JP2004529605A - Dermatophagoides nucleic acid molecules, proteins and uses thereof - Google Patents

Abstract

The present invention relates to high molecular weight Dermatophagoides proteins, nucleic acid molecules encoding such proteins, and therapeutic and diagnostic agents derived from such proteins. The present invention relates to novel proteins having a molecular weight of about 60 (kd or kD), 70 kD, or about 98 kD to about 109 kD. Such proteins comprise at least one epitope of a Dermatophagoides family of mite protein allergens and are herein designated as Der HMW-map proteins. The invention also provides proteins that are fragments or peptides of the full-length or mature proteins, as well as antibodies, mimetopes, or muteins of any of such proteins.

Description

これらの結果は、ダニアレルゲンに感受性であることが既知のネコの数匹からの血清が、本発明のｍａｐＡタンパク質、ｍａｐＢタンパク質またはｍａｐＣタンパク質に特異的に結合するＩｇＥ抗体を含むことを示す。さらに、ｍａｐＡタンパク質、ｍａｐＢタンパク質またはｍａｐＣタンパク質に結合するＩｇＥを含むいくつかの血清はまた、全てのＤ．ｐｔｅｒｏｎｙｓｓｉｕｓ抽出物に結合するＩｇＥ抗体を含む。コントロール血清は、本発明のｍａｐＡタンパク質、ｍａｐＢタンパク質またはｍａｐＣタンパク質のいずれかに結合するＩｇＥ抗体を含まなかった。
【０１８８】
（実施例７）
本実施例は、イヌにおいて高感受性応答を誘導するＤ．ｆａｒｉｎａｅ ＨＭＷ−ｍａｐ組成物の能力を例証する。
【０１８９】
実施例１に記載されるＤ．ｆａｒｉｎａｅ ＨＭＷ−ｍａｐ組成物が粉末ダニアレルギー応答に感受性である動物においてアレルギー応答を誘導し得るか否かを決定するために、粉末ダニアレルギーに対する臨床的兆候を活性に示すイヌ（本明細書中、アトピー性イヌという）に対して、皮膚試験を実施した。正常なイヌは、ダニアレルギーの症状を示さないがダニアレルギー応答に感受性であり得るイヌを含む。各イヌ（すなわち、４匹の正常イヌおよび４匹のアトピー性イヌ）を、外側胸部／腹部領域で剃毛し、その領域中の異なる部位に、実施例１に記載される方法によって分離された約１：５０，０００希釈のＤ．ｆａｒｉｎａｅ粗抽出物を用いて約２μｇの精製したＤ．ｆａｒｉｎａｅ ＨＭＷ−ｍａｐ組成物および／またはコントロール溶液（すなわち、ネガティブコントロールとしての生理食塩水、およびポジティブコントロールとしての１：１０００希釈のヒスタミン）とともに皮内注射した。４匹の正常イヌ全ておよび４匹のアトピー性イヌ全てが、Ｄ．ｆａｒｉｎａｅ全抽出物を受けた。正常イヌの３匹およびアトピー性イヌの２匹は、Ｄ．ｆａｒｉｎａｅ ＨＭＷ−ｍａｐ組成物を受けた。８匹のイヌ全てが、ネガティブコントロールサンプルおよびポジティブコントロールサンプルの両方を受けた。注射当たりの総容量は、５０マイクロリットル（μｌ）であり、生理食塩水中に希釈された組成物およびコントロールを有した。これらの注射を、単回の注射として投与した。
【０１９０】
全ての注射部位を、１５分時のミリメートル（ｍｍ）で客観的に測定し、そしてコントロールサンプルと比較した場合に（＋）または（−）のいずれかでスコア付けした。客観的スコア付けを、Ｏｈｉｏ Ｓｔａｔｅ Ｕｎｉｖｅｒｓｉｔｙ，Ｃｏｌｕｍｂｕｓ，ＯＨのＡｎｄｒｅｗ Ｈｉｌｌｉｅｒ，Ｄ．Ｖ．Ｍ．によって実施した。結果を、表２に示す。
（表２）
【０１９１】
【表２】

これらの結果は、Ｄ．ｆａｒｉｎａｅ ＨＭＷ−ｍａｐ組成物がアトピー性イヌを含むイヌにおいて即時高感受性応答を誘導し得たことを示す。従って、ＨＭＷ−ｍａｐ組成物は、アトピー性イヌを含むイヌにおいて高感受性応答を誘導するために十分アレルギー性である。
【０１９２】
表３は、以下の実験の結果を記載する。ＨＭＷ−ｍａｐ組成物に対するＩｇＥを、以下の３群のイヌの血清においてＥＬＩＳＡを使用して測定した：Ｄ．ｆａｒｉｎａｅアレルギー性のイヌ（ＨＤＭ−ＡＤ）、（他のアレルゲンに対して）アトピー性であるがＨＤＭアレルギー性ではないイヌ（ＡＤ）、およびナイーブなイヌ。３群のイヌをまた、Ｄ．ｆａｒｉｎａｅ全抽出物およびＨＭＷ−ｍａｐ組成物に対する皮内皮膚試験によって試験した。
【０１９３】
（表３．Ｄ．ｆａｒｉｎａｅアレルギー性のイヌ、アトピー性であるがＨＤＭ−アレルギー性ではないイヌおよびナイーブなイヌにおける、Ｄ．ｆａｒｉｎａｅ全抽出物およびＨＭＷ−ｍａｐ組成物についての皮膚試験およびＥＬＩＳＡデータ）
【０１９４】
【表３Ａ】

【０１９５】
【表３Ｂ】

Ｄ．ｆａｒｉｎａｅ全抽出物に対してＥＬＩＳＡによってポジティブであった全てのイヌはまた、ＨＭＷ−ｍａｐ組成物アレルゲンに対してもポジティブであった。Ｄ．ｆａｒｉｎａｅ全抽出物に対してＥＬＩＳＡによってネガティブであった８匹のイヌについて、８匹中７匹がまた、ＨＭＷ−ｍａｐ組成物に対してもネガティブであった。
【０１９６】
（実施例８）
本実施例は、本発明のＤｅｒ ＨＭＷ−ｍａｐ組成物をコードする核酸分子の単離を記載する。
【０１９７】
Ｄｅｒ ＨＭＷ−ｍａｐ組成物核酸分子を、以下のように同定しそして単離した。
【０１９８】
（Ａ．Ｄｅｒｍａｔｏｐｈａｇｏｉｄｅｓ ｆａｒｉｎａｅ ｃＤＮＡライブラリーの調製）
Ｄｅｒｍａｔｏｐｈａｇｏｉｄｅｓ ｆａｒｉｎａｅ ｃＤＮＡライブラリーを、以下のように調製した。総ＲＮＡを、約２ｇの瞬間凍結しかつ破砕した粉末家ダニから、Ｃｈｏｍｚｙｎｓｋｉら、１９８７、Ａｎａｌ．Ｂｉｏｃｈｅｍ．１６２，１５６−１５９によって記載されるものと類似の酸−グアニジウム−フェノール−クロロホルム法を使用して抽出した。ポリＡ^＋選択されたＲＮＡを、製造業者によって推奨される方法に従って、ｍＲＮＡ Ｐｕｒｉｆｉｃａｔｉｏｎ Ｋｉｔ（Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ，Ｎｅｗａｒｋ，ＮＪから入手可能）を使用するオリゴ−ｄＴセルロースクロマトグラフィーによって総ＲＮＡ調製物から分離した。ｃＤＮＡライブラリーを、ＳｔｒａｔａｇｅｎｅのＺＡＰ−ｃＤＮＡ Ｓｙｎｔｈｅｓｉｓ Ｋｉｔプロトコルを使用して、λ−Ｕｎｉ−ＺＡＰ^ＴＭＸＲベクター（Ｓｔｒａｔａｇｅｎｅから入手可能）中に構築した。約５μｇのポリＡ^＋ＲＮＡを使用して、Ｄｅｒｍａｔｏｐｈａｇｏｉｄｅｓ ｆａｒｉｎａｅ ｃＤＮＡライブラリーを生成した。
【０１９９】
（Ｂ．ＰＣＲプライマーの調製）
さらなるＮ末端アミノ酸配列分析を、実施例２に上記した方法に従って実施した。３４アミノ酸のＮ末端アミノ酸部分配列を推測し、そして以下のアミノ酸配列を有する配列番号２４によって示す：
【０２００】
【化１】

（ここで、「Ｘａａ」は、任意のアミノ酸残基を示す）。配列番号４のアミノ酸配列（実施例２に上記した）および配列番号２４を使用して、合成オリゴヌクレオチドプライマーを設計した。以下のヌクレオチド配列：
【０２０１】
【化２】

を有する配列番号２４由来のセンスプライマーＤｅｒｆ１（配列番号２５と称する）（ここで、ＹはＣまたはＴを示し、ＲはＡまたはＧを示し、そしてＮはＡ、Ｃ、ＴまたはＧを示す）、または以下のヌクレオチド配列：
【０２０２】
【化３】

を有する配列番号４由来のセンスプライマーＤｅｒｆ２（配列番号２６と称する）を、以下のヌクレオチド配列：
【０２０３】
【化４】

を有する配列番号４由来のアンチセンスプライマーＤｅｒｆ３（配列番号２７と称する）、または以下のヌクレオチド配列：
【０２０４】
【化５】

を有する配列番号２４由来のアンチセンスプライマーＤｅｒｆ４（配列番号２８と称する）と組合わせて使用した。
【０２０５】
次いで、前述のプライマーを使用して、Ｄｅｒｆ ｃＤＮＡライブラリーを、標準的ポリメラーゼ連鎖反応増幅（ＰＣＲ）技術を用いてスクリーニングした。これらのプライマーにハイブリダイズするｃＤＮＡを同定する試み全てが、失敗した。
【０２０６】
（Ｃ．抗Ｄｅｒ ＨＭＷ−ｍａｐ組成物抗体を使用するＤ．ｆａｒｉｎａｅ ｃＤＮＡライブラリーの免疫スクリーニング）
ＰＣＲ法を使用したｃＤＮＡクローン単離の試みが失敗したので、本発明者らは、以下のように生成した抗血清を使用してＤ．ｆａｒｉｎａｅ ｃＤＮＡライブラリーをスクリーニングした。実施例１に上記した方法に従って単離したタンパク質を抗原の供給源として使用して、本明細書中で抗Ｄｅｒ ＨＭＷ−ｍａｐ組成物抗体といわれるウサギポリクローナル抗体を生成した。ウサギポリクローナル抗体の調製を、標準的技術を使用して実施した。
【０２０７】
約７．５ｍｌのＥｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ（ＸＬ１ Ｂｌｕｅ、Ｏ．Ｄ．_６００＝０．５）を、Ｄｅｒｍａｔｏｐｈａｇｏｉｄｅｓ ｆａｒｉｎａｅ ＺＡＰ−ｃＤＮＡライブラリー（１．８×１０^９ｐｆｕ／ｍｌ）からの３．０×１０^４ｐｆｕのファージとともに３７℃で１５分間インキュベートし、３０ｍｌのＬｕｒｉａ−Ｂｅｒｔａｎｉ（ＬＢ）培地アガープレート（１５０ｍｍ）にプレートした。このプレートを、３７℃で一晩インキュベートした。次いで、各プレートを、ＩＰＴＧ（１０ｍＭ）処理したニトロセルロースフィルターで３７℃で約４時間オーバーレイした。次いで、このフィルターを取り外し、０．１％ Ｔｗｅｅｎを含むＴｒｉｓ緩衝化生理食塩水（ｐＨ７．５）（ＴＢＳＴ）で５分間洗浄した。このフィルターを、ＴＢＳＴ中に１％脱脂粉乳、１％ＢＳＡ、２％ヤギ血清および０．１５％ゼラチンを調製した溶液で、室温にて２時間ブロックした。次いで、フィルターを、１：１０００希釈の抗Ｄｅｒ ＨＭＷ−ｍａｐ組成物抗体を含む上記のブロッキング溶液とともに４℃で一晩インキュベートした。次いで、この混合物を、西洋ワサビペルオキシダーゼに結合体化したロバ抗ウサギＩｇＧ抗体（Ｊａｃｋｓｏｎ ＩｍｍｕｎｏＲｅｓｅａｒｃｈ，Ｗｅｓｔ Ｇｒｏｖｅ，ＰＮより入手可能）とともに、室温で２時間インキュベートした。全てのフィルターを、各インキュベーションの間に、ＴＢＳＴ中に含まれたブロッキング溶液で洗浄（１洗浄あたり３×１５分）した。次いで、全てのフィルターを、Ｔｒｉｓ緩衝化生理食塩水（ｐＨ７．５）で室温にて５分間の最後の洗浄に付した。免疫複合体化プラークを、顕色化溶液（Ｋｉｒｋｅｇａａｒｄ＆Ｐｅｒｒｙ ＬａｂｏｒａｔｏｒｉｅｓからのＴＭＢ Ｐｅｒｏｘｉｄａｓｅ Ｓｕｂｓｔｒａｔｅ／ＴＭＢ Ｐｅｒｏｘｉｄａｓｅ Ｓｏｌｕｔｉｏｎ／ＴＭＢ Ｍｅｍｂｒａｎｅ Ｅｎｈａｎｃｅｒを１／１／０．１の容量比で）にこのフィルターを浸して着色反応を生成することによって同定した。１２３プラークを同定し、５０プラークをさらに、上記と同様な免疫スクリーニング条件下で２回以上精製した。
【０２０８】
（Ｄ．精製したファージプラグのＰＣＲスクリーニング）
次いで、前出の免疫スクリーニング研究において同定したファージプラグをさらに、８Ｂ項で上記したプライマーを使用するＰＣＲ増幅によって分析した。配列番号２５、配列番号２６、配列番号２７および配列番号２８によって同定された４つのプライマーの混合物を使用して、５０プラークからのＤＮＡを増幅した。ＰＣＲ増幅を、標準的技術を使用して行った。得られたＰＣＲ増幅産物の１つは、約７００ヌクレオチドのフラグメントを構成した。このＰＣＲ産物を、ＩｎＶｉｔｒｏｇｅｎ，Ｃｏｒｐ．，ＴＡ^ＴＭクローニングベクター（Ｉｎ Ｖｉｔｒｏｇｅｎ，Ｃｏｒｐ．によって提供される手順）にクローニングし、標準的技術を使用するＤＮＡ配列分析に供した。７００ｂｐのＰＣＲ産物中にコードされる配列を含むことを決定された、精製したファージ由来のファージミドを回収し、標準的技術を使用するＤＮＡ配列分析に供した。
【０２０９】
約１７５２ヌクレオチド挿入物を含むクローンを単離し、本明細書中でｎＤｅｒｆ９８_１７５２と呼ぶ。核酸配列を、標準的技術を使用してｎＤｅｒｆ９８_１７５２から得て、核酸配列配列番号１４を有するコード鎖および核酸配列配列番号１６を有する相補鎖から構成されるｎＤｅｒｆ９８_１７５２と呼ばれるＤｅｒｍａｔｏｐｈａｇｏｉｄｅｓ ｆａｒｉｎａｅ核酸分子を得た。配列番号１４の翻訳は、ｎＤｅｒｆ９８_１７５２核酸分子がアミノ酸配列配列番号１５を有する全長ｆｌｅａタンパク質の約５５５アミノ酸（本明細書中でＰＤｅｒｆ９８_５５５と呼ぶ）をコードすることを示唆する。ここで、開始コドンは、配列番号１４のヌクレオチド１〜ヌクレオチド３にわたり、終止コドンは、配列番号１４のヌクレオチド１６６６〜ヌクレオチド１６６８にわたるオープンリーディングフレームが予想される。ＰＤｅｒｆ９８_５５５のアミノ酸配列は、核酸配列配列番号１７を有するコード鎖および核酸配列配列番号１９を有する相補鎖を有する核酸分子ｎＤｅｒｆ９８_１６６５によってコードされる。配列番号１８としても示されるＰＤｅｒｆ９８_５５５は、推定分子量約６３．２ｋＤおよび推定ｐＩ約５．３３を有する。配列番号１５の分析は、およそのアミノ酸１〜およそのアミノ酸１９にわたるシグナルペプチドの存在を示唆する。ＰＤｅｒｆ９８_５３６と本明細書中で示される、提唱した成熟タンパク質は、約５３６アミノ酸を含み、この配列は、配列番号２１として本明細書中に示され、そして、配列番号２０（コード鎖）および配列番号２２（相補鎖）によって示される、ｎＤｅｒｆ９８_１６０８と本明細書中で示される核酸分子によってコードされる。ｆｌｅａ ＰＤｅｒｆ９８_５３６のアミノ酸配列（すなわち、配列番号２１）は、ＰＤｅｒｆ９８_５３６が推定分子量６１．２ｋＤおよび推定ｐＩ約５．２６を有することを予測する。
【０２１０】
アミノ酸配列配列番号１５とＧｅｎＢａｎｋに報告されたアミノ酸配列との比較は、配列番号１５がＡｎｏｐｈｅｌｅｓ ｇａｍｂｉａｅ由来のキチナーゼタンパク質（ＧｅｎＢａｎｋ登録番号２６５４６０２）に最大の相同性（すなわち、約４２％の同一性）を示したことを示す。核酸配列配列番号１７とＧｅｎＢａｎｋに報告された核酸配列との比較は、配列番号１７が、配列番号１７とＣｈｅｌｏｎｕｓ ｓｐ．ｖｅｎｏｍキチナーゼｍＲＮＡ（ＧｅｎＢａｎｋ登録番号Ｕ１０４２２）との間で最大の相同性（すなわち、約５８％の同一性）を示したことを示す。
【０２１１】
（実施例９）
本実施例は、ダニアレルゲンに対してアレルギー性であることが既知のイヌ由来のＩｇＥに結合する６０ｋＤタンパク質の精製、およびこの６０ｋＤタンパク質由来の部分アミノ酸配列を記載する。
【０２１２】
（Ａ．６０ｋＤタンパク質の精製）
Ｄ．ｆａｒｉｎａｅ抽出物を調製し、実施例１に上記された方法に従ってＳｅｐｈａｃｒｙｌ Ｓ−１００カラムで分画した。排除ピーク（画分２９〜３５）後の画分を、Ｓｅｐｈａｃｒｙｌ Ｓ−１００カラムから回収し、そしてプールした。次いで、プールした画分を、１０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ８）で１：１に希釈し、Ｑ−ｓｅｐｈａｒｏｓｅカラムに適用して実施例１で上記した方法を使用して画分を得た。０．４Ｍ Ｔｒｉｓ−ＨＣｌで溶出した画分を濃縮し、実施例１で上記した方法を使用してＴＭＳ２５０逆相ＨＰＬＣカラムによってさらに精製した。この各文中のタンパク質を、実施例１において１２％ゲルでのタンパク質分離について記載した同様の方法を使用して、１４％Ｔｒｉｓ−グリシンＳＤＳ−ＰＡＧＥによって分離した。染色したゲルを、図２に示す。約５０％ Ｂ（９０％アセトニトリル中０．０５％ＴＡ）で溶出した、約９０％の純度の約６０ｋＤの分子量を有するタンパク質を同定した（図２、レーン４）。示した６０ｋｄタンパク質の分子量を、ＢｉｏＲａｄ Ｍｕｌｔｉ−Ａｎａｌｙｓｔ／ＰＣ Ｖｅｒｓｉｏｎ １．１プログラムおよびＭａｒｋ−１２タンパク質分子量マーカーを使用して、５６．１１ｋｄであると見積もった。約６０ｋｄタンパク質は、本明細書中でｍａｐＤタンパク質という。
【０２１３】
（Ｂ．６０ｋｄタンパク質より得たＮ末端部分配列および内部配列）
上記のパートＡから溶出したタンパク質を、ＰＶＤＦ上にブロットし、これを、Ｃｏｏｍａｓｓｉｅ Ｒ−２５０で染色し、標準的手順を使用して脱色した。約６０ｋｄのバンドに対応するタンパク質を抽出し、当業者に公知の技術を使用するＮ末端アミノ酸配列決定に供した。約２５アミノ酸のＮ末端アミノ酸部分配列を、このタンパク質について推定し、配列番号２３として本明細書中で示されるアミノ酸配列を、以下：
【０２１４】
【化６】

であると推定した（ここで、Ｘａａは任意のアミノ酸をいう）。
【０２１５】
この６０ｋｄの領域に対応するタンパク質をまた、内部アミノ酸配列データを得るために、蛋白質分解切断に供した。６０ｋｄタンパク質の消化および逆相クロマトグラフィーを、実施例１に記載したように実施した。４つの蛋白質分解フラグメントを、単離および配列決定し、これらを、本明細書中でｍａｐ（１３）、ｍａｐ（１４）、ｍａｐ（１５）およびｍａｐ（１６）という。
【０２１６】
ｍａｐ（１３）のＮ末端部分アミノ酸配列を、以下：
【０２１７】
【化７】

として決定し、これをまた、配列番号２９と示した。ｍａｐ（１４）のＮ末端部分アミノ酸配列を、以下：
【０２１８】
【化８】

として決定し、これをまた、配列番号３０と示した。ｍａｐ（１５）のＮ末端部分アミノ酸配列を、以下：
【０２１９】
【化９】

として決定し、これをまた、配列番号３１と示した。ｍａｐ（１６）のＮ末端部分アミノ酸配列を、以下：
【０２２０】
【化１０】

として決定し、これをまた、配列番号３２と示した。
【０２２１】
（実施例１０）
本実施例は、Ｄ．ｆａｒｉｎａｅ ６０ｋＤ（ｍａｐＤ）アレルゲンの一部をコードする核酸分子の単離および配列決定を記載する。
【０２２２】
実施例８で先に記載したように、Ｄ．ｆａｒｉｎａｅライブラリーを調製した。Ｄ．ｆａｒｉｎａｅ ６０ｋＤタンパク質（配列番号２３）について推定されるＮ末端アミノ酸配列より、縮重合成オリゴヌクレオチドプライマーを設計した：配列番号２３の約３〜約１１のアミノ酸残基に対応するセンスプライマーであるプライマー１は、配列番号４６としても識別される配列：
【０２２３】
【化１１】

を有し、ここで、Ｈは、ＡまたはＣまたはＴを示し、Ｎは、ＡまたはＣまたはＧまたはＴを示し、そしてＹは、ＣまたはＴを示す。Ｄ．ｆａｒｉｎａｅライブラリー由来のフラグメントのＰＣＲ増幅を、標準的技術を使用して行った。配列番号４６（プライマー１）、および配列番号４７として示されるＭ１３順方向ユニバーサルプライマー：
【０２２４】
【化１２】

の組合わせを使用して、ＰＣＲ増幅産物を生成した。
【０２２５】
第二の入れ子（ｎｅｓｔｅｄ）ＰＣＲ反応を、第一のＰＣＲ反応の産物に対して行った。６０ｋＤタンパク質内部アミノ酸配列（配列番号３１）のおよそのアミノ酸残基１〜アミノ酸残基１０にわたる領域に対応する、合成オリゴヌクレオチドを合成した。このプライマー（プライマー２）は、配列番号４８として示される以下の核酸配列：
【０２２６】
【化１３】

を有し、ここで、Ｒは、ＡまたはＧを示す。プライマー２（配列番号４８）、および配列番号４９として示されるＴ７標準プライマー：
【０２２７】
【化１４】

の組合わせを使用して、ＰＣＲ増幅産物を生成した。生じたＰＣＲ産物を、標準的技術を使用するＤＮＡ配列分析に供した。
【０２２８】
このＰＣＲ産物を配列決定し、５１０ヌクレオチドを含むことを見出し、これは、ｎＤｅｒ６０_５１０として識別される。ｎＤｅｒｆ６０_５１０のコード鎖のヌクレオチド配列を、本明細書中で配列番号４３として示し、そしてその相補体を、配列番号４５として示す。配列番号４３の翻訳は、ｎＤｅｒｆ６０_５１０が、配列番号４４に示されるアミノ酸配列を有する約１７０アミノ酸の部分Ｄ．ｆａｒｉｎａｅ ６０ｋＤタンパク質（本明細書中でＰＤｅｒｆ６０_１７０と呼ぶ）をコードすることを示唆する。ここで、開始コドンは配列番号４３のおよそのヌクレオチド１〜およそのヌクレオチド３にわたり、終止コドンは、配列番号４３のおよそのヌクレオチド５０８〜およそのヌクレオチド５１０にわたるオープンリーディングフレームを推定する。ＰＤｅｒｆ６０_１７０は、推定分子量約１９．２ｋＤおよび推定ｐＩ約６．５１を有する。
【０２２９】
核酸分子ｎＤｅｒｆ６０_５１０をプローブとして使用して、全長Ｄ．ｆａｒｉｎａｅ ６０ｋＤタンパク質またはより大きな部分のＤ．ｆａｒｉｎａｅ ６０ｋＤタンパク質に対応するタンパク質をコードする核酸分子を単離した。実施例８の上記に示した手順を使用して、標準的技術を使用して^３２Ｐで放射標識した核酸配列番号４３を用いて、全Ｄ．ｆａｒｉｎａｅライブラリーをスクリーニングした。６×ＳＳＣ、５×Ｄｅｎｈａｒｄｔ溶液、０．５％ ＳＤＳ、１００ｍｇ／ｍｌ ｓｓＤＮＡ中で５５℃にて約３６時間、ハイブリダイゼーションを行った。フィルターを、２×ＳＳＣ、０．２％ ＳＤＳ中で５５℃にて１洗浄あたり３０分間３回洗浄した後、０．２×ＳＳＣ、０．２％ ＳＤＳ中で約３０分間最終洗浄した。
【０２３０】
ＰＣＲ増幅を、最初のファージプラグに対して行った。配列番号４６として示されるプライマー１、および配列番号４９として示されるＴ７標準プライマーをプライマーとして使用して、ＰＣＲ産物を生成した。この１．６キロベースのＰＣＲ産物を予備的配列分析することによって、これがＰＤｅｒｆ６０全長タンパク質をコードする完全配列を含む核酸配列を示すことが示された。
【０２３１】
配列番号４４のアミノ酸配列ＰＤｅｒｆ６０_１７０とＧｅｎＢａｎｋに報告されたアミノ酸配列との比較は、ＰＤｅｒｆ６０_１７０がＡｐｈａｎｏｄｉｄｉｕｍ ａｌｂｕｍ．由来のキチナーゼタンパク質前駆体（ＧｅｎＢａｎｋ登録番号Ｐ３２４７０）と最も高い相同性（すなわち約３９％の同一性）を示したことを示す。核酸配列配列番号４３は、ＧｅｎＢａｎｋに提出された配列のいずれにも顕著な相同性を示さなかった。
【０２３２】
（実施例１１）
本実施例は、Ｄｅｒｍａｔｏｐｈａｇｏｉｄｅｓ ｐｔｅｒｏｎｙｓｓｉｕｓ ９８ｋＤアレルゲンタンパク質をコードする核酸分子の単離を記載する。
【０２３３】
Ｄ．ｆａｒｉｎａｅ ＨＭＷ−ｍａｐ組成物をコードする３２−Ｐ標識したｃＤＮＡを用いるハイブリダイゼーションによって、Ｄ．ｆａｒｉｎａｅ ９８ｋＤアレルゲン（ｍａｐＢ）に高い相同性を有する核酸分子を、Ｄ．ｐｔｅｒｏｎｙｓｓｉｕｓ ｃＤＮＡライブラリーより単離した。
【０２３４】
Ｄ．ｐｔｅｒｏｎｙｓｓｉｕｓ ｃＤＮＡライブラリーを、以下のように調製した。総ＲＮＡを、約２ｇのＤ．ｐｔｅｒｏｎｙｓｓｉｕｓダニから、Ｃｈｏｍｚｙｎｓｋｉら、１９８７、Ａｎａｌ．Ｂｉｏｃｈｅｍ．１６２：ｐｐ１５６−１５９によって記載される酸−グアニジウム−フェノール−クロロホルム法を使用して抽出した。ポリＡ＋選択されたＲＮＡを、製造業者によって推奨される方法に従って、ｍＲＮＡ Ｐｕｒｉｆｉｃａｔｉｏｎ Ｋｉｔ（Ｐｈａｒｍａｃｉａ，Ｎｅｗａｒｋ，ＮＪから入手可能）を使用するオリゴ−ｄＴセルロースクロマトグラフィーによって、総ＲＮＡ調製物から分離した。全Ｄ．ｐｔｅｒｏｎｙｓｓｉｕｓ ｃＤＮＡライブラリーを、ＳｔｒａｔａｇｅｎｅのＺＡＰ−ｃＤＮＡ Ｓｙｎｔｈｅｓｉｓ Ｋｉｔプロトコルを使用して、λ−Ｕｎｉ−ＺＡＰ^ＴＭＸＲベクター（Ｓｔｒａｔａｇｅｎｅ，Ｌａ Ｊｏｌｌａ，ＣＡから入手可能）中に構築した。約５ミリグラム（ｍｇ）のポリＡ＋ＲＮＡを使用して、Ｄ．ｐｔｅｒｏｎｙｓｓｉｕｓ ｃＤＮＡライブラリーを生成した。
【０２３５】
ｃＤＮＡ Ｓｙｎｔｈｅｓｉｓ Ｋｉｔ（Ｓｔｒａｔａｇｅｎｅより入手可能）に記載されるプロトコルの改変を使用して、３２Ｐ−標識した、Ｄ．ｆａｒｉｎａｅ ９７ｋＤ ＭａｐＢアレルゲン（すなわち、配列番号１７）をコードするｃＤＮＡで、全Ｄ．ｐｔｅｒｏｎｙｓｓｉｕｓ ｃＤＮＡライブラリーを二連のプラークリフト（ｐｌａｑｕｅ ｌｉｆｔ）を使用してスクリーニングした。ハイブリダイゼーションを、６×ＳＳＣ（処方について、Ｓａｍｂｒｏｏｋら、同書を参照のこと）、５×Ｄｅｎｈａｒｄｔ溶液（処方について、Ｓａｍｂｒｏｏｋら、同書を参照のこと）、０．５％ ドデシル硫酸ナトリウム（ＳＤＳ）（Ｓｉｇｍａより入手可能）、および１００ｍｇ／ｍｌの一本鎖ＤＮＡ（Ｓｉｇｍａより入手可能）中で、５５℃にて約３６時間行った。２×ＳＳＣ、０．２％ ＳＤＳ中で５５℃にてフィルターを３回洗浄し（１洗浄あたり約３０分間）、続いて、０．２×ＳＳＣ、０．２％ ＳＤＳ中で５５℃にて約３０分間フィルターを最終洗浄した。Ｄ．ｐｔｅｒｏｎｙｓｓｉｕｓ ９７ｋＤアレルゲン（ｍａｐＢ）をコードする、Ｄ．ｐｔｅｒｏｎｙｓｓｉｕｓ核酸分子のプラーク精製したクローンを、ＺＡＰ−ｃＤＮＡ Ｓｙｎｔｈｅｓｉｓ Ｋｉｔ（Ｓｔｒａｔａｇｅｎｅより入手可能）に記載されるインビボ切除プロトコルに従って、ＥｘＡｓｓｉｓｔ^ＴＭヘルパーファージおよびＳＯＬＲ^ＴＭ Ｅ．ｃｏｌｉを用いて二本鎖組換え分子に変換した。Ｄ．ｐｔｅｒｏｎｙｓｓｉｕｓクローンを含むプラスミドを、標準的技術を使用するＤＮＡ配列分析に供した。ＤＮＡ配列分析（分子量および等電点（ｐＩ）の決定を含む）を、ＧＣＧ^ＴＭプログラムを使用して行った。
【０２３６】
約１６２１ヌクレオチド挿入物を含むクローンを単離した。これは、本明細書中でｎＤｅｒｐ９８_１６２１と呼ぶ、配列番号３４として示されるコード鎖および配列番号３６として示される相補鎖を有する全長コード領域を含む。明白な開始コドンおよび終止コドンは、それぞれ配列番号３４のヌクレオチド１４〜ヌクレオチド１６、およびヌクレオチド１５４１〜ヌクレオチド１５４３にわたる。推定のポリアデニル化シグナル（５’ＡＡＴＡＡＡ３’）は、配列番号３４のヌクレオチド１５８４〜ヌクレオチド１５８９にわたる領域に位置する。
【０２３７】
配列番号３４の翻訳は、配列番号３５として示されるアミノ酸配列であり、ＰＤｅｒｐ９８_５０９として示される、約５０９アミノ酸のタンパク質を生じる。ＰＤｅｒｐ９８_５０９をコードするコード領域からなる核酸分子は、本明細書中でｎＤｅｒｐ９８_１５２７として呼ばれ、その核酸配列は、配列番号３７（コード鎖）および配列番号３９（相補鎖）として示される。本明細書中で配列番号３８としても示されるＰＤｅｒｐ９８_５０９のアミノ酸配列は、推定分子量約５８．９ｋＤおよび推定ｐＩ約５．６１を有する。ＰＤｅｒｐ９８_５０９の分析は、およそのアミノ酸１〜およそのアミノ酸１９にわたるシグナルペプチドの存在を示唆する。提唱した成熟タンパク質（本明細書中でＰＤｅｒｐ９８_４９０として示される）は、約４９０アミノ酸を含み、そして本明細書中で配列番号４１として示される。ＰＤｅｒｐ９８_４９０のアミノ酸配列によって、このタンパク質が、推定分子量約５６．８ｋＤおよび推定ｐＩ約５．４９、ならびに２つのアスパラギン連結グリコシル化部位（それぞれ、およそのアミノ酸１１５〜およそのアミノ酸１１７、およびおよそのアミノ酸２４０〜アミノ酸２４２に伸展する）を有することを予想する。ＰＤｅｒｐ９８_４９０をコードする核酸分子は、配列番号４０によって示されるコード鎖および配列番号４２によって示される相補鎖を有するｎＤｅｒｐ９８_１４７０として公知である。
【０２３８】
ＢＬＡＳＴ検索を、以前に記載されるように実施した。ＰＤｅｒｐ９８_５０９（配列番号３５）は、Ｍａｎｄｕｃａ ｓｅｘｔａキチナーゼ（ＳｗｉｓｓＰｒｏｔ登録番号ｐ３６３６２）とアミノ酸レベルで最も高い相同性を示し、約３４％の同一性を有した。ｎＤｅｒｐ９８_１６２１（配列番号３４）は、Ｃｈｅｌｏｎｕｓ ｓｐ．キチナーゼ（登録番号Ｕ１０４２２）に核酸レベルで最も高い相同性を示し、約４９％の同一性を有した。Ｄ．ｆａｒｉｎａｅの９８ｋＤのアレルゲンタンパク質についてのコード領域に対応するｃＤＮＡ領域と、Ｄ．ｐｔｅｒｏｎｙｓｓｉｕｓの９８ｋＤのアレルゲンタンパク質についてのコード領域に対応するｃＤＮＡ領域との比較は、約８４％の同一性を示す。
【０２３９】
（実施例１４）
本実施例は、Ｄ．ｆａｒｉｎａｅ ＨＭＷ−ｍａｐ組成物の、ダニアレルゲンに対してアレルギー性であることが公知の、ヒトから単離されたヒト血清中のヒトＩｇＥへの結合を示す。
【０２４０】
ＲＡＳＴすなわちラスト法（ｒａｄｉｏ−ａｌｌｅｒｇｏ−ａｂｓｏｒｂｅｎｔ ｔｅｓｔ）と呼ばれる技術を使用した。なぜなら、ヒト血清中に存在するヒトＩｇＥの量が非常に低いためである。ＲＡＳＴを、本質的に、Ａａｌｂｅｒｓｅ，ＲＣら（１９８１）Ｊ．Ａｌｌｅｒｇｙ Ｃｌｉｎ Ｉｍｍｕｎ．６８：３５６〜３６４頁において記載されるように実施した。単位ＩＵ／ｍｌを計算するために、標準曲線を、Ｄｅｒｐ２に対する十分に特徴付けられたキメラヒト／マウスＩｇＥモノクローナル抗体（ヒトＩｇＥ／モノクローナル抗Ｄｅｒｐ２、Ｓｃｈｕｕｒｍａｎら（１９９７）Ｊ Ａｌｌｅｒｇｙ Ｃｌｉｎ Ｉｍｍｕｎｏｌ．９９：５４５〜５５０頁の手順に従う）のいくつかの希釈物を用いてＲＡＳＴを行なうことによって導き出した。
【０２４１】
手短に言うと、５０μｇのＨＭＷ−ｍａｐ組成物（実施例１に記載されるように精製した）を、製造業者のプロトコルに従って、５０ｍｇのＣＮＢｒ活性型Ｓｅｐｈａｒｏｓｅ４Ｂ（Ｐｈａｒｍａｃｉａ，Ｐｉｓｃａｔａｗａｙ，ＮＪより入手可能）に結合させた。ヒト血清を、全ダニＤ．ｆａｒｉｎａｅ抽出物についてポジティブなＲＡＳＴに基づいて選択し（全部で１７個の異なるサンプル）、ポジティブなＲＡＳＴ数は、１ＩＵ／ｍｌより大きい。２つのネガティブ（０．３ＩＵ未満）コントロール血清もまた含んだ。
【０２４２】
各々の個体の血清サンプルを試験するために、０．５ｍｇのＤ．ｆａｒｉｎａｅ ＨＭＷ−ｍａｐ組成物結合Ｓｅｐｈａｒｏｓｅを、総容量３００μｌのＰＢＳ−Ｔ（０．１％容量／容量Ｔｗｅｅｎ−２０（Ｓｉｇｍａより入手可能）を添加したリン酸緩衝化生理食塩水）中５０μｌの血清と共にインキュベートした。インキュベーションは、振盪しながら２７℃で一晩行なった。インキュベーション後、結合したＳｅｐｈａｒｏｓｅをＰＢＳ−Ｔで５回洗浄した。総容量７５０μｌ中（４．５％（ｖ／ｖ）ウシ血清および０．５％（ｖ／ｖ）ヒツジ血清を含むＰＢＳ−Ｔ中で希釈した）放射性標識した（^１２５−ヨウ素）ヒツジ抗ヒトＩｇＥ（標準的な放射ヨウ素化標識プロトコルによって作製された）を添加し、そして２７℃で一晩インキュベートした。インキュベーション後、結合したＳｅｐｈａｒｏｓｅを、ＰＢＳ−Ｔで４回洗浄し、そしてγカウンターでカウントしてＨＭＷ−ｍａｐ組成物結合Ｓｅｐｈａｒｏｓｅに結合した放射性標識したヒツジ抗ヒトＩｇＥの量を決定した。結果を表４に示す。
【０２４３】
（表４．Ｄ．ｆａｒｉｎａｅ由来のＨＭＷ−ｍａｐ組成物へのヒトＩｇＥの結合）
【０２４４】
【表４】

