JP3618342B2 - Allergen of the protein of the species of Cynodon dactylon - Google Patents

Description

MAbの生産およびスクリーニング
Balb/Cマウスを50μｇのCyn ｄ Ｉ（ロトフォー［Rotofor］上で分離された、バイオ−ラド［Biorad］、カリフォルニア州リッチモンド）で腹腔内免疫化することによって、抗Cyn ｄ ＩのMAbを得た。RIBI（RIBIイムノケム（Immunochem）、米国モンタナ州ハミルトン）を、４回の免疫化の第１においてアジュバントとして使用した。残りの腹腔内免疫化は生理的食塩水の中であった。ハイブリドーマの融合および成長は、本質的にHarlowおよびLane、1988、Antibodies:A Laboratory Manual、Cold Spring Harbor Laboratoryに記載されている通りであった。単一の細胞のクローニングは制限希釈によった。ハイブリドーマ生産性抗Cynd I抗体は、ELISAアッセイにより同定した。CAPS緩衝液（6.67mM NaCO₃ 35mM NaHCO₃ pH9.6）中で希釈した30μｇのギョウギシバ（Bermuda grass）の花粉の抽出物で、ELISAプレートを一夜被覆した。次いで、ウェルをTPBS（0.1％のツイーン20を含有するPBS）で３回洗浄し、そして１％のBSAを含有するPBS（PBS/BSA）で30分間ブロックした。100μｌの一次抗体を各ウェルに添加し、そして60分間インキュベーションし、次いで洗浄し（上のように）そしてβ−gal標識化抗マウスIg（PBS/BSA中の1/250希釈、60分）中でインキュベーションした。洗浄後、200μｌの蛍光性基質の４−メチルウンベリフェリル−Ｂ−Ｄ−ガラクトシド（MUG）を各ウェルに添加し、そして37℃において30分間インキュベーションした。次いで、プレートをフルオロカウント（fluoroCount）96フルオロメーター（ファーマシア［Pharmacia］）で読んだ。
この方法により陽性であった抗体を3A2、4D2、1D1および3C2と表示し、そしてSDS−PAGEにより分割したギョウギシバ（Bermuda grass）の花粉のタンパク質のウェスタンブロット上のCyn ｄ Ｉへの結合について試験した。
cDNAライブラリーおよび免疫学的スクリーニング
本質的にHerrinおよびMichaels（1984）に記載されているようにグリール・ラボラトリーズ（Greer Laboratories）米国ノースカロライナ州レノイル、から購入したギョウギシバ（Bermuda grass）の花粉から、ポリ（Ａ＋）mRNAを分離した。cDNAをファーマシア（Pharmacia）cDNA合成既知の技術を使用して合成し、そしてベクターのラムダ−gt11のEcoR I部位の中にクローニングした。プレートしたcDNAライブラリーをIPTGで含浸したニトロセルロースのフィルターでオーバーレイすることによって、ファージのプラークからの組換えタンパク質をニトロセルロースのフィルターに移した。次いで、これらのフィルターを抗Cynd I MAbの混合物中でインキュベーションした。組換えタンパク質へのMAbの結合を前述したように可視化した。抗Cyn ｄ Ｉ MAbと結合したプラーク生成タンパク質を単離し、そして精製した。
cDNAクローンの単離
ギョウギシバ（Bermuda grass）の花粉のcDNAを、前述したように、最初に主としてMAb3.2を含有する抗Cynd Iハイブリドーマの上澄み液の混合物でスクリーニングし、そして30の陽性のcDNAをプラーク精製した。次いで、これらのクローンを抗Cyn ｄ Ｉ MAb3A2、4D2、3C2および1D1への結合について試験した。第１ラウンドのスクリーニング後に選抜したすべてのクローンは、MAb3A2に対して特異的な組換え融合タンパク質を生産した。MAbへのクローンの結合を第14図に示し、そして表２に要約した。ここで分離されたcDNAクローンは組換え融合タンパク質に対して示されたMAbの結合に基づいてC yn ｄ Ｉをエンコードすると、結論される。MAb1D1は他のMAbよりも非常に高いバックグラウンドの結合を有し、その結合を非常にいっそう主観的とした。

cDNAクローンのヌクレオチド配列およびアミノ酸配列
クローン２、３、18、21、22、23および33（表２参照）を、それらの抗体親和性に基づくそれ以上の研究のために選択した。クローン２、３、18、21、22、23および33をファージから分離し、そしてpGEM−4Z（プロメガ［Promega］）またはブルースクリプト（Bluescript）（ストラタジーン［Stratagene］）ベクターの中にサブクローニングした。T7ポリメラーゼ（ファーマシア）を使用して連鎖停止法（Sangerら、Proc.Natl.Acad.Sci.（1977）、74:5460−5463）により実施した二本鎖配列決定により、DNA配列を決定した。これらのクローンのヌクレオチド配列および推定されたアミノ酸配列を第１図（クローン２）、第２図（クローン18）、第15図（クローン３）、第16図（クローン22）および第17図（クローン23）に示す。
配列決定したすべてのクローンは、とくにオープンリーディングフレーム（ORF）において、互いとの相同性を示す。さらに、配列決定したすべてのクローンとLolp I、すなわち、主要なライグラスのアレルゲンとの間の有意なヌクレオチド配列の相同性が存在する。しかしながら、配列決定したクローンはヌクレオチドおよび推定されたアミノ酸配列の相同性に基づいて３つのグループに分割した、クローン２に最も類似する配列をもつもの（すなわち、クローン３）、クローン18に最も類似する配列をもつもの（すなわち、クローン21および33）、およびクローン22に最も類似する配列をもつもの（すなわち、クローン23）。クローン18および２のORFによりエンコードされる推定されたアミノ酸配列を、Lolp Iの推定されたアミノ酸配列と比較した（Perezら、1991、前掲;Griffithら、1991、前掲）（第６図）。Lolp Iとクローン18との間に67％およびLolp Iとクローン２との間に72％のアミノ酸の相同性が存在する。クローン２および18の推定されたアミノ酸配列は、互いに83％の同一性（87％の相同性）を有する。
実施例２
Cynd Iの５′末端のクローニング
二本鎖cDNAを、ほぼ４μｇの花粉RNA（グリール・ラボラトリーズ、米国ノースカロライナ州レノイル）から、cDNA合成システムプラスキット（BRL、米国マリイランド州ベセスダ）を使用して合成した。フェノール抽出およびエタノール沈殿後、cDNAをT4DNAポリメラーゼ（プロメガ、米国ウイスコンシン州マディソン）で平滑末端とし、そしてエタノール沈殿し、自己アニーリングした、AT,5′−GGGTCTAGAGGTACCGTCCGATC−GATCATT−３′，およびAL,5′−ｐ−AATGATCGATGCT−３′、すなわち、変更されたアンカード（Anchored）PCR（Marshら、1986;RouxおよびDhanarajan、1990;Rafnarら、1991）反応において使用のためのオリゴヌクレオチド、に結合した。１μｇのオリゴヌクレオチドAP、５′−GGGTCTAGAGGTACCGTCCG−３′，およびCD−5,5′−GATGTGCTCGTAGTTCTT−３′、すなわち、アミノ酸配列KNYEHIに相当するCynd Iの非コーディング鎖配列に基づくオリゴヌクレオチドのプライマー、の各々でリンカー結合したcDNA（20μｌの反応からの５μｌ）から、Cynd Iのアミノ末端をエンコードするcDNAを増幅した。10μｌの10×種々のdNTP含有緩衝液、１μｇの各プライマー、cDNA、0.5μｌのアンプリタク（Amplitaq）DNAポリメラーゼを含有する反応混合物中で、ジーン・アンプ（Gene Amp）DNA増幅キット（パーキン・エルマー・セツス［Perkin Elmer Cetus］、米国コネチカット州ノーウォーク）を使用して、MJリサーチ・インコーポレーテッド（Reseach,Inc.）（米国マサチュセッツ州ケンブリッジ）からのプログラム可能なコントローラーの中で、一次ポリメラーゼ連鎖反応（PCR）を実施し、そして蒸留水で100μｌにした。25ラウンドの増幅は、94℃における１分間の変性、65℃において1.5分間の鋳型へのプライマーのアニーリング、および72℃において２分間の連鎖延長から成っていた。次いで、この一次増幅の５％（５μｌ）を、１μｇの各CD−4,5′−GGGGATCCGAGGCCGT−CCTTGAAG−３′、アミノ酸IFKDGLに相当する非コーディング鎖配列に基づくCD−５に関してネスチングしたCynd Iのオリゴヌクレオチドのプライマー、およびAPを使用する二次増幅において、上のようにして、使用した。すべてのオリゴヌクレオチドはリサーチ・ジェネティックス・インコーポレーテッド（Research Genetics,Inc.）（アラバマ州ハンツビレ）により合成された。オリゴヌクレオチドのプライマーAP、ATおよびALは従来記載された（Rafnarら、1991;Morgensternら、1991;Rogersら、1991）。CD−４の最初の８ヌクレオチドを添加して、クローニングの目的のBamH I制限部位をつくった。
増幅されたDNAを順次のクロロホルム、フェノール、およびクロロホルムの抽出により回収し、次いで−20℃において0.5体積の7.5酢酸アンモニウムおよび1.5体積のイソプロパノールで沈澱させた。沈澱および70％エタノールで洗浄後、DNAを15μｌの反応混合物中でXba IおよびBamH Iで同時に消化し、そして調製用３％シープラーク（SeaPlaque）の低融点のアガロースゲル（FMCコーポレーション、米国メイン州ロックランド）を通して電気泳動させた。適当なサイズのDNAのバンドを臭化エチジウム（EtBr）の染色により可視化し、切除し、そしてセクエナーゼ（Sequenase）キット（U.S.バイオケミカルス、米国オハイオ州クレブランド）を使用するジデオキシDNA配列決定（Sangerら、（1977）、Proc.Natl.Acad.Sci.USA、74:5460−5463）のために適当に消化したM13mp19の中に結合した。２つのクローン14a1および14c1がこのライゲーションから得られ、完全に配列決定され、そしてインフレームのイニシエイターのメチオニンを含有することが発見された。14a1配列のヌクレオチド28〜30によりエンコードされるメチオニン（第６図）は、最も好ましくは、開始コドンを表す。なぜなら、取り囲む配列は共通の植物配列５′−AACAATGGC−３′（Lutckeら、（1987）EMBO J.6:43−48）と密接に合致し、そしてちょうど上流にインフレームの終止コドンが存在するからである。14c1（第７図）は２つの潜在的なインフレームのメチオニンを含有したが、ヌクレオチド27−29によりエンコードされるメチオニンはイニシエイターのメチオニンである最もある。なぜなら、取り囲む配列は、ヌクレオチド42〜45によりエンコードされるメチオニンより、コンセンサス植物配列５′−AACAATGGC−３′（Lutckeら、前掲）と密接に合致からである（78％対56％の合致）。さらに、配列の対応するヌクレオチド27〜29はクローン14alのそれと同一である。クローン14a1およびクローン14c1の両者の配列は、最長のCyn ｄ Ｉクローン、すなわち、クローン18との17ヌクレオチドのオーバーラップを有した。成熟Cynd Iのアミノ末端、クローン14a1によりエンコードされるNH2−AIGDKPGPNITATGNKWLEAKATFYGおよびクローン14clによりエンコードされるNH2−AIGDKPGPNITATGSKWLEAKATFYG−は、Cyn ｄ Ｉについて従来発表された２つのタンパク質配列:NH2−AMGDKPGP?ITATYGDKWLDAKATFYG（Matthiesenら1988,前掲Matthiesenら,1990,前掲Matthiesenら,1991,前掲）およびNH2−AIGDKPGPKITATY??KWLEAKAT（Singhら,1990,前掲）．と比較することによって同定することができた。これにより、クローン14a1および14c1は、それぞれ、22および26アミノ酸のリーダー配列を有することが示された。これらのリーダー配列を切断すると、Cyn ｄ Ｉタンパク質の成熟形態が得られるであろう。第９図に示すようにCyn ｄ I.14の配列をクローン18にそれらのオーバーラップにおいて結合することによって、Cyn ｄ I.18と表示するCyn d Ｉの潜在的な全長のアミノ酸配列（第９図）をつくった。両者の場合において、Cyn ｄ Ｉの成熟形態は、26.7kDaの計算分子量をもつ246アミノ酸であると予測される。
実施例３
