JP5576585B2 - Phosphorylated protein immunoassay reagent - Google Patents
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Description
本発明は、蛍光共鳴エネルギー転移（FRET）を利用したリン酸化タンパク質免疫測定用試薬に関する。 The present invention relates to a phosphorylated protein immunoassay reagent using fluorescence resonance energy transfer (FRET).
タンパク質の翻訳後修飾として、リン酸化、糖鎖付加、脂質付加などが知られ、これらの修飾によって、タンパク質の機能、細胞内での局在、細胞外への分泌などが制御されている。タンパク質の翻訳後修飾反応の中で、最も代表的なものがタンパク質のリン酸化である。リン酸化は細胞分裂や細胞内のシグナル伝達、酵素の活性調節、高次構造の維持など様々な生命現象に関係することが明らかとなっている。タンパク質の可逆的リン酸化は、細胞内外間のシグナル伝達において重要な役割を果たし、細胞シグナル伝達経路に関わる多くのタンパク質は、キナーゼによってリン酸化され、ホスファターゼによって脱リン酸化される。また、多くの疾病が特定タンパク質の異常なリン酸化/脱リン酸化と関連していることが証明されている。従って、タンパク質のリン酸化状態における異常を迅速かつ正確に検出することは、細胞内または細胞間のシグナル伝達事象を理解するのに大いに役立ち、様々な疾病状態を効果的に診断できる方法の開発にも寄与すると考えられる。 As post-translational modifications of proteins, phosphorylation, sugar chain addition, lipid addition, and the like are known. By these modifications, protein functions, intracellular localization, extracellular secretion, and the like are controlled. The most typical post-translational modification reaction of proteins is protein phosphorylation. Phosphorylation has been shown to be involved in various biological phenomena such as cell division, intracellular signal transduction, enzyme activity regulation, and maintenance of higher order structures. Protein reversible phosphorylation plays an important role in signal transduction between inside and outside the cell, and many proteins involved in cell signaling pathways are phosphorylated by kinases and dephosphorylated by phosphatases. Many diseases have also been demonstrated to be associated with abnormal phosphorylation / dephosphorylation of specific proteins. Therefore, the rapid and accurate detection of abnormalities in the phosphorylation state of proteins greatly helps to understand intracellular or intercellular signaling events, and the development of methods that can effectively diagnose various disease states. Will also contribute.
リン酸化タンパク質の分析は、従来から32P標識や、抗リン酸化チロシン抗体を利用するELISA法やウェスタンブロット法によって行われてきた。しかしながら、リン酸化タンパク質は微量でしかも不安定なため、その定量は非常に困難であり、また、これらの免疫化学的方法では、抗体の結合及びその後の洗浄などの煩雑な作業が必要である。 The analysis of phosphorylated proteins has been conventionally performed by 32 P labeling, ELISA method using anti-phosphotyrosine antibody or Western blotting. However, since the amount of phosphorylated protein is very small and unstable, its quantification is very difficult, and these immunochemical methods require complicated operations such as antibody binding and subsequent washing.
一方、「蛍光エネルギー転移」（FRET：Fluorescence resonance energy transfer）という現象を用いてタンパク間相互作用や構造変化を解析する技術がある。FRETは、２種類の蛍光物質（ドナーとアクセプター）間に見られる現象であり、ドナーの蛍光スペクトルとアクセプターの吸収スペクトルがオーバーラップし、かつ、両者が100オングストローム程度以内の距離に存在していると、ドナー色素の励起エネルギーがアクセプターに移動し、直接は励起していないアクセプター色素の方から蛍光が発せられる現象をいう（非特許文献１参照）。これまでFRETを免疫化学的方法に利用した測定系について種々の検討が行われている。例えば、免疫測定に用いる抗体と、測定対象である抗原のそれぞれに、互いにFRETを起こす蛍光物質を結合させて共存させたものを免疫測定用試薬として用い、被験物質を測定する手法が報告されている（特許文献１参照）。この方法では、前記免疫試薬を構成する抗体と抗原の間に抗原抗体反応による結合が生じている状態では、それぞれに結合した蛍光物質間でFRETが生じ、一方、該抗原と競合する物質（被験物質）が存在する状態では、前記抗体と被験物質との間で抗原抗体反応が生じることによってFRETが減少するので、そのFRETシグナルの変化を指標に被験物質の有無や量を分析することができる。しかしながら、この方法では、例えば、被験物質が微量である場合、免疫測定用試薬と被験物質を接触する前後において、FRETシグナルの変化が小さく、精度よく検出できない。また、FRETを起こすドナー発色団とアクセプター発色団との間に、リン酸化される部位を有する基質ドメインとリン酸化認識ドメインがリンカー配列を介して結合している直列融合ユニットからなるプローブを用いて、タンパク質のリン酸化・脱リン酸化を検出する方法が提案されているが（特許文献２）、この方法は細胞内のリン酸化酵素の活性をモニタリングしているだけであって、細胞内に内在するするリン酸化タンパク質を検出するものではない。 On the other hand, there is a technique for analyzing protein-protein interactions and structural changes using a phenomenon called “fluorescence resonance energy transfer” (FRET). FRET is a phenomenon observed between two types of fluorescent materials (donor and acceptor), where the donor's fluorescence spectrum and the acceptor's absorption spectrum overlap, and both exist within a distance of about 100 angstroms. And a phenomenon in which the excitation energy of the donor dye moves to the acceptor and fluorescence is emitted from the acceptor dye that is not directly excited (see Non-Patent Document 1). Various studies have been conducted on measurement systems using FRET for immunochemical methods. For example, a method for measuring a test substance using an immunoassay reagent in which an antibody used for immunoassay and an antigen to be measured are combined with a fluorescent substance that causes FRET to coexist with each other has been reported. (See Patent Document 1). In this method, in the state in which the antibody constituting the immunoreagent and the antigen are bound by an antigen-antibody reaction, FRET occurs between the fluorescent substances bound to each of the antigens, while the substance that competes with the antigen (test In the state where (substance) is present, FRET is reduced by an antigen-antibody reaction between the antibody and the test substance, so that the presence or amount of the test substance can be analyzed using the change in the FRET signal as an index. . However, in this method, for example, when the amount of the test substance is very small, the change in the FRET signal is small before and after contacting the immunoassay reagent and the test substance, and cannot be detected with high accuracy. In addition, a probe comprising a serial fusion unit in which a substrate domain having a site to be phosphorylated and a phosphorylated recognition domain are bonded via a linker sequence between a donor chromophore that causes FRET and an acceptor chromophore is used. A method for detecting phosphorylation / dephosphorylation of a protein has been proposed (Patent Document 2), but this method only monitors the activity of a phosphorylase in the cell, and is endogenous to the cell. It does not detect phosphorylated proteins.
本発明の目的は、FRETを利用した測定系において、測定対象となるリン酸化タンパク質が微量であっても、大きなFRETシグナル変化を生じ、リン酸化タンパク質を迅速かつ高感度に検出できる手段を提供することにある。 An object of the present invention is to provide a means capable of detecting a phosphorylated protein rapidly and with high sensitivity by causing a large FRET signal change even in a measurement system using FRET even if the amount of phosphorylated protein to be measured is very small. There is.