Ｄ．ｆａｒｉｎａｅ全ダニ抽出物に対して感受性を示した患者の約７５％（１５人中１１人）は、ＨＭＷ−ｍａｐ組成物抗原に対して感受性であり、このことは、ＨＭＷ−ｍａｐ組成物抗原が、Ｄ．ｆａｒｉｎａｅ感受性ヒトに対する主要な抗原であることを示す。ＨＭＷ−ｍａｐ組成物に対する感受性は、０．５ＩＵ以上のＲＡＳＴとして規定された。
【０２４５】
（実施例１５）
本実施例は、実施例１に記載されるＤ．ｆａｒｉｎａｅ ＨＭＷ−ｍａｐ組成物が糖タンパク質を含むことを実証する。
【０２４６】
実施例１に従って調製された約５．４μｇのＤ．ｆａｒｉｎａｅ ＨＭＷ−ｍａｐ組成物を、ＳＤＳ ＰＡＧＥに適用し、そして電気泳動を標準的な技術に従って行なった。このタンパク質を、標準的な技術に従ってニトロセルロース膜にブロットし、そして糖タンパク質を、製造業者のプロトコルを使用して、ＤＩＧ^ＴＭグリシン検出キット（Ｂｏｅｈｒｉｎｇｅｒ Ｍａｎｎｈｅｉｍ，Ｉｎｄｉａｎａｐｏｌｉｓ，ＩＮより入手可能）を使用して検出した。ＨＭＷ−ｍａｐ領域に対応する領域は、キットを用いて陽性反応を示し、このことは、ＨＭＷ−ｍａｐ組成物が糖タンパク質を含むことを示す。
【０２４７】
（実施例１６）
本実施例は、Ｄ．ｆａｒｉｎａｅ ＨＭＷ−ｍａｐ組成物が、化学的手段および酵素的手段の両方によって、アミノ酸残基が取り除かれる場合でさえ、アレルゲンとしてのその特徴を保持することを示す。この結果は、主なエピトープが、ＨＭＷ−ｍａｐ組成物上のＮ連結グリコシル化部位またはＯ連結グリコシル化部位に結合した多糖を含む、炭水化物エピトープであり得ることを示唆する。
【０２４８】
（Ａ．化学的手段によるタンパク質除去（タンパク質のβ除去））
１２μｇ（マイクログラム）のＨＭＷ−ｍａｐ組成物（実施例１に記載されるように精製された）を、１００μｌ（マイクロリットル）の蒸留した脱イオン水中に溶解した。この混合物に、５μｌの１０Ｍ（モーラー）ＮａＯＨおよび３．８ｍｇ（ミリグラム）ＮａＢＨ_４（Ｓｉｇｍａより入手可能）を添加して、０．５Ｍ ＮａＯＨおよび１Ｍ ＮａＢＨ_４の最終濃度を得た。この反応混合物を、５０℃で３０分間加熱し、次いで冷却し、そして１００μｌのアセトンを添加した。この混合物に、十分な量（すなわち、約１５０μｌ）のＤｏｗｅｘ５０（Ｈ＋）（Ｐｈａｒｍａｃｉａより入手可能）を添加して、わずかに酸性の溶液を作製した。Ｄｏｗｅｘ５０は、タンパク質を吸収および除去し、上清に任意の糖部分を残す。この混合物を、マイクロ遠心分離機で遠心分離し、そして１００μｌの水で３回洗浄した。遠心分離からの上清を合わせて、乾燥するまで蒸発させて、メタノール：ＨＣｌ溶液（１０００：１ ｖ／ｖ）で５回洗浄し、各洗浄後、乾燥するまで蒸発させて塩を取り除く。この混合物を、１００μｌの水に溶解し、そして一部（２０μｌ）を標準的な技術を使用するＳＤＳ−ＰＡＧＥによって分析し、そしてクマシーブルー染色および銀染色の両方を使用して、化学的に処理されたサンプル中のタンパク質の量を決定した。クマシーブルー染色および銀染色のいずれによってもタンパク質は検出されず、このことは、タンパク質の除去を示す。タンパク質上の任意の糖部分は、これらの条件によって影響されなかった。
【０２４９】
各サンプルからの残渣の残りを、実施例４に記載されるように、ＥＬＩＳＡ分析に供した。手短に言うと、１００ｎｇのＨＭＷ−ｍａｐ組成物のβ除去サンプルまたは非β除去サンプルのいずれかをＩｍｍｕｌｏｎプレートにコーティングし、そしてＤ．ｆａｒｉｎａｅ感受性イヌ血清プール、Ｄ．ｆａｒｉｎａｅ感受性ネコ血清プール、および（ＥＬＩＳＡによって測定される場合）Ｄ．ｆａｒｉｎａｅ感受性または非感受性のいずれかである種々の別々のイヌ血清を用いて、ＥＬＩＳＡを、実施例４に記載されるように実行した。結果を表５に示す。
【０２５０】
（表５．ＨＭＷ−ｍａｐ組成物およびβ除去ＨＭＷ−ｍａｐ組成物（これは、炭水化物のみである）に対するイヌ血清およびネコ血清の反応性）
【０２５１】
【表５】

表５からの結果は、β除去ＨＭＷ−ｍａｐ組成物サンプルが、Ｄ．ｆａｒｉｎａｅ ＨＭＷ−ｍａｐ組成物に感受性であるイヌ血清およびネコ血清由来のＩｇＥを結合する能力をまだ保持することを示し、このことは、タンパク質に結合したグリカンが、ＨＭＷ−ｍａｐ組成物アレルゲンタンパク質の主要なエピトープを構成することを示す。
【０２５２】
（Ｂ．酵素的手段によるタンパク質除去）
１４μｇのＨＭＷ−ｍａｐ組成物（実施例１に記載されるように精製した）を、１μｇ エンドプロテイナーゼＫ（Ｓｉｇｍａより入手可能）で消化して、分子のタンパク質部分を除去する。消化反応を、５６℃で２４時間行ない、その後、反応物中のエンドプロテイナーゼを、煮沸水中で１０分間熱変性させた。
【０２５３】
この反応物の一部を、標準的な技術を使用するＳＤＳ−ＰＡＧＥによって分析し、そしてクマシーブルー染色および銀染色の両方を使用して、酵素的に消化したサンプル中のタンパク質の存在を検出した。ＨＭＷ−ｍａｐ組成物は、クマシーブルー染色および銀染色のいずれによっても検出されず、このことは、ＨＭＷ−ｍａｐ組成物の除去を示す。タンパク質上のグリコシル化部位を介して結合したグリカンはいずれも、これらの条件によって影響されなかった。
【０２５４】
酵素的に消化した反応物の残りを、実施例４に記載される様式で、ＥＬＩＳＡによって試験した。手短に言うと、１００ｎｇのＨＭＷ−ｍａｐ組成物のプロテイナーゼＫ消化サンプルまたはプロテイナーゼＫ未消化サンプルのいずれかをＩｍｍｕｌｏｎプレートにコーティングし、そしてＥＬＩＳＡを、（ＥＬＩＳＡによって測定される場合）Ｄ．ｆａｒｉｎａｅ感受性または非感受性のいずれかである種々の別々のイヌ血清を用いて、実施例４に記載されるように実行した。結果を表６に示す。
【０２５５】
（表６．ＨＭＷ−ｍａｐ組成物およびエンドプロテイナーゼＫ消化ＨＭＷ−ｍａｐ組成物に対するイヌ血清の反応性）
【０２５６】
【表６】