ChomczynskiおよびSacchi（1987）Analyt.Biochem.162:156−159のチオシアン酸グアニジニウム法の変法を使用して、シノドン・ダクチロン（Cynodon dactylon）の花粉からRNAを分離した。9mlのチオシアン酸グアニジニウム緩衝液（0.05％トリスHCl［pH7.0］、0.05体積のβ−メルカプトエタノール、0.1容量％のナトリウムラウロイルサルコシン）を使用して液体窒素中で、花粉を粉砕した。次いで花粉の溶液をフェノール（10ml）とともに10分間震盪し、その後10mlのクロロホルム：イソアミルアルコールを添加し、そしてこの混合物をさらに20分間震盪した。この混合物を7,000×ｇで25分間遠心し、そして水性相を集めた。
水性相をフェニル：クロロホルム：イソアミルアルコール25:24:1で再抽出し、次いで界面が明瞭になるまで2,000×ｇで遠心した。次いで水性相を3mlのCsClクッション（5.7M EDTA;密度＝1.71g/ml）を下に配置したクイックシールの超遠心管の中にデカンテーションし、そしてベックマン（Beckman）Ti70.1ローター（Beckman L8−70超遠心機；ベックマン・インスツルメンツ（Beckman Instruments）、カリフォルニア州フラートン）中の遠心した（20時間、40,000rpm、20℃）。遠心後、ペレットの中のRNAを0.005％SDSの中に再懸濁させ、フェノール／クロロホルムで抽出し、そして−20℃において一夜沈澱させた。
製造業者の使用説明書に従いファーマシア（Pharmacia）mRNA精製キットを使用して、ポリA⁺RNAを分離した。
0.8μｇのmRNAを70℃に0.5μｇのオリゴ−dTプライマー（ファーマシア、ニュージャージイ州ピスカタェイ）とともに加熱することによって、第１鎖cDNAを調製した。mRNAを氷上で冷却した後、５×第１鎖結合および25UのRNアシンリボヌクレアーゼ阻害剤を添加した。次いで、この混合物を42℃に１時間加熱した。最終の反応条件は、25μｌの最終体積の50mMトリスHCl、pH8.3、50mM KCl、10mM MgCl₂、0.5mMスペルミジン、10mM DTT、4mMピロリン酸ナトリウム、1mM各dATP、dCTP、dGTPおよびTTP、25U RNアシンリボヌクレアーゼ阻害剤および15μAMV逆転写酵素／μgRNA（プロメガcDNA合成キット、プロメガ、ウイスコンシン州マディソン）であった。Cy n ｄ ＩをエンコードするcDNA配列を、パーキン−エルマー・セツス（Perkin−Elmer Cetus）の遺伝子増幅キット（U.S.バイオケミカルス、オハイオ州クレブランド）を使用して増幅した。５μｌ（25％）の第１鎖cDNA合成生産物を10×緩衝液と混合して2mM MgCl₂、50mM KCl、10mMトリス−HCl、１μｇのオリゴヌクレオチドのプライマーCD15′Ｎ、５′−GGGAATTCGCCATCGGCG−ACAAG−CCAG−３′、１μｇのオリゴヌクレオチドのプライマーCDI3′B18,5′−CCCTGCAGATG−GAGGATCATCGTCTC−３′0.2mM dNTPおよび2.5単位のTaqDNAポリメラーゼ（ファーマシア、ニュージャージイ州ピスカタェイ）の最終緩衝液濃度にした。CD15′Ｎのヌクレオチド１〜８を添加して、クローニングの目的のためのEcoR Iエンドヌクレアーゼ制限部位をつくったが、ヌクレオチド９〜27はCyn ｄ Ｉのアミノ酸１〜６（AIGDKP）をエンコードする第６図におけるクローン14a1のヌクレオチド107〜125に相当する（表１、第８図および第９図）。CD13′B18のヌクレオチド１〜８を添加して、クローニングの目的のためのPst Iエンドヌクレアーゼ制限部位をつくったが、ヌクレオチド９〜26はクローン18のヌクレオチド604〜621に対して相補的な非コーディング鎖配列に相当する（第２図）。
PCRをパーキン−エルマー・セツス（Perkin−Elmer Cetus）サーマル・サイクラー（Perkin−Elmer、コネチカット州ノーウォーク）の中で実施し、そして５サイクルの変性（94℃、１分）、アニーリング（45℃、1.5分）および延長（72℃、３分）および引き続く20サイクルの変性（94℃、１分）、アニーリング（55℃、1.5分）および延長（72℃、３分）から成っていた。最終の延長反応は72℃において10分間実施した。増幅した生産物をフェノール抽出、クロロホルム抽出、次いで−20℃において0.5体積の7.5Mの酢酸アンモニウムおよび1.5体積のイソプロパノールを使用する沈澱により回収した。反応生産物をDNAポリメラーゼのクレノー断片で平滑末端とし、次いでEcoR Iで切断し、そしてEcoR IおよびHinc IIで消化したブルースクリプト（Bluescript）ベクターの中にクローニングした。クローンCD1をジデオキシ連鎖停止法（Sanger、前掲）により、実施例１に記載するように、配列決定し、そして第18図に示すCyn ｄ Ｉのヌクレオチド配列および推定されたアミノ酸配列を含有することが発見された。
実施例４
二本鎖cDNAを調製し、そして実施例２に記載するように一次PCR反応においてオリゴヌクレオチドのプライマーCD−13およびCD−15を使用して増幅した。CD−13は配列５′−TTTCTAGAGCCATCGGCGACAAGCCAGGG−CCC−３′を有したが、ヌクレオチド14はＣまたはＧであったであろう。CD−13のヌクレオチド１〜８を加えて、クローニングの目的のためのXho I制限部位をつくった。残りのヌクレオチドはアミノ酸Ala（Ile/Met）−GlyAspLysProGlyProをエンコードし、ここでアミノ酸２はIleまたはMetであったであろう。（Cynd I aおよびCyn ｄ I bのアミノ酸１〜８（表１）。CD−15は配列5'−GCGTACTTCACGAGCAGCGCCAG−GTAATT−3'を有し、これはアミノ酸AsnTyrLeuAlaLeuLeuValLysAla（第５図におけるクローン２（C2）およびクローン３（C3）の番号を付したアミノ酸159〜168）をエンコードするコーディング鎖配列に対して相補的な非コーディング鎖配列に相当した。一次反応の５％を二次PCRにおいて、実施例２に記載するように、オリゴヌクレオチドのプライマーCD−13およびCD−16を使用して増幅した。CD−16は配列5'−TTGAATTCGACACGGCGGAACTGCAGCAT−3'を有し、ここでヌクレオチド12はＧまたはＡであったであろう。CD−16のヌクレオチド１〜８を加えて、クローニングの目的のためのEcoR I制限部位をつくった。ヌクレオチド９〜29は、アミノ酸MetLeuGlnPheArgArgVal（第５図においてC2およびC3の番号を付したアミノ酸132〜138）をエンコードするコーディング鎖配列に対して相補的な非コーディング鎖配列に相当する。
PCR増幅を実施例２に記載するように実施した。増幅した生産物を回収し、適当に消化し、そして実施例２に記載するような配列決定のためにpUCの中に結合した。C yn ｄ Ｉクローンとして同定される配列を有するKAT−39−１と表示するクローンが単離された。KAT−39−１のヌクレオチド配列および推定されたアミノ酸配列を第19図に示す。このクローンはCyn ｄ ＩのクローンC2およびC3の伸長である。オリゴヌクレオチドCD−15およびCD−16は、Cyn ｄ ＩクローンC18およびその相同体中の対応するをもつ単一の不一致をそれらの３′末端を有する。したがって、クローンC2またはC3のみか、あるいは密接な族の構成員が増幅されるであろう。Cyn ｄ I.2/3（全長）と表示するKAT−39−１およびCyn ｄ I.2/3の複合配列は、Cyn ｄ I.CD1およびCyn ｄ I.18と比較して第20図に示されている。
本発明をその好ましい態様を参照して説明したが、他の態様は同一の結果を達成することができる。本発明に対する変化および変更は当業者にとおて明らかであり、そしてすべてのこのような変更および本発明の真の精神および範囲に入る同等の態様は添付する請求の範囲の中に包含されることを意図する。 Background of the Invention
Bermuda grass (Bermuda grass)Cynodon dactylo n) Is an important source of pollen allergens in many regions of the world, especially in tropical and subtropical climates. These allergens have been studied by a number of means, which include the following: Immunoblotting of IgE (Ford D. and Baldo, BA, J. Allergy Clin. Immunol. 79: 711 -720 (1987); Shen HD et al., Clin. Allergy 18: 401-409 (1988), column chromatography (Orren, A. and Dowdle, S. Afr. Med. J. 51: 586 (1977); Matthiesen et al. J. Allergy Clin. Immunol. 81: 266 (Ab) (1988)), and immunoelectrophoresis (Matthiesen et al., Supra, 1988).
The major allergen of pollen allergen in grass grass (Bermuda grass) was identified as a protein with a molecular weight (MW) in the range of 30-34 kD, which is more than 76% of individuals allergic to grass grass (Bermuda grass) Binds to IgE from many sera (Ford and Baldo (1987) supra; Shen et al. (1988) supra, and Cynd I (Kahn and Marsh, (1986) Mol. Immunol. 23: 1281-1288; Marsh et al. (1988) Ann. Allergy, 60: 499-504, Matthiesen et al., Supra).Cyn d  I is a member of the group I family of allergens (Kahn and Marsh, (1986) supra, these allergens are a number of taxonomically related grasses such as ryegrass (Lolp I), nakahagusa (Poa p  I) and timothy (Phl p  I) (Standring et al., 1987 Intl. Arch. Allergy and Aplied. Immunol. 83: 96-103; Esch and Klapper, (1987), J. Allergy Clin. Immunol. 79: 489-495; Matthiesen and Lowenstein (1991). Clin. Exp. Allrgy, 21, 209-320, however, the allergens of Bermuda grass show limited cross-reactivity of antibodies with other grasses (March et al.). , Supra, Berstein et al. (1976) J. Allergy Clin. Immunol. 57: 141-152.Cyn d  I was shown to be different from closely related group I homologs of grasses (Matthiesen and Lowenstein (1991) supra).Cyn d  The first 27 amino acids at the N-terminus of I have been determined ((Matthiesen et al., 1988, supra; Matthiesen et al. (1990) Eptopes of Atopic Allergens, Brussel, UCB Institute of Allergu, 9-13; Singh et al., Monographs in Allergy (1990), 28: 101-120; Matthiesen and Lowenstein, (1991), supra).