本発明者らは、上記課題を解決するため鋭意検討を重ねた結果、FRETを生じさせる２種の蛍光物質（ドナー蛍光タンパク質とアクセタプター蛍光タンパク質）のそれぞれにロイシンジッパーモチーフを付加すると、それらのロイシンジッパー相互作用によってFRETがオン状態におけるFRETシグナルが増強され、その結果、競合反応によりFRETがオフ状態になったときに大きなFRETシグナルの変化が得られることを見出し、本発明を完成させるに至った。 As a result of intensive studies to solve the above problems, the present inventors have added leucine zipper motifs to each of two types of fluorescent substances (donor fluorescent protein and acceptor fluorescent protein) that generate FRET. As a result of the zipper interaction, the FRET signal was enhanced when the FRET was turned on, and as a result, a large change in the FRET signal was obtained when the FRET was turned off by a competitive reaction, and the present invention was completed. .
即ち、本発明は以下の発明を包含する。
（１） ドナー蛍光タンパク質-ロイシンジッパーモチーフをリン酸化ペプチドにフレキシブルリンカーを介して結合させた第１複合体と、アクセプター蛍光タンパク質-ロイシンジッパーモチーフを抗リン酸化タンパク質抗体にフレキシブルリンカーを介して結合させた第２複合体とから構成される、リン酸化タンパク質免疫測定用試薬。
That is, the present invention includes the following inventions.
(1) A first complex in which a donor fluorescent protein-leucine zipper motif is bound to a phosphorylated peptide via a flexible linker, and an acceptor fluorescent protein-leucine zipper motif is bound to an anti-phosphorylated protein antibody via a flexible linker. A phosphorylated protein immunoassay reagent comprising the second complex.
（２） アクセプター蛍光タンパク質-ロイシンジッパーモチーフをリン酸化ペプチドにフレキシブルリンカーを介して結合させた第１複合体と、ドナー蛍光タンパク質-ロイシンジッパーモチーフを抗リン酸化タンパク質抗体にフレキシブルリンカーを介して結合させた第２複合体とから構成される、リン酸化タンパク質免疫測定用試薬。 (2) A first complex in which an acceptor fluorescent protein-leucine zipper motif is bound to a phosphorylated peptide via a flexible linker, and a donor fluorescent protein-leucine zipper motif bound to an anti-phosphorylated protein antibody via a flexible linker. A phosphorylated protein immunoassay reagent comprising the second complex.
（３） 前記リン酸化ペプチドが、測定対象のリン酸化タンパク質のリン酸化モチーフを含むものである、（１）または（２）に記載のリン酸化タンパク質免疫測定用試薬。
（４） 前記抗リン酸化タンパク質抗体が、測定対象のリン酸化タンパク質のリン酸化モチーフを特異的に認識するものである、（１）または（２）に記載のリン酸化タンパク質免疫測定用試薬。
(3) The phosphorylated protein immunoassay reagent according to (1) or (2), wherein the phosphorylated peptide contains a phosphorylated motif of a phosphorylated protein to be measured.
(4) The reagent for phosphorylated protein immunoassay according to (1) or (2), wherein the anti-phosphorylated protein antibody specifically recognizes a phosphorylated motif of a phosphorylated protein to be measured.
（５） 前記ドナー蛍光タンパク質とアクセタプター蛍光タンパク質との間に、蛍光共鳴エネルギー転移（FRET： Fluorescence resonance energy transfer）が生じる、（１）〜（４）のいずれかに記載のリン酸化タンパク質免疫測定用試薬。
（６） 前記リン酸化タンパク質がMAPキナーゼであって、リン酸化ペプチドがDHTGFL(pT)E(pY)であることを特徴とする、（１）〜（５）のいずれかに記載のリン酸化タンパク質免疫測定用試薬。
(5) The phosphorylated protein immunoassay according to any one of (1) to (4), wherein fluorescence resonance energy transfer (FRET) occurs between the donor fluorescent protein and the acceptor fluorescent protein. reagent.
(6) The phosphorylated protein according to any one of (1) to (5), wherein the phosphorylated protein is MAP kinase and the phosphorylated peptide is DHTGFL (pT) E (pY) Reagent for immunoassay.
（７） 前記リン酸化タンパク質がMAPキナーゼであって、リン酸化ペプチドがDHTGFL(pT)E(pY)Vであることを特徴とする、（１）〜（５）のいずれかに記載のリン酸化タンパク質免疫測定用試薬。
（８） （１）〜（７）のいずれかに記載のリン酸化タンパク質免疫測定用試薬を試料と接触させ、接触前後のFRETシグナルの変化を指標として、当該試料中のリン酸化タンパク質を測定する方法。
(7) The phosphorylation according to any one of (1) to (5), wherein the phosphorylated protein is MAP kinase and the phosphorylated peptide is DHTGFL (pT) E (pY) V. Reagent for protein immunoassay.
(8) The phosphorylated protein immunoassay reagent according to any one of (1) to (7) is brought into contact with a sample, and the phosphorylated protein in the sample is measured using a change in the FRET signal before and after the contact as an index. Method.
本発明のリン酸化タンパク質免疫測定用試薬は、試料中の微量なリン酸化タンパク質を、FRETの原理に基づいて迅速かつ高感度に測定できる。MAPキナーゼ（MAPK）をはじめとするリン酸化タンパク質は、様々な転写因子を活性化することにより細胞内の情報伝達に重要な役割を果たし、細胞の増殖・分化にも関わっている。また、タンパク質のリン酸化の異常は、ガン、糖尿病、免疫疾患などに関わっていることが知られており、組織・細胞中のリン酸化タンパク質を解析することは、上記の疾病状態を効果的に診断する上でも役立つ。従って、本発明のリン酸化タンパク質免疫測定用試薬は、細胞内外間のシグナル伝達機構に関する基礎研究のみならず、様々な疾患に対する診断・創薬などの臨床応用に有用である。 The reagent for immunoassay of phosphorylated protein of the present invention can measure a small amount of phosphorylated protein in a sample rapidly and with high sensitivity based on the principle of FRET. Phosphorylated proteins such as MAP kinase (MAPK) play an important role in intracellular signal transduction by activating various transcription factors, and are also involved in cell proliferation and differentiation. In addition, protein phosphorylation abnormalities are known to be related to cancer, diabetes, immune diseases, etc. Analyzing phosphorylated proteins in tissues and cells effectively improves the above disease states. It is also useful for diagnosis. Accordingly, the phosphorylated protein immunoassay reagent of the present invention is useful not only for basic research on signal transduction mechanisms between inside and outside cells but also for clinical applications such as diagnosis and drug discovery for various diseases.
以下、本発明を詳細に説明する。
１．リン酸化タンパク質免疫測定用試薬
本発明のリン酸化タンパク質免疫測定用試薬(以下、「免疫測定用試薬」という）は、ドナー蛍光タンパク質-ロイシンジッパーモチーフをリン酸化ペプチドにフレキシブルリンカーを介して結合させた第１複合体と、アクセプター蛍光タンパク質-ロイシンジッパーモチーフを抗リン酸化タンパク質抗体にフレキシブルリンカーを介して結合させた第２複合体とから構成される。
Hereinafter, the present invention will be described in detail.