表６からの結果は、プロテイナーゼＫ消化ＨＭＷ−ｍａｐ組成物サンプルが、Ｄ．ｆａｒｉｎａｅ ＨＭＷ−ｍａｐ組成物に感受性であるイヌ血清およびネコ血清由来のＩｇＥを結合する能力をまだ保持することを示し、このことは、タンパク質に結合したグリカンが、ＨＭＷ−ｍａｐ組成物上の主要なエピトープを構成することを示唆する。
【０２５７】
（実施例１７）
本実施例は、ＨＭＷ−ｍａｐ組成物からＮ連結グリカンを除去する試みを記載する。
【０２５８】
ＨＭＷ−ｍａｐ組成物（２μｇ）（実施例１におけるように精製された）を、製造業者の指示に従って、Ｎ−グリコシダーゼＦ（Ｂｏｅｈｒｉｎｇｅｒ−Ｍａｎｎｈｅｉｍより入手可能）を用いて消化した。消化物を、ＳＤＳ−ＰＡＧＥによって分析し、そして標準的なプロトコルに従って染色した。２μｇ Ｆｅｔｕｉｎ（Ｓｉｇｍａより入手可能）を、ポジティブなＮ連結グリコシル化タンパク質コントロールとして使用した。ＳＤＳ−ＰＡＧＥの分析は、インタクトなｍａｐ Ｂタンパク質および消化したｍａｐ Ｂタンパク質の分子量の間に見かけの差異は存在しないことを示した。ポジティブコントロールのｆｅｔｕｉｎは、Ｎ−グリコシダーゼＦでの消化後に分子量の減少を示さなかった。この結果は、ＨＭＷ−ｍａｐ組成物上にＮ連結グリカンが存在しないこと、またはＨＭＷ−ｍａｐ組成物上に小さいサイズのＮグリカンのみが存在することを示す。
【０２５９】
（実施例１８）
本実施例は、全長Ｄｅｒｍａｔｏｐｈａｇｏｉｄｅｓ ｆａｒｉｎａｅの６０ｋＤアレルゲンをコードする核酸分子の単離および配列決定を記載する。
【０２６０】
この核酸分子を、実施例１０に記載されるＤｅｒｍａｔｏｐｈａｇｏｉｄｅｓ ｆａｒｉｎａｅの６０ｋＤアレルゲンの一部をコードする^３２Ｐ−標識されたｃＤＮＡとハイブリダイズする能力によって、Ｄｅｒｍａｔｏｐｈａｇｏｉｄｅｓ ｆａｒｉｎａｅのｃＤＮＡライブラリーから単離した。
【０２６１】
Ｄｅｒｍａｔｏｐｈａｇｏｉｄｅｓ ｆａｒｉｎａｅ ｃＤＮＡ ライブラリーを、以下のように調製した。全ＲＮＡを、Ｃｈｏｍｚｙｎｓｋｉら、１９８７、Ａｎａｌ．Ｂｉｏｃｈｅｍ．１６２，１５６〜１５９によって記載される方法に類似の、酸グアニジニウム−フェノール−クロロホルム法を用いて、約２グラムのＤ．ｆａｒｉｎａｅダニから抽出した。ポリＡ^＋選択したＲＮＡを、製造者によって推奨される方法に従って、ｍＲＮＡ精製キット（Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ，Ｎｅｗａｒｋ，ＮＪから入手可能）を用いて、オリゴｄＴセルロースクロマトグラフィーによって、全ＲＮＡ調製物から分離した。全ダニｃＤＮＡライブラリーを、ＳｔｒａｔａｇｅｎｅのＺＡＰ−ｃＤＮＡ Ｓｙｎｔｈｅｓｉｓ Ｋｉｔプロトコルを用いて、ｌａｍｂｄａ−Ｕｎｉ−ＺＡＰ^ＴＭ ＸＲベクター（Ｓｔｒａｔａｇｅｎｅから入手可能）中に構築した。約５μｇのポリＡ^＋ＲＮＡを使用して、Ｄ．ｆａｒｉｎａｅ ｃＤＮＡライブラリーを生成した。
【０２６２】
ｃＤＮＡ Ｓｙｎｔｈｅｓｉｓ Ｋｉｔ中に記載されるプロトコルの改変を用いて、^３２Ｐ−標識されたｃＤＮＡ ｎＤｅｒｆ６０_５１０を有する複数のプラークリフトを用いて、全ダニｃＤＮＡライブラリーをスクリーニングした。ハイブリダイゼーションを、６×ＳＳＣ、５×Ｄｅｎｈａｒｄｔ溶液、０．５％ＳＤＳ、１００ｍｇ／ｍｌのｓｓＤＮＡで、５２℃で１８時間行った。フィルターを、各洗浄あたり、２×ＳＳＣ、０．２％のＳＤＳ中、５５℃で３０分で、２回洗浄した後、約０．２×ＳＳＣを用いる以外は同じ緩衝液中で、３０分間最終の洗浄を行った。Ｄ．ｆａｒｉｎａｅの６０ｋＤアレルゲンをコードする核酸分子のプラーク精製クローンを、ＺＡＰ−ｃＤＮＡ Ｓｙｎｔｈｅｓｉｓ Ｋｉｔ（Ｓｔｒａｔａｇｅｎｅから入手可能）中に記載されるインビボでの切り出しプロトコルに従って、ＥｘＡｓｓｉｓｔ^ＴＭヘルパーファージおよびＳＯＬＲ^ＴＭ Ｅ．ｃｏｌｉを用いて、二本鎖組換え分子へと変換し、これを本明細書中において、ｎＤｅｒｆ６０_１４５５として示す。二本鎖プラスミドＤＮＡを、アルカリ溶解プロトコル（例えば、Ｓａｍｂｒｏｏｋら、前出に記載される）を用いて調製した。
【０２６３】
（実施例１９）
本実施例は、本発明のＤ．ｆａｒｉｎａｅ核酸分子の配列決定を記載する。
【０２６４】
ｎＤｅｒｆ６０_１４５５を含むプラスミドを、Ｓａｎｇｅｒジデオキシ鎖停止法によって、Ａｍｐｌｉ Ｔａｑ ＤＮＡポリメラーゼ、ＦＳ（Ｐｅｒｋｉｎ Ｅｌｍｅｒ Ｃｏｒｐｏｒａｔｉｏｎ，Ｎｏｒｗａｌｋ，ＣＴから入手可能）を用いたＰＲＩＳＭ^ＴＭ Ｒｅａｄｙ Ｄｙｅ Ｔｅｒｍｉｎａｔｏｒ Ｃｙｃｌｅ Ｓｅｑｕｅｎｃｉｎｇ Ｋｉｔを用いて、配列決定した。ＰＣＲ伸長を、ＧｅｎｅＡｍｐ^ＴＭ ＰＣＲ Ｓｙｓｔｅｍ ９６００（Ｐｅｒｋｉｎ−Ｅｌｍｅｒから入手可能）中で行った。過剰の色素ターミネーターを、製造者の標準的なプロトコルに従って、Ｃｅｎｔｒｉｆｌｅｘ^ＴＭ Ｇｅｌ Ｆｉｌｔｒａｔｉｏｎ Ｃａｒｔｒｉｄｇｅ （Ａｄｖａｎｃｅｄ Ｇｅｎｅｔｉｃｓ Ｔｅｃｈｎｏｌｏｇｉｅｓ Ｃｏｒｐｏｒａｔｉｏｎ，Ｇａｉｔｈｅｒｓｂｕｒｇ，ＭＤから入手可能）を用いて、伸長産物から除去した。サンプルを、ＡＢＩプロトコルに従って再懸濁し、そして、Ｐｅｒｋｉｎ−Ｅｌｍｅｒ ＡＢＩ ＰＲＩＳＭ^ＴＭ ３７７ Ａｕｔｏｍａｔｅｄ ＤＮＡ Ｓｅｑｕｅｎｃｅｒにかけた。配列の編集およびオープンリーディングフレームの決定を含むＤＮＡ配列分析を、ＧＣＧ^ＴＭプログラム（Ｇｅｎｅｔｉｃｓ Ｃｏｍｐｕｔｅｒ Ｇｒｏｕｐ，Ｍａｄｉｓｏｎ，ＷＩから入手可能）を用いて行った。分子量および当電点（ｐＩ）の決定を含むタンパク質配列分析を、ＧＣＧ^ＴＭプログラムを用いて行った。
【０２６５】
完全なｎＤｅｒｆ６０_１４５５核酸分子の約１４５５ヌクレオチドコンセンサス配列を決定した；２つの相補鎖の配列が、配列番号５０（コード鎖）および配列番号５２（相補鎖）として存在する。この２つの相補的な鎖の配列を、配列番号５０（コード鎖）および配列番号５２（相補鎖）として示す。ｎＤｅｒｆ６０_１４５５配列は、全長のコード領域を含む。明らかな開始コドンおよび停止コドン範囲は、それぞれ、配列番号５０の１４〜１６および１４００〜１４０２のヌクレオチドにわたる。推定ポリアデニル化シグナル（５’ＡＡＴＡＡＡ３’）は、配列番号５０のヌクレオチド約１４０８〜１４１３にわたる領域内に配置される。
【０２６６】
配列番号５０の翻訳は、ＰＤｅｒｆ６０_４６２と示される、４６２アミノ酸のタンパク質を生じ、このアミノ酸配列は、配列番号５１に示される。ＰＤｅｒｆ６０_４６２をコードするコード領域からなる核酸分子は、本明細書中で、ｎＤｅｒｆ６０_１３８６といい、この核酸配列は、配列番号５３（コード鎖）および配列番号５４（相補鎖）に示される。ＰＤｅｒｆ６０_４６２のアミノ酸配列（すなわち、配列番号５１）は、ＰＤｅｒｆ６０_４６２が、約５２．１ｋＤの推定分子量を有し、約５．７３の推定ＰＩを有することを予測する。配列番号５１の分析は、アミノ酸１〜アミノ酸２５にわたるアミノ酸の範囲によってコードされるシグナルペプチドの存在を示唆する。提唱された成熟タンパク質は、本明細書中でＰＤｅｒｆ６０_４３７と示され、このタンパク質は、本明細書中で配列番号５６と示される約４３７アミノ酸を含む。ＰＤｅｒｆ６０_４３７のアミノ酸配列（すなわち、配列番号５６）は、ＰＤｅｒｆ６０_４６２が、約５０．０ｋＤの推定分子量を有し、約５．６１の推定ＰＩ、およびアミノ酸３１３〜アミノ酸３１５にわたる１つの推定アスパラギン連結グリコシル化部位を有することを予測する。この成熟タンパク質をコードする核酸分子を、配列番号５５と示し、その逆位相補体を、配列番号５７と示す。
【０２６７】
ＢＬＡＳＴｐ検索を、以下に従って行った：Ａｌｔｓｃｈｕｌら，（１９９０），Ｊ．Ｍｏｌ．Ｂｉｏｌ．２１５：４０３〜４１０；およびＡｌｔｓｃｈｕｌら，（１９９７），Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｒｅｓ． ２５： ３３８９−３４０２。タンパク質検索を、配列番号５１を用いて行い、この配列番号５１は、キチナーゼ分子に対して有意な相同性を示した。アミノ酸レベルでの相同性検索のスコア一致が最も高いのは、ＰＩＲ受託番号Ａ５３９１８：Ｃｈｅｌｏｎｕｓ ｓｐキチナーゼ前駆体であり、これは、配列番号５１と約３２％同一であった。ヌクレオチドレベルで、この検索を、配列番号５３を用いて行い、この配列番号５３は、データベース中の任意の配列と有意な類似性を示さなかった。配列分析を、上述のようにＧＣＧ ＧＡＰプログラムを用いて行った。
【０２６８】
（実施例２０）
本実施例は、Ｄ．ｆａｒｉｎａｅ ＨＭＷ−ｍａｐ組成物（これは、Ｄｅｒ ｆ １５ともいわれる）の特徴付けをさらに記載する。
【０２６９】
実施例８の核酸分子ｎＤｅｒｆ９８_１７５２を、適切な発現ベクターに挿入し、Ｅ．ｃｏｌｉおよびＰ．ｐａｓｔｏｒｓ中で発現させた。得られたタンパク質（ＰＤｅｒｆ９８_５５５）が、Ｅ．ｃｏｌｉまたはＰ．ｐａｓｔｏｒｉｓ中で発現される場合、実施例４に記載されるように産生した、感作されたイヌ血清は、組換えタンパク質を認識できなかった。このことは、実施例１のネイティブなＤ．ｆａｒｉｎａｅ ＨＭＷ−ｍａｐ組成物（これはまた、ネイティブなＤｅｒｆ１５ともいう）が使用される場合に得られる陽性の結果と対照的である；実施例４を参照のこと。
【０２７０】
Ｅ．ｃｏｌｉ中で発現されるタンパク質の非反応性は、実施例１６に示される結果と一致し、この結果は、ネイティブなＨＭＷアレルゲンが、アミノ酸が除去された後でさえ、アレルゲンとしての特性を保持していることを示した。
【０２７１】
これらの結果を全て合わせると、主要なエピトープは、分子または他の二次的改変のいくつかの炭化水素領域であることが示唆される。
【０２７２】
ネイティブなＤｅｒｆ１５タンパク質の抗原性は、過ヨウ素酸処理の後に失われず；一般的に、炭化水素エピトープは、さらなる基でさらに置換されたエピトープ、またはジェミナルでないヒドロキシル基を有する珍しい糖を有するエピトープを除いて、過ヨウ素酸塩によって破壊される。
【０２７３】
ネイティブなＤｅｒｆ１５抗原を、炭化水素含量に関して分析した。炭化水素の実質的な量が見い出された（約３０重量％）。特に、約２．８重量％の抗原から構成されるマンノース；ガラクトース約２３．２％；グルコース約４．３％（グリコシル残基の存在は、グルコースがしばしば糖タンパク質を含むため、仮の値であるとみなされなければならない）；そして検出可能なレベルでのＨｅｘＮＡｃ；さらなる観察により、ＨｅｘＮＡｃがＧｌｃＮＡｃおよびＧａｌＮＡｃであることが明らかとなった。
【０２７４】
ネイティブなＤｅｒｆ１５タンパク質を、ＮａＢＨ_４存在下にて塩基で処理して、Ｐ−４サイズ排除クロマトグラフィーによって分析した。Ｄｅｒｆ１５中に存在するＯ−連結オリゴ糖によって、このカラムが役に立たないことを見い出した。この結果は、非常に大きなＯ−連結オリゴ糖、またはオリゴ糖上のサルフェートのような酸性基の存在のいずれかと一致する。サルフェートの存在または非存在をより直接的に決定するための試みは、あいまいな結果を与えた。
【０２７５】
Ｄｅｒｆ１５を、ｐＨ４、ｐＨ５、およびｐＨ７で、３７℃にて一晩処理した。次いで、得られたサンプルを、このタンパク質に対する抗体またはＤｅｒｆ１５と反応性であることが公知のイヌ血清でプローブした。ｐＨ５およびｐＨ７で処理したサンプルにおいて、イヌ抗血清エピトープの全てが破壊されたが、ｐＨ４で処理されたサンプルにおいて、いくらか活性が残った。抗Ｄｅｒｆ１５抗体は、脱グリコシル化が起こったかのように、全てのｐＨで、Ｄｅｒｆ１５の分子量が減少したことを示し、ｐＨ４では元の物質がいくらか残っている。この変化が、Ｄｅｒｆ１５タンパク質による自己触媒であったかまたは化学的に生じたか否かは、知られていない；理論に縛られないが、エピトープの喪失がこのような温和な条件下で生じるため、自己触媒作用が起こったと考えられる。
【０２７６】
（実施例２１）
本実施例は、ネコ血清中のネコＩｇＥに対する、いくつかのイエダニ（ＨＤＭ）アレルゲンの結合を記載する。
【０２７７】
イエダニに対するネコのＩｇＥ応答のアレルゲンプロフィールは、イヌのものとは異なっているようである。Ｈｅｓｋａ’ｓ Ｖｅｔｅｒｉｎａｒｙ Ｄｉａｇｎｏｓｔｉｃ Ｌａｂｏｒａｔｏｒｉｅｓ（ＶＤＬ）に２０００年１月に提出されたネコ血清に対するＩｇＥ試験の結果の検査は、全てのアレルゲン特異的なＩｇＥ陽性のネコの４０％が、抗ＨＤＭ ＩｇＥを有したことを示した。これらのネコの全ては、Ｄ．ｆａｒｉｎａｅおよびＤ．ｐｔｅｒｏｎｙｓｓｉｎｕｓの両方に対して陽性であった。Ｄ．ｆａｒｉｎａｅに関して陽性であることが公知の８８の血清を、Ｄｅｒｆ１、Ｄｅｒｆ２、Ｄｅｒｆ１５、および６０ｋＤアレルゲンの高度に精製された調製物に対して、ＥＬＩＳＡによってアッセイした。このアッセイにおいて、３２％のネコが、Ｄｅｒｆ１について陽性であり、４２％が、Ｄｅｒｆ２について陽性であり、６８％がＤｅｒｆ１５について陽性であり、そして８６％が、６０ｋＤアレルゲンについて陽性であった。
【０２７８】
本発明の種々の実施形態が詳細に記載されているが、これらの実施形態の改変および適応が、当業者に思い浮かぶことは、明らかである。しかし、このような改変および適応が、添付の特許請求の範囲に記載されるような、本発明の範囲内であることが明らかに理解されるべきである。
【図面の簡単な説明】
【図１】
図１は、１２％Ｔｒｉｓ−グリシンＳＤＳ−ＰＡＧＥにより分離された高分子量のＤｅｒ ｆタンパク質を示す。
【図２】
図２は、１４％Ｔｒｉｓ−グリシンＳＤＳ−ＰＡＧＥにより分離された約６０ｋＤのＤｅｒ ｆタンパク質を示す。[0001]
(Field of the Invention)
The present invention relates to high molecular weight Dermatophagoides proteins, nucleic acid molecules, and therapeutic and diagnostic agents derived from such proteins.
[0002]
(Background of the Invention)
Immunoglobulin E (IgE), which mediates allergic symptoms, afflicts many animals. IgE antibody products in animals can induce pathogenic IgE responses, including, for example, atopic diseases, asthma and rhinitis. Allergens are proteins or peptides that are characterized by their ability to induce a pathogenic IgE response in susceptible individuals.
[0003]
House dust mite (eg, Dermatophagoides farinae and Dermatophagoides pteronyssinus; Der f and Der p, respectively) allergens are major causative agents associated with IgE-mediated etiology. Previous investigators have reported two major groups of house dust mite allergy in humans (Group I (Der fI and Der pI, Mr 25,000) and Group 2 (Der fII and Der pII, Mr 14,000); Chapman et al., Allergy, 52, pp. 37-379, 1997). Previous investigators have disclosed nucleotide and / or amino acid sequences for: Der fI, Der fII, Der pI, and Der pII, US Pat. No. 5,552,142 (Thomas et al., Published September 3, 1996), US Patent No. 5,460,977 (Ando et al., Published October 24, 1995), PCT Patent Application Publication No. WO 95/28424 (Chen et al., Published October 26, 1995). ), US Patent No. 5,433,948 (Thomas et al., Issued July 18, 1995), PCT Patent Application Publication No. WO93 / 08279 (Garmen et al., Issued March 4, 1993) or Chapman, supra. Der pIII, PCT Patent Application Publication No. WO 95/15976 (Thomas et al., 1995). Der pVII, PCT Patent Application Publication No. WO 94/20614 (Thomas et al., September 15, 1994); 40 kilodalton (kd) Der f allergen, US Pat. No. 5,405,758 (Oka). Et al., April 11, 1995); U.S. Pat. No. 5,314,991 (Oka et al., May 24, 1994); 70-kd Der f allergen, a heat shock protein (Hsp70); Aki et al., J. . Biochem. 115, 435-440, 1994; or Noli et al., Vet. Immunol. Immunopath. Vol. 52, pp. 147-157, 1996; and 98-k Der f paramyosin-like allergen, Tsai et al. Allergy Clin. Immunol. 102, 295-303, 1998. These published sequences disclose neither the tick allergy nucleic acid molecules nor the proteins of the invention, and also the relevance of such proteins that are immunoreactive with IgE antibodies in dog, cat or human serum. Neither suggest nor predict.
[0004]
The products and processes of the present invention are needed in the field of providing specific detection and treatment of mite allergy.
[0005]
(Summary of the Invention)
The present invention relates to novel proteins having a molecular weight of about 60 (kd or kD) 70 kD or about 98 kD to about 109 kD. Such proteins comprise at least one epitope of a Dermatophagoides mite protein allergen and are designated herein as Der HMW-map proteins. Preferred proteins are Dermatophagoides farinae or Dermatophagoides pteronyssius proteins. The invention also provides proteins that are fragments or peptides of the full-length or mature proteins, as well as any antibodies, mimetopes, or muteins of such proteins. The invention also provides nucleic acid molecules encoding any of such proteins, as well as the complements thereof. The invention also provides methods for obtaining such proteins, nucleic acid molecules, antibodies, mimetopes, or muteins, as well as methods for using such compounds in diagnostic or therapeutic applications. The present invention also relates to reagents containing non-proteinaceous epitopes that bind to mite allergic dog or cat IgE, as well as antibodies raised against such epitopes. The present invention also relates to therapeutic compositions or assay kits comprising such non-proteinaceous epitopes, as well as methods for identifying and / or desensitizing animals susceptible to an allergic response to ticks, the method comprising: Includes the use of non-proteinaceous epitopes.
[0006]
One embodiment of the invention is at least one of the following isolated nucleic acid molecules: (a) a nucleic acid molecule comprising at least about 150 nucleotides, wherein such a nucleic acid molecule comprises 1 × SSC and Hybridizes to a nucleic acid molecule comprising at least one of the following nucleic acid sequences in a solution containing 0% formamide at a temperature of about 50 ° C.): SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, A protein comprising the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 45, and SEQ ID NO: 33 And (b) a nucleic acid molecule comprising any fragment of the nucleic acid molecule of (a), wherein Instrument comprises at least about 15 nucleotides). The present invention also includes recombinant molecules, recombinant viruses and cells containing such nucleic acid sequences, and methods of producing them.
[0007]
Another embodiment of the invention is an isolated protein encoded by at least one of the following nucleic acid molecules: (a) a nucleic acid molecule comprising at least about 150 nucleotides, wherein such a nucleic acid molecule is Hybridizes to a nucleic acid molecule comprising at least one of the following nucleic acid sequences in a solution containing 1 × SSC and 0% formamide at a temperature of about 50 ° C.): SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 A nucleic acid sequence encoding a protein comprising the amino acid sequence of SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 45, and SEQ ID NO: 33; and (b) any fragment of the nucleic acid molecule of (a). A nucleic acid molecule, wherein the fragment comprises at least about 15 nucleotides. An isolated protein of the invention can also be encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions to the complement of a nucleic acid molecule that encodes a protein having at least one of the following amino acid sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 41 and SEQ ID NO: 44. The invention also includes antibodies that selectively bind to a protein of the invention, as well as methods of producing and using such proteins or antibodies.
[0008]
The invention also includes therapeutic compositions for treating an allergic response to mites. Such therapeutic compositions comprise at least one of the following desensitizing compounds: (a) an isolated nucleic acid molecule of the invention; (b) an isolated mite allergic protein of the invention; ) Mimetopes of such mite allergic proteins; muteins of such mite allergic proteins; (e) antibodies to such mite allergic proteins; and (f) inhibitors of the binding of mite allergic proteins to IgE. Also included are methods of desensitizing a host animal to an allergic response to mites. Such methods include administering a therapeutic composition of the present invention to an animal.
[0009]
One embodiment of the present invention is an assay kit that tests whether an animal is susceptible to or has an allergic response to mites. Such a kit comprises an isolated protein of the invention, as well as means for determining whether the animal is susceptible to, or has, the allergic response. Such means include the use of such proteins to identify animals that are susceptible to or have an allergic response to mites. The invention also includes a method of identifying an animal susceptible to or having an allergic response to mites. Such methods include the following steps: (a) contacting the isolated protein of the present invention with an animal antibody; and (b) determining the formation of an immune complex between the protein and the antibody. (Where the formation of the immune complex indicates that the animal is susceptible to or has this allergic response to ticks).
[0010]
The invention includes a reagent comprising a non-proteinaceous epitope having at least one of the following identified properties: (a) the epitope is resistant to β-elimination of the peptide; (b) the epitope is protein kinase K. Is resistant to digestion; and (c) the epitope is responsive to a test designed to detect glycosylated proteins. Such epitopes bind to at least one of the following antibodies: dog IgE from dogs allergic to ticks, and cat IgE from cats allergic to ticks. Also included are isolated antibodies that selectively bind such non-proteinaceous epitopes and derivatives of such epitopes.
[0011]
The present invention also relates to therapeutic compositions and assay kits comprising the non-proteinaceous epitopes of the invention, and methods for identifying and / or desensitizing animals susceptible to an allergic response to mites, the method comprising Includes the use of proteinaceous epitopes.
[0012]
(Detailed description of the invention)
The present invention provides an isolated protein having a molecular weight in the range of about 60 kilodaltons (kD) to about 109 kilodaltons, which is useful for mites of the genus Dermatophagoides (especially Dermatophagoides farinae species and / or Dermatophagoides spteronyss sp.). At least one epitope of a protein allergen. Such a protein is referred to herein as a Der HMW-map protein. The invention further includes methods for isolating and identifying nucleic acid molecules encoding Der HMW-map protein, antibodies to Der HMW-map protein, and inhibitors of Der HMW-map protein activity. As used herein, the term "isolated Der HMW-map protein" refers to Dermatophagoides, more preferably Dermatophagoides farinae and / or Dermatophagoides pteronyssius, which are themselves derived from their natural sources. Or Der HMW-map protein (e.g., produced using recombinant nucleic acid technology or chemical synthesis). The invention includes methods for detecting immunoglobulins that specifically bind to Der HMW-map protein, methods for treating the pathogenesis of mite allergens, and in other applications as disclosed below. The use of proteins and antibodies is also included. The products and processes of the present invention are advantageous. Because they allow the detection of anti-Der HMW-map antibodies in the body fluids of animals and the inhibition of Der HMW-map protein activity involved in IgE or disease.
[0013]
One embodiment of the present invention is an isolated Dermatophagoides allergic composition comprising: (a) a composition produced by a method comprising: (1) a Dermatophagoides extract on a gel filtration column. (2) collecting the rejected protein from the gel filtration column and applying the rejected protein to an anion exchange column; and (3) about 0.3 M Tris-HCl (pH 8). Eluting the protein bound to the anion exchange column having an anion exchange column to obtain a Dermatophagoides allergic composition; and (b) a composition comprising a peptide of the protein produced with respect to step (a), wherein the allergic composition The composition comprises: binding to IgE; B Enabling biological functions, including stimulating lymphocyte responses and stimulating T lymphocyte responses). Such Dermatophagoides allergic compositions are also referred to herein as Der HMW-map compositions. Suitable gel filtration columns include any gel filtration columns that can exclude proteins having a molecular weight between about 50 kD and about 150 kD. Preferred gel filtration columns include, but are not limited to, Sephacryl S-100 columns. Suitable anion exchange columns include any anion exchange column capable of binding proteins having a pI of less than about pI6. Preferred anion exchange columns include, but are not limited to, Q-Sepharose columns. As used herein, "stimulating a B lymphocyte response" refers to a humoral response in an animal that is preferentially induced in an animal by a Der HMW-map of the invention and is associated with the activity of a B lymphocyte. Refers to an increase in the immune response. As used herein, "stimulating a T lymphocyte response" refers to a humoral response in an animal that is preferentially induced in an animal by a Der HMW-map of the invention and is associated with T lymphocyte activity. Refers to an increase in the immune response.
[0014]
One embodiment of the present invention is an isolated protein comprising a Der HMW-map protein. The term (a) ("a" or "an") entity specifically refers to one or more entities. For example, (a) protein, (a) nucleic acid molecule, (a) antibody, (a) inhibitor, (a) compound, or (a) therapeutic composition is "one or more" or "at least one", respectively. "One or more" or "at least one" nucleic acid molecule, "one or more" or "at least one" antibody, "one or more" or "at least one" inhibitor, "one "Or more" or "at least one" compound, or "one or more" or "at least one" therapeutic composition. As such, the terms "a" (or "an"), "one or more", and "at least one" may be used interchangeably herein. The terms “comprising,” “containing,” and “having” specifically refer to being able to be used interchangeably. According to the present invention, an isolated protein or a biologically pure protein is a protein that has been removed from its natural environment. As such, "isolated" and "biologically pure" need not reflect the degree to which the protein has been purified. An isolated protein of the present invention can be obtained from its natural source, can be produced using recombinant DNA technology, or can be produced by chemical synthesis.
[0015]
As used herein, a Der HMW-map protein can be a full-length protein, or any homolog of such a protein. As used herein, a protein can be a polypeptide or a peptide (the terms are used by those skilled in the art). Preferably, the Der HMW-map protein is recognized by an IgE antibody in the presence of a major histocompatibility complex (MHC) molecule from an animal exhibiting an IgE-mediated pathogenesis for the Der HMW-map protein (ie, At least one of the Der HMW-map proteins comprising at least one of the epitopes recognized by antibodies on the surface of B lymphocytes and / or recognized by T cell receptors. Including some.
[0016]
The peptides of the invention may bind to IgE, desensitize animals to mite allergens, stimulate B lymphocyte responses, and / or stimulate T lymphocyte responses. Includes HMW-map protein. Preferably, the peptides of the present invention comprise a B lymphocyte epitope, or a T lymphocyte epitope. Peptides having a B lymphocyte epitope can bind to the antibody. A peptide having a T lymphocyte epitope can bind to an MHC molecule in such a way that the peptide can stimulate T lymphocytes via a T cell receptor. In accordance with the present invention, a peptide comprising a B lymphocyte epitope can be from about 4 residues to about 50 residues in length, preferably from about 5 residues to about 20 residues in length. In accordance with the present invention, a peptide comprising a T lymphocyte epitope can be from about 4 to about 20 residues in length, preferably from about 8 to about 16 residues in length.
[0017]
The Der HMW-map proteins (including homologs) of the present invention can be identified in a simple manner by the ability of the protein to induce an allergic response to the Der HMW-map protein. Examples of Der HMW-map protein homologs include Der HMW-map proteins, where amino acids are deleted (eg, a truncated version of the protein such as a peptide), inserted, converted, substituted, and / or Or has been derivatized (eg, by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitoylation, amidation, and / or addition of glycerophosphatidylinositol) such that the homolog is naturally occurring Der Allergic response to HMW-map protein can be induced.
[0018]
Der HMW-map protein homologs can be the result of natural allelic alterations or natural mutations. The Der HMW-map protein homologues of the present invention also include genes that encode proteins, using classical or recombinant nucleic acid techniques that result in direct modifications to the protein or, for example, random or targeted mutations. Can be produced using techniques known in the art, including but not limited to modifications to
[0019]
One embodiment of the present invention is the Der HMW-map gene, which has SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 45, and the complement of any of these nucleic acid sequences. These nucleic acid sequences are further described herein. For example, the nucleic acid sequence (SEQ ID NO: 14) is a nucleic acid molecule nDerf98 of the Der HMW-map gene. ₁₇₅₂ (This generation is disclosed in the Examples) shows the degenerate sequence of the coding strand of the cDNA (complementary DNA) set forth herein. Nucleic acid molecule nDerf98 ₁₇₅₂ Clearly contains the full-length coding region. The complement of SEQ ID NO: 14 (shown herein by SEQ ID NO: 16) refers to the nucleic acid sequence of a strand that is complementary to the strand having SEQ ID NO: 14, which can be readily determined by one skilled in the art. Similarly, the nucleic acid sequence complement of any nucleic acid sequence of the present invention refers to the nucleic acid sequence of a nucleic acid strand that is complementary to (ie, capable of forming a duplex with) the referenced strand. . SEQ ID NO: 14 (as well as the other nucleic acid and protein sequences set forth herein) provides a definitive description of the nucleic acid molecule encoding the Der HMW-map protein of the present invention, as nucleic acid sequencing techniques are not totally error free. It should be noted that the nucleic acid sequence is unique.
[0020]
In another embodiment, the Der HMW-map gene, or nucleic acid molecule, is an allelic variant that is similar, but not identical, to SEQ ID NO: 14 or SEQ ID NO: 16, or any other Der referred to herein. It may be an HMW-map nucleic acid sequence. For example, an allelic variant of the Der HMW-map gene comprising SEQ ID NO: 14 or SEQ ID NO: 16 occurs at the locus (s) of the genome that is essentially the same as the gene comprising SEQ ID NO: 14 and SEQ ID NO: 16. Although they have genes, they have similar but not identical sequences, for example, due to natural variations caused by mutation or recombination. Since natural selection is typically a selection for mutations that affect function, allelic variants (ie, corresponding to or alleles of the cited nucleic acid sequence) are usually Encodes a protein having an activity similar to that of the protein encoded by the gene to be compared. An allelic variant of a gene or nucleic acid molecule can also include a change in the 5 ′ or 3 ′ untranslated region of the gene (eg, in regulating a control region) or involve alternative splicing of the early translation. Thus, exons can be alternated in parallel. Allelic variants are well known to those of skill in the art and are predicted to occur naturally in certain dust mites (eg, Dermatophagoides). Because the representative genome is diploid, and sexual reproduction results in allelic reclassification.
[0021]
In one embodiment of the invention, the isolated Der HMW-map protein is encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions to a gene encoding a Der HMW-map protein. The minimal size of a Der HMW-map protein of the invention is capable of forming stable hybrids (ie, hybridizing under stringent hybridization conditions) with the complementary sequence of a nucleic acid molecule encoding the corresponding native protein. A) sufficient size to be encoded by the nucleic acid molecule. The size of a nucleic acid molecule encoding such a protein depends on the composition of the nucleic acid and the percent homology between the Der HMW-map nucleic acid molecule and the complementary nucleic acid sequence. The range of homology required to form a stable hybrid under stringent conditions is that homologous sequences are scattered throughout a given nucleic acid molecule or clustered in distinct regions of a given nucleic acid molecule It will be readily understood that this can vary depending on whether it is done (ie, localized).
[0022]
The minimum size of a nucleic acid molecule that can form a stable hybrid with the gene encoding the Der HMW-map protein is typically at least about 12 nucleotides to about 15 nucleotides long when the nucleic acid molecule is GC rich. And when it is AT-rich, it is at least about 15 nucleotides to about 17 bases in length. The minimum size of a nucleic acid molecule used to encode a Der HMW-map protein homolog of the present invention is from about 12 nucleotides to about 18 nucleotides in length, preferably about 12 nucleotides in length, about 15 nucleotides in length. Or about 18 nucleotides in length. Thus, the minimum length of a Der HMW-map protein homolog of the present invention is from about 4 to about 6 amino acids in length. There is no limit other than the particular limit on the minimum size of a nucleic acid molecule encoding a Der HMW-map protein of the present invention. This is because the nucleic acid molecule of the present invention can include a gene, an entire gene, or a part of a plurality of genes. The preferred size of a protein encoded by a nucleic acid molecule of the invention depends on whether a full-length, fusion, multivalent, or functional protein of such a protein is desired. Preferably, the preferred size of the protein encoded by the nucleic acid molecule of the invention is a portion of the protein that elicits an immune response, which is about 30 amino acids, more preferably about 35 amino acids, and even more preferably , About 44 amino acids in length.
[0023]
Stringent hybridization conditions are determined based on defined physical properties of the gene, to which the nucleic acid molecules will hybridize and can be mathematically defined. Stringent hybridization conditions are experimental parameters that enable one of ordinary skill in the art to identify significant similarities between heterologous nucleic acid molecules. These conditions are well known to those skilled in the art. See, eg, Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press, and Meinkoth et al., 1984, Anal. Biochem. 138, 267-284. As described in detail in the references, determination of hybridization conditions includes determination of ionic strength (M (moles / liter)), hybridization temperature (° C.), concentration of nucleic acid helix destabilizing agent (eg, formamide), It involves the manipulation of a set of variables including the average length (n) of the shortest hybrid duplex, and the percentage of the G + C composition of the fragment to which the unknown nucleic acid molecule hybridizes. For nucleic acid molecules of at least about 150 nucleotides, these variables can be substituted into standard mathematical equations to determine the melting temperature (ie, T.sub.T) of a given nucleic acid molecule. _m ) Is calculated. As defined by the following equation, T _m Is the temperature at which two complementary nucleic acid molecule strands dissociate, which is considered to be 100% complementary between the two strands:
T _m = 81.5 ° C + 16.6 log M + 0.41 (% of G + C)-500 / n-0.61 (% of formamide). For nucleic acid molecules smaller than about 50 nucleotides, the hybrid stability is determined by the dissociation temperature (T _d ) (Defined as the temperature at which 50% of the duplexes dissociate). For these smaller molecules, stability at standard ionic strength is defined by the following equation:
T _d = 4 (G + C) +2 (A + T).
[0024]
T _d Temperatures lower than 5 ° C. are used to determine hybridization between perfectly matched molecules.
[0025]
Also, how many base pair mismatches (ie, the differences between the two nucleic acid molecules being compared) include the non-complementarity of the base at a given position, and gaps are defined for any given nucleic acid molecule being compared. Due to the insertion or deletion of one or more bases at a position, the T for nucleic acid molecules of different sizes _m Or T _d It is also well known to those skilled in the art that it may affect For example, T _m Decreases about 1 ° C. for each 1% mismatched base pair for hybrids greater than about 150 bp, and T _d Decreases about 5 ° C. for each mismatched base pair for hybrids smaller than about 50 bp. Conditions for hybrids between about 50 to about 150 base pairs can be determined empirically and without undue experimentation using standard laboratory procedures well known to those skilled in the art. These simple procedures require one of skill in the art to understand that hybridization conditions (eg, by changing salt concentration, formamide concentration, or temperature) are such that only nucleic acid hybrids having less than a particular% base pair mismatch will hybridize. Allows setting of It is generally understood by those skilled in the art that stringent hybridization conditions are experimental conditions that allow hybridization between molecules having less than about 30% base pair mismatches (ie, about 70% or more identity). Is done. One of skill in the art can readily determine whether a given nucleic acid molecule to be tested is less than or greater than about 50 nucleotides, and therefore selects the appropriate formula to determine hybridization conditions. One skilled in the art will be able to determine whether a nucleic acid molecule will hybridize to a given gene under stringent hybridization conditions and, similarly, allow the nucleic acid molecule to have the desired amount of base pair mismatches. To hybridize under the conditions designed to do so.
[0026]
Hybridization reactions often involve attaching the nucleic acid molecule to be hybridized to a solid support, such as a membrane, followed by a labeled nucleic acid molecule (typically referred to as a probe) suspended in a hybridization solution. ) And a step of hybridization. Examples of common hybridization reaction techniques include, but are not limited to, well-known Southern and Northern blotting procedures. Typically, the actual hybridization reaction will be performed under non-stringent conditions (ie, lower temperature and / or higher salt concentration), and then increase the stringency to achieve the desired stringency. This is achieved by washing the membrane in a solution at high temperature and / or lower salt concentration.
[0027]
For example, if one of skill in the art desires to identify a nucleic acid molecule that hybridizes under stringent hybridization conditions to an approximately 150 bp long nucleic acid molecule of Dermatophagoides farinae and / or Dermatophagoides pteronyssius, the following conditions are preferably used: Can be done. The average G + C content of the DNA of Dermatophagoides farinae and Dermatophagoides pteronyssius is about 39%. An unknown nucleic acid molecule is attached to the support membrane and the 150 bp probe is labeled (eg, using a radioactive tag). Hybridization reactions can be performed at a temperature of about 37 ° C. (low stringency conditions) in a solution containing 2 × SSC and 0% formamide. Solutions of different concentrations of SSC were prepared by combining a stock solution of 20 × SSC (175.3 g NaCl and about 88.2 g sodium citrate (pH 7) in 1 liter of water) to obtain the desired concentration of SSC. By dilution, it can be made by those skilled in the art. To achieve high stringency hybridization, one skilled in the art will calculate the wash conditions required to tolerate up to 30% base pair mismatch. For example, the complete hybrid T in a wash solution containing 1 × SSC and 0% formamide _m Is about 80 ° C .:
81.5 ° C + 16.6 log (0.15M) + (0.41 x 39)-(500/150)-(0.61 x 0) = 80.4 ° C.
Thus, to achieve hybridization with a nucleic acid molecule having about 30% base pair mismatch, washing of the hybridization is performed at a temperature of about 50 ° C. Thus, it is within the skill of the artisan to calculate additional hybridization temperatures based on the desired% base pair mismatch, formula and G / C content disclosed herein. For example, a nucleic acid molecule to be tested for hybridization to a nucleic acid molecule of the invention having a sequence as specified herein is longer than 150 nucleotides, so that a hybridization reaction that allows up to 30% base pair mismatches. T for _m It is understood by those skilled in the art that does not vary significantly from 50 ° C.
[0028]
In addition, it is known in the art that there are commercially available computer programs for determining the degree of similarity between two nucleic acid sequences. These computer programs include various known methods for determining the percent identity between hybrid nucleic acid molecules and the number and length of gaps. A preferred method for determining the percent identity between amino acid sequences and also between nucleic acid sequences involves analysis using one or more commercially available computer programs designed to compare and analyze nucleic acid or amino acid sequences. . These computer programs include, but are not limited to: GCG ^TM (Available from Genetics Computer Group, Madison, WI), DNAsis ^TM (Available from Hitachi Software, San Bruno, CA) and MacVector ^TM (Available from Eastman Kodak Company, New Haven, CT). A preferred method for determining the percent identity between amino acid sequences and also between nucleic acid sequences involves the use of the Compare function by maximum matching in the DNAsis version 2.1 program with default parameters.
[0029]
One embodiment of the invention comprises a Der HMW-map protein. In one embodiment, the Der HMW-map protein of the present invention, when presented on reduced 12% Tris glycine SDS-PAGE, as a band with a molecular weight of about 98 kD to about 109 kD, as shown in FIG. Including proteins that move. This band in FIG. 1 is obtained when the protein is collected from Dermatophagoides farinae mites using the method described in detail in Example 1. Preferably, the Der HMW-map proteins of the present invention include proteins having a molecular weight ranging from about 90 kD to about 120 kD, and more preferably, from about 98 kD to about 109 kD. Preferred Der HMW-map proteins of the present invention include mapA and mapB. This identification is described in the Examples section.
[0030]
In another embodiment, the Der HMW-map protein of the present invention is a protein that, when presented on reduced 14% Tris glycine SDS-PAGE, migrates as a band of approximately 60 kD molecular weight, as shown in FIG. including. This band in FIG. 2 is obtained when the protein is collected from a Dermatophagoides farinae mite using the method described in detail in Example 9. Preferably, the Der HMW-map protein of the present invention comprises a protein having a molecular weight of about 60kD. Preferred Der HMW-map proteins of the present invention include mapD. This identification is described in the Examples section.
[0031]
In another embodiment, preferred Der HMW-map proteins are at least about 50 nucleotides or about 150 nucleotides, and preferably under conditions that permit up to about 40% base pair mismatch, more preferably about Under conditions that allow up to 35% base pair mismatch, more preferably under conditions that allow up to about 30% base pair mismatch, more preferably under conditions that allow up to about 25% base pair mismatch, more preferably Under conditions that allow up to about 20% base pair mismatch, more preferably under conditions that allow up to about 15% base pair mismatch, more preferably under conditions that allow up to about 10% base pair mismatch, and More preferably, SEQ ID NO: 16, SEQ ID NO: No. 19, SEQ ID NO: 22, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 45, and a nucleic acid sequence encoding a protein comprising the amino acid sequence of SEQ ID NO: 33, and a complement thereof. Includes proteins encoded by nucleic acid molecules that hybridize to nucleic acid molecules.
[0032]
Another embodiment of the invention comprises a Der HMW-map protein encoded by a nucleic acid molecule selected from the group consisting of: a nucleic acid molecule comprising at least about 150 nucleotides, wherein at least about 150 nucleotides SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 42 in a solution containing 1 × SSC and 0% formamide at a temperature of about 50 ° C. A nucleic acid selected from the group consisting of a complement of a nucleic acid sequence encoding a protein comprising the amino acid sequence of SEQ ID NO: 45 and SEQ ID NO: 33, and a nucleic acid molecule comprising a fragment of any of the above nucleic acid molecules comprising at least about 15 nucleotides Hybridize to the sequence.
[0033]
Yet another preferred Der HMW-map protein of the invention is the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 40, SEQ ID NO: 43 and / or SEQ ID NO: 33 Preferably, it is at least about 60% identical, more preferably at least about 65% identical, more preferably at least about 70% identical, more preferably Is encoded by a nucleic acid molecule that is at least about 75% identical, more preferably at least about 80% identical, more preferably at least about 85% identical, more preferably at least about 90% identical, and even more preferably at least about 95% identical. Including the protein to be purified. Fragments of such proteins are also preferred. Percent identity, as used herein, is determined by using the Compare function by maximum matching in the program DNAsis version 2.1 with default parameters.
[0034]
Further preferred Der HMW-map proteins of the present invention include: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, a protein having the amino acid sequence of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, and SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, sequence No. 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO 18, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44 A protein comprising a homolog of a protein having an amino acid sequence, wherein such homologs are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 29, SEQ ID NO: No. 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44 Comprising at least one epitope to induce disease response protein. Similarly, a nucleic acid sequence encoding a protein comprising the nucleic acid sequence of SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 40, SEQ ID NO: 43 and / or the amino acid sequence of SEQ ID NO: 33 Or proteins encoded by nucleic acid molecules encoded by their homologs are also preferred.
[0035]
A preferred isolated protein of the present invention is a protein encoded by at least one of the following nucleic acid molecules: nDerf98 ₁₇₅₂ , NDerf98 ₁₆₆₅ , NDerf98 ₁₆₀₈ , NDerp98 ₁₆₂₁ , NDerp98 ₁₅₂₇ , NDerp98 ₁₄₇₀ , NDerf60 ₅₁₀ Or allelic variants of any of these nucleic acid molecules. Another preferred isolated protein is encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 40, SEQ ID NO: 43; Or proteins encoded by allelic variants of any of these listed nucleic acid molecules.
[0036]
SEQ ID NO: 14 (nDerf98 ₁₇₅₂ The translation of PDerf98 herein ₅₅₅ This amino acid sequence is presented in SEQ ID NO: 15, and the first in-frame codon is believed to extend from nucleotide 1 to nucleotide 3 of SEQ ID NO: 14. The complement of SEQ ID NO: 14 is presented herein as SEQ ID NO: 16. PDerf98 ₅₅₅ The amino acid sequence of nDerf98 ₁₆₆₅ Which has the coding strand set forth in SEQ ID NO: 17 and the complementary strand set forth in SEQ ID NO: 19. Analysis of SEQ ID NO: 15 suggests the presence of a signal peptide ranging from approximately amino acid 1 to approximately amino acid 19. In the present specification, PDerf98 ₅₃₆ The proposed mature protein, referred to as containing about 536 amino acids. This sequence is represented herein as SEQ ID NO: 21 and represented by nDerf98 herein, represented by SEQ ID NO: 20 (coding strand) and SEQ ID NO: 22 (complementary strand). ₁₆₀₈ Encoded by a nucleic acid molecule referred to as
[0037]
SEQ ID NO: 34 (nDerp98 ₁₆₂₁ Translation of the PDerp98 herein ₅₀₉ A protein of about 509 amino acids results, the amino acid sequence of which is presented in SEQ ID NO: 35, and the first in-frame codon is believed to extend from nucleotide 14 to nucleotide 16 of SEQ ID NO: 34. The complement of SEQ ID NO: 34 is presented herein as SEQ ID NO: 36. PDerpf98 ₅₀₉ The amino acid sequence of nDerp98 ₁₅₂₇ Which has the coding strand set forth in SEQ ID NO: 37 and the complementary strand set forth in SEQ ID NO: 39. Analysis of SEQ ID NO: 35 suggests the presence of a signal peptide ranging from approximately amino acid 1 to approximately amino acid 19. In this specification, PDerp98 ₄₉₀ The proposed mature protein, referred to as containing about 490 amino acids. This sequence is represented herein as SEQ ID NO: 41 and represented by nDerp98 herein, represented by SEQ ID NO: 40 (coding strand) and SEQ ID NO: 42 (complementary strand). ₁₄₇₀ Encoded by a nucleic acid molecule referred to as
[0038]
D. SEQ ID NO: 43 (nDerf60), which is a nucleic acid molecule encoding a part of the farinae 60-kD antigen protein. ₅₁₀ Translation of PDerf60 herein ₁₇₀ This amino acid sequence is presented as SEQ ID NO: 44, and the first in-frame codon is believed to extend from nucleotide 1 to nucleotide 3 of SEQ ID NO: 43. The complementary sequence to SEQ ID NO: 43 is represented herein as SEQ ID NO: 45.
[0039]
A preferred Der HMW-map protein of the present invention is PDerf98 ₅₅₅ And at least about 45%, preferably at least about 50%, more preferably at least about 55%, even more preferably at least about 60%, even more preferably at least about 65%, even more preferably at least about 70%, More preferably, it comprises proteins that are at least about 75%, even more preferably at least about 80%, even more preferably at least about 85%, even more preferably at least about 90%, and even more preferably about 95% identical. More preferred is PDerf98 ₅₅₅ , PDerf98 ₅₃₆ , PDerp98 ₅₀₉ , PDerp98 ₄₉₀ And / or PDerf60 ₁₇₀ A Der HMW-map protein comprising: ₅₅₅ , PDerf98 ₅₃₆ , PDerp98 ₅₀₉ , PDerp98 ₄₉₀ And / or PDerf60 ₁₇₀ Is a protein that is encoded by an allelic variant of a nucleic acid molecule that encodes the protein.
[0040]
Other preferred Der HMW-map proteins of the invention include SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: No. 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32 SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 41, and / or SEQ ID NO: 44 and at least about 45%, preferably at least about 50%, more preferably at least about 55%, and even more. Preferably at least about 60%, even more preferably at least about 65%, even more preferably at least about 70%, even more preferably Is a protein comprising an amino acid sequence that is at least about 75%, even more preferably at least about 80%, even more preferably at least about 85%, even more preferably at least about 90%, and even more preferably about 95% identical. Including. More preferred are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: No. 12, Sequence No. 13, Sequence No. 15, Sequence No. 18, Sequence No. 21, Sequence No. 23, Sequence No. 24, Sequence No. 29, Sequence No. 30, Sequence No. 31, Sequence No. 32, Sequence No. 33, Sequence No. 35 A Der HMW-map protein comprising the amino acid sequence of SEQ ID NO: 38, SEQ ID NO: 41, and / or SEQ ID NO: 44; and SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 2 3, having an amino acid sequence of SEQ ID NO: 24, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 41, and / or SEQ ID NO: 44 Der HMW-map protein encoded by an allelic variant of a nucleic acid molecule encoding the HMW-map protein.
[0041]
In one embodiment of the invention, the Der HMW-map protein comprises the amino acids SEQ ID NO: 15, SEQ ID NO: 35, and / or SEQ ID NO: 44 (SEQ ID NO: 15, SEQ ID NO: 35, and / or SEQ ID NO: 44). (Including, but not limited to, proteins consisting of amino acid sequences, fragments thereof, fusion proteins, and multivalent proteins), and encodes proteins having the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 35, and / or SEQ ID NO: 44. And proteins encoded by allelic variants of the nucleic acid molecule.
[0042]
In one embodiment, a preferred Der HMW-map protein is at least about 35 amino acids in length, preferably at least about 50 amino acids in length, more preferably at least about 100 amino acids in length, more preferably at least about 200 amino acids in length, even more preferably It includes an amino acid sequence at least about 250 amino acids in length. In this embodiment, a preferred Der HMW-map protein of the invention has an amino acid sequence comprising at least a portion of SEQ ID NO: 15. In another embodiment, preferred Der HMW-map proteins include the full length protein (ie, the protein encoded by the full length coding region).
[0043]
A further preferred Der HMW-map protein of the present invention is nDerf98 ₁₇₅₂ , NDerf98 ₁₆₆₅ , NDerf98 ₁₆₀₈ , NDerp98 ₁₆₂₁ , NDerp98 ₁₅₂₇ , NDerp98 ₁₄₇₀ , And nDerf60 ₅₁₀ And Der HMW-map proteins encoded by allelic variants of such nucleic acid molecules.
[0044]
Also preferred is a nucleic acid sequence encoding a protein comprising the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 40, SEQ ID NO: 43 and / or SEQ ID NO: 33, And a Der HMW-map protein encoded by a nucleic acid molecule having a nucleic acid sequence comprising at least a portion of an allelic variant of these nucleic acid molecules.
[0045]
In another embodiment, a preferred Der HMW-map protein of the invention has at least about 12 nucleotides, preferably at least about 16 nucleotides, more preferably at least about 18 nucleotides, more preferably at least about 20 nucleotides, more preferably Is at least about 25 nucleotides, more preferably at least about 50 nucleotides, more preferably at least about 100 nucleotides, more preferably at least about 350 nucleotides, more preferably at least about 450 nucleotides, more preferably at least about 500 nucleotides. Encoded by a nucleic acid molecule comprising nucleotides, and even more preferably, at least about 800 nucleotides. Included in this embodiment is nDerf98 ₁₇₅₂ , NDerp98 ₁₆₂₁ And / or nDerf60 ₅₁₀ Or a Der HMW-map protein encoded by an allelic variant of these nucleic acid molecules. In yet another embodiment, a preferred Der HMW-map protein of the invention is a nucleic acid molecule comprising an apparently full length Der HMW-map coding region (ie, a nucleic acid molecule encoding an apparently full length Der HMW-map protein). Coded by
[0046]
One embodiment of a Der HMW-map protein of the invention is a fusion protein comprising a Der HMW-map protein containing domain linked to one or more fusion segments. Suitable fusion segments for use in the present invention include: a segment capable of enhancing protein stability; a segment capable of acting as an immunopotentiator for enhancing an immune response to Der HMW-map protein, a Der HMW-map protein And / or segments that can aid in the purification of Der HMW-map protein (eg, by affinity chromatography). Suitable fusion segments can be of any size having the desired function (eg, conferring increased stability, conferring increased immunogenicity on the protein, reducing the IgE response, and / or simplifying protein purification). Can be a domain. The fusion segment can be attached to the amino and / or carboxyl terminus of the Der HMW-map protein containing domain of the protein and is sensitive to cleavage to allow for easy recovery of the Der HMW-map protein. possible. The fusion protein is preferably cultured in a recombinant cell transformed with a fusion nucleic acid molecule encoding a protein comprising a fusion segment attached to either the carboxyl and / or amino terminus of the Der HMW-map protein containing domain. Generated by Preferred fusion segments include: metal binding domains (eg, poly-histidine segments); immunoglobulin binding domains (eg, protein A; protein G; T cells; B cells; antibodies to Fc receptors or complement proteins) A binding domain); a sugar binding domain (eg, a maltose binding domain); a “tag” domain (eg, galactosidase, strep tag peptide, other domains that can be purified using a compound that binds to this domain (eg, a monoclonal antibody) And / or a linker and an enzyme domain (e.g., an alkaline phosphatase domain linked to a Der HMW-map protein by a linker). More preferred fusion segments include: a metal binding domain (eg, a poly-histidine segment); a maltose binding domain; a strep tag peptide (eg, a peptide available from Biometer, Tampa, FL); a phage T7 S10 peptide .
[0047]
In another embodiment, a Der HMW-map protein of the invention also comprises at least one additional protein segment that can desensitize an animal from one or more allergens. Such multivalent desensitized proteins can be produced by culturing cells transformed with a nucleic acid molecule comprising two or more nucleic acid domains, wherein the two or more nucleic acid domains are Are linked together in such a way that they are expressed as multivalent desensitizing compounds, including at least two desensitizing compounds capable of desensitizing an animal from an allergen.
[0048]
Examples of multivalent desensitizing compounds include, but are not limited to: caused by one or more allergens (eg, plant allergens, animal allergens, parasite allergens, or ectoparasite allergens) A Der HMW-map protein of the invention conjugated to one or more compounds that desensitize to allergy. Allergens include, but are not limited to: Grasses, Meadow Fescue, Curly Dock, Plantain, Mexican Firebush, Mexican Firbush s Quarter, pigweed, ragweed, sage, elm, elm, cocklebur, box elder, box elder, walnut, cotwort, cotwow ), Birch, cedar, oak, mulberry-derived plants (pant) allergens, cockroaches Dermatophagoides, Alternaria, Aspergillus, Cladosporium, Fusarium, Helminthosporium, Mucor, Penicillium, Pullularia, Rhizopus, Rhizopus, Trichophyte, Rhizophrenia, Rhizophrenia, Rhizophrenia, Rhizophrenia, Rhizophrenia, Rhinoceros (Black fly), horse flies, horn fly, horn flies, tsetse flies, stable fly, myiasis-causing fly and biting gnats, ants, spider mites, Winged insects (including winged insects) and leech The next ectoparasite allergen.
[0049]
The present invention also includes mimetopes of the Der HMW-map proteins of the present invention. As used herein, the mimetope of the Der HMW-map protein of the present invention refers to the activity of such a Der HMW-map protein (eg, binding to induce an immune response to the Der HMW-map protein). Ability to mimic the ability to mimic the Der HMW-map protein, often because the mimetope has a structure that mimics the Der HMW-map protein. However, it should be noted that the mimetope need not have a similar structure to the Der HMW-map protein, as long as the mimetope functionally mimics the Der HMW-map protein. A mimetope can be, but is not limited to: a peptide modified to reduce its susceptibility to degradation; anti-idiotype and / or catalytic antibodies, or fragments thereof; Non-proteinaceous immunogenic moieties (eg, carbohydrate structures); synthetic or natural organic or inorganic molecules (including nucleic acids); and / or any other peptidomimetic compound. The mimetopes of the invention can be designed using computer generated structures for the Der HMW-map proteins of the invention. Mimetopes also generate random samples of molecules (eg, oligonucleotides, peptides or other organic molecules) and use such samples with corresponding binding partners (eg, anti-Der HMW-map protein antibodies). And by screening by affinity chromatography techniques. Mimetopes can also be obtained, for example, by rational drug design. In a rational drug design procedure, the three-dimensional structure of a compound of the invention may be analyzed, for example, by nuclear magnetic resonance (NMR) or X-ray crystallography. The three-dimensional structure can then be used to predict the structure of a potential mimetope, for example, by computer modeling. The predicted mimetope structure can then be generated, for example, by chemical synthesis, recombinant DNA technology, or by isolating the mimetope from a natural source. Specific examples of mimetopes of the Der HMW-map protein include anti-idiotypic antibodies, Selex ^TM Oligonucleotides generated using the technology, peptides identified by random screening of peptide libraries, and proteins identified by phage display technology. Preferred mimetopes are peptidomimetic compounds that are structurally and / or functionally similar to the Der HMW-map protein of the invention, and in particular to an epitope of the Der HMW-map protein that induces an immune response.
[0050]
The present invention also includes muteins of the Der HMW-map proteins of the present invention. As used herein, mutein refers to a specific homolog of the Der HMW-map protein in which desired amino acid residues have been substituted or removed. Preferred muteins of the present invention include Der HMW-map protein homologs in which amino acid residues have been altered to reduce the anaphylactic response by the animal when the mutein is administered to the animal at a therapeutic dose. More preferred muteins of the invention include Der HMW-map protein homologs in which one or more cysteine residues of the Der HMW-map protein have been substituted or removed. Methods for producing muteins are known to those of skill in the art, and such methods are disclosed herein. Preferably, muteins are produced using recombinant techniques.
[0051]
Another embodiment of the present invention is an isolated nucleic acid molecule comprising a Der HMW-map nucleic acid molecule. The distinguishing features of such nucleic acid molecules have been described previously. The nucleic acid molecules of the present invention may include the isolated native Der HMW-map gene or a homolog thereof, the latter described in more detail below. A nucleic acid molecule of the invention may include one or more regulatory regions, full-length or partial coding regions, or a combination thereof. The minimum size of a nucleic acid molecule of the invention is large enough to allow the formation of a stable hybrid with the complementary sequence of another nucleic acid molecule (ie, hybridization under stringent hybridization conditions). .
[0052]
According to the present invention, an isolated nucleic acid molecule is a nucleic acid molecule that has been removed from its natural environment (ie, has been manipulated by a human), and the isolated nucleic acid molecule is DNA, RNA, or It may include derivatives of either DNA or RNA. Thus, "isolated" does not reflect the degree to which the nucleic acid molecule has been purified. An isolated Der HMW-map nucleic acid molecule of the invention or a homolog thereof can be isolated from its natural source, or can be obtained by recombinant DNA technology (eg, polymerase chain reaction (PCR) amplification or cloning) or chemical synthesis. Can be generated using Isolated Der HMW-map nucleic acid molecules and homologs thereof include, for example, naturally occurring allelic variants and nucleotides in a manner that does not substantially interfere with the ability to encode the Der HMW-map protein of the present invention. Nucleic acid molecules that have been modified by insertions, deletions, substitutions, and / or inversions may be included.
[0053]
Der HMW-map nucleic acid molecule homologs can be prepared by a number of methods known to those skilled in the art (eg, Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press; see Sambrook et al., Ibid.). Can be used and generated. For example, various techniques can be used to modify nucleic acid molecules, including classical mutagenesis techniques and recombinant DNA techniques (eg, site-directed mutagenesis, chemical treatment, restriction enzymes). Cleavage, ligation of nucleic acid fragments, PCR amplification, synthesis of oligonucleotide mixtures, and ligation of mixtures to "build" a mixture of nucleic acid molecules, and combinations thereof). Nucleic acid molecule homologs may be isolated by hybridization with a Der HMW-map nucleic acid molecule or by the function of a protein encoded by the nucleic acid molecule (e.g., the ability to elicit an immune response against at least one epitope of the Der HMW-map protein, Or the ability to effect Der HMW-map activity).
[0054]
An allelic variant typically encodes a protein that has an activity similar to that of the protein encoded by the gene to which the variant is being compared. An allelic variant can also include changes in the 5 'or 3' untranslated regions (eg, regulatory control regions) of the gene. Allelic variants are well known to those of skill in the art and are expected to be found in certain house dust mites. Because the genome is diploid and / or between two or more groups of house dust mites. The invention also includes variants resulting from laboratory operations, such as, but not limited to, variants produced during polymerase chain reaction amplification.
[0055]
An isolated nucleic acid molecule of the present invention may include a nucleic acid sequence encoding at least one Der HMW-map protein of the present invention, examples of such proteins being disclosed herein. Although the phrase "nucleic acid molecule" mainly refers to a physical nucleic acid molecule and the phrase "nucleic acid sequence" mainly refers to the sequence of nucleotides for the nucleic acid molecule, the two phrases may be used interchangeably. In particular, with respect to nucleic acid molecules or nucleic acid sequences capable of encoding a Der HMW-map protein, they may be used interchangeably.
[0056]
Preferred nucleic acid molecules of the invention, when administered to an animal, are capable of desensitizing the animal from an allergic reaction caused by a Der HMW-map allergen. As disclosed in more detail below, such nucleic acid molecules can be or encode antisense RNA, molecules capable of forming triple helices, ribozymes, or other nucleic acid based drug compounds. In a further embodiment, a nucleic acid molecule of the invention may encode a desensitizing protein (eg, a Der HMW-map protein of the invention), wherein the nucleic acid molecule is, for example, by direct injection (ie, as a DNA reagent). Or in a vehicle (eg, a recombinant viral or cellular reagent) to the animal.
[0057]
One embodiment of the invention is an isolated nucleic acid molecule that hybridizes under stringent hybridization conditions to a Der HMW-map gene. Stringent hybridization conditions refer to standard hybridization conditions described herein. Preferred nucleic acid molecules of the invention are those which, under stringent hybridization conditions, have SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, No. 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 An isolated nucleic acid molecule that hybridizes to a gene encoding a protein comprising an amino acid sequence comprising SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 41, and / or SEQ ID NO: 44. Including. A more preferred nucleic acid molecule of the present invention comprises, under stringent hybridization conditions, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, which hybridizes with the complement of a nucleic acid sequence encoding a protein comprising an amino acid sequence comprising SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 41, and / or SEQ ID NO: 44. Nucleic acid molecules.