The presence of pollen allergens of Bermuda grass in the environment causes hay fever and seasonal asthma in many individuals and continues to have a significant socio-economic impact on Western societies. The available spectrum of drugs, including antihistamines and steroids, has led to improvements in the treatment of allergic diseases, but has practically adverse side effects associated with long-term use. These problems have regained interest in immunotherapy of allergic diseases. Immunotherapy requires injecting allergen extracts to desensitize the patient to allergic reactions (Bousquet, J. and Michel, FB (1989) Allergy and Clin Immol. News 1: 7-10). Unfortunately, pollen preparations used as allergens are multivalent and inferior in quality, eventually crude extracts are frequently used at high concentrations and are potentially lethal including anaphylaxis Product expressed from synthetic peptides based on cloned genes, fragments thereof or allergen sequences can be quality controlled, characterized and standardized, and optimally Does not bind IgE, thus providing a safe medium for treatment.
Summary of invention
The present invention relates to allergens of the major proteins of the Cynodon dactylon species (Cyn d  I), or a nucleic acid sequence encoding at least one fragment thereof or a functional equivalent of such a nucleic acid sequence. The present invention also provides expression vectors comprising such nucleic acid sequences and host cells transformed with them. The invention is further produced in isolated, recombinant, chemical or syntheticallyCyn d  I or a fragment thereof is provided. IsolatedCyn d  I or fragments thereof are useful for diagnosing and treating susceptibility of individuals in Bermuda grass to pollen allergens.
[Brief description of the drawings]
Figure 1 was derived from a cDNA clone designated as clone 2 (C2).Cyn d  A nucleotide sequence encoding I andCyn d  1 shows the deduced partial amino acid sequence of I.
FIG. 2 was derived from a cDNA clone designated clone 18 (C18).Cyn d  2 shows the partial nucleotide sequence encoding I and the deduced partial amino acid sequence of Cynd I.
FIG. 3 shows a comparison of the nucleic acid sequences of clones 2 and 18.
FIG. 4 shows a comparison of the deduced amino acid sequences of clones 2 and 18.
FIG.Cyn d  7 clones encoding I: estimation of clone 18 (C18), clone 22 (C22), clone 23 (C23) clone 2 (C2), clone 3 (C3), clone 21 (C21), and clone 33 (C33) A comparison of the resulting amino acid sequences is shown.
FIG. 6 was derived from a cDNA clone designated clone 14a1.Cyn d  A partial nucleotide sequence encoding I andCyn d  1 shows the deduced partial amino acid sequence of I.
FIG. 7 shows a partial nucleotide sequence encoding a partial and deduced partial amino acid sequence derived from a cDNA clone designated clone 14c1.
FIG. 8 was predicted from the complex of clones 14a1 and 14c1Cyn d  Display as I.14Cyn d  The partial amino acid sequence of I is shown.
Figure 9Cyn d  Display as I.18Cyn d  1 shows the predicted full-length amino acid sequence of I.
Figure 10Cyn d  Display as I.2 / 3Cyn d  1 shows the predicted partial amino acid sequence of I.
FIG. 11a shows the SDS-PAGE separation of protein fractions obtained by primary preparative isoelectric focusing (IEF) of these proteins on Rotofor.
FIG. 11b shows a Western blot of isolated protein screened with MAb 3.2.
FIG. 12a shows the separation by SDS-PAGE of the protein fraction obtained by re-fractionation on the Rotofor of fractions 10-13 pooled from the crude pollen extract.
FIG. 12b shows the separation by SDS-PAGE of the protein fraction obtained by re-fractionation on the Rotofor of fractions 15-20 pooled from the crude pollen extract.
FIG. 13 is a diagram of natural, isolated by SDS-PAGE and probed with IgE from the serum of an allergic individual of Bermuda grass.Cyn d  I a andCyn d  Western blot of Ib is shown.
Figure 14Cyn d  The binding of MAbs 1D1, 3A3, 3C2 and 4D2 to cDNA from the Iλgt11 library is shown. The number on the overlay corresponds to the number of cDNA clones.
FIG. 15 was derived from a cDNA clone designated as clone 3.Cyn d  A partial nucleotide sequence encoding I andCyn d  1 shows the deduced partial amino acid sequence of I.
FIG. 16 was derived from a cDNA clone designated clone 22Cyn d  A partial nucleotide sequence encoding I andCyn d  1 shows the deduced partial amino acid sequence of I.
FIG. 17 was derived from a cDNA clone designated clone 23Cyn d  A partial nucleotide sequence encoding I andCyn d  1 shows the deduced partial amino acid sequence of I.
Figure 18 was derived from a full-length cDNA clone designated CD1.Cyn d  The nucleotide sequence of I and the deduced amino acid sequence are shown.
FIG. 19 was derived from a cDNA clone designated KAT-39-1.Cyn d  The partial nucleotide of I and the deduced amino acid sequence are shown.
Figure 20Cyn d  I.18,Cyn d  I.CD1 andCyn d  Display as I.2 / 3 (full length)Cyn d  A comparison of the predicted full-length amino acid sequences of the I mature protein is shown.
Detailed description of the invention
The present inventionCyn d  I, ie, a nucleic acid sequence encoding the major allergen found in pollen of Bermuda grass, or a functional equivalent thereof.Cyn d  I appears to be a closely related allergen family. As defined herein, an “allergen family” is a protein that is related in function and amino acid sequence, but encoded by genes at separate loci. Members of each family can have polymorphisms where nucleotide variations can occur at a given locus. Nucleic acid sequence polymorphisms can result in amino acid polymorphisms, but this is not always the case when the code for the nucleotide encoding the amino acid is degenerate.Cyn d  The nucleic acid sequence encoding I may vary among individual plants of Bermuda grass due to natural allelic variations. Any and all such nucleotide variations and resulting amino acid polymorphisms are encompassed within the scope of the invention.
Derived from a cDNA clone designated clone 2;Cyn d  The partial nucleic acid sequence encoding I has the sequence shown in FIG. As shown in FIG.Cyn d  The partial nucleic acid sequence encoding I consists of 435 bases. The 3'-untranslated region starts at base 436 and extends to base 662. Encoded by clone 2 (C2)Cyn d  The deduced partial amino acid sequence of I is also shown in FIG.
FIG. 2 shows the partial nucleic acid sequence and deduced amino acid sequence for the second cDNA clone, designated clone 18 (C18). Shown in Figure 2Cyn d  The nucleic acid sequence encoding I consists of 600 nucleotides encoding 200 putative amino acids. The 3'-untranslated region starts at base 601 and extends to base 775.
As shown in FIG. 3, the coding sequences for clone 2 and clone 18 are clearly homologous, but the 3'-untranslated region is much more divergent. As this suggests, clones 2 and 18 areCyn d  Separate members of the I gene family can be encoded.
As shown in FIG. 4, the deduced amino acid sequence encoded by clone 2 and clone 18 has 88.2% homology (84.1% identity). There are 22 amino acid differences in the 143 amino acid overlap estimated from the two clones, 6 of which are conservative substitutions and 16 are non-conservative substitutions. The partial protein encoded by clone 18 is 2 amino acids longer at the carboxy terminus than the partial protein encoded by clone 2 (FIG. 4). Amino acid homology was demonstrated using software contained in PCGENE (Intelligenetics, Mountain View, Calif.).
Described in Example 1Cyn d  A comparison of the deduced amino acid sequences encoded by seven cDNA clones derived from the I library is shown in FIG. The amino acid sequence encoded by these cDNA clones labeled C2, C3, C21, C22, C23 and C33 isCyn d  I shown aligned with the deduced amino acid sequence encoded by clone 18 (C18), the longest clone derived from the cDNA library. As shown in FIGS. 5 and 6, the overlapping portions of the amino acid sequences encoded by clones 18, 22, 23, 21 and 33 are identical. As this suggests, clones 18, 22, 23, 21 and 23 are identical.Cyn d  An example of a member of the I gene family. However, clones 22 and 23 are shorter amino acids than clone 18 and have a different 3'-untranslated region (Fig. 2, Fig. 16 and Fig. 17). This is because clones 22 and 23Cyn d  It may be suggested to represent separate members of the I gene family. Alternatively, they may represent differently spliced forms of the same family member.
As shown in FIG. 5, there are only 5 amino acids between the deduced amino acid sequences encoded by clones 2 and 3. Therefore, clones 2 and 3Cyn d  May represent polymorphism of members of the I gene familyCyn d  Members of the I gene family belong to clones 18, 21 and 33Cyn d  Different from members of the I gene family. Clones 2 and 3 areCyn d  Assuming that the polymorphisms of the members of the I gene family are represented in fact, they are shown in Figure 10.Cyn d  Display as I.2 / 3Cyn d  The predicted partial amino acid sequence of I can be generated from the amino acid sequence encoded by clones 2 and 3.
FIG. 6 shows the nucleotide sequence of cDNA clone 14a1 and its deduced amino acid sequence. This clone is isolated from PCR as described in Example 2, and the amino acid sequence it encodes is partially encoded by clone 18.Cyn d  Corresponds to the amino moiety of a Group I member. There is a 19 nucleotide overlap between the 3 'end of clone 14a1 and the 5' end of clone 18. Clone 14a1 was amplified in PCR using oligonucleotide primers based on the non-coding strand sequence of clone 18, as described in Example 2. The methionine encoded by nucleotides 41-43 of clone 14a1 is presumed to represent the first amino acid of the translated protein. This is the first methionine encoded after the in-frame stop codon at nucleotides 11-13 of clone 14a1, and the initiation of protein translation does not occur 5 'of methionine encoded by nucleotides 41-43 of clone 14a1 Indicates. The nucleotide sequence surrounding the putative initiator methionine is 78% consistent with the consensus sequence 5'AACAATGGC-3 '(Lutcke et al., 1987, EMBO J. 6: 43-48) for protein initiation in plants. MatureCy n d  A 22 amino acid leader sequence is present before the start of the N-terminus of the I protein (indicated by amino acid 1 in FIG. 6)Cyn d  The N-terminus (first 27 amino acids) of the I protein has already been identified (Matthiesen et al., 1988, J. Allergy Clin. Immunol. 81: 226 Shing et al., 1990, Monogr. Allergy, 28: 101-120; Matthiesen et al., 1991, J. Allergy Clin. Immunol. 88: 763-774).
FIG. 7 shows the nucleotide sequence of cDNA clone 14c1 and its deduced amino acid sequence. This clone is also isolated from PCR as described in Example 2, and the amino acid sequence encoding it is partially encoded by clone 18.Cyn d  Corresponds to the amino moiety of a Group I member. This clone is homologous to clone 14a1, but has one amino acid difference from clone 14a1 in the sequence of mature tyrosine phosphatase (matureCyn d  The N-terminus of I tyrosine phosphatase is indicated by amino acid 1 in FIG. 7). Clone 14c1 has a nucleotide difference in the leader sequence encoding 7 amino acids that differs from clone 14a1, including an insert of 12 nucleotides encoding an additional 4 amino acids. The composite sequences of 14a1 and 14cl, including the potential polymorphisms of these clones, are shown in FIG.Cyn d  Display in I.14.