1. Phosphorylated protein immunoassay reagent The phosphorylated protein immunoassay reagent of the present invention (hereinafter referred to as "immunoassay reagent") has a donor fluorescent protein-leucine zipper motif bound to a phosphorylated peptide via a flexible linker. It consists of a first complex and a second complex in which an acceptor fluorescent protein-leucine zipper motif is bound to an anti-phosphorylated protein antibody via a flexible linker.
また、本発明の免疫測定用試薬は、上記の第１複合体のドナー蛍光タンパク質と第２複合体中のアクセプター蛍光タンパク質を入れ換えてもよく、従って、本発明の別の態様の免疫測定用試薬は、アクセプター蛍光タンパク質-ロイシンジッパーモチーフをリン酸化ペプチドにフレキシブルリンカーを介して結合させた第１複合体と、ドナー蛍光タンパク質-ロイシンジッパーモチーフを抗リン酸化タンパク質抗体にフレキシブルリンカーを介して結合させた第２複合体とから構成される。 In addition, the immunoassay reagent of the present invention may replace the donor fluorescent protein in the first complex and the acceptor fluorescent protein in the second complex, and thus the immunoassay reagent of another aspect of the present invention. Has a first complex in which an acceptor fluorescent protein-leucine zipper motif is bound to a phosphorylated peptide via a flexible linker, and a donor fluorescent protein-leucine zipper motif is bound to an anti-phosphorylated protein antibody via a flexible linker. It is comprised from a 2nd composite_body | complex.
本発明において、測定対象とするリン酸化タンパク質の種類は、特に限定はされないが、セリン／スレオニンキナーゼの代表であるMAPキナーゼ系（MAPキナーゼカスケード）のMEK（MAPキナーゼ・キナーゼ）、MAPK（MAPキナーゼ）、extracellular signal-regulated kinases (ERK 1/2）、c-Jun N-terminal Kinases (JNK1-3)、p38（α/β/δ/γ)、RSK、Elk-1、MNKなど、あるいは、チロシンキナーゼの代表であるレセプターチロシンキナーゼのEGFレセプターファミリー(erbB1、 erbB2、 erbB3、 erbB4)、STATファミリー（STAT1，STAT3，STAT5）などが挙げられる。 In the present invention, the type of phosphorylated protein to be measured is not particularly limited, but is representative of serine / threonine kinases, MAP kinase system (MAP kinase cascade) MEK (MAP kinase kinase), MAPK (MAP kinase) ), Extracellular signal-regulated kinases (ERK 1/2), c-Jun N-terminal Kinases (JNK1-3), p38 (α / β / δ / γ), RSK, Elk-1, MNK, or tyrosine The EGF receptor family of receptor tyrosine kinases (erbB1, erbB2, erbB3, erbB4), the STAT family (STAT1, STAT3, STAT5), which are representative of kinases, can be mentioned.
本発明の免疫測定用試薬を構成する第１複合体（または第２複合体）におけるドナー蛍光タンパク質と第２複合体（または第１複合体）におけるアクセプター蛍光タンパク質は、蛍光共鳴エネルギー転移（FRET： Fluorescence resonance energy transfer）が起こるものであれば特に限定はされない。ここで、FRETは、励起波長が異なる２つの蛍光色素（ドナー及びアクセプター）間で励起エネルギーが移動する現象をいう。ドナーの励起波長の光を照射した場合、ドナーとアクセプターが近距離にあるときはFRETが起こり、ドナーの蛍光は減少してアクセプターの蛍光が増大するが、両者が離れるとFRETが起こらなくなり、ドナーの蛍光が増大してアクセプターの蛍光が減少する。ここで、FRETにおける「ドナー」とは、励起光を吸収しそのエネルギーの一部分は蛍光として発光し、他の一部分は近接するアクセプターに移動する蛍光物質をいい、FRETにおける「アクセプター」とは、直接励起光を受けなくても、ドナーからのエネルギーを吸収し蛍光を発する蛍光物質をいう。本発明においては、蛍光物質として、蛍光タンパク質を用いるが、蛍光強度を増強させることなどを目的として遺伝子工学的手法により改変された蛍光タンパク質であってもよい。好ましいドナー蛍光タンパク質とアクセプター蛍光タンパク質の組み合わせとしては、例えば、CFP（Cyan fluorescent protein）とYFP（yellow fluorescent proteinのペア、GFP（green fluorescent protein）とRFP（red fluorescent protein)のペアが挙げられる。 The donor fluorescent protein in the first complex (or second complex) constituting the immunoassay reagent of the present invention and the acceptor fluorescent protein in the second complex (or first complex) are fluorescent resonance energy transfer (FRET). There is no particular limitation as long as fluorescence resonance energy transfer) occurs. Here, FRET refers to a phenomenon in which excitation energy moves between two fluorescent dyes (donor and acceptor) having different excitation wavelengths. When irradiated with light at the excitation wavelength of the donor, FRET occurs when the donor and the acceptor are at a short distance, and the fluorescence of the donor decreases and the fluorescence of the acceptor increases. The fluorescence of the acceptor increases and the fluorescence of the acceptor decreases. Here, the “donor” in FRET is a fluorescent substance that absorbs excitation light, a part of its energy is emitted as fluorescence, and the other part moves to a nearby acceptor. The “acceptor” in FRET is directly A fluorescent substance that absorbs energy from a donor and emits fluorescence without receiving excitation light. In the present invention, a fluorescent protein is used as the fluorescent substance, but it may be a fluorescent protein modified by a genetic engineering technique for the purpose of enhancing the fluorescence intensity. Preferred combinations of donor fluorescent protein and acceptor fluorescent protein include, for example, a pair of CFP (Cyan fluorescent protein) and YFP (yellow fluorescent protein) and a pair of GFP (green fluorescent protein) and RFP (red fluorescent protein).
CFP、YFP、GFP、RFP、またはこれらの改変体は市販品から自由に入手できる。CFPとしてはECFP、YFPとしてはEYFP、Phi-Yellow、GFPとしてはEGFP、Cop-Green、RFPとしてはDsRed2、HcRed1、HcRed-Tandemなどが挙げられる。 CFP, YFP, GFP, RFP, or a variant thereof can be freely obtained from commercial products. Examples of CFP include ECFP, YFP includes EYFP, Phi-Yellow, GFP includes EGFP, Cop-Green, and RFP includes DsRed2, HcRed1, and HcRed-Tandem.
上記の第１複合体（または第２複合体）中のドナー蛍光タンパク質と第２複合体（または第１複合体）中のアクセプター蛍光タンパク質にそれぞれ結合させるロイシンジッパーモチーフとしては、互いに相補的な関係にあって、結合しうるロイシンジッパーモチーフであれば特に制限はされないが、Jun-LzipとJun-Lzip同士の組み合わせが好適に使用できる。 The leucine zipper motif that binds to the donor fluorescent protein in the first complex (or the second complex) and the acceptor fluorescent protein in the second complex (or the first complex) is complementary to each other. Thus, there is no particular limitation as long as it is a leucine zipper motif that can be bound, but a combination of Jun-Lzip and Jun-Lzip can be preferably used.
