JPWO2019159934A1 - 融合タンパク質、及びそれを用いた高密度リポタンパク質の測定キット - Google Patents
融合タンパク質、及びそれを用いた高密度リポタンパク質の測定キット Download PDFInfo
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Abstract
融合タンパク質は、レクチン様酸化LDL受容体:LOX−1へ結合するLOX−1結合タンパク配列に、アポリポタンパク質A1:ApoA Iの全タンパク配列又は部分フラグメントタンパク配列が、直接又はスペーサを介して連結されている。
検体試料中の変性高密度リポタンパク質の測定方法は、LOX−1と抗ApoA I抗体とに対して、変性高密度リポタンパク質が結合することによる変性高密度リポタンパク質の測定方法であって、この融合タンパク質を変性高密度リポタンパク質の標準品として用いつつ検体試料中の変性高密度リポタンパク質を、光学的検知及び/又は放射線量検知で、前記標準品との比較によって求めるというものである。
Description
LOX−1タンパク質と抗ApoA I抗体の両方に親和性を持つタンパク質を得るため、先ず、抗LOX−1抗体遺伝子(可変領域(Fv領域))とApoA I遺伝子からなる融合タンパク質遺伝子を作製した。Fv型抗体遺伝子は、マウスモノクローナル抗ヒトLOX−1抗体(#10−1)産生ハイブリドーマのcDNAからクローニングした可変領域遺伝子をもとに作製したanti−LOX−1−Fv遺伝子を用いた(”LOX-1-MT1-MMP axis is crucial for RhoA and Rac1 activation induced by oxidized low-density lipoprotein in endothelial cells.”, Sugimoto K, et al., Cardiovasc Res, Vol.84, p127-136, 2009に記載)。ApoA I遺伝子はヒト肝臓cDNAよりクローニングした。
ヒトApoA I全長遺伝子(GenBank accession no. NM000039.2; 1,239 bp)を、Human MTC Panel II (Clontech)の肝臓cDNAライブラリーを鋳型とし、プライマーセットApoAI-F (CACCATGAAAGCTGCGGTGCTGACCTTG)(配列番号2), ApoAI-R (CTGGGTGTTGAGCTTCTTAGTGTAC)(配列番号3)、PrimeSTAR HS DNA Polymerase (TAKARA#R010A)を用いてPCRを行い、ApoA I全長遺伝子を得た。次に、この全長遺伝子をpcDNA Gateway Directional TOPO Expression kit (Invitrogen)を用いてpcDNA6.2/V5/GW/D-TOPO vector (Invitorgen)にサブクローニングし、ABI PRISM Cycle sequencing kitにより塩基配列を確認した。
Fv型抗LOX−1抗体のC末端側とApoA IのN末端側とがリンカー配列を介して連結された融合タンパク質を、以下の手順により作製した。
上記のFv型抗LOX−1抗体発現ベクターを鋳型とし、フォワードプライマー(Fv-H-Fプライマー:CACCATGGATTTTGGGCTGATTTTTTTTA)(配列番号4)及び3’末端にリンカー配列の一部を含むリバースプライマー(Fv-L-Linker-Rプライマー:ACCAGAGCCGCCGCCGCCGCTACCACCACCACCTTTCAACTCCAGCTTGGTCCC)(配列番号5)、PrimeSTAR HS DNA Polymerase (TAKARA#R010A)を用いてPCRを行い、Fv型抗LOX−1抗体遺伝子を再増幅した。
また、上記で調製したApoA I全長遺伝子を鋳型とし、リンカー配列の一部を5’末端に含むフォワードプライマー(Linker-ApoAI-Fプライマー:AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCCGGCATTTCTGGCAGCAAGATGAAC)(配列番号6)及びリバースプライマー(ApoAI-Rプライマー:CTGGGTGTTGAGCTTCTTAGTGTACTC)(配列番号7)、PrimeSTAR HS DNA Polymerase (TAKARA#R010A)を用いてPCRを行い、ApoA Iをコードする遺伝子を再増幅した。
これらを、overlap−extension PCR法を用いることにより、Fv型抗体とApoA I(55-801bp)をそれぞれリンカー配列を介してVLのC末端側とApoA IのN末端側を連結させ、この融合タンパク質遺伝子をpEF6-V5-His vector (Invitrogen)に挿入した。
続いて、KOD-Plus- Mutagenesis Kit (TOYOBO#SMK-101)、Linker Rプライマー(GGATCCACCACCACCAGAGCCGCCG)(配列番号8)、ApoA1-31aa Fプライマー(CCCTGGGATCGAGTGAAGGACCTGG)(配列番号9)を用いて、ApoA Iの翻訳後切断部位Gln24(“cDNA cloning of human apoA-I: amino acid sequence of preproapoA-I.”, Law SW., et al., Biochem Biophys Res Commun, Vol.112, p257-264, 1983 参照)を含む19-30番目のアミノ酸配列を欠失させた。ABI PRISM Cycle sequencing kitにより塩基配列を確認し、これをタンパク質発現系に用いた。
融合タンパク質の発現はExpi293 Expression System (Invitrogen)を用いて行い、培地中に分泌された融合タンパク質をNi Sepharose excel resin (GE)を用いてHis精製した。得られたタンパク質はPBS中で透析した後、0.22μmのフィルターろ過滅菌を行い実験に使用した。