[0058]
More preferred nucleic acid molecules of the invention include isolated nucleic acid molecules selected from the group consisting of nucleic acid molecules comprising at least about 150 nucleotides, wherein the nucleic acid molecule comprising at least about 150 nucleotides comprises 1 × SSC and SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37 at a temperature of about 50 ° C. in a solution containing 0% formamide SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 45, and / or a nucleic acid sequence encoding a protein comprising the amino acid sequence of SEQ ID NO: 33, and a complement thereof. Hybridizes to a more selected nucleic acid sequence.
[0059]
The invention also encompasses fragments of any of the nucleic acid molecules disclosed herein. In accordance with the present invention, fragments may range in size between a length smaller than the sequence identified by the SEQ ID NOs of the present invention and the minimum size of the oligonucleotide as defined herein. , Any nucleic acid molecule or nucleic acid sequence. For example, the size of a fragment of the invention can be any size that is less than about 1752 nucleotides in length and greater than 11 nucleotides.
[0060]
In one embodiment of the invention, a preferred Der HMW-map nucleic acid molecule comprises an isolated nucleic acid molecule, wherein the isolated nucleic acid molecule is at least about 50 nucleotides or at least about 150 nucleotides and An isolated nucleic acid molecule can be isolated under conditions that allow for up to about 40% base pair mismatch, more preferably under conditions that allow up to about 35% base pair mismatch, and more preferably about 30% or less. %, More preferably under conditions that allow up to about 25% base pair mismatch, and more preferably under conditions that allow up to about 20% base pair mismatch. More preferably, under conditions that allow up to about 15% base pair mismatch, more preferably, up to about 10% base pair mismatch. SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22 under acceptable conditions, and even more preferably under conditions permitting about 5% or less base pair mismatch. SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 45, and nucleic acid sequence encoding a protein comprising the amino acid sequence of SEQ ID NO: 33, and Hybridizes to a nucleic acid sequence selected from the group consisting of the complements of
[0061]
Another embodiment of the present invention includes a nucleic acid molecule comprising at least about 150 base pairs, wherein the nucleic acid molecule comprises SEQ ID NO: 14 in a solution comprising 1 × SSC and 0% formamide at a temperature of about 50 ° C. SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, Sequence Hybridizes to a nucleic acid sequence selected from the group consisting of the nucleic acid sequence of No. 45 and / or a protein comprising the amino acid sequence of SEQ ID No. 33, and a complement thereof. A further preferred nucleic acid molecule of the invention comprises a fragment of the isolated nucleic acid molecule comprising at least about 150 base pairs, wherein the nucleic acid molecule is in a solution comprising 1 × SSC and 0% formamide at a temperature of about 50 ° C. , SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: , SEQ ID NO: 43, SEQ ID NO: 45, and a nucleic acid sequence encoding a protein comprising the amino acid sequence of SEQ ID NO: 33, and a nucleic acid sequence selected from the group consisting of complements thereof.
[0062]
Further preferred Der HMW-map nucleic acid molecules of the invention include an isolated nucleic acid molecule that is at least about 50 nucleotides or at least about 150 nucleotides, wherein the isolated nucleic acid molecule comprises SEQ ID NO: 14, SEQ ID NO: 16, Nucleic acid sequences of SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 45 , And a nucleic acid sequence encoding a protein comprising the amino acid sequence of SEQ ID NO: 33, and a nucleic acid sequence selected from the group consisting of the complements thereof, preferably at least about 60%, and more preferably at least about 65%. At least about 70% identical, more preferably at least about 70% identical, more preferably at least about 70% identical 75% identical, more preferably at least about 80% identical, more preferably at least about 85% identical, more preferably at least about 90% identical, and even more preferably at least about 95% identical. Certain nucleic acid sequences. Fragments of any of such nucleic acid molecules are also preferred. Percent identity can be determined using the Compare by Maximum Match function using the default parameters within the program DNAsis Version 2.1.
[0063]
One embodiment of the present invention relates to the nucleic acid molecule nDerf98. ₁₇₅₂ , NDerf98 ₁₆₆₅ , And nDerf98 ₁₆₀₈ , NDerp98 ₁₆₂₁ , NDerp98 ₁₅₂₇ , NDerp98 ₁₄₇₀ And / or nDerf60 ₅₁₀ Or all or part of an allelic variant of these nucleic acid molecules. Another preferred nucleic acid molecule of the present invention is SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, Nucleic acid sequences encoding proteins comprising the nucleic acid sequences of SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 45, and / or the amino acid sequence of SEQ ID NO: 33, and allelic modification of nucleic acid molecules having these nucleic acid sequences Preferably, such homologs comprise SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, Sequence No. 18, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 41, and / or It encodes, or is complementary to, a nucleic acid molecule encoding at least one epitope that elicits an immune response against a protein having the amino acid sequence of SEQ ID NO: 44. Such nucleic acid molecules can include, in addition to the nucleotides included in the above SEQ ID NOs, nucleotides, such as, for example, a full length gene, a full length coding region, a nucleic acid molecule encoding a fusion protein, or a multivalent defense compound. An encoding nucleic acid molecule, but is not limited thereto.
[0064]
In one embodiment, the Der HMW-map nucleic acid molecule of the invention is PDerf98. ₅₅₅ , PDerp98 ₅₀₉ And / or PDerf60 ₁₇₀ And at least about 45%, preferably at least about 50%, more preferably at least about 55%, even more preferably at least about 60%, even more preferably at least about 65%, even more preferably at least about 70%, More preferably, a protein that is at least about 75%, even more preferably at least about 80%, even more preferably at least about 85%, even more preferably at least about 90%, and even more preferably at least about 95% identical Code. Still more preferred is PDerf98 ₅₅₅ , PDerf98 ₅₃₆ , PDerp98 ₅₀₉ , PDerp98 ₄₉₀ And / or PDerf60 ₁₇₀ And / or allelic variants of such nucleic acid molecules.
[0065]
In another embodiment, the Der HMW-map nucleic acid molecule of the invention comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, sequence No. 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 41, and / or SEQ ID NO: 44, at least about 45%, preferably at least about 50%, more preferably at least about 55%, More preferably at least about 60%, even more preferably at least about 65%, even more preferably at least about 70%, even more preferably at least about 60% Encodes a protein having an amino acid sequence that is 75%, even more preferably at least about 80%, even more preferably at least about 85%, even more preferably at least about 90%, and even more preferably at least about 95% identical I do. The present invention also relates to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: No. 12, Sequence No. 13, Sequence No. 15, Sequence No. 18, Sequence No. 21, Sequence No. 23, Sequence No. 24, Sequence No. 29, Sequence No. 30, Sequence No. 31, Sequence No. 32, Sequence No. 33, Sequence No. 35 HMW-map nucleic acid molecules encoding proteins having at least a portion of SEQ ID NO: 38, SEQ ID NO: 41, and / or SEQ ID NO: 44, and allelic variants of Der HMW-map nucleic acid molecules encoding proteins having these sequences Genetic variants, including nucleic acid molecules, that have been modified to match the codon usage characteristics of the cell in which they are expressed, are also included.
[0066]
In another embodiment, a preferred Der HMW-map nucleic acid molecule is at least about 35 amino acids in length, preferably at least about 50 amino acids in length, more preferably at least about 100 amino acids in length, more preferably at least about 200 amino acids in length, even more preferably. Encodes a Der HMW-map protein comprising at least about 250 amino acids in length.
[0067]
By knowing the nucleic acid sequence of a particular Der HMW-map nucleic acid molecule of the invention, one of skill in the art can, for example, (a) make a copy of those nucleic acid molecules and (b) produce at least one such nucleic acid molecule. (E.g., nucleic acid molecules containing full length genes, full length coding regions, regulatory control sequences, truncated coding regions) and (c) obtaining other Der HMW-map nucleic acid molecules. it can. Such nucleic acid molecules can be obtained in a variety of ways, including screening an appropriate expression library using the antibodies of the present invention; Conventional cloning techniques using the oligonucleotide probes of the invention; and PCR amplification of appropriate libraries or DNA using the oligonucleotide primers of the invention. Preferred libraries for screening or amplifying nucleic acid molecules include the Dermatophagoides farinae library and / or the Dermatophagoides pteronyssius library (eg, the libraries disclosed in the Examples herein). Techniques for cloning and amplifying genes are disclosed, for example, in Sambrook et al. (Ibid).
[0068]
The present invention also provides, under stringent hybridization conditions, other nucleic acid molecules of the invention (preferably, other nucleic acid molecules that are longer) (eg, nucleic acid molecules including Der HMW-map nucleic acid molecules, or other Der HMW-map nucleic acid molecules). Nucleic acid molecules (including HMW-map nucleic acid molecules) that are oligonucleotides that are capable of hybridizing to complementary regions. The oligonucleotides of the present invention can be RNA, DNA, or any derivative. The minimum size of such an oligonucleotide is that required to form a stable hybrid between the oligonucleotide and a complementary sequence on a nucleic acid molecule of the invention. Preferred oligonucleotides of the invention have a maximum size of preferably about 200 nucleotides, more preferably about 150 nucleotides, and even more preferably about 100 nucleotides. The invention includes oligonucleotides that can be used, for example, as probes to identify nucleic acid molecules.
[0069]
One embodiment of the present invention encompasses a recombinant vector, wherein the recombinant vector is capable of delivering at least one isolated nucleic acid molecule of the present invention into a host cell. In a state of being inserted into the vector. Such a vector comprises a heterologous nucleic acid sequence, which is not naturally found in close proximity to the nucleic acid molecule of the invention, and preferably is in a species other than the species from which the nucleic acid molecule of the invention is derived. Derived nucleic acid sequence. The vector can be either RNA or DNA, can be either prokaryotic or eukaryotic, and is typically a virus or a plasmid. Recombinant vectors can be used in cloning, sequencing, and / or other manipulations of the Der HMW-map nucleic acid molecules of the invention.
[0070]
One type of recombinant vector (referred to herein as a recombinant molecule) comprises a nucleic acid molecule of the present invention operably linked to an expression vector. The phrase "operably linked" refers to the insertion of a nucleic acid molecule into an expression vector in a manner that allows the nucleic acid molecule to be expressed when transformed into a host cell. As used herein, an expression vector is a DNA or RNA vector capable of transforming a host cell and effecting the expression of a particular nucleic acid molecule. Preferably, the expression vector is also capable of replicating in the host cell. An expression vector can be either prokaryotic or eukaryotic, and the expression vector is typically a virus or a plasmid. The expression vector of the present invention functions in the recombinant cell of the present invention (including bacterial cells, fungal cells, endophytic cells, insect cells, other animal cells, and plant cells) (ie, directs gene expression). ) And any vector. Preferred expression vectors of the invention are capable of directing gene expression in bacterial cells, yeast cells, insect cells, and mammalian cells, and more preferably, in the cell types disclosed herein.
[0071]
In particular, the expression vectors of the present invention include regulatory sequences (eg, transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with recombinant cells and that control the expression of a nucleic acid molecule of the present invention). including. In particular, the recombinant molecules of the present invention include a transcription control sequence. A transcription control sequence is a sequence that controls the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those that control transcription initiation (eg, promoter sequences, enhancer sequences, operator sequences, and repressor sequences). Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells of the present invention. A variety of such transcription control sequences are known to those skilled in the art. Preferred transcription control sequences include sequences that function in bacterial cells, yeast cells, insect cells, and mammalian cells, for example, tac promoter, lac promoter, trp promoter, trc promoter, oxy-pro promoter, omp / lpp. Promoter, rrnB promoter, bacteriophage λ promoter (eg, λP _L Promoter and λP _R Promoters and fusions containing such promoters), bacteriophage T7 promoter, T7lac promoter, bacteriophage T3 promoter, bacteriophage SP6 promoter, bacteriophage SP01 promoter, metallothionein promoter, alpha mating factor promoter, Pichia alcohol oxidase promoter, alpha Viral subgenomic promoter (eg, Sindbis virus subgenomic promoter), antibiotic resistance gene promoter, baculovirus promoter, Heliothis zae insect virus promoter, vaccinia virus promoter, herpes virus promoter, raccoon poxvirus promoter, other poxvirus promoters Promoter, cytomegalovirus promoter (eg, the immediate early promoter), simian virus 40 promoter, retrovirus promoter, actin promoter, retrovirus long terminal repeat promoter, rous sarcoma virus promoter, heat shock promoter, phosphate and nitrate Transcription control sequences include, but are not limited to, other sequences capable of controlling gene expression in prokaryotic or eukaryotic cells. Additional suitable transcription control sequences include tissue-specific promoters and enhancers, and lymphokine-inducible promoters (eg, promoters induced by interferons or interleukins). The transcription control sequences of the present invention can also include naturally occurring transcription control sequences that are naturally associated with dogs or cats.
[0072]
Suitable and preferred nucleic acid molecules for inclusion in the recombinant vectors of the present invention are nucleic acid molecules as disclosed herein. Preferred nucleic acid molecules for inclusion in a recombinant vector, especially a recombinant molecule, include nDerf98 ₁₇₅₂ , NDerf98 ₁₆₆₅ , NDerf98 ₁₆₀₈ , NDerp98 ₁₆₂₁ , NDerp98 ₁₅₂₇ , NDerp98 ₁₄₇₀ , And nDerf60 ₅₁₀ Is included.
[0073]
The recombinant molecule of the present invention also comprises a secretory signal (ie, a signal segment nucleic acid sequence) such that (a) the expressed Der HMW-map protein of the present invention can be secreted from the cell producing the protein. And / or (b) a fusion sequence which results in expression of the nucleic acid molecule of the invention as a fusion protein. Examples of suitable signal segments include any signal segment that can direct the secretion of a protein of the invention. Preferred signal segments include tissue plasminogen activator (t-PA) signal segment, interferon signal segment, interleukin signal segment, growth hormone signal segment, histocompatibility signal segment and viral envelope glycoprotein signal segment, and native Signal segments include, but are not limited to. Suitable fusion segments encoded by the fusion segment nucleic acids are disclosed herein. Further, the nucleic acid molecules of the invention can be linked to a fusion segment (eg, a ubiquitin fusion segment) that directs the encoded protein to the proteosome. Recombinant molecules may also include intervening and / or untranslated sequences around and / or within the nucleic acid sequences of the nucleic acid molecules of the invention.
[0074]
Another embodiment of the present invention encompasses a recombinant cell, including a host cell transformed with one or more recombinant molecules of the present invention. Transformation of a nucleic acid molecule into a cell can be accomplished by any method that can insert a nucleic acid molecule into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. Recombinant cells can remain single cells or can grow into tissues, organs, or multicellular organisms. The transformed nucleic acid molecule of the present invention can remain extrachromosomal in the transformed (ie, recombinant) cell in such a way that the ability of the nucleic acid molecule to be expressed is maintained, Alternatively, it may be integrated at one or more sites in the chromosome of the cell. Preferred nucleic acid molecules for transforming a cell include the Der HMW-map nucleic acid molecules disclosed herein. Particularly preferred nucleic acid molecules for transforming cells include nDerf98 ₁₇₅₂ , NDerf98 ₁₆₆₅ , NDerf98 ₁₆₀₈ , NDerp98 ₁₆₂₁ , NDerp98 ₁₅₂₇ , NDerp98 ₁₄₇₀ , And nDerf60 ₅₁₀ Is included.
[0075]
Suitable host cells for transformation include any cell that can be transformed with a nucleic acid molecule of the present invention. The host cell can be a non-transformed cell or a nucleic acid molecule encoding at least one nucleic acid molecule (eg, one or more proteins of the invention and / or other proteins useful for producing a multivalent vaccine). ) May have already been transformed. A host cell of the invention can produce the Der HMW-map protein of the invention endogenously (ie, naturally), or produce such a protein after being transformed with at least one nucleic acid molecule of the invention. You can either. A host cell of the invention can be any cell capable of producing at least one protein of the invention and includes bacterial cells, fungal cells (including yeast), other insect cells, other animal cells And plant cells. Preferred host cells include bacterial cells, mycobacterial cells, yeast cells, parasite cells, insect cells and mammalian cells. More preferred host cells include Salmonella, Escherichia, Bacillus, Listeria, Saccharomyces, Spodoptera, Mycobacteria, Trichoplusia, BHK (infant hamster kidney) cells, MDCK cells (infectious cells of canine normal necrositis virus C), MDCK cells (for normal canine herpes virus C virus). Cells (normal cat kidney cell line for feline herpes virus culture), CV-1 cells (eg, African monkey kidney cell line used to culture raccoon poxvirus), COS (eg, COS-7) ) Cells, and Vero cells. Particularly preferred host cells include Escherichia coli (including an E. coli K-12 derivative); Salmonella typhi; Salmonella typhimurium (UK-1). _X 3987 and SR-11 _X Spodoptera frugiperda; Trichoplusia ni; BHK cells; MDCK cells; CRFK cells; CV-1 cells; COS cells; Vero cells and non-tumorigenic mouse myoblast G8 cells (eg, ATCC CRL 1246). Additional suitable mammalian cell hosts include other kidney cell lines, other fibroblast cell lines (eg, human, mouse, or chick embryo fibroblast cell lines), myeloma cell lines, Chinese hamster ovary cells, mice NIH / 3T3 cells, LMTK ³¹ And / or HeLa cells.
[0076]
The recombinant cells are preferably produced by transforming a host cell with one or more recombinant molecules, each recombinant molecule operably linked to an expression vector comprising one or more transcription control sequences. It comprises one or more nucleic acid molecules of the invention. The phrase "operably linked" refers to inserting a nucleic acid molecule into an expression vector in such a way that it can be expressed when the nucleic acid molecule is transformed into a host cell.
[0077]
A recombinant molecule of the present invention is a molecule that can include at least one of any of the above nucleic acid molecules operably linked to at least one of the optional transcription control sequences, wherein the transcription control sequence is , Can effectively regulate the expression of the nucleic acid molecule in the transformed cell. These examples are disclosed herein.
[0078]
A recombinant cell of the invention includes any cell transformed with at least one of the Der HMW-map nucleic acid molecules of the invention. Suitable and preferred Der HMW-map nucleic acid molecules as well as suitable and preferred recombinant molecules for transforming cells are disclosed herein.
[0079]
Recombinant DNA technology involves, for example, manipulating the copy number of nucleic acid molecules in a host cell, the efficiency with which those nucleic acid molecules are transcribed, the efficiency with which the resulting transcript is translated, and the efficiency of post-translational modifications. Can be used to improve the expression of the transformed nucleic acid molecule. Recombinant techniques useful for increasing expression of a nucleic acid molecule of the invention include, but are not limited to: operable linkage of a nucleic acid molecule to a high copy number plasmid, one or more host cells. Integration of nucleic acid molecules into chromosomes, addition of vector stability sequences to plasmids, substitution or modification of transcription control signals (eg, promoters, operators, enhancers), translation control signals (eg, ribosome binding sites, Shine-Dalgarno sequences) ), Modification of the nucleic acid molecules of the invention to correspond to the codon usage of the host cell, deletion of sequences that destabilize transcripts, and growth of recombinant cells from recombinant enzyme production during fermentation. The use of control signals to decouple time. The activity of the expressed recombinant proteins of the invention can be improved by fragmenting, modifying or derivatizing nucleic acid molecules encoding such proteins.
[0080]
The isolated Der HMW-map proteins of the present invention can be produced in various ways, including the production and recovery of native proteins, the production and recovery of recombinant proteins, and the chemical synthesis of these proteins. No. In one embodiment, an isolated protein of the invention is produced by culturing cells capable of expressing the protein under conditions effective to produce the protein, and recovering the protein. You. Preferred cells for culturing are the recombinant cells of the present invention. Effective culture conditions include, but are not limited to, effective media conditions, bioreactor conditions, temperature conditions, pH conditions, and oxygen conditions that allow for protein production. An effective medium refers to any medium in which cells are cultured to produce a Der HMW-map protein of the present invention. Such media typically includes an aqueous medium having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients (eg, vitamins). The cells of the invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and Petri plates. The culturing can be performed at a temperature, pH and oxygen content appropriate for the recombinant cells. Such culture conditions are within the skill of those in the art.
[0081]
Depending on the vector and host system used for production, the resulting protein of the present invention may remain in the recombinant cell; may be secreted into the fermentation medium; It can either be secreted into space (eg, the periplasmic space of E. coli); or it can be retained on the outer surface of the cell or virus membrane. The phrase "recover protein", as well as similar phrases, refers to recovering the entire fermentation medium containing the protein, and need not include the additional steps of separation or purification. The proteins of the present invention can be prepared using various standard protein purification techniques (eg, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A). (Including, but not limited to, chromatography, isoelectric focusing and differential solubilization). The proteins of the present invention can preferably be recovered in "substantially pure" form. As used herein, "substantially pure" refers to a purity that allows for the effective use of the protein as a therapeutic composition or diagnostic. For example, a therapeutic composition for an animal should not exhibit substantial toxicity and should preferably be able to desensitize the animal to be treated.
[0082]
The present invention also provides isolated (ie, removed from their natural environment) antibodies (ie, anti-Der HMW-map) that selectively bind to a Der HMW-map protein of the invention or its mimetope. Protein antibodies). As used herein, the term "selectively binds" to Der HMW-map means that the antibody of the invention preferentially binds to a specified protein of the invention or a mimetope thereof. Ability. Binding can be measured using various methods standard in the art, including enzyme immunoassays (eg, ELISA), immunoblot assays, and the like; see, eg, Sambrook et al., Ibid. The anti-Der HMW-map protein antibody preferably binds selectively to the portion of the Der HMW-map protein that elicits an immune response in the animal.
[0083]
An isolated antibody of the invention can include an antibody in a body fluid, such as, but not limited to, serum, or an antibody that has been purified to various degrees. The antibodies of the present invention can be polyclonal or monoclonal. Functional equivalents of such antibodies (eg, antibody fragments and engineered antibodies, including single-chain or chimeric antibodies capable of binding more than one epitope) are also included in the invention.
[0084]
A preferred method for producing the antibodies of the present invention comprises: (a) administering an effective amount of the protein, peptide or mimetope thereof to an animal to produce the antibodies; and (b) recovering those antibodies. Performing the steps of: In another method, the antibodies of the invention are produced recombinantly, using techniques such as those disclosed above, to produce a Der HMW-map protein of the invention. Antibodies raised against the defined protein or mimetope may be advantageous. This is because such antibodies, when used in therapeutic compositions, are substantially free of antibodies to other substances that might otherwise cause interference or side effects in diagnostic assays.
[0085]
The antibodies of the present invention have various potential uses within the scope of the present invention. For example, such antibodies may be (a) used as a tool for detecting mite allergens (particularly Der HMW-map proteins); (b) as a tool for screening expression libraries; and / or (c) It can be used to recover the desired protein of the invention from a mixture of protein and other contaminants. The antibodies of the present invention also inhibit, for example, the binding of Der HMW-map protein to IgE, which specifically binds to Der HMW-map, and prevent immune complex formation, thereby reducing the hypersensitivity response to mite allergens. Can be used to reduce.
[0086]
The Der HMW-map protein of the present invention may be comprised in a chimeric molecule, wherein the chimeric molecule comprises at least a portion of a Der HMW-map protein that induces an immune response in an animal, and a second molecule. The second molecule has the chimeric molecule bound to the substrate in such a way that a portion of the Der HMW-map protein can bind IgE in essentially the same manner as the Der HMW-map protein not bound to the substrate. To be able to Examples of suitable second molecules include a portion of an immunoglobulin molecule or another ligand having a suitable binding partner that can be immobilized on a substrate (eg, biotin and avidin, or a metal binding protein and metal). (Eg, His), or a sugar-binding protein and a sugar (eg, maltose)).
[0087]
The Der HMW-map protein of the present invention may be included in a formulation and is referred to herein as a Der HMW-map protein formulation. For example, a Der HMW-map protein can be combined with a buffer and / or carrier in which the Der HMW-map protein is solubilized. Suitable buffers and carriers are known to those skilled in the art. Examples of suitable buffers include any buffer that can function to selectively bind an antibody that specifically binds to a Der HMW-map protein, such as a phosphate buffer. Saline, water, saline, phosphate buffer, bicarbonate buffer, HEPES buffer (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffered saline), TES buffer Liquid (Tris-EDTA buffered saline), Tris buffer and TAE buffer (Tris-acetic acid-EDTA). Examples of carriers include, but are not limited to, polymer matrices, toxoids, and serum albumin (eg, bovine serum albumin). The carrier can be mixed with the Der HMW-map protein in a manner that does not substantially interfere with the ability of the Der HMW-map protein to selectively bind to an antibody that specifically binds to the Der HMW-map protein, or It can be conjugated (ie, attached) to a Der HMW-map protein.
[0088]
The Der HMW-map protein of the present invention can be produced by a cell containing the Der HMW-map protein. Preferred Der HMW-map protein-bearing cells include recombinant cells containing a nucleic acid molecule encoding the Der HMW-map protein of the present invention.
[0089]
In addition, the Der HMW-map protein formulations of the present invention may comprise not only Der HMW-map protein, but also one or more additional antigens or antibodies useful in desensitizing animals to allergies or preventing or treating mite allergen pathogens. May be included. As used herein, antigen refers to any molecule that can be selectively bound by an antibody. As used herein, an allergen refers to any antigen that can stimulate the production of antibodies involved in an allergic response in an animal. As used herein, selective binding of a first molecule to a second molecule refers to the ability of the first molecule to bind to the third molecule as compared to the ability of the first molecule to bind to the third molecule. Refers to the ability to preferentially bind (eg, have higher affinity, higher avidity) to a second molecule. This first molecule need not necessarily be the natural ligand of the second molecule. The allergens of the present invention are preferably of mite origin and are mite associated allergens, including but not limited to other insect and plant allergens.
[0090]
In accordance with the present invention, virtually any substance can act as an antigen and elicit an antibody response (ie, can function as an epitope). For example, antibodies can be raised in response to a carbohydrate epitope (a saccharide and / or a polysaccharide (a so-called glycosylated protein) attached to a protein). However, saccharides and / or polysaccharides can act alone as antigens in the absence of proteins. The terminal and internal sugars of the carbohydrate moiety can act as epitopes. Polysaccharides can exist as branches, in which case the epitope can have sugars that are not consecutive but spatially adjacent. Unusual, insect-specific sugars not normally found in mammalian proteins can be present in glycoproteins derived from insect nucleic acid molecules, and these unusual sugars are recognized by the mammalian immune system It may include an epitope.
[0091]
One embodiment of the present invention relates to the ability to bind to IgE in animals that are allergic to mites, to desensitize animals to mite allergens, to stimulate B lymphocyte responses, And / or a reagent comprising a non-protein epitope capable of stimulating a T lymphocyte response. Such epitopes (referred to herein as Der NP epitopes) may be present as part of a Der HMW-map protein of the invention, or may be isolated from a Der HMW-map protein of the invention. Such epitopes are, for example, those of the D. cerevisiae produced according to Example 1. present on proteins contained in the farinae HMW-map composition. A Der NP epitope of the invention can be isolated from its natural source or can be produced synthetically. Such an epitope may be, but need not be, linked to a carrier or other molecule. A Der NP epitope has at least one of the following distinguishing characteristics: (a) the epitope is resistant to β-elimination of the peptide; (b) the epitope is proteinase K digested And (c) the epitope is reactive to a test designed to detect glycosylated proteins. Preferred Der NP epitopes have all of these identified features. Der NP epitopes can selectively bind to dog or cat IgE allergic to mites. Without being bound by theory, it is believed that the Der NP epitope includes a carbohydrate moiety that is apparently free of N-linked glycans. The identification of the structural features of such epitopes can be determined by one skilled in the art. In one embodiment, an isolated antibody that selectively binds to a Der NP epitope is provided. The present invention also provides for derivatives of a Der NP epitope (ie, mimics the activity of such an epitope (eg, is a mimetope of a Der NP epitope)), against a native (ie, naturally occurring) Der Np epitope. A compound capable of binding to the antibody obtained.
[0092]
Reagents containing a Der NP epitope of the invention can be used in various ways according to the invention. Such a reagent can be a desensitizing compound or a detection reagent for testing for susceptibility or sensitivity to mite allergy. In one embodiment, a therapeutic composition of the invention comprises a reagent comprising a Der NP epitope. In another embodiment, an assay kit of the invention comprises a reagent comprising a Der NP epitope. One embodiment of the invention is a method of identifying an animal susceptible to or having an allergic response to a tick. Such methods include contacting a reagent comprising the Der NP epitope with an antibody in an animal, and determining the formation of an immune complex between the reagent and the antibody, wherein the formation of the immune complex is Indicates that the animal is susceptible to or has the allergic response. Another embodiment of the present invention is a method for desensitizing a host animal to an allergic response to mites. Such methods include administering to the animal a therapeutic composition that includes a reagent that includes a Der NP epitope as a desensitizing compound.
[0093]
Another embodiment of the present invention is a Der HMW-map protein lacking a Der NP epitope. Without being bound by theory, such proteins are better desensitizing compounds. This is because such proteins are expected to have reduced ability to bind IgE. Such proteins can be produced, for example, by removing the Der NP epitope from the native Der HMW-map protein, or by producing the protein recombinantly (eg, in E. coli).
[0094]
One embodiment of the present invention is an in vivo test that can detect whether an animal is hypersensitive to Der HMW-map protein. The in vivo hypersensitivity test of the present invention is particularly useful for identifying animals that are susceptible to or have an allergy to mite allergens. A suitable in vivo hypersensitivity test of the present invention may be, but is not limited to, a skin test, which comprises administering an effective amount of a formulation comprising a Der HMW-map protein or a mimetope thereof (eg, a skin test). Internal injection or surface scratching). Methods for performing the skin tests of the present invention are known to those of skill in the art and are briefly disclosed herein.
[0095]
Suitable formulations for use in in vivo skin tests include Der HMW-map protein, homologues of Der HMW-map protein and / or mimetopes of Der HMW-map protein.
[0096]
The appropriate amount of Der HMW-map protein protein formulation for use in the skin test of the present invention will vary widely depending on the allergenicity of the formulation used in the test and the site at which the product is to be delivered. Can change. An appropriate amount of a Der HMW-map protein formulation for use in the skin test of the present invention may form a reaction, such as a detectable wheal or hardening (hardness) resulting from an allergic reaction to the formulation. Quantity. The preferred amount of Der HMW-map protein for use in the skin test of the present invention is about 1 x 10 ^-8 Micrograms (μg) to about 100 μg, more preferably about 1 × 10 ^-7 μg to about 10 μg, and even more preferably about 1 × 10 ^-6 It ranges from μg to about 1 μg Der HMW-map protein. It is understood by those skilled in the art that such amounts will vary depending on the allergenicity of the protein being administered.
[0097]
According to the present invention, the Der HMW-map protein of the present invention may be combined with an immunopotentiator, such as a carrier or adjuvant of the present invention, as defined in detail below. However, a new aspect of the invention is that the Der HMW-map protein of the invention can induce a hypersensitivity response in the absence of immunopotentiators, especially in dogs.
[0098]
The skin test of the present invention further comprises the step of administering a control solution to the animal. The control solution may include a negative control solution and / or a positive control solution. The positive control solution of the present invention contains an effective amount of at least one compound known to induce a hypersensitivity response when administered to an animal. Preferred compounds for use as a positive control solution include, but are not limited to, histamine. The negative control solution of the present invention may include a solution known to not induce a hypersensitivity response when administered to an animal. As such, a negative control solution may comprise a solution having a compound that is essentially unable to induce a hypersensitivity response, or may comprise a buffer (eg, saline) used to prepare the formulation. It may include a solution that simply has. An example of a preferred negative control solution is phenolated phosphate buffered saline (available from Greer Laboratories, Inc., Lenoir, NC).
[0099]
Hypersensitivity of an animal to one or more formulations of the present invention measures the response resulting from the administration of one or more experimental and control samples to the animal (eg, wheal, hardening or hardness; techniques known to those of skill in the art). Use) and by comparing the response to the experimental sample with the response resulting from the administration of one or more control solutions. Preferred devices for intradermal injection include individual syringes. Preferred devices for scratching include devices that allow administration of many samples at a time. The hypersensitivity of an animal is determined by determining whether the response resulting from administration of a formulation of the invention is greater than the response resulting from administration of a negative control, and / or the response resulting from administration of the formulation. By determining whether it is at least about the same size as the response resulting from administration of the positive control solution. As such, an animal is hypersensitive to the experimental sample if the experimental sample produces a response greater than or equal to the size of the wheal caused by administration of the positive control sample to the animal. Conversely, if the experimental sample produces a response similar to that produced by administration of the negative control sample to the animal, the animal is not hypersensitive to the experimental sample.
[0100]
Preferred wheal sizes for the assessment of animal hypersensitivity are diameters ranging from about 16 mm to about 8 mm, more preferably from about 15 mm to about 9 mm, and even more preferably from about 14 mm to about 10 mm.
[0101]
Preferably, the ability of the animal to produce an immediate or no hypersensitivity response to the formulation of the invention is from about 2 minutes to about 30 minutes after administration of the sample, more preferably From about 10 minutes to about 25 minutes after administration of the sample, and even more preferably about 15 minutes after administration of the sample.
[0102]
Preferably, the ability of the animal to produce a delayed-type hypersensitivity response to the formulation of the invention and the inability to produce the hypersensitivity response is from about 18 hours to about 30 hours after administration of the sample, more preferably About 20 hours to about 28 hours after administration of the sample, and even more preferably about 24 hours after administration of the sample, by measuring sclerosis and / or erythema. A delayed type hypersensitivity response may also be determined using other techniques (e.g., using techniques known to those skilled in the art, to determine the extent of cellular invasion at the site of administration during the period directly defined above). ) Can be measured.
[0103]
In a preferred embodiment, the skin test of the present invention comprises the steps of intradermally injecting an effective amount of a formulation comprising Der HMW-map protein into an animal at a predetermined site, and applying an effective amount of a control solution to the same animal at different sites. Intradermal injection is included. It is within the skill of the art to use a device that can deliver multiple samples simultaneously at many sites (preferably allowing for simultaneous evaluation of many formulations). Preferred Der HMW-map proteins for use in skin tests include the full length protein. A preferred positive control sample can be a sample containing histamine. A preferred negative control sample can be a sample containing a diluent.
[0104]
Suitable and preferred animals for testing for hypersensitivity to Der HMW-map protein using the skin test of the present invention are disclosed herein. Particularly preferred animals for testing in the skin test of the present invention include humans, dogs, cats and horses, with humans, dogs and cats being even more preferred. As used herein, canine refers to any member of the canine family, including domestic dogs, wild dogs, and zoo dogs. Examples of dogs include, but are not limited to, domestic dogs, wild dogs, foxes, wolves, jackals and coyotes. As used herein, cat refers to any member of the family Felidae, including domestic cats, wild cats and zoo cats. Examples of cats include, but are not limited to, cats, lions, tigers, leopards, leopards, panthers, pumas, bobcats, lynxes, jaguars, cheetahs and servals in the home. As used herein, horse refers to any member of the equid family, including horses, donkeys, mules, and zebras.
[0105]
One embodiment of the invention is a method of detecting an antibody that binds to a Der HMW-map protein (referred to herein as an anti-Der HMW-map antibody) in vitro. The method comprises the steps of: (a) combining the isolated HMW-map protein and a composition containing a putative anti-Der HMW-map antibody to form a Der HMW-map protein: antibody complex And (b) detecting the presence of the antibody by detecting the Der HMW-map protein: antibody complex. The presence of such a Der HMW-map protein: antibody complex indicates that the animal is producing antibodies to mite allergens. Preferred anti-Der HMW-map antibodies for detection include antibodies having the IgE or IgG isotype. Preferred anti-Der HMW-map antibodies for detection include cat, dog, horse, and human, with cat, dog and human being particularly preferred.
[0106]
As used herein, the term "contacting" refers in this case to combining or mixing the putative antibody-containing composition and the Der HMW-map protein. The formation of a complex between the Der HMW-map protein and the antibody indicates that the Der HMW-map protein selectively binds to the antibody to form a stable complex that can be measured (ie, detected). Ability to do. As used herein, the term selectively binds to an antibody refers to the Der HMW-map of the present invention without substantially binding to other antibodies that do not specifically bind to the Der HMW-map protein. Refers to the ability of a protein to bind preferentially to an antibody. Binding between the Der HMW-map protein and the antibody is effected under conditions suitable for forming a complex; such conditions (eg, appropriate concentrations, buffers, temperatures, reaction times), and the like. Methods for optimizing such conditions are known to those skilled in the art, and examples are disclosed herein. Examples of complex formation conditions are also described, for example, in Sambrook et al. , (Ibid.).
[0107]
As used herein, the term “detecting complex formation” refers to determining whether any complex has formed (ie, assaying for the presence (ie, presence) of the complex). That). Once the complex is formed, the amount of complex formed is determined, but not required. Complex formation, or selective binding, between the Der HMW-map protein and the antibody in the composition can be accomplished using a variety of methods standard in the art (see, eg, Sambrook et al., Ibid.). Measured (ie, detected, determined). These examples are disclosed herein.
[0108]
In one embodiment, the putative antibody-containing composition of the method of the invention comprises a biological sample from an animal. Suitable biological samples include, but are not limited to, body fluid compositions or cell compositions. Body fluid refers to any fluid that can be collected (ie, obtained) from an animal, such as blood, serum, plasma, urine, tears, aqueous humor, cerebrospinal fluid ( CSF), saliva, lymph, nasal secretions, milk, and feces. Such compositions of the invention may be pre-treated to remove at least some of the non-IgE or non-IgG isotypes of immunoglobulins and / or other proteins (eg, albumin) present in body fluids, It is not necessary. Such removal involves contacting the body fluid with a substance such as an antibody that specifically binds to the lectin jacalin or the constant region of an IgA immunoglobulin (ie, an anti-IgA isotype antibody) to remove the IgA antibody. And / or affinity purification of the IgE or IgG antibody from other components of the body fluid by, for example, exposing the body fluid to, for example, concanavalin A or protein G, respectively. In another embodiment, the composition comprises a collected bodily fluid that has been pre-processed to concentrate immunoglobulins contained in the bodily fluid. For example, immunoglobulins contained in body fluids can be precipitated from other proteins using ammonium sulfate. A preferred composition of the present invention is serum.
[0109]
In another embodiment, the antibody-containing composition of the method of the present invention includes an IgE or IgG producing cell. Such cells may have IgE or IgG bound to the cell surface and / or may secrete IgE or IgG. Examples of such cells include myeloma cells. IgE or IgG can be bound to the surface of cells, either directly to the membrane of the cell or to a molecule (eg, an antigen) bound to the surface of the cell.
[0110]
The complex can be detected in various ways, including but not limited to the use of one or more of the following assays: enzyme-linked immunoassay, radioimmunoassay, fluorescence immunoassay, chemiluminescence assay, Lateral flow assays, agglutination assays, particle-based assays (eg, using particles such as, but not limited to, magnetic particles or particles such as plastic polymers (eg, latex or polystyrene beads)), immunoprecipitation Assay, BioCore ^TM Assays (eg, using colloidal gold), and immunoblotting assays (eg, Western blot). Such assays are well-known to those skilled in the art. Assays can be used to give qualitative or quantitative results, depending on how the results can be used. For example, some assays, such as agglutination, particle separation, and immunoprecipitation, can be visually observed without the need for detectable markers (eg, unaided or densitometric or spectrophotometric, such as By any of the machines).
[0111]
In other assays, the detectable marker Der HMW-map protein, an antibody bound to the Der HMW-map protein, or a reagent that selectively binds to the Der HMW-map protein or an antibody bound to the Der HMW-map protein ( Conjugation (i.e., binding), described in more detail below, assists in detecting complex formation. Examples of detectable markers include, but are not limited to, radioactive labels, enzymes, fluorescent labels, chemiluminescent labels, chromogenic labels or ligands. A ligand refers to a molecule that selectively binds to another molecule. Preferred detectable markers include, but are not limited to: fluorescein, radioisotopes, phosphatases (eg, alkaline phosphatase), biotin, avidin, peroxidase (eg, horseradish peroxidase), and biotin-related compounds Or an avidin-related compound (eg, streptavidin or ImmunoPure® NeutrAvidin commercially available from Pierce, Rockford, IL).
[0112]
In one embodiment, the complex is detected by contacting the putative antibody-containing composition with a Der HMW-map protein conjugated to a detectable marker. Suitable detection markers for conjugation to Der HMW-map protein include, but are not limited to, radioactive labels, fluorescent labels, enzyme labels, chemiluminescent labels, chromogenic labels or ligands. The detectable marker is conjugated to the Der HMW-map protein such that it does not block the ability of the Der HMW-map protein to bind to the antibody to be detected.
[0113]
In another embodiment, the Der HMW-map protein: antibody complex is detected by contacting the putative antibody-containing composition with the Der HMW-map protein, and then contacting the complex with an indicator molecule. You. Suitable indicator molecules of the present invention include molecules that can bind to either the Der HMW-map protein or an antibody that binds to the Der HMW-map protein. Thus, indicator molecules can include, for example, antigens and antibodies, depending on which part of the Der HMW-map protein: antibody complex is detected. Preferred indicator molecules that are antibodies include, for example, anti-IgE antibodies, anti-IgG antibodies, and a putative antibody-containing composition that binds to a Der HMW-map protein, but is known to bind to a different epitope on the Der HMW-map protein. Antibodies other than the antibody identified in the product. Preferred lectins include those that bind to high mannose groups. The indicator molecule itself can be bound to a detectable marker of the invention. For example, the antibodies can be conjugated to biotin, horseradish peroxidase, alkaline phosphatase or fluorescein.
[0114]
In one preferred embodiment, the Der HMW-map protein: antibody conjugate is selected from a combination of the conjugate with an indicator molecule that selectively binds an IgE antibody (referred to herein as an anti-IgE reagent) or an IgG antibody. It is detected by contact with a specifically binding indicator molecule (herein referred to as an anti-IgG reagent). Examples of such anti-IgE or anti-IgG antibodies include secondary antibodies that are anti-isotype antibodies (eg, antibodies that selectively bind to the constant region of IgE or IgG), antibody-binding bacterial surface proteins (eg, protein A). Or protein G), antibody-binding cells (eg, B cells, T cells, natural killer cells, polymorphonuclear leukocytes, monocytes or macrophage cells), antibody-binding eukaryotic cell surface proteins (eg, Fc receptors), and Antibody binding complement proteins include, but are not limited to. Preferred indicator molecules include, but are not limited to: anti-cat IgE antibodies, anti-cat IgG antibodies, anti-dog IgE antibodies, anti-dog IgG antibodies, anti-human IgE antibodies, and anti-human IgG antibodies. As used herein, an anti-IgE or anti-IgG antibody includes not only whole antibodies, but also any subunits or portions thereof that can selectively bind to an IgE or IgG heavy chain constant region. obtain. For example, anti-IgE reagents or anti-IgG reagents include Fab fragments or F (ab ') ₂ Fragments, both of which are described in Janeway et al. , Immunobiology, the Immun System in Health and Disease, Garland Publishing, Inc. , NY, 1996.
[0115]
In another preferred embodiment, the Der HMW-map protein: antibody conjugate comprises a Der HMW-map protein and a different epitope other than the epitope at which the antibody in the putative antibody-containing composition binds to the Der HMW-map protein. It is detected by contact with an indicator molecule that selectively binds to the map protein.
[0116]
In one embodiment, the complex is formed in solution and can be detected. In another embodiment, a complex is formed, wherein one or more members of the complex are immobilized on (eg, coated on) the substrate. Immobilization techniques are known to those skilled in the art. Suitable substrate materials include plastic, glass, gel, celluloid, paper, PVDF (poly-vinylidene-fluoride), nylon, nitrocellulose, and particulate matter (eg, latex, polystyrene, nylon, nitrocellulose, agarose and magnetic Resin), but is not limited thereto. Suitable shapes for the substrate material include wells (eg, microtiter dish wells), plates, dipsticks, beads, sidestream devices, membranes, filters, tubes, dishes, celluloid-type matrices, magnetic particles, and other particles Substances include, but are not limited to. Particularly preferred substrates include ELISA plates, dipsticks, radioimmunoassay plates, agarose beads, plastic beads, latex beads, immunoblot membranes and immunoblot papers. In one embodiment, the substrate (eg, particulate matter) can include a detectable marker.
[0117]
A preferred method for detecting antibodies that bind to Der HMW-map protein is an immunosorbent assay. The immunosorbent assay of the present invention includes a capture molecule and an indicator molecule. The capture molecules of the invention bind to IgE or IgG in such a way that the IgE or IgG is immobilized on the substrate. As such, the capture molecule is preferably immobilized on the substrate of the invention, after which the capture molecule is exposed to the putative IgE-containing composition or putative IgG-containing composition. The indicator molecule of the present invention detects the presence of IgE or IgG bound to the capture molecule. Thus, the indicator molecule is preferably not immobilized on the same substrate as the capture molecule prior to exposing the capture molecule to the putative IgE-containing composition or putative IgG-containing composition.
[0118]
A preferred immunosorbent assay involves any of the following steps: (a) After immobilizing the Der HMW-map protein on a substrate, the Der HMW-map protein and a putative IgE-containing composition or putative IgG. Contacting the Der HMW-map protein-immobilized substrate to form a Der HMW-map protein-immobilized substrate; and (b) contacting the Der HMW-map protein with a putative IgE-containing composition or a putative IgG-containing composition. Binding the putative IgE-containing composition or putative IgG-containing composition to the substrate prior to forming the putative IgE-containing composition-attached substrate or putative IgG-containing composition-binding substrate. Preferably, the substrate includes an uncoated substrate, a Der HMW-map protein-immobilized substrate, an anti-IgE antibody-immobilized substrate, and an anti-IgG antibody-immobilized substrate.
[0119]
Both the capture molecule and the indicator molecule of the invention can bind to IgE, IgG or Der HMW-map proteins. Preferably, the capture molecule binds to a region of the IgE, IgG or Der HMW-map protein that is different from the indicator molecule, whereby the capture molecule simultaneously binds to the IgE, IgG or Der HMW-map protein simultaneously with the indicator molecule. Make it possible. The use of a reagent as a capture or indicator molecule depends on whether the molecule is immobilized on a substrate when exposed to an IgE, IgG or Der HMW-map protein. For example, the Der HMW-map protein of the present invention is used as a capture molecule when the Der HMW-map protein is bound to a substrate. Alternatively, Der HMW-map protein is used as an indicator molecule when Der HMW-map protein is not bound to a substrate. Suitable molecules for use as capture or indicator molecules include, but are not limited to, a Der HMW-map protein of the invention, an anti-IgE or anti-IgG antibody reagent of the invention.
[0120]
An immunosorbent assay of the invention can include a second molecule or other binding molecule that can detect the presence of one or more layers and / or types of indicator molecules. For example, a non-tagged (ie, not conjugated to a detectable marker) secondary antibody that selectively binds to an indicator molecule can be a tagged antibody that selectively binds to the secondary antibody. (Ie, conjugated to a detectable marker). Appropriate secondary, tertiary and other secondary or tertiary molecules can be selected by those skilled in the art. Preferred secondary molecules of the invention include antigens, anti-IgE idiotype antibodies (ie, antibodies that bind to an epitope unique to anti-IgE antibodies), anti-IgE isotype antibodies, anti-IgG idiotype antibodies (ie, anti-IgG antibodies). Antibodies that bind to a unique epitope), and anti-IgG isotype antibodies. Preferred tertiary molecules can be selected by one skilled in the art based on the characteristics of the secondary molecule. The same strategy applies to subsequent hierarchies.
[0121]
In one embodiment, the Der HMW-map protein is used as a capture molecule by being immobilized on a substrate (eg, a microtiter dishwell or dipstick). The biological sample collected from the animal is applied to a substrate and allowed to bind to the substrate, allowing Der HMW-map protein: antibody complex formation (i.e., if the IgE or IgG in the sample is Incubate under conditions appropriate (ie, sufficient) to bind to the Der HMW-map protein immobilized above. Excess unbound material (i.e., material from a biological sample that is not bound to the Der HMW-map protein), if present, is present under conditions that retain the antigen: antibody complex bound to the substrate. Is removed from the substrate. Preferred conditions are generally disclosed in Sambrook et al. (Ibid). An indicator molecule capable of selectively binding to IgE or IgG bound to the antigen is added to the substrate and incubated to form a complex between the indicator molecule and the Der HMW-map protein: antibody complex. . Excess indicator molecules are removed, a chromogen is added if necessary, and the substrate is subjected to a detection device for analysis. A preferred indicator molecule for this embodiment is an anti-IgG antibody for detecting an IgG antibody bound to the Der HMW-map protein, or an anti-IgE antibody for detecting an IgE antibody bound to the Der HMW-map protein. Preferably, the anti-IgG or anti-IgE antibody is conjugated to biotin, a fluorescent label or an enzyme label.
[0122]