また、第１複合体、第２複合体中のフレキシブルリンカーは、第１複合体中のリン酸化ペプチドと第２複合体中の抗リン酸化抗体とが抗原抗体反応により結合した際にFRETが起きるように、蛍光タンパク質の位置を適宜調節し、また、各複合体に十分な柔軟性を付与するものである。フレキシブルリンカーは、通常、５〜２０アミノ酸残基のポリペプチドが好ましく、１５〜２０アミノ酸残基のポリペプチドがさらに好ましい。 The flexible linker in the first complex and the second complex causes FRET when the phosphorylated peptide in the first complex and the anti-phosphorylated antibody in the second complex are bound by an antigen-antibody reaction. As described above, the position of the fluorescent protein is appropriately adjusted, and sufficient flexibility is imparted to each complex. The flexible linker is usually preferably a polypeptide having 5 to 20 amino acid residues, and more preferably a polypeptide having 15 to 20 amino acid residues.
第１複合体を調製する場合は、まず、フレキシブルリンカー、ドナー蛍光タンパク質（またはアクセプター蛍光タンパク質）、ロイシンジッパーの融合タンパク質を調製し、この融合タンパク質にリン酸化ペプチドを結合させればよい。融合タンパク質とリン酸化ペプチドとの結合は、融合タンパク質中のS-S結合を還元してなるチオール基と反応しうるマレイミド基を結合させることによる両者のカップリング反応によって行うことができる。 When preparing the first complex, first, a fusion protein of a flexible linker, donor fluorescent protein (or acceptor fluorescent protein), and leucine zipper may be prepared, and a phosphorylated peptide may be bound to the fusion protein. The fusion protein and the phosphorylated peptide can be bound by a coupling reaction of both by binding a maleimide group capable of reacting with a thiol group obtained by reducing the S—S bond in the fusion protein.
第２複合体を調製する場合も同様に、まず、フレキシブルリンカー、アクセプター蛍光タンパク質（またはドナー蛍光タンパク質）、ロイシンジッパーの融合タンパク質を調製し、この融合タンパク質に前記リン酸化ペプチドに結合しうる抗リン酸化タンパク質抗体を結合させればよい。融合タンパク質と抗リン酸化タンパク質抗体との結合は、融合タンパク質中のS-S結合を還元してなるチオール基と反応しうるマレイミド基を結合させることによる両者のカップリング反応によって行うことができる。 Similarly, in the case of preparing the second complex, first, a flexible linker, an acceptor fluorescent protein (or donor fluorescent protein), and a leucine zipper fusion protein are prepared, and the anti-phosphorus that can bind to the phosphorylated peptide to the fusion protein. An oxidized protein antibody may be bound. The fusion protein and the anti-phosphorylated protein antibody can be bound by a coupling reaction of both by binding a maleimide group capable of reacting with a thiol group formed by reducing the S—S bond in the fusion protein.
融合タンパク質とリン酸化ペプチド（または抗リン酸化タンパク質抗体）とのカップリング反応は、上記のようなマレイミド法のほか、抗体とプロテインＧ、プロテインＡなどの相互作用、ビオチン・アビジン相互作用などの結合反応を利用して行ってもよい。 The coupling reaction between the fusion protein and the phosphorylated peptide (or anti-phosphorylated protein antibody) is not limited to the maleimide method as described above, but also includes the interaction between the antibody and protein G, protein A, etc., biotin / avidin interaction, etc. You may carry out using reaction.
融合タンパク質の調製は、例えば、リン酸化ペプチド（または抗リン酸化タンパク質抗体）とのカップリング反応をマレイミド法で行う場合、チオレドキシン、フレキシブルリンカー、蛍光タンパク質、ロイシンジッパーそれぞれのDNAフレームが一致するように連結してこれを発現ベクターに導入し、この発現ベクターで大腸菌等の適当な宿主を形質転換し、得られた形質転換体を培養し、その培養物から目的とする融合タンパク質を採取することにより行うことができる。培養後、融合タンパク質が菌体内又は細胞内に生産される場合には、菌体又は細胞を破砕することにより該タンパク質を抽出する。また、融合タンパク質が菌体外又は細胞外に生産される場合には、培養液をそのまま使用するか、遠心分離等により菌体又は細胞を除去する。その後、タンパク質の単離精製に用いられる一般的な生化学的方法、例えば硫酸アンモニウム沈殿、ゲルクロマトグラフィー、イオン交換クロマトグラフィー、アフィニティークロマトグラフィーなどを単独で又は適宜組み合わせて用いることにより、前記培養物中から融合タンパク質を単離精製することができる。 For example, when the coupling reaction with a phosphorylated peptide (or anti-phosphorylated protein antibody) is carried out by the maleimide method, the DNA frames of thioredoxin, flexible linker, fluorescent protein, and leucine zipper are matched. By ligating and introducing this into an expression vector, transforming a suitable host such as E. coli with this expression vector, culturing the resulting transformant, and collecting the desired fusion protein from the culture It can be carried out. When the fusion protein is produced in the microbial cells or cells after culturing, the protein is extracted by disrupting the microbial cells or cells. When the fusion protein is produced outside the cells or cells, the culture solution is used as it is, or the cells or cells are removed by centrifugation or the like. Thereafter, by using general biochemical methods used for protein isolation and purification such as ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc. alone or in appropriate combination, The fusion protein can be isolated and purified from
第１複合体中のリン酸化ペプチドは、対応する第２複合体中の抗リン酸化タンパク質抗体への抗原抗体反応による結合性を保持したものであれば特に限定はされず、該抗体の産生を誘起する免疫原として用いることができる抗原分子そのもの、抗原決定基を保持した抗原断片、及び免疫原性を有しないハプテンを包含する。また、当該リン酸化ペプチドは必ずしも抗リン酸化タンパク質抗体の産生を誘起する免疫原やその断片に限定されるものではなく、当該抗体と交差反応するものであってもよい。このようなリン酸化ペプチドとしては、測定対象とするリン酸化タンパク質のリン酸化モチーフを含むものであれば特に限定はされず、測定対象とするリン酸化タンパク質の種類に応じて適宜設計できる。例えば、リン酸化モチーフ部分を含んでアミノ酸数が３〜１５個のアミノ酸配列からなるものが好ましい。また、第１複合体中のリン酸化ペプチドは、被検物質と同一の物質であってもよいし、対応する抗リン酸化タンパク質抗体と抗原抗体反応するもの（すなわち、同一又は類似のエピトープを有するもの）であれば被検物質と異なる物質であってもよい。 The phosphorylated peptide in the first complex is not particularly limited as long as it retains the binding property by the antigen-antibody reaction to the anti-phosphorylated protein antibody in the corresponding second complex. It includes antigen molecules themselves that can be used as inducing immunogens, antigen fragments retaining antigenic determinants, and non-immunogenic haptens. The phosphorylated peptide is not necessarily limited to an immunogen or a fragment thereof that induces production of an anti-phosphoprotein antibody, and may cross-react with the antibody. Such a phosphorylated peptide is not particularly limited as long as it contains a phosphorylated motif of a phosphorylated protein to be measured, and can be appropriately designed according to the type of phosphorylated protein to be measured. For example, those consisting of an amino acid sequence containing 3 to 15 amino acids including a phosphorylated motif portion are preferred. In addition, the phosphorylated peptide in the first complex may be the same substance as the test substance, or one that undergoes an antigen-antibody reaction with the corresponding anti-phosphorylated protein antibody (that is, has the same or similar epitope) A substance different from the test substance.