The sequences of clones 14a1 and 14c1 areCyn d  Useful in the generation of predicted full-length nucleic acid sequences encoding I.Cyn d  The predicted full-length nucleotide sequence encoding I has the formula:
L₁NYX
Where L can be derived from₁Is a nucleic acid sequence of 0-300 nucleotides,Cyn d  Contains nucleotides encoding the leader sequence of the I protein and can also contain 5'-untranslated region nucleotides, N is a nucleic acid sequence of up to 600 nucleotides and matureCyn d  Contains nucleotides encoding the amino terminal portion of I, Y is clone 2, clone 18, clone 3, clone 22 or clone 23 or matureCyn d  Part of the nucleic acid sequence of any polymorphic form of those clones encoding the I protein, and X is a nucleic acid sequence of 0 to 600 nucleotides, said nucleic acid sequence beingCyn d  Contains the nucleotide of the 3 'untranslated portion of I. For example, L₁Contains the 5'-untranslated region of clone 14a1, nucleotides 1 to 106 of clone 14a1 shown in FIG.Cyn d  The nucleic acid sequence represented by the nucleotides of clone 14a1 encoding the leader sequence of I (nucleotides 41-106 shown in FIG. 6) can be included. L₁Also includes nucleotides 1 to 103 of clone 14c1 shown in FIG. 7 including the 5′-untranslated region of clone 14c1 andCyn d  The nucleic acid sequence represented by the nucleotide of clone 14c1 (nucleotides 28-103 shown in FIG. 7) encoding the leader sequence of I can be included. L₁Is alsoCyn d  Clone 14a1 nucleotides (nucleotides 41 to 106 shown in FIG. 6) encoding only the leader sequence portion of I orC yn d  It can be a nucleic acid sequence comprising the nucleotide of clone 14c1 (nucleotides 28-103 shown in FIG. 7) encoding only the leader sequence portion of I or any polymorphic form thereof. One is matureCyn d  When generating a nucleic acid sequence encoding I, L₁Is 0 and X is 0 and the formula is simply NY. N is preferably full length or matureCyn d  A nucleic acid sequence encoding the I protein.
As shown in FIG.Cyn d  Display as I.18Cyn d  The predicted full-length amino acid sequence for I isCyn d  The amino acid sequence shown in FIG. 8 labeled I.14 can be generated by conjoining amino acid residues 53 to 246 of clone 18 shown in FIG. The predicted complex of the mature protein is in this case shown in FIG.Cyn d  It consists of amino acids 1 to 246 of I.18 and will have a predicted molecular weight of approximately 26.7 kDa without post-translational changes. As used herein, “maturity”Cyn d  I proteinCyn d  Does not include the amino acid sequence of the leader portion of the I protein. Polymorphisms or potential polymorphisms are indicated by superscripts and subscripts in all applicable drawings discussed herein.
PCR was used to generate full-length clones as described in Example 3 and shown in FIG. Using an oligonucleotide primer based on nucleotides 107-125 of clone 14a1 (FIG. 6) and nucleotides 604-621 of clone 18 (FIG. 2), the full-length clone shown in FIG. 18 and designated as clone CD1 Was generated from PCR. The deduced amino acid sequence of clone CD1 excludes the 2 amino acid sequence as described above and as shown in FIG.Cyn d  Display as I.18Cyn d  Corresponds to the full-length amino acid sequence of the predicted complex of members of the I protein family. The deduced amino acid sequence of clone CD1 shown in FIG. 18 and FIG.Cyn d  I.CD1 is displayed.Cyn d  I.CD1 is shown in Fig. 9.Cyn d  Substantially identical to the predicted composite sequence represented by I.18Cyn d  I protein. Host cells transformed with a vector consisting of the cDNA insert of clone CD1 are assigned accession numbers to ATCC. It was commissioned.
Cyn d  The amino acid sequence of the full length of another predicted complex labeled I.2 / 3 (full length) is shown in FIG. A portion of this sequence includes nucleotides 178-206 (identical to the corresponding nucleotide sequence of clone 3 (FIG. 15)) of clone 2 (FIG. 1) and nucleotides 107-130 of clone 14a1 (FIG. 6). Generated from PCR using oligonucleotide primers based on essentially the same nucleotidesCyn d  Inferred from the I clone. This clone is designated as clone KAT-39-1. The deduced amino acid sequence of clone KAT-39-1 is shown in FIG.Cy n d  Overlaps with part of the predicted partial amino acid sequence of I.2 / 3Cyn d  1 represents the partial amino acid sequence of I. Therefore, the nucleic acid sequence and the deduced amino acid sequence of clone KAT-39-1Cyn d  The combined sequence formed by combining the I.2 / 3 nucleic acid sequence and the deduced amino acid sequence is shown in FIG.Cyn d  Predicted to display I.2 / 3 (full length)Cyn d  1 represents the nucleic acid sequence and deduced amino acid sequence of a complex of members of the family I protein. Figure 20Cyn d  I.18 andCyn d  The amino acid sequence of the composite sequence labeled I.2 / 3 (full length), andCyn d  I. Comparison of the full-length amino acid sequence deduced from the full-length cDNA clone labeled CD1, CD1.
The aboveCyn d  Nucleic acid encoding allergen of protein ICynodon dac tylon) Can be obtained from any part of the plant.C yn d  The nucleic acid encoding I can be obtained from genomic DNA.Cyn d  Nucleic acids encoding I can be obtained using the methods disclosed herein or other suitable techniques for gene isolation and cloning.
Cyn d  Fragments of nucleic acid sequences that encode fragments of I are also within the scope of the invention. Fragments within the scope of the present invention are immune responses in mammals, preferably humans, such as stimulation of minimal amounts of IgE; induction of IgG and IgM antibody production; or T cell responses, such as induction of proliferation and / or Or induce lymphokine secretion and / or induction of T cell anergyCyn d  Includes a fragment encoding part I.Cyn d  The aforementioned fragment of I is referred to herein as an antigenic fragment. Fragments within the scope of the present invention are alsoCyn d  Includes fragments that can hybridize with nucleic acids from other plant species for use in screening protocols to detect allergens that are cross-reactive with I. As used herein,Cyn d  A fragment of the nucleic acid sequence encoding ICyn d  I and / or maturityCyn d  A nucleotide sequence having fewer bases than the nucleotide sequence encoding the entire amino acid sequence of I is referred to. In general,Cyn d  The nucleic acid sequence encoding one or more fragments of I will be selected from the bases encoding the mature protein, but in certain cases, one or more than one from the leader sequence portion of the nucleic acid sequence of the invention It is desirable to select all or part of the fragment. The nucleic acid sequences of the invention also include linker sequences, modified restriction endonuclease sites andCyn d  Other sequences useful for cloning, expression or purification of I or fragments thereof can be included.
The present invention provides expression vectors and host cells transformed to express the nucleic acid sequences of the present invention.Cyn d  The nucleic acid encoding I, or at least one fragment thereof, is a bacterium such as E. coli (E.coli), Insect cells (baculovirus), yeast, or mammalian cells such as Chinese hamster ovary cells (CHO). Suitable expression vectors, promoters, enhancers, and other expression control elements can be found in Sambrook et al., Molecular C1oning: A Laboratory Manua1, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989). Can be found. Other suitable expression vectors, promoters, enhancers, and other expression elements are known to those skilled in the art. Expression in mammalian, yeast or insect cells leads to partial or complete glycosylation and interchain or intrachain of the recombinant material. Suitable vectors for expression in yeast are YepSec1 (Baldari et al. (1987) EMBO J. 6: 229-234); pMFa (Kurjan and Herskowitz (1982) Cell 30: 933-943); JRY88 (Schultz et al. ( 1987) Gene 54: 113-123) and pYES2 (Invitrogen Corporation, San Diego, Calif.). These vectors are freely available. Baculovirus and mammalian expression systems are also available. For example, the baculovirus system is commercially available for expression in insect cells (PharMingen, San Diego, Calif.), While the pMSG vector is commercially available for expression in mammalian cells. (Pharmacia, Piscathay, NJ).
For expression in E. coli, suitable expression vectors are, among others, pTRC (Amann et al. (1988) Gene 69: 301-315; pGEX (Amrad Corporation, Melbourne, Australia). PMAL (NE Biolabs, Bebery, Mass.)); PRIT5 (Pharmacia, Piscathay, NJ); pET-11d (Novagen, Madison, Wis.) Jammeel et al. (1990) J. Virol. 64 : 3963-3966; and pSEM (Knapp et al. (1990) Bio Techniques 8: 3963-3966). For example, the use of pTRC and pET-11d will lead to the expression of non-fusion proteins. The use of pMAL, pRIT5, pSEM and pGEX can be used to express allergens fused to maltose E binding protein (pMAL), protein A (pRIT5), truncated β-galactosidase (PSEM), or glutathione S-transferase (pGEX). Will lead.Cyn d  I, when one or more fragments thereof are expressed as a fusion protein,Cyn d  It is particularly advantageous to introduce an enzyme cleavage site at the fusion junction with I or a fragment thereof. ThenCyn d  I or a fragment thereof can be recovered from the flavin protein by enzymatic cleavage at the enzyme site and biochemical purification using conventional techniques of protein and peptide purification. Suitable enzymatic cleavage sites include sites for blood clotting factor Xa or thrombin, and suitable enzymes and protocols for cleavage for these sites are described, for example, by Sigma Chemical Company, St. Louis, Missouri. And NE Biolabs, commercially available from Bevelly, Massachusetts. Different vectors may also be used, for example, for induction of IPTG (PRTC, Amann et al. (1988) supra; pET-11d, Novagen [Novagen] Madison, Wis.) Or temperature induction (pRIT5, Pharmacia, New Jersey). It has different promoter regions that allow constitutive or inducible expression with Piscatay). It may also be appropriate to express recombinant Cynd I in different E. coli hosts with altered ability to degrade recombinantly expressed proteins (eg, US Patent No. 4,758,512). Alternatively, it may be advantageous to alter the nucleic acid sequence to use codons preferentially utilized by E. coli, where such nucleic acid alteration affects the amino acid sequence of the expressed protein. Will not give.
Host cells can be transformed to express the nucleic acid sequences of the invention using conventional techniques, such as calcium phosphate or calcium chloride coprecipitation, DEAE-dextran mediated transfection, or electroporation. Suitable methods for host cell transformation can be found in Sambrook et al., Supra, and other laboratory texts. The nucleic acid sequences of the invention can also be synthesized using standard techniques.
The present invention also includes a process:Cyn d  A host cell transformed with a DNA sequence encoding I or at least one fragment thereof is cultured in a suitable medium andCy n d  I or a medium containing at least one fragment thereof is produced; and the mixture is purified to obtain substantially pureCyn d  I or at least one fragment thereof;Cyn d  A method of producing I or at least one fragment thereof is provided.Cyn d  A host cell transformed with an expression vector containing DNA encoding I or at least one fragment thereof is cultured in a medium appropriate for the host cell.Cyn d  I proteins and peptides can be prepared using techniques known in the art for peptide and protein purification, such as ion exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis andCyn d  Immunopurification using antibodies specific for I or fragments thereof can be used to purify from cell culture media, host cells or both. The terms isolated and purified are used interchangeably herein and are substantially free of cellular material or medium when produced by recombinant DNA techniques, or chemical precursors, or chemically synthesized. As such, it refers to peptides, proteins, protein fragments, and nucleic acid sequences that are substantially free of other chemicals. Thus, the isolated peptides of the present invention are produced by recombinant DNA technology or chemically synthesized and are substantially free of cellular material, media, chemical precursors or other chemicals.
Another aspect of the present invention is:Cyn d  Synthesized in a host cell transformed with a DNA sequence encoding all or part of I or chemically synthesizedCyn d  Purified or chemically synthesized in a host cell transformed with I or at least one fragment thereof and a nucleic acid sequence of the inventionCyn d  A preparation consisting of the I protein or at least one fragment thereof is provided. In a preferred embodiment of the invention,Cy n d  I protein is at least matureCyn d  Produced in host cells transformed with the I protein.