In one embodiment, the anti-IgE or anti-IgG antibody (ie, an isotype-specific antibody or an idiotype-specific antibody) is immobilized on a substrate (eg, a microtiter dishwell or dipstick). Is used as a capture molecule. A biological sample collected from an animal is applied to the substrate and the appropriate bound to allow the formation of an anti-IgE antibody: IgE complex or an anti-IgG antibody: IgG complex, respectively, bound to the substrate. Incubate under conditions. Excess unbound material, if present, is removed from the substrate under conditions that retain the anti-IgE antibody: IgE complex or anti-IgG antibody: IgG complex bound to the substrate. Der HMW-map protein is added to the substrate and incubated to allow the formation of a complex between the Der HMW-map protein and an anti-IgE antibody: IgE or anti-IgG antibody: IgG complex. You. Preferably, the Der HMW-map protein is conjugated to a detectable marker, preferably biotin, an enzyme label or a fluorescent label. Excess Der HMW-map protein is removed, a chromogen is added if necessary, and the substrate is subjected to a detection device for analysis.
[0123]
In one embodiment, the immunosorbent assays of the invention do not utilize a capture molecule. In this embodiment, the biological sample collected from the animal is applied to a substrate (eg, a microtiter dishwell or dipstick) and conditions appropriate to allow binding of IgE or IgG to the substrate. Incubate underneath. Any IgE or IgG present in the body fluid is immobilized on the substrate. Excess unbound material, if present, is removed from the substrate under conditions that retain the binding of IgE or IgG to the substrate. Der HMW-map protein is added to the substrate and incubated to allow for the formation of a complex between the Der HMW-map protein and IgE or IgG. Preferably, the Der HMW-map protein is conjugated to a detectable marker, preferably biotin, an enzyme label or a fluorescent label. Excess Der HMW-map protein is removed, a chromogen is added if necessary, and the substrate is subjected to a detection device for analysis.
[0124]
Another preferred method for detecting IgE or IgG is the sidestream assay, an example of which is U.S. Patent No. 5,424,193 by Pronovost et al, issued June 13, 1995; Imrich et al. U.S. Patent No. 5,415,994 issued May 16, 1995; WO94 / 29696 by Miller et al (published December 22, 1994); and WO94 / 01775 by Pawlak et al (January 1994). Published on 20th). In one embodiment, the biological sample is placed in a side stream device comprising the following components: (a) a support structure defining a flow path; (b) conjugated to a Der HMW-map protein. A labeling reagent comprising the labeled beads (the labeling reagent is injected into the support structure in the labeling zone); and (c) a capture reagent comprising an IgE binding composition or an IgG binding composition. The capture reagent is located downstream of the labeling reagent in a capture zone fluidly connected to the labeling zone in a manner that allows the labeling reagent to flow from the labeling zone to the capture zone. The support structure includes a substance that does not impede the flow of beads from the labeling zone to the capture zone. Suitable materials for use as the support structure include ionic (ie, anionic or cationic) materials. Examples of such materials include, but are not limited to, nitrocellulose (NC), PVDF, carboxymethylcellulose (CM). The support structure defines a flow path that is lateral and divided into zones (ie, a label zone and a capture zone). The device may further comprise a sample receiving zone located along the flow path, more preferably upstream of the labeling reagent. The flow path in this support structure contacts a portion of the support structure downstream of the capture zone, preferably at the end of the flow path, with an adsorbent capable of absorbing excess reagent from the labeling zone and the capture zone. It is produced by causing
[0125]
In this embodiment, a biological sample is applied to a sample receiving zone comprising a portion of the support structure. The labeling zone receives sample from a sample receiving zone directed downstream by a flow path. The labeling zone contains a labeling reagent that binds IgE or IgG, or both. A preferred labeling reagent is a Der HMW-map protein conjugated to a plastic bead substrate (eg, latex beads), either directly or via a linker. The substrate also includes a detectable marker, preferably a chromogenic marker. Typically, the labeling reagent is injected into the support structure by drying or lyophilization. The sample structure also comprises a capture zone downstream of the label zone. This capture zone receives the labeling reagent from a labeling zone directed downstream by a flow path. The capture zone contains a capture reagent that immobilizes IgE and / or IgG conjugated to the Der HMW-map protein in the capture zone (in this case, the anti-IgE and / or IgG antibodies described above, or both). . The capture reagent is preferably fixed to the support structure by drying or freeze-drying. The labeling reagent accumulates in the capture zone, and this accumulation is assessed visually or by an optical detection device.
[0126]
In another embodiment, the sidestream device used to detect IgE or IgG comprises: (a) a support structure defining a flow path; (b) an anti-IgE or anti-IgG antibody as described above; Or (c) a capture reagent comprising Der HMW-map protein, wherein the labeling reagent comprises a labeling reagent, wherein the labeling reagent comprises a labeling reagent. (Located downstream of the labeling reagent in a capture zone fluidly connected to the labeling zone in a manner that can flow from the zone to the capture zone). The apparatus also preferably comprises a sample receiving zone located along the flow path, preferably upstream of the labeling reagent. The device also preferably includes an adsorbent located at the end of the flow path.
[0127]
Animals that are hypersensitive to Der HMW-map protein are identified by comparing the level of immune complex formation using a sample of the body fluid with the level of immune complex formation using a control sample. An immune complex refers to a complex containing an antibody and a Der HMW-map protein (ie, a Der HMW-map protein: antibody complex). Thus, an immune complex is formed when a positive control sample is used, but not when a negative control sample is used. Thus, if the body fluid sample results in more immune complex formation than the immune complex formation using the positive control sample, the animal from which the body fluid was collected is overly sensitive to the Der HMW-map protein bound to the substrate. It is. Conversely, if the body fluid sample results in an immune complex formation similar to the immune complex formation with the negative control sample, the animal from which the body fluid was collected is over-sensitive to the Der HMW-map protein bound to the substrate. is not.
[0128]
It is within the scope of the present invention that two or more different skin tests and / or in vitro tests can be used for diagnostic purposes. For example, the immediate overreaction of an animal to the Der HMW-map protein can be tested using an in vitro immunosorbent assay that can detect IgE antibodies specific for the Der HMW-map protein in the body fluid of the animal. Most animals that show delayed hypersensitivity to Der HMW-map protein also show immediate hypersensitivity to allergens, while a few animals that show delayed hypersensitivity to allergens show immediate hypersensitivity to this allergen Does not show symptoms. In such cases, according to the negative results from the IgE-specific in vitro test, delayed hypersensitivity to Der HMW-map protein in animals can be tested using the skin test of the present invention.
[0129]
The invention also includes a kit for detecting an antibody that specifically binds to a Der HMW-map protein based on each of the disclosed detection methods. One embodiment is a kit for detecting a Der HMW-map protein specific antibody, the kit comprising a means for detecting Der HMW-map protein and IgE and / or IgG. Suitable means of detection include compounds disclosed herein that bind to either the Der HMW-map protein or IgE and / or IgG. Preferred kits of the present invention include antibodies capable of selectively binding to IgE or IgG disclosed herein and / or compounds capable of binding to a detectable marker conjugated to a Der HMW-map protein (e.g., If the detectable marker is biotin, the detection means includes avidin, streptavidin and ImmunoPure® NeutrAvidin).
[0130]
Another preferred kit of the present invention is an allergen kit, which contains the Der HMW-map protein and an allergen commonly detected in the same environment as the mite allergen. Suitable and preferred mite-related allergens for use with the kits of the present invention include the tick-related allergens disclosed herein.
[0131]
A preferred kit of the present invention includes a kit in which Der HMW-map protein is immobilized on a substrate. If the kit includes a Der HMW-map protein and another allergen, the kit includes one or more compositions, each composition including one allergen. In this way, each allergen can be tested separately. The kit may also include more than one diagnostic reagent for IgE or IgG, or other compounds disclosed herein. Kits used in a side-stream assay format are particularly preferred. It is within the scope of the present invention that the sidestream assay kit may include one or more sidestream assay devices. A plurality of sidestream devices may be joined together at one end of each device, thereby creating a fan-like structure.
[0132]
Another aspect of the invention involves treating an animal susceptible to or having a tick allergy with a Der HMW-map protein formulation of the invention. According to the invention, the term treatment may refer to the modulation of a hypersensitivity response by an animal to mite allergens. Modulation can include, for example, immunomodulation of cells involved in an animal's hypersensitive response. Immunomodulation can include modulating the activity of molecules typically involved in the immune response, such as antibodies, antigens, major histocompatibility gene molecules (MHCs) and molecules that react simultaneously with MHC molecules. In particular, immunomodulation refers to the modulation of an antigen: antibody interaction that results in an inflammatory response, immunosuppression and tolerance of the cells involved in the hypersensitivity response. Immunosuppression refers to inhibiting the immune response, for example, by killing specific cells involved in the immune response. Immunotolerization refers to inhibiting the immune response by anerizing (ie, reducing T cell responsiveness to an antigen) specific cells involved in the immune response.
[0133]
One embodiment of the present invention is a therapeutic composition comprising a desensitizing compound capable of inhibiting an immune response to a Der HMW-map protein of the present invention. Such desensitizing compounds include blocking compounds, tolerogens and / or suppressor compounds. Blocking compounds include compounds that can modulate antigen: antibody interactions that can produce an inflammatory response, tolerogens are compounds that can tolerize an animal, and suppressor compounds can immunosuppress an animal. The desensitizing compounds of the present invention can be soluble or membrane-bound. Membrane-bound desensitizing compounds can associate with biological membranes, including cells, liposomes, planar membranes or micelles. The soluble desensitizing compounds of the present invention are useful for the following purposes: (1) IgE: inhibiting type I hypersensitivity reactions by blocking antigen-mediated mast cell degranulation; (2) Inhibiting the type III hypersensitivity response by blocking IgG: antigen complex formation leading to cell complement destruction; and (3) Type IV hypersensitivity by blocking T helper cell stimulation of cytokine secretion by macrophages Inhibiting a reaction. The membrane-bound desensitizing compounds of the present invention are useful for the following purposes: (1) Type II hypersensitivity by blocking IgG: antigen complex formation on the cell surface leading to complement destruction of the cell Inhibiting a response; (2) inhibiting a type II hypersensitivity response by blocking IgG-regulated signaling in immune cells; and (3) blocking T cell killing of antigen bearing cells. Inhibit type IV hypersensitivity reactions by doing so. Examples of desensitizing compounds include, but are not limited to, the muteins, mimetopes and antibodies of the present invention, and the compounds of the present invention that inhibit the binding between a protein of the present invention and IgE. Other inhibitors.
[0134]
The desensitizing compound of the present invention can also be covalently linked to a ligand molecule that can target the desensitizing compound to specific cells involved in a hypersensitivity response to Der HMW-map protein. Suitable ligands used to link desensitizing compounds include, for example, immunoglobulin molecules, cytokines, lectins, heterologous allergens, CD8 molecules or major histocompatibility molecule molecules (eg, MHC class I or MHC class II molecules) At least a part thereof. Preferred portions of immunoglobulin molecules for linking to desensitizing compounds include variable regions capable of binding to immune cell-specific surface molecules and constant regions capable of binding to Fc receptors on immune cells (particularly IgE Constant region). Preferred CD8 molecules include at least the extracellular functional domain of the α chain of CD8. An immune cell refers to a cell involved in an immune response (particularly, a cell having an MHC class I molecule or an MHC class II molecule). Preferred immune cells include antigen presenting cells, T cells and B cells.
[0135]
In one embodiment, a therapeutic composition of the invention comprises a Der HMW-map protein of the invention, a mimetope or mutein thereof, or a Der HMW-map nucleic acid molecule of the invention. Suitable therapeutic compositions of the invention for treating mite allergy comprise a Der HMW-map protein, its mimetope or mutein, or a Der HMW-map nucleic acid molecule of the invention. Preferred therapeutic compositions include: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 under stringent hybridization conditions SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 30, SEQ ID NO: 31, Sequence SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 41 and SEQ ID NO: 44, hybridizing with the complement of a nucleic acid molecule encoding an amino acid sequence selected from the group consisting of Nucleic acid molecule encoding mite allergenic protein; mimetope of mite allergenic protein; mutein of mite allergenic protein And an isolated nucleic acid molecule selected from the group consisting of: a nucleic acid molecule comprising at least about 150 nucleotides, wherein the nucleic acid molecule comprising at least about 150 nucleotides comprises 1 × SSC and 0% formamide SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39 in solution at a temperature of about 50 ° C. , SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 45, and a nucleic acid sequence encoding a protein comprising the amino acid sequence of SEQ ID NO: 33 and its complement. A fragment of any of the foregoing nucleic acid molecules, comprising at least about 150 nucleotides. Nucleic acid molecules containing Preferred Der HMW-map muteins comprise at least a portion of a Der HMW-map protein, wherein an appropriate number of cysteine residues have been removed or replaced with non-cysteine residues, so that The modified Der HMW-map protein is not toxic to animals (eg, does not cause anaphylaxis).
[0136]
In another embodiment, a therapeutic composition of the present invention encodes a Der HMW-map protein that can be administered to the animal in a manner that allows expression of the nucleic acid molecule to the Der HMW-map protein in the animal. Nucleic acid molecules. Nucleic acid molecules can be delivered to an animal in a variety of ways, including but not limited to: (a) nucleic acid molecules that are naked (ie, not packaged in a virus coat or cell membrane) (eg, (Eg, as a naked DNA or RNA molecule, as taught in, for example, Wolf et al., 1990, Science 247, 1465-1468), or (b) packaged as a recombinant virus or cell. Administering the nucleic acid molecule (ie, the nucleic acid molecule is delivered by a viral or cell vehicle).
[0137]
A naked nucleic acid molecule of the invention includes a nucleic acid molecule of the invention, and preferably includes a recombinant molecule of the invention that is preferably capable of replication or otherwise amplifying. A naked nucleic acid of the invention can include one or more nucleic acid molecules of the invention, eg, having one or more internal ribosome entry sites, eg, in the form of a bicistronic recombinant molecule. Preferred naked nucleic acid molecules comprise at least a portion of a viral genome (ie, a viral vector). Preferred viral vectors include viral vectors based on alphavirus, poxvirus, adenovirus, herpesvirus, picornavirus, and retrovirus, including alphaviruses (eg, Sindbis virus or Semlikivirus), species-specific herpes. Particularly preferred are viral vectors based on viruses and species-specific poxviruses. Any suitable transcription control sequence may be used, including those disclosed as being suitable for protein production. Particularly preferred transcription control sequences include the immediate early (preferably in the context of intron-A) cytomegalovirus, the Rous sarcoma virus long terminal repeat, and tissue-specific transcription control sequences, and when using viral vectors. And transcription control sequences that are endogenous to the viral vector. Incorporation of "strong" poly (A) sequences is also preferred.
[0138]
The naked nucleic acid molecules of the present invention can be administered by various methods. Suitable delivery methods include, e.g., intramuscular injection, subcutaneous injection, intradermal injection, intradermal ablation, particle bombardment, oral and nasal application, intramuscular injection, intradermal injection, intradermal ablation and particle Bombardment is preferred, and intramuscular injection is even more preferred. Preferred single doses of naked DNA molecules range from about 1 nanogram (ng) to about 1 milligram (mg), as can be determined by one of skill in the art, depending on the route of administration and / or method of delivery. Examples of methods of administration are described, for example, in US Pat. No. 5,204,253 (Bruner et al., Issued Apr. 20, 1993), PCT Publication No. WO 95/19799 (published Jul. 27, 1995, by McCabe). And PCT Publication No. WO 95/05853 (published March 2, 1995, by Carson et al.). The naked DNA molecules of the present invention can be included in an aqueous vehicle (eg, phosphate buffered saline) and / or with a carrier (eg, a lipid-based vehicle) or can contain fine particles (eg, (Eg, gold particles).
[0139]
A recombinant virus of the invention comprises a recombinant molecule of the invention that is packaged in a virus coat and that can be expressed in an animal after administration. Preferably, the recombinant molecule is defective in packaging and / or encodes an attenuated virus. Numerous recombinant viruses can be used, including, but not limited to, viruses based on alphavirus, poxvirus, adenovirus, herpes virus, picornavirus, and retrovirus. Preferred recombinant viruses are viruses based on alphavirus (eg, Sindbis virus), raccoon poxvirus, species-specific herpes virus and species-specific poxvirus. One example of a method for producing and using an alphavirus recombinant virus is disclosed in PCT Publication No. WO 94/17813 (published August 18, 1994 by Xiong et al.).
[0140]
When administered to an animal, the recombinant virus of the invention can infect cells in a recipient animal and reduce the Der HMW-map protein-mediated biological response in that animal. Directed to the production of For example, a recombinant virus comprising a Der HMW-map nucleic acid molecule of the invention can be produced according to a protocol that results in the production of a protein or RNA in the animal in an amount sufficient to reduce the Der HMW-map protein-mediated biological response. Is administered. A preferred single dose of the recombinant virus of the present invention is about 1 × 10 5 per kilogram of animal body weight. ⁴ ~ About 1 × 10 ⁷ Viral plaque forming units (pfu). The administration protocol is similar to the administration protocol described herein for protein-based compositions, with subcutaneous, intramuscular, intranasal and oral routes of administration being preferred.
[0141]
The recombinant cell vaccine of the present invention includes the recombinant cell of the present invention that expresses at least one protein of the present invention. Preferred recombinant cells for this embodiment include Salmonella, E. coli, Listeria, Mycobacterium, S. coli. frugiperda, yeast (including Saccharomyces cerevisiae and Pichia pastoris), BHK, CV-1, myoblast G8, COS (eg, COS-7), Vero, MDCK and CRCK recombinant cells. The recombinant cell vaccines of the present invention can be administered in a variety of ways, but they can be administered orally (preferably, at about 10 ⁸ Cells to about 10 ¹² May be administered (at doses that span the range of cells). The administration protocol is similar to the administration protocol described herein for protein-based vaccines. Recombinant cell vaccines can include whole cells, cells with detached cell walls, or cell lysates.
[0142]
The efficacy of the therapeutic composition of the present invention in desensitizing an animal to mite allergy can be determined by the in vivo skin test method described herein, the detection of cellular immune activity in the treated animal, or The determination of the level of IgE that specifically binds to the Der HMW-map protein of the present invention can be tested in a variety of ways, including but not limited to. Methods for determining cellular immune activity and IgE levels in animals are known to those skilled in the art. In one embodiment, the therapeutic compositions can be tested in animal models such as dogs, cats, rabbits, and mice, and can also be tested in humans. Such techniques are known to those skilled in the art.
[0143]
Preferred nucleic acid molecules for use with the therapeutic compositions of the present invention include any of the Der HMW-map nucleic acid molecules disclosed herein, specifically SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, sequence SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 45, and / or amino acid sequence Nucleic acid sequences encoding a protein comprising SEQ ID NO: 33, as well as the complements thereof.
[0144]
Recombinant cells useful in the therapeutic compositions of the present invention include the recombinant cells of the present invention that include a Der HMW-map protein of the present invention. Preferred recombinant cells for this embodiment include Salmonella, E. coli, Listeria, Mycobacterium, S.E. frugiperda, yeast (including Saccharomyces cerevisiae), BHK, CV-1, myoblast G8, COS (for example, COS-7), Vero, MDCK, and recombinant cells of CRRF. The recombinant cells of the present invention can be administered in a variety of ways, but are preferably ⁸ ~ About 10 ¹² It has the advantage that it can be administered orally in doses in the range of cells. The administration protocol is similar to the protocol described herein for the protein composition. Recombinant cells can include whole cells, cells with detached cell walls, or cell lysates.
[0145]
One embodiment of the present invention is a method of immunotherapy, comprising administering to an animal an effective amount of a therapeutic composition comprising a Der HMW-map protein of the present invention. Suitable therapeutic compositions and modes of administration are disclosed herein. According to the present invention, the therapeutic compositions and methods of the present invention can be used to prevent or alleviate symptoms associated with mite allergen pathogenesis.
[0146]
The efficacy (efficacy) of the therapeutic compositions of the present invention in producing an allergic response to Der HMW-map protein can be determined by standard methods for determining Der HMW-map protein-mediated immunity (immediate hypersensitivity, delayed hypersensitivity, antibody dependence (Including, but not limited to, inflammatory cytotoxicity (ADCC), immune complex activity, mitogenic activity, histamine release assays, and other methods such as those described in Janeway et al., Ibid.) I can do it.
[0147]
The present invention also includes therapeutic compositions that include one or more therapeutic compounds of the present invention. Examples of such therapeutic compounds include, for example, other allergens disclosed herein.
[0148]
Therapeutic compositions of the invention can be formulated in excipients to which the animal to be treated can be resistant. Examples of such excipients include water, saline, Ringer's solution, dextran solution, Hanks solution, and other aqueous solutions of physiologically balanced salts. Non-aqueous vehicles such as fixed oils, sesame oil, ethyl oleate, or triglycerides can also be used. Other useful formulations include suspensions containing a thickening agent (eg, sodium carboxymethylcellulose, sorbitol, or dextran). Excipients may also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability. Examples of buffers include phosphate buffer, bicarbonate buffer, and Tris buffer, while examples of preservatives (preservatives) include thimerosal, o-cresol, formalin, and benzyl alcohol. Can be Standard formulations can be either injectable liquids or solids which can be incorporated into a suitable liquid as a suspension or solution for injection. Thus, in non-liquid formulations, excipients may include dextrose, human serum albumin, preservatives (preservatives) and the like, to which sterile water or saline may be added prior to administration.
[0149]
In one embodiment of the invention, the therapeutic composition may include an adjuvant. Adjuvants are agents that can enhance an animal's immune response to a specific antigen. Suitable adjuvants include, but are not limited to: cytokines, chemokines, and compounds that induce the production of cytokines and chemokines (eg, granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), colony stimulating factor (CSF), Flt-3 ligand, erythropoietin (EPO), interleukin 2 (IL-2), interleukin-3 (IL-3) ), Interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 10 (IL-10), interleukin 12 (IL- 2), interferon γ, interferon γ-inducible factor I (IGIF), transforming growth factor β, RANTES (regulated upon activation, normal T cell expressed and presumably secreted (normal T cells regulated during activation) Expressed and possibly secreted)), macrophage inflammatory proteins (eg, MIP-1α, and MIP-1β), and Leishmania elongation initiation factor (LEIF); bacterial components (eg, endotoxin, specifically superantigen Aluminum-based salts; calcium-based salts; silica; polynucleotides; toxoids; serum proteins, virus coat (shell) proteins; Lock copolymer adjuvant (for example, Titermax of Hunter ^TM Adjuvant (Vaxcel ^TM , Inc. Norcross, GA), Ribi adjuvant (Ribi ImmunoChem Research, Inc., Hamilton, MT); and saponins and their derivatives (for example, Quil A (Superfos Biosector A / S, Denmark protein of the present invention). Using the methods described herein, it can be delivered in the form of the protein itself or in the form of a nucleic acid molecule encoding such a protein.
[0150]
In one embodiment of the invention, the therapeutic composition can include a carrier. Carriers include compounds that increase the half-life of the therapeutic composition in the treated animal. Suitable carriers include, but are not limited to, polymeric sustained release vehicles, biodegradable implants, liposomes, bacteria, viruses, other cells, oils, esters, and glycols.
[0151]
One embodiment of the present invention is a sustained release formulation that is capable of slow release of a composition of the present invention to an animal. As used herein, a sustained release formulation comprises a composition of the present invention in a sustained release vehicle. Suitable sustained release vehicles include biocompatible polymers, other polymeric matrices, capsules, microcapsules, microparticles, bolus preparations, osmotic pumps, diffusion devices, liposomes, lipospheres, and transdermal delivery Systems include, but are not limited to. Other sustained release formulations of the present invention include liquids that form a solid or gel in situ (in situ) upon administration to an animal. Preferred sustained release formulations are biodegradable (ie, bioerodible).
[0152]
Preferred sustained release formulations of the present invention release a therapeutic composition of the present invention into the blood of an animal at a constant rate sufficient to achieve a therapeutic dose level of the composition for attenuating mite allergy in an animal. I can do it. As used herein, mite allergy refers to the cellular response that occurs when a mite allergen contacts an animal. For example, IgE, which specifically binds mite allergens, becomes bound to the Fcε receptor, which is a biological mediator (eg, histamine, prostaglandin, and / or protease) that can trigger clinical symptoms of allergy. Produces an Fcε receptor-mediated biological response, including the release of The therapeutic composition is preferably released over a time interval ranging from about 1 to about 12 months. Preferred sustained release formulations of the present invention are preferably for at least about 1 month, more preferably for at least 3 months, even more preferably for at least 6 months, even more preferably for at least about 9 months, and even more preferably Can provide treatment for at least about 12 months.
[0153]
The therapeutic compositions of the present invention can be sterilized by conventional methods that do not produce proteolysis, such as filtration, and / or can be lyophilized.
[0154]
The therapeutic compositions of the present invention can be administered to any animal susceptible to mite allergy, as described herein. Acceptable protocols for administering the therapeutic compositions of the present invention in an effective manner can vary with the particular dosage size, number of doses, frequency of dose administration, and mode of administration. Determining such a protocol can be accomplished by one skilled in the art. An effective dose refers to a dose that can treat an animal for hypersensitivity to mite allergens. Effective doses may vary, for example, depending on the therapeutic composition used, and the size and type of the recipient animal. Effective doses for immunomodulating an animal for mite allergens include doses administered over a period of time that can alleviate the hypersensitivity response by the animal to mite allergens. For example, a first tolerizing dose can include an amount of a therapeutic composition of the invention that produces a minimal hypersensitivity response when administered to a hypersensitive animal. The second tolerizing dose may include a greater amount than the first dose of the same therapeutic composition. Effective tolerizing doses may include increasing concentrations of the therapeutic composition necessary to tolerize the animal such that the animal does not have a hypersensitivity response to exposure to mite allergens. An effective dose for desensitizing an animal is that of a therapeutic composition of the invention sufficient to block the animal from having a hypersensitive response to exposure to mite allergens present in the animal's environment. Concentration may be included. An effective desensitizing amount can include repeated doses having a concentration of the therapeutic composition that produces a minimal hypersensitivity response when administered to a hypersensitive animal.
[0155]
An appropriate single dose is one that, when administered one or more times over an appropriate time interval, can treat the animal for hypersensitivity to mite allergens. For example, a preferred single dose of a mite allergen, or mimetope therapeutic composition is from about 0.5 ng to about 1 g of the therapeutic composition per kilogram of animal body weight. Further treatment with the therapeutic composition can be administered about one day to one year after the original administration. Further treatment with the therapeutic composition is preferably administered if the animal is no longer protected from a hypersensitivity response to mite allergen. Particular dosages and schedules can be developed by those skilled in the art based on the parameters discussed above. Modes of administration can include, but are not limited to, subcutaneous, intradermal, intravenous, nasal, oral, transdermal, and intramuscular routes.
[0156]
The therapeutic compositions of the present invention can be used in combination with other compounds that can modify the sensitivity of an animal to mite allergens. For example, animals may be sensitized by systemic or anti-inflammatory agents (eg, antihistamines, anti-steroidal, anti-inflammatory, and reagents that drive the switching of the immunoglobulin heavy chain class from IgE to IgG). It can be treated with a compound that can alter the function of cells involved in the response, which compound attenuates allergic reactions. Suitable compounds useful for altering the function of cells involved in the hypersensitivity response include, but are not limited to: antihistamine, cromolyn sodium, theophylline, cyclosporin A, adrenaline, cortisone, Compounds that can modulate cell signaling, compounds that can modulate adenosine 3 ′, 5′-cyclic phosphate (cAMP) activity, and compounds that block IgE activity (eg, IgE-derived peptides or IgE-specific Fc receptors; A peptide derived from IgE or an antibody specific to an IgE-specific Fc receptor, or an antibody capable of blocking the binding of IgE to the Fc receptor).
[0157]
The compositions of the present invention may be administered to any animal that has or is hypersensitive to mite allergen sensitivity. Animals that are preferred to be treated include mammals and birds, including cats, dogs, horses, humans, and other pets, working animals, and / or economic food animals. Particularly preferred animals for protection are cats and dogs.
[0158]
Another aspect of the present invention includes a method for using the formulations of the present invention to prescribe treatment for animals susceptible to or having hypersensitivity to mite allergens. Preferred methods for prescribing a treatment for mite allergic hypersensitivity include, for example, (1) an effective amount of a formulation comprising a mite allergen of the invention or a mimetope thereof (suitable and preferred formulations are described herein). (2) intradermally injecting an effective amount of a control solution into a second site of the animal; and (3) injecting an effective amount of a control solution into a second site of the animal. Measuring and comparing the wheal size resulting from the injection with the wheal size resulting from the injection of the control solution to assess whether the animal has mite allergic hypersensitivity; and (4) mite allergic hypersensitivity Prescribing treatment for
[0159]
An alternative preferred method for prescribing a treatment for mite allergic hypersensitivity comprises: (1) providing a first portion of a body fluid sample obtained from the animal to be tested with an effective amount of a formulation comprising mite allergen or a mimetope thereof. (A suitable and preferred formulation is disclosed herein) to form a first immune complex solution; (2) contacting with a positive control antibody to form a second immune complex Forming a solution; (3) the animal has mite allergic hypersensitivity by measuring and comparing the amount of immune complex formation in the first immune complex solution and the second immune complex solution Assessing whether or not; and (4) prescribing a treatment for mite allergic hypersensitivity. It should be noted that similar methods can be used to prescribe treatment for allergies using the mite allergen formulations disclosed herein.
[0160]
Another aspect of the invention includes a method for monitoring an animal susceptible to or having hypersensitivity to mite allergens using a formulation of the invention. The in vivo and in vitro tests of the present invention can be used to test animals for mite allergic hypersensitivity before and after any treatment for mite allergic hypersensitivity. Preferred methods for monitoring the treatment of mite allergic hypersensitivity, which may also be applied to monitor the treatment of other allergies, include: (1) an effective amount of a formulation comprising mite allergen or a mimetope thereof. (A suitable and preferred formulation is disclosed herein) intradermally at a first site in an animal; (2) applying an effective amount of a control solution to a second site in the animal. Intra-injection; and (3) determining whether the animal is desensitized to mite allergen by measuring and comparing the wheal size resulting from the injection of the formulation with the wheal size resulting from the injection of the control solution. Deciding whether or not it is not.
[0161]
An alternative preferred method for monitoring treatment for mite allergic hypersensitivity, which may also be applied to monitor the treatment of other allergies, comprises: (1) fluid obtained from the animal being tested A first portion of the sample is contacted with an effective amount of a formulation comprising a mite allergen or mimetope thereof (a suitable and preferred formulation is disclosed herein) to form a first immune complex solution. (2) contacting with a positive control antibody to form a second immune complex solution; and (3) immunocomplex in the first immune complex solution and the second immune complex solution. Determining whether the animal is desensitized to mite allergen by measuring and comparing the amount of body formation.
[0162]
The invention also includes antibodies that can selectively bind to mite allergens or mimetopes thereof. Such antibodies are referred to herein as anti-mite allergen antibodies. As used herein, the term "selectively binds" refers to the ability of such an antibody to preferentially bind to mite allergen or its mimetope. In particular, the invention includes antibodies that can selectively bind to a Der HMW-map protein. Binding can be measured using various methods known to those of skill in the art, including immunoblot assays, immunoprecipitation assays, enzyme immunoassays (eg, ELISA), radioimmunoassays, immunofluorescent antibody assays, and immunoelectron microscopy; See, Sambrook et al., Ibid.
[0163]
The antibodies of the present invention can be either polyclonal or monoclonal antibodies. The antibodies of the present invention include functional equivalents, such as antibody fragments and engineered antibodies (single-chain antibodies, which can selectively bind to at least one epitope of the protein or mimetope used to obtain the antibody). Including))). Preferred antibodies are raised in response to the Der HMW-map protein or its mimetope. More preferred Der HMW-map proteins from which antibodies are raised are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 41, and / or SEQ ID NO: 44, or at least a portion of a protein having a homolog thereof. Preferably, the antibody of the present invention has about 10 to the Der HMW-map protein of the present invention. ³ M ^-1 ~ About 10 ¹² M ^-1 Has a single-site binding affinity of
[0164]
A preferred method for producing an antibody of the invention comprises administering an effective amount of a Der HMW-map protein or a mimetope thereof to an animal to produce the antibody, and collecting the antibody. Antibodies raised against defined products or mimetopes may have advantages. This is because such antibodies are substantially free of antibodies to other substances that might otherwise interfere with diagnostic assays or cause side effects when used in therapeutic compositions.
[0165]
The antibodies of the present invention have a variety of potential uses, which are within the scope of the present invention. For example, such antibodies can be used as (a) a vaccine to passively immunize an animal to protect it from mite allergic hypersensitivity and (b) used as a positive control in a test kit. And / or (c) may be used as a means to recover the desired mite allergen from a mixture of proteins and other contaminants.
[0166]
The following examples are provided for illustrative purposes and are not intended to limit the scope of the invention.
[0167]
(Example)
It should be noted that this example includes a number of molecular biological, microbiological, immunological and biochemical techniques that would be known to those skilled in the art. Disclosures of such techniques can be found, for example, in Sambrook et al. (Ibid.) And related references.
[0168]
(Example 1)
This example describes the identification of a high molecular weight protein that binds to IgE from dogs known to be allergic to mite allergens.
[0169]
Approximately 5.5 grams (g) of frozen wet Dermatophagoides farinae (Derf) mite (available from Bayer Allergy, Spokane, WA) was added to 30 ml of phosphate buffered saline (PBS) or 0.1 M Tris. -HCl, pH 8 (each containing a complete protease inhibitor (available from Boehringer Mannheim, Indianapolis, IN)) with a round bottom glass homogenizer to obtain a crude Der f extract. Was. The resulting supernatants were collected and each was concentrated in a Centriprep 30 concentrator (available from Amicon, Beverly, Mass.) By centrifugation at 16,000 × g for about 30 minutes. The concentrated supernatant was applied to a separate Sephacryl S-100 column (2.7 × 70 cm; available from Pharmacia, Piscataway, NJ) in PBS or 0.1 M Tris-HCl (pH 8), respectively. Fractions excluded from each column were pooled. When using PBS, the fraction was dialyzed against 10 mM Tris-HC (pH 8). The fractions were applied to a separating Q-Sepharose column (2.5 × 5 cm; available from Pharmacia). When a fraction containing 0.1 M Tris-HCl (pH 8) was used, the Q-Sepharose column was pre-equilibrated in 10 mM Tris-HCl (pH 8). Approximately 45 ml of 10 mM Tris-HCl (pH 8), then 0.1 M Tris-HCl (pH 8), then 0.2 M Tris-HCl (pH 8), then 0.3 M Tris-HCl (pH 8), then 0 Elution was carried out sequentially with 0.4 M Tris-HCl (pH 8) and then with 0.5 M Tris-HCl (pH 8). Fractions were collected from each elution step. Each fraction was isolated from IgE antibodies present in dog serum isolated from dogs of known allergy to mite allergen (herein referred to as mite allergic dog antiserum or mite allergic antiserum). The presence of binding proteins was analyzed by Western blot. Specifically, the proteins contained in the fractions were digested by 12% Tris-glycine SDS-PAGE and then blotted on nitrocellulose. The blot was incubated with a serum pool from a dog known to be allergic to mite allergen, diluted 1:20 with standard buffer. The blot was incubated and then washed using standard procedures. The blot was then incubated with the mouse monoclonal anti-dog IgE antibody DEI38 (1 mg / ml; 1: 1000 dilution). The blot was incubated and then washed using standard procedures. The blot was then incubated with a donkey anti-mouse IgG antibody conjugated to horseradish peroxidase (1: 1000 dilution; available from Jackson Labs, Maine). The presence of HRP-conjugated antibody bound to the blot was detected using standard techniques. A protein of about 70 kD was identified in the 0.2 M Tris-HCl (pH 8) fraction, and a protein of about 98 kD and about 109 kD was identified in the 0.3 M Tris-HCl (pH 8) fraction.
[0170]
The above fraction eluted with 0.3 M Tris-HCl (pH 8) was concentrated in a Centriprep 30 concentrator and then diluted in 20 mM Na-Ac (pH 5.6). The eluted fraction was then applied to a PolyCat A HPLC cation exchange column (available from PolyLC, Columbia, MD). The column was eluted with about 10 ml of 20 mM Na-Ac, pH 5.6, followed by about 45 ml of a 0-0.5 M linear gradient of NaCl in 20 mM Na-Ac, pH 5.6. Elution was performed using a flow rate of about 1 ml / min. Fractions were collected from the elution procedure and assayed for the presence of high molecular weight proteins using the mite allergic antisera and Western blot protocol described above. Fractions containing high molecular weight proteins were pooled. Trifluoroacetic acid (TFA) was added to a concentration of about 0.05%. This solution was applied to a TSK-Gel TMS-250 Cl reverse phase column (commercially available from TosoHaas, Montgomeryville, PA) pre-equilibrated with 80% solvent A and 20% solvent B. Solvent A contains about 0.05% TFA in water and solvent B contains about 0.05% TFA in 90% acetonitrile in water. The column was eluted with about 5 ml of 20% solvent B, then eluted with a linear gradient of about 20% to about 70% solvent B at a flow rate of 0.6 ml / min. The protein eluted from this column was resolved by 12% Tris-glycine PAGE. The gel was stained with Coomassie Blue. The stained gel is shown in FIG. Lane 1 contains the Mark-12 protein molecular weight marker (available from Novex, San Diego, CA), lane 2 contains the protein eluted from the reversed phase column, and lane 3 contains the SeeBlue ^TM Protein molecular weight markers (available from Novex). Two major proteins were identified in the eluate. The molecular weight of this protein is determined by BioRad ^TM Multi-Analyst ^TM / PC Image System (available from BioRad Corp.). The higher molecular weight protein in lane 2 of FIG. 1 was determined to be about 109 kD and is referred to herein as mite allergen protein A (mapA). The lower molecular weight protein in lane 2 of FIG. 1 was determined to be about 98 kD and is referred to herein as mite allergen protein B (mapB). The purity of the combined protein was higher than 85% purity, ie less than 15% impurities. This purified eluate is referred to herein as D.E. It is referred to as a farinae high molecular weight map (HMW-map) composition.
[0171]
(Example 2)
In the present embodiment, N-terminal sequencing of proteins in farinae HMW-map compositions is described.
[0172]
The protein contained in the 0.3 M Tris-HCl (pH 8) fraction obtained as described above in Example 1 was separated by SDS-PAGE using a 12% Tris-glycine polyacrylamide-SDS gel, and subsequently separated. And stained with Coomassie. Proteins were blotted onto PVDF, stained with Coomassie R-250, and decolorized using standard procedures. The proteins corresponding to the approximately 98 kD band and the approximately 109 kD band were excised and separately subjected to N-terminal amino acid sequencing using techniques known to those skilled in the art. A partial N-terminal amino acid sequence of about 14 amino acids was deduced for both proteins and these sequences were determined to be identical. The N-terminal amino acid sequence is shown herein as SEQ ID NO: 1 having the amino acid sequence: Ser Ile Lys Arg Asp His Asn Asp Tyr Ser Lys Asn Pro Met.
[0173]
D. The protein in the farinae HMW-map composition was also subjected to proteolytic cleavage to obtain internal amino acid sequence data. In detail, this D. The farinae HMW-map composition was cut using the endoproteinase Asp-N (available from Boehringer Mannheim Biochemical, Indianapolis, IN) using methods standard in the art. The digested protein was then purified by the method of Stoney et al., Enzymatic Digestion of Proteins and HPLC Peptide Isolation (A Practical Guide to Protein and PeptidePulse Quantification Scheme by Microelectronics, Peptide, Pharmaceuticals, Pharmaceuticals, Pharmaceuticals, Pharmaceuticals, Pharmaceuticals, Pharmaceuticals, Pharmaceuticals) And separated by HPLC. Twelve proteolytic fragments were isolated and referred to herein as map (1), map (2), map (3), map (4), map (5), map (6), map (6). (7), map (8), map (9), map (10), map (11) and map (12).
[0174]
The N-terminal partial amino acid sequence of map (1) was determined to be Asp Tyr Glu Tyr Pro Gly Ser Arg Leu Gly Asn Pro Lys Ala Pro Leu Tyr Lys Arg Pro and is also referred to as SEQ ID NO: 2. The N-terminal partial amino acid sequence of map (2) was determined to be Asp Ile Pro His His Pro Thr Asn Ile His Lys Tyr Leu Val Cys Glu Ser Val Asn Gly Gly, and is also referred to as SEQ ID NO: 3. The N-terminal partial amino acid sequence of map (3) was determined to be Asp Pro Ala Lys Gly Met Ser Pro Pro Gly Phe Ile Val Gly Glu Glu Gly Val Leu Ser and is also referred to as SEQ ID NO: 4. The N-terminal partial amino acid sequence of map (4) was determined to be Asp Glu Lys Asn Ser Plu Glu Cys Ile Leu Gly Pro and is also referred to as SEQ ID NO: 5. The N-terminal partial amino acid sequence of map (5) was determined to be Asp Ala Phe Glu Pro His Gly Tyr Leu Leu Thr Ala Ala Val Ser Pro Gly Lys and is also referred to as SEQ ID NO: 6. The N-terminal partial amino acid sequence of map (6) was determined to be Asp Lys Gln Asn Tyr Leu Ala Leu Val Arg Glu Leu Lys and is also referred to as SEQ ID NO: 7. The N-terminal partial amino acid sequence of map (7) is Asp Met Ala Gln Asn Tyr Lys Tyr Arg Gln Gln Phe Ile Gln Ser Val Leu Asn Asn Gly Ala, which is also referred to as Arg Arg. The N-terminal partial amino acid sequence of map (8) is Asp Glu Xaa Asn Val Met Xaa Tyr Val Leu Tyr Thr Met His Tyr Tyr Leu Asn Asn Gly Ala, which is also referred to as A9, which is also referred to as Ar9, which is also referred to as Ar9. Xaa represents any amino acid). The N-terminal partial amino acid sequence of map (9) was determined to be Asp Lys Leu Val Met Gly Val Pro Pro Tyr Gly Arg Ala Xaa Ser Ile Glu and is also referred to as SEQ ID NO: 10 (where Xaa is an arbitrary amino acid). Is shown). The N-terminal partial amino acid sequence of map (10) was determined to be Asp Ile Pro His His Pro Thr Asn Ile His Lys Tyr Leu Val Cys Glu Ser Val Asn Gly and is also referred to as SEQ ID NO: 11. The N-terminal partial amino acid sequence of map (11) was determined to be Asp Tyr Ala Lys Asn Pro Lys Arg Ile Val Cys Ile Val Gly Thr Glu Gly Val Leu Ser and is also referred to as SEQ ID NO: 12. The N-terminal partial amino acid sequence of map (12) was determined to be Asp Pro Ala Lys Gly Met Ser Pro Pro Gly He Ile Val Gly Glu Glu Gly Val Leu Ser and is also referred to as SEQ ID NO: 13. map (1), map (2), map (3), map (4), map (5), map (6), map (7), map (8), map (9), map (10), Since the amino acid sequences for map (11), map (12) and map (13) were generated from a mixture of mapA and mapB proteins, these sequences do not necessarily represent a partial sequence of the signal protein. .
[0175]
(Example 3)
This example describes the purification of a 70 kD protein that binds IgE from dogs known to be allergic to mite allergens.
[0176]
The fraction described in Example 1 eluted with 0.2 M Tris-HCl (pH 8) was concentrated in a Centriprep 30 concentrator and then eluted with 20 mM Na-Ac (pH 5.6). The diluted protein was then applied to a PolyCat A HPLC cation exchange column. The column was eluted with about 10 ml of 20 mM Na-Ac, pH 5.6, followed by about 45 ml of a linear gradient of 0-0.5 M NaCl in 20 mM Na-Ac, pH 5.6 buffer. Was eluted using a flow rate of about 1 ml / min. Fractions were collected from the elution procedure and assayed for the presence of the 70 kD protein using the mite allergic antiserum and Western blot protocol described above. The fractions containing the 70 kD protein were pooled. Trifluoroacetic acid (TFA) was added to a concentration of about 0.05%. This solution was applied to a TSK-Gel TMS-250Cl reverse phase column that had been pre-equilibrated with 80% solvent A and 20% solvent B. Solvent A contains about 0.05% TFA in water and solvent B contains about 0.05% TFA in 90% acetonitrile in water. The column was eluted with about 3 ml of 20% solvent B and then with a linear gradient of about 20% to about 70% of solvent B at a flow rate of 0.6 ml / min. An approximately 70 kD protein of greater than 90% purity was obtained. This approximately 70 kD protein is referred to herein as mapC.
[0177]
The N-terminal sequence of the region on SDS-PAGE corresponding to this 70 kD protein (mapC) was obtained as described in Example 2. The N-terminal amino acid sequence of about 21 amino acids was deduced at an 80% confidence level, and is represented herein as SEQ ID NO: 33, which is: Gln Ser Arg Asp Arg Asn Asp Lys Pro Tyr Xaa Ile It has an amino acid sequence of Val Lys Lys Lys Lys Lys Ala Leu Asp.
[0178]
(Example 4)
This example demonstrates the ability of D. cerevisiae against dog IgE in dog serum isolated from dogs known to be allergic to mite allergens. The binding of the farinae HMW-map composition (ie, including mapA and mapB) is described.
[0179]
Multiple wells of an Immulon II microtiter plate were prepared at approximately 100 nanograms / well (ng / well) diluted in CBC buffer. coated with the farinae HMW-map composition, which was isolated according to the method described above in Example 1. The plate was incubated overnight at 4 ° C. After incubation, the D.C. The solution containing the farinae HMW-map composition was removed from the plate and the plate was blotted dry. The plate was then incubated at room temperature with about 200 μl / well of 4.0% fetal calf serum (PBSTFCS) in phosphate buffered saline (PBS) with 0.05% Tween-20. Blocked for about 1 hour. The plate was then washed four times with 0.05% Tween-20 in PBS (PBST) using an automatic washer (available from Dynatech, Chantilly, VA). Approximately 100 μl (per well) of a 1:10 dilution of a serum sample isolated from a different dog with known sensitivity to mite allergen in the intradermal skin test was added to the plate. A negative control group of sera was also added to a plate containing a combination of sera from six dogs (available from Harlan Bioproducts, Indianapolis, IN) raised in a blocking facility. Some wells did not receive dog serum, and therefore, background binding levels could be determined. The plate was incubated for about 1 hour at room temperature and then washed four times with PBST. Approximately 100 μl of a 1: 4000 dilution of 40 μg / ml biotinylated human FcεRα chain protein (produced as described in Frank et al., WO 98/23964 (published November 24, 1997)) contained in PBSTFCS ( Per well) was added. The plate was incubated at room temperature for about 1 hour and then washed four times with PBST. Approximately 100 μl of approximately 0.25 μg / ml streptavidin (available from Kirkegaard and Perry Laboratories (KPL), Gaithersburg, MD; diluted in PBST) conjugated to horseradish peroxidase received an experimental or control sample. Added to each well. The plate was then incubated for 1 hour at room temperature and washed four times with PBST. About 100 μl of TMB substrate (available from KPL) pre-warmed to room temperature was added to each well. The plate was then incubated for about 10 minutes at room temperature, then about 100 μl / well of stop solution (available from KPL) was added. The optical density (OD) of the wells was read at 450 nm on a Spectramax Microtiter Plate (available from Molecular Devices Inc.) reader within 10 minutes of adding the stop solution.
[0180]
O.D. obtained using the negative control sample wells and background wells. D. The reading is 0. D. Met. Serum from 5 of the 26 mite allergen-sensitive dogs was approximately 2,000 O.D. D. And about 3,200O. D. Between O. D. Generated read. Serum from three other mite allergen-sensitive dogs was approximately 1,000 O.D. D. And 2,000O. D. Between O. D. Generated read. Serum from three other mite allergen-sensitive dogs was approximately 500 O.D. D. And 1,000O. D. Between O. D. A reading occurred. Serum from seven other mite allergen-sensitive dogs was approximately 200O. D. And 500O. D. Between O. D. A reading occurred. Activity from six other mite allergen-sensitive dogs was 50O.D. D. O. less than D. Generated read. Thus, these results indicate that serum from dogs known to be sensitive to mite allergens contains IgE antibodies that specifically bind to the mapA and mapB proteins of the invention.
[0181]
(Example 5)
This example demonstrates a 70 kD D.E. against canine IgE in dog serum isolated from dogs known to be allergic to mite allergens. The binding of the farinae protein is described.
[0182]
Multiple wells of an Immulon II microtiter plate were prepared at approximately 100 ng / well 70 kD D.I. diluted in CBC buffer. Farinae protein (referred to herein as mapC), which was isolated according to the methods described above in Examples 1 and 3, was coated. The plate was incubated overnight at 4 ° C. The plate was blocked and washed using the method described in Example 4. Approximately 100 μl (per well) of a 1:10 dilution of a serum sample isolated from a different dog with known sensitivity to mite allergen in the intradermal skin test was added to the plate. A negative control sample was also added to the plate containing the SPF serum sample (serum from a dog that was maintained in a blocking facility and was therefore never exposed to mite allergen). Some wells did not receive dog serum, and therefore, background binding levels could be determined. The plate was incubated for about 1 hour at room temperature and then washed four times with PBST. Biotinylated human FcεRα chain protein was then added and the presence of IgE bound to the plate was detected using the method described in Example 4.
[0183]
O.D. obtained using the negative control sample wells and background wells. D. The reading is 0. D. Met. Serum from three of the 26 mite allergen-sensitive dogs was approximately 1,500 O.D. D. And about 2,700O. D. Between O. D. Generated read. Serum from five other mite allergen-sensitive dogs was approximately 800 O.D. D. And about 1,500O. D. Between O. D. Generated read. Sera from four other mite allergen-sensitive dogs had about 500 O.D. D. And about 800O. D. Between O. D. A reading occurred. Serum from six other mite allergen-sensitive dogs was approximately 200O. D. And about 500O. D. Between O. D. A reading occurred. The activity from eight other mite allergen-sensitive dogs was 50O. D. O. less than D. Generated read. Thus, these results indicate that sera from dogs known to be sensitive to mite allergens contain IgE antibodies that specifically bind to the mapC protein of the invention.
[0184]
(Example 6)
This example demonstrates the binding of the mapA, mapB, or mapC protein to cat IgE in cat serum isolated from cats, as demonstrated by in vitro testing for hypersensitivity to mite allergens. Describe.
[0185]
Multiple wells of an Immulon II microtiter plate were prepared with approximately 100 ng / well of D.I. farinae HMW-map composition (isolated according to the method described above in Example 1) and 70 kD Coated with farinae protein (isolated according to the method described above in Example 3). The other wells of this plate are either 400 ng / well of total Dermatophagoides pteronyssius extract (available from Greer Laboratories, Inc., Lenoir, NC; concentrated 8-fold prior to use) or total D.I. Coated with farinae extract (available from Miles, Inc., Elkhart, IN). All samples were diluted in CBC buffer. The plate was incubated overnight at 4 ° C. The plate was blocked and washed using the method described in Example 4. Approximately 100 μl (per well) of a 1:10 dilution of serum samples from different cats known to be sensitive to mite allergens in an in vitro allergen test in PBSTFCS was added to the plate. Seven control cats (# 15, # 16, # 17, # 18, # 19, # 20, and # 21) (these were shown by in vitro tests to be insensitive to mite allergen dust) ) Were also tested. Some wells do not receive cat serum, so background binding levels can be determined. The plate was incubated for about 1 hour at room temperature and then washed four times with PBST. Biotinylated human FcεRα chain protein was then added and the presence of IgE bound to the plate was detected using the method described in Example 4.
[0186]
The results are shown in Table 1 below. All values are O.D. D. Indicates 1,000 times the value. HDM refers to cats that are sensitive to dust mite allergens (by serologic testing (ie, ELISA against all D. farinae extracts)).
(Table 1)
[0187]
[Table 1]

