JP2005532820A - ノミおよびダニのオクトパミンレセプター核酸分子、タンパク質およびそれらの使用 - Google Patents
ノミおよびダニのオクトパミンレセプター核酸分子、タンパク質およびそれらの使用 Download PDFInfo
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- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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Abstract
Description
本発明は、ノミおよびダニのオクトパミンレセプター核酸分子、このような核酸分子によってコードされるタンパク質、このようなタンパク質に対して惹起された抗体、およびこのようなタンパク質のインヒビターに関する。本発明はまた、このようなタンパク質、核酸分子、抗体、および阻害組成物を得るための方法を包含する。本発明はまた、このようなインヒビターを含む治療用組成物、ならびにその使用を包含する。
ノミおよびダニの動物外寄生は、ペットの飼い主の健康上および経済的な関心事である。ノミおよびダニは、細菌、ウイルス、原生動物寄生体およびリケッチアを含むがこれらに限定されない種々の感染性因子を運搬することが公知である。そのため、ノミおよびダニは、それらが動物上にいる場合だけでなく、動物の一般的環境にいる場合にも問題となる。
本発明は、ノミおよびダニのオクトパミンレセプタータンパク質;ノミおよびダニのオクトパミンレセプタータンパク質をコードする核酸分子;このようなタンパク質に対して惹起された抗体(すなわち、抗ノミオクトパミンレセプター抗体および抗ダニオクトパミンレセプター抗体);このようなタンパク質または抗体のミメトープ;ならびにノミおよび/またはダニのオクトパミンレセプター活性を阻害する化合物(すなわち、阻害性化合物またはインヒビター)を提供する。
本発明は、ノミおよびダニのオクトパミンレセプター核酸分子、このような核酸分子によってコードされるタンパク質、このようなタンパク質に対して惹起された抗体、およびこのようなタンパク質のインヒビターを提供する。本明細書中で用いられる場合、ノミおよびダニのオクトパミンレセプター核酸分子およびこのような核酸分子によってコードされるタンパク質はまた、それぞれ、本発明のオクトパミンレセプター核酸分子およびオクトパミンレセプタータンパク質とも呼ばれる。本発明のノミおよびダニのオクトパミンレセプター核酸分子およびオクトパミンレセプタータンパク質は、ノミもしくはダニから単離されてもよく、または組換えもしくは合成によって調製されてもよい。本発明のノミおよびダニのオクトパミンレセプター核酸分子は、RNAもしくはDNAまたはそれらの改変形態であり得、そして二本鎖もしくは一本鎖であり得る;核酸分子の例としては、相補性DNA(cDNA)分子、ゲノムDNA分子、合成DNA分子、メッセンジャーRNAについての特異的タグであるDNA分子、および対応するmRNA分子が挙げられるがこれらに限定されない。それゆえ、本発明のノミまたはダニの核酸分子は、このような核酸分子が含まれる染色体全体を言及することを意図しないが、しかし、本発明のノミまたはダニのオクトパミンレセプター核酸分子は、このような核酸分子によってコードされるオクトパミンレセプタータンパク質の産生を制御する調節領域のような全ての領域(例えば、転写制御領域、翻訳制御領域または翻訳後制御領域であるがこれらに限定されない)ならびにそれらのコード領域、および任意のイントロンまたは非翻訳コード領域を含み得る。本明細書中で用いられる場合、語句「ダニオクトパミンレセプタータンパク質」とは、ダニオクトパミンレセプター核酸分子によってコードされるタンパク質をいい、そして語句「ノミオクトパミンレセプタータンパク質」とは、ノミオクトパミンレセプター核酸分子によってコードされるタンパク質をいう。
Tm=81.5℃+16.6logM+0.41(%G+C)−500/n−0.61(%ホルムアミド)。
約50ヌクレオチドよりも小さい核酸分子については、ハイブリッド安定性は、二重鎖の50%が解離する温度として規定される解離温度(Td)によって規定される。これらのより小さな分子について、標準的なイオン強度での安定性は、以下の式によって規定される:
Td=4(G+C)+2(A+T)。
Tdより5℃低い温度が使用されて、完全にマッチする分子の間でのハイブリダイゼーションが検出される。
81.5℃+16.6log(.15M)+(0.41×30)−(500/150)−(0.61×0)=76.8℃。
従って、約20%塩基対ミスマッチを有する核酸分子とのハイブリダイゼーションを達成するために、ハイブリダイゼーション洗浄は、56.8℃以下の温度にて行われる。ノミDNAとのハイブリダイゼーションのための洗浄条件を計算するために、当業者は、ダニと比較して、ノミのG+C含有量における差異に起因して式を適合させて、79.6℃の洗浄温度にする。従って、所望のパーセンテージの塩基対ミスマッチ、本明細書中に開示される式およびG/C含有量に基づいて、さらなるハイブリダイゼーション温度を計算することは、当業者の技術範囲内である。例えば、本明細書中に特定される配列を有する本発明のダニ核酸分子に対するハイブリダイゼーションについて試験されるべき核酸分子が、150ヌクレオチドより長くなる場合、20%までの塩基対ミスマッチを可能にするハイブリダイゼーション反応のためのTmは、56.8℃から大きく変化しないことは、当業者によって理解される。同様に、約10%の塩基対ミスマッチを有する核酸分子とのハイブリダイゼーションを達成するために、ハイブリダイゼーション洗浄は、66.8℃以下の温度において行われ、そして約5%の塩基対ミスマッチを有する核酸分子とのハイブリダイゼーションを達成するために、ハイブリダイゼーション洗浄は、71.8℃以下の温度において行われる。