Cyn d  Fragments of I can be obtained, for example, by screening peptides synthesized from corresponding fragments of the nucleic acid sequences of the present invention that encode the peptides or chemically synthesized using techniques known in the art. Can get. Peptide fragments of allergens can be obtained by selecting fragments of the desired length that do not have peptide overlap, or by selecting overlapping fragments of the desired length that can be produced recombinantly or synthetically. Can be obtained. Fragments can be tested to determine antigenicity (eg, the ability of the fragment to elicit an immune response). Such a fragment is referred to herein as an antigenic fragment. T cell responses, eg, stimulation (ie proliferation or lymphokine secretion) can be induced and / or T cell anergy can be induced.Cyn d  Allergen fragments of the I protein are particularly desirable. Does not bind to immunoglobulin E (IgE) or binds to IgE to a degree substantially less than the allergen of the protein from which the fragment is derivedCyn d  The fragment of I is also particularly desirable. The main complication of standard immunotherapy is a systemic response, such as anaphylaxis. Immunoglobulin E is a mediator of anaphylactic reactions resulting from antigen binding and cross-reactivity to IgE on mast cells or basophils and release of mediators (eg, histamine, serotonin, eosinophils, chemotactic factors). Thus, anaphylaxis can be avoided by using fragments that do not bind to IgE, or if the fragment binds to IgE, such binding is a mediator from mast cells or basophils (eg, histamine, etc.) Resulting in liberation. Moreover, fragments with minimal IgE stimulating activity are particularly desirable for therapeutic effects. Minimal IgE stimulating activity refers to IgE stimulating activity that is less than the production of IgE stimulated by allergens of the protein of whole grass grass. Preferred fragments of the present invention include, but are not limited to, the following: as shown in FIG.Cyn d  A fragment derived from amino acids 5-246, 10-246, 20-246 and 25-246 of I.18; and as shown in FIG.Cyn d  I. Fragments derived from amino acids 5-246, 10-246, 20-246 and 25-246 of CD1; shown in FIG.Cyn d  I. A fragment derived from 2/3 (full length) of amino acids 5-244, 10-244, 20-244 and 25-244.
Cyn d  I and preferred antigenic fragments thereof can alter an individual's allergic response to an allergen when administered to an individual susceptible to pollen in Belgian grass, and preferably the individual's B cells, T, to the allergen The cellular response or the response of both B and T cells can be altered. As used herein, allergens of pollen protein of the grass grass, for example,Cyn d  Changes in the allergic response of individuals susceptible to I are determined by standard clinical procedures (see, eg, Varney et al., British Medical Journal 302: 265-269), and are non-responsive or symptomatic to allergens. For example, a reduction in asthma symptoms induced by pollen of grass grass (Bermuda grass). As referred to herein, reducing symptoms includes reducing symptoms in an individual's allergic response to an allergen after a regimen of treatment using a protein or peptide of the invention. This reduction in symptoms can be determined subjectively (ie, the patient feels more comfortable when exposed to allergens) or clinically, for example, using standard tests Can do.Cyn d  Initial screening for IgE binding to I or fragments thereof can be by scratch or intradermal skin tests on laboratory animal or human volunteers, or by in vitro systems such as RAST (Radioallergic Saw Radioallergosorbent test), RAST inhibition, ELISA assay, radioimmunoassay (RIA), or histamine release.
Antigenic fragments of the present invention having T cell stimulating activity and consisting of at least one T cell epitope are particularly desirable. T cell epitopes are believed to be involved in the prevention or perpetuation of immune responses to protein allergens that cause clinical symptoms of allergies. These T cell epitopes are thought to trigger early events at the level of T helper cells by binding to the appropriate HLA molecules on the surface of antigen presenting cells and stimulating a subpopulation of related T cells. It is done. These events lead to T cell proliferation, lymphokine secretion, local inflammatory response, recrementation of additional immune cells to the site, and activation of the B cell cascade leading to antibody production. One isotype of these antibodies, IgE, is fundamentally important for the development of allergic symptoms, and its production depends on the nature of the secreted lymphokines in the cascade of events, at the level of T helper cells. , Affected early. A T cell epitope is a basic element or smallest unit of recognition by a T cell receptor, where the epitope consists of amino acids essential for receptor recognition and is adjacent and / or non-contiguous in the amino acid sequence of the protein. Can be adjacent. Amino acid sequences that mimic the amino acid sequence of a T cell epitope and alter the allergic response to a protein allergen are within the scope of the invention.
Of the invention comprising at least one T cell epitopeCyn d  Patient exposure to I or antigenic fragments tolerates an appropriate subpopulation of T cells so that the patient becomes unresponsive to protein allergens and does not participate in stimulating the immune response upon such exposure. Or it can be anergy. Further, the present invention comprises at least one T cell epitope.Cyn d  Administration of I or an antigenic fragment can alter the secretion profile of lymphokine compared to exposure to a naturally found protein allergen or a portion thereof (eg, decreased IL-4 and / or IL -2 increase). further,Cyn d  Exposure to I or such antigenic fragments can affect a subpopulation of T cells that normally participate in the response to allergens, so that these T cells are one or more of the normal exposures to allergens. Are attracted towards one or more sites of therapeutic administration of the fragments away from the site (eg, nasal mucosa, skin, and lungs). This redistribution of T cell subpopulations can reduce or reduce the individual's immune system's ability to stimulate a normal response at the site of normal exposure to allergens.
Cyn d  I and fragments or portions derived from it (peptides) can be used in methods of diagnosing, treating and preventing an allergic response to pollen in Bermuda grass. Thus, the present invention has been separatedCyn d  A therapeutic composition is provided comprising I or at least one fragment thereof and a pharmaceutically acceptable carrier or diluent.Cyn d  I or at least one fragment thereof is preferably produced in a cell transformed to express an allergen of the protein or fragment thereof, or is produced synthetically. Administration of the therapeutic composition of the present invention to the individual to be desensitized can be performed using known techniques.Cyn d  I or a fragment thereof can be administered to an individual in combination with, for example, a suitable diluent, carrier and / or adjuvant. Pharmaceutically acceptable diluents include physiological saline or aqueous buffer. Pharmaceutically acceptable carriers include polyethylene glycol (Wie et al. (1981) Intl. Arch. Allergy and Aplied. Immunol. 64: 84-99) and liposomes (Strejan et al. (1984) J. Neuroimmunol. 7:27). Is included. For the purpose of inducing T cell anergy, the therapeutic composition is preferably administered in a non-immunogenic form, for example, it does not contain an adjuvant. Such compositions will generally be administered by injection (such as subcutaneously or intravenously), oral administration, inhalation, transdermal application, or rectal administration. The therapeutic composition of the present invention reduces an individual's susceptibility to pollen (Bermuda grass) pollen (i.e., reduces allergic response) in a regimen of treating individuals sensitive to pollen (Bermuda grass) pollen. Effective dose) and over time. The effective amount of the therapeutic composition is determined by factors such as the degree of sensitivity of the individual to pollen of grass grass, age, sex, and body weight of the grass, and pollen allergen or fragment thereof of grass grass Will vary according to the ability to elicit an antigenic response in the individual.
Cyn d  The cDNA encoding I (or the mRNA that transcribed it) or a portion thereof is used to identify similar sequences in any of various or types of plants, and thus a protein allergen cDNA or mRNA or its Sequences with sufficient homology to hybridize to a portion can be identified or used to “select”. For example, the cDNA of the present invention has low stringency in DNA from temperate grasses such as ryegrass, nakahasa, timothy and camogaya, and DNA from other grasses such as Bahia grass. Hybridization can be performed under conditions. Sequences with sufficient homology (generally greater than 40%) can be selected for other evaluations using the methods described herein. Alternatively, high stringency conditions can be used. In this method, using the DNA of the present invention in other types of plants, preferably related families, genera or species,Cyn d  Sequences encoding polypeptides having an amino acid sequence similar to the amino acid sequence of I can be identified, and thus allergens in other species can be identified. Thus, the present invention is an allergen of Bermuda grass.Cyn d  In addition to I, it also includes other allergens encoded by DNA that hybridize to the DNA of the present invention. The invention further encompasses isolated allergens or fragments thereof that exclude protein allergens or fragments thereof from the genus Lolium, which include, for example, antibody cross-reactivity or protein allergens. Or fragments thereof of the present invention.Cyn d  By other immunological assays capable of binding to antibodies specific for I or fragments, or isolated antigenic proteins or fragments thereof stimulate T cells specific for the proteins and peptides of the invention T cell cross-reactivityCyn d  Immunologically related to I or a fragment thereof. The present invention also providesCyn d  Have greater than 73% homology with I, orCyn d  Allergens of proteins having a homology greater than 90% with I or fragments thereof.
The protein or peptide encoded by the cDNA of the present invention can use, for example, a “purified” allergen. Such purified allergens are useful in the standardization of allergen extracts, which are the primary reagents for diagnosis and treatment of sensitivity to pollen in bermuda grass. further,Cyn d  By using proteins based on the nucleic acid sequence of I or fragments thereof, anti-peptide antisera, polyclonal antibodies or monoclonal antibodies can be made by standard methods. These sera or polyclonal or monoclonal antibodies can be used to standardize allergen extracts or can be used in the purification of natural or recombinant protein allergens.
Cyn d  By using I and synthetic or recombinantly produced and isolated antigenic fragments thereof, a consistent, well-defined composition and biological activity preparation is made and for therapeutic purposes (E.g., to change the allergic response sensitive to pollen in grass grass. Administration of such peptides or proteins can include, for example,Cyn d  B cell response to I,Cyn d  The response of T cells to I or both responses can be altered. The isolated peptides can also be used to study the mechanism of immunotherapy for pollen allergy in Bermuda grass and to design modified derivatives or analogs useful in immunotherapy.
For purposes of increasing solubility and enhancing therapeutic or prophylactic efficacy, or stability (eg, ex vivo shelf life, and resistance to proteolytic degradation in vivo)Cyn d  The structure of I or a fragment thereof can be modified. The amino acid sequence has been modified, for example, altered by amino acid substitutions, deletions to reduce immunogenicity and / or allergenicity, or with the addition of components of the same purposeCyn d  I or modified fragments thereof can be produced. For example, amino acid residues essential for the function of a T cell epitope can be determined by known techniques (eg, measuring the presence or absence of each residue substitution and T cell reactivity). Residues that have been shown to be essential can be modified (eg, substitution with other amino acids whose presence has been shown to enhance T cell reactivity), as well as to T cell reactivity. Undesired residues can be altered (eg, by replacing with other amino acids whose incorporation enhances T cell reactivity but does not reduce binding to the relevant MHC). To enhance stability and / or reactivity,Cyn d  I or a fragment thereof can also be modified to incorporate one or more polymorphisms into the amino acid sequence of a protein allergen derived from a variant of a natural allele. In addition, D-amino acids, unnatural amino acids or non-amino acid analogs can be substituted or added to produce modified proteins or fragments within the scope of the present invention. further,Cyn d  I or fragments thereof can be modified by the polyethylene glycol (PEG) method of A. Sehon and co-workers (Wie et al., Supra) to produce peptides conjugated to PEG.Cyn d  Modifications of I or fragments thereof may also be reduced / alkylated (Tarr: Methods of Protein Microcharacterization, edited by JESilver, Humana Press, Clifton, NJ, pp. 155-194 (1986)); acylation (Tarr, Esterification (Tarr, supra); Chemical coupling to a suitable carrier (Mishell and Shiigi, Ed., Selected Methods in Cellular Immunology, WH Freeman, San Francisco, Calif. (1981); US Pat. No. 4,939,239); or Includes mild formalin treatment (Marsh, International Archieves of Allergy and Applied Immunology 41: 199-215 (1971)).
Cyn d  Site-directed mutagenesis of DNA encoding I or a fragment thereof can be used to modify the structure. Such methods include PCR (Ho et al., Gene 77: 51-59 (1989)) or total synthesis of mutated genes (Hostomsky, Z. et al., Biochem. Biophys. Res. Comm. 161: 1056-1063). (1989)). To enhance bacterial expression, the above method is used in combination with other procedures to replace the plant codon in the DNA construct encoding the peptide with E. coli (E.coli) Can be replaced with the one used preferentially.