These results indicate that sera from several cats known to be sensitive to mite allergens contain IgE antibodies that specifically bind to the mapA, mapB or mapC proteins of the invention. In addition, some sera containing IgE binding to the mapA, mapB or mapC proteins are also found in all D.A. and IgE antibodies that bind to P. teronyssius extract. Control sera did not contain IgE antibodies that bind to any of the mapA, mapB or mapC proteins of the invention.
[0188]
(Example 7)
This example demonstrates that D. elegans induces a hypersensitive response in dogs. 3 illustrates the performance of a farinae HMW-map composition.
[0189]
D. described in Example 1. To determine whether a farinae HMW-map composition can induce an allergic response in an animal susceptible to a powdered mite allergic response, dogs exhibiting clinical signs of powdered mite allergy, Skin test was carried out on the atopic dog). Normal dogs include dogs that do not show symptoms of mite allergy but may be susceptible to a mite allergic response. Each dog (ie, 4 normal dogs and 4 atopic dogs) was shaved in the outer thoracic / abdominal region and separated at different sites in that region by the method described in Example 1. D. at a dilution of about 1: 50,000. Approximately 2 μg of purified D. farinae extract was used. It was injected intradermally with the farinae HMW-map composition and / or control solution (i.e., saline as a negative control and histamine at a 1: 1000 dilution as a positive control). All 4 normal dogs and all 4 atopic dogs had farinae whole extract was received. Three of the normal dogs and two of the atopic dogs were farinae HMW-map composition. All eight dogs received both negative and positive control samples. The total volume per injection was 50 microliters (μl), with the composition and controls diluted in saline. These injections were administered as a single injection.
[0190]
All injection sites were objectively measured in millimeters (mm) at 15 minutes and scored as either (+) or (-) when compared to control samples. Objective scoring was performed as described by Ohio State University, Columbia, OH, Andrew Hillier, D .; V. M. It was carried out by. Table 2 shows the results.
(Table 2)
[0191]
[Table 2]

