JP2007505163A - Sars感染の画像解析可能な動物モデル - Google Patents
Sars感染の画像解析可能な動物モデル Download PDFInfo
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Abstract
Description
本出願は、米国特許法第119条(e)項のもとで、米国仮特許出願第60/473,691号、出願日2003年5月27日、からの優先権の利益を主張するものであり、これは参考としてその全体を本明細書に含めるものとする。
本発明は、蛍光タンパク質のようなマーカーを発現するように操作された組換えコロナウイルスを使用する。モデル生物に前記の組換えコロナウイルスを感染させることによって、当業者は、宿主動物においてウイルス複製の進行を調べるために組換えウイルスを使用することができる。さらに、組換えコロナウイルスモデル系は、コロナウイルスの複製を遅延または抑制する抗ウイルス薬を同定するアッセイとして有用である。
本発明は、蛍光タンパク質のようなマーカーを発現するよう操作された組換えコロナウイルスを使用する。モデル生物に前記組換えコロナウイルスを感染させることによって、当業者は、組換えウイルスを使用して、宿主動物におけるウイルスの複製の進行を調べることができる。その上、組換えコロナウイルスモデル系は、コロナウイルス複製を遅らせ、または抑制する抗ウイルス薬を同定するためのアッセイとして有用である。
GFPトランスジェニックマウスで増殖したRFP発現腫瘍に基づく腫瘍-宿主相互作用に関するデュアルカラー蛍光イメージングモデルが、腫瘍血管新生、および腫瘍におけるリンパ球浸潤を含めた、腫瘍-間質相互作用のデュアルカラー可視化を可能にすることが、報告されている。ニワトリβ-アクチンプロモーターおよびサイトメガロウイルスエンハンサーのもとで、GFPを発現するトランスジェニックマウスを宿主として使用した(Okabe, M., et al., FEBS Lett (1997) 407:315-319)。このトランスジェニック系統から得られる組織はすべて、青色励起光の下で緑色の蛍光を発する。RFP発現B16F0(B16F0-RFP)マウスメラノーマ細胞は、pLNCX2-DsRed-2-RFPプラスミドを用いて形質導入された。B16F0-RFP腫瘍およびGFP発現宿主細胞を、同時にクリアに画像化することができた。高解像度デュアルカラー画像によって、腫瘍細胞および宿主細胞を1つ1つの細胞レベルに至るまで解像することが可能となった。線維芽細胞、腫瘍浸潤リンパ球、樹状細胞、血管および毛細血管を含めた宿主細胞は、RFP発現腫瘍細胞から容易に区別することができた。このデュアルカラー蛍光イメージング系は、腫瘍増殖時および腫瘍血管新生時の腫瘍-宿主相互作用を理解するための研究を促進するはずである。デュアルカラーキメラ系も、治療および診断/分析目的で、腫瘍浸潤リンパ球、および他の、腫瘍と相互作用する宿主間質細胞を分析して分離する有力な手段を与える。このモデルの原理は、本発明のデュアルカラーイメージング可能なRFP-MHV-GFP-宿主感染モデルにおいて使用される。
ウイルスおよび細胞:de Haanらの方法(Virol. (2002) 296:177-189)に従う。MHV-A59温度感受性(ts)変異株LA16、プラーククローニングされたMHV-JHM、および元のプラーククローニングされたMHV-JHMを19回無希釈継代した後に得られたウイルスサンプル(JHM19th)を使用する。RNAトランスフェクションおよびウイルスの増殖のためにマウスDBT細胞を使用する。
ウイルス特異的細胞内RNAの調製およびノーザン(RNA)ブロッティング:ウイルス特異的RNAをウイルス感染細胞から抽出する。細胞内RNA 1.5mgを変性させ、ホルムアルデヒド含有1%アガロースゲルで電気泳動する。分離されたRNAをナイロンフィルター上にブロットした。フィルター上のRNAをMHV RNAのさまざまな部分に特異的な32P-標識プローブとハイブリダイズする。
感染マウスをさまざまな投薬計画で治療し、薬物の有無でウイルスの複製を評価する。ウイルスの複製を減少させることに成功した薬物を治療薬として有望な候補として同定する。
Claims (12)
- 蛍光タンパク質と結合した標識コロナウイルスタンパク質もしくはその断片。
- タンパク質が構造タンパク質もしくは非構造タンパク質である、請求項1に記載の標識コロナウイルスタンパク質。
- 構造タンパク質が、ヌクレオカプシド リン酸化タンパク質、スパイク糖タンパク質、膜糖タンパク質、スモールエンベロープタンパク質もしくは血球凝集素-エステラーゼ糖タンパク質からなる一群から選択される、請求項2に記載の標識コロナウイルスタンパク質。
- 構造タンパク質が、SARSスパイク糖タンパク質(配列番号)である、請求項2に記載の標識コロナウイルスタンパク質。
- 構造タンパク質が、SARSスモールエンベロープタンパク質(配列番号)である、請求項2に記載の標識コロナウイルスタンパク質。
- 構造タンパク質が、SARSマトリックスタンパク質(配列番号)である、請求項2に記載の標識コロナウイルスタンパク質。
- 蛍光タンパク質が緑色もしくは赤色タンパク質である、請求項1に記載の標識コロナウイルスタンパク質。
- 請求項1〜7のいずれか1つに記載の標識コロナウイルスタンパク質をコードしているコロナウイルスを含んでなる感染の、画像解析可能な動物モデル。
- 蛍光タンパク質を発現する宿主である、請求項8に記載の画像解析可能な動物モデル。
- 動物モデルがトランスジェニック緑色蛍光タンパク質発現マウスを含んでなる、請求項9に記載の画像解析可能な動物モデル。
- 抗ウイルス薬をスクリーニングする方法であって、
テスト群の動物および対照群の動物を提供すること(ここで、各群の動物は請求項8〜10のいずれか1つに記載の動物モデルを含んでなる);
テスト群に抗ウイルス薬候補を投与すること;
テスト群および対照群によって生じる蛍光発光をモニターすること;
テスト群によって生じる蛍光発光を対照群と比較すること;および
対照群と比較してテスト群で蛍光を減少させる抗ウイルス薬候補を選択すること、
を含んでなる前記方法。 - 有効な抗ウイルス性ワクチンをスクリーニングする方法であって、
テスト群の動物および対照群の動物を提供すること(ここで、各群の動物は請求項8〜10のいずれか1つに記載の動物モデルを含んでなる);
テスト群に抗ウイルス性ワクチン候補を投与すること;
テスト群および対照群によって生じる蛍光発光をモニターすること;
テスト群によって生じる蛍光発光を対照群と比較すること;および
対照群と比較してテスト群で蛍光を減少させる抗ウイルス性ワクチン候補を選択すること、
を含んでなる前記方法。
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