound that reduces the level of expression of the gene as compared to a control that does not A method for screening a therapeutic drug for an allergic disease, comprising the following steps.
(1) contacting a candidate substance with a protein encoded by a gene comprising the nucleotide sequence of SEQ ID NO: 1 and / or a protein functionally equivalent to the protein;
(2) a step of measuring the activity of the protein, and (3) a step of selecting a compound that reduces the activity of the protein as compared to a control not contacted with a candidate compound. A remedy for allergic diseases, comprising as an active ingredient a compound obtainable by the screening method according to any one of claims 7, 9, and 10. A therapeutic agent for an allergic disease, comprising a gene comprising the nucleotide sequence of SEQ ID NO: 23 or a part thereof as an antisense DNA as a main component. A polynucleotide comprising a nucleotide sequence of a gene comprising the nucleotide sequence of SEQ ID NO: 1, or an oligonucleotide having a nucleotide sequence complementary to a complementary strand thereof and having a length of at least 15 nucleotides; A kit for screening a candidate compound for a therapeutic agent for an allergic disease, comprising a cell that expresses a gene containing the nucleotide sequence of An antibody that recognizes a peptide containing the amino acid sequence of the protein encoded by the gene containing the nucleotide sequence of SEQ ID NO: 1, and a cell that expresses the gene containing the nucleotide sequence of SEQ ID NO: 1, A kit for screening a candidate therapeutic compound for a disease. As a model animal of a transgenic non-human vertebrate allergic disease in which the expression intensity of a gene containing the nucleotide sequence of SEQ ID NO: 1 and / or a gene functionally equivalent to this gene in airway epithelial cells is increased. Use of.

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