JP2017145221A - Tauタンパク質凝集阻害剤 - Google Patents
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Abstract
Description
<実施形態1>
<概要>
<構成>
<試験>
低温真空法によって得た黒ガリンガル乾燥粉末2gを40mLの50%エタノール水溶液(50%EtOH)に懸濁し、80℃のウォーターバス中で1時間抽出した。その後、10,000rpmで15分間, 20℃で遠心分離後、上清を回収し各抽出液とした。50%EtOH抽出液中のエキス固形分濃度は、7.40 mg/mL(50%EtOH)であった。
Tauタンパク質は、His-tag融合タンパク質として発現するプラスミドベクター(3R MBD: 3repeat-microtubule binding domain)を用いて培養、精製したものを用いる。チオフラビンT(ThT)蛍光強度を測定することにより、Tauタンパク質凝集を評価する。
Tauタンパク質は凝集するとβ-sheet構造を形成するとされている。チオフラビンT(ThT)はタンパク質のβ-sheet構造を認識して蛍光を発するため、この特徴を利用してTauタンパク質の凝集が確認できる。さらに蛍光強度を比較することにより、凝集の程度を比較することができる。(参考文献1「Okuyama K, Nishiura C, Mizushima F, Minoura K, Sumida M, Taniguchi T, et al (2008).Linkage-dependent contribution of repeat peptides to self-aggregation of three- or four-repeat microtubule-binding domains in tau protein. FEBS J. 275(7): 1529-39」、参考文献2「Sugino E, Nishiura C, Minoura K, In Y, Sumida M, Taniguchi T, et al (2009).Three-/four-repeat-dependent aggregation profile of tau microtubule-binding domain clarified by dynamic light scattering analysis. Biochem Biophys Res Commun. 385(2): 236-40」)
プラスミドベクターをタンパク質大量発現用大腸菌BL21(DE3)に形質転換し、作成したグリセロールストックを用いる。(参考文献3「Yao TM, Tomoo K, Ishida T, Hasegawa H, Sasaki M, Taniguchi T (2003).Aggregation analysis of the microtubule binding domain in tau protein by spectroscopic methods. J Biochem. 134(1): 91-9」)
≪方法≫
培養・回収した菌体を、菌体量1Lあたり10 mLのBuffer(50 mM Tris-HCl、50 mM NaCl pH7.6)で懸濁し、超音波破砕を行う(超音波破砕:1秒+氷冷:2秒 を40回)。この懸濁液を遠心分離した後、上清を回収し、アフィニティークロマトグラフィー(Ni Chelating)を用いて単離精製を行う。
・結合Buffer:50 mM Tris-HCl/500 mM NaCl/10 mM Imidazole、pH7.6
・溶出Buffer:50 mM Tris-HCl/500 mM NaCl/100 mM Imidazole、pH7.6
凝集反応:
1.5 mlマイクロチューブ(BIO-BIK)に、以下の順に試薬及びTau溶液を添加する。
(1)50 mM Tris-HCl (pH7.6) 150 μL
(2)被験物質、陰性対照(Waterまたは50%EtOH) 10 μL
(3)100 μM Tau (3R-MBD) 20 μL
(4)100 μM Heparin 20 μL
Total 200 μL
上記を添加後、ボルテックスミキサーで撹拌し、アルミホイルで覆って遮光後37℃、16時間インキュベートする。
インキュベート終了後、各反応液を96 well plateへ135 μLずつ添加し(n=3)、Perkin Elmer ARVO Wallac1420にて自家蛍光を測定する。(励起波長:440nm、測定波長:486 nm)さらにこの反応液に100 μM ThT溶液を15 μLずつ添加し、プレートミキサーにかけた後、同波長で測定する。
陰性対照の測定値を100%として各被験物質の測定値を%換算し、さらに100%からこの%換算値を引いて凝集阻害率とする。
下記の表2は、蒸留水(DW)で抽出した黒ガリンガル抽出液の濃度を200 mg/mLから30 mg/mLまで下げながら得た3つの試料についての凝集阻害率を示している。また、図1の上のグラフは、各試料の凝集阻害率を示したものである。いずれの濃度においてもTauタンパク質凝集阻害作用を確認することができる。
<実施形態2>
<概要>
<試験>
チオフラビンT(ThT)蛍光強度を測定することにより、Aβタンパク質凝集を評価する。
Aβタンパク質は凝集するとβ-sheet構造を形成するとされている。チオフラビンT(ThT)はタンパク質のβ-sheet構造を認識して蛍光を発するため、この特徴を利用してAβタンパク質の凝集が確認できる。さらに蛍光強度を比較することにより、凝集の程度を比較することができる。(参考文献4「Hudson SA, Ecroyd H, Kee TW, Carver JA. (2009) The thioflavin T fluorescence assay for amyloid fibril detection can be biased by the presence of exogenous compounds FEBS J.; 276 (20): 5960-72.」、参考文献5「HARRY LEVINE. (1992) Thioflavine T interaction with synthetic Alzheimer's disease β-amyloid peptides:Detection of amyloid aggregation in solution. Department of Neuroscience Pharmacology, Parke-Davis Pharmaceutical Research Division, Warner-Lambert Company, Ann Arbor, Michigan 48106-1047」)
≪3.1 Aβタンパク質の準備≫
Aβタンパク質1-42:
インキュベート直前にAβタンパク質1-42(ペプチド研 大阪 4349-v Lot:611204)に0.1% NH3水を240 μL加え溶解後(500 μM)、PBSを960μL加える。この溶液をPBSにて20 μMに希釈する。
凝集反応:
反応系終濃度の100倍の被験物質溶液を蒸留水または50%EtOHで希釈し作成し、これをPBSにて50倍希釈する。384well plateに調製した各被験物質溶液を25μL添加し、さらに希釈したAβタンパク質1-42を25uLずつ添加する(n=3)。撹拌後アルミホイルで遮光し、37 ℃で24 時間インキュベートする。なお、黒ガリンガル抽出物は実施形態1の試験と同様に抽出した。また、溶媒としてエタノールの他に摂氏80度の蒸留水(DW)による抽出も行った。
インキュベート終了後、Perkin Elmer ARVO Wallac1420にて自家蛍光を測定する(励起389 nm、測定488 nm)。さらに反応液に6 μM ThT溶液を50 μLずつ添加し、プレートミキサーに30秒かけた後、同波長で測定する。
陰性対照の測定値を100%として各被験物質の測定値を%換算し、さらに100%からこの%換算値を引いて凝集阻害率とする。
下記の表4は、蒸留水(DW)で抽出した黒ガリンガル抽出液の濃度を200 mg/mLから10 mg/mLまで下げながら得た4つの試料についての凝集阻害率を示している。表5は50%エタノールで抽出した場合の凝集阻害率である。また、図2は、各試料の凝集阻害率をグラフとして示したものである。
<効果>
Claims (7)
- 黒ガリンガル(Kaempheria parviflora)抽出物を有効成分として含有するTauタンパク質凝集阻害剤。
- Aβタンパク質の凝集抑制作用を有する請求項1に記載のTauタンパク質凝集阻害剤。
- 請求項1又は2に記載のTauタンパク質凝集阻害剤を含有する医薬品。
- 請求項1又は2に記載のTauタンパク質凝集阻害剤を含有する食品。
- 請求項1又は2に記載のTauタンパク質凝集阻害剤を含有する医薬部外品。
- 請求項1又は2に記載のTauタンパク質凝集阻害剤を含有する食品添加物。
- 請求項1又は2に記載のTauタンパク質凝集阻害剤を含有する化粧品。
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