第２複合体中の抗リン酸化タンパク質抗体は、第１複合体中のリン酸化ペプチドのリン酸化モチーフを特異的に認識し、当該ペプチドと抗原抗体反応による結合性を保持するものであればいかなるものでもよく、必ずしも該抗原を免疫原として得られる抗体に限定されない。また、当該抗体には、Fab、Fab'、F(ab')2、scFvなどの抗原結合性断片も含まれる。なお、本発明の免疫測定用試薬は、試料中の被検物質を競合法により免疫測定を行うものであるから、用いる抗体は、被検物質とも抗原抗体反応するものである。 Any anti-phosphorylated protein antibody in the second complex can be used as long as it specifically recognizes the phosphorylated motif of the phosphorylated peptide in the first complex and retains the binding property of the peptide by the antigen-antibody reaction. It is not necessarily limited to antibodies obtained using the antigen as an immunogen. The antibody also includes antigen-binding fragments such as Fab, Fab ′, F (ab ′) 2 and scFv. In addition, since the reagent for immunoassay of the present invention performs immunoassay on a test substance in a sample by a competitive method, the antibody to be used is an antigen-antibody reaction with the test substance.
２．リン酸化タンパク質の測定
本発明の免疫測定用試薬を用いてリン酸化タンパク質を測定する原理を、リン酸化MAPKを例として図１に基づいて説明する。
本発明の免疫測定用試薬は、競合物質(リン酸化MAPK)が存在しない状態では、第１複合体中のリン酸化ペプチド（DHTGFL(pT)E(pY)またはDHTGFL(pT)E(pY)V）と、第２複合体中の抗リン酸化タンパク質抗体（抗MAPK抗体）との間に抗原抗体反応による結合が生じ、かつ、ドナー蛍光タンパク質とアクセプター蛍光タンパク質にそれぞれに付加しているロイシンジッパーモチーフの相互作用が働いている（図１の左図）。この状態では、ドナー蛍光タンパク質の励起波長を照射すると、ドナー蛍光タンパク質とアクセプター蛍光タンパク質の間が近接しているため蛍光共鳴エネルギー転移(FRET)が生じ（FRETオン）、アクセプター蛍光タンパク質の蛍光が観察される。この反応系に、競合物質(リン酸化MAPK)が存在すると、その競合物質は第１複合体のリン酸化ペプチドと競合的に第２複合体の抗体部位に結合する（図１の右図）。そのため、被検試料中の競合物質の量に応じて第１複合体と第２複合体の抗原抗体反応が解消され、その結果、FRETが解消し（FRETオフ）、アクセプターの蛍光は減少してドナーの蛍光が増大する。従って、本発明の免疫測定用試薬を試料と接触させた際に、FRETシグナルの変化を観察することにより、試料中の被検物質（すなわち、測定対象とするリン酸化タンパク質）の有無やその存在量を測定することができる。なお、「測定」には検出と定量の両者が包含される。
2. Measurement of Phosphorylated Protein The principle of measuring phosphorylated protein using the immunoassay reagent of the present invention will be described based on FIG. 1 using phosphorylated MAPK as an example.
In the absence of a competitor (phosphorylated MAPK), the immunoassay reagent of the present invention is a phosphorylated peptide (DHTGFL (pT) E (pY) or DHTGFL (pT) E (pY) V in the first complex. ) And the anti-phosphorylated protein antibody (anti-MAPK antibody) in the second complex are bound by the antigen-antibody reaction and added to the donor fluorescent protein and the acceptor fluorescent protein respectively. (Fig. 1 left). In this state, when the excitation wavelength of the donor fluorescent protein is irradiated, fluorescence resonance energy transfer (FRET) occurs (FRET on) due to the close proximity between the donor fluorescent protein and the acceptor fluorescent protein, and the fluorescence of the acceptor fluorescent protein is observed. Is done. When a competitor (phosphorylated MAPK) is present in this reaction system, the competitor binds competitively with the phosphorylated peptide of the first complex to the antibody site of the second complex (right diagram in FIG. 1). Therefore, the antigen-antibody reaction between the first complex and the second complex is eliminated according to the amount of the competitor in the test sample, and as a result, FRET is eliminated (FRET off), and the acceptor fluorescence decreases. Donor fluorescence increases. Therefore, when the immunoassay reagent of the present invention is brought into contact with the sample, the presence or absence of the test substance (that is, the phosphorylated protein to be measured) in the sample is observed by observing the change in the FRET signal. The amount can be measured. “Measurement” includes both detection and quantification.
本発明の免疫測定用試薬を用いた測定は、該免疫測定用試薬を試料と接触させた後、ドナー蛍光タンパク質の励起波長の光を照射し、それにより生じる蛍光の波長を測定することにより行う。免疫測定用試薬と試料との接触時間は、特に限定されないが、通常、
０分〜60分程度が例示できる。また、使用する試薬の濃度は、予想される被検物質濃度等に応じて適宜設定することができるが、通常、1nM〜10μM程度である。
The measurement using the immunoassay reagent of the present invention is carried out by bringing the immunoassay reagent into contact with the sample, irradiating light of the excitation wavelength of the donor fluorescent protein, and measuring the wavelength of the resulting fluorescence. . The contact time between the immunoassay reagent and the sample is not particularly limited.
Examples are about 0 to 60 minutes. The concentration of the reagent to be used can be appropriately set according to the expected concentration of the test substance and the like, but is usually about 1 nM to 10 μM.
以下、実施例により本発明をさらに具体的に説明する。但し、本発明はこれらに限定されるものではない。
（実施例１） 免疫測定用試薬の調製
（1) 蛍光融合タンパク質発現ベクターの作製
N末端から、チオレドキシン（Trx）、フレキシブルリンカー（FL4）、蛍光タンパク質（ECFPまたはEYFP）、ロイシンジッパー（LzipJun）からなる融合タンパク質の発現ベクターは、既報（Analytical Chemistry 2002年74号p5786-5792）に記載の抗ヒトアルブミン抗体-蛍光タンパク質-ロイシンジッパー融合タンパク質発現ベクターから抗体遺伝子を除去することにより作製した。
Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these.
(Example 1) Preparation of immunoassay reagent (1) Preparation of fluorescent fusion protein expression vector
The fusion protein expression vector consisting of thioredoxin (Trx), flexible linker (FL4), fluorescent protein (ECFP or EYFP), and leucine zipper (LzipJun) from the N-terminus has been reported in the previous report (Analytical Chemistry 2002 No. 74, p5786-5792). It was prepared by removing the antibody gene from the described anti-human albumin antibody-fluorescent protein-leucine zipper fusion protein expression vector.