Cynd I peptide that alters an individual's response to pollen of beetle grass (Bermuda grass) when administered in sufficient quantities to individuals sensitive to pollen of beetle grass (Bermuda grass) using currently available structural information Can be designed. This is, for example,Cyn d  Produce peptides to be examined for their ability to influence the B cell and / or T cell response in individuals susceptible to pollen sensitivity to pollen of Bermuda grass (through an expression system or synthesized) And) by selecting the appropriate B or T cell epitope recognized by the cell. The proteins, peptides or antibodies of the invention can also be used to detect or diagnose the sensitivity of pollen allergy to bermuda grass pollen. For example, this can be done as follows: blood or blood products from an individual to be assessed for sensitivity to pollen in Bermuda grass,Cyn d  I isolated antigenic fragment, or isolatedCyn d  Combining I with components in the blood (eg, antibodies, T cells, B cells) and one or more fragments or proteins under appropriate conditions and the extent to which such binding occurs decide.
Currently, it also triggers an allergic response in individuals susceptible to pollen in grass grass (Bermuda grass)Cy n d  It is possible to design factors or drugs that can block or inhibit the ability of I. Such factors are, for example, the resistances with which they relate.Cyn d  It can be designed in such a way as to bind to I-IgE, thus preventing IgE-allergen binding and subsequent degranulation of mast cells. Alternatively, such factors can bind to cellular components of the immune system, resulting in suppression or desensitization of the allergic response to pollen in Bermuda grass. This non-limiting example is an allergic response to pollen in grass grass using suitable B and T cell epitope peptides, or modifications thereof, based on the cDNA / protein constructs of the present invention. It is to suppress. This is done in an in vitro study using blood components from individuals sensitive to pollen in grass grass by defining the peptide structure of the T cell epitopes that affect B and T cell function. can do.
The DNA used in any embodiment of the present invention can be a cDNA obtained as described herein, or any oligonucleotide having all or part of the sequence represented herein. It can be a sequence, or a functional equivalent thereof. Such oligonucleotide sequences can be generated chemically or mechanically by known techniques. A functional equivalent of an oligonucleotide sequence is high relative to a complementary oligonucleotide to which the sequence (or corresponding portion of the sequence) hybridizes, or a sequence complementary to the nucleic acid sequence (or portion of the corresponding sequence). Those that can hybridize and / or encode products (eg, polypeptides or peptides) that have the same functional properties of the product encoded by the sequence (or corresponding portion of the sequence). Whether a functional equivalent must meet one or both criteria will depend on its application (eg, if it is used only as an oligo probe, it will (It needs to be satisfied, and when it should be used for the production of Cynd I, it only needs to satisfy the second standard.)
The following examples further illustrate the present invention.
Example 1
Preparation of Cynd I isolated pollen extract for protein sequencing and MAb production
Bermuda grass pollen was purchased from Greer Laboratories, Lenoir, North Carolina, USA. To prepare soluble protein loaded on Rotofor, 3g by shaking 5g of pollen pollen with 10ml of 10mM phosphate buffer (PBS) for 1 hour at 4 ° C. Extracted once. After each extraction, the mixture was centrifuged (2500 rpm, 10 minutes) and the supernatant was collected. After three extractions, the supernatants were pooled and filtered through a 3 mm Whatman filter.
Isoelectric focusing electrophoresis (IEF) for preparation
Preparative IEF in Rotofor (Biorad, Richmond, Calif.) Is described in detail in Egan et al. (1988) Analyt. Biochem. 172, 488-494. Briefly, 5 ml of ampholyte solution (Bio-lyte, pH range 3-10; 40%) was added to the pollen extract and the volume was adjusted to 50 ml with distilled water. This mixture was loaded into a Rotofor cell and run at 4 ° C. and a constant power of 12 W. After 4 hours, 20 fractions were collected and their pH determined. Fractions containing the protein of interest were identified with MAb 3.2 on immunoblots after SDS-PAGE. This MAb raised to purified Lolp I, but was found to cross-react with group 1 homologues from nine other gramineous plants, including Bermuda grass (Kahn and Marsh, 1986, Mol. Immunol. 23, 1281-1288). Fractions containing the protein of interest were pooled and re-fractionated in Rotofor under the same conditions as above except that the samples were focused for 2.5 hours. The pH of each fraction was determined.
SDS-PAGE and Western blotting
The proteins in the Rotofor fraction were resolved by electrophoresis on a 10-15% gradient SDS-polyacrylamide gel under reducing conditions. Conditions for electrophoresis were essentially as described in Singh and Knox, Int. Archs Appl. Immun. 78: 300-304 (1985). Molecular weight (MW) was determined using a low molecular weight (MW) standard from Pharmacia. Proteins on polyacrylamide gels were visualized using Coomassie Brilliant Blue R250.
The protein was nitrocellulose (Schleicher & Schuell, 0.45 μm) overnight at 120 mA and 4 ° C. according to Towbin et al. (1979); Proc. Natl. Acad. Sci. USA 76, 4350-4354; Moved. After transferring the protein, nonspecific binding sites were obtained by incubation of the Western blot in powdered milk [10% in TBS (Tris buffered saline: 150 mM NaCl / 10 mM Tris HCl, pH 7.5)]. Blocked.
In resolution by SDS-PAGE of the fraction obtained by preparative IEF,Cyn d  I was found to focus in fractions 10-20 in the pH range of 6-10. These fractions contained 31-32 kD protein bound to MAb3.2. The protein in fractions 10-13 (32 kD) bound to MAb3.2 had a slightly higher MW than that in fractions 15-20 (31 kD) (FIGS. 11a-11b). Intermediate fraction 14 contained both proteins bound to MAb 3.2. These proteinsCyn d  I a (32kD) andCyn d  I b (31 kD) was displayed.
Cyn d  Fractions 10-13 of FIG. 11a containing Ia were pooled and refractionated.Cyn d  Ib was found in all fractions of Figure 12a, but was superior to the protein components of fractions 13-20 (Figure 12a). These fractions have a pH of 6.5;Cyn d  I had a p I indication of a.
Pool and re-fractionate fractions 15-20 in Figure 11aC yn d  Ib was purified.Cyn d  Ib was found in all fractions of Figure 12b, but was superior to the protein profiles of fractions 1-12 (Figure 12b). These fractions have a pH of 7.4;Cyn d  I had an indication of p I of b.
Immunoblot analysis
Western blots were incubated in serum of MAb 3.2 or allergic individuals. MAb3.2 was diluted 1: 1000 in PBS containing 0.5% BSA. Binding of MAb was followed by incubation in solution of peroxidase labeled anti-mouse Ig antibody (Dakopatts Corporation, Carpinteria, Calif., USA) and subsequent as described in Singh and Knox (1985) supra. Visualized by addition of enzyme substrate. Human serum was diluted 1: 4 in 150 mM PBS containing 0.5% BSA. IgE binding¹²⁵Visualization was by incubation of blots in I-labeled anti-human IgE (Kallestad (diluted 1: 6 in PBS / BSA)) followed by autoradiography. PurifiedCyn d  I a and I b were evaluated for their ability to bind IgE from the serum of allergic individuals (FIG. 13). Both fractions were bound to IgE from the serum of an allergic individual of Bermuda grass.
NH₂-Sequencing of terminal amino acids
Cyn d  I proteinCyn d  I a andCyn d  I b was isolated as described above and using 10 mM CAPS 10% methanol (pH 11.0) as transfer buffer (Ward et al., 1990) (3 [cyclohexylamino] -1-propanesulfonic acid). Visualization by electrotransfer on polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, Mass., USA), followed by staining with Coomassie Brilliant Blue R250, methanol acetate water (50:10: 40, v / v / v) and washed well with deionized water. Both Cynd I a andCyn d  Ib protein NH₂The terminal amino acid sequence was determined as described by Ward et al. (1990).
Separated by RotoforCyn d  The I protein was also purified by reverse phase HPLC and the 31 kD protein NH₂-The terminal amino acid sequence was determined.
TwoCyn d  I component is their NH₂-Show small amino acid sequence variations in the terminal region, andCyn d  There is a homology between I and Lolp I from ryegrass (Table 1).

MAb production and screening
Balb / C miceCyn d  By intraperitoneal immunization with I (Biorad, Richmond, CA) isolated on Rotofor.Cyn d  I MAb was obtained. RIBI (RIBI Immunochem, Hamilton, Mont., USA) was used as an adjuvant in the first of four immunizations. The remaining intraperitoneal immunization was in saline. Hybridoma fusion and growth were essentially as described in Harlow and Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory. Single cell cloning was by limiting dilution. Hybridoma-producing anti-Cynd I antibody was identified by ELISA assay. CAPS buffer (6.67 mM NaCO_Three  35 mM NaHCO_Three  The ELISA plate was coated overnight with 30 μg of pollen extract of Bermuda grass diluted in pH 9.6). The wells were then washed 3 times with TPBS (PBS containing 0.1% Tween 20) and blocked with PBS containing 1% BSA (PBS / BSA) for 30 minutes. 100 μl of primary antibody is added to each well and incubated for 60 minutes, then washed (as above) and in β-gal labeled anti-mouse Ig (1/250 dilution in PBS / BSA, 60 minutes) Incubated with After washing, 200 μl of fluorescent substrate 4-methylumbelliferyl-BD-galactoside (MUG) was added to each well and incubated at 37 ° C. for 30 minutes. The plate was then read on a fluoroCount 96 fluorometer (Pharmacia).
Antibodies that were positive by this method are labeled 3A2, 4D2, 1D1, and 3C2, and on Western blots of pollen protein of grass grass that was resolved by SDS-PAGECyn d  Tested for binding to I.
cDNA library and immunological screening
Poly (A +) mRNA was isolated from pollen from grass grass purchased from Greer Laboratories, Lenoir, North Carolina, USA essentially as described in Herrin and Michaels (1984). The cDNA was synthesized using known techniques of Pharmacia cDNA synthesis and cloned into the EcoR I site of the vector lambda-gt11. Recombinant protein from phage plaques was transferred to a nitrocellulose filter by overlaying the plated cDNA library with a nitrocellulose filter impregnated with IPTG. These filters were then incubated in a mixture of anti-Cynd I MAbs. The binding of MAb to the recombinant protein was visualized as described above. AntiCyn d  Plaque-producing protein bound to I MAb was isolated and purified.
Isolation of cDNA clones
The pollen cDNA of Bermuda grass was first screened with a mixture of anti-Cynd I hybridoma supernatants containing mainly MAb 3.2 as described above, and 30 positive cDNAs were plaque purified. These clones are thenCyn d  I tested for binding to MAbs 3A2, 4D2, 3C2 and 1D1. All clones selected after the first round of screening produced a recombinant fusion protein specific for MAb3A2. The binding of the clones to MAb is shown in FIG. 14 and summarized in Table 2. The cDNA clone isolated here is based on the binding of MAb shown against the recombinant fusion protein.C yn d  It is concluded that I is encoded. MAb1D1 has a much higher background binding than the other MAbs, making the binding much more subjective.

Nucleotide and amino acid sequences of cDNA clones
Clones 2, 3, 18, 21, 22, 23 and 33 (see Table 2) were selected for further studies based on their antibody affinity. Clones 2, 3, 18, 21, 22, 23 and 33 were isolated from the phage and subcloned into pGEM-4Z (Promega) or Bluescript (Stratagene) vectors. DNA sequence was determined by double-stranded sequencing performed by the chain termination method (Sanger et al., Proc. Natl. Acad. Sci. (1977), 74: 5460-5463) using T7 polymerase (Pharmacia) . The nucleotide sequences and deduced amino acid sequences of these clones are shown in Fig. 1 (Clone 2), Fig. 2 (Clone 18), Fig. 15 (Clone 3), Fig. 16 (Clone 22) and Fig. 17 (Clone). 23).