These results are described in 2 shows that the farinae HMW-map composition was able to induce an immediate hypersensitive response in dogs, including atopic dogs. Thus, the HMW-map composition is sufficiently allergic to induce a hypersensitive response in dogs, including atopic dogs.
[0192]
Table 3 describes the results of the following experiments. IgE against the HMW-map composition was measured using ELISA in the following three groups of dog sera: farinae allergic dogs (HDM-AD), atopic (but not allergen) but not HDM allergic dogs (AD), and naive dogs. Three groups of dogs were also given D.D. The farinae total extract and the HMW-map composition were tested by an intradermal skin test.
[0193]
Table 3. Skin tests and ELISA data for D. farinae total extract and HMW-map compositions in D. farinae allergic dogs, atopic but not HDM-allergic dogs and na ナ イ ve dogs.
[0194]
[Table 3A]

[0195]
[Table 3B]

D. All dogs that were positive by ELISA for farinae total extract were also positive for the HMW-map composition allergen. D. Of the eight dogs that were negative by ELISA for farinae total extract, seven out of eight dogs were also negative for the HMW-map composition.
[0196]
(Example 8)
This example describes the isolation of a nucleic acid molecule encoding a Der HMW-map composition of the invention.
[0197]
Der HMW-map composition nucleic acid molecules were identified and isolated as follows.
[0198]
(A. Preparation of Dermatophagoides farinae cDNA Library)
A Dermatophagoides farinae cDNA library was prepared as follows. Total RNA was obtained from approximately 2 g of flash frozen and crushed house dust mites by Chomzynski et al., 1987, Anal. Biochem. Extracted using a similar acid-guanidium-phenol-chloroform method as described by 162,156-159. Poly A ⁺ Selected RNA was separated from total RNA preparations by oligo-dT cellulose chromatography using the mRNA Purification Kit (available from Pharmacia Biotech, Newark, NJ) according to the method recommended by the manufacturer. The cDNA library was subjected to λ-Uni-ZAP using Stratagene's ZAP-cDNA Synthesis Kit protocol. ^TM Constructed in XR vector (available from Stratagene). About 5 μg of poly A ⁺ RNA was used to generate a Dermatophagoides farinae cDNA library.
[0199]
(B. Preparation of PCR primer)
Further N-terminal amino acid sequence analysis was performed according to the method described above in Example 2. The N-terminal amino acid subsequence of 34 amino acids is deduced and is shown by SEQ ID NO: 24 having the following amino acid sequence:
[0200]
Embedded image

(Where "Xaa" indicates any amino acid residue). Using the amino acid sequence of SEQ ID NO: 4 (described above in Example 2) and SEQ ID NO: 24, a synthetic oligonucleotide primer was designed. The following nucleotide sequence:
[0201]
Embedded image

(SEQ ID NO: 25) wherein Y indicates C or T, R indicates A or G, and N indicates A, C, T or G Or the following nucleotide sequence:
[0202]
Embedded image

A sense primer Derf2 (referred to as SEQ ID NO: 26) derived from SEQ ID NO: 4 having the following nucleotide sequence:
[0203]
Embedded image

Or the antisense primer Derf3 from SEQ ID NO: 4 (designated SEQ ID NO: 27), or the following nucleotide sequence:
[0204]
Embedded image

And used in combination with the antisense primer Derf4 (referred to as SEQ ID NO: 28) derived from SEQ ID NO: 24 having
[0205]
The Derf cDNA library was then screened using standard polymerase chain reaction amplification (PCR) techniques using the primers described above. All attempts to identify cDNAs that hybridized to these primers failed.
[0206]
(C. Immunoscreening of D. farinae cDNA library using anti-Der HMW-map composition antibody)
Since an attempt to isolate a cDNA clone using the PCR method failed, we used the antiserum generated as follows to obtain a DNA clone. The farinae cDNA library was screened. The protein isolated according to the method described above in Example 1 was used as a source of antigen to generate a rabbit polyclonal antibody, referred to herein as an anti-Der HMW-map composition antibody. Preparation of rabbit polyclonal antibodies was performed using standard techniques.
[0207]
About 7.5 ml of Escherichia coli (XL1 Blue, OD. ₆₀₀ = 0.5) with a Dermatophagoides farinae ZAP-cDNA library (1.8 × 10 ⁹ 3.0 × 10 from pfu / ml) ⁴ Incubated with pfu phage at 37 ° C. for 15 minutes and plated on 30 ml Luria-Bertani (LB) medium agar plate (150 mm). The plate was incubated overnight at 37 ° C. Each plate was then overlaid on a nitrocellulose filter treated with IPTG (10 mM) at 37 ° C. for about 4 hours. The filter was then removed and washed with Tris-buffered saline (pH 7.5) containing 0.1% Tween (TBST) for 5 minutes. The filter was blocked with a solution of 1% skim milk powder, 1% BSA, 2% goat serum and 0.15% gelatin in TBST for 2 hours at room temperature. The filters were then incubated overnight at 4 ° C. with the above blocking solution containing the anti-Der HMW-map composition antibody at a 1: 1000 dilution. This mixture was then incubated for 2 hours at room temperature with donkey anti-rabbit IgG antibody conjugated to horseradish peroxidase (available from Jackson ImmunoResearch, West Grove, PN). All filters were washed (3 × 15 minutes per wash) between each incubation with the blocking solution contained in TBST. All filters were then subjected to a final wash for 5 minutes at room temperature with Tris-buffered saline (pH 7.5). The immunocomplexed plaques were stained with a color developing solution (TMB Peroxidase Substrate / TMB Peroxidase Solution / TMB Membrane Enhancer from Kirkegaard & Perry Laboratories, and the filter was immersed in this filter at a volume ratio of 1/1 / 0.1). Identified. 123 plaques were identified and 50 plaques were further purified two or more times under the same immunoscreening conditions as described above.
[0208]
(D. PCR screening of purified phage plug)
The phage plugs identified in the above immunoscreening studies were then further analyzed by PCR amplification using the primers described above in Section 8B. DNA from 50 plaques was amplified using a mixture of the four primers identified by SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28. PCR amplification was performed using standard techniques. One of the resulting PCR amplification products constituted a fragment of about 700 nucleotides. This PCR product was obtained from InVitrogen, Corp. , TA ^TM It was cloned into a cloning vector (a procedure provided by In Vitrogen, Corp.) and subjected to DNA sequence analysis using standard techniques. Phagemids from the purified phage, determined to contain the encoded sequence in the 700 bp PCR product, were recovered and subjected to DNA sequence analysis using standard techniques.
[0209]
A clone containing the approximately 1752 nucleotide insert was isolated and described herein as nDerf98. ₁₇₅₂ Call. The nucleic acid sequence can be converted to nDerf98 using standard techniques. ₁₇₅₂ From the coding strand having the nucleic acid sequence SEQ ID NO: 14 and the complementary strand having the nucleic acid sequence SEQ ID NO: 16 ₁₇₅₂ A Dermatophagoides farinae nucleic acid molecule was obtained. The translation of SEQ ID NO: 14 is from nDerf98 ₁₇₅₂ The nucleic acid molecule comprises about 555 amino acids of the full length flea protein having the amino acid sequence SEQ ID NO: 15 (PDerf98 herein) ₅₅₅ Code). Here, an open reading frame extending from nucleotide 1 to nucleotide 3 of SEQ ID NO: 14 and an end codon extending from nucleotide 1666 to nucleotide 1668 of SEQ ID NO: 14 is expected. PDerf98 ₅₅₅ Is a nucleic acid molecule nDerf98 having a coding strand having the nucleic acid sequence SEQ ID NO: 17 and a complementary strand having the nucleic acid sequence SEQ ID NO: 19. ₁₆₆₅ Coded by PDerf98, also shown as SEQ ID NO: 18 ₅₅₅ Has an estimated molecular weight of about 63.2 kD and an estimated pI of about 5.33. Analysis of SEQ ID NO: 15 indicates the presence of a signal peptide spanning from about amino acid 1 to about amino acid 19. PDerf98 ₅₃₆ The proposed mature protein, shown herein as comprises about 536 amino acids, the sequence is shown herein as SEQ ID NO: 21, and SEQ ID NO: 20 (coding strand) and SEQ ID NO: 22. NDerf98, indicated by (complementary strand) ₁₆₀₈ And encoded by the nucleic acid molecule set forth herein. flea PDerf98 ₅₃₆ The amino acid sequence of SEQ ID NO: 21 (ie, SEQ ID NO: 21) ₅₃₆ Has an estimated molecular weight of 61.2 kD and an estimated pI of about 5.26.
[0210]
Comparison of amino acid sequence SEQ ID NO: 15 with the amino acid sequence reported in GenBank shows that SEQ ID NO: 15 has the greatest homology (ie, about 42% identity) to a chitinase protein from Anopheles gambiae (GenBank accession number 2654602). Indicates what was shown. A comparison of the nucleic acid sequence SEQ ID NO: 17 with the nucleic acid sequence reported in GenBank shows that SEQ ID NO: 17 differs from SEQ ID NO: 17 and Chelonus sp. 4 shows that it exhibited the greatest homology (ie, approximately 58% identity) with the venom chitinase mRNA (GenBank accession number U10422).
[0211]
(Example 9)
This example describes the purification of a 60 kD protein that binds to IgE from a dog known to be allergic to mite allergen, and the partial amino acid sequence from this 60 kD protein.
[0212]
(A. Purification of 60 kD protein)
D. The farinae extract was prepared and fractionated on a Sephacryl S-100 column according to the method described above in Example 1. Fractions after the exclusion peak (fractions 29-35) were collected from a Sephacryl S-100 column and pooled. The pooled fractions were then diluted 1: 1 with 10 mM Tris-HCl (pH 8) and applied to a Q-sepharose column to obtain fractions using the method described in Example 1. The fraction eluted with 0.4 M Tris-HCl was concentrated and further purified by a TMS250 reverse phase HPLC column using the method described above in Example 1. The proteins in each sentence were separated by 14% Tris-glycine SDS-PAGE using the same method described in Example 1 for protein separation on a 12% gel. The stained gel is shown in FIG. A protein with a molecular weight of about 60 kD of about 90% purity, eluted at about 50% B (0.05% TA in 90% acetonitrile) was identified (FIG. 2, lane 4). The molecular weight of the indicated 60 kd protein was estimated to be 56.11 kd using the BioRad Multi-Analyst / PC Version 1.1 program and the Mark-12 protein molecular weight marker. The approximately 60 kd protein is referred to herein as a mapD protein.
[0213]
(B. N-terminal partial sequence and internal sequence obtained from 60 kd protein)
The protein eluted from Part A above was blotted onto PVDF, which was stained with Coomassie R-250 and decolorized using standard procedures. The protein corresponding to the band at about 60 kd was extracted and subjected to N-terminal amino acid sequencing using techniques known to those skilled in the art. An N-terminal amino acid subsequence of about 25 amino acids was deduced for this protein, and the amino acid sequence shown herein as SEQ ID NO: 23 was replaced with:
[0214]
Embedded image

(Where Xaa refers to any amino acid).
[0215]
The protein corresponding to this 60 kd region was also subjected to proteolytic cleavage to obtain internal amino acid sequence data. Digestion and reverse phase chromatography of the 60 kd protein were performed as described in Example 1. The four proteolytic fragments were isolated and sequenced and are referred to herein as map (13), map (14), map (15) and map (16).
[0216]
The N-terminal partial amino acid sequence of map (13) is as follows:
[0219]
Embedded image

As shown in SEQ ID NO: 29. The N-terminal partial amino acid sequence of map (14) is as follows:
[0218]
Embedded image

And this was also set forth as SEQ ID NO: 30. The N-terminal partial amino acid sequence of map (15) is as follows:
[0219]
Embedded image

And this was also set forth as SEQ ID NO: 31. The N-terminal partial amino acid sequence of map (16) is as follows:
[0220]
Embedded image

And this was also set forth as SEQ ID NO: 32.
[0221]
(Example 10)
In the present embodiment, The isolation and sequencing of a nucleic acid molecule encoding a part of the farinae 60 kD (mapD) allergen is described.
[0222]
As described above in Example 8, D.I. A farinae library was prepared. D. From the deduced N-terminal amino acid sequence for the farinae 60 kD protein (SEQ ID NO: 23), a degenerate synthetic oligonucleotide primer was designed: Primer 1, a sense primer corresponding to amino acid residues from about 3 to about 11 of SEQ ID NO: 23 Is a sequence also identified as SEQ ID NO: 46:
[0223]
Embedded image

Where H denotes A or C or T, N denotes A or C or G or T, and Y denotes C or T. D. PCR amplification of fragments from the farinae library was performed using standard techniques. SEQ ID NO: 46 (primer 1) and M13 forward universal primer shown as SEQ ID NO: 47:
[0224]
Embedded image

Was used to generate PCR amplification products.
[0225]
A second nested PCR reaction was performed on the products of the first PCR reaction. Synthetic oligonucleotides were synthesized that corresponded to the region spanning approximately amino acid residues 1 to 10 of the 60 kD protein internal amino acid sequence (SEQ ID NO: 31). This primer (primer 2) has the following nucleic acid sequence shown as SEQ ID NO: 48:
[0226]
Embedded image

Wherein R represents A or G. Primer 2 (SEQ ID NO: 48), and T7 standard primer shown as SEQ ID NO: 49:
[0227]
Embedded image