具体的には、発現ベクターpET32からなるpET32/Trx-ScFv（No.13）-FL4-EYFP-Lzip（Jun）およびpET32/Trx-ScFv（No.11）-FL4-ECFP-Lzip（Jun）を制限酵素EcoRV（TAKARA）およびHindIII（TAKARA）で処理し、1%アガロースゲル電気泳動にかけ、抗アルブミン抗体ScFv断片以外のベクター部分を切り出し精製した。なお、上記FL4は、フレキシブルリンカー（GGGGS)4を示す。次に、チオレドキシン、フレキシブルリンカー、蛍光タンパク質、ロイシンジッパーが正確に翻訳されるようにHind IIIリンカー（TAKARA）を導入し、ライゲーションすることによって2種類の発現ベクター、発現ベクターA：pET32/Trx-FL4-ECFP-Lzip（Jun）および発現ベクターB：pET32/Trx-FL4-EYFP-Lzip（Jun）を構築した。 Specifically, pET32 / Trx-ScFv (No.13) -FL4-EYFP-Lzip (Jun) and pET32 / Trx-ScFv (No.11) -FL4-ECFP-Lzip (Jun) comprising the expression vector pET32 It was treated with restriction enzymes EcoRV (TAKARA) and HindIII (TAKARA), and subjected to 1% agarose gel electrophoresis, and the vector part other than the anti-albumin antibody ScFv fragment was cut out and purified. FL4 represents flexible linker (GGGGS) 4. Next, by introducing and ligating Hind III linker (TAKARA) so that thioredoxin, flexible linker, fluorescent protein, and leucine zipper are correctly translated, two expression vectors, expression vector A: pET32 / Trx-FL4 -ECFP-Lzip (Jun) and expression vector B: pET32 / Trx-FL4-EYFP-Lzip (Jun) was constructed.
(2) 蛍光融合タンパク質Trx-FL4-ECFP-Lzip(Jun)およびTrx-FL4-EYFP-Lzip(Jun）の発現及び精製
(1)にて作製した発現ベクターAおよび発現ベクターBを用いて、大腸菌Origami(DE3)（Novagen社）の形質転換を行った。形質転換した大腸菌を5ml LB（50μｇ/mlアンピシリン、15μg/mlカナマイシン、12.5μg/mlテトラサイクリン）培地にて30℃で一晩振とう培養した。翌日、培養細胞を1.5l LB培地に植え継ぎ、30℃で培養を続けた。培養液の濁度がO.D.600が約0.5に達したところ（約６時間培養）で、0.1M IPTGを1.5ml添加し、温度を16℃に下げ、一晩培養を続けた。翌日、遠心分離により菌体を回収し、菌体破砕液(50mM NaH2PO4-NaOH、300mM NaCl、10mM imidazole, pH8.0)を各サンプルに40ml加え、菌体を縣濁させた。その後、この菌体液を遮光した状態で-80℃と室温に交互に置き、凍結融解を2回おこなった。凍結融解後のサンプルは、超音波処理により菌体を破砕し、破砕後のサンプルを遠心分離することにより菌体破砕上清を回収した。
(2) Expression and purification of fluorescent fusion proteins Trx-FL4-ECFP-Lzip (Jun) and Trx-FL4-EYFP-Lzip (Jun)
Using expression vector A and expression vector B prepared in (1), E. coli Origami (DE3) (Novagen) was transformed. The transformed E. coli was cultured with shaking at 30 ° C. overnight in 5 ml LB (50 μg / ml ampicillin, 15 μg / ml kanamycin, 12.5 μg / ml tetracycline) medium. On the next day, the cultured cells were inoculated into 1.5 l LB medium and continued to be cultured at 30 ° C. When the turbidity of the culture reached OD600 of about 0.5 (culture for about 6 hours), 1.5 ml of 0.1M IPTG was added, the temperature was lowered to 16 ° C., and the culture was continued overnight. On the next day, the cells were collected by centrifugation, and 40 ml of a cell disruption solution (50 mM NaH 2 PO 4 -NaOH, 300 mM NaCl, 10 mM imidazole, pH 8.0) was added to each sample to suspend the cells. Thereafter, this bacterial cell solution was alternately placed at -80 ° C. and room temperature in a light-shielded state, and freeze-thawed twice. The freeze-thawed sample was crushed by sonication, and the crushed sample was centrifuged to recover the crushed cell supernatant.
目的タンパク質は、Hisタグ精製および陰イオン交換カラム精製により回収した。ニッケル-NTAアガロースカラム（キアゲン）2mlに菌体破砕上清を10ml添加した。次に、カラムを40mlの洗浄液（50mM NaH2PO4-NaOH、300mM NaCl、20mM imidazole、pH8.0）にて洗浄し、5mlの溶出液（50mM NaH2PO4-NaOH、300mM NaCl、250mM imidazole、pH8.0）を添加することにより目的タンパク質を溶出させた。回収したサンプルは、陰イオン交換カラムの吸着液（20mM Tris-HCl、pH8.0）に対して一晩透析した。翌日、サンプルをMonoQカラム（ファルマシア）に吸着させ、洗浄液（20mM Tris-HCl、pH8.0）をカラムのベッドボリュームの20倍量流した後、NaClの濃度勾配によりカラムに吸着したサンプルを溶出させた。回収した各フラクションの蛍光活性を測定し、十分な蛍光活性を有するフラクションを集め、目的タンパク質溶液とした。さらに、このタンパク質溶液をリン酸緩衝液に対して透析した。透析後のサンプルは使用するまで-80℃で保存した。 The target protein was recovered by His tag purification and anion exchange column purification. 10 ml of the cell disruption supernatant was added to 2 ml of a nickel-NTA agarose column (Qiagen). Next, the column was washed with 40 ml of washing solution (50 mM NaH 2 PO 4 -NaOH, 300 mM NaCl, 20 mM imidazole, pH 8.0) and 5 ml of eluent (50 mM NaH 2 PO 4 -NaOH, 300 mM NaCl, 250 mM imidazole). , PH 8.0) was added to elute the target protein. The collected sample was dialyzed overnight against an anion exchange column adsorbent (20 mM Tris-HCl, pH 8.0). The next day, the sample was adsorbed on a MonoQ column (Pharmacia), and a washing solution (20 mM Tris-HCl, pH 8.0) was flowed 20 times the bed volume of the column, and then the sample adsorbed on the column was eluted by a NaCl concentration gradient. It was. The fluorescence activity of each collected fraction was measured, and fractions having sufficient fluorescence activity were collected and used as a target protein solution. Furthermore, this protein solution was dialyzed against a phosphate buffer. The dialyzed sample was stored at −80 ° C. until use.
(3) 抗リン酸化MAPK抗体-蛍光融合タンパク質複合体の作製
抗リン酸化MAPK抗体IgG（Sigma）3mgを1.5mlの酢酸ナトリウム緩衝液に溶解後、60μgのペプシン（Sigma）を添加し、37℃で16時間ペプシン消化した。ペプシン消化したサンプルは１N NaOHで直ちに中和し、Superdex200（16/60）カラムでF(ab')2断片を精製した。精製したF(ab')2溶液に20mMの2-メルカプトエタノール（2ME）を添加し、37℃、1.5時間処理することにより、Fab'を調製した。さらに、Sephadex-G25カラムで2MEを除去した後に、1,11-maleimidotetraethyleneglycol（BM(PEO)4）（ピアス）をFab'の100倍量（モル比）添加し、30℃で1時間保温した。反応溶液をSephadex-G25カラムで精製し、未反応の1,11-bismaleimidotetraethyleneglycolを除去することによりマレイミド-Fab'を調製した。
(3) Preparation of anti-phosphorylated MAPK antibody-fluorescent fusion protein complex 3 mg of anti-phosphorylated MAPK antibody IgG (Sigma) was dissolved in 1.5 ml of sodium acetate buffer, and then 60 μg of pepsin (Sigma) was added at 37 ° C. Digested with pepsin for 16 hours. The pepsin-digested sample was immediately neutralized with 1N NaOH and the F (ab ′) 2 fragment was purified on a Superdex200 (16/60) column. Fab ′ was prepared by adding 20 mM 2-mercaptoethanol (2ME) to the purified F (ab ′) 2 solution and treating at 37 ° C. for 1.5 hours. Furthermore, after removing 2ME with a Sephadex-G25 column, 1,11-maleimidotetraethyleneglycol (BM (PEO) 4) (Pierce) was added 100 times as much as Fab '(molar ratio) and kept at 30 ° C for 1 hour. The reaction solution was purified by Sephadex-G25 column, and unreacted 1,11-bismaleimidotetraethyleneglycol was removed to prepare maleimide-Fab ′.
一方、上記(2)で調製したTrx-FL4-EYFP-Lzip(Jun)タンパク質溶液に20mMのdithiothreitol（DTT）を添加し、室温で一晩処理後、Sephadex-G25カラムにより還元Trx-FL4-EYFP-Lzip (Jun）タンパク質を精製した。次に、上記マレイミド-Fab'溶液と還元Trx-FL4-EYFP-Lzip(Jun）タンパク質溶液を等量（モル比）混合し、30℃で1時間保温することにより、抗リン酸化MAPK抗体-蛍光融合タンパク質複合体を作製した。さらに、カップリングしたサンプルに、蛍光タンパク質の5倍量（モル比）のN-ethylmaleimide（NEM）を添加することにより、カップリング反応を停止した。反応後のサンプルは、Superdex200（16/60）カラムを用いて、未反応のマレイミド-Fab'、Trx-FL4-EYFP-Lzip(Jun）、NEMを除去し、抗リン酸化MAPK抗体-蛍光融合タンパク質複合体を精製した。 On the other hand, 20 mM dithiothreitol (DTT) was added to the Trx-FL4-EYFP-Lzip (Jun) protein solution prepared in (2) above, treated at room temperature overnight, and then reduced with a Sephadex-G25 column to reduce Trx-FL4-EYFP. -Lzip (Jun) protein was purified. Next, the above-mentioned maleimide-Fab 'solution and reduced Trx-FL4-EYFP-Lzip (Jun) protein solution are mixed in an equal amount (molar ratio) and incubated at 30 ° C. for 1 hour, thereby anti-phosphorylated MAPK antibody-fluorescence A fusion protein complex was made. Furthermore, the coupling reaction was stopped by adding 5 times (molar ratio) N-ethylmaleimide (NEM) of the fluorescent protein to the coupled sample. The sample after the reaction was removed using the Superdex200 (16/60) column to remove unreacted maleimide-Fab ', Trx-FL4-EYFP-Lzip (Jun), NEM, and anti-phosphorylated MAPK antibody-fluorescence fusion protein The complex was purified.
(4)リン酸化MAPKペプチド-蛍光融合タンパク質複合体の作製
リン酸化MAPKペプチドとして、N末端に標識用のマレイミド基を導入したペプチド３種（マレイミド-DHTGFL(pT)E(pY)VA、マレイミド-DHTGFL(pT)E(pY)V、マレイミド-DHTGFL(pT)E(pY)）を化学合成し、カップリング反応用のリン酸緩衝液に溶解した。一方、上記(2)で調製したTrx-FL4-ECFP-Lzip(Jun)タンパク質溶液に20mMのDTTを添加し、室温で一晩処理後、Sephadex-G25カラムにより還元Trx-FL4-ECFP-Lzip(Jun)を精製した。次に、大過剰のマレイミド-リン酸化MAPKペプチド溶液と還元Trx-FL4-ECFP-Lzip(Jun)タンパク質溶液を混合し、30℃で1時間保温することにより、リン酸化MAPKペプチド-蛍光融合タンパク質複合体を作製した。さらに、カップリングしたサンプルに、蛍光タンパク質の5倍量（モル比）のN-ethylmaleimide（NEM）を添加することにより、カップリング反応を停止した。反応後のサンプルは、Superdex200（16/60）カラムを用いて、未反応のマレイミド-ペプチド、NEMを除去し、リン酸化MAPKペプチド-蛍光融合タンパク質複合体を精製した。
(4) Preparation of phosphorylated MAPK peptide-fluorescent fusion protein complex Three types of phosphorylated MAPK peptides (maleimide-DHTGFL (pT) E (pY) VA, maleimide-) with a maleimide group for labeling introduced at the N-terminus DHTGFL (pT) E (pY) V, maleimide-DHTGFL (pT) E (pY)) were chemically synthesized and dissolved in a phosphate buffer for coupling reaction. On the other hand, 20 mM DTT was added to the Trx-FL4-ECFP-Lzip (Jun) protein solution prepared in (2) above, treated overnight at room temperature, and then reduced with a Sephadex-G25 column to reduce Trx-FL4-ECFP-Lzip ( Jun) was purified. Next, a large excess of maleimide-phosphorylated MAPK peptide solution and reduced Trx-FL4-ECFP-Lzip (Jun) protein solution are mixed and incubated at 30 ° C for 1 hour, thereby phosphorylated MAPK peptide-fluorescent fusion protein complex The body was made. Furthermore, the coupling reaction was stopped by adding 5 times (molar ratio) N-ethylmaleimide (NEM) of the fluorescent protein to the coupled sample. The sample after the reaction was purified using a Superdex200 (16/60) column to remove unreacted maleimide-peptide and NEM, and purified the phosphorylated MAPK peptide-fluorescent fusion protein complex.
（実施例２） 最適なリン酸化MAPKペプチドの選択
実施例１(3)および(4)で作製した抗リン酸化MAPK抗体-蛍光融合タンパク質複合体と３種のリン酸化MAPKペプチド-蛍光融合タンパク質複合体を40nMになるように、アッセイ用緩衝液（50mM Tris-HCl、50mM NaCl、0.1%ウシ血清アルブミン、pH8.0）に溶解し、37℃で10分間反応させた。次に、混合液を蛍光光度計のセルに注入し、ここにリン酸化MAPKペプチドDHTGFL(pT)E(pY)VATを終濃度で40nMになるように添加し、添加10分後の蛍光スペクトル変化を測定した。蛍光スペクトル測定は蛍光光度計Shimadzu RF-5300PC（島津）を用い、433nmで励起し、450〜600nmの蛍光スペクトルを測定した（室温）。その結果、DHTGFL(pT)E(pY)VAペプチド-蛍光融合タンパク質複合体（図２）では、リン酸化ペプチド添加による大きなFRETシグナル変化は確認できなかったが、DHTGFL(pT)E(pY)Vペプチド-蛍光融合タンパク質複合体（図３）およびDHTGFL(pT)E(pY)ペプチド-蛍光融合タンパク質複合体（図４）では、競合反応に伴う迅速なFRETシグナル変化が確認された。以上の結果から、リン酸化MAPKペプチドとしてDHTGFL(pT)E(pY)VペプチドまたはDHTGFL(pT)E(pY)ペプチドを用いることにより迅速なリン酸化MAPKペプチドの検出が可能であることが示された。
(Example 2) Selection of optimal phosphorylated MAPK peptide Antiphosphorylated MAPK antibody-fluorescent fusion protein complex and three phosphorylated MAPK peptide-fluorescent fusion protein complexes prepared in Examples 1 (3) and (4) The body was dissolved in an assay buffer (50 mM Tris-HCl, 50 mM NaCl, 0.1% bovine serum albumin, pH 8.0) so as to be 40 nM, and reacted at 37 ° C. for 10 minutes. Next, the mixture was injected into a fluorometer cell, and phosphorylated MAPK peptide DHTGFL (pT) E (pY) VAT was added to a final concentration of 40 nM, and the
（実施例３）In vitroにおけるリン酸化MAPKタンパク質の検出
実施例１(3)および(4)で作製した抗リン酸化MAPK抗体-蛍光タンパク質複合体とDHTGFL(pT)E(pY)ペプチド-蛍光融合タンパク質複合体を40nMになるように、アッセイ用緩衝液（50mM Tris-HCl、50mM NaCl、0.1%ウシ血清アルブミン、pH8.0）に溶解し、37℃で10分間反応させた。次に、混合液を蛍光光度計のセルに注入し、ここにグルタチオンSトランスフェラーゼ融合リン酸化MAPK（GST-pERK2：Upstate）を終濃度で360nMになるように添加し、添加直後から60分間の蛍光スペクトルを測定した。蛍光スペクトル測定は蛍光光度計Shimadzu RF-5300PC（島津）を用い、433nmで励起し、450〜600nmの蛍光スペクトルを測定した（37℃）。
(Example 3) Detection of phosphorylated MAPK protein in vitro Antiphosphorylated MAPK antibody-fluorescent protein complex and DHTGFL (pT) E (pY) peptide-fluorescent fusion prepared in Examples 1 (3) and (4) The protein complex was dissolved in an assay buffer (50 mM Tris-HCl, 50 mM NaCl, 0.1% bovine serum albumin, pH 8.0) so as to be 40 nM, and reacted at 37 ° C. for 10 minutes. Next, the mixed solution is injected into a fluorometer cell, and glutathione S transferase-fused phosphorylated MAPK (GST-pERK2: Upstate) is added to a final concentration of 360 nM, followed by fluorescence for 60 minutes immediately after the addition. The spectrum was measured. The fluorescence spectrum was measured using a fluorometer Shimadzu RF-5300PC (Shimadzu), excited at 433 nm, and measured at 450 to 600 nm (37 ° C.).
その結果、抗原であるGST-pERK2を添加した直後から、アクセプターであるEYFPの蛍光強度（525nm）が減少する蛍光スペクトル変化が観察された。ドナーであるECFPの蛍光強度（475nm）とアクセプターであるEYFPの蛍光強度（525nm）の比（Fluorescence ratio I(475nm)/(525nm))をFRETの指標とし、その経時変化を測定すると、競合反応に伴うFRET ratioは約10分程度で大きく変化し、迅速な抗原検出が可能であることが確認された（図５）。 As a result, immediately after addition of the antigen GST-pERK2, a change in the fluorescence spectrum was observed in which the fluorescence intensity (525 nm) of the acceptor EYFP decreased. The ratio of the fluorescence intensity of ECFP as a donor (475 nm) to the fluorescence intensity of EYFP as an acceptor (525 nm) (Fluorescence ratio I (475 nm) / (525 nm)) is used as an index of FRET. The FRET ratio accompanying the change greatly changed in about 10 minutes, confirming that rapid antigen detection was possible (FIG. 5).
（実施例４）生細胞内におけるリン酸化MAPKタンパク質の検出
観察用のHela細胞は、10%のウシ血清を含むDMEM培地にて、37℃、5% 二酸化炭素濃度の培養条件下で培養し、アッセイ前日から無血清のDMEM培地にて培養した。実施例１(3)および(4)で作製した抗リン酸化MAPK抗体-蛍光融合タンパク質複合体とDHTGFL(pT)E(pY)ペプチド-蛍光融合タンパク質複合体を4μMになるように混合し、15,000rpm、15分間遠心分離した。遠心分離後、回収した上清をインジェクションニードルに分注し、ニードルをマイクロインジェクション装置に設置した。次に、蛍光顕微鏡IX81（オリンパス）の観察ステージにHela細胞をセットし、マイクロインジェクション（エッペンドルフ）により、蛍光プローブ（上記抗リン酸化MAPK抗体-蛍光融合タンパク質複合体とDHTGFL(pT)E(pY)ペプチド-蛍光融合タンパク質複合体）を生細胞内に導入した。
(Example 4) Detection of phosphorylated MAPK protein in living cells Hela cells for observation were cultured in a DMEM medium containing 10% bovine serum under culture conditions of 37 ° C and 5% carbon dioxide concentration. From the day before the assay, the cells were cultured in serum-free DMEM medium. The anti-phosphorylated MAPK antibody-fluorescent fusion protein complex prepared in Example 1 (3) and (4) and the DHTGFL (pT) E (pY) peptide-fluorescent fusion protein complex were mixed to 4 μM, and 15,000 Centrifugation at rpm for 15 minutes. After centrifugation, the collected supernatant was dispensed into an injection needle, and the needle was placed in a microinjection device. Next, the Hela cells were set on the observation stage of the fluorescence microscope IX81 (Olympus), and by microinjection (Eppendorf), the fluorescent probe (anti-phosphorylated MAPK antibody-fluorescent fusion protein complex and DHTGFL (pT) E (pY) The peptide-fluorescent fusion protein complex) was introduced into living cells.
導入後、終濃度100ng/mlのEGF （Epidermal Ggrowth Factor)を添加し、添加後30分間のFRET現象を30秒間隔のコマ撮り撮影した。FRET ratioの変化は、解析ソフトMetaFluor（モレキュラーデバイス）により解析した。その結果、EGF添加後、約30分で顕著なFRET ratioの変化が確認された（図６）。この結果から、本測定系によれば、EGF添加によって起こる、EGFRのリン酸化、ひいては、EGFRのシグナル伝達系の下流メディエーターである細胞内MAPKのリン酸化の検出をFRET ratioの変化によって検出できるといえる。 After the introduction, EGF (Epidermal Ggrowth Factor) at a final concentration of 100 ng / ml was added, and the FRET phenomenon for 30 minutes after the addition was photographed at 30-second intervals. The change in FRET ratio was analyzed by analysis software MetaFluor (molecular device). As a result, a remarkable change in FRET ratio was confirmed about 30 minutes after the addition of EGF (FIG. 6). From this result, according to this measurement system, it is possible to detect the phosphorylation of EGFR, and consequently the phosphorylation of intracellular MAPK, which is a downstream mediator of the EGFR signal transduction system, by the change of FRET ratio. I can say that.
Claims (4)
ギー転移（FRET： Fluorescence resonance energy transfer）が生じる、請求項１または２に記載のリン酸化MAPキナーゼの免疫測定用試薬。 The reagent for immunoassay of phosphorylated MAP kinase according to claim 1 or 2 , wherein fluorescence resonance energy transfer (FRET) occurs between the donor fluorescent protein and the acceptor fluorescent protein.
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