All clones sequenced show homology with each other, especially in the open reading frame (ORF). Furthermore, there is significant nucleotide sequence homology between all sequenced clones and Lolp I, the major ryegrass allergen. However, the sequenced clones have the most similar sequence to clone 2 (ie, clone 3), most similar to clone 18, divided into three groups based on nucleotide and deduced amino acid sequence homology. Those with sequences (ie clones 21 and 33) and those with sequences most similar to clone 22 (ie clone 23). The deduced amino acid sequence encoded by the ORF of clones 18 and 2 was compared to the deduced amino acid sequence of Lolp I (Perez et al., 1991, supra; Griffith et al., 1991, supra) (FIG. 6). There is 67% amino acid homology between Lolp I and clone 18 and 72% between Lolp I and clone 2. The deduced amino acid sequences of clones 2 and 18 have 83% identity (87% homology) with each other.
Example 2
Cloning of the 5 'end of Cynd I
Double-stranded cDNA was synthesized from approximately 4 μg of pollen RNA (Grill Laboratories, Lenoir, NC, USA) using a cDNA synthesis system plus kit (BRL, Bethesda, MA, USA). After phenol extraction and ethanol precipitation, the cDNA was blunt-ended with T4 DNA polymerase (Promega, Madison, Wis., USA), ethanol precipitated, self-annealed, AT, 5'-GGGTCTAGAGGTACCGTCCGATC-GATCATT-3 ', and AL, 5' Conjugated to p-AATGATCGATGCT-3 ′, an oligonucleotide for use in a modified Anchored PCR (Marsh et al., 1986; Roux and Dhanarajan, 1990; Rafnar et al., 1991) reaction. 1 μg of oligonucleotide AP, 5′-GGGTCTAGAGGTACCGTCCG-3 ′, and CD-5,5′-GATGTGCTCGTAGTTCTT-3 ′, an oligonucleotide primer based on the noncoding strand sequence of Cynd I corresponding to the amino acid sequence KNYEHI From each linker-linked cDNA (5 μl from a 20 μl reaction), the cDNA encoding the amino terminus of Cynd I was amplified. In a reaction mixture containing 10 μl of 10 × various dNTP-containing buffers, 1 μg of each primer, cDNA, 0.5 μl of Amplitaq DNA polymerase, Gene Amp DNA amplification kit (Perkin Elmer. In the programmable controller from Reseach, Inc. (Cambridge, Mass., USA), the primary polymerase chain reaction (Perkin Elmer Cetus, Norwalk, Conn., USA) PCR) was performed and made up to 100 μl with distilled water. The 25 rounds of amplification consisted of denaturation at 94 ° C for 1 minute, primer annealing to template at 65 ° C for 1.5 minutes, and chain extension at 72 ° C for 2 minutes. 5% (5 μl) of this primary amplification was then nested for 1 μg of each CD-4,5′-GGGGATCCGAGGCCGT-CCTTGAAG-3 ′, CD-5 based on the non-coding strand sequence corresponding to the amino acid IFKDGL. Used as above in secondary amplification using oligonucleotide primers and AP. All oligonucleotides were synthesized by Research Genetics, Inc. (Huntsville, Alabama). Oligonucleotide primers AP, AT and AL have been previously described (Rafnar et al., 1991; Morgenstern et al., 1991; Rogers et al., 1991). The first 8 nucleotides of CD-4 were added to create a BamHI restriction site for cloning purposes.
Amplified DNA was recovered by sequential extraction of chloroform, phenol, and chloroform and then precipitated with 0.5 volumes of 7.5 ammonium acetate and 1.5 volumes of isopropanol at -20 ° C. After precipitation and washing with 70% ethanol, the DNA was simultaneously digested with Xba I and BamH I in a 15 μl reaction mixture and a preparative 3% SeaPlaque low melting point agarose gel (FMC Corporation, Maine, USA) Electrophoresis through Rockland). Appropriately sized DNA bands were visualized by ethidium bromide (EtBr) staining, excised, and dideoxy DNA sequencing (Sanger et al.) Using the Sequenase kit (US Biochemicals, Cleveland, Ohio, USA). (1977), Proc. Natl. Acad. Sci. USA, 74: 5460-5463). Two clones 14a1 and 14c1 were obtained from this ligation, fully sequenced and found to contain the in-frame initiator methionine. The methionine encoded by nucleotides 28-30 of the 14a1 sequence (FIG. 6) most preferably represents the start codon. Because the surrounding sequence closely matches the common plant sequence 5'-AACAATGGC-3 '(Lutcke et al. (1987) EMBO J.6: 43-48) and there is an in-frame stop codon just upstream. Because. Although 14c1 (FIG. 7) contained two potential in-frame methionines, the methionine encoded by nucleotides 27-29 is most likely the initiator methionine. Because the surrounding sequence is from the methionine encoded by nucleotides 42-45, the consensus plant sequence 5'-AACA.ATGFrom a close match with GC-3 '(Lutcke et al., Supra) (78% vs. 56% match). Furthermore, the corresponding nucleotides 27-29 of the sequence are identical to that of clone 14al. The sequences of both clone 14a1 and clone 14c1Cyn d  It had a 17 nucleotide overlap with the I clone, ie clone 18. The amino terminus of mature Cynd I, NH2-AIGDKPGPNITATGNKWLEAKATFYG encoded by clone 14a1 and NH2-AIGDKPGPNITATGSKWLEAKATFYG- encoded by clone 14cl areCyn d  Two previously published protein sequences for I: NH2-AMGDKPGP? ITATYGDKWLDAKATFYG (Matthiesen et al. 1988, supra Matthiesen et al., 1990, supra Matthiesen et al., 1991, supra) and NH2-AIGDKPGPKITATY ?? KWLEAKAT (Singh et al., 1990, supra). . Could be identified. This indicated that clones 14a1 and 14c1 had leader sequences of 22 and 26 amino acids, respectively. When these leader sequences are cleaved,Cyn d  A mature form of the I protein will be obtained. As shown in FIG.Cyn d  By joining the sequence of I.14 to clone 18 in their overlap,Cyn d  Display as I.18Cyn A potential full-length amino acid sequence of dI was generated (FIG. 9). In both cases,Cyn d  The mature form of I is predicted to be 246 amino acids with a calculated molecular weight of 26.7 kDa.
Example 3
RNA was isolated from the pollen of Cynodon dactylon using a modification of the guanidinium thiocyanate method of Chomczynski and Sacchi (1987) Analyt. Biochem. 162: 156-159. Pollen was ground in liquid nitrogen using 9 ml guanidinium thiocyanate buffer (0.05% Tris HCl [pH 7.0], 0.05 volume β-mercaptoethanol, 0.1 volume% sodium lauroyl sarcosine). The pollen solution was then shaken with phenol (10 ml) for 10 minutes, after which 10 ml chloroform: isoamyl alcohol was added and the mixture was shaken for an additional 20 minutes. The mixture was centrifuged at 7,000 × g for 25 minutes and the aqueous phase was collected.
The aqueous phase was re-extracted with phenyl: chloroform: isoamyl alcohol 25: 24: 1 and then centrifuged at 2,000 × g until the interface was clear. The aqueous phase was then decanted into a quick seal ultracentrifuge tube with a 3 ml CsCl cushion (5.7 M EDTA; density = 1.71 g / ml) placed underneath and a Beckman Ti70.1 rotor (Beckman L8 -70 ultracentrifuge; centrifuge (20 hours, 40,000 rpm, 20 ° C.) in Beckman Instruments, Fullerton, Calif.). After centrifugation, the RNA in the pellet was resuspended in 0.005% SDS, extracted with phenol / chloroform and precipitated overnight at -20 ° C.
PolyA using a Pharmacia mRNA purification kit according to the manufacturer's instructions.⁺RNA was isolated.
First strand cDNA was prepared by heating 0.8 μg of mRNA to 70 ° C. with 0.5 μg of oligo-dT primer (Pharmacia, Piscathay, NJ). After cooling the mRNA on ice, 5x first strand binding and 25 U RNacin ribonuclease inhibitor were added. The mixture was then heated to 42 ° C. for 1 hour. Final reaction conditions were 25 μl final volume of 50 mM Tris HCl, pH 8.3, 50 mM KCl, 10 mM MgCl.₂, 0.5 mM spermidine, 10 mM DTT, 4 mM sodium pyrophosphate, 1 mM each dATP, dCTP, dGTP and TTP, 25 U RNacin ribonuclease inhibitor and 15 μAMV reverse transcriptase / μgRNA (Promega cDNA Synthesis Kit, Promega, Madison, Wis.) It was.Cy n d  The cDNA sequence encoding I was amplified using the Perkin-Elmer Cetus gene amplification kit (U.S. Biochemicals, Cleveland, Ohio). Mix 5 μl (25%) first strand cDNA synthesis product with 10 × buffer to give 2 mM MgCl 2₂, 50 mM KCl, 10 mM Tris-HCl, 1 μg oligonucleotide primer CD15′N, 5′-GGGAATTCGCCATCGGCG-ACAAG-CCAG-3 ′, 1 μg oligonucleotide primer CDI3′B18,5′-CCCTGCAGATG-GAGGATCATCGTCTC-3 ′ A final buffer concentration of 0.2 mM dNTPs and 2.5 units Taq DNA polymerase (Pharmacia, Piscathay, NJ) was used. CD15′N nucleotides 1-8 were added to create an EcoR I endonuclease restriction site for cloning purposes, but nucleotides 9-27Cyn d  Corresponds to nucleotides 107-125 of clone 14a1 in FIG. Nucleotides 1-8 of CD13'B18 were added to create a Pst I endonuclease restriction site for cloning purposes, but nucleotides 9-26 were non-coding complementary to nucleotides 604-621 of clone 18. Corresponds to the chain sequence (Fig. 2).
PCR was performed in a Perkin-Elmer Cetus thermal cycler (Perkin-Elmer, Norwalk, Conn.) And 5 cycles of denaturation (94 ° C., 1 min), annealing (45 ° C., 1.5 minutes) and extension (72 ° C., 3 minutes) followed by 20 cycles of denaturation (94 ° C., 1 minute), annealing (55 ° C., 1.5 minutes) and extension (72 ° C., 3 minutes). The final extension reaction was performed at 72 ° C. for 10 minutes. The amplified product was recovered by phenol extraction, chloroform extraction, and then precipitation at −20 ° C. using 0.5 volumes of 7.5 M ammonium acetate and 1.5 volumes of isopropanol. The reaction product was blunted with a Klenow fragment of DNA polymerase, then cut with EcoR I and cloned into a Bluescript vector digested with EcoR I and Hinc II. Clone CD1 was sequenced by the dideoxy chain termination method (Sanger, supra) as described in Example 1 and shown in FIG.Cyn d  It was discovered to contain the nucleotide sequence of I and the deduced amino acid sequence.