この実施例は、cDNA末端の迅速増幅(RACE pool)による、ノミゲノムDNAならびにノミの頭部および神経索のcDNAプールの調製を記載する。
この実施例は、ノミのオクトパミンレセプター核酸分子の、クローニング、配列決定、および発現を記載する。
この実施例は、タグ付加したノミオクトパミンレセプタータンパク質の発現を記載する。
この実施例は、cDNA末端の高速増幅によるダニゲノムDNAならびにダニ頭部および神経索DNAプール(RACEプール)の調製を記載する。
本実施例は、ダニオクトパミンレセプター核酸分子のクローニングおよび配列決定を記載する。
Claims (20)
- 以下:
(a)少なくとも50ヌクレオチドの長さの核酸分子であって、以下:
(1)37℃の温度で、核酸へリックス不安定化化合物の非存在下において2×SSCを含む溶液中でハイブリダイズすること、および
(2)66.8℃の温度で、へリックス不安定化剤の非存在下において1×SSCを含む溶液で洗浄すること、を包含する条件下で、配列番号32、配列番号35、配列番号38および配列番号41からなる群より選択される核酸配列を有する核酸分子とハイブリダイズし、ここで、該少なくとも50ヌクレオチド核酸分子が、オクトパミンに結合するタンパク質をコードする、核酸分子;ならびに
(b)該(a)の核酸分子に完全に相補的な核酸分子、
からなる群より選択される、単離された核酸分子。 - 以下:
(a)少なくとも35ヌクレオチド長の核酸分子であって、以下:
(1)37℃の温度で、核酸へリックス不安定化剤の非存在下において2×SSCを含む溶液中でハイブリダイズすること、および
(2)74.6℃の温度で、へリックス不安定化剤の非存在下において1×SSCを含む溶液で洗浄すること、を包含する条件下で、配列番号2、配列番号5、配列番号8および配列番号13からなる群より選択される核酸配列を有する核酸分子とハイブリダイズし、ここで、該少なくとも35ヌクレオチド核酸分子が、オクトパミンに結合するタンパク質をコードする、核酸分子;ならびに
(b)該(a)の核酸分子に完全に相補的な核酸分子、
からなる群より選択される、単離された核酸分子。 - 以下:
(a)配列番号30、配列番号33、配列番号36および配列番号39からなる群より選択される核酸配列を有する核酸分子、ならびに配列番号30、配列番号33、配列番号36および配列番号39からなる群より選択される核酸配列に対して少なくとも95%同一である改変体であって、ここで、該核酸分子が、オクトパミンに結合するタンパク質をコードする、核酸分子;
(b)(a)の核酸分子のフラグメントを含む核酸分子であって、該フラグメントが、少なくとも50ヌクレオチドの長さである、核酸分子;ならびに
(c)(a)または(b)の核酸分子に完全に相補的な核酸分子、
からなる群より選択される、単離された核酸分子。 - 以下:
(a)配列番号1、配列番号3、配列番号6および配列番号11からなる群より選択される核酸配列を有する核酸分子、ならびに配列番号1、配列番号3、配列番号6および配列番号11からなる群より選択される核酸配列に対して少なくとも95%同一である改変体であって、ここで、該核酸分子が、オクトパミンに結合するタンパク質をコードする、核酸分子;
(b)(a)の核酸分子のフラグメントを含む核酸分子であって、該フラグメントが、少なくとも35ヌクレオチドの長さである、核酸分子;ならびに
(c)(a)または(b)の核酸分子に完全に相補的な核酸分子、
からなる群より選択される、単離された核酸分子。 - 配列番号31、配列番号34、配列番号37および配列番号40からなる群より選択されるアミノ酸配列を含む単離されたタンパク質、ならびに配列番号31、配列番号34、配列番号37および配列番号40からなる群より選択されるアミノ酸配列に対して少なくとも95%同一である改変体であって、ここで、該タンパク質改変体が、オクトパミンに結合する、単離されたタンパク質。
- 配列番号4、配列番号7および配列番号12からなる群より選択されるアミノ酸配列を含む単離されたタンパク質、ならびに配列番号4、配列番号7および配列番号12からなる群より選択されるアミノ酸配列に対して少なくとも95%同一である改変体であって、ここで、該タンパク質改変体が、オクトパミンに結合する、単離されたタンパク質。
- 請求項1〜4のいずれか1項に記載の単離された核酸分子によりコードされるタンパク質を生産する方法であって、該方法は、該タンパク質をコードする核酸分子で形質転換した細胞を培養する工程を包含する、方法。
- 転写制御配列に作動可能に連結された、請求項1〜4に記載の核酸分子を含む組換え分子。
- 請求項1〜4に記載の核酸分子を含む組換えウイルス。
- 請求項1〜4に記載の核酸分子を含む組換え細胞。
- 請求項1および3に記載の核酸分子であって、該核酸分子は、配列番号30、配列番号33、配列番号36および配列番号39からなる群より選択される核酸配列を含む、核酸分子。
- 請求項2および4に記載の核酸分子であって、該核酸分子は、配列番号1、配列番号3、配列番号6および配列番号11からなる群より選択される核酸配列を含む、核酸分子。
- 請求項1および3に記載の核酸分子であって、該核酸分子は、配列番号31、配列番号34、配列番号37および配列番号40からなる群より選択されるアミノ酸配列を含むタンパク質をコードする、核酸分子。
- 請求項2および4に記載の核酸分子であって、該核酸分子は、配列番号4、配列番号7および配列番号12からなる群より選択されるアミノ酸配列を含むタンパク質をコードする、核酸分子。
- ダニのオクトパミンレセプター活性のインヒビターを検出する方法であって、該方法は、以下:
(a)請求項5または6に記載の単離されたオクトパミンレセプタータンパク質を、推定の阻害性化合物の非存在下で、該タンパク質が、ダニのオクトパミンレセプタータンパク質活性を有する条件下で、該化合物と接触させる工程、および
(b)該推定の阻害性化合物が、ダニのオクトパミンレセプタータンパク質活性を阻害するか否かを決定する工程、
