JP5278942B2 - タンパク質短寿命化ペプチドをコードする遺伝子及びその使用方法 - Google Patents
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Description
項1. 以下の(1)〜(4)のいずれか塩基配列を有する、タンパク質短寿命化ペプチドをコードする遺伝子:
(1) 配列番号1の第1105〜1230番目の塩基で表される塩基配列;
(2) 配列番号2の第369〜410番目のアミノ酸で表されるアミノ酸配列をコードする塩基配列;
(3) 配列番号2の第369〜410番目のアミノ酸で表されるアミノ酸配列において、1又は複数個のアミノ酸が置換、付加、欠失若しくは挿入され、且つ配列番号2の第369〜410のアミノ酸で表されるペプチドと実質的に同一のタンパク質短寿命化活性を有するペプチドをコードする塩基配列;
(4) 上記(1)〜(3)の塩基配列とストリンジェントな条件でハイブリダイズし、且つ配列番号2の第369〜410番目のアミノ酸で表されるペプチドと実質的に同一のタンパク質短寿命化活性を有するペプチドをコードする塩基配列。
項2. プロモーター配列と、前記プロモーターの制御下にレポータータンパク質をコードする塩基配列及び項1に記載の遺伝子の塩基配列とを有する遺伝子構築物。
項3. レポータータンパク質がルシフェラーゼである、項2に記載の遺伝子構築物。
項4. 項2又は3のいずれかに記載の遺伝子構築物を含む発現ベクター。
項5. 項2〜4のいずれかに記載の遺伝子構築物を、一過的又は安定的に発現するように導入された生物又はその一部。
項6. 項5に記載の生物又はその一部を異なる条件下で培養し、レポータータンパク質のシグナルを測定し、プロモーターの転写活性を比較することからなる、プロモーター活性の分析方法。
項7. 項6に記載の生物又はその一部を被検化合物の存在下及び非存在下で培養し、レポータータンパク質のシグナルを測定し、プロモーターの転写活性を比較することからなる、前記被検化合物のプロモーターに対する影響を評価する方法。
項8. 以下の工程を含む、標的タンパク質を短寿命化させる方法:
(1) 項1に記載の遺伝子を、標的タンパク質をコードする遺伝子と連結させる工程、
(2) 工程(1)で得た組換え遺伝子を、発現ベクターに取り込む工程、
(3) 工程(2)で得た発現ベクターを宿主細胞に導入し、発現させる工程。
実施例1
ヒカリコメツキルシフェラーゼcDNAと、カルパイン蛋白質のうち60bpから951bpに分割したペプチド配列部分を連結したルシフェラーゼ群を作製した(図1)。なお、コントロール実験としてマウスオルニチンデカルボキシラーゼのPEST配列を連結した短寿命型ルシフェラーゼを用いた。これらを時計遺伝子マウスBmal1プロモーター下流に挿入したベクター群(Luc‐hCAPN‐C1、‐C2、‐C3、‐C4、‐C5、‐C6、 ‐C7、‐C8、‐C9、‐C12)を作製した。
各ベクター群1μgを、35mm培養ディッシュに播種した培養繊維芽細胞NIH3T3にリポフェクション法(リポフェクトアミンプラス)により導入し、24時間37℃で培養後、100μMのシクロヘキシミドを含むDMEM培地を加え、アトー社製AB2500クロノスにセットし、1分間隔で発光量を測定した。PEST配列を連結した短寿命型ルシフェラーゼをコントロールとして同様な方法で測定した。図2は測定した結果の例である。コントロールであるPEST配列を連結した短寿命型ルシフェラーゼは、最大発光強度から徐々に発光量が減衰し、約5時間で半分の発光量となった。代表的な発光の短い融合物としてLuc‐hCAPN‐C2、‐C9があるが、これらの場合、PEST配列を導入したものに比べ、3倍以上早くの発光活性が現象した。
Claims (8)
- 以下の(1)〜(2)のいずれか塩基配列を有する、タンパク質短寿命化ペプチドをコードする遺伝子:
(1) 配列番号1の第1105〜1230番目の塩基で表される塩基配列;
(2) 配列番号2の第369〜410番目のアミノ酸で表されるアミノ酸配列をコードする塩基配列; - プロモーター配列と、前記プロモーターの制御下にレポータータンパク質をコードする塩基配列及び請求項1に記載の遺伝子の塩基配列とを有する遺伝子構築物。
- レポータータンパク質がルシフェラーゼである、請求項2に記載の遺伝子構築物。
- 請求項2又は3のいずれかに記載の遺伝子構築物を含む発現ベクター。
- 請求項2〜4のいずれかに記載の遺伝子構築物を、一過的又は安定的に発現するように導入された非ヒト生物又はその一部あるいはヒトの培養細胞。
- 請求項5に記載の非ヒト生物又はその一部あるいはヒトの培養細胞を異なる条件下で培養し、レポータータンパク質のシグナルを測定し、プロモーターの転写活性を比較することからなる、プロモーター活性の分析方法。
- 請求項5に記載の非ヒト生物又はその一部あるいはヒトの培養細胞を被検化合物の存在下及び非存在下で培養し、レポータータンパク質のシグナルを測定し、プロモーターの転写活性を比較することからなる、前記被検化合物のプロモーターに対する影響を評価する方法。
- 以下の工程を含む、標的タンパク質を短寿命化させる方法:
(1) 請求項1に記載の遺伝子を、標的タンパク質をコードする遺伝子と連結させる工程、
(2) 工程(1)で得た組換え遺伝子を、発現ベクターに取り込む工程、
(3) 工程(2)で得た発現ベクターを宿主細胞に導入し、発現させる工程。
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