まず、ヒトEDTA血漿からKBr密度勾配遠心法により分離したHDLまたはLDLを、濃度が3mg/mlとなるようにPBSで調整し、硫酸銅を7.5μMとなるように添加した後、37℃、5%CO2インキュベーター内で16時間インキュベーションした。続いて、この溶液を、2mM EDTAを含有する0.15M塩化ナトリウム溶液を外液として透析し、ヒト酸化HDL、酸化LDLとした。
受容体LOX−1に、この酸化HDLが結合するかどうかをLOX−1タンパク質と抗ApoA I抗体を用いたサンドイッチELISAにより検討した。
組換えヒトLOX−1(61−273aa)または対照としてLOX−1と同じファミリーに属するタンパク質Dectin−1を、ELISAプレートに4℃で終夜固相化した。PBSで洗浄した後、3%BSA含有HEPESバッファー(10mM HEPES,150mM NaCl,pH7.4)によりブロッキングした。PBSで3回洗浄後、酸化HDL、HDL、酸化LDL、LDLをそれぞれ添加しインキュベートした。PBSで洗浄した後、ニワトリ抗ApoA Iモノクローナル抗体(chicken monoclonal anti-ApoB antibody)あるいはHRP標識抗ApoA Iポリクローナル抗体(Sheep polyclonal anti-ApoB antibody-HRP)を添加した。ニワトリ抗ApoA Iモノクローナル抗体を用いる場合には、ニワトリ抗ApoA Iモノクローナル抗体を添加し、室温で1時間反応させた後、PBSで洗浄した後、二次抗体HRP-labeled Donkey anti-chicken IgYを添加し、室温で1時間インキュベートした。
抗体反応後PBSで5回洗浄し、TMB solutionをプレートに添加し室温で反応させた。2M硫酸で反応を停止させ、450nmの吸光度をSpectraMax 340PC384 (Molecular Devices)を用いて測定し、結合した酸化HDLを検出した。
前記の試験例1で作製した人工酸化HDLと、前記調製例1で作製した融合タンパク質とを夫々用い、前記試験例1の方法に準じ、人工酸化HDL又は融合タンパク質のLOX−1及び抗ApoA I抗体への結合を検討した。その結果を、図3中に、●で示す。図3から明らかな通り、作製した融合タンパク質は、LOX−1と抗ApoA I抗体に同時に結合し、同図中、△で示すように、LOX−1を固相化していない場合には検出されなかった。このように、融合タンパク質も人工酸化HDLと同様の方法で、ELISAで検出できることが分かった。
ヒト血漿より調製したHDLを人工的に酸化させた酸化HDLについて、ロット間の違いにより測定値にどの程度影響するのかを、LOX−1と抗ApoA I抗体を用いたサンドイッチELISA(上記(試験例1))により検討した。また、本実施例で作製した融合タンパク質でのロット間の違いも同様に検討し、融合タンパク質の有用性を検証した。その結果を、図4に示す。図中、〇、●、▲はロットが異なる人工酸化HDL又は融合タンパク質を示す。人工酸化LDL(oxHDL)は3つの異なるロットで反応性に差が見られた。一方、融合タンパク質ではロット間でほとんど差はみられなかった。
前記試験例1の方法に準じ、ヒト血液中の変性HDL検出を試みた。測定検体には、健常人ボランティア6名から採取したEDTA血漿を用いた。
結果を図5に示す。すなわち、LOX−1と抗ApoA I抗体を用いたサンドイッチELISAにより、ヒト血液中の変性HDLを検出できることが示された。
Claims (13)
- LOX−1へ結合するLOX−1結合タンパク配列に、ApoA Iの全タンパク配列又は部分フラグメントタンパク配列が、直接又はスペーサを介して連結されていることを特徴とする融合タンパク質。
- 前記LOX−1結合タンパク配列が、抗LOX−1抗体、又はそれのLOX−1結合ドメイン配列であることを特徴とする請求項1に記載の融合タンパク質。
- 前記抗LOX−1抗体、又はLOX−1結合ドメイン配列が、LOX−1に抗原抗体反応により結合する抗体への活性部位を有していることを特徴とする請求項2に記載の融合タンパク質。
- 前記抗LOX−1抗体が、前記活性部位を、Fv型抗体の活性部位とすることを特徴とする請求項3に記載の融合タンパク質。
- 前記ApoA Iの部分フラグメントタンパク配列が、ヒトApoA Iにおけるアミノ酸番号31−267aaの領域を含むものである請求項1〜4の何れかに記載の融合タンパク質。
- 請求項1〜5の何れかに記載の融合タンパク質をコードするものであることを特徴とする核酸。
- 請求項6に記載の核酸が導入されていることを特徴とするベクター。
- 請求項7に記載のベクターを有することを特徴とする細胞。
- LOX−1と抗ApoA I抗体とに対して、変性高密度リポタンパク質が結合することによる変性高密度リポタンパク質の測定方法であって、請求項1〜3の何れかに記載の融合タンパク質を変性高密度リポタンパク質の標準品として用いつつ前記標準品と検体試料中の変性高密度リポタンパク質とを、光学的検知及び/又は放射線量検知で、前記標準品との比較によって求めることを特徴とする検体試料中の変性高密度リポタンパク質の測定方法。
- LOX−1と抗ApoA I抗体とに対して、変性高密度リポタンパク質が結合することによる変性高密度リポタンパク質の測定キットであって、請求項1〜3の何れかに記載の融合タンパク質を変性高密度リポタンパク質の標準品として用いつつ、光学的検知及び/又は放射線量検知で測定するためのものであることを特徴とする変性高密度リポタンパク質測定キット。
- 検体試料中の前記変性高密度リポタンパク質の濃度及び質を、光学的検知及び/又は放射線量検知で、前記標準品との比較によって求めるためのものであることを特徴とする請求項10に記載の変性高密度リポタンパク質の測定キット。
- LOX−1及び/又は抗ApoA I抗体をさらに含む請求項10〜11の何れかに記載の変性高密度リポタンパク質の測定キット。
- 前記検体試料が、全血、血漿又は血清であることを特徴とする請求項10〜12の何れかに記載の変性高密度リポタンパク質の測定キット。
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