を包含する、方法。 - 請求項5または6に記載のタンパク質に選択的に結合する、単離された抗体。
- 請求項5に記載のタンパク質であって、該タンパク質は、配列番号30、配列番号33、配列番号36および配列番号39からなる群より選択される核酸配列を有する核酸分子によってコードされる、タンパク質。
- 請求項5に記載のタンパク質であって、該タンパク質は、配列番号31、配列番号34、配列番号37および配列番号40からなる群より選択されるアミノ酸配列を含む、タンパク質。
- 請求項6に記載のタンパク質であって、該タンパク質は、配列番号1、配列番号3、配列番号6および配列番号11からなる群より選択される核酸配列を有する核酸分子によってコードされる、タンパク質。
- 請求項6に記載のタンパク質であって、該タンパク質は、配列番号4、配列番号7および配列番号12からなる群より選択されるアミノ酸配列を含む、タンパク質。
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US31940202P | 2002-07-17 | 2002-07-17 | |
US42660102P | 2002-11-15 | 2002-11-15 | |
PCT/US2003/021706 WO2004009762A2 (en) | 2002-07-17 | 2003-07-11 | Flea and tick octopamine receptor nucleic acid molecules, proteins and thereof |
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JP2009091604A Pending JP2009159980A (ja) | 2002-07-17 | 2009-04-03 | ノミおよびダニのオクトパミンレセプター核酸分子、タンパク質およびそれらの使用 |
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US (2) | US7419793B2 (ja) |
EP (1) | EP1543325A4 (ja) |
JP (2) | JP2005532820A (ja) |
AU (2) | AU2003265269B2 (ja) |
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US5344776A (en) * | 1991-03-28 | 1994-09-06 | The United States Of America As Represented By The Department Of Health And Human Services | DNA encoding an insect octopamine receptor |
US6063610A (en) | 1996-11-12 | 2000-05-16 | Heska Corporation | Carboxylesterase nucleic acid molecules, proteins and uses thereof |
WO1999021891A1 (en) | 1997-10-27 | 1999-05-06 | Baylor College Of Medicine | Invertebrate octopamine receptor |
AU775084B2 (en) | 1999-04-09 | 2004-07-15 | Heska Corporation | Flea head, nerve cord, hindgut and malpighian tubule nucleic acid molecules, proteins and uses thereof |
CA2425872A1 (en) * | 2000-12-01 | 2002-06-06 | Heska Corporation | Flea gaba receptor subunit nucleic acid molecules, proteins and uses thereof |
US7419793B2 (en) | 2002-07-17 | 2008-09-02 | Heska Corporation | Flea octopamine receptor nucleic acid molecules, proteins and uses thereof |
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2003
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EP1543325A2 (en) | 2005-06-22 |
US7932371B2 (en) | 2011-04-26 |
WO2004009762A9 (en) | 2004-04-01 |
AU2010200627A1 (en) | 2010-03-11 |
US20090017494A1 (en) | 2009-01-15 |
AU2003265269B2 (en) | 2009-11-26 |
CA2492498A1 (en) | 2004-01-29 |
WO2004009762A2 (en) | 2004-01-29 |
US7419793B2 (en) | 2008-09-02 |
US20050287529A1 (en) | 2005-12-29 |
WO2004009762A3 (en) | 2004-05-27 |
EP1543325A4 (en) | 2008-02-20 |
CA2492498C (en) | 2013-09-10 |
AU2003265269A1 (en) | 2004-02-09 |
JP2009159980A (ja) | 2009-07-23 |
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