Example 4
Double stranded cDNA was prepared and amplified using oligonucleotide primers CD-13 and CD-15 in the primary PCR reaction as described in Example 2. CD-13 had the sequence 5'-TTTCTAGAGCCATCGGCGACAAGCCAGGG-CCC-3 ', but nucleotide 14 would have been C or G. Nucleotides 1-8 of CD-13 were added to create an Xho I restriction site for cloning purposes. The remaining nucleotides encode the amino acid Ala (Ile / Met) -GlyAspLysProGlyPro, where amino acid 2 would have been Ile or Met. (Cynd I a andCyn d  Amino acids 1-8 of Ib (Table 1). CD-15 has the sequence 5′-GCGTACTTCACGAGCAGCGCCAG-GTAATT-3 ′, which encodes the amino acids AsnTyrLeuAlaLeuLeuValLysAla (amino acids 159-168 numbered clone 2 (C2) and clone 3 (C3) in FIG. 5). Corresponding to the non-coding strand sequence complementary to the coding strand sequence to be performed. 5% of the primary reaction was amplified in the secondary PCR using oligonucleotide primers CD-13 and CD-16 as described in Example 2. CD-16 has the sequence 5'-TTGAATTCGACACGGCGGAACTGCAGCAT-3 ', where nucleotide 12 would have been G or A. Nucleotides 1-8 of CD-16 were added to create an EcoR I restriction site for cloning purposes. Nucleotides 9-29 correspond to the non-coding strand sequence complementary to the coding strand sequence encoding amino acids MetLeuGlnPheArgArgVal (amino acids 132-138 numbered C2 and C3 in FIG. 5).
PCR amplification was performed as described in Example 2. The amplified product was recovered, digested appropriately and ligated into pUC for sequencing as described in Example 2.C yn d  A clone designated KAT-39-1 having the sequence identified as the I clone was isolated. The nucleotide sequence of KAT-39-1 and the deduced amino acid sequence are shown in FIG. This cloneCyn d  Extension of clones C2 and C3 of I. Oligonucleotides CD-15 and CD-16 areCyn d  I clone C18 and its homologues have a single mismatch with their 3 'ends with corresponding. Thus, only clone C2 or C3, or close family members will be amplified.Cyn d  KAT-39-1 and I.2 / 3 (full length)Cyn d  The composite array of I.2 / 3 isCyn d  I.CD1 andCyn d  Compared to I.18, it is shown in FIG.
Although the invention has been described with reference to its preferred embodiments, other embodiments can achieve the same results. Variations and modifications to the present invention will be apparent to those skilled in the art, and all such modifications and equivalent embodiments falling within the true spirit and scope of the present invention are intended to be included within the scope of the appended claims. Intended.

Claims (31)

Encodes the allergen Cyn d I of the pollen protein of Candida, where the allergens of the protein are Cyn d I.18, Cyn d I.CD1 and Cyn d I.2 / 3 as shown in FIG. An isolated nucleic acid comprising an amino acid sequence selected from the group consisting of: formula:
L ₁ NYX
Wherein L ₁ is a nucleic acid sequence of 0-300 nucleotides, said nucleic acid sequence comprises nucleotides encoding Cyn d I leader sequence, N is a nucleic acid sequence consisting of up to 600 nucleotides, said nucleic acid sequence is mature contains nucleotides encoding the amino-terminal portion of Cyn d I, Y is clone 2 shown in FIG. 1, clone 18 shown in FIG. 2, clone 3 shown in FIG. 15, clone 22 or a shown in FIG. 16 17 is a nucleic acid sequence portions partial clone 2 3 shown in FIG., and X is a nucleic acid sequence of 0 to 600 nucleotides, said nucleic acid sequence contains a nucleotide 3 'untranslated portion of Cyn d I, and L ₁ and X can be 0,
An isolated nucleic acid encoding the allergen Cyn d I of the pollen protein of Candidae. formula:
L ₁ NYX
Wherein L ₁ is a nucleic acid sequence of 0-300 nucleotides, said nucleic acid sequence comprises nucleotides encoding Cyn d I leader sequence, N is a nucleic acid sequence consisting of up to 600 nucleotides, said nucleic acid sequence is mature contains nucleotides encoding the amino-terminal portion of Cyn d I, Y is clone 2 shown in FIG. 1, clone 18 shown in FIG. 2, clone 3 shown in FIG. 15, clone 22 or a shown in FIG. 16 17 is a nucleic acid sequence portions partial clone 2 3 shown in FIG., and X is a nucleic acid sequence of 0 to 600 nucleotides, said nucleic acid sequence contains a nucleotide 3 'untranslated portion of Cyn d I, and N Of the nucleic acid sequence does not overlap the 5 ′ end of the Y nucleic acid sequence and L ₁ and X can be 0.
The isolated nucleic acid of claim 2 having The isolated nucleic acid according to claim 3, wherein L ₁ is a nucleic acid sequence represented by nucleotides 1 to 106 of clone 14a1 shown in Fig. 6 or nucleotides 1 to 103 of clone 14c1 shown in Fig. 7. N is the nucleic acid sequence represented by nucleotides 107 to 244 of clone 14a1 shown in FIG. 6 or the nucleic acid sequence represented by nucleotides 104 to 243 of clone 14c1 shown in FIG. 7, and Y is the clone shown in FIG. 5. The isolated nucleic acid of claim 4, wherein the nucleic acid sequence is represented by 18 nucleotides 1-603, and the X nucleic acid sequence contains nucleotides 604-775 of clone 18 shown in FIG. N is the nucleic acid sequence represented by nucleotides 107 to 247 of clone 14a1 shown in FIG. 6 or the nucleic acid sequence represented by nucleotides 104 to 246 of clone 14c1 shown in FIG. 7, and Y is the clone shown in FIG. 5. The isolated nucleic acid of claim 4, wherein the nucleic acid sequence is represented by 22 nucleotides 1-594, and the X nucleic acid sequence contains nucleotides 595-802 of clone 22 shown in FIG. N is the nucleic acid sequence represented by nucleotides 107 to 246 of clone 14a1 shown in FIG. 6 or the nucleic acid sequence represented by nucleotides 104 to 245 of clone 14c1 shown in FIG. 7, and Y is the clone shown in FIG. 5. The isolated nucleic acid of claim 4, wherein the nucleic acid sequence is represented by 23 nucleotides 1-595, and the X nucleic acid sequence contains nucleotides 596-832 of clone 23 shown in FIG. The isolated nucleic acid according to claim 3, wherein L ₁ consists of a nucleic acid sequence represented by nucleotides 41 to 106 of clone 14a1 shown in Fig. 6 or nucleotides 28 to 103 of clone 14c1 shown in Fig. 7. N is a nucleic acid sequence represented by nucleotides 107 to 244 of clone 14a1 shown in FIG. 6 or nucleotides 104 to 243 of clone 14c1 shown in FIG. 7, and Y is nucleotides 1 to 603 of clone 18 shown in FIG. The isolated nucleic acid of claim 8, wherein X is 0. N is the nucleic acid sequence represented by nucleotides 107 to 247 of clone 14a1 shown in FIG. 6 or nucleotides 104 to 246 of clone 14c1 shown in FIG. 7, and Y is nucleotides 1 to 594 of clone 22 shown in FIG. The isolated nucleic acid of claim 8, wherein X is 0. N is a nucleic acid sequence represented by nucleotides 107 to 246 of clone 14a1 shown in FIG. 6 or nucleotides 104 to 245 of clone 14c1 shown in FIG. 7, and Y is nucleotides 1 to 595 of clone 23 shown in FIG. The isolated nucleic acid of claim 8, wherein X is 0. L ₁ is 0, N is a nucleic acid sequence represented by nucleotides 107 to 244 of clone 14a1 shown in FIG. 6 or nucleotides 104 to 243 of clone 14c1 shown in FIG. 7, and Y is shown in FIG. 4. The isolated nucleic acid of claim 3, wherein the nucleic acid sequence is represented by nucleotides 1-603 of clone 18 and X is 0. L ₁ is 0, N is the nucleic acid sequence represented by nucleotides 107 to 247 of clone 14a1 shown in FIG. 6 or nucleotides 104 to 246 of clone 14c1 shown in FIG. 7, and Y is shown in FIG. 4. The isolated nucleic acid of claim 3, wherein the nucleic acid sequence is represented by nucleotides 1 to 594 of clone 22 and X is 0. L ₁ is 0, N is a nucleic acid sequence represented by nucleotides 107 to 246 of clone 14a1 shown in FIG. 6 or nucleotides 104 to 245 of clone 14c1 shown in FIG. 7, and Y is shown in FIG. 4. The isolated nucleic acid of claim 3, wherein the nucleic acid sequence is represented by nucleotides 1-595 of clone 23 and X is zero. 4. The isolated nucleic acid of claim 3, wherein Y is the nucleic acid sequence represented by nucleotides 1 to 438 of clone 2 shown in FIG. 4. Y is the nucleic acid sequence represented by nucleotides 1-417 of clone 3 shown in FIG. 15, and X is the nucleic acid sequence represented by nucleotides 418-594 of clone 3 shown in FIG. An isolated nucleic acid according to 1. 4. The isolated nucleic acid of claim 3, wherein L ₁ is 0, Y is the nucleic acid sequence represented by nucleotides 1 to 438 of clone 2 shown in FIG. 1, and X is 0. L ₁ is 0, Y is a nucleic acid sequence represented by nucleotides 1-417 of clone 3 shown in FIG. 15, The isolated nucleic acid of claim 3. Comprising the nucleic acid sequence of clone CD1 shown in FIG. 18, it encodes the allergen Cyn d I protein pollen bermudagrass, isolated nucleic acid. Comprising the amino acid sequence of the mature Cyn d I.CD1 shown in FIG. 20, it encodes the allergen Cyn d I protein pollen bermudagrass, isolated nucleic acid. An isolated nucleic acid encoding the allergen Cyn d I of the pollen protein of Cyprus, comprising the amino acid sequence of Cyn d I.2 / 3 (full length ) shown in FIG. An expression vector comprising the nucleic acid sequence according to claim 1, 2, 3, 19 or 21. 21. A host cell transformed to express a protein or peptide encoded by the nucleic acid sequence of claim 1, 2, 3, 19, or 20. 請 Motomeko 1,2,3,19 or encoded by the nucleic acid sequence set forth in 20, of proteins isolated behaved Shiba pollen allergen Cyn d I. Amino acids 1-246 of Cyn d I.18 shown in FIG. 20, amino acids Cyn d I.2 / 3 shown in FIG. 20 and amino acids 1-246 up for Cyn d I.CD1 shown in FIG. 20 (full length) 1-24 4, ing comprise an amino acid sequence represented by amino acid selected from the group consisting of proteins of pollen isolated Bermuda grass allergen Cyn d I. Isolated allergen Cyn d I protein of bermudagrass, Contact and pharmaceutically acceptable carrier or diluent comprising the composition of claim 25. A blood sample obtained from an individual, and allergens isolated protein of claim 25, the combination under conditions appropriate for binding of the allergen of the protein and blood components, and such Measuring in the individual the susceptibility to the allergen Cyn d I of a pollen protein of C. elegans, comprising measuring the extent to which the binding occurs. T-cell function, measuring the extent to which binding occurs by the evaluating the combinations bond, or those with an antibody to the protein present in the blood, The method of claim 27. Allergy in said individual against an allergen Cyn d I pollen bermudagrass according to an amount sufficient as claimed in claim 25 to produce a response of allergy in an individual comprising administering to said individual, and the allergen of the pollen of the bermudagrass A method of determining an individual's susceptibility to an allergen of pollen pollen, comprising measuring the occurrence of a response of Host cells transformed with nucleic acid according to claim 1 or 2, in a suitable medium to produce a mixture of medium containing allergen Cyn d I of proteins in pollen cells and bermudagrass cultured; and comprising generating a substantially pure Cyn d I purification of the previous SL mixture, method for producing an allergen Cyn d I protein pollen bermudagrass. Amino acid 5-246,10-246,20-246 of Cyn d I.18 shown in full in FIG. 20, 25-246, amino acids Cyn d I.CD1 5-246,10-246,20- 248 , 25-246 and Cyn d I.2 / 3 (full length) amino acids 5-244, 10-244, 20-244 , 25-244 amino acids selected from the group consisting of pollen allergen Cyn d Isolated peptide of I.

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