Was used to generate PCR amplification products. The resulting PCR product was subjected to DNA sequence analysis using standard techniques.
[0228]
The PCR product was sequenced and found to contain 510 nucleotides, indicating that nDer60 ₅₁₀ Identified as nDerf60 ₅₁₀ The nucleotide sequence of the coding strand of is shown herein as SEQ ID NO: 43, and its complement is shown as SEQ ID NO: 45. The translation of SEQ ID NO: 43 is based on nDerf60 ₅₁₀ Has a portion of about 170 amino acids having the amino acid sequence set forth in SEQ ID NO: 44. farinae 60 kD protein (PDerf60 herein) ₁₇₀ Code). Here, the start codon presumes an open reading frame spanning from about nucleotide 1 to about nucleotide 3 of SEQ ID NO: 43 and the stop codon spanning from about nucleotide 508 to about nucleotide 510 of SEQ ID NO: 43. PDerf60 ₁₇₀ Has an estimated molecular weight of about 19.2 kD and an estimated pI of about 6.51.
[0229]
Nucleic acid molecule nDerf60 ₅₁₀ Was used as a probe to generate farinae 60 kD protein or a larger portion of A nucleic acid molecule encoding a protein corresponding to the farinae 60 kD protein was isolated. Using the procedure set forth above in Example 8, using standard techniques ³² Using nucleic acid SEQ ID NO: 43 radiolabeled with P, all D.P. The farinae library was screened. Hybridization was carried out in 6 × SSC, 5 × Denhardt solution, 0.5% SDS, 100 mg / ml ssDNA at 55 ° C. for about 36 hours. The filters were washed three times for 30 minutes per wash at 55 ° C. in 2 × SSC, 0.2% SDS for 30 minutes, followed by a final wash in 0.2 × SSC, 0.2% SDS for about 30 minutes.
[0230]
PCR amplification was performed on the first phage plug. PCR products were generated using primer 1 as SEQ ID NO: 46 and the T7 standard primer as SEQ ID NO: 49 as primers. Preliminary sequence analysis of this 1.6 kb PCR product indicated that it exhibited the nucleic acid sequence including the complete sequence encoding the PDerf60 full length protein.
[0231]
Amino acid sequence PDerf60 of SEQ ID NO: 44 ₁₇₀ And the amino acid sequence reported in GenBank were compared to PDerf60 ₁₇₀ Is Aphanodium album. 4 shows that it showed the highest homology (ie, about 39% identity) with the chitinase protein precursor from the original (GenBank accession number P32470). Nucleic acid sequence SEQ ID NO: 43 did not show significant homology to any of the sequences submitted to GenBank.
[0232]
(Example 11)
This example describes the isolation of a nucleic acid molecule encoding a Dermatophagoides pteronyssius 98 kD allergen protein.
[0233]
D. Hybridization with a 32-P labeled cDNA encoding a F. farinae HMW-map composition resulted in a D. tertiary HMW-map composition. A nucleic acid molecule having high homology to the F. farinae 98 kD allergen (mapB) was identified as pteronyssius cDNA library.
[0234]
D. A pteronyssius cDNA library was prepared as follows. About 2 g of D.E. pteronyssius mite, Chonzynski et al., 1987, Anal. Biochem. 162: Extracted using the acid-guanidium-phenol-chloroform method described by pp 156-159. Poly A + selected RNA was separated from total RNA preparation by oligo-dT cellulose chromatography using the mRNA Purification Kit (available from Pharmacia, Newark, NJ) according to the method recommended by the manufacturer. All D. The Pteronyssius cDNA library was constructed using the Stratagene ZAP-cDNA Synthesis Kit protocol with λ-Uni-ZAP. ^TM Constructed in the XR vector (available from Stratagene, La Jolla, CA). Using about 5 milligrams (mg) of poly A + RNA, A Pteronyssius cDNA library was generated.
[0235]
Using a modification of the protocol described in the cDNA Synthesis Kit (available from Stratagene), 32P-labeled D.C. cDNA encoding the F. farinae 97 kD MapB allergen (ie, SEQ ID NO: 17); The pteronyssius cDNA library was screened using a duplicate plaque lift. Hybridization was performed with 6 × SSC (for formulation, see Sambrook et al., Ibid), 5 × Denhardt solution (for formulation, Sambrook et al., Ibid), 0.5% sodium dodecyl sulfate (SDS) ( Sigma) and 100 mg / ml single-stranded DNA (available from Sigma) at 55 ° C. for about 36 hours. Wash the filters three times at 55 ° C. in 2 × SSC, 0.2% SDS (about 30 minutes per wash), followed by 55 ° C. in 0.2 × SSC, 0.2% SDS. The filter was finally washed for about 30 minutes. D. pteronyssius, which encodes a 97 kD allergen (mapB); Plasterically purified clones of P. teronysius nucleic acid molecules were subjected to the ExAssist method according to the in vivo excision protocol described in the ZAP-cDNA Synthesis Kit (available from Stratagene). ^TM Helper phage and SOLR ^TM E. FIG. It was converted to a double-stranded recombinant molecule using E. coli. D. Plasmids containing the Pteronyssius clone were subjected to DNA sequence analysis using standard techniques. DNA sequence analysis (including determination of molecular weight and isoelectric point (pI)) was performed by GCG ^TM Made using the program.
[0236]
Clones containing an approximately 1621 nucleotide insert were isolated. This is referred to herein as nDerp98. ₁₆₂₁ And a full length coding region having the coding strand shown as SEQ ID NO: 34 and the complementary strand shown as SEQ ID NO: 36. Distinct start and stop codons range from nucleotide 14 to nucleotide 16 and nucleotide 1541 to nucleotide 1543 of SEQ ID NO: 34, respectively. The putative polyadenylation signal (5'AATAAA3 ') is located in the region spanning nucleotide 1584 to nucleotide 1589 of SEQ ID NO: 34.
[0237]
The translation of SEQ ID NO: 34 is the amino acid sequence shown as SEQ ID NO: 35, and PDerp98 ₅₀₉ Yields a protein of about 509 amino acids, denoted as PDerp98 ₅₀₉ The nucleic acid molecule consisting of the coding region encoding is referred to herein as nDerp98. ₁₅₂₇ And the nucleic acid sequences are shown as SEQ ID NO: 37 (coding strand) and SEQ ID NO: 39 (complementary strand). PDerp98, also designated herein as SEQ ID NO: 38 ₅₀₉ Has an estimated molecular weight of about 58.9 kD and an estimated pI of about 5.61. PDerp98 ₅₀₉ Analysis suggests the presence of a signal peptide ranging from approximately amino acids 1 to approximately amino acids 19. The proposed mature protein (PDerp98 herein) ₄₉₀ ) Comprises about 490 amino acids and is designated herein as SEQ ID NO: 41. PDerp98 ₄₉₀ Shows that the protein has a predicted molecular weight of about 56.8 kD and a predicted pI of about 5.49, and two asparagine-linked glycosylation sites (about amino acid 115 to about amino acid 117, and about amino acid 240 to 240, respectively). (Extending to amino acid 242). PDerp98 ₄₉₀ Has a coding strand represented by SEQ ID NO: 40 and a complementary strand represented by SEQ ID NO: 42. ₁₄₇₀ It is known as
[0238]
BLAST searches were performed as described previously. PDerp98 ₅₀₉ (SEQ ID NO: 35) showed the highest homology at the amino acid level with Manduca sexta chitinase (SwissProt accession number p36362), with approximately 34% identity. nDerp98 ₁₆₂₁ (SEQ ID NO: 34) is a Chelonus sp. It showed the highest homology to the chitinase (Accession No. U10422) at the nucleic acid level, with about 49% identity. D. a cDNA region corresponding to the coding region for the 98 kD allergen protein of F. farinae; Comparison with the cDNA region corresponding to the coding region for the 98 kD allergen protein of Pteronyssius shows about 84% identity.
[0239]
(Example 14)
In the present embodiment, Figure 2 shows the binding of the farinae HMW-map composition to human IgE in human serum isolated from humans, which is known to be allergic to mite allergens.
[0240]
A technique called RAST, the last method (radio-allergo-absorbent test), was used. This is because the amount of human IgE present in human serum is very low. RAST is essentially described in Aalberse, RC et al. Allergy Clin Immun. 68: 356-364. To calculate unit IU / ml, a standard curve was generated using a well-characterized chimeric human / mouse IgE monoclonal antibody against Derp2 (human IgE / monoclonal anti-Derp2, Schuurman et al. (1997) J Allergy Clin Immunol. 99: 545-45). (Following the procedure on page 550) was performed by performing a RAST with several dilutions.
[0241]
Briefly, 50 μg of the HMW-map composition (purified as described in Example 1) was combined with 50 mg of CNBr activated Sepharose 4B (available from Pharmacia, Piscataway, NJ) according to the manufacturer's protocol. Combined. Human serum was used for whole mite D. Selection based on the positive RAST for the farinae extract (17 different samples in total), the number of positive RAST is greater than 1 IU / ml. Two negative (less than 0.3 IU) control sera were also included.
[0242]
To test a serum sample of each individual, 0.5 mg of D.E. farinae HMW-map composition-conjugated Sepharose with 50 μl serum in a total volume of 300 μl PBS-T (phosphate buffered saline with 0.1% volume / volume Tween-20 (available from Sigma)). Incubated. Incubation was performed overnight at 27 ° C. with shaking. After incubation, the bound Sepharose was washed 5 times with PBS-T. Radiolabeled (diluted in PBS-T with 4.5% (v / v) bovine serum and 0.5% (v / v) sheep serum) in a total volume of 750 μl ( ¹²⁵ -Iodine) Sheep anti-human IgE (generated by a standard radioiodination labeling protocol) was added and incubated overnight at 27 ° C. After incubation, the bound Sepharose was washed four times with PBS-T and counted in a gamma counter to determine the amount of radiolabeled sheep anti-human IgE bound to the HMW-map composition-bound Sepharose. Table 4 shows the results.
[0243]
(Table 4. Binding of human IgE to HMW-map composition from D. farinae)
[0244]
[Table 4]

D. Approximately 75% (11/15) of the patients who were sensitive to the farinae whole mite extract were sensitive to the HMW-map composition antigen, indicating that the HMW-map composition antigen was , D. It is a major antigen for farinae-sensitive humans. Sensitivity to the HMW-map composition was defined as a RAST of 0.5 IU or more.
[0245]
(Example 15)
This embodiment is similar to D.O. 7 demonstrates that the farinae HMW-map composition comprises a glycoprotein.
[0246]
About 5.4 μg of D.I. The farinae HMW-map composition was applied to SDS PAGE and electrophoresis was performed according to standard techniques. The protein is blotted to nitrocellulose membrane according to standard techniques, and the glycoprotein is analyzed by DIG using the manufacturer's protocol. ^TM Detection was performed using a glycine detection kit (available from Boehringer Mannheim, Indianapolis, IN). The region corresponding to the HMW-map region showed a positive reaction using the kit, indicating that the HMW-map composition contains a glycoprotein.
[0247]
(Example 16)
In the present embodiment, It is shown that the farinae HMW-map composition retains its characteristics as an allergen even when amino acid residues are removed by both chemical and enzymatic means. This result suggests that the major epitope may be a carbohydrate epitope, including a polysaccharide linked to an N-linked or an O-linked glycosylation site on the HMW-map composition.
[0248]
(A. Removal of protein by chemical means (β removal of protein))
12 μg (microgram) of the HMW-map composition (purified as described in Example 1) was dissolved in 100 μl (microliter) of distilled deionized water. To this mixture, 5 μl of 10 M (molar) NaOH and 3.8 mg (milligram) NaBH ₄ (Available from Sigma) was added and 0.5M NaOH and 1M NaBH ₄ Was obtained. The reaction mixture was heated at 50 ° C. for 30 minutes, then cooled and 100 μl of acetone was added. To this mixture, a sufficient amount (ie, about 150 μl) of Dowex 50 (H +) (available from Pharmacia) was added to make a slightly acidic solution. Dowex 50 absorbs and removes proteins, leaving any sugar moieties in the supernatant. The mixture was centrifuged in a microcentrifuge and washed three times with 100 μl of water. Combine the supernatants from the centrifugations, evaporate to dryness and wash 5 times with a methanol: HCl solution (1000: 1 v / v), after each wash evaporate to dryness to remove salts. The mixture is dissolved in 100 μl water and an aliquot (20 μl) is analyzed by SDS-PAGE using standard techniques and chemically treated using both Coomassie blue and silver stains The amount of protein in the sample was determined. No protein was detected by either Coomassie blue or silver staining, indicating protein removal. Any sugar moieties on the protein were not affected by these conditions.
[0249]
The remainder of the residue from each sample was subjected to an ELISA analysis as described in Example 4. Briefly, 100 ng of either the β- or non-β-depleted sample of the HMW-map composition was coated on Immulon plates, and D.C. farinae-sensitive dog serum pool, F. farinae-sensitive cat serum pool, and (as measured by ELISA). An ELISA was performed as described in Example 4 using various separate canine sera that were either sensitive or insensitive to farinae. Table 5 shows the results.
[0250]
Table 5. Reactivity of dog serum and cat serum to HMW-map and β-depleted HMW-map compositions, which are carbohydrates only
[0251]
[Table 5]

The results from Table 5 indicate that the β-removed HMW-map composition sample was show that they still retain the ability to bind IgE from dog and cat sera that are sensitive to the farinae HMW-map composition, indicating that glycans bound to the protein are a major component of the HMW-map composition allergen protein. It constitutes a novel epitope.
[0252]
(B. Protein removal by enzymatic means)
14 μg of the HMW-map composition (purified as described in Example 1) is digested with 1 μg of endoproteinase K (available from Sigma) to remove the protein portion of the molecule. The digestion reaction was performed at 56 ° C. for 24 hours, after which the endoproteinase in the reaction was heat denatured in boiling water for 10 minutes.
[0253]
A portion of this reaction was analyzed by SDS-PAGE using standard techniques, and both Coomassie blue and silver staining were used to detect the presence of protein in the enzymatically digested sample . The HMW-map composition was not detected by either Coomassie blue staining or silver staining, indicating removal of the HMW-map composition. Any glycans attached via glycosylation sites on the protein were not affected by these conditions.
[0254]
The remainder of the enzymatically digested reaction was tested by ELISA in the manner described in Example 4. Briefly, 100 ng of either the proteinase K digested sample or the proteinase K undigested sample of the HMW-map composition was coated on Immulon plates, and the ELISA was performed (as measured by ELISA). Performed as described in Example 4 using a variety of separate canine sera that were either sensitive or insensitive to farinae. Table 6 shows the results.
[0255]
(Table 6. Reactivity of dog serum to HMW-map composition and endoproteinase K digested HMW-map composition)
[0256]
[Table 6]

The results from Table 6 show that the proteinase K digested HMW-map composition sample was D.E. It is shown that the ability to bind IgE from dog serum and cat serum that is sensitive to the farinae HMW-map composition is still retained, indicating that the glycans attached to the protein are the major components on the HMW-map composition. Suggests that it constitutes an epitope.
[0257]
(Example 17)
This example describes an attempt to remove N-linked glycans from a HMW-map composition.
[0258]
HMW-map composition (2 μg) (purified as in Example 1) was digested with N-glycosidase F (available from Boehringer-Mannheim) according to the manufacturer's instructions. Digests were analyzed by SDS-PAGE and stained according to standard protocols. 2 μg Fetuin (available from Sigma) was used as a positive N-linked glycosylated protein control. SDS-PAGE analysis showed that there was no apparent difference between the molecular weights of intact map B protein and digested map B protein. The positive control fetuin did not show a decrease in molecular weight after digestion with N-glycosidase F. This result indicates that no N-linked glycans are present on the HMW-map composition, or that only small sized N-glycans are present on the HMW-map composition.
[0259]
(Example 18)
This example describes the isolation and sequencing of a nucleic acid molecule encoding a full-length Dermatophagoides farinae 60 kD allergen.
[0260]
This nucleic acid molecule encodes a portion of the Dermatophagoides farinae 60 kD allergen described in Example 10. ³² It was isolated from a Dermatophagoides farinae cDNA library by its ability to hybridize to P-labeled cDNA.
[0261]
A Dermatophagoides farinae cDNA library was prepared as follows. Total RNA was prepared as described in Chonzynski et al., 1987, Anal. Biochem. Using an acid guanidinium-phenol-chloroform method similar to the method described by 162, 156-159, about 2 grams of D.E. farinae mite. Poly A ⁺ Selected RNA was separated from total RNA preparations by oligo dT cellulose chromatography using an mRNA purification kit (available from Pharmacia Biotech, Newark, NJ) according to the method recommended by the manufacturer. The whole mite cDNA library was subjected to lambda-Uni-ZAP using Stratagene's ZAP-cDNA Synthesis Kit protocol. ^TM Constructed in XR vector (available from Stratagene). About 5 μg of poly A ⁺ Using RNA, A farinae cDNA library was generated.
[0262]
Using a modification of the protocol described in the cDNA Synthesis Kit, ³² P-labeled cDNA nDerf60 ₅₁₀ The entire mite cDNA library was screened using multiple plaque lifts with Hybridization was performed with 6 × SSC, 5 × Denhardt solution, 0.5% SDS, 100 mg / ml ssDNA at 52 ° C. for 18 hours. Filters were washed twice at 55 ° C. for 30 minutes in 2 × SSC, 0.2% SDS for each wash, followed by 30 minutes in the same buffer except using about 0.2 × SSC. A final wash was performed. D. Plaque-purified clones of the nucleic acid molecule encoding the F. farinae 60 kD allergen were prepared according to the in vivo excision protocol described in the ZAP-cDNA Synthesis Kit (available from Stratagene) using the ExAssist system. ^TM Helper phage and SOLR ^TM E. FIG. E. coli was used to convert it into a double-stranded recombinant molecule, which is referred to herein as nDerf60. ₁₄₅₅ As shown. Double-stranded plasmid DNA was prepared using an alkaline lysis protocol (eg, as described in Sambrook et al., Supra).
[0263]
(Example 19)
This embodiment describes the D.D. Describes the sequencing of a farinae nucleic acid molecule.
[0264]
nDerf60 ₁₄₅₅ PRISM using Ampli Taq DNA polymerase, FS (available from Perkin Elmer Corporation, Norwalk, CT) by the Sanger dideoxy chain termination method. ^TM The sequence was determined using the Ready Dye Terminator Cycle Sequencing Kit. PCR extension to GeneAmp ^TM Performed in a PCR System 9600 (available from Perkin-Elmer). Excess dye terminator is added to Centriflex according to the manufacturer's standard protocol. ^TM The gel was removed from the extension product using Gel Filtration Cartridge (available from Advanced Genetics Technologies Corporation, Gaithersburg, MD). Samples were resuspended according to the ABI protocol, and the Perkin-Elmer ABI PRISM ^TM 377 Automated DNA Sequencer. DNA sequence analysis, including sequence editing and open reading frame determination, was performed by GCG. ^TM This was done using a program (available from Genetics Computer Group, Madison, WI). Protein sequence analysis, including determination of molecular weight and isoelectric point (pI), was performed by GCG ^TM Performed using the program.
[0265]
Complete nDerf60 ₁₄₅₅ An approximately 1455 nucleotide consensus sequence of the nucleic acid molecule was determined; the sequences of the two complementary strands are present as SEQ ID NO: 50 (coding strand) and SEQ ID NO: 52 (complementary strand). The sequences of the two complementary strands are shown as SEQ ID NO: 50 (coding strand) and SEQ ID NO: 52 (complementary strand). nDerf60 ₁₄₅₅ The sequence includes the full length coding region. The apparent start and stop codon ranges extend from nucleotides 14 to 16 and 1400 to 1402 of SEQ ID NO: 50, respectively. The putative polyadenylation signal (5'AATAAA3 ') is located within the region spanning nucleotides about 1408-1413 of SEQ ID NO: 50.
[0266]
Translation of SEQ ID NO: 50 is PDerf60 ₄₆₂ Which results in a protein of 462 amino acids, the amino acid sequence of which is shown in SEQ ID NO: 51. PDerf60 ₄₆₂ The nucleic acid molecule consisting of the coding region encoding is referred to herein as nDerf60. ₁₃₈₆ This nucleic acid sequence is shown in SEQ ID NO: 53 (coding strand) and SEQ ID NO: 54 (complementary strand). PDerf60 ₄₆₂ Of SEQ ID NO: 51 (ie, SEQ ID NO: 51) ₄₆₂ Would have an estimated molecular weight of about 52.1 kD and an estimated PI of about 5.73. Analysis of SEQ ID NO: 51 indicates the presence of a signal peptide encoded by a range of amino acids ranging from amino acid 1 to amino acid 25. The proposed mature protein is referred to herein as PDerf60. ₄₃₇ The protein comprises about 437 amino acids, designated herein as SEQ ID NO: 56. PDerf60 ₄₃₇ Of SEQ ID NO: 56 (ie, SEQ ID NO: 56) ₄₆₂ Has a predicted molecular weight of about 50.0 kD, a predicted PI of about 5.61, and one putative asparagine-linked glycosylation site spanning amino acids 313 to 315. The nucleic acid molecule encoding this mature protein is shown as SEQ ID NO: 55, and its inverted complement is shown as SEQ ID NO: 57.
[0267]
BLASTp searches were performed according to the following: Altschul et al., (1990), J. Am. Mol. Biol. 215: 403-410; and Altschul et al., (1997), Nucleic Acids Res. 25: 3389-3402. A protein search was performed using SEQ ID NO: 51, which showed significant homology to the chitinase molecule. The highest homology search score match at the amino acid level was PIR accession number A53918: Chelonus sp chitinase precursor, which was approximately 32% identical to SEQ ID NO: 51. At the nucleotide level, this search was performed using SEQ ID NO: 53, which did not show significant similarity to any of the sequences in the database. Sequence analysis was performed using the GCG GAP program as described above.
[0268]
(Example 20)
In the present embodiment, The characterization of the farinae HMW-map composition (also referred to as Der f 15) is further described.
[0269]
The nucleic acid molecule nDerf98 of Example 8 ₁₇₅₂ Was inserted into an appropriate expression vector. coli and P. coli. Pastors were expressed. Obtained protein (PDerf98 ₅₅₅ ) Is E. coli or P. coli. When expressed in P. pastoris, sensitized dog sera produced as described in Example 4 failed to recognize the recombinant protein. This is the same as the native D.F. In contrast to the positive results obtained when a farinae HMW-map composition (also referred to as native Derfl5) is used; see Example 4.
[0270]
E. FIG. The non-reactivity of the protein expressed in E. coli is consistent with the results shown in Example 16, indicating that the native HMW allergen retains its properties as an allergen even after amino acids have been removed. Showed that.
[0271]
Taken together, these results suggest that the major epitope is some hydrocarbon region of the molecule or other secondary modification.
[0272]
The antigenicity of the native Derfl5 protein is not lost after periodate treatment; generally, carbohydrate epitopes exclude epitopes further substituted with additional groups or epitopes with rare sugars having non-geminal hydroxyl groups. And is destroyed by periodate.
[0273]
The native Derfl5 antigen was analyzed for hydrocarbon content. Substantial amounts of hydrocarbons were found (about 30% by weight). In particular, mannose composed of about 2.8% by weight of antigen; galactose about 23.2%; glucose about 4.3% (the presence of glycosyl residues is a tentative value since glucose often contains glycoproteins). HexNAc at detectable levels; further observations revealed that HexNAc was GlcNAc and GalNAc.
[0274]
NaBH ₄ Treated with base in the presence and analyzed by P-4 size exclusion chromatography. The column was found useless due to the O-linked oligosaccharides present in Derf15. This result is consistent with either very large O-linked oligosaccharides, or the presence of acidic groups such as sulfate on the oligosaccharide. Attempts to determine the presence or absence of sulfate more directly have given ambiguous results.
[0275]
Derf15 was treated at pH 4, pH 5, and pH 7 at 37 ° C. overnight. The resulting sample was then probed with an antibody against this protein or canine serum known to be reactive with Derfl5. All of the canine antiserum epitopes were destroyed in the samples treated at pH 5 and pH 7, but some activity remained in the samples treated at pH 4. The anti-Derfl5 antibody shows a decrease in the molecular weight of Derfl5 at all pHs, as if deglycosylation had occurred, and at pH 4 some original material remains. It is not known whether this change was autocatalytic by the Derfl5 protein or caused chemically; without being bound by theory, the loss of epitopes occurs under such mild conditions and therefore, the autocatalytic It is considered that the effect has occurred.
[0276]
(Example 21)
This example describes the binding of some house dust mite (HDM) allergens to cat IgE in cat serum.
[0277]
The allergen profile of the cat's IgE response to house dust mites appears to be different from that of the dog. Examination of the results of the IgE test on cat serum submitted to Heska's Veterinary Diagnostic Laboratories (VDL) in January 2000 revealed that 40% of all allergen-specific IgE-positive cats had anti-HDM IgE. That was shown. All of these cats are farinae and D.M. pteronyssinus was positive. D. 88 sera known to be positive for farinae were assayed by ELISA against highly purified preparations of Derfl, Derfl, Derfl5, and 60 kD allergen. In this assay, 32% of the cats were positive for Derfl, 42% were positive for Derf2, 68% were positive for Derfl5, and 86% were positive for the 60kD allergen.
[0278]
While various embodiments of the present invention have been described in detail, it is evident that modifications and adaptations of these embodiments will occur to those skilled in the art. However, it should be clearly understood that such modifications and adaptations are within the scope of the present invention, as set forth in the appended claims.
[Brief description of the drawings]
FIG.
FIG. 1 shows high molecular weight Derf proteins separated by 12% Tris-glycine SDS-PAGE.
FIG. 2
FIG. 2 shows an approximately 60 kD Derf protein separated by 14% Tris-glycine SDS-PAGE.

Claims (30)

An isolated nucleic acid molecule, comprising:
(A) a nucleic acid molecule comprising at least about 150 nucleotides, wherein the nucleic acid molecule comprises SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 16 in a solution containing 1 × SSC and 0% formamide at a temperature of about 50 ° C. 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 45 A nucleic acid molecule comprising at least about 150 nucleotides, which hybridizes to a nucleic acid sequence encoding a nucleic acid sequence encoding a protein comprising the amino acid sequence of SEQ ID NO: 33 and a complement thereof, and (b) (a A) a nucleic acid molecule comprising at least about 15 nucleotides of any of the nucleic acid molecules of No, nucleic acid molecule,
A nucleic acid molecule selected from the group consisting of: The nucleic acid molecule of claim 1, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding a Der HMW-map protein. A nucleic acid molecule of claim 1, wherein the nucleic acid _{molecule, nDer98 1752, nDerf98 1665, nDerf98} 1608, nDerp98 1621, nDerp98 1527, is selected from the group consisting of _{NDerp98 1470} and NDerf60 _510, nucleic acid molecules. 2. The nucleic acid molecule according to claim 1, wherein the nucleic acid molecule comprises:
(A) SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42 A nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 43, SEQ ID NO: 45; and (b) a nucleic acid molecule comprising an allelic variant of the nucleic acid molecule of (a);
A nucleic acid molecule selected from the group consisting of: 2. The nucleic acid molecule according to claim 1, wherein the nucleic acid molecule comprises:
(A) SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 35, Sequence A nucleic acid molecule comprising a nucleic acid sequence encoding a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 38, 41 and 44; and (b) encoding a protein having the amino acid sequence of (a) A nucleic acid molecule, including an allelic variant of the nucleic acid molecule;
A nucleic acid molecule selected from the group consisting of: A recombinant molecule comprising the nucleic acid molecule according to claim 1. A recombinant virus comprising the nucleic acid molecule according to claim 1. A recombinant cell comprising the nucleic acid molecule according to claim 1. An isolated protein, wherein the isolated protein comprises:
(A) a nucleic acid molecule comprising at least about 150 nucleotides, wherein the nucleic acid molecule comprises SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 19 in a solution comprising 1 × SSC and 0% formamide at a temperature of about 50 ° C. 22, a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 45, and a complement of a nucleic acid sequence encoding a protein comprising the amino acid sequence of SEQ ID NO: 33 A nucleic acid molecule comprising at least about 150 nucleotides that hybridizes to the nucleic acid molecule; and (b) a nucleic acid molecule comprising any fragment of the nucleic acid molecule of (a), wherein the fragment comprises at least about 15 nucleotides. molecule,
An isolated protein encoded by a nucleic acid selected from the group consisting of: 10. The protein of claim 9, wherein the protein elicits an immune response against a Der HMW-map protein when administered to an animal. 10. The protein of claim 9, wherein the protein is:
(A) a nucleic acid sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 40, SEQ ID NO: 43, and a protein comprising the amino acid sequence of SEQ ID NO: 33 A protein encoded by a nucleic acid molecule having a coding strand of a nucleic acid sequence encoding the nucleic acid molecule; and (b) a protein encoded by a nucleic acid molecule comprising an allelic variant of the nucleic acid molecule comprising any of the nucleic acid molecules of (a). ,
A protein selected from the group consisting of: 10. The protein of claim 9, wherein the protein is:
(A) SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 35, Sequence A protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 38, SEQ ID NO: 41, and SEQ ID NO: 44; and (b) an allelic variant of a nucleic acid molecule encoding a protein comprising any of the amino acid sequences of (a) A protein, encoded by
A protein selected from the group consisting of: An isolated antibody that selectively binds to the protein of claim 9. 10. The protein of claim 9, wherein said protein selectively binds to IgE. 10. The protein of claim 9, wherein the protein comprises an epitope having at least one identified property, wherein the property comprises:
(A) the epitope is resistant to β-elimination of the peptide;
(B) the epitope is resistant to proteinase-K digestion; and (c) the epitope is selected from the group consisting of properties that are reactive to tests designed to detect glycosylated proteins. Wherein the epitope binds to IgE, wherein the IgE is selected from the group consisting of dog IgE from a dog allergic to ticks and cat IgE from a cat allergic to ticks. ,protein. A therapeutic composition for treating an allergic reaction to a tick, wherein the therapeutic composition comprises:
(A) the isolated protein of claims 9-12;
(B) a mimetope of the mite allergic protein;
(C) the mite allergic protein mutein;
(D) an isolated nucleic acid molecule according to claims 1-5;
A therapeutic composition comprising a desensitizing compound selected from the group consisting of: (e) an antibody against the mite allergic protein; and (f) an inhibitor of the binding of the mite allergic protein to IgE. 17. The composition of claim 16, wherein the composition further comprises a component selected from the group consisting of an excipient, an adjuvant, and a carrier. 17. The composition of claim 16, wherein said desensitizing compound is a naked nucleic acid molecule. An assay kit for testing whether an animal is susceptible to a tick or has an allergic reaction, the kit comprising:
(A) an isolated protein according to claims 9 to 12; and (b) means for determining whether the animal is susceptible or has an allergic reaction, said means comprising: An assay kit comprising using said protein to identify an animal that is susceptible to a tick or has an allergic reaction. A method for identifying an animal susceptible to a tick or having an allergic reaction, the method comprising the following steps:
(A) contacting the isolated protein of claims 9-12 with an animal antibody; and (b) determining the formation of an immune complex between the protein and the antibody. Wherein the formation of the immune complex is indicative of the animal being susceptible or having the allergic reaction.
Including, methods. A method for desensitizing a host animal to an allergic reaction to mites, the method comprising administering to the animal a therapeutic composition according to claim 16. 22. The method according to claim 21, wherein the protein is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, Sequence No. 32, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 41, and SEQ ID NO: 44. 22. The method of claim 21, wherein the therapeutic composition further comprises a component selected from the group consisting of an excipient, an adjuvant, and a carrier. A method for producing a mite allergic protein, comprising the following steps:
A nucleic acid molecule comprising at least about 150 nucleotides, wherein the nucleic acid molecule comprises SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 22, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 45 and a complement of a nucleic acid sequence encoding a protein comprising the amino acid sequence of SEQ ID NO: 33; and any of the nucleic acid molecules Wherein the fragment is transformed with a nucleic acid molecule selected from the group consisting of nucleic acid molecules comprising at least about 150 nucleotides that hybridizes to a nucleic acid molecule comprising at least about 15 nucleotides. Culturing the cells. A reagent comprising a non-protein epitope having at least one identified property, wherein the property comprises:
(A) the epitope is resistant to β-elimination of the peptide;
(B) the epitope is resistant to proteinase-K digestion; and (c) the epitope is selected from properties that are reactive to a test designed to detect glycosylated proteins. Wherein the epitope binds to IgE, wherein the IgE is selected from the group consisting of dog IgE from a dog allergic to ticks and cat IgE from a cat allergic to ticks. An isolated antibody that selectively binds to the epitope of claim 25. 26. A therapeutic composition for treating an allergic reaction to mites, wherein the therapeutic composition comprises a desensitizing compound comprising the reagent of claim 25. 26. An assay kit for testing whether an animal is susceptible to mites or has an allergic reaction, said kit comprising a reagent according to claim 25 and said animal being susceptible. Or means for determining whether or not it has an allergic reaction, said means comprising using the reagent to identify animals that are susceptible to or have an allergic reaction to mites. Assay kit. A method for identifying an animal that is susceptible to a tick or has an allergic reaction, the method comprising:
(A) contacting the reagent of claim 25 with an animal antibody; and (b) determining an immune complex between the reagent and the antibody, wherein the formation of the immune complex comprises: Indicating that the animal is susceptible or has an allergic reaction,
A method comprising: 27. A method for desensitizing a host animal to an allergic reaction to mites, the method comprising administering to the animal a therapeutic composition comprising a desensitizing compound comprising the reagent of claim 25. A method comprising: