JP2004333232A - Separating method, detecting method, and screening method for target component - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To perform separation, collection, detection, and screening of a target component with the target component and a bonded molecule of target-components kept in conditions closer to organism conditions when performing the separation, collection, detection, and screening of the target component by using a structural body formed by causing a carrier to carry, in an immobilized manner, molecules having bonding affinity with the target component. <P>SOLUTION: In this method, target-component bonded molecules 4 are used for separating, collecting, detecting, and screening the target component. What is made by at least partly coating the surface of a carrier substrate 1 (magnetic body) with polyhydroxyalkanoate 2, a polymeric material having high bio-affinity, is used as the carrier for immobilizing the bonded molecules 4. On the surface of the carrier where conditions closer to the living conditions are maintained, the bonded molecules carried on the surface are bonded to the target component. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

【００５９】
（式中、Ｒ１およびａは、下記するＲ１とａとの組み合わせからなる群より選択されるいずれか一つの組み合わせで表される：
Ｒ１は、水素原子（Ｈ）であり、ａは、３〜１０の整数のいずれかである；
Ｒ１は、ハロゲン原子であり、ａは、１〜１０の整数のいずれかである；
Ｒ１は、発色団であり、ａは、１〜１０の整数のいずれかである；
Ｒ１は、カルボキシ基あるいはその塩であり、ａは、１〜１０の整数のいずれかである；あるいは、
Ｒ１は、下記：
【００６０】
【化２】

【００６１】
１，２−エポキシエチル基であり、ａは、１〜７の整数のいずれかである。）
化学式［１］：
【００６２】
【化３】

【００６３】
（式中、
ｂは、０〜７の整数のいずれかを表し、
Ｒ２は、水素原子（Ｈ）、ハロゲン原子、−ＣＮ、−ＮＯ_２、−ＣＦ_３、−Ｃ_２Ｆ_５、−Ｃ_３Ｆ_７からなる群から選択されるいずれか一つを表す。）
化学式［３］：
【００６４】
【化４】

【００６５】
（式中、
ｃは、１〜８の整数のいずれかを表し、
Ｒ３は、水素原子（Ｈ）、ハロゲン原子、−ＣＮ、−ＮＯ_２、−ＣＦ_３、−Ｃ_２Ｆ_５、−Ｃ_３Ｆ_７からなる群から選択されるいずれか一つを表す。）
化学式［４］：
【００６６】
【化５】

【００６７】
（式中、
ｄは、０〜７の整数のいずれかを表し、
Ｒ４は、水素原子（Ｈ）、ハロゲン原子、−ＣＮ、−ＮＯ_２、−ＣＦ_３、−Ｃ_２Ｆ_５、−Ｃ_３Ｆ_７からなる群から選択されるいずれか一つを表す。）
化学式［５］：
【００６８】
【化６】

【００６９】
（式中、
ｅは、１〜８の整数のいずれかを表し、
Ｒ５は、
水素原子（Ｈ）、ハロゲン原子、−ＣＮ、−ＮＯ_２、−ＣＦ_３、−Ｃ_２Ｆ_５、−Ｃ_３Ｆ_７、−ＣＨ_３、−Ｃ_２Ｈ_５、−Ｃ_３Ｈ_７からなる群から選択されるいずれか一つを表す。）
化学式［６］：
【００７０】
【化７】

【００７１】
（式中、ｆは、０〜７の整数のいずれかを表す。）
化学式［７］：
【００７２】
【化８】

【００７３】
（式中、ｇは、１〜８の整数のいずれかを表す。）
化学式［８］：
【００７４】
【化９】

【００７５】
（式中、
ｈは、１〜７の整数のいずれかを表し、
Ｒ６は、水素原子（Ｈ）、ハロゲン原子、−ＣＮ、−ＮＯ_２、−ＣＯＯＲ’、−ＳＯ_２Ｒ”、−ＣＨ_３、−Ｃ_２Ｈ_５、−Ｃ_３Ｈ_７、−ＣＨ（ＣＨ_３）_２、−Ｃ（ＣＨ_３）_３からなる群から選択されるいずれか一つを表し、
ここで、
Ｒ’は、水素原子（Ｈ）、Ｎａ、Ｋ、−ＣＨ_３、−Ｃ_２Ｈ_５からなる群から選択されるいずれか一つであり、
Ｒ”は、−ＯＨ、−ＯＮａ、−ＯＫ、ハロゲン原子、−ＯＣＨ_３、−ＯＣ_２Ｈ_５からなる群から選択されるいずれか一つである。）
化学式［９］：
【００７６】
【化１０】

【００７７】
（式中、
ｉは、１〜７の整数のいずれかを表し、
Ｒ７は、水素原子（Ｈ）、ハロゲン原子、−ＣＮ、−ＮＯ_２、−ＣＯＯＲ’、−ＳＯ_２Ｒ”からなる群から選択されるいずれか一つを表し、
ここで、
Ｒ’は、水素原子（Ｈ）、Ｎａ、Ｋ、−ＣＨ_３，−Ｃ_２Ｈ_５からなる群から選択されるいずれか一つであり、
Ｒ”は、−ＯＨ、−ＯＮａ、−ＯＫ、ハロゲン原子、−ＯＣＨ_３、−ＯＣ_２Ｈ_５からなる群から選択されるいずれか一つである。）
化学式［１０］：
【００７８】
【化１１】

【００７９】
（式中、ｊは、１〜９の整数のいずれかを表す。）
なお、前記ハロゲン原子の具体例として、フッ素、塩素、臭素などを挙げることができる。また、前記発色団は、化学式［１］で示されるモノマーユニットに対応する、３−ヒドロキシアシルＣｏＡ体が、ＰＨＡ合成酵素の触媒作用を受け得るものである限り、特に限定はされないが、ＰＨＡ合成酵素による高分子合成時における立体障害などを考慮すると、３−ヒドロキシアシルＣｏＡ 分子内において、ＣｏＡと結合するカルボキシ基と発色団との間の離反を保つ上では、側鎖として、炭素数１〜５のアルキレン鎖、例えば、直鎖のアルキレン鎖を有する方が望ましい。一方、該発色団の光吸収波長が可視域にあれば、かかるＰＨＡを被覆してなる磁性複合体は、着色したものが得られ、また、該発色団の光吸収波長が可視域以外にあっても、かかるＰＨＡは、種々の電子材料としての利用に供することができる。上記化学式［１］で示されるモノマーユニットにおける発色団の例として、ニトロソ、ニトロ、アゾ、ジアリールメタン、トリアリールメタン、キサンテン、アクリジン、キノリン、メチン、チアゾール、インダミン、インドフェノール、ラクトン、アミノケトン、ヒドロキシケトン、スチルベン、アジン、オカサジン、チアジン、アントラキノン、フタロシアニン、インジゴイドなどを挙げることができる。
【００８０】
本発明の方法で利用する、磁性複合体において、その表面を被覆しているＰＨＡの具体的な例として、さらに、下記化学式［１１］〜化学式［１８］で表されるモノマーユニットを少なくとも含むＰＨＡを例示することができる。
【００８１】
化学式［１１］：
【００８２】
【化１２】

【００８３】
（式中、
Ｒ１は、ビニル基であり、
ａは、１〜１０の整数のいずれかである。）
化学式［１２］：
【００８４】
【化１３】

【００８５】
（式中、
ｂは、０〜７の整数のいずれかを表し、
Ｒ２は、ＣＨ_３基、Ｃ_２Ｈ_５基、Ｃ_３Ｈ_７基、ビニル基、１，２−エポキシエチル基、ＣＯＯＲ_２１（ここで、Ｒ_２１は、Ｈ原子、Ｎａ原子、Ｋ原子のいずれか一つを表す）からなる群から選択されるいずれか一つを表す。
なお、ＰＨＡ中に、化学式［１２］で示されるモノマーユニット複数種が存在する場合、各モノマーユニット毎に、そのｂとＲ２とは、独立して上記の意味を表す。）
化学式［１３］：
【００８６】
【化１４】

【００８７】
（式中、
ｃは、１〜８の整数のいずれかを表し、
Ｒ３は、ＣＨ_３基、Ｃ_２Ｈ_５基、Ｃ_３Ｈ_７基、ＳＣＨ_３基からなる群から選択されるいずれか一つを表す。
なお、ＰＨＡ中に、化学式［１３］で示されるモノマーユニット複数種が存在する場合、各モノマーユニット毎に、そのｃとＲ３とは、独立して上記の意味を表す。）
化学式［１４］：
【００８８】
【化１５】

【００８９】
（式中、
ｄは、０〜８の整数のいずれかを表し、
Ｒ４は、
ｄが０の場合、Ｈ原子、ＣＮ基、ＮＯ_２基、ハロゲン原子、ＣＨ_３基、Ｃ_２Ｈ_５基、Ｃ_３Ｈ_７基、ＣＦ_３基、Ｃ_２Ｆ_５基またはＣ_３Ｆ_７基からなる群から選択されるいずれか一つを表し、
ｄが１〜８の場合、ＣＨ_３基、Ｃ_２Ｈ_５基、Ｃ_３Ｈ_７基からなる群から選択されるいずれか一つを表す。
なお、ＰＨＡ中に、化学式［１４］で示されるモノマーユニット複数種が存在する場合、各モノマーユニット毎に、そのｄとＲ４とは、独立して上記の意味を表す。）
化学式［１５］：
【００９０】
【化１６】

【００９１】
（式中、ｅは、１〜８の整数のいずれかを表す。）
化学式［１６］：
【００９２】
【化１７】

【００９３】
（式中、
ｆは、１〜８の整数のいずれかを表し、
Ｒ６は、ＣＨ_３基、Ｃ_２Ｈ_５基、Ｃ_３Ｈ_７基、（ＣＨ_３）_２−ＣＨ基または（ＣＨ_３）_３−Ｃ基からなる群から選択されるいずれか一つを表す。
なお、ＰＨＡ中に、化学式［１６］で示されるモノマーユニット複数種が存在する場合、各モノマーユニット毎に、そのｆとＲ６とは、独立して上記の意味を表す。）
化学式［１７］：
【００９４】
【化１８】

【００９５】
（式中、
ｇは、１〜８の整数のいずれかを表し、
Ｒ７は、Ｈ原子、ハロゲン原子、ＣＮ基、ＮＯ_２基、ＣＯＯＲ_７１（ここで、Ｒ_７１は、Ｈ、Ｎａ、Ｋ、ＣＨ_３、Ｃ_２Ｈ_５のいずれかを表す）、ＳＯ_２Ｒ_７２（ここで、Ｒ_７２は、−ＯＨ、−ＯＮａ、−ＯＫ、ハロゲン原子、−ＯＣＨ_３、−ＯＣ_２Ｈ_５のいずれかを表す）、ＣＨ_３基、Ｃ_２Ｈ_５基、Ｃ_３Ｈ_７基、（ＣＨ_３）_２−ＣＨ基または（ＣＨ_３）_３−Ｃ基からなる群から選択されるいずれか一つを表す。
なお、ＰＨＡ中に、化学式［１７］で示されるモノマーユニット複数種が存在する場合、各モノマーユニット毎に、そのｇとＲ７とは、独立して上記の意味を表す。）
化学式［１８］：
【００９６】
【化１９】

【００９７】
（式中、
ｇは、１〜８の整数のいずれかを表し、
Ｒ７は、Ｈ原子、ハロゲン原子、ＣＮ基、ＮＯ_２基、ＣＯＯＲ_７１（ここで、Ｒ_７１は、Ｈ、Ｎａ、Ｋ、ＣＨ_３、Ｃ_２Ｈ_５のいずれかを表す）、ＳＯ_２Ｒ_７２（ここで、Ｒ_７２は、−ＯＨ、−ＯＮａ、−ＯＫ、ハロゲン原子、−ＯＣＨ_３、−ＯＣ_２Ｈ_５のいずれかを表す）、ＣＨ_３基、Ｃ_２Ｈ_５基、Ｃ_３Ｈ_７基、（ＣＨ_３）_２−ＣＨ基または（ＣＨ_３）_３−Ｃ基からなる群から選択されるいずれか一つを表す。
なお、ＰＨＡ中に、化学式［１８］で示されるモノマーユニット複数種が存在する場合、各モノマーユニット毎に、そのｇとＲ７とは、独立して上記の意味を表す。）
本発明において利用されるＰＨＡとして、上記する化学式［１］〜化学式［１８］で示されるモノマーユニットを複数種含むランダム共重合体やブロック共重合体を用いることも可能であり、かかる複数種のモノマーユニットを含むＰＨＡでは、各モノマーユニットや含まれる官能基の特性を利用したＰＨＡの物性制御や複数の機能の付与、官能基間の相互作用を利用した新たな機能の発現等が可能となる。
【００９８】
さらには、例えば、磁性微粒子の表面にＰＨＡの被覆を行う際、ＰＨＡ合成酵素の基質である３−ヒドロキシアシルＣｏＡの種類や濃度などを経時的に変化させることによって、作製されるＰＨＡの組成も経時的に変化させることができる。従って、構造体の形状が粒状であれば、表面のＰＨＡ被覆は、内側から外側に向かう方向に、また、構造体の形状が平面状であれば、表面のＰＨＡ被覆は、その平面と垂直な方向に、被覆されているＰＨＡのモノマーユニット組成を変化させることも可能である。
【００９９】
例えば、最終的に作製される構造体の形態を、核となる磁性体に対して、その構造体の最表面には、該磁性体と親和性の低いＰＨＡによる被覆がなされているものにする必要がある際には、先ず、核となる磁性体の表面を、該磁性体と親和性の高いＰＨＡで被覆して、次いで、その磁性体と親和性の高いＰＨＡのモノマーユニット組成から、目的とするＰＨＡのモノマーユニット組成へと、内側から外側に向かう方向、あるいは、垂直方向に、組成が変化する構造、例えば、多層構造あるいはグラディエント構造とすることで、核となる磁性体と、その表面を被覆するＰＨＡ層との結合を強固にしたＰＨＡ被膜を形成することが可能となる。
【０１００】
また、３−ヒドロキシプロピオン酸ユニット、３−ヒドロキシ−ｎ−酪酸ユニット、３−ヒドロキシ−ｎ−吉草酸ユニット、４−ヒドロキシ−ｎ−酪酸ユニット、さらには、炭素数６〜１４のヒドロキシアルカン酸ユニットからなるホモポリマー、あるいはこれらの複数のユニットからなるコポリマーなども、本発明の方法において利用可能である。さらには、必要に応じて、一旦ＰＨＡを酵素合成した後、あるいは、酵素合成中に、さらに化学修飾等を施してもよい。
【０１０１】
また、被覆に用いるＰＨＡの分子量制御、および得られるＰＨＡ被覆膜の親水性向上という観点から、反応溶液中に水酸基を有する化合物を適宜添加してもよい。
【０１０２】
本発明の方法において、前記の目的で反応溶液中に添加する水酸基を有する化合物は、アルコール類、ジオール類、トリオール類、アルキレングリコール類、ポリエチレングリコール類、ポリエチレンオキサイド類、アルキレングリコールモノエステル類、ポリエチレングリコールモノエステル類、ポリエチレンオキサイドモノエステル類化合物から選ばれる少なくとも１種類であるが、さらに詳しく述べると以下の通りである。すなわち、好適に利用できるアルコール類、ジオール類およびトリオール類化合物は、炭素数３〜１４の直鎖状または分岐のアルコール、ジオール、トリオールである。好適に利用できるアルキレングリコール類およびアルキレングリコールモノエステル類化合物は、そのアルキレングリコールの炭素鎖は、炭素数２〜１０の直鎖状または分岐状構造を有している化合物である。また、好適に利用できるポリエチレングリコール類、ポリエチレンオキサイド類、ポリエチレングリコールモノエステル類、ポリエチレンオキサイドモノエステル類化合物は、その数平均分子量が１００〜２０，０００の範囲であるものである。
【０１０３】
上記目的で反応溶液中に添加する、水酸基を有する化合物の添加濃度は、ＰＨＡ合成酵素による３−ヒドロキシアシルＣｏＡの重合反応が阻害されない濃度であれば、特に制限はないが、好ましくは、ＰＨＡ合成酵素と３−ヒドロキシアシルＣｏＡとを含む反応溶液に対して、０．０１％〜１０％（ｗ／ｖ）の範囲、さらに好ましくは、０．０２％〜５％（ｗ／ｖ）の範囲で添加することがよく、その添加方法は、反応初期に一括して添加する方法、反応時間内に、数回に分けて反応溶液中に添加する方法のいずれでもよい。
【０１０４】
なお、化学式［１２］で示されるモノマーユニットにおいて、Ｒ２として、カルボキシ基（ＣＯＯＲ_２１）を含むモノマーユニットは、この化学式［１２］で示され、Ｒ２がビニル基である、すなわち、側鎖末端にビニルフェニル基を有するモノマーユニットから、そのビニル基の二重結合部分を選択的に酸化開裂することで、カルボキシ基へ変換することで製造することができ、結果として、化学式［１２］で示されるモノマーユニットの側鎖末端にカルボキシフェニル基を有するユニットを含むＰＨＡが得られる。
【０１０５】
前記のビニル基をカルボキシ基へと変換する方法、すなわち、炭素−炭素の二重結合を酸化剤を用いて、酸化開裂してカルボン酸を得る方法としては、例えば、過マンガン酸塩を用いる方法（Ｊ． Ｃｈｅｍ． Ｓｏｃ．， Ｐｅｒｋｉｎ． Ｔｒａｎｓ． １， ８０６ （１９７３））、重クロム酸塩を用いる方法（Ｏｒｇ． Ｓｙｎｔｈ．， ４， ６９８ （１９６３））、過ヨウ素酸塩を用いる方法（Ｊ． Ｏｒｇ． Ｃｈｅｍ．， ４６， １９ （１９８１））、硝酸を用いる方法（特開昭５９−１９０９４５号広報）、オゾンを用いる方法（Ｊ． Ａｍ． Ｃｈｅｍ． Ｓｏｃ．， ８１， ４２７３ （１９５９））等が知られており、さらに、ＰＨＡに関しては、前述のＭａｃｒｏｍｏｌｅｃｕｌａｒ Ｃｈｅｍｉｓｔｒｙ， ４， ２８９−２９３ （２００１）に、ＰＨＡの側鎖末端の炭素−炭素二重結合を、酸化剤として過マンガン酸カリウムを用いて、反応を酸性条件下で行うことで、カルボン酸を得る方法が報告されており、本発明において、前記ビニル基をカルボキシ基へと変換する反応を行う際、これらの酸化開裂方法を参考とすることができる。
【０１０６】
前記ビニル基をカルボキシ基へと変換する反応に際して、酸化剤として用いる前記過マンガン酸塩としては、過マンガン酸カリウムが一般的である。過マンガン酸塩の使用量は、酸化開裂反応が化学量論的反応であるため、Ｒ２としてビニル基を有する化学式［１２］で示されるモノマーユニット１モルに対して、通常１モル当量以上、好ましくは、２〜４モル当量使用するのがよい。
【０１０７】
酸化剤として過マンガン酸カリウムを用いる、酸化開裂反応において、反応系を酸性条件下にするためには、通常、硫酸、塩酸、酢酸、硝酸などの各種の無機酸や有機酸が用いられる。しかしながら、硫酸、硝酸、塩酸などの酸を用いた場合、ＰＨＡの主鎖のエステル結合が切断され、分子量低下を引き起こす恐れがある。そのため、酸性条件下にする際、酢酸を用いることが好ましい。反応系に添加される、酸の添加量は、Ｒ２としてビニル基を有する化学式［１２］で示されるモノマーユニット１モルに対して、通常、０．２〜２００モル当量、好ましくは、０．４〜１００モル当量の範囲に選択する。反応系に添加される、酸の添加量が０．２モル当量に満たない場合には、酸化開裂反応は低収率となり、２００モル当量を超える場合には、添加した酸による分解物が副生するため、いずれの場合も好ましくない。また、酸化開裂反応を促進する目的で、クラウン−エーテルを用いることができる。この場合、クラウン−エーテルと過マンガン酸塩とが、錯体を形成する結果、その反応活性が増大する効果が得られる。前記目的で利用可能なクラウン−エーテルとして、ジベンゾ−１８−クラウン−６−エーテル、ジシクロ−１８−クラウン−６−エーテル、１８−クラウン−６−エーテルが、一般的に用いられる。反応系へのクラウン−エーテルの添加量は、過マンガン塩１モルに対して、通常、１．０〜２．０モル当量の範囲、好ましくは、１．０〜１．５モル当量の範囲に選択することが望ましい。
【０１０８】
本発明において、前記酸化反応を行う際、Ｒ２としてビニル基を有する化学式［１２］で示すユニットを含むＰＨＡで被覆された構造体、および過マンガン酸塩と酸を、一括して最初からともに仕込んで反応させてもよく、あるいは、それぞれ、連続的に、もしくは、断続的に反応系内に加えながら、反応させてもよい。あるいは、過マンガン酸塩のみを先に反応系に溶解、もしくは懸濁させておき、続いて、ＰＨＡで被覆された構造体と酸とを、連続的に、もしくは、断続的に反応系内に加えて反応させてもよく、ＰＨＡで被覆された構造体のみを先に反応系に懸濁させておき、続いて、過マンガン酸塩と酸とを、連続的に、もしくは断続的に反応系内に加えて反応させてもよい。さらには、ＰＨＡで被覆された構造体と酸を先に仕込んでおき、続いて、過マンガン酸塩を連続的に、もしくは断続的に反応系内に加えて反応させてもよく、過マンガン酸塩と酸を先に仕込んでおき、続いて、ＰＨＡで被覆された構造体を連続的に、もしくは断続的に反応系内に加えて反応させてもよく、ＰＨＡで被覆された構造体と過マンガン酸塩を先に仕込んでおき、続いて、酸を連続的に、もしくは断続的に反応系内に加えて反応させてもよい。
【０１０９】
Ｒ２としてビニル基を有する化学式［１２］で示すユニットを含むＰＨＡで被覆された構造体を対象とする、酸化剤として過マンガン酸カリウムを用いる、酸化開裂反応では、反応温度は、通常、−２０〜４０℃の範囲、好ましくは、０〜３０℃の範囲に選択するのがよい。反応速度は、Ｒ２としてビニル基を有する化学式［１２］で示すユニットと過マンガン酸塩との量論比、ならびに反応温度に依存し、加えて、前記ビニル基をカルボキシ基へと変換する目標比率に応じて、反応時間は選択されるものであるが、かかる目標比率を概ね１００％と選択する際には、反応時間を、通常、２〜４８時間とするのがよい。
【０１１０】
同様の酸化開裂反応を、Ｒ１がビニル基である化学式［１１］で示すユニットに適用すると、側鎖末端のビニル基をカルボキシ基へと変換することも可能である。
【０１１１】
また、化学式［１７］で示されるモノマーユニット、あるいは化学式［１８］で示されるモノマーユニットを含むＰＨＡは、一旦化学式［８］で示されるモノマーユニットを含むＰＨＡを作製した後、そのスルファニル基（−Ｓ−）を選択的に酸化して、スルフィニル基（−ＳＯ−）、あるいはスルホニル基（−ＳＯ_２−）へと変換することで製造することができる。かかるスルファニル基（−Ｓ−）の選択的な酸化は、例えば、過酸化化合物による酸化処理を施すことで達成でき、その際、スルファニル基（−Ｓ−）の酸化に寄与し得るものであれば、いかなる種類の過酸化化合物をも用いることが可能である。なお、酸化効率、ＰＨＡ主鎖骨格ならびにＰＨＡに含まれる他のモノマーユニットへの影響、処理の簡便さ等を考慮した場合、特に、過酸化水素、過炭酸ナトリウム、メタクロロ過安息香酸、過蟻酸、過酢酸からなる群から選択される過酸化化合物を用いることが好ましい。
【０１１２】
例えば、スルファニル基（−Ｓ−）の酸化に利用する過酸化化合物として、メタクロロ過安息香酸（ＭＣＰＢＡ）を用いると、化学式［８］で示されるモノマーユニット中のスルファニル基（−Ｓ−）に対する酸化は、化学量論的に進行するため、化学式［１７］で示されるモノマーユニット、あるいは化学式［１８］で示されるモノマーユニットの含有比率の制御がし易い。また、その反応条件が温和であるため、ＰＨＡ主鎖骨格の切断や活性部位の架橋反応等、不要な副次反応が起こり難い。
【０１１３】
一般的な反応条件として、スルファニル基（−Ｓ−）をスルフィニル基（−ＳＯ−）まで選択的に酸化するためには、ＰＨＡ中に含有されるスルファニル基（−Ｓ−）を含む化学式［８］で示されるモノマーユニットのユニットモル量、１モルに対して、ＭＣＰＢＡを若干過剰量、具体的には、１．１〜１．４モル量の範囲に選択し、クロロホルム中、温度を０℃〜３０℃の範囲に選択して、反応を行う。前記の反応条件範囲においては、反応時間を１０時間程度とすると、理論値のほぼ９０％、２０時間程度とすると、理論値のほぼ１００％まで、スルフィニル基（−ＳＯ−）へと酸化を進行させることができる。また、スルファニル基（−Ｓ−）を全てスルホニル基（−ＳＯ_２−）まで酸化するためには、ＰＨＡ中に含有されるスルファニル基（−Ｓ−）を含む化学式［８］で示されるモノマーユニットのユニットモル量、１モルに対して、ＭＣＰＢＡを２モルより若干過剰量、具体的には、２．１〜２．４モル量の範囲に選択し、前記と同様の溶媒、温度、時間条件を選択して、反応を行えばよい。かかる過酸化化合物として、ＭＣＰＢＡを用いる酸化処理では、スルファニル基（−Ｓ−）にＭＣＰＢＡ１分子が作用して、スルフィニル基（−ＳＯ−）へと変換し、さらに、ＭＣＰＢＡ１分子が作用して、スルホニル基（−ＳＯ_２−）へと段階的に酸化が進行するが、スルファニル基（−Ｓ−）からスルフィニル基（−ＳＯ−）への変換は、スルフィニル基（−ＳＯ−）からスルホニル基（−ＳＯ_２−）への変換より、より高い反応性を示す。
【０１１４】
また、Ｒ２として１，２−エポキシエチル基を有する化学式［１２］で示すユニットは、Ｒ２としてビニル基を有する化学式［１２］で示すユニットに対して、側鎖末端ビニルフェニル基中のビニル基の二重結合部分を選択的に部分酸化して、エポキシ基の導入を行うことで製造することができる。すなわち、Ｒ２としてビニル基を有する化学式［１２］で示すユニットを含むＰＨＡに対して、ビニル基に選択的な酸化処理を施すことで、Ｒ２として１，２−エポキシエチル基を有する化学式［１２］で示すユニットを含むＰＨＡが得られる。
【０１１５】
前記ビニル基から１，２−エポキシエチル基へとエポキシ化を行う酸化処理についても、例えば、過酸化化合物を利用することができ、ビニル基の選択的な部分酸化に寄与し得るものであれば、いかなる種類の過酸化化合物をも用いることが可能である。その際、酸化効率、ＰＨＡ主鎖骨格ならびにＰＨＡに含まれる他のモノマーユニットへの影響、処理の簡便さ等を考慮した場合、特に、過酸化水素、過炭酸ナトリウム、メタクロロ過安息香酸、過蟻酸、過酢酸からなる群から選択される過酸化化合物を用いることが好ましい。ビニル基から１，２−エポキシエチル基へとエポキシ化を行う酸化処理に過酸化化合物を用いる場合、その反応条件は、過酸化化合物による前記スルファニル基の選択的な部分酸化処理における反応条件を参考とすることができる。
【０１１６】
本発明の方法で利用されるポリヒドロキシアルカノエートの分子量は、数平均分子量として、１，０００から１０，０００，０００程度とするのが望ましい。
【０１１７】
なお、本発明において、ＰＨＡ被覆された構造体の作製工程に利用される、ＰＨＡ合成酵素により合成されるＰＨＡは、一般に、Ｒ体のみから構成されるアイソタクチックなポリマーである。
【０１１８】
本発明の方法で利用される、磁性体を核とする構造体に含有される磁性体の量は、１〜８０重量％の範囲、好ましくは５〜７０重量％の範囲、さらに好ましくは１０〜６０重量％の範囲に選択する。構造体中の磁性体含有量が１重量％より少ないと、磁気性能が不足して磁性体含有構造体としての性能が不十分となる恐れがあり、また、磁性体含量が８０重量％を超えると、核とする磁性体に対して、その表面を被覆するＰＨＡの含有比率が相対的に低下するため、表面に十分なＰＨＡ被覆層を設けることで達成される、かかる構造体本来の機能が損なわれ、実用性能の面で満足できなくなる恐れがある。
【０１１９】
本発明の方法に利用される、粒状構造体の粒子径は、その個別的な用途等に応じて適宜選定されるが、通常、０．０２〜１００μｍの範囲、好ましくは０．０５〜２０μｍの範囲に選択する。
【０１２０】
＜標的成分、ならびに標的成分に対して結合親和力を有する分子＞
本発明の方法が対象とする「標的成分」は、生理学的に有用であり、多くの場合、検体試料中では、その他の物質と混在しているか、あるいは、他の物質の混在がない、単一体であっても、広範囲に低濃度で存在しているものである。従って、検体試料中に含有されている、「標的成分」のみを、分離・回収、検出、およびスクリーニングする手段・方法が求められている。
【０１２１】
本発明の方法では、前記目的のために、「標的成分」のみを捕捉する上で好適に利用可能な、「標的成分に対して結合親和力を有する分子（以下、「標的成分結合分子」と記載する場合もある）」を用いる。
【０１２２】
本発明の方法が対象とする「標的成分」ならびに「標的成分に対して結合親和力を有する分子」の具体的な例として、核酸、タンパク質、ペプチド、糖鎖、脂質、および低分子化合物、あるいはこれらの複合体、さらには、これらの分子を部分的に含んでなる物質を挙げることができる。
【０１２３】
ここで、「核酸」は、デオキシリボ核酸、リボ核酸、オリゴヌクレオチド、ポリヌクレオチド、アプタマー、リボザイム等を包括する。
【０１２４】
ここで、「タンパク質」は、糖タンパク質、リポタンパク質、膜タンパク質、および標識タンパク質、ならびに低分子量ペプチドなどの天然および人工的に誘導された異形分子を包括する。
【０１２５】
当該タンパク質が免疫反応体であれば、例えば、抗体、抗原、ハプテン、あるいはこれらの錯体が可能であり、特に、「抗体」の場合、モノクローナル抗体またはポリクローナル抗体、組換えタンパク質型抗体または天然型抗体、キメラ抗体、ハイブリッド抗体混合物（単数あるいは複数）、単鎖抗体提示ファージ抗体（単鎖抗体を提示しているファージ全体を含む）、ならびに抗体およびタンパク質の融合体、あるいはこれらのハイブリッド混合物も包含される。当該タンパク質が触媒反応体の場合は、天然酵素、遺伝子改変により作製された変異酵素、ポリエチレングリコール等の合成分子と複合化された半人工酵素、非天然アミノ酸が導入された半人工酵素等を包括する。
【０１２６】
前記「抗体」として、通常、ＩｇＧ（免疫グロブリンＧ、あるいはイムノグロブリンＧ）が挙げられるが、ペプシン、パパインなどの消化酵素、あるいはジチオトレイトール、２−スルファニルエタノール（２−メルカプトエタノール）などの還元剤を用いて、Ｆ（ａｂ’）_２、Ｆａｂ’、Ｆａｂ、Ｆｖなどの低分子化処理が施されたものであってもよい。また、ＩｇＧだけでなく、ＩｇＭ、あるいは前記ＩｇＧと同様の処理で低分子化したＩｇＭ由来のフラグメントであってもよい。また、モノクローナル抗体、ポリクローナル抗体のいずれも、本発明における「標的成分結合分子」として適用できる。「標的成分結合分子」として、モノクロール抗体を用いる際には、Ｂ型肝炎ウイルス表面抗原のように繰り返し構造を有するタンパク質や、ＣＡ１９−９抗原のように分子内に複数のエピトープが存在する抗原に対しては、各エピトープに対する反応性を示すモノクローナル抗体複数種を併用することもできる。また、認識エピトープの異なるものを２種類以上組み合わせても使用できる。
【０１２７】
一方、「抗原」としては、例えば、タンパク質、ポリペプチド、ステロイド、多糖類、脂質、花粉、遺伝子工学的に産生された組換えタンパク質、薬物など種々のものが挙げられる。すなわち、本発明において対象とする「抗原」は、人、あるいは動物に対して、抗体産生を惹起する能力のある全ての物質のうち、例えば、診断等特別の目的の下に選択された単一あるいは複数の物質、ないしはそれらを含有する混合物である。
【０１２８】
「ペプチド」は、主に、本発明においては、その分子量とは無関係にタンパク質の断片を指す。
【０１２９】
「低分子化合物」は、受容体に認知可能である低分子量の、好ましくは有機分子である。通常、低分子化合物は、タンパク質に対する特異結合化合物であり、その多くは、生理活性物質あるいは薬剤候補物質あって、特に、抗原性の低分子化合物は、「ハプテン」と称される場合もある。
【０１３０】
「糖鎖」としては、グルコース、マンノース、Ｎ−アセチルグルコサミン、フコース、ガラクトース、グルクロン酸、Ｎ−アセチルグルコサミン、シアル酸から選択される単糖ユニットが複数（数個から数十個）連なった、直鎖および分岐のオリゴマーおよびポリマーを挙げることができる。また、本発明の方法では、これらの糖鎖とタンパク質の複合体である糖タンパク質、脂質との複合体である糖脂質、さらには、これら糖鎖が生体細胞表面に提示されている場合は、細胞膜表面に糖鎖を提示している細胞そのもの、あるいは細胞膜断片も含まれる。
【０１３１】
「脂質」としては、生物の神経組織、形質膜、ミトコンドリア、ミクロソーム、細胞核等細胞内オルガネラの膜として存在する複合脂質、組織境界脂質膜等の天然脂質（アシルグリセロール）や、タンパク質との複合体であるリポタンパク質、レシチン（ホスファジチルコリン）のようなリン脂質やその脂質二重膜カプセルとしてのリポソーム等を挙げることができる。
【０１３２】
＜磁性体＞
本発明の方法において、構造体の核として用いる磁性体としては、その表面にＰＨＡ合成酵素を固定化することのできるものであれば、磁性材料の種類、形状、大きさに関しては、その個別的な用途に応じて、適宜選択して用いることができる。すなわち、ＰＨＡ合成酵素の固定化方法や、作製した構造体の応用の形態等に応じて、磁性体の種類や構造を適宜選択して用いることができる。
【０１３３】
本発明の方法で利用する、構造体を構成する磁性体の例として、例えば、磁性を有する金属または金属化合物が挙げられ、さらに具体的には、四三酸化鉄（Ｆｅ_３Ｏ_４）、γ−重三二酸化鉄（γ−Ｆｅ_２Ｏ_３）、ＭｎＺｎフェライト、ＮｉＺｎフェライト、ＹＦｅガーネット、ＧａＦｅガーネット、Ｂａフェライト、Ｓｒフェライト等各種フェライト、鉄、マンガン、コバルト、ニッケル、クロムなどの金属、鉄、マンガン、コバルト、ニッケルなどの合金を挙げることができるが、これらに限定されるものではない。ここで、例えば、磁性複合体上に生体物質を固定する場合、あるいは、磁性複合体を生体内に投与する場合には、生体に対する適合性の良好なマグネタイト（Ｆｅ_３Ｏ_４）のほか、必要に応じて、マグネタイトの金属元素の一部を少なくとも１種類の他の金属元素で置換した各種フェライト組成などが好適に適用可能である。これら磁性体の形状は、生成条件によって変化し、多面体、８面体、６面体、球状、棒状、鱗片状等などがあるが、異方性の少ない構造が、構造体とした際、その表面を被覆するＰＨＡ層が示す機能の安定発現のためにはより好ましい。本発明の構造体を構成する磁性体の一次粒子の粒子径は、その用途に応じて適宜選択可能であるが、例えば、０．００１〜１０μｍの範囲内の粒径を有する粒子を用いるとよい。
【０１３４】
また、本発明の方法で利用する、構造体を構成する磁性体として、超常磁性を有するものについても好ましく用いることができる。例えば、フェライトの粒子径が２０ｎｍ程度以下と小さい場合には、フェライトは熱擾乱影響を受け超常磁性を示すようになり、残留磁化や保磁力を持たなくなる。超常磁性であっても、外部磁界を印加することにより、磁気的操作が可能であり、また、超常磁性であれば、残留磁化や保磁力を持たないので、外部磁界が印加されていない際に、残留磁化に起因する磁気的な凝集の生じる恐れもない。
【０１３５】
さらには、本発明の方法で利用する磁性体は、金属または金属化合物を含むマトリックスなどのような複合材料であってもよく、そのマトリックスは、有機または無機の各種材料から構成されるものである。フェライトめっきのような手法で、有機高分子表面を磁性体により被覆した材料、有機高分子中に磁性体が分散した材料等も、その表面の一部に磁性体が露出した形態であれば、利用可能である。
【０１３６】
なお、本発明の方法に用いる磁性体は、粒子状、平板状、フィルム状として、用いることができるが、本発明の方法における、磁気的操作による分離の具体的な手法を考慮する場合、粒子状が好ましく、さらには、液体試料中への分散を想定すると、その粒子径は、０．００１〜１０μｍ程度の微粒子であることがより好ましい。
【０１３７】
＜ＰＨＡ磁性構造体の作製＞
本発明の方法で利用するＰＨＡ磁性構造体の製造方法は、磁性体にＰＨＡ合成酵素を固定化する工程と、該固定化ＰＨＡ合成酵素を用いて、３−ヒドロキシアシルＣｏＡに対する酵素反応により、ＰＨＡを合成する工程とを含むものである。
【０１３８】
磁性体にＰＨＡ合成酵素を固定化する方法としては、該ＰＨＡ合成酵素の活性が保持され得るものであり、かつ、目的とする磁性体に対して適用可能なものであれば、通常行われている酵素固定化方法の中から、任意に選択される方法を用いることができる。例えば、ＰＨＡ合成酵素を固定化する方法として、共有結合法、イオン吸着法、疎水吸着法、物理的吸着法、アフィニティ吸着法、架橋法、格子型包括法などを例示することができるが、特に、イオン吸着や疎水吸着を利用した固定化方法を用いると、簡便である。
【０１３９】
一般に、ＰＨＡ合成酵素などの酵素タンパク質は、アミノ酸が多数結合したポリペプチドであり、リシン、ヒスチジン、アルギニン、アスパラギン酸、グルタミン酸などの側鎖上にイオン性基を有するアミノ酸残基によって、イオン吸着体としての性質を示し、また、アラニン、バリン、ロイシン、イソロイシン、メチオニン、トリプトファン、フェニルアラニン、プロリンなどの側鎖として疎水性基を有するアミノ酸残基によって、あるいは、全体として、有機高分子であるという点で、疎水吸着体としての性質を有している。従って、程度の差はあるが、イオン性や疎水性、もしくはイオン性と疎水性の両方の性質を有する固体表面に吸着させることが可能である。
【０１４０】
主にイオン吸着法によってＰＨＡ合成酵素を固定化する方法を適用する際、その核には、イオン性官能基を表面に発現しているコアを用いればよく、例えば、カオリナイト，ベントナイト、タルク、雲母等の粘土鉱物、アルミナ、二酸化チタン等の金属酸化物、シリカゲル、ヒドロキシアパタイト、リン酸カルシウムゲル等の不溶性無機塩などをコアとして用いることができる。また、これらを主要な成分とする無機顔料、イオン交換樹脂、キトサン、ポリアミノポリスチレン等の、イオン性官能基を有する重合体も、イオン吸着性コアとして用いることができる。
【０１４１】
本発明に利用するＰＨＡ磁性構造体において、基材が磁性体の場合、例えば、フェライトなどの金属酸化物においては、その表面に水酸基が存在しており、ＰＨＡ合成酵素表面のカルボキシ基との水素結合による固定化法などが好適に用いうる。
【０１４２】
一方、主に疎水吸着によってＰＨＡ合成酵素を固定化する方法を適用する際、その核には、表面が非極性であるコアを用いればよく、例えば、スチレン系ポリマー、アクリル系ポリマー、メタクリル系ポリマー、ビニルエステル類、ビニル系ポリマーなど、イオン性官能基を表面に発現していない、もしくは疎水性官能基を表面に発現している多くの重合体をコアとして用いることができる。具体的には、芳香環を複数有するアゾ顔料や縮合多環のフタロシアニン系顔料、アントラキノン系顔料等の有機顔料、カーボンブラックなどは疎水吸着性である。また、疎水吸着法は、親油化処理を施した磁性体にも適用することが可能である。
【０１４３】
イオン吸着法または疎水吸着法によるＰＨＡ合成酵素のコアへの固定化は、該ＰＨＡ合成酵素とコアとを所定の反応液中で混合することによって達成される。このとき、該ＰＨＡ合成酵素がコアの表面に均等に吸着されるよう、反応容器を適当な強度で振盪あるいは攪拌することが望ましい。
【０１４４】
反応液のｐＨや塩濃度、温度によって、コアおよびＰＨＡ合成酵素の表面電荷の正負や電荷量、疎水性が変化するので、用いるコアの性質に合わせて、酵素活性上許容される範囲内で溶液の調整を行うことが望ましい。例えば、コアが主にイオン吸着性である場合には、塩濃度を下げることにより、コアとＰＨＡ合成酵素との吸着に寄与する電荷量を増やすことができる。また、ｐＨを調整する事により、両者の反対電荷を増やすことができる。コアが主に疎水吸着性である場合には、塩濃度を上げることによって、両者の疎水性を増やすことができる。また、予め電気泳動やぬれ角等を測定し、コアやＰＨＡ合成酵素の荷電状態や疎水性を調べることで、吸着に適した溶液条件の設定をすることもできる。さらに、コアとＰＨＡ合成酵素との吸着量を直接測定して条件を求めることもできる。吸着量の測定は、例えば、コアが分散された溶液に濃度既知のＰＨＡ合成酵素溶液を添加し、吸着処理を行った後、溶液中のＰＨＡ合成酵素濃度を測定し、差し引き法により吸着酵素量を求める等の方法を用いればよい。
【０１４５】
イオン吸着法や疎水吸着法では酵素を固定化し難いコア材質の場合には、操作の煩雑さや酵素の失活の可能性を考慮した上で、共有結合法によってもかまわない。例えば、芳香族アミノ基を有するコア材質（固体粒子）をジアゾ化し、これに酵素をジアゾカップリングする方法や、カルボキシ基、アミノ基を有するコア材質（固体粒子）とＰＨＡ合成酵素の間にペプチド結合を形成させる方法、ハロゲン基（ハロゲノアルキル基）を有するコア材質（固体粒子）とＰＨＡ合成酵素のアミノ基等との間でアルキル化する方法、臭化シアンで活性化した多糖類コア粒子とＰＨＡ合成酵素のアミノ基を反応させる方法、コア材質（固体粒子）のアミノ基と酵素のアミノ基との間を架橋する方法、アルデヒド基またはケトン基を有する化合物とイソシアニド化合物の存在下、カルボキシ基，アミノ基を有するコア材質（固体粒子）とＰＨＡ合成酵素とを反応させる方法、ジスルフィド基（−Ｓ−Ｓ−）を有するコア材質（固体粒子）とＰＨＡ合成酵素のスルファニル基（−ＳＨ）との間で交換反応させる方法などがある。
【０１４６】
また、アフィニティ吸着によって、コア材質（固体粒子）の表面にＰＨＡ合成酵素を吸着してもよい。アフィニティ吸着とは、生体高分子とそれに特異的な親和力を示す、リガンドと呼ばれる特定物質との間の生物学的吸着で、例えば、酵素と基質、抗体と抗原、レセプターとアセチルコリン等の情報物質、ｍＲＮＡとｔＲＮＡなどがある。一般に、アフィニティ吸着を利用して、酵素タンパク質を固定化する方法では、酵素の基質や反応生成物、拮抗阻害剤、補酵素、アロステリックエフェクター等をリガンドとして固体表面に結合させ、このリガンドと添加した酵素タンパク質との結合を介して、固体表面にアフィニティ吸着させる方法を採用する。しかしながら、ＰＨＡ合成酵素に対しては、例えば、基質である３−ヒドロキシアシルＣｏＡをリガンドとして用いた場合、該ＰＨＡ合成酵素中のＰＨＡ合成を触媒する活性部位がリガンドとの結合により塞がれてしまうため、ＰＨＡを合成できなくなるという問題を生じる。しかしながら、他の生体高分子をＰＨＡ合成酵素に予め融合させ、該生体高分子のリガンドを利用して、アフィニティ吸着法を適用することによって、アフィニティ吸着による固定化後も、ＰＨＡ合成酵素自体のＰＨＡ合成活性を維持することができる。なお、ＰＨＡ合成酵素と生体高分子との融合は遺伝子工学的手法によって行ってもよく、また、生体高分子をＰＨＡ合成酵素に化学的に結合させてもよい。用いる生体高分子としては、生体高分子に対するリガンドの入手容易で、そのリガンドがコアに容易に結合できるものであればいかなるものでも構わないが、遺伝子組換えによって融合物を発現させる場合には、融合させる生体高分子はタンパク質であることが好ましい。具体的には、形質転換によって、融合パートナーのＧＳＴを発現する遺伝子配列にＰＨＡ合成酵素の遺伝子配列を繋げた遺伝子を導入した大腸菌を用いて、ＧＳＴとＰＨＡ合成酵素との融合タンパク質を生産し、この融合タンパク質をＧＳＴのリガンドであるグルタチオンを結合したＳｅｐｈａｒｏｓｅに添加することで、融合タンパク質型のＰＨＡ合成酵素をＳｅｐｈａｒｏｓｅ上にアフィニティ吸着させることができる。
【０１４７】
また、磁性体に対して結合能を有するアミノ酸配列を含むペプチドをＰＨＡ合成酵素に融合して提示させ、該磁性体に対して結合能を有するアミノ酸配列のペプチド部分と、磁性体との結合性に基づいて、該磁性体表面にＰＨＡ合成酵素を固定化することもできる。
【０１４８】
磁性体に対する結合能を有するアミノ酸配列は、例えば、ランダム・ペプチド・ライブラリーのスクリーニングによって決定することができる。特に、例えば、Ｍ１３系ファージの表面蛋白質（例えば。ｇｅｎｅＩＩＩ蛋白質）のＮ末端側遺伝子にランダム合成遺伝子を連結して調製されたファージ・ディスプレイ・ペプチド・ライブラリーを好適に用いることができるが、その場合、磁性体に対する結合能を有するアミノ酸配列を決定するには、次のような手順をとる。すなわち、磁性体あるいは該磁性体を構成する少なくとも一成分に対して、ファージ・ディスプレイ・ペプチド・ライブラリーを添加することによって接触させ、その後、洗浄により結合ファージと非結合ファージを分離する。磁性体結合ファージを酸などにより溶出し、緩衝液で中和した後、大腸菌に感染させ、磁性体結合ファージを増幅する。この選別を複数回繰り返すと、目的の磁性体に高い結合能を示す複数のクローンが濃縮される。ここで、単一なクローンを得るため、再度大腸菌に感染させた状態で培地プレート上にコロニーを作らせる。それぞれの単一コロニーを液体培地で培養した後、培地上清中に存在するファージをポリエチレングリコール等で沈澱精製し、そのクローンが有する合成遺伝子の塩基配列を解析すれば、磁性体に対する結合能を有するペプチドの構造（アミノ酸配列）を知ることができる。
【０１４９】
上記スクリーニング方法により選別される、磁性体に対する結合能を有するペプチドのアミノ酸配列をコードする合成遺伝子は、通常の遺伝子工学的手法を用いて、ＰＨＡ合成酵素の遺伝子に融合して利用される。磁性体に対する結合能を有するペプチドは、ＰＨＡ合成酵素のＮ末端あるいはＣ末端に連結して発現することができる。また、適当なスペーサー配列を挿入して発現することもできる。スペーサー配列としては、約３〜約４００アミノ酸が好ましく、また、スペーサー配列はいかなるアミノ酸を含んでもよい。最も好ましくは、スペーサー配列は、ＰＨＡ合成酵素の機能を妨害せず、また、前記磁性体に対する結合能を有するペプチドを介して、融合タンパク質型ＰＨＡ合成酵素が磁性体に結合する際、結合能を有するペプチドによる結合を妨害しないものである。
【０１５０】
上記方法により作製された固定化ＰＨＡ合成酵素は、そのままでも用いることができるが、さらに、凍結乾燥等を施した上で保存し、その都度、酵素反応液用媒体に添加して、使用することもできる。
【０１５１】
固定化ＰＨＡ合成酵素による３−ヒドロキシアシルＣｏＡの重合によって、ＰＨＡが合成される反応において放出されるＣｏＡ量が、１分間に１μｍｏｌとなるＰＨＡ合成酵素量を１単位（Ｕ）としたとき、磁性体に固定するＰＨＡ合成酵素の量は、例えば、磁性体がカプセル構造体のコアである場合、磁性体１ ｇ当たり、１０ 単位（Ｕ）〜１，０００単位（Ｕ）の範囲、望ましくは、５０ 単位（Ｕ）〜５００単位（Ｕ）の範囲内に設定するとよい。
【０１５２】
前記の固定化ＰＨＡ合成酵素は、所望のＰＨＡの原料となる３−ヒドロキシアシルＣｏＡを含む水系反応液中に添加され、磁性体表面に固定化したＰＨＡ合成酵素によってＰＨＡを合成することにより、磁性体の表面が合成されるＰＨＡにより被覆された構造体を形成する。その際、水系反応液は、固定化ＰＨＡ合成酵素の活性を発揮させ得る条件に調整された反応系に構成するべきであり、例えば、通常、ｐＨ５．５〜ｐＨ９．０の範囲、好ましくは、ｐＨ７．０〜ｐＨ ８．５の範囲となるよう、緩衝液によりｐＨ調整を行う。ただし、使用する固定化ＰＨＡ合成酵素の至適ｐＨやｐＨ安定性によっては、上記のｐＨ範囲以外に条件を設定することも除外されない。緩衝液の種類は、使用する固定化ＰＨＡ合成酵素の活性を発揮させ得るものであれば、設定するｐＨ領域等に応じて、適宜選択して用いることができるが、例えば、一般の生化学反応に用いられる緩衝液、具体的には、酢酸バッファー、リン酸バッファー、リン酸カリウムバッファー、３−（Ｎ−モルフォリノ）プロパンスルフォン酸（ＭＯＰＳ）バッファー、Ｎ−トリス（ヒドロキシメチル）メチル−３−アミノプロパンスルフォン酸（ＴＡＰＳ）バッファー、トリス塩酸バッファー、グリシンバッファー、２−（シクロヘキシルアミノ）エタンスルフォン酸（ＣＨＥＳ）バッファーなどを用いるとよい。緩衝液の濃度も、使用する固定化ＰＨＡ合成酵素の活性を発揮させ得るものであれば、特に限定はされないが、通常、５．０ ｍＭ〜１．０ Ｍの範囲、好ましくは０．１ Ｍ〜０．２ Ｍの濃度のものを使用するとよい。反応温度は、使用するＰＨＡ合成酵素の特性に応じて、適宜設定するものであるが、通常、４℃〜５０℃の範囲、好ましくは、２０℃〜４０℃の範囲に設定するとよい。ただし、使用するＰＨＡ合成酵素の至適温度や耐熱性によっては、上記の範囲以外に条件を設定することも除外されない。反応時間は、合成すべきＰＨＡの被覆層の厚さに応じて、選択すべきものであり、また、使用するＰＨＡ合成酵素の安定性等にもよるが、通常、１分間〜２４時間の範囲、好ましくは、３０分間〜３時間の範囲内で適宜選択して設定する。反応液中の基質３−ヒドロキシアシルＣｏＡ濃度は、合成すべきＰＨＡの被覆層の組成、厚さに応じて、適宜選択するべきものであり、また、使用する固定化ＰＨＡ合成酵素の活性を発揮させ得る範囲内で、適宜設定するものであるが、通常、０．１ ｍＭ〜１．０ Ｍ、好ましくは０．２ ｍＭ〜０．２ Ｍの範囲内で設定するとよい。なお、反応液中における基質３−ヒドロキシアシルＣｏＡ濃度が高い場合、一般に、反応系のｐＨが低下する傾向にあるため、基質３−ヒドロキシアシルＣｏＡ濃度を高く設定する場合は、ｐＨの変動を抑制するため、前記の緩衝液濃度も高めに設定することが好ましい。
【０１５３】
また、上記工程において、水系反応液中の基質３−ヒドロキシアシルＣｏＡの種類や濃度などの組成を経時的に変化させることによって、形成される構造体の形状が粒状であれば内側から外側に向かう方向に、構造体の形状が平面状であれば垂直方向に、磁性体表面を被覆するＰＨＡの膜厚方向におけるモノマーユニット組成を変化させることができる。
【０１５４】
このＰＨＡのモノマーユニット組成の変化した構造体の形態として、例えば、ＰＨＡ被膜の組成変化が連続的で、内側から外側に向かう方向、もしくは垂直方向に組成の勾配を形成した１層のＰＨＡが磁性体を被覆した形態を挙げることができる。組成の勾配を形成した１層のＰＨＡの製造方法としては、例えば、ＰＨＡを合成しながら、反応液中に別組成の基質３−ヒドロキシアシルＣｏＡを添加するなどの方法によればよい。
【０１５５】
また別の形態として、ＰＨＡ被膜の組成変化が段階的で、組成の異なるＰＨＡが磁性体を多層に被覆した形態を挙げることができる。この組成の異なるＰＨＡ多層被覆を製造方法としては、ある３−ヒドロキシアシルＣｏＡの組成でＰＨＡを合成した後、遠心分離などによって調製中の構造体を反応液から一旦回収し、回収された調製中の構造体表面に、異なる３−ヒドロキシアシルＣｏＡの組成からなる反応液を再度添加して、組成の異なるＰＨＡを積層するなどの方法を適用よればよい。
【０１５６】
＜標的成分結合分子のＰＨＡ磁性構造体への担持＞
ＰＨＡ磁性構造体の表面に、標的成分に対して結合親和力を有する分子（以下、標的成分結合分子と記載する。）を担持する方法としては、該表面を被覆するＰＨＡと標的成分結合分子との疎水性、イオン性、ファンデルワールス力等の物理的親和力による物理吸着によることもできるが、再現性や安定性を考慮すると、該ＰＨＡ側鎖の官能基と、標的成分結合分子の有する官能基とを、そのまま、あるいは変換・修飾・活性化試薬の存在下に結合させて、不可逆的な共有結合を介在させることがより望ましい。
【０１５７】
本発明の方法に用いるＰＨＡ磁性構造体の一形態として、該表面を被覆するＰＨＡの側鎖上にエポキシ基を有するＰＨＡ磁性構造体を用いることができる。該エポキシ基は、標的成分結合分子が有するアミノ基（−ＮＨ_２）あるいはスルファニル基（−ＳＨ）と直接的に共有結合を形成することができる。結合形成に試薬を必要とせず、共有結合の形成が可能であることから、用いる標的成分結合分子として、酵素タンパク質等の変性を起こし易いタンパク質や、Ａタンパク質、Ｇタンパク質といった抗体（Ｆｃ）受容体タンパク質等の担持に有効である。さらには、該表面を被覆するＰＨＡ上にイミノ二酢酸（ＩＤＡ）を担持し、Ｎｉ^２＋等の金属イオンを添加することにより、ヒスタグ化された標的タンパク質の効率的回収に利用することが可能である。本エポキシ基との反応を利用して、共有結合を形成する方法は、標的成分結合分子が薬剤候補物質のような低分子化学物質である場合にも、該低分子化学物質がアミノ基（−ＮＨ_２）あるいはスルファニル基（−ＳＨ）を有していれば、勿論応用することができる。
【０１５８】
該エポキシ基含有ＰＨＡ磁性構造体は、エポキシ基に対して、１０〜１００倍モル量の水酸化アンモニウム、あるいはヘキサメチレンジアミン塩酸塩等を用いて、アルカリ条件下で反応させることにより、アミノ基を有するＰＨＡ磁性構造体に変換することができる。該アミノ基は、標的成分結合分子がタンパク質やペプチドの場合は、その主鎖カルボキシ末端やアスパラギン酸、グルタミン酸といった、該タンパク質やペプチドが有するアミノ酸残基の側鎖カルボキシ基との間で、ＮＨＳ（Ｎ−ヒドロキシスクシンイミド）等の架橋化剤によりアミド結合を形成させることができる。その場合、標的成分結合分子側のカルボキシ基をＮＨＳ処理により活性化エステルとする必要があるので、ＮＨＳ処理を施した際、標的成分結合分子の機能が十分保持されるか否かを、予め確認しておく必要がある。本アミド結合形成を利用する担持方法は、標的成分結合分子が薬剤候補物質のような低分子化学物質である場合にも、該低分子化学物質がカルボキシ基を有していれば、勿論応用することができる。
【０１５９】
さらに、該アミノ基は、標的成分結合分子が、糖鎖やレクチン等の糖タンパク質、リポ多糖等の糖脂質の場合には、その糖鎖中のアルデヒド構造（ホルミル基：−ＣＨＯ）との間で、シッフ塩基（−ＣＨ＝Ｎ−）形成および還元的アミネーションによる安定な結合を形成することができる。本糖鎖とアミノ基との反応を利用する共有結合の形成方法は、糖鎖中にアルデヒド構造を導入する、糖鎖部分の部分的酸化により促進され、また、そのＦｃ部分に糖鎖を有するＩｇＧ等の抗体分子の担持にも応用可能である。本アミノ基とアルデヒド構造（ホルミル基：−ＣＨＯ）との方法は標的成分結合分子が薬剤候補物質のような低分子化学物質である場合にも、該低分子化学物質がアルデヒド構造（ホルミル基：−ＣＨＯ）を有しているか、部分的酸化によりアルデヒド構造（ホルミル基：−ＣＨＯ）を導入することが可能であれば、勿論応用することができる。
【０１６０】
さらに、該アミノ基は、マレイミド誘導体、ピリジルジチオ化合物、ヨウ素／臭素アセチル化合物の存在下で、スルファニル基（−ＳＨ）を有する標的成分結合分子と結合させることができる。このアミノ基とスルファニル基（−ＳＨ）との反応の条件は、マレイミド誘導体を用いる場合は、０．１Ｍリン酸ナトリウム（ｐＨ６．５−７．５）中で、４℃から室温で２−４時間、ピリジルジチオ化合物を用いる場合は、ＰＢＳ緩衝液（ｐＨ７．５）中で、室温にて１５−２０時間程度、ヨウ素／臭素アセチル化合物を用いる場合には、０．０５Ｍホウ酸ナトリウム溶液（ｐＨ８．３）中で、遮光下に室温にて、１時間の反応が基本的な反応条件となるが、該反応条件は、標的成分結合分子の種類、その後の目的に応じて適宜変更することも可能である。
【０１６１】
本発明の方法に用いるＰＨＡ磁性構造体への担持の別の一形態として、該ＰＨＡの側鎖にカルボキシ基を有するＰＨＡ磁性構造体を利用するものを挙げることができる。該カルボキシ基は、標的成分結合分子が、タンパク質やペプチドの場合は、その主鎖アミノ末端や、リジン、アルギニンといった該タンパク質やペプチドが有するアミノ酸残基側鎖上のアミノ基との間で、ＮＨＳ（Ｎ−ヒドロキシスクシンイミド）等の架橋化剤によって、アミド結合を形成することができる。このアミド結合形成反応は、ＰＨＡのカルボキシ基をＮＨＳ処理により予め活性化エステルとすることにより、アミド結合の形成速度・頻度を向上したものである。また、本ＰＨＡ側鎖のカルボキシ基を利用するアミド結合の形成方法は、標的成分結合分子がＤＮＡやオリゴヌクレオチドである場合には、通常の既知の方法で、その末端をアミノ化したＤＮＡ、オリゴヌクレオチドを用いることで、上記の反応方法によって、ＰＨＡ上に担持することができる。本ＰＨＡ側鎖のカルボキシ基を利用するアミド結合の形成方法は、標的成分結合分子が薬剤候補物質のような低分子化学物質である場合にも、該低分子化学物質がアミノ基を有していれば、勿論応用することができる。
【０１６２】
本発明の方法に用いるＰＨＡ磁性構造体への担持の別の一形態として、該ＰＨＡの側鎖にクロロ基（−Ｃｌ）、ブロモ基（−Ｂｒ）、フルオロ基（−Ｆ）といったハロゲンを有するＰＨＡ磁性構造体を利用するものを挙げることができる。該ハロゲンがクロロ基やブロモ基の場合、スルファニル基（−ＳＨ）を有する標的成分結合分子との間で、温和な条件により、スルフィド結合（−Ｓ−）を形成することができる。
【０１６３】
その他、上記の活性官能基を用いて、表面にＰＨＡを被覆した構造体に担持したい「標的成分結合分子」と特異的に結合する物質（例えば、「標的成分結合分子」が抗体の場合には、プロテインＡやプロテインＧなど）を、その表面に結合させた後、「標的成分結合分子」と特異的に結合する物質に対して、目的の「標的成分結合分子」を特異的に結合させることにより、構造体上に担持する方法も挙げられるし、「標的成分結合分子」を、さらに修飾した後、同様の方法で担持することも可能である。後者の例としては、カルボキシ型ＰＨＡ磁性構造体の表面に、試薬ＮＨＳ−ｉｍｉｎｏｂｉｏｔｉｎ（Ｐｉｅｒｃｅ製）を用いて、ビオチンを担持し、一方、アビジンあるいはストレプトアビジンで修飾した標的成分結合分子をビオチンに特異的に結合させることで、構造体上に担持する方法が挙げられる。利用可能な親和性結合対の例として、他に、レクチンと糖、ハプテンと抗体、タンパク質ＡあるいはＧと抗体Ｆｃ、他の特異結合タンパク質対、フェニルボロン酸とサリチルヒドロキサム酸、あるいは、互いに反応はするが、一般に、タンパク質とは反応しないその他の化学的部分対が挙げられる。
【０１６４】
構造体上への非特異吸着を防止するため、担持される標的成分結合分子の活性を損失しないような「ブロッキング剤」で、担持がなされていない構造体表面部分をコーティングすると好ましい。この「ブロッキング処理」に適したブロッキング剤として、コラーゲン、ゼラチン（特に、冷水魚皮ゼラチン）、スキムミルク、ＢＳＡ等の血清タンパク質および、タンパク質とは反応しない疎水性部分およびも親水性部分を含む数多くの化合物が挙げられる。
【０１６５】
＜標的成分結合分子担持ＰＨＡ磁性構造体と標的成分との接触および結合＞
本発明の方法における、ＰＨＡ磁性構造体上に担持された標的成分結合分子と標的成分との接触は、通常、水性媒体中で行われるが、標的成分がある種の薬剤候補物質のような難水溶性分子である場合には、アルコールやアセトン、ＤＭＳＯ（ジメチルスルホキシド）やＤＭＦ（ジメチルホルムアミド）といった極性溶媒や、ＴｗｅｅｎやＴｒｉｔｏｎ、ＳＤＳといった界面活性剤を添加し、さらには、トルエンやキシレン、ヘキサンのような非極性溶媒を加えて、エマルジョン系で接触を行い、結合反応を促進してもよい。但し、これらの溶媒、界面活性剤を用いる場合には、その添加濃度は、担持されている標的成分結合分子の親和結合機能が損なわれないような濃度範囲に選択することが必要である。
【０１６６】
本発明の方法における、ＰＨＡ磁性構造体上に担持された標的成分結合分子と標的成分との接触、結合を促進するために、標的成分結合分子の親和結合機能が損なわれない程度において、加熱手段や攪拌手段を用いることも可能であり、その際、超音波等の手段を用いることも可能である。
【０１６７】
また、本発明の方法で利用する構造体は、特には、磁性構造体であるので、その表面に担持された標的成分結合分子と標的成分との接触、結合を促進する手段として、磁力による操作を利用することも可能である。磁力による操作を施す場合、磁力を発揮する構造体（永久磁石や電磁石等：以下まとめて磁石と記載する場合もある）を用い、磁力の発揮、解放を繰り返す操作を施すことができる。電磁石を用いる場合、スイッチ切換えにより、電磁石に対する通電と遮断を反復して、磁性構造体の捕捉および解放を行う。磁石を用いた操作の一例としては、プローブ状の電磁石を反応容器内に挿入し、スイッチの切換え、電源のオン・オフ等により電磁石に対する通電と遮断を反復して、磁性構造体の捕捉・解放を繰り返す方法がある。また、別の一例として、磁石を容器側部の外側に配置して、スイッチの切換え、電源のオン・オフ、あるいは磁石自身の反応容器との距離の調節により、印加される磁場強度を反復的に変動させ、磁性構造体の捕捉・解放を繰り返す方法がある。
【０１６８】
なお、本発明の方法でいう標的成分と標的成分結合分子との「結合」は、結合対の要素、すなわち、化学的あるいは物理的作用により一方の分子が他方の分子に特異的に結合することである。周知の抗原・抗体反応による、抗原と抗体間の結合はもとより、ビオチンとアビジン、炭水化物とレクチン、核酸・ヌクレオチドの相補配列間、作動体と受容体分子、酵素補助因子と酵素、酵素抑制剤と酵素、ペプチド配列とその配列あるいはタンパク質全体に特異な抗体、ポリマーの酸と塩基、色素とタンパク質バインダー、ペプチドと特異タンパク質バインダー（リボヌクレアーゼ、Ｓ−ペプチドおよびリボヌクレアーゼＳ−タンパク質）、糖とホウ酸、および結合アッセイにおける分子会合を可能とする親和力を備えた同様の分子対の間における結合が挙げられるが、これに限定するものではない。さらに、結合対として、組換え技術あるいは分子工学により製造される分析物類似体あるいは結合要素など、元の結合要素の類似体である要素を挙げることができる。結合要素が免疫反応体であれば、例えば、抗体、抗原、ハプテンあるいはこれらの錯体が可能であり、「抗体」を用いる場合、単クローン抗体あるいはポリクローナル抗体、組換えタンパク質型抗体あるいは天然型抗体、キメラ抗体、混合物（単数あるいは複数）、単鎖抗体提示ファージ抗体（ファージ全体を含む）、単鎖抗体を提示するその断片（単数あるいは複数）、ならびに抗体およびタンパク質の結合要素の混合物でよい。
【０１６９】
近年、進化分子工学の発達により、ランダムなオリゴヌクレオチド・ライブラリーから蛋白質等の標的分子に対して高いアフィニティーを有する核酸分子、すなわちアプタマー（核酸抗体と称される場合もある）をスクリーニングする技術（“ｓｙｓｔｅｍａｔｉｃ ｅｖｏｌｕｔｉｏｎ ｏｆ ｌｉｇａｎｄｓ ｂｙｅｘｐｏｎｅｎｔｉａｌ ｅｎｒｉｃｈｍｅｎｔ”； ＳＥＬＥＸ ｏｒ ｉｎ ｖｉｔｒｏ ｓｅｌｅｃｔｉｏｎ）が開発された。このアプタマーを用いるスクリーニング方法を用いて、抗体よりも迅速且つ容易な高アフィニティー・リガンドの調製が数多く報告されている（例えば、Ｎａｔｕｒｅ， ３５５： ５６４ （１９９２） 、国際特許出願ＷＯ９２／１４８４３号公報、特開平８−２５２１００号公報、特開平９−２１６８９５号公報等）。
【０１７０】
あるいは、タンパク質である核酸の転写因子と、ある特定の塩基配列を含む核酸との結合に関しても、疾病発生の原因究明や、さらには、効果的な診断・治療への応用も期待されている。
【０１７１】
本発明の方法が対象とする「結合」には、勿論、このような核酸−タンパク質間の親和性結合も包含される。
【０１７２】
また、本発明の方法が対象とする「結合」には、永久的あるいは一次的を問わず、あらゆる物理的付着あるいは化学的付着、あるいは密接な特異的・選択的な会合を含む。一般に、イオン結合の相互作用、水素結合、疎水力、ファンデルワールス力などにより、対象のリガンド分子と受容体との間を物理的に付着させることができる。「結合」の相互作用は、結合により化学変化が起こる場合のように短い可能性がある。これは、結合成分が酵素であり、その分析物「標的成分結合分子」が酵素用基質である場合に一般的である。さらには、化学的連結が、永久的あるいは可逆的結合である可能性もある。結合は、特に異なる条件下において、特異的となる可能性がある。
【０１７３】
実際に、本発明の方法における、ＰＨＡ磁性構造体上に担持された標的成分結合分子と標的成分との接触および結合工程の一例としては、標的成分結合分子を担持するＰＨＡ磁性構造体を、組織や細胞ホモジェネートあるいは血清などの流体といった天然タンパク質を含む生物学的試料に接触させて、その天然タンパク質をＰＨＡ磁性構造体表面に担持する標的成分結合分子に特異的に吸着・結合させる工程である。
【０１７４】
また、該工程の別の一例としては、周知のファージ・ディスプレイ抗体選択法で用いられているように、標的成分結合分子であるタンパク質を担持する磁性構造体を用いて、適したバクテリオファージの表面に提示される抗体部分を選択的に結合することができる。
【０１７５】
また、該工程の別の一例としては、ハイブリドーマ上澄み液、ファージ・ディスプレイなどの流体など、受容体を含有した生物学的試料に接触させて、生物学的試料中に含まれる受容体を、特異的にＰＨＡ磁性構造体上に担持された標的成分結合分子に吸着させる工程も考え得る。
【０１７６】
＜標的成分結合磁性複合体の磁気分離および洗浄＞
磁性複合体（標的成分結合分子が担持されている磁性構造体）に標的成分を結合させた後、標的成分結合磁性複合体を分離および洗浄する方法においては、磁力を発揮する構造体（永久磁石や電磁石等）を用い、磁力の発揮、解放を繰り返すことにより、磁気分離の操作を施す。電磁石を用いる場合、スイッチ切換えにより、電磁石に対する通電と遮断を行って、標的成分結合磁性複合体の捕捉および解放を行う。
【０１７７】
実施形態の一例としては、プローブ型磁石を用いて、容器内で標的成分結合磁性複合体を捕捉した後、残余する液体を容器から除去する。洗浄液をその容器内に投入して、プローブ上にまだ捕捉されている標的成分結合磁性複合体を洗浄する。あるいは、標的成分結合磁性複合体が捕捉されたプローブ型磁石を反応溶液から取り出し、洗浄液へと移動させ、洗浄操作を行うこともできる。
【０１７８】
別の方法として、磁石を容器側部の外側に配置して、液体の交換時に標的成分結合磁性複合体を容器内側部に付着させておくこともできる。この外部磁石を取り除くと、標的成分結合磁性複合体は解放され、交換された液体（洗浄液）と混ざり合うことで洗浄が行われる。
【０１７９】
なお、本磁気分離の操作には、磁性微粒子を操作する用途で市販されている、数多くの磁選機を利用することも可能である。市販されている磁選機の例としては、ＤＹＮＡＬ社製 ＤＹＮＡＬ ＭＣＰ、 Ｓｅｒｏｎｏ Ｄｉａｇｎｏｓｔｉｃｓ社製 ＭＡＩＡ Ｍａｇｎｅｔｉｃ Ｓｅｐａｒａｔｏｒ、 宝酒造社製Ｍａｇｎｅｔｉｇｈｔ Ｓａｐａｒａｔｉｏｎ Ｓｔａｎｄ、 Ａｄｖａｎｃｅｄ Ｍａｇｎｅｔｉｃｓ社製 ＢｉｏＭａｇ Ｓｅｐａｒａｔｏｒ、等が挙げられる。
【０１８０】
＜標的成分の磁性構造体からの溶離・遊離＞
本発明の方法においては、その必要に応じて、標的成分結合磁性複合体を単離した後、結合している標的成分を標的成分結合分子から溶離・遊離することもできる。標的成分結合分子と標的成分とが、タンパク質−タンパク質の組み合わせである場合は、通常の解離条件（ｐＨ２、４Ｍグアニジン、２Ｍチオシアン酸アンモニウム、あるいは１％ＳＤＳなど）で遊離することができるが、その際、遊離する標的成分タンパク質は変性を受けていることが多い。遊離した標的成分タンパク質が仮に変性していても、電気泳動やＨＰＬＣで精製を施す場合は、特に問題はないが、標的成分タンパク質本来の立体構造等を検証したい場合には、透析等の復元工程が必要な場合がある。
【０１８１】
＜標的成分の検出＞
本発明の方法で用いる、標的成分の検出方法として、通常の比色法、蛍光法、化学発光法、ラジオアイソトープ法等、イムノアッセイ、ハイブリダイゼーション・アッセイで用いることのできる如何なる方法も、採用することが可能である。さらには、標的成分結合分子から上記の方法で溶離・遊離された標的成分は、同様の方法を用いて分析することもできるし、さらに、標的成分がＤＮＡである場合は、ＰＣＲ等の手法で増幅した上で、シーケンサー等で塩基配列を検証することも可能であるし、標的成分がタンパク質である場合は、酵素分解した後、その酵素消化断片を、二次元電気泳動やＨＰＬＣにより分離した後、エレクトロスプレイ・イオン化法−ＭＳ分析を行うことも可能であるし、また、標的成分タンパク質について、直接あるいは酵素分解した後、マトリックス支援レーザ脱離イオン化法−飛行時間型質量分析法（ＭＡＬＤＩ ＴｏＦ−ＭＳ）で分析することも可能である。
【０１８２】
勿論、核磁気共鳴スペクトロスコピー（ＮＭＲ）や赤外吸収スペクトロスコピー（ＩＲ）、紫外吸収スペクトロスコピー（ＵＶ）のような、その他のスペクトル分析を単独で、あるいは組み合わせて、標的成分の検出を行うことも可能である。
【０１８３】
【実施例】
以下、実施例により、本発明をさらに具体的に説明する。ただし、以下に述べる実施例は、本発明にかかる最良の実施形態の一例ではあるが、本発明の技術的範囲はこれら実施例に限定されるものではない。
【０１８４】
参考例１． ＰＨＡ合成酵素生産能を有する形質転換体の作製
ＹＮ２株を１００ ｍｌのＬＢ培地（１％ポリペプトン（日本製薬（株）製）、０．５％酵母エキス（Ｄｉｆｃｏ社製）、０．５％塩化ナトリウム、ｐＨ７．４）中で３０℃、一晩培養した後、マーマーらの方法により染色体ＤＮＡを分離回収した。得られたＹＮ２株の染色体ＤＮＡを制限酵素Ｈｉｎｄ ＩＩＩで完全分解した。クローニング・ベクターには、ｐＵＣ１８を使用し、制限酵素Ｈｉｎｄ ＩＩＩで切断した。末端の脱リン酸処理（Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ、 １、 ５７２、 （１９８９）； Ｃｏｌｄ Ｓｐｒｉｎｇ ＨａｒｂｏｒＬａｂｏｒａｔｏｒｙ 出版）の後、ＤＮＡライゲーション・キット Ｖｅｒ． ＩＩ（宝酒造（株）製）を用いて、ベクターの切断部位（クローニング・サイト）と染色体ＤＮＡのＨｉｎｄ ＩＩＩ完全分解断片とを連結した。この染色体ＤＮＡ断片を組み込んだプラスミド・ベクターを用いて、大腸菌（Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ）ＨＢ１０１株を形質転換し、ＹＮ２株のＤＮＡライブラリーを作製した。
【０１８５】
次に、ＹＮ２株のＰＨＡ合成酵素遺伝子を含むＤＮＡ断片を選択するため、コロニー・ハイブリダイズ用のプローブ調製を行った。配列番号：２および配列番号：３の塩基配列からなるオリゴヌクレオチドを合成し（アマシャム・ファルマシア・バイオテク（株））、このオリゴヌクレオチドをプライマーに用いて、染色体ＤＮＡをテンプレートとしてＰＣＲを行った。ＰＣＲ増幅されてきたＤＮＡ断片を、コロニー・ハイブリダイズ用のプローブとして用いた。プローブの標識化は、市販の標識酵素系ＡｌｋＰｈｏｓ・Ｄｉｒｅｃｔ（アマシャム・ファルマシア・バイオテク（株）製）を利用して行った。得られた標識化プローブを用いて、ＹＮ２株の染色体ＤＮＡライブラリーからコロニー・ハイブリダイゼーション法によって、ＰＨＡ合成酵素遺伝子を含む組換えプラスミドを有する大腸菌菌株を選抜した。
【０１８６】
選抜した菌株から、アルカリ法によってプラスミドを回収することで、ＹＮ２株由来のＰＨＡ合成酵素遺伝子を含むＤＮＡ断片を得ることができた。ここで取得した遺伝子ＤＮＡ断片を、不和合性グループであるＩｎｃＰ、ＩｎｃＱ、あるいはＩｎｃＷの何れにも属さない広宿主域複製領域を含むベクターｐＢＢＲ１２２（Ｍｏ ＢｉＴｅｃ）に組み換えた。この組み換えプラスミドをシュードモナス・チコリアイＹＮ２ｍｌ株（ＰＨＡ合成能欠損株）にエレクトロポレーション法により挿入して、形質転換したところ、ＹＮ２ｍｌ株のＰＨＡ合成能が復帰し、相補性を示した。従って、選抜された遺伝子ＤＮＡ断片は、シュードモナス・チコリアイＹＮ２ｍｌ株内において、ＰＨＡ合成酵素に翻訳可能な、ＰＨＡ合成酵素遺伝子領域を含むことが確認される。
【０１８７】
この遺伝子ＤＮＡ断片について、サンガー法により塩基配列を決定した。その結果、決定された塩基配列中には、それぞれペプチド鎖をコードする、配列番号：４および配列番号：５で示される塩基配列が存在することが確認された。これらのＰＨＡ合成酵素遺伝子について、染色体ＤＮＡをテンプレートとしてＰＣＲを行い、ＰＨＡ合成酵素遺伝子の完全長を再調製した。すなわち、配列番号：４で示される塩基配列のＰＨＡ合成酵素遺伝子に対する、上流側プライマー（配列番号：６）および下流側プライマー（配列番号：７）と、配列番号：５で示される塩基配列のＰＨＡ合成酵素遺伝子に対する、上流側プライマー（配列番号：８）および下流側プライマー（配列番号：９）とを、それぞれ合成した（アマシャム・ファルマシア・バイオテク（株））。
【０１８８】
これらのプライマー対を用いて、配列番号：４ならびに配列番号：５で示される塩基配列のそれぞれについて、ＰＣＲ増幅を行い、ＰＨＡ合成酵素遺伝子の完全長を増幅した（ＬＡ−ＰＣＲキット；宝酒造（株）製）。次に、得られたＰＣＲ増幅断片および発現ベクターｐＴｒｃ９９Ａを制限酵素Ｈｉｎｄ ＩＩＩで切断し、脱リン酸化処理（Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ， １巻， ５７２頁， １９８９年； Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ出版）した後、この発現ベクターｐＴｒｃ９９Ａの切断部位に、両末端の不用な塩基配列を除いたＰＨＡ合成酵素遺伝子の完全長を含むＤＮＡ断片を、ＤＮＡライゲーション・キット Ｖｅｒ． ＩＩ（宝酒造（株）製）を用いて連結した。
【０１８９】
得られた組換えプラスミドで、大腸菌（Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ ＨＢ１０１：宝酒造）を塩化カルシウム法により形質転換した。得られた組換え体を培養し、組換えプラスミドの増幅を行い、組換えプラスミドをそれぞれ回収した。配列番号：４の遺伝子ＤＮＡを保持する組換えプラスミドを、ｐＹＮ２−Ｃ１、配列番号：５の遺伝子ＤＮＡを保持する組換えプラスミドを、ｐＹＮ２−Ｃ２とした。ｐＹＮ２−Ｃ１、ｐＹＮ２−Ｃ２で、大腸菌（Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ ＨＢ１０１ｆＢ ｆａｄＢ欠損株）を塩化カルシウム法により形質転換し、それぞれの組換えプラスミドを保持する組換え大腸菌株、ｐＹＮ２−Ｃ１組換え株、ｐＹＮ２−Ｃ２組換え株を得た。
【０１９０】
参考例２． ＰＨＡ合成酵素の生産１
ｐＹＮ２−Ｃ１に対して、上流側プライマーとなる、オリゴヌクレオチド（配列番号：１０）および下流側プライマーとなる、オリゴヌクレオチド（配列番号：１１）をそれぞれ設計・合成した（アマシャム・ファルマシア・バイオテク（株））。このオリゴヌクレオチドをプライマーとして、ｐＹＮ２−Ｃ１をテンプレートとしてＰＣＲを行い、上流にＢａｍＨＩ制限部位、下流にＸｈｏＩ制限部位を有するＰＨＡ合成酵素遺伝子の完全長を増幅した（ＬＡ−ＰＣＲキット；宝酒造（株）製）。
【０１９１】
同様に、ｐＹＮ２−Ｃ２に対して、上流側プライマーとなる、オリゴヌクレオチド（配列番号：１２）および下流側プライマーとなる、オリゴヌクレオチド（配列番号：１３）をそれぞれ設計・合成した（アマシャム・ファルマシア・バイオテク（株））。このオリゴヌクレオチドをプライマーとして、ｐＹＮ２−Ｃ２をテンプレートとしてＰＣＲを行い、上流にＢａｍＨＩ制限部位、下流にＸｈｏＩ制限部位を有するＰＨＡ合成酵素遺伝子の完全長を増幅した（ＬＡ−ＰＣＲキット；宝酒造（株）製）。
【０１９２】
精製したそれぞれのＰＣＲ増幅産物をＢａｍＨＩおよびＸｈｏＩにより消化し、プラスミドｐＧＥＸ−６Ｐ−１（アマシャム・ファルマシア・バイオテク（株）製）の対応する部位に挿入した。これらのベクターを用いて、大腸菌（ＪＭ１０９）を形質転換し、発現用菌株を得た。発現用菌株の確認は、Ｍｉｎｉｐｒｅｐ（Ｗｉｚａｒｄ Ｍｉｎｉｐｒｅｐｓ ＤＮＡ Ｐｕｒｉｆｉｃａｔｉｏｎ Ｓｙｓｔｅｍｓ、ＰＲＯＭＥＧＡ社製）を用いて、大量に調製したプラスミドＤＮＡをＢａｍＨＩ、ＸｈｏＩで処理して得られるＤＮＡ断片によって行った。得られた菌株を、ＬＢ−Ａｍｐ培地１０ ｍｌで一晩プレ・カルチャーした後、その培養物０．１ ｍｌを、１０ ｍｌのＬＢ−Ａｍｐ培地に添加し、３７℃、１７０ ｒｐｍで３時間振とう培養した。その後、ＩＰＴＧを添加（終濃度 １ ｍＭ）し、３７℃で４〜１２時間培養を続けた。
【０１９３】
ＩＰＴＧ誘導した大腸菌を集菌（８，０００×ｇ， ２分、４℃）し、１／１０量の４℃リン酸緩衝生理食塩水（ＰＢＳ；８ ｇ ＮａＣｌ，１．４４ ｇ Ｎａ_２ＨＰＯ_４，０．２４ ｇ ＫＨ_２ＰＯ_４，０．２ ｇ ＫＣｌ，１，０００ ｍｌ精製水）に再懸濁した。凍結融解およびソニケーションにより菌体を破砕し、遠心（８，０００×ｇ， １０分、４℃）して固形夾雑物を取り除いた。目的の発現タンパク質が上清に存在することを、ＳＤＳ−ＰＡＧＥで確認した後、誘導され発現されたＧＳＴ融合タンパク質をグルタチオン・セファロース４Ｂ（アマシャム・ファルマシア・バイオテク（株）製）で精製した。使用するグルタチオン・セファロースは、予め非特異的吸着を抑える処理を行った。すなわち、グルタチオン・セファロースを同量のＰＢＳで３回洗浄（８，０００×ｇ、 １分、４℃）した後、４％ウシ血清アルブミン含有ＰＢＳを同量加えて、４℃で１時間処理した。処理後、同量のＰＢＳで２回洗浄し、１／２量のＰＢＳに再懸濁した。前処理したグルタチオン・セファロース ４０μｌを、無細胞抽出液１ ｍｌに添加し、４℃で静かに攪拌した。前記の手順で、融合タンパク質ＧＳＴ−ＹＮ２−Ｃ１および融合タンパク質ＧＳＴ−ＹＮ２−Ｃ２をグルタチオン・セファロースに吸着させた。吸着後、遠心（８，０００×ｇ、１分、４℃）してグルタチオン・セファロースを回収し、４００μｌのＰＢＳで３回洗浄した。その後、１０ ｍＭグルタチオン４０μｌを添加し、４℃で１時間攪拌して、吸着した融合タンパク質を溶出した。遠心（８，０００×ｇ、２分、４℃）して上清を回収した後、ＰＢＳに対して透析し、ＧＳＴ融合タンパク質を精製した。ＳＤＳ−ＰＡＧＥにより、ＧＳＴ融合タンパク質由来のシングル・バンドを確認した。
【０１９４】
各ＧＳＴ融合タンパク質５００μｇをＰｒｅＳｃｉｓｓｉｏｎプロテアーゼ（アマシャム・ファルマシア・バイオテク（株）製、５Ｕ）で消化した後、グルタチオン・セファロースに通してプロテアーゼとＧＳＴとを除去した。フロー・スルー分画をさらに、ＰＢＳで平衡化したセファデックスＧ２００カラムにかけ、発現タンパク質ＹＮ２−Ｃ１およびＹＮ２−Ｃ２の最終精製物を得た。ＳＤＳ−ＰＡＧＥにより、それぞれ６０．８ｋＤａ、および６１．５ｋＤａのシングル・バンドを確認した。
【０１９５】
該組換え型酵素を生体溶液試料濃縮剤（みずぶとりくん ＡＢ−１１００、アトー（株）製）を用いて濃縮し、１０ Ｕ／ｍｌの精製酵素溶液を得た。なお、各精製酵素活性は、前述の方法で測定した。また、試料中のタンパク質濃度は、マイクロＢＣＡタンパク質定量試薬キット（ピアスケミカル社製）によって測定した。表１に、各精製酵素の活性測定の結果を示す。
【０１９６】
【表１】

【０１９７】
参考例３．Ｐ ＨＡ合成酵素の生産２
Ｐ９１株、Ｈ４５株、ＹＮ２株またはＰ１６１株を、酵母エキス（Ｄｉｆｃｏ社製）０．５％、オクタン酸０．１％とを含むＭ９培地２００ｍｌに植菌して、３０℃、１２５ストローク／分で振盪培養した。２４時間後、菌体を遠心分離（１０，０００×ｇ、４℃、１０分間）によって回収し、０．１Ｍ トリス塩酸バッファー（ｐＨ８．０） ２００ ｍｌに再懸濁して、再度遠心分離することによって洗浄した。洗浄後の菌体を、０．１Ｍ トリス塩酸バッファー（ｐＨ８．０）２．０ ｍｌに再懸濁し、超音波破砕機にて破砕した後、遠心分離（１２，０００×ｇ、４℃、１０分間）して上清を回収して粗酵素を得た。
各精製酵素活性は前述の方法で測定し、表２に測定結果を示す。
【０１９８】
【表２】

【０１９９】
参考例４． 磁性体の調製
硫酸第一鉄水溶液中に、鉄イオンに対してｌ．０〜１．１当量の苛性ソーダ溶液を混合し、水酸化第一鉄を含む水溶液を調製した。この水溶液のｐＨを８前後に維持しながら、空気を吹き込み、８０〜９０℃で酸化反応を行い、種晶を生成させるスラリー液を調製した。
【０２００】
次いで、このスラリー液に、当初のアルカリ量（苛性ソーダのナトリウム成分）に対して、０．９〜１．２当量となるよう硫酸第一鉄水溶液を加えた後、スラリー液をｐＨ８前後に維持して、空気を吹き込みながら酸化反応を進める。酸化反応後に、生成した磁性酸化鉄粒子を洗浄、濾過、乾燥し、凝集している粒子を解砕し、平均粒径が０．１μｍの粒状の磁性体１を得た。
【０２０１】
参考例５． エポキシ型ＰＨＡ磁性構造体微粒子の作製１
ｐＹＮ２−Ｃ１組換え株由来のＰＨＡ合成酵素溶液（１０ Ｕ／ｍｌ）１０質量部に、参考例４で作製した磁性体を１質量部、ＰＢＳ ３９質量部を添加し、３０℃にて３０分間緩やかに振盪して、ＰＨＡ合成酵素を磁性体表面に吸着させた。これを遠心分離（１０，０００×ｇ、４℃、１０分間）し、沈澱をＰＢＳ溶液に懸濁し、再度遠心分離（１０，０００×ｇ、４℃、１０分間）して固定化ＰＨＡ合成酵素を得た。
【０２０２】
前記固定化ＰＨＡ合成酵素を０．１ Ｍリン酸バッファー（ｐＨ７．０）４８質量部に懸濁し、（Ｒ，Ｓ）−３−ヒドロキシ−５−フェノキシバレリルＣｏＡ（３−フェノキシプロパナールとブロモ酢酸エチルとのＲｅｆｏｒｍａｔｓｋｙ反応で得られた３−ヒドロキシ−５−フェノキシ吉草酸エステルを加水分解して３−ヒドロキシ−５−フェノキシ吉草酸を得た後、Ｅｕｒ． Ｊ． Ｂｉｏｃｈｅｍ．， ２５０， ４３２−４３９ （１９９７）に記載の方法で調製）０．８質量部、（Ｒ，Ｓ）−３−ヒドロキシ−７，８−エポキシオクタノイルＣｏＡ（Ｉｎｔ． Ｊ． Ｂｉｏｌ． Ｍａｃｒｏｍｏｌ．， １２， ８５−９１（１９９０）に記載の方法で合成した３−ヒドロキシ−７−オクテン酸の不飽和部分を３−クロロ安息香酸でエポキシ化したのち、 Ｅｕｒ． Ｊ． Ｂｉｏｃｈｅｍ．， ２５０， ４３２−４３９ （１９９７）に記載の方法で調製）０．２質量部、ウシ血清アルブミン（Ｓｉｇｍａ社製）０．１質量部を添加し、３０℃で２時間緩やかに振盪して、ＰＨＡ磁性構造体微粒子を得た。
【０２０３】
上記の試料１０μｌをスライドグラス上に採取し、１％ナイルブルーＡ水溶液１０μｌを添加し、スライドグラス上で混合した後、カバーグラスを載せ、蛍光顕微鏡（３３０〜３８０ ｎｍ励起フィルタ、４２０ ｎｍロングパス吸収フィルタ、（株）ニコン製）観察を行った。その結果、いずれの試料においても、ＰＨＡ磁性構造体微粒子表面が蛍光を発していることが確認された。従って、該ＰＨＡ磁性構造体微粒子は、確かにＰＨＡにより表面を被覆されていることが示された。
【０２０４】
さらに、試料の一部を遠心分離（１０，０００×ｇ、４℃、１０分間）により回収し、真空乾燥した後、クロロホルムに懸濁し、６０℃で２０時間攪拌して、外被を成すＰＨＡを抽出した。この抽出液について、^１Ｈ−ＮＭＲ分析を行った（使用機器：ＦＴ−ＮＭＲ：Ｂｒｕｋｅｒ ＤＰＸ４００、測定核種：^１Ｈ，使用溶媒：重クロロホルム（ＴＭＳ入り））。その結果、該ＰＨＡ磁性構造体微粒子の外被ＰＨＡの組成およびユニット％は、３−ヒドロキシ−５−フェノキシ吉草酸ユニット：７５％，３−ヒドロキシ−７，８−エポキシオクタン酸ユニット：２５％であることが確認された。
【０２０５】
参考例６． ビニルフェニル型ＰＨＡ磁性構造体微粒子の作製
ｐＹＮ２−Ｃ１組換え株由来のＰＨＡ合成酵素溶液（１０ Ｕ／ｍｌ）１０質量部に湿式法で合成した粒子径０．３μｍのマグネタイト微粒子「マグネタイトＥＰＴ５００」（戸田工業（株）製）を１質量部、ＰＢＳ ３９質量部を添加し、３０℃にて３０分間緩やかに振盪して、ＰＨＡ合成酵素を微粒子表面に吸着させた。これを遠心分離（１０，０００×ｇ、４℃、１０分間）し、沈澱をＰＢＳ溶液に懸濁し、再度遠心分離（１０，０００×ｇ、４℃、１０分間）して、固定化ＰＨＡ合成酵素を得た。
【０２０６】
上記固定化ＰＨＡ合成酵素を０．１ Ｍリン酸バッファー（ｐＨ７．０）４８質量部に懸濁し、（Ｒ）−３−ヒドロキシ−５−フェニルバレリルＣｏＡ（Ｅｕｒ． Ｊ． Ｂｉｏｃｈｅｍ．， ２５０， ４３２−４３９ （１９９７）に記載の方法で調製）０．８質量部、（Ｒ）−３−ヒドロキシ−５−（４−ビニルフェニル）バレリルＣｏＡ（Ｅｕｒ． Ｊ． Ｂｉｏｃｈｅｍ．， ２５０， ４３２−４３９ （１９９７）に記載の方法で調製）０．２質量部、ウシ血清アルブミン（Ｓｉｇｍａ社製）０．１質量部を添加し、３０℃で２時間緩やかに振盪して、ＰＨＡ磁性構造体微粒子を得た。
【０２０７】
上記の試料１０μｌをスライドグラス上に採取し、１％ナイルブルーＡ水溶液１０μｌを添加し、スライドグラス上で混合した後、カバーグラスを載せ、蛍光顕微鏡（３３０〜３８０ ｎｍ励起フィルタ、４２０ ｎｍロングパス吸収フィルタ、（株）ニコン製）観察を行った。その結果、ＰＨＡ磁性構造体微粒子の表面が蛍光を発していることが確認され、該ＰＨＡ磁性構造体微粒子は、確かにＰＨＡにより表面を被覆されていることが判った。
【０２０８】
さらに、試料の一部を遠心分離（１０，０００×ｇ、４℃、１０分間）により回収し、真空乾燥した後、クロロホルムに懸濁し、６０℃で２０時間攪拌して外被を成すＰＨＡを抽出した。この抽出液について、^１Ｈ−ＮＭＲ分析を行った（使用機器：ＦＴ−ＮＭＲ：Ｂｒｕｋｅｒ ＤＰＸ４００、測定核種：^１Ｈ，使用溶媒：重クロロホルム（ＴＭＳ入り））。その結果、該ＰＨＡ磁性構造体微粒子の外被ＰＨＡの組成およびユニット％は、３−ヒドロキシ−５−フェニル吉草酸ユニット：８３％，３−ヒドロキシ−５−（４−ビニルフェニル）吉草酸ユニット：１７％であることが確認された。
【０２０９】
参考例７． エポキシ型ＰＨＡ磁性構造体微粒子の作製２
参考例６で製造したビニルフェニル型ＰＨＡ磁性構造体微粒子について、そのビニル基のエポキシ化反応を行った。ビニルフェニル型ＰＨＡ磁性構造体微粒子の１質量部を四つ口丸底フラスコに加え、蒸留水６質量部を加えて攪拌した。フラスコ内を４０℃に加温し、これに過酢酸３０％のヘキサン溶液１質量部を連続的に滴下し、攪拌下４０℃で５時間反応させた。反応は、ＰＨＡ磁性構造体微粒子同士が凝集することなく進行した。反応終了後、室温まで冷却し、反応液をろ過することで、ＰＨＡ磁性構造体微粒子を回収した。この回収したＰＨＡ磁性構造体微粒子は蒸留水に再懸濁した後、遠心分離（３０００×ｇ、４℃、３０分間）した。分離した後、ＰＨＡ磁性構造体微粒子を蒸留水に再懸濁し、再度遠心分離を行って洗浄した。さらに、この洗浄操作を３回繰り返した。その後、真空乾燥することで、下記組成のＰＨＡ磁性構造体微粒子を得た。
【０２１０】
さらに、試料の一部を遠心分離（１０，０００×ｇ、４℃、１０分間）により回収し、真空乾燥した後、クロロホルムに懸濁し、６０℃で２０時間攪拌して、外被を成すＰＨＡを抽出した。この抽出液について、^１Ｈ−ＮＭＲ分析を行った（使用機器：ＦＴ−ＮＭＲ：Ｂｒｕｋｅｒ ＤＰＸ４００、測定核種：^１Ｈ，使用溶媒：重クロロホルム（ＴＭＳ入り））。この測定結果から計算した、外被ＰＨＡに含まれる各側鎖ユニットのユニット％は、３−ヒドロキシ−５−フェニル吉草酸ユニット：８５％、３−ヒドロキシ−５−（４−ビニルフェニル）吉草酸ユニット：１１％、３−ヒドロキシ−５−（４−（１，２−エポキシエチル）フェニル）吉草酸ユニット：４％であった。この反応系が不均一反応であるため、微粒子表面のＰＨＡはエポキシ化され、被覆内部では、未反応ＰＨＡのままに残されていると考えられる。
【０２１１】
参考例８． カルボキシ型ＰＨＡ磁性構造体微粒子の作製
参考例６で製造したビニルフェニル型ＰＨＡ磁性構造体微粒子について、そのビニル基の酸化開裂反応を行った。ビニルフェニル型ＰＨＡ磁性構造体微粒子の１０質量部を三つ口フラスコに移して、過酸化水素５０ｐｐｍを添加した３００重量部の蒸留水を加えた。オゾンを１重量部／時間の割りで吹き込み、３時間室温で攪拌した。反応は、磁性構造体微粒子同士が凝集することなく進行した。反応終了後、反応液をろ過することで、ＰＨＡ磁性構造体微粒子を回収した。ＰＨＡ磁性構造体微粒子は、蒸留水に再懸濁した後、遠心分離（３０００×ｇ、４℃、３０分間）を行って、残余する過酸化水素水を洗浄した。さらに、この洗浄操作を２回繰り返した。その後、真空乾燥することで、下記する組成のＰＨＡ磁性構造体微粒子を得た。
【０２１２】
さらに、試料の一部を遠心分離（１０，０００×ｇ、４℃、１０分間）により回収し、真空乾燥した後、クロロホルムに懸濁し、６０℃で２０時間攪拌して外被を成すＰＨＡを抽出した。この抽出液について、^１Ｈ−ＮＭＲ分析を行った（使用機器：ＦＴ−ＮＭＲ：Ｂｒｕｋｅｒ ＤＰＸ４００、測定核種：^１Ｈ，使用溶媒：重クロロホルム（ＴＭＳ入り））。この測定結果から計算した外被ＰＨＡに含まれる各側鎖ユニットのユニット％は、３−ヒドロキシ−５−フェニル吉草酸ユニット：８４％、３−ヒドロキシ−５−（４−ビニルフェニル）吉草酸ユニット：１１％、３−ヒドロキシ−５−（４−カルボキシフェニル）吉草酸ユニット：５％であった。この反応系が不均一反応であるため、微粒子表面のＰＨＡはエポキシ化され、被覆内部では、未反応ＰＨＡのままに残されていると考えられる。
【０２１３】
参考例９． ブロモ型ＰＨＡ磁性構造体微粒子の作製
ＹＮ２−Ｃ１組換え株由来のＰＨＡ合成酵素溶液（１０ Ｕ／ｍｌ）１０質量部に、参考例４で作製した磁性体を１質量部、ＰＢＳ ３９質量部を添加し、３０℃にて３０分間緩やかに振盪してＰＨＡ合成酵素を磁性体表面に吸着させた。これを遠心分離（１０，０００×ｇ、４℃、１０分間）し、沈澱をＰＢＳ溶液に懸濁し、再度遠心分離（１０，０００×ｇ、４℃、１０分間）して、固定化ＰＨＡ合成酵素を得た。
【０２１４】
上記固定化ＰＨＡ合成酵素を０．１ Ｍリン酸バッファー（ｐＨ７．０）４８質量部に懸濁し、（Ｒ，Ｓ）−３−ヒドロキシ−５−フェノキシバレリルＣｏＡ（３−フェノキシプロパナールとブロモ酢酸エチルとのＲｅｆｏｒｍａｔｓｋｙ反応で得られた３−ヒドロキシ−５−フェノキシ吉草酸エステルを加水分解して３−ヒドロキシ−５−フェノキシ吉草酸を得た後、Ｅｕｒ． Ｊ． Ｂｉｏｃｈｅｍ．， ２５０， ４３２−４３９ （１９９７）に記載の方法で調製）０．８質量部、（Ｒ）−３−ヒドロキシ−８−ブロモオクタノイルＣｏＡ（Ｅｕｒ ．Ｊ． Ｂｉｏｃｈｅｍ．， ２５０， ４３２−４３９（ １９９７） に記載の方法で調製）０．２質量部、ウシ血清アルブミン（Ｓｉｇｍａ社製）０．１質量部を添加し、３０℃で２時間緩やかに振盪して、ＰＨＡ磁性構造体微粒子を得た。
【０２１５】
上記のＰＨＡ磁性構造体微粒子の１０μｌをスライドグラス上に採取し、１％ナイルブルーＡ水溶液１０μｌを添加し、スライドグラス上で混合した後、カバーグラスを載せ、蛍光顕微鏡（３３０〜３８０ ｎｍ励起フィルタ、４２０ ｎｍロングパス吸収フィルタ、（株）ニコン製）観察を行った。その結果、磁性構造体の粒子表面が蛍光を発していることが確認された。従って、該ＰＨＡ磁性構造体微粒子は、確かにＰＨＡにより表面を被覆されていること示された。
【０２１６】
さらに、該ＰＨＡ磁性構造体微粒子の一部を遠心分離（１０，０００×ｇ、４℃、１０分間）により回収し、真空乾燥した後、クロロホルムに懸濁し、６０℃で２０時間攪拌して、外被を成すＰＨＡを抽出した。この抽出液について、^１Ｈ−ＮＭＲ分析を行った（使用機器：ＦＴ−ＮＭＲ：Ｂｒｕｋｅｒ ＤＰＸ４００、測定核種：^１Ｈ，使用溶媒：重クロロホルム（ＴＭＳ入り））。この測定結果から計算した外被ＰＨＡに含まれる各側鎖ユニットのユニット％は、３−ヒドロキシ−５−フェノキシ吉草酸ユニット：８９％、３−ヒドロキシ−８−ブロモオクタン酸ユニット：１１％であった。
【０２１７】
実施例１． エポキシ型ＰＨＡ磁性構造体微粒子上に担持された抗ＡＦＰ抗体によるＡＦＰの免疫検出
１． エポキシ型ＰＨＡ磁性構造体微粒子への抗体の担持
リン酸緩衝液（０．１Ｍ；ｐＨ７．４）中に、参考例５において作製されたエポキシ型ＰＨＡ磁性構造体微粒子を均一に分散させ（理論濃度５−１０×１０^８個／ｍＬ）、同様のリン酸緩衝液に溶解した抗ＡＦＰ（α−フェトプロテイン）抗体を、前記ＰＨＡ磁性構造体微粒子と抗体の最終比率が５−１０μｇ／１０^７となるよう加え、ピペッティングにより穏やかに攪拌した。
【０２１８】
ロータリー・シェーカーで３０℃１時間反応させた後、ブロッキングのためにウシ血清アルブミン（ＢＳＡ）を最終濃度０．３％になるように加え、さらに、１５時間反応を行うことにより、エポキシ型ＰＨＡ磁性構造体微粒子表面に担持された抗体を得た。
【０２１９】
２．標的成分（抗原：ＡＦＰ）との反応
５ｍＬ容エッペンドルフチューブ（ＢＳＡ処理）中、１で作製した抗ＡＦＰ抗体担持エポキシ型ＰＨＡ磁性構造体微粒子の分散液５００μＬに、濃度１μｇ／ｍＬのＡＦＰ溶液２０μＬを混合し、３７℃で３０分間反応させた。このチューブを磁石に接して、磁性構造体微粒子を捕捉し、上清をデカンテーションにより除去した。その後、チューブ中に０．０４％ＮａＣｌ溶液２ｍＬを加え攪拌した。前記と同様に、磁石により磁性構造体微粒子を捕捉し、上清をデカンテーションにより除去した。この洗浄操作を３回繰り返した。
【０２２０】
３．酵素標識二次抗体との反応
Ｊ． Ｉｍｍｕｎｏａｓｓａｙ， ４， ２０９（１９８３）およびＢｉｏｃｈｅｍｉｓｔｒｙ， １１（１２）， ２２９１ （１９７２）に記載の方法に従って、アルカリ・ホスファターゼ標識抗ＡＦＰ抗体Ｆａｂ’を作製し、最終濃度濃度０．１μｇ／ｍＬになるように、０．１Ｍトリス−塩酸緩衝液（２％ ＢＳＡ、１ｍＭ ＭｇＣｌ_２ 、０．１ｍＭ ＺｎＣｌ_２ 含有；ｐＨ７．５）と混合・溶解し、その５００μＬを３．で得られた抗原ＡＦＰ結合磁性複合体微粒子に加え、３７℃１０分間、反応させた。その後、２．と同様の方法により、酵素標識二次抗体を反応させた抗原ＡＦＰ結合磁性複合体微粒子の洗浄を行った。
【０２２１】
４．検出
３．で得られた酵素標識二次抗体を反応させた抗原ＡＦＰ結合磁性複合体微粒子を含むチューブに、５００μＬのグリシン−ＮａＯＨ緩衝液（０．１Ｍ：ｐＨ１０．３：ＭｇＣｌ_２（１ｍＭ）および卵白アルブミン（２５０ｍｇ／Ｌ）を含む）を加え、３７℃で５分間反応させた後、４−ニトロフェニルリン酸（最終濃度５．５ｍＭ）を含む前記緩衝液５００μｌを加え、３７℃で６０分間反応させた。反応終了後、１Ｍ ＮａＯＨ水溶液５００μＬを加えて、反応を停止し、紫外・可視吸光度を測定した。その結果、標識酵素アルカリ・ホスファターゼによる反応産物に由来する４０５ｎｍの吸収が検出され、実際に、抗原ＡＦＰが回収されていることが確認された。
【０２２２】
実施例２． セロペンタオース担持アミノ化ＰＨＡ磁性構造体微粒子を用いたコンカナバリンＡの回収
１．エポキシ型ＰＨＡ磁性構造体微粒子のアミノ化
参考例７で作製されたエポキシ型ＰＨＡ磁性構造体微粒子に、２，２’−（エチレンジオキシ）−ジメチルアミンを加え、３０℃で１５時間反応を行いアミノ化を行った。反応終了後、ビス−２−メトキシエチルエーテルで５回洗浄し、残留アミンを除去した後、蒸留水でさらに３回洗浄することにより、アミノ化ＰＨＡ磁性構造体微粒子を得た。
【０２２３】
２．アミノ化ＰＨＡ磁性構造体微粒子へのセロペンタオースの担持
工程１．で得られたアミノ化ＰＨＡ磁性構造体微粒子を、実施例１と同様にリン酸緩衝液中に均一に分散させ、セロペンタオース（Ｄ−（＋）−Ｃｅｌｌｏｐｅｎｔａｏｓｅ：Ｓｉｇｍａ社製）を予め過ヨウ素酸ナトリウムで非還元末端を酸化することにより、アルデヒド構造（−ＣＨＯ：ホルミル基）を生成させたものと３０℃で３０時間攪拌し、セロペンタオースをアミノ化ＰＨＡ磁性構造体微粒子上への担持を行った。その後、微粒子表面に残留する余剰のアミノ基を保護するため、無水ブタンジエン酸を加えて反応させ、蒸留水で３回洗浄することにより、セロペンタオース担持アミノ化ＰＨＡ磁性構造体微粒子を得た。
【０２２４】
３．コンカナバリンＡの回収
前記リン酸緩衝液にコンカナバリンＡ（Ｃｏｎｃａｎａｖａｌｉｎ Ａ：Ｓｉｇｍａ社製）とＢＳＡを溶解させ、２．で得られたセロペンタオース担持アミノ化ＰＨＡ磁性構造体微粒子を加えて、３０℃で１５時間反応した後、磁力により微粒子を回収した。回収した微粒子を、ドデシル硫酸ナトリウム（ＳＤＳ）で処理し、溶出液のＳＤＳ−ＰＡＧＥ分析を行ったところ、１０４ｋＤａにほぼ単一のバンドが見られ、コンカナバリンＡが回収されたことが示された。
【０２２５】
実施例３．カルボキシ型ＰＨＡ磁性構造体微粒子を用いたＤＮＡ断片のスクリーニング
１．モデル核酸Ｍ１３ｐ１８ｓｓＤＮＡの合成とアミノ化
配列番号：１４に示す、大腸菌Ｍ１３ファージｍｐ１８一本鎖ＤＮＡに相補的な塩基配列を有する２０量体オリゴヌクレオチドをＤＮＡ自動合成機（ＡＢＩ社製３８１Ａ）で合成した。
配列番号：１４： ５’−ＧＴＴＧＴＡＡＡＡＣＧＡＣＧＧＣＣＡＧＴ−３’
その後、通常のアミダイド試薬に変わり、アミノ基を導入したデオキシウリジル酸誘導体モノマー（化合物［１９］）を用いて、上記２０量体オリゴヌクレオチドの５’側にアミノ基（−ＮＨ_２）を導入した（化合物［２０］）。常法により、ＣＰＧサポートからの切り出し、脱保護基、高速液体クロマトグラフィー（ＨＰＬＣ）による精製を行った。
【０２２６】
【化２０】

【０２２７】
【化２１】

【０２２８】
２．アミノ化プローブ・オリゴヌクレオチドのカルボキシ型ＰＨＡ磁性構造体微粒子への担持
参考例８で得られたカルボキシ型ＰＨＡ磁性構造体微粒子を、予め０．０１Ｍ水酸化ナトリウム溶液で洗浄し、得られた微粒子に対し、理論上カルボキシ基の２モル倍程度のＥＤＣ（１−エチル−３−（３−ジエチルアミノプロピル）−カルボジイミド−塩酸）および式［２０］に示すアミノ化オリゴヌクレオチドを加えて、４℃で１８時間反応を行い、プローブ・オリゴヌクレオチドを担持したＰＨＡ磁性構造体微粒子を得た。
【０２２９】
３．ターゲット・モデルオリゴヌクレオチドの合成と標識化
上記配列番号：１４のオリゴヌクレオチドに対して、完全相補的な２０量体オリゴヌクレオチド（配列番号：１５）、１塩基ミスマッチの（配列番号：１６）、２塩基ミスマッチの（配列番号：１７）オリゴヌクレオチドを、それぞれ自動合成機で合成し、常法により精製した。
配列番号１５： ５’−ＡＣＴＧＧＣＣＧＴＣＧＴＴＴＴＡＣＡＡＣ−３’
配列番号１６： ５’−ＡＣＴＧＧＣＣＧＴＣＣＴＴＴＴＡＣＡＡＣ−３’
配列番号１７： ５’−ＡＣＴＧＧＣＧＧＴＣＧＴＴＡＴＡＣＡＡＣ−３’
精製したモデルオリゴヌクレオチド三種を、それぞれ工程１と同様の方法で、デオキシウリジル酸誘導体モノマー（化合物［１９］）を用いて、５’末端をアミノ化した。
【０２３０】
これとは別に、特許第０３３６８０１１号公報に開示の方法に従って、シアニン色素（化合物［２１］）１７０ｍｇを５ｍｌの乾燥ＤＭＦに溶解し、これに乾燥ピリジン５０μｌを加えた。さらに、ＤＳＣ（ジスクシイミジルカーボネート）１２８ｍｇを加えた後、暗所、室温で２０時間攪拌した。反応混液にジエチルエーテル１５０ｍｌを加え、析出した沈澱を集め、ジエチルエーテルで洗った後乾燥させた。得られた活性エステル体（化合物［２２］）を、そのままターゲット・モデルオリゴヌクレオチド（完全相補：配列番号１５）の標識に用いた。
【０２３１】
【化２２】

【０２３２】
【化２３】

【０２３３】
また、同様の方法でアズレン色素活性エステルである化合物［２３］で、上記の１塩基ミスマッチの（配列番号：１６）、２塩基ミスマッチの（配列番号：１７）オリゴヌクレオチドのアミノ化物を標識した（化合物［２４］）。
【０２３４】
【化２４】

【０２３５】
【化２５】

【０２３６】
４．完全相補オリゴヌクレオチドのスクリーニング
工程２で得られたプローブ・オリゴヌクレオチド担持ＰＨＡ磁性構造体微粒子と、工程３で得られた標識化ターゲット・オリゴヌクレオチドを、プローブ／ターゲット量比が理論上１／１０になるよう調整し、８０℃で２分間保持した後、時間をかけて室温に戻した（ハイブリダイゼーション条件）。
【０２３７】
磁力により微粒子を分離し、７８０ｎｍのレーザーで励起して、蛍光光度計で測定したところ、化学式［２１］の化合物に由来する、８２０ｎｍ付近にピークを持つ蛍光が見られ、配列番号：１５のターゲットＤＮＡが、選択的に回収されていることが確認された。
【０２３８】
実施例４．抗アルキルフェノール抗体担持カルボキシ型ＰＨＡ磁性構造体微粒子を用いたノニルフェノールの回収
１．抗アルキルフェノール抗体のカルボキシ型ＰＨＡ磁性構造体微粒子への担持
参考例８で作製したカルボキシ型ＰＨＡ磁性構造体微粒子のＰＨＡ側鎖上のカルボキシ基を、常法に従って、Ｎ−エチル−Ｎ’−（ジメチルアミノプロピル）カルボジイミド（ＥＤＣ）およびＮ−ヒドロキシスクシンイミド（ＮＨＳ）で活性化し、理論上２倍量の抗アルキルフェノール抗体（和光純薬製）を反応させて、抗アルキルフェノール抗体を担持したＰＨＡ磁性構造体微粒子を得た。
【０２３９】
２．ノニルフェノールの回収、検出
和光純薬製「アルキルフェノールＥＬＩＳＡキット」の手法に準じ、ノニルフェノール標準原液と抗原酵素複合体溶解液を混合し、工程１で得られた抗アルキルフェノール抗体担持ＰＨＡ磁性構造体微粒子を加えて、容器の外側から密着させた電磁石のスイッチ切換えにより、反応を促進した。本操作を３０℃で３０分間行った後、電磁石のスイッチをオンにした状態で微粒子を容器内壁面に捕捉し、反応液を除去した。次いで、容器に洗浄液を加え、電磁石のスイッチ切換えにより、洗浄を促進した。本洗浄工程を、洗浄液を置換して５分ずつ３回行った後、発色基質溶液を加えて、吸光度を測定したところ４５０ｎｍに強い吸収が見られ、本方法により、ノニルフェノールが回収され、その検出が行えることが示された。
【０２４０】
実施例５．長鎖アルカン担持ブロモ型ＰＨＡ磁性構造体微粒子を用いたリポソームの分離・回収
１．ドデカンチオールを用いたブロモ型ＰＨＡ磁性構造体微粒子へのドデカン担持
１−ドデカンチオールをヘキサンに溶解し、ヨウ化ナトリウム、炭酸カリウムおよびジエチルアミンを加えて、参考例９で作製したブロモ型ＰＨＡ磁性構造体微粒子と混合し、室温で２０時間攪拌することで、スルフィド結合（−Ｓ−）を介したドデカン担持ＰＨＡ磁性構造体微粒子を得た。
【０２４１】
２．蛍光リポソームの回収・検出
Ｓｉｇｍａ社が製造販売している「Ｌｉｏｐｓｏｍｅ Ｋｉｔ」を用い、そのプロトコルに従って、内部にＦＩＴＣを含んだリポソームを作製し、工程１で得られたドデカン担持ＰＨＡ磁性構造体微粒子と攪拌・混合した。混合１５分後、プローブ型電磁石を容器内に装入し、プローブ型電磁石用電源スイッチをオンにして、微粒子をプローブ型電磁石上に捕捉し、次いで、反応容器より洗浄液の入った容器へ移し、電源スイッチをオフにして微粒子を解放し、１５分攪拌した。この洗浄操作を３回繰り返した後、蛍光光度計で測定したところ、ＦＩＴＣに基づく５２０ｎｍに極大を有する蛍光が確認され、本方法により、リポソームが効率よく回収され、検出できることが示された。
【０２４２】
実施例６．コンセンサス結合配列遺伝子断片担持カルボキシ型ＰＨＡ磁性構造体微粒子を用いた転写因子タンパク質の分離・回収
塩基性のロイシンジッパーを持った転写因子であるＡＴＦ−２とそのコンセンサス結合配列との親和性を利用して、ＡＴＦ−２の分離・回収を試みた。
【０２４３】
１．末端チオール型ＡＴＦ−２コンセンサス結合配列ＤＮＡ断片の合成
ＤＮＡ自動合成機を用いて、配列番号：１８の一本鎖核酸を合成した。なお、配列番号：１８の一本鎖ＤＮＡの５’末端には、ＤＮＡ自動合成機での合成時にチオール・モディファイア（Ｔｈｉｏｌ−Ｍｏｄｉｆｉｅｒ）（グレンリサーチ（ＧｌｅｎＲｅｓｅａｒｃｈ）社製）を用いることによって、スルファニル基（−ＳＨ）を導入した（化合物［２５］）。続いて、通常の脱保護によりＤＮＡを回収し、高速液体クロマトグラフィーにて精製し、以下の実験に用いた。
配列番号：１８： ５’−ＴＧＡＣＡＴＣＡ−３’
【０２４４】
【化２６】

【０２４５】
２．末端チオール化ＤＮＡ断片のカルボキシ型ＰＨＡ磁性構造体微粒子への担持
参考例８で作製したカルボキシ型ＰＨＡ磁性構造体微粒子のＰＨＡ側鎖上のカルボキシ基を、常法に従って、Ｎ−エチル−Ｎ’−（ジメチルアミノプロピル）カルボジイミド（ＥＤＣ）およびＮ−ヒドロキシスクシンイミド（ＮＨＳ）で活性化し、ホウ酸緩衝液（ｐＨ８．５：ＮａＯＨにて調整）に溶解した２−（２−ピリジニルジチオ）エタンアミン（ＰＤＥＡ）により、チオールに対して活性化した後、工程１で得られた末端チオール化ＤＮＡ断片を加えて、担持を行った。なお、反応終了後、ＰＨＡ磁性構造体微粒子上に残留する活性基は、システイン−ＮａＣｌで不活性化した。
【０２４６】
３．結合反応、標識化
クロンテック社のプロトコルに従い、ＡＦＴ−２のＰＢＳ溶液中に工程２で得られたＤＮＡ断片担持ＰＨＡ磁性構造体微粒子を導入し、実施例４と同様の方法で、磁石によりＤＮＡ断片とＡＦＴ−２の親和性に起因する結合反応を促進した。反応終了後、実施例４と同様の方法で洗浄し、常法によりＦＩＴＣ標識された抗ＡＦＴ−２抗体（ポリクローナル）溶液中で、抗原・抗体反応を行った。
【０２４７】
４．洗浄および検出
抗原・抗体反応終了後、実施例４の方法で、磁石を用いた操作により洗浄を行い、蛍光検出を行ったところ、標識ＦＩＴＣに基づく５２０ｎｍに極大を有する蛍光が確認され、本方法により、転写因子タンパク質−コンセンサス配列ＤＮＡ断片の親和性結合に基づいた回収がなされ、検出できることが示された。
【０２４８】
【発明の効果】
本発明では、担体表面を被覆する高分子材料層として、生体親和性の高いポリマー材料である、ポリヒドロキシアルカノエート、例えば、３−ヒドロキシアルカン酸ユニットを含有するポリヒドロキシアルカノエートを用いることにより、より生体条件に近い条件で、検体中の標的物質の分離、検出を効率よく行うことができる。
【０２４９】
【配列表】

【図面の簡単な説明】
【図１】本発明において利用される、担体（磁性体）表面を被覆するＰＨＡ上に標的成分に対して結合親和力を有する分子を担持した、標的成分結合分子担持ＰＨＡ構造体の構成を模式的に示す図である。
【図２】本発明における、標的成分結合分子担持ＰＨＡ構造体に対する、標的成分と標的成分結合分子との選択的な結合形成過程を模式的に示す図である。
【図３】本発明における、標的成分との選択的な結合を形成した標的成分結合分子担持ＰＨＡ磁性構造体の、磁力を発揮する構造体を利用した磁気的分離過程を模式的に示す図である。
【符号の説明】
１ 担体基体（磁性体）
２ ポリヒドロキシアルカノエート被覆層
３ ポリヒドロキシアルカノエート中の標的成分に対して結合親和力を有する分子を選択的に担持する部位
４ 標的成分に対して結合親和力を有する分子
５ 標的成分
６、７ 標的成分以外の混在成分
８ 磁力を発揮する構造体（永久磁石や電磁石等）[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for separating a specific target component, a method for detecting the target component, or a method for screening for a specific target component contained in a sample, and more specifically, a method for screening a specific target component. Utilizing a molecule having a specific affinity for, for example, a naturally-derived or artificially modified nucleic acid molecule, protein and peptide, sugar chain, lipid, low-molecular compound, and a complex thereof, On the surface of the carrier is formed a structure supporting the molecule having the specific affinity, and by performing the binding between the structure and a specific target component, the specific content contained in the sample The present invention relates to a method for selectively separating, detecting or screening a target component, and a device which can be exclusively used for carrying out the method.
[0002]
[Background Art]
When separating / collecting, detecting, and screening target components contained in a specimen sample, particularly, a target component that is effective for medical treatment / diagnosis or has industrial value, for example, probe molecules, etc. Various separation / recovery, detection, and screening methods using microparticles having a micrometer to nanometer size have been developed and used as carriers for supporting molecules having a binding affinity for a target component. In particular, the method using magnetic fine particles (hereinafter, referred to as magnetic fine particles) as the carrier has an advantage that the magnetic fine particles can be easily separated and recovered by magnetic force when separating and recovering the carrier from the specimen sample. Have. Therefore, many development examples of the method using the magnetic fine particles can be cited.
[0003]
Many of the magnetic fine particles used as carriers for supporting the above-described probe molecules and the like are used in a form in which the surface is coated with an organic polymer for the purpose of improving the stability and controlling the magnetism.
[0004]
As one area of application of magnetic fine particles coated with an organic polymer to a carrier, immunodetection / diagnosis methods utilizing an antigen-antibody reaction have been conventionally developed.
[0005]
As one example, JP-A-07-151755 discloses a method of performing immunodetection using magnetic substance-containing polystyrene latex particles having an average particle size of 0.7 μm manufactured by Rhone Poulin Co., Ltd. as a carrier.
[0006]
Japanese Patent Application Laid-Open No. Hei 10-221341 discloses an immunoassay method using tosylated magnetic fine particles (Dynabead M-280, average particle size 2.8 μm) manufactured by Nippon Dynal. Similarly, Japanese Patent Application Laid-Open No. 09-229936 discloses an immunoassay method and an apparatus using magnetic fine particles Dynabeads M-450 uncoated, manufactured by Dynal, particle size: 4.5 μm, 3% (w / v). ing.
[0007]
A further area of application of the magnetic fine particles coated with an organic polymer to a carrier is a method for testing and diagnosing nucleic acid molecules such as DNA.
[0008]
Japanese Patent Application Laid-Open No. Hei 05-281230 discloses that, as magnetic fine particles for a carrier, XP-6006 manufactured by Dyno Industry AS (Norway) is used. Are disclosed.
[0009]
In addition, as a further area of application of magnetic fine particles coated with an organic polymer, methods for separating and recovering target components have been developed.
[0010]
JP-A-09-304385 discloses a method and an apparatus for separating and recovering basophils using Dynabeads M-450 Uncoated (Dynal: Dynal) (diameter of magnetic fine particles themselves: 45 μm). .
[0011]
JP-A-10-068731 discloses that magnetic fine particles manufactured by Rhone-Poulenc are used as magnetic fine particles for forming a covalent bond with an immunologically active substance or a nucleic acid, and are used in a liquid. A method for magnetically separating a test component is disclosed.
[0012]
U.S. Pat. Nos. 4,230,685, 3,970,518, 5,508,164, 5,567,326, and 4,018,886. The gazette also discloses a method in which magnetic fine particles are bound to a target component and used for treating and separating the target component bound to the magnetic fine particles.
[0013]
U.S. Pat. No. 5,900,481 discloses that a DNA is bound to a magnetic fine particle by using a coated magnetic fine particle to perform an operation on the DNA.
[0014]
U.S. Pat. No. 5,834,197 discloses a method for capturing certain strains from a liquid using coated magnetic microparticles. To these beads, a labeled antibody that has a selective affinity for the antigen is added, and the target antigen is sandwiched, and the magnetic particles can be detected so that the antigen that reacts with the labeled antibody can be easily detected and recovered. Methods for attaching various labels have been disclosed.
[0015]
In addition to the above-mentioned patent documents, non-patent documents including disclosure on manipulation of various molecules bound to magnetic fine particles include Analytical Chemistry 68 (13): 2122-6 (1996) and Nucleic Acids Research 23 (16): 3126. To 31 (1995).
[0016]
In addition to the above-described magnetic fine particles, examples of magnetic fine particles that are already commercially available for use in a method of detecting, collecting, and screening a target component include ferromagnetic particles manufactured by Spherotech. Particle, Seradyn, Sera-Mag, and Bangs Laboratory, Estapor.
[0017]
[Patent Document 1]
JP-A-07-151755
[Patent Document 2]
JP-A-10-221341
[Patent Document 3]
JP-A-09-229936
[Patent Document 4]
JP 05-281230 A
[Patent Document 5]
JP-A-09-304385
[Patent Document 6]
JP-A-10-068731
[Patent Document 7]
U.S. Pat. No. 4,230,685
[Patent Document 8]
U.S. Pat. No. 3,970,518
[Patent Document 9]
U.S. Pat. No. 5,508,164
[Patent Document 10]
U.S. Pat. No. 5,567,326
[Patent Document 11]
U.S. Pat. No. 4,018,886
[Patent Document 12]
U.S. Pat. No. 5,900,481
[Patent Document 13]
U.S. Pat. No. 5,834,197
[Non-patent document 1]
Analytical Chemistry 68 (13): 2122-6 (1996)
[Non-patent document 2]
Nucleic Acids Research 23 (16): 3126-31 (1995)
[0018]
[Problems to be solved by the invention]
In target component separation / recovery methods, detection methods, and screening methods, the target component of interest often has a physiological activity or is effective for medical treatment and diagnosis in many cases. It is desirable to operate in an environment that does not impair the conditions. However, in the above-described method used for the carrier containing the magnetic fine particles, the coating layer component that coats or coats the surface of the magnetic fine particles is a synthetic polymer such as styrene, acrylic, and vinyl. However, problems such as non-specific adsorption and deterioration of functions due to leakage of residual monomer components in the synthetic polymer still occur. Furthermore, not only the target component, but also the target component-binding molecule carried and immobilized on the carrier containing the magnetic fine particles is often a component derived from a living body or a derivative thereof. It cannot be said that the non-natural polymer surface used in the coating layer does not adversely affect the binding function of these target component binding molecules to the target component.
[0019]
The present invention solves the above-mentioned problems, and uses a structure in which a target component-binding molecule is supported and immobilized on a carrier to separate, collect, detect, and screen a target component. It is possible to carry out separation / collection, detection, and screening of target components while maintaining the target components as well as target component-binding molecules supported and immobilized on a carrier under conditions closer to biological conditions. To provide a way to:
[0020]
[Means for Solving the Problems]
The present inventors have conducted intensive studies in order to solve the above-mentioned problems.When performing separation / recovery, detection, and screening of target components, the target component-binding molecules are supported and immobilized on a carrier to be used. By using a polymer material with high biocompatibility instead of the conventional non-natural polymer as the polymer coating layer provided on the surface of the carrier, the conditions closer to biological conditions can be maintained Was found. Furthermore, as a high biocompatible polymer material used for a polymer coating layer provided on the surface of a carrier, polyhydroxyalkanoate (hereinafter, PHA), which is a polymer enzymatically polymerized in biological cells such as microorganisms, is used. (May be abbreviated in some cases), it has been found that more preferable results can be obtained.
[0021]
On the other hand, the present inventors have already made polyhydroxyalkanoate, a polymer enzymatically polymerized in biological cells such as microorganisms, into a PHA synthase immobilized and supported on an artificial structure by using a precursor monomer of the PHA. (Japanese Patent Application Laid-Open No. 2002-327046) has found a method for enzymatically performing a polymerization reaction by contacting 3-hydroxyacyl CoA, which is described in Japanese Patent Application Laid-Open No. 2002-327046. And JP-A-2003-11312).
[0022]
The present inventors can apply this technique to a carrier containing a magnetic substance, for example, to prepare a structure in which the surface of the carrier containing the magnetic substance is coated with the PHA. Since the PHA used for the coating layer itself is a material excellent in biodegradability and biocompatibility, a desired target component binding molecule is supported on a carrier containing a magnetic substance coated with such a PHA. The present invention has been verified to be able to satisfactorily separate / collect, detect, and screen target components having high utility values in the fields of biochemistry, diagnosis, medicine, and drug discovery. It was completed.
[0023]
That is, the method for separating a target component according to the present invention comprises:
A method for separating a target component contained in a sample,
A step in which a molecule having a binding affinity for the target component prepares a carrier immobilized on its surface,
Mixing the carrier and the sample,
The step of binding the target component contained in the sample and the molecule having the binding affinity immobilized on the carrier surface, which is mixed in the mixing step,
The binding step, through binding with the molecule having the binding affinity, the target component immobilized on the carrier, a step of separating from the sample together with the carrier from the sample,
The method for separating a target component, wherein the carrier is at least partially coated with a polyhydroxyalkanoate.
[0024]
At this time, a carrier containing a magnetic substance can be used as the carrier. For example, as the polyhydroxyalkanoate, a polyhydroxyalkanoate containing a 3-hydroxyalkanoic acid unit can be suitably used. When using a carrier comprising a magnetic substance, through the bond with the molecule having the binding affinity, in the step of separating the target component immobilized on the carrier from the specimen together with the carrier,
Preferably, a step of applying a magnetic field to the carrier containing the magnetic material to magnetically separate the carrier from the sample is preferably employed.
[0025]
On the other hand, for the method of separating a target component according to the present invention, the molecule having a binding affinity for the target component, immobilization on the surface of the carrier,
As the polyhydroxyalkanoate, which is coated on at least a part of the surface of the carrier, an epoxy group, an amino group, a carboxy group, and a polyhydroxyalkanoate having at least one functional group selected from the group consisting of halogen molecules Using
It is preferable that a molecule having a binding affinity for the target component is immobilized by utilizing at least one of the functional groups of the polyhydroxyalkanoate.
[0026]
Further, in the method for separating a target component according to the present invention, the molecule having a binding affinity for the target component, immobilized on the carrier surface,
It can be suitably applied to a molecule containing at least one selected from the group consisting of nucleic acids, proteins, peptides, sugar chains, lipids, low molecular weight compounds, and complexes thereof. In addition, the target component separated from the sample,
Nucleic acids, proteins, peptides, sugar chains, lipids, low molecular weight compounds, and when it is a component containing one or more selected from the group consisting of a complex thereof, the method for separating a target component according to the present invention, This is a more preferable method.
[0027]
In the method for separating a target component according to the present invention, the shape of the carrier to be used can be arbitrarily selected.For example, the shape of the carrier containing the magnetic substance may be granular, Although generally desirable, it is also possible to select the shape of the carrier containing the magnetic material into a plate shape or a film shape depending on the application. The magnetic material in the carrier containing the magnetic material is desirably a magnetic material made of a metal or a metal compound having magnetism.
[0028]
Further, the method for detecting a target component according to the present invention,
A method for detecting a target component contained in a sample,
A step in which a molecule having a binding affinity for the target component prepares a carrier immobilized on its surface,
Mixing the carrier and the sample,
The step of binding the target component contained in the sample and the molecule having the binding affinity immobilized on the carrier surface, which is mixed in the mixing step,
The binding step, through the binding with the molecule having the binding affinity, the step of selectively detecting the target component immobilized on the carrier,
A method for detecting a target component, wherein the carrier is at least partially coated with a polyhydroxyalkanoate.
[0029]
At this time, the step of selectively detecting the target component immobilized on the carrier through binding with the molecule having the binding affinity by the binding step,
By the binding step, through the binding with the molecule having the binding affinity, the target component immobilized on the carrier, the step of separating from the sample together with the carrier from the sample,
A step of selectively detecting the target component immobilized on the carrier through binding with the molecule having the binding affinity, which is separated from the sample together with the carrier. Is preferred.
[0030]
Also in the method for detecting a target component according to the present invention, a carrier containing a magnetic substance can be used as the carrier. For example, as the polyhydroxyalkanoate, a polyhydroxyalkanoate containing a 3-hydroxyalkanoic acid unit can be suitably used. When using a carrier comprising a magnetic substance, through the bond with the molecule having the binding affinity, in the step of separating the target component immobilized on the carrier from the specimen together with the carrier,
Preferably, a step of applying a magnetic field to the carrier containing the magnetic material to magnetically separate the carrier from the sample is preferably employed.
[0031]
On the other hand, in the method for detecting a target component according to the present invention, various spectroscopic techniques can be adopted as a means for detecting the target component. For example, the target component immobilized on the carrier can be selectively used. In the step of detecting
As a detection method, one detection method selected from the group consisting of a colorimetric method, a fluorescence method, a chemiluminescence method, and a radioisotope method, or a combination of two or more detection methods can be used. Alternatively, in the step of selectively detecting the target component immobilized on the carrier,
As a detection method, one detection method selected from the group consisting of nuclear magnetic resonance spectroscopy, mass spectroscopy, infrared absorption spectroscopy, and ultraviolet absorption spectroscopy, or a combination of two or more detection methods may be used. it can.
[0032]
In addition, the target component screening method according to the present invention,
A method for screening a target component contained in a medium,
The screening is directed to a mixed sample in which a mixture containing the target component is contained in the medium,
A step in which a molecule having a binding affinity for the target component prepares a carrier immobilized on its surface,
Mixing the carrier and the mixed sample,
The step of binding the target component contained in the mixed sample and the molecule having the binding affinity immobilized on the carrier surface, which is mixed in the mixing step,
The binding step, through the binding with the molecule having the binding affinity, the target component immobilized on the carrier, the step of separating from the mixed sample together with the carrier,
A method for screening a target component, wherein the carrier is at least partially coated with a polyhydroxyalkanoate.
[0033]
At this time, a carrier containing a magnetic substance can be used as the carrier. For example, as the polyhydroxyalkanoate, a polyhydroxyalkanoate containing a 3-hydroxyalkanoic acid unit can be suitably used. When using a carrier comprising a magnetic material, in the step of separating the target component immobilized on the carrier from the mixed sample together with the carrier, through binding with the molecule having the binding affinity. ,
It is preferable to adopt a step of applying a magnetic field to the carrier containing the magnetic material to magnetically separate the carrier from the mixed sample.
[0034]
In the present invention, the polyhydroxyalkanoate means a polyhydroxyalkanoate containing a hydroxyalkanoic acid unit as a constituent unit. In addition, in describing the embodiments of the present invention described below, a form using a polyhydroxyalkanoate containing a 3-hydroxyalkanoic acid unit as a polyhydroxyalkanoate is employed as a specific example. Any polyhydroxyalkanoate may be used without limitation, as long as it is included in the polyhydroxyalkanoate satisfying the above definition, without being limited to the form using a polyhydroxyalkanoate containing a 3-hydroxyalkanoic acid unit. can do.
[0035]
BEST MODE FOR CARRYING OUT THE INVENTION
First, the features of the present invention will be outlined with reference to FIGS.
[0036]
The method for separating / collecting, detecting, and screening a target component according to the present invention uses a molecule having a binding affinity for the target component as a means for selectively collecting the target component. Molecules 4 having a binding affinity (target component binding molecules) 4 are preliminarily supported and immobilized on the surface of a carrier, and are used in the form of a target component binding molecule supporting structure. More specifically, the carrier to be used is selected to have a structure in which a coating layer made of an organic polymer material is provided on the surface of the carrier base material. In this case, the coating layer made of the organic polymer material provided on the surface of the carrier base material is selected. As shown in FIG. 1, for example, a substrate made of a magnetic material is used as a carrier substrate 1, and at least a part of the surface of the carrier substrate 1 is a polyhydroxy polymer material having high biocompatibility. It is configured to be covered with alkanoate 2. When the carrier substrate contains a magnetic material, a magnetic carrier provided with a coating of polyhydroxyalkanoate (hereinafter sometimes referred to as PHA) may be referred to as a “PHA magnetic structure”. When the target component binding molecule 4 is carried on a carrier having a coating with polyhydroxyalkanoate 2 such as the PHA magnetic structure, the target component is bound to the target component during coating with polyhydroxyalkanoate 2. It utilizes a selectively loadable site 3 for an affinity molecule 4. When a substrate made of a magnetic material is used as the carrier substrate 1, the step of supporting the target component binding molecule 4 on the PHA magnetic structure prepares the PHA magnetic structure supporting the target component binding molecule.
[0037]
Next, a step of mixing a target component-binding molecule-supporting PHA-coated carrier and a liquid containing the target component 5 dissolved or dispersed in a medium, specifically, a specimen or a mixed sample is provided, As shown in FIG. 2, the target component 5 contained in the specimen or the mixed sample is brought into contact with the target component binding molecule 4 carried on the carrier, and the binding between the target component binding molecule 4 and the target component 5 is performed. Is completed, the target component 5 is immobilized on the carrier. On the other hand, the components 6 and 7 other than the target component contained in the specimen or the mixed sample do not cause the binding to the target component binding molecule 4 and the component 6 other than the target component with respect to the PHA coating the carrier surface. , 7 are less likely to adhere non-selectively, and even if non-selective adhesion of components 6 and 7 other than the target component occurs, by performing a simple cleaning operation, the non-selective adherence of the target component Components 6 and 7 other than the above can be easily removed.
[0038]
After the target component 5 is immobilized on the carrier, the carrier is recovered and separated together with the carrier using, for example, solid-liquid separation means. Is similarly recovered and separated. As illustrated in FIG. 3, when a magnetic body is used as a carrier substrate 1 in a carrier used as a carrier substrate 1, a magnetic body is applied when a magnetic field is applied by a structure 8 (a permanent magnet, an electromagnet, or the like) that exerts a magnetic force. Due to the magnetic attraction between the base and the structure 8 that exerts a magnetic force, the carrier is integrated on the structure 8 (permanent magnet, electromagnet, or the like) that exerts a magnetic force.
[0039]
In the method for separating and recovering target components, the detection method, and the screening method according to the present invention, as described above, the support of the target component binding molecule is a high biocompatible polymer material, polyhydroxyalkanoate. Separation / recovery of target components while maintaining conditions closer to biological conditions, since the binding between the target component and the target component binding molecule occurs on the surface portion coated with the polyhydroxyalkanoate. , Detection and screening can be performed.
[0040]
In the method for separating and recovering a target component and the method for detecting a target component according to the present invention, components other than the target component may not be present in the sample in some cases. That is, although the target component is present alone in the medium, but the target component is present over a wide range at a low concentration, after immobilizing the target component on a carrier, and recovering the carrier together with the carrier, In the carrier group to be separated / recovered, the target component has a high abundance ratio and is concentrated, which makes its detection easier.
[0041]
The polyhydroxyalkanoate: 1 and its supporting portion: 3 are described in <Polyhydroxyalkanoate> and <Supporting target component binding molecule on PHA magnetic structure>. In the section>, for the molecule having a binding affinity for the target component: 4 and for the target component 5, in the section for <the target component and the molecule having a binding affinity for the target component>, a structure exhibiting magnetism: No. 8 will be described later in detail in the section of <Magnetic Separation and Washing of Target Component-Binding Magnetic Structure>.
[0042]
Hereinafter, the present invention will be described in more detail.
[0043]
<Polyhydroxyalkanoate>
The polyhydroxyalkanoate that can be used in the method of the present invention is a short-chain-length polyhydroxyalkanoate whose monomer unit, 3-hydroxyalkanoic acid unit, has 4 or 5 carbon atoms. PHA: hereinafter sometimes abbreviated as scl-PHA); not only polyhydroxybutyrate (PHB) having 4 carbon atoms or polyhydroxyvalerate (PHV) having 5 carbon atoms, but also 6 carbon atoms. Polyhydroxyalkanoate composed of 3-hydroxyalkanoic acid units having a medium-chain-length (hereinafter sometimes referred to as “mcl”) of about to 12 (hereinafter sometimes abbreviated as “mcl-PHA”) Is included. Further, in addition to the above-mentioned polyhydroxyalkanoate comprising a monomer unit having an alkyl group in a side chain (hereinafter sometimes abbreviated as “usual-PHA”), a wider range of applications, for example, application as a functional polymer In consideration of the above, various substituents other than the alkyl group (for example, phenyl group, unsaturated hydrocarbon group, ester group, aryl group, cyano group, halogenated hydrocarbon group, epoxy group (-O-), etc.) Polyhydroxyalkanoates containing monomer units having a monomer introduced into the side chain (hereinafter sometimes abbreviated as unusual-PHA), or copolymers containing a plurality of these monomer units in an arbitrary unit ratio are also usable in the present invention. Method can be used.
[0044]
The PHA used in the method of the present invention is not particularly limited as long as it is a PHA that can be synthesized by a PHA synthase (for example, various mcl-PHAs and unusual-PHAs). As described above, PHA synthase is an enzyme that catalyzes the final step in a PHA synthesis reaction system in an organism, and therefore, any PHA that is known to be synthesized in an organism is the same. Is biosynthesized by the catalytic action of Furthermore, in the present invention, for all types of PHA that have been confirmed to be biosynthesized in an organism, 3-hydroxyacyl CoA corresponding to a monomer unit contained in a desired PHA is immobilized on a magnetic substance. By acting on the obtained PHA synthase, a desired PHA can be generated, and a structure in which the surface of the magnetic material is coated can be produced.
[0045]
In the present invention, the PHA synthase used in the step of producing a desired PHA and producing a structure covering the surface of the magnetic substance is appropriately selected from a group of microorganisms producing the synthase depending on the PHA to be synthesized. A microorganism produced by a selected microorganism or a transformant into which a PHA synthase gene derived from the microorganism has been introduced can be used.
[0046]
The biosynthesis of scl-PHA is based on (R) -3-hydroxypropionyl-CoA, (R) -3 produced from various carbon sources in the body of scl-PHA-producing bacteria through various metabolic pathways in vivo. The reaction is carried out by a polymerization reaction using a PHA synthase using at least one of -hydroxybutyryl-CoA and (R) -3-hydroxyvaleryl-CoA as a substrate. In the present invention, the enzyme that catalyzes the polymerization reaction of scl-PHA is particularly referred to as scl-PHA synthase. Among the scl-PHA synthases, for example, a PHA synthase that performs biosynthesis of PHB is usually called a PHB synthase (also called PHB polymerase or PHB synthase).
[0047]
The scl-PHA synthase used in the present invention is a gene set produced by a microorganism appropriately selected from microorganisms producing the synthase or a transformant into which a scl-PHA synthase gene derived from the microorganism is introduced. Recombinant scl-PHA synthase can be used. As the microorganism producing the scl-PHA synthase, for example, a microorganism known as a PHB or PHB / V producing bacterium can be used. Examples of the microorganisms having the PHB and PHB / V productivity include Aeromonas sp., Alcaligenes sp., Chromatium sp., Comamonas sp., And Methylobacterium. (Methylobacterium sp.), Paracoccus sp., Pseudomonas sp., And the like are known. In addition, the present inventors separately isolated strains, Bulkholderia cepacia KK01 strain, Ralstonia eutropha TB64 strain (Ralstonia eutroSCL-PHA TB64), and Alcaligenes TL2 esga strain (Alc). sp.TL2) can be used. The KK01 strain has a deposit number of FERM BP-4235, the TB64 strain has a deposit number of FERM BP-6933, and the TL2 strain has a deposit number of FERM BP-6913. Based on the “Budapest Treaty on the International Recognition of Deposits”, the International Depositary Agency, National Institute of Advanced Industrial Science and Technology (AIST) Patent Biological Depositary Center (formerly Ministry of Economy, Trade and Industry, National Institute of Advanced Industrial Science and Technology) Microbial Deposit Center).
[0048]
In addition to the wild-type strain producing scl-PHA synthase described above, a transformant can also be used to produce scl-PHA synthase. Cloning of a scl-PHA synthase gene derived from a wild strain, production of an expression vector, and creation of a transformant by genetic recombination can be performed according to a conventional method. Regarding the cloning of the scl-PHA synthase gene, the PHB synthase gene (phbC) of Alcaligenes eutrophus has already been cloned and reported. In addition, the present inventors have completed the cloning of phbC of Burkholderia cepacia KK01 strain, and also completed the cloning of phbC of Ralstonia eutropha TB64 strain. A transformant producing scl-PHA synthase can be created by introducing a vector containing the phbC gene into a host microorganism. A vector containing the phbC gene used for transformation is prepared, for example, by inserting the phbC gene into a plasmid vector, a phage vector, or the like. As a host, for example, Escherichia coli is widely used.
[0049]
For the production of scl-PHA synthase, the scl-PHA-producing bacteria described above can be used alone or in combination of two or more as needed.
[0050]
In addition, a desired scl-PHA synthase can be produced using a transformant into which the scl-PHA synthase gene derived from the scl-PHA producing bacterium has been introduced. For a transformant using a bacterium such as Escherichia coli as a host, a natural medium or a synthetic medium such as an LB medium or an M9 medium can be used as a medium for culturing the transformant. In addition, the culture temperature is selected in the range of 25 to 37 ° C., and the microorganism is cultured aerobically for 8 to 27 hours to increase the microorganism. Thereafter, the cells are collected, and the scl-PHA synthase accumulated in the cells can be collected. If necessary, antibiotics such as kanamycin, ampicillin, tetracycline, chloramphenicol, and streptomycin may be added to the medium, depending on the marker gene and various resistance genes contained in the expression vector. When an inducible promoter is used for transcription of the inserted scl-PHA synthase gene in the expression vector, when the transformant is cultured, an inducer corresponding to the promoter is added to the culture medium. May be added to promote expression. Examples of available inducers include isopropyl-β-D-thiogalactopyranoside (IPTG), tetracycline, indoleacrylic acid (IAA), and the like.
[0051]
In the present invention, as the scl-PHA synthetase, a crude cell lysate of a microorganism or a crude enzyme such as an ammonium sulfate salted-out product obtained by precipitating and recovering a protein component from a cell lysate with ammonium sulfate or the like may be used. Further, purified enzymes purified by various methods may be used. The scl-PHA synthase can be used by appropriately adding a stabilizer such as a metal salt, glycerin, dithiothreitol, EDTA, bovine serum albumin (BSA) and an activator, if necessary.
[0052]
In the step of separating and purifying the scl-PHA synthase from the cultured microbial cells, any separation and purification method can be used as long as the enzyme activity of the scl-PHA synthase is retained. For example, after culturing, microbial cells obtained by collecting cells are crushed using a French press, an ultrasonic crusher, lysozyme, various surfactants and the like. Affinity chromatography, cation or anion exchange chromatography is performed on the crude enzyme solution obtained as a supernatant by centrifuging the cell lysate of the microorganism, or the ammonium sulfate salting-out product prepared from the crude enzyme solution. A purified enzyme can be obtained by applying purification means such as gel filtration or the like, alone or in appropriate combination. In particular, recombinant proteins are expressed in the form of a fusion protein in which a "tag" such as a histidine residue is bound to the N-terminus or C-terminus, and the desired fusion protein is selectively expressed through the affinity resin via this tag. Purification can be performed more easily by using the technique of binding to. In order to separate the target enzyme protein from the affinity resin to which the fusion protein is selectively bound, the "tag" portion is cleaved with a protease such as thrombin or blood coagulation factor Xa, the pH is lowered, the binding competition is reduced. As the agent, a method such as adding a high concentration of imidazole may be used. Further, when using an expression vector such as pTYB1 (manufactured by New England Biolab), binding to an affinity resin via a tag is performed using “ Intein Is included in the reaction, a reducing substance such as dithiothreitol is added, and the mixture is cut under reducing conditions. Examples of fusion partners of the fusion protein that enable purification by affinity chromatography include, in addition to the histidine tag, glutathione S-transferase (GST), chitin binding domain (CBD), maltose binding protein (MBP), or Thioredoxin (TRX) is also known. For example, a GST fusion protein can be purified by a GST affinity resin.
[0053]
Further, as a microorganism that produces a PHA synthase, for example, a bacterium that produces mcl-PHA or unusual-PHA can be used. Examples of such microorganisms having the ability to produce mcl-PHA and unusual-PHA include the aforementioned Pseudomonas oleovorans, Pseudomonas reginovorans, Pseudomonas sp. 61-3 strain, Pseudomonas putida KT2442 strain, Pseudomonas aeruginosa, and the like. Pseudomonas putida P91 strain, Pseudomonas chicoryi H45 strain, Pseudomonas chicoryi YN2 strain, and Pseudomonas chicory JN. (Pseudomonas jessenii P161) and other Pseudomonas microorganisms. Burkholderia sp. OK3 strain described in 2001-78753 (Burkholderia sp. OK3, accession number: FERM P-17370), and Burkholderia sp. OK4 strain described in JP-A-2001-69968 (Burkholderia sp. OK4, accession number: FERM P-17371). In addition, microorganisms belonging to the genus Aeromonas sp., The genus Comamonas sp., And producing mcl-PHA or unusual-PHA can also be used.
[0054]
The P91 strain has a deposit number of FERM BP-7373, the H45 strain has a deposit number of FERM BP-7374, the YN2 strain has a deposit number of FERM BP-7375, and the P161 strain has a deposit number of FERM BP-7375. No.-7376, based on the “Budapest Treaty on the International Recognition of Deposits of Microorganisms under Patent Procedures”, the International Depository Agency, National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary (formerly Ministry of Economy, Trade and Industry) It has been internationally deposited at the National Institute of Biotechnology and Industrial Technology (NIBH) Patent Microorganisms Depositary.
[0055]
Further, these PHA-producing microorganisms and PHA synthases derived from the PHA-producing microorganisms are described in detail in JP-A-2002-327046 and JP-A-2003-11312, and thus should be referred to.
[0056]
Specific examples of the PHA coating the surface of the magnetic composite used in the method of the present invention include a PHA containing at least a monomer unit represented by the following chemical formulas [1] to [10]. can do.
[0057]
Chemical formula [1]:
[0058]
Embedded image

[0059]
(Wherein, R1 and a are represented by any one combination selected from the group consisting of the following combinations of R1 and a:
R1 is a hydrogen atom (H), and a is any of integers from 3 to 10;
R1 is a halogen atom, and a is any integer from 1 to 10;
R1 is a chromophore, and a is any integer from 1 to 10;
R1 is a carboxy group or a salt thereof, and a is any integer of 1 to 10;
R1 is:
[0060]
Embedded image

[0061]
A is a 1,2-epoxyethyl group, and a is any of integers from 1 to 7. )
Chemical formula [1]:
[0062]
Embedded image

[0063]
(Where
b represents any one of integers from 0 to 7,
R2 represents a hydrogen atom (H), a halogen atom, -CN, -NO ₂ , -CF ₃ , -C ₂ F ₅ , -C ₃ F ₇ Represents any one selected from the group consisting of )
Chemical formula [3]:
[0064]
Embedded image

[0065]
(Where
c represents any of integers from 1 to 8,
R3 represents a hydrogen atom (H), a halogen atom, -CN, -NO ₂ , -CF ₃ , -C ₂ F ₅ , -C ₃ F ₇ Represents any one selected from the group consisting of )
Chemical formula [4]:
[0066]
Embedded image

[0067]
(Where
d represents any of integers from 0 to 7,
R4 represents a hydrogen atom (H), a halogen atom, -CN, -NO ₂ , -CF ₃ , -C ₂ F ₅ , -C ₃ F ₇ Represents any one selected from the group consisting of )
Chemical formula [5]:
[0068]
Embedded image

[0069]
(Where
e represents any of integers from 1 to 8,
R5 is
Hydrogen atom (H), halogen atom, -CN, -NO ₂ , -CF ₃ , -C ₂ F ₅ , -C ₃ F ₇ , -CH ₃ , -C ₂ H ₅ , -C ₃ H ₇ Represents any one selected from the group consisting of )
Chemical formula [6]:
[0070]
Embedded image

[0071]
(In the formula, f represents any one of integers from 0 to 7.)
Chemical formula [7]:
[0072]
Embedded image

[0073]
(In the formula, g represents any one of integers 1 to 8.)
Chemical formula [8]:
[0074]
Embedded image

[0075]
(Where
h represents any of integers from 1 to 7,
R6 represents a hydrogen atom (H), a halogen atom, -CN, -NO ₂ , -COOR ', -SO ₂ R ", -CH ₃ , -C ₂ H ₅ , -C ₃ H ₇ , -CH (CH ₃ ) ₂ , -C (CH ₃ ) ₃ Represents any one selected from the group consisting of
here,
R ′ is a hydrogen atom (H), Na, K, —CH ₃ , -C ₂ H ₅ Any one selected from the group consisting of
R ″ is —OH, —ONa, —OK, a halogen atom, —OCH ₃ , -OC ₂ H ₅ Any one selected from the group consisting of )
Chemical formula [9]:
[0076]
Embedded image

[0077]
(Where
i represents any of integers from 1 to 7,
R7 represents a hydrogen atom (H), a halogen atom, -CN, -NO ₂ , -COOR ', -SO ₂ R "represents any one selected from the group consisting of:
here,
R ′ is a hydrogen atom (H), Na, K, —CH ₃ , -C ₂ H ₅ Any one selected from the group consisting of
R ″ is —OH, —ONa, —OK, a halogen atom, —OCH ₃ , -OC ₂ H ₅ Any one selected from the group consisting of )
Chemical formula [10]:
[0078]
Embedded image

[0079]
(In the formula, j represents any one of integers from 1 to 9.)
Note that specific examples of the halogen atom include fluorine, chlorine, and bromine. The chromophore is not particularly limited as long as the 3-hydroxyacyl-CoA compound corresponding to the monomer unit represented by the chemical formula [1] can be catalyzed by a PHA synthase. Taking into account steric hindrance and the like during polymer synthesis by the enzyme, in order to keep separation between the carboxy group bound to CoA and the chromophore in the 3-hydroxyacyl CoA molecule, the side chain has 1 to 1 carbon atoms. It is desirable to have 5 alkylene chains, for example, a linear alkylene chain. On the other hand, if the light absorption wavelength of the chromophore is in the visible range, the magnetic composite coated with the PHA can be colored, and the light absorption wavelength of the chromophore may be outside the visible range. However, such a PHA can be used for various electronic materials. Examples of the chromophore in the monomer unit represented by the chemical formula [1] include nitroso, nitro, azo, diarylmethane, triarylmethane, xanthene, acridine, quinoline, methine, thiazole, indamine, indophenol, lactone, aminoketone, and hydroxy. Ketone, stilbene, azine, ocasazine, thiazine, anthraquinone, phthalocyanine, indigoid and the like can be mentioned.
[0080]
As a specific example of the PHA coating the surface of the magnetic composite used in the method of the present invention, PHA further containing at least a monomer unit represented by the following chemical formulas [11] to [18] Can be exemplified.
[0081]
Chemical formula [11]:
[0082]
Embedded image

[0083]
(Where
R1 is a vinyl group,
a is any of integers from 1 to 10. )
Chemical formula [12]:
[0084]
Embedded image

[0085]
(Where
b represents any one of integers from 0 to 7,
R2 is CH ₃ Group, C ₂ H ₅ Group, C ₃ H ₇ Group, vinyl group, 1,2-epoxyethyl group , COOR ₂₁ (Where R ₂₁ Represents any one of H, Na and K atoms).
When a plurality of types of monomer units represented by the chemical formula [12] are present in the PHA, b and R2 independently represent the above-mentioned meanings for each monomer unit. )
Chemical formula [13]:
[0086]
Embedded image

[0087]
(Where
c represents any of integers from 1 to 8,
R3 is CH ₃ Group, C ₂ H ₅ Group, C ₃ H ₇ Group, SCH ₃ Represents any one selected from the group consisting of groups;
When a plurality of types of monomer units represented by the chemical formula [13] are present in the PHA, c and R3 independently represent the above-mentioned meanings for each monomer unit. )
Chemical formula [14]:
[0088]
Embedded image

[0089]
(Where
d represents any one of integers from 0 to 8,
R4 is
When d is 0, H atom, CN group, NO ₂ Group, halogen atom, CH ₃ Group, C ₂ H ₅ Group, C ₃ H ₇ Group, CF ₃ Group, C ₂ F ₅ Group or C ₃ F ₇ Represents any one selected from the group consisting of groups,
When d is 1 to 8, CH ₃ Group, C ₂ H ₅ Group, C ₃ H ₇ Represents any one selected from the group consisting of groups;
When a plurality of types of monomer units represented by the chemical formula [14] are present in the PHA, d and R4 independently represent the above-mentioned meanings for each monomer unit. )
Chemical formula [15]:
[0090]
Embedded image

[0091]
(In the formula, e represents any one of integers 1 to 8.)
Chemical formula [16]:
[0092]
Embedded image

[0093]
(Where
f represents any of integers from 1 to 8,
R6 is CH ₃ Group, C ₂ H ₅ Group, C ₃ H ₇ Group, (CH ₃ ) ₂ -CH group or (CH ₃ ) ₃ Represents any one selected from the group consisting of —C groups.
When a plurality of types of monomer units represented by the chemical formula [16] are present in the PHA, f and R6 independently represent the above-mentioned meanings for each monomer unit. )
Chemical formula [17]:
[0094]
Embedded image

[0095]
(Where
g represents any of integers from 1 to 8,
R7 is an H atom, a halogen atom, a CN group, NO ₂ Group, COOR ₇₁ (Where R ₇₁ Is H, Na, K, CH ₃ , C ₂ H ₅ ), SO ₂ R ₇₂ (Where R ₇₂ Represents -OH, -ONa, -OK, a halogen atom, -OCH ₃ , -OC ₂ H ₅ ), CH ₃ Group, C ₂ H ₅ Group, C ₃ H ₇ Group, (CH ₃ ) ₂ -CH group or (CH ₃ ) ₃ Represents any one selected from the group consisting of —C groups.
When a plurality of types of monomer units represented by the chemical formula [17] are present in the PHA, g and R7 independently represent the above-mentioned meanings for each monomer unit. )
Chemical formula [18]:
[0096]
Embedded image

[0097]
(Where
g represents any of integers from 1 to 8,
R7 is an H atom, a halogen atom, a CN group, NO ₂ Group, COOR ₇₁ (Where R ₇₁ Is H, Na, K, CH ₃ , C ₂ H ₅ ), SO ₂ R ₇₂ (Where R ₇₂ Represents -OH, -ONa, -OK, a halogen atom, -OCH ₃ , -OC ₂ H ₅ ), CH ₃ Group, C ₂ H ₅ Group, C ₃ H ₇ Group, (CH ₃ ) ₂ -CH group or (CH ₃ ) ₃ Represents any one selected from the group consisting of —C groups.
When a plurality of types of monomer units represented by the chemical formula [18] are present in the PHA, g and R7 independently represent the above-mentioned meanings for each monomer unit. )
As the PHA used in the present invention, a random copolymer or a block copolymer containing a plurality of types of monomer units represented by the above-mentioned chemical formulas [1] to [18] can be used. In a PHA containing a monomer unit, it is possible to control the physical properties of the PHA using the characteristics of each monomer unit and the contained functional group, to provide a plurality of functions, and to express a new function using an interaction between the functional groups. .
[0098]
Furthermore, for example, when coating the surface of the magnetic fine particles with PHA, the composition of PHA produced by changing the type and concentration of 3-hydroxyacyl-CoA, which is a substrate of PHA synthase, with time is also changed. It can be changed over time. Therefore, if the shape of the structure is granular, the PHA coating on the surface is in a direction from the inside to the outside, and if the structure is planar, the PHA coating on the surface is perpendicular to the plane. It is also possible to vary the monomer unit composition of the coated PHA in the direction.
[0099]
For example, the form of a structure finally manufactured is such that a magnetic material serving as a nucleus is coated with PHA having a low affinity for the magnetic material on the outermost surface of the structure. When necessary, first, the surface of a magnetic substance serving as a nucleus is coated with a PHA having a high affinity for the magnetic substance, and then the monomer unit composition of the PHA having a high affinity for the magnetic substance is determined. A magnetic substance that becomes a nucleus by forming a structure in which the composition changes in a direction from the inside to the outside or in a vertical direction, for example, a multilayer structure or a gradient structure, to a PHA monomer unit composition, and a surface thereof. It is possible to form a PHA film having a strong bond with the PHA layer covering the PHA.
[0100]
Further, a 3-hydroxypropionic acid unit, a 3-hydroxy-n-butyric acid unit, a 3-hydroxy-n-valeric acid unit, a 4-hydroxy-n-butyric acid unit, and a hydroxyalkanoic acid unit having 6 to 14 carbon atoms A homopolymer consisting of: or a copolymer consisting of a plurality of these units can also be used in the method of the present invention. Further, if necessary, chemical modification or the like may be further performed after once synthesizing the PHA or during the enzymatic synthesis.
[0101]
From the viewpoint of controlling the molecular weight of PHA used for coating and improving the hydrophilicity of the obtained PHA coating film, a compound having a hydroxyl group may be appropriately added to the reaction solution.
[0102]
In the method of the present invention, the compound having a hydroxyl group to be added to the reaction solution for the purpose described above includes alcohols, diols, triols, alkylene glycols, polyethylene glycols, polyethylene oxides, alkylene glycol monoesters, and polyethylene. It is at least one selected from glycol monoesters and polyethylene oxide monoester compounds. The details are as follows. That is, the alcohol, diol and triol compounds that can be suitably used are linear or branched alcohols, diols and triols having 3 to 14 carbon atoms. Alkylene glycols and alkylene glycol monoester compounds that can be suitably used are compounds in which the carbon chain of the alkylene glycol has a linear or branched structure having 2 to 10 carbon atoms. The polyethylene glycols, polyethylene oxides, polyethylene glycol monoesters, and polyethylene oxide monoester compounds which can be suitably used have a number average molecular weight in the range of 100 to 20,000.
[0103]
The concentration of the compound having a hydroxyl group to be added to the reaction solution for the above purpose is not particularly limited as long as the polymerization reaction of the 3-hydroxyacyl-CoA by the PHA synthase is not inhibited. With respect to the reaction solution containing the enzyme and 3-hydroxyacyl-CoA, the amount is in the range of 0.01% to 10% (w / v), more preferably in the range of 0.02% to 5% (w / v). It is preferable to add the compound in a batch manner at the beginning of the reaction, or to add it to the reaction solution in several portions within the reaction time.
[0104]
In the monomer unit represented by the chemical formula [12], a carboxy group (COOR ₂₁ ) Is represented by the chemical formula [12], and R2 is a vinyl group, that is, a monomer unit having a vinylphenyl group at a side chain terminal is selectively converted to a double bond portion of the vinyl group. Oxidative cleavage allows production by conversion to a carboxy group, and as a result, a PHA containing a unit having a carboxyphenyl group at the side chain terminal of the monomer unit represented by the chemical formula [12] is obtained.
[0105]
As a method for converting the vinyl group into a carboxy group, that is, a method for obtaining a carboxylic acid by oxidative cleavage of a carbon-carbon double bond using an oxidizing agent, for example, a method using a permanganate (J. Chem. Soc., Perkin. Trans. 1, 806 (1973)), a method using a dichromate (Org. Synth., 4, 698 (1963)), a method using a periodate (J. Chem., 46, 19 (1981)), a method using nitric acid (JP-A-59-190945), and a method using ozone (J. Am. Chem. Soc., 81, 4273 (1959)). And the like. Further, with regard to PHA, the above-mentioned Macromolecular Chemical Chemistry, 4, 289-293 (2) 01), a method of obtaining a carboxylic acid by carrying out a reaction under acidic conditions using potassium permanganate as an oxidizing agent at a carbon-carbon double bond at the side chain terminal of PHA, In the present invention, when performing the reaction for converting the vinyl group into a carboxy group, these oxidative cleavage methods can be referred to.
[0106]
In the reaction for converting the vinyl group into a carboxy group, potassium permanganate is generally used as the permanganate used as an oxidizing agent. Since the oxidative cleavage reaction is a stoichiometric reaction, the amount of permanganate used is usually at least 1 molar equivalent, preferably at least 1 molar equivalent, per 1 mol of the monomer unit represented by the chemical formula [12] having a vinyl group as R2. Is preferably used in an amount of 2 to 4 molar equivalents.
[0107]
In order to make the reaction system acidic under the oxidative cleavage reaction using potassium permanganate as an oxidizing agent, various inorganic acids and organic acids such as sulfuric acid, hydrochloric acid, acetic acid and nitric acid are usually used. However, when an acid such as sulfuric acid, nitric acid, or hydrochloric acid is used, an ester bond in the main chain of PHA is cleaved, which may cause a decrease in molecular weight. Therefore, it is preferable to use acetic acid under acidic conditions. The amount of the acid added to the reaction system is usually 0.2 to 200 molar equivalents, preferably 0.4 to 1 mol of the monomer unit represented by the chemical formula [12] having a vinyl group as R2. Select in the range of ~ 100 molar equivalents. When the amount of the acid added to the reaction system is less than 0.2 molar equivalent, the oxidative cleavage reaction has a low yield, and when it exceeds 200 molar equivalents, the decomposition product due to the added acid is In any case, it is not preferable. Further, a crown-ether can be used for the purpose of accelerating the oxidative cleavage reaction. In this case, as a result of forming a complex between the crown-ether and the permanganate, the effect of increasing the reaction activity is obtained. As the crown-ether usable for the above purpose, dibenzo-18-crown-6-ether, dicyclo-18-crown-6-ether, 18-crown-6-ether is generally used. The amount of the crown ether added to the reaction system is usually in the range of 1.0 to 2.0 molar equivalents, preferably in the range of 1.0 to 1.5 molar equivalents, per 1 mol of the permanganate salt. It is desirable to choose.
[0108]
In the present invention, when the oxidation reaction is performed, a PHA-coated structure including a unit represented by the chemical formula [12] having a vinyl group as R2, and a permanganate and an acid are charged together from the beginning. The reaction may be performed continuously or intermittently while being added to the reaction system. Alternatively, only the permanganate alone is dissolved or suspended in the reaction system first, and then the PHA-coated structure and the acid are continuously or intermittently introduced into the reaction system. In addition, only the PHA-coated structure may be suspended in the reaction system first, and then the permanganate and the acid are continuously or intermittently reacted with each other. The reaction may be performed in addition to the above. Furthermore, the PHA-coated structure and the acid may be charged first, and then the permanganate may be continuously or intermittently added to the reaction system and reacted. The salt and the acid may be charged first, and then the PHA-coated structure may be added continuously or intermittently to the reaction system to cause a reaction. The manganate may be charged first, and then the acid may be added continuously or intermittently to the reaction system for reaction.
[0109]
In an oxidative cleavage reaction using potassium permanganate as an oxidizing agent for a PHA-coated structure containing a unit represented by the chemical formula [12] having a vinyl group as R2, the reaction temperature is usually -20. It is good to select in the range of -40C, preferably in the range of 0-30C. The reaction rate depends on the stoichiometric ratio between the unit represented by the chemical formula [12] having a vinyl group as R2 and the permanganate, and the reaction temperature, and additionally, the target ratio for converting the vinyl group into a carboxy group. The reaction time is selected depending on the reaction time. However, when the target ratio is selected to be approximately 100%, the reaction time is usually preferably 2 to 48 hours.
[0110]
When the same oxidative cleavage reaction is applied to the unit represented by the chemical formula [11] in which R1 is a vinyl group, the vinyl group at the side chain terminal can be converted to a carboxy group.
[0111]
Further, a PHA containing a monomer unit represented by the chemical formula [17] or a monomer unit represented by the chemical formula [18] is prepared by first preparing a PHA containing a monomer unit represented by the chemical formula [8], and then preparing the sulfanyl group (− S-) is selectively oxidized to form a sulfinyl group (-SO-) or a sulfonyl group (-SO ₂ It can be manufactured by converting to-). Such selective oxidation of the sulfanyl group (-S-) can be achieved by, for example, performing an oxidation treatment with a peroxide compound, and if it can contribute to the oxidation of the sulfanyl group (-S-) at that time. Any type of peroxide compound can be used. In consideration of the oxidation efficiency, the influence on the PHA main chain skeleton and other monomer units contained in the PHA, and the simplicity of the treatment, in particular, hydrogen peroxide, sodium percarbonate, metachloroperbenzoic acid, performic acid, It is preferable to use a peroxide compound selected from the group consisting of peracetic acid.
[0112]
For example, when metachloroperbenzoic acid (MCPBA) is used as a peroxide compound used for oxidizing a sulfanyl group (-S-), oxidation to a sulfanyl group (-S-) in a monomer unit represented by a chemical formula [8] is performed. Proceeds stoichiometrically, so that it is easy to control the content ratio of the monomer unit represented by the chemical formula [17] or the monomer unit represented by the chemical formula [18]. In addition, since the reaction conditions are mild, unnecessary secondary reactions such as cleavage of the PHA main chain skeleton and cross-linking reaction of the active site hardly occur.
[0113]
As a general reaction condition, in order to selectively oxidize a sulfanyl group (-S-) to a sulfinyl group (-SO-), a chemical formula [8] containing a sulfanyl group (-S-) contained in PHA is used. The amount of MCPBA is slightly excessive, specifically, in the range of 1.1 to 1.4 mol per mol of the monomer unit represented by the formula (1), and the temperature is adjusted to 0 ° C in chloroform. The reaction is performed by selecting a temperature in the range of 30 ° C. In the above reaction conditions, oxidation proceeds to about 90% of the theoretical value when the reaction time is about 10 hours, and to about 100% of the theoretical value when the reaction time is about 20 hours, to a sulfinyl group (-SO-). Can be done. Further, all the sulfanyl groups (-S-) are converted to sulfonyl groups (-SO ₂ In order to oxidize to −), MCPBA is slightly more than 2 moles per 1 mole of the monomer unit represented by the chemical formula [8] containing the sulfanyl group (—S—) contained in PHA. The reaction may be carried out by selecting an excess amount, specifically in the range of 2.1 to 2.4 mol, and selecting the same solvent, temperature and time conditions as described above. In an oxidation treatment using MCPBA as such a peroxide compound, one molecule of MCPBA acts on a sulfanyl group (-S-) to convert it to a sulfinyl group (-SO-), and one molecule of MCPBA acts on the sulfonyl group (-SO-). Group (-SO ₂ -), The conversion proceeds from the sulfinyl group (-SO-) to the sulfonyl group (-SO-). ₂ Shows higher reactivity than conversion to-).
[0114]
Further, the unit represented by the chemical formula [12] having a 1,2-epoxyethyl group as R2 is different from the unit represented by the chemical formula [12] having a vinyl group as R2 in that the vinyl group in the side chain terminal vinyl phenyl group is It can be produced by selectively oxidizing a double bond portion and introducing an epoxy group. That is, by selectively oxidizing a vinyl group on a PHA containing a unit represented by the chemical formula [12] having a vinyl group as R2, the chemical formula [12] having a 1,2-epoxyethyl group as R2 A PHA including a unit represented by the following formula is obtained.
[0115]
Regarding the oxidation treatment for epoxidizing the vinyl group to a 1,2-epoxyethyl group, for example, a peroxide compound can be used as long as it can contribute to the selective partial oxidation of the vinyl group. Any type of peroxide compound can be used. At that time, considering the oxidation efficiency, the influence on the PHA main chain skeleton and other monomer units contained in the PHA, the simplicity of the treatment, etc., particularly, hydrogen peroxide, sodium percarbonate, metachloroperbenzoic acid, and formic acid It is preferable to use a peroxide compound selected from the group consisting of peracetic acid and peracetic acid. When a peroxide compound is used in the oxidation treatment for epoxidation from a vinyl group to a 1,2-epoxyethyl group, the reaction conditions refer to the reaction conditions in the selective partial oxidation treatment of the sulfanyl group with a peroxide compound. It can be.
[0116]
The molecular weight of the polyhydroxyalkanoate used in the method of the present invention is desirably about 1,000 to 10,000,000 as a number average molecular weight.
[0117]
In the present invention, the PHA synthesized by the PHA synthase used in the step of producing the PHA-coated structure is generally an isotactic polymer composed of only the R-form.
[0118]
The amount of the magnetic substance contained in the magnetic substance-based structure used in the method of the present invention is in the range of 1 to 80% by weight, preferably in the range of 5 to 70% by weight, more preferably 10 to 70% by weight. Select in the range of 60% by weight. When the content of the magnetic substance in the structure is less than 1% by weight, the magnetic performance may be insufficient and the performance as the magnetic substance-containing structure may be insufficient, and the content of the magnetic substance exceeds 80% by weight. Since the content ratio of PHA covering the surface with respect to the magnetic material serving as the nucleus relatively decreases, the original function of such a structure achieved by providing a sufficient PHA coating layer on the surface is achieved. There is a possibility that it will be impaired and that the practical performance will not be satisfactory.
[0119]
The particle size of the granular structure used in the method of the present invention is appropriately selected depending on the individual use thereof, but is usually in the range of 0.02 to 100 μm, preferably 0.05 to 20 μm. Select a range.
[0120]
<Target component and molecule having binding affinity for target component>
The “target component” targeted by the method of the present invention is physiologically useful, and is often mixed with other substances or is not mixed with other substances in a specimen sample. Even if it is one, it is present at a low concentration over a wide range. Therefore, there is a need for a means / method for separating / collecting, detecting, and screening only a “target component” contained in a specimen sample.
[0121]
In the method of the present invention, for the purpose, a molecule having a binding affinity for a target component (hereinafter, referred to as a “target component binding molecule”) that can be suitably used for capturing only the “target component” is described. May be used) ".
[0122]
Specific examples of the “target component” and the “molecule having a binding affinity for the target component” targeted by the method of the present invention include nucleic acids, proteins, peptides, sugar chains, lipids, and low-molecular compounds, or And a substance partially containing these molecules.
[0123]
Here, the “nucleic acid” includes deoxyribonucleic acid, ribonucleic acid, oligonucleotide, polynucleotide, aptamer, ribozyme and the like.
[0124]
Here, “protein” encompasses glycoproteins, lipoproteins, membrane proteins, and labeled proteins, as well as naturally and artificially derived variant molecules such as low molecular weight peptides.
[0125]
If the protein is an immunoreactant, for example, an antibody, an antigen, a hapten, or a complex thereof can be used. In particular, in the case of an “antibody,” a monoclonal antibody or a polyclonal antibody, a recombinant protein antibody, or a natural antibody , Chimeric antibodies, hybrid antibody mixture (s), phage antibodies displaying single chain antibodies (including whole phage displaying single chain antibodies), and fusions of antibodies and proteins, or hybrid mixtures thereof. You. When the protein is a catalytic reactant, it includes natural enzymes, mutant enzymes produced by genetic modification, semi-artificial enzymes complexed with synthetic molecules such as polyethylene glycol, and semi-artificial enzymes into which unnatural amino acids are introduced. I do.
[0126]
Examples of the "antibody" include IgG (immunoglobulin G or immunoglobulin G), and digestion enzymes such as pepsin and papain, or reduction such as dithiothreitol and 2-sulfanylethanol (2-mercaptoethanol). Using the agent, F (ab ') ₂ , Fab ′, Fab, Fv, etc. In addition, not only IgG but also IgM or a fragment derived from IgM whose molecular weight has been reduced by the same treatment as that for IgG may be used. In addition, any of a monoclonal antibody and a polyclonal antibody can be applied as the “target component binding molecule” in the present invention. When a monoclonal antibody is used as the “target component binding molecule”, a protein having a repeating structure such as a hepatitis B virus surface antigen or an antigen having a plurality of epitopes in the molecule such as a CA19-9 antigen , A plurality of types of monoclonal antibodies exhibiting reactivity to each epitope may be used in combination. Further, two or more kinds of different recognition epitopes may be used in combination.
[0127]
On the other hand, examples of the “antigen” include various substances such as proteins, polypeptides, steroids, polysaccharides, lipids, pollen, recombinant proteins produced by genetic engineering, and drugs. That is, the “antigen” of the present invention is a single substance selected for a special purpose such as diagnosis among all substances capable of inducing antibody production to humans or animals. Alternatively, it is a plurality of substances or a mixture containing them.
[0128]
"Peptide" mainly refers to fragments of a protein, regardless of their molecular weight, in the present invention.
[0129]
"Low molecular weight compounds" are low molecular weight, preferably organic molecules, that are recognizable by the receptor. In general, low molecular weight compounds are specific binding compounds for proteins, most of which are physiologically active substances or drug candidate substances, and particularly, antigenic low molecular weight compounds are sometimes referred to as “haptens”.
[0130]
As the “sugar chain”, a plurality (several to several tens) of monosaccharide units selected from glucose, mannose, N-acetylglucosamine, fucose, galactose, glucuronic acid, N-acetylglucosamine, and sialic acid are connected. Mention may be made of linear and branched oligomers and polymers. Further, in the method of the present invention, glycoproteins that are complexes of these sugar chains and proteins, glycolipids that are complexes with lipids, and further, when these sugar chains are presented on the surface of a living cell, The cell itself presenting a sugar chain on the cell membrane surface, or a cell membrane fragment is also included.
[0131]
The term “lipid” includes natural lipids (acylglycerols) such as complex lipids existing as membranes of intracellular organelles such as neural tissues, plasma membranes, mitochondria, microsomes and cell nuclei of living organisms, and lipids such as tissue boundary lipid membranes, and complexes with proteins. And liposomes as a lipid bilayer capsule thereof, such as lipoproteins and lecithin (phosphaditylcholine).
[0132]
<Magnetic body>
In the method of the present invention, the type, shape and size of the magnetic material are individually determined as long as the magnetic material used as the core of the structure can immobilize PHA synthase on its surface. It can be appropriately selected and used according to various applications. That is, the type and structure of the magnetic substance can be appropriately selected and used depending on the method of immobilizing the PHA synthase, the form of application of the produced structure, and the like.
[0133]
Examples of the magnetic material constituting the structure used in the method of the present invention include, for example, a metal or a metal compound having magnetism, and more specifically, triiron tetroxide (Fe ₃ O ₄ ), Γ-iron trioxide (γ-Fe ₂ O ₃ ), Various ferrites such as MnZn ferrite, NiZn ferrite, YFe garnet, GaFe garnet, Ba ferrite, Sr ferrite, metals such as iron, manganese, cobalt, nickel and chromium, and alloys such as iron, manganese, cobalt and nickel. It is possible, but not limited to these. Here, for example, when a biological substance is immobilized on a magnetic composite or when a magnetic composite is administered in vivo, magnetite (Fe ₃ O ₄ In addition to the above, various ferrite compositions in which a part of the metal element of magnetite is replaced by at least one other metal element can be suitably applied as necessary. The shapes of these magnetic materials vary depending on the generation conditions, and include polyhedrons, octahedrons, hexahedrons, spheres, rods, scales, and the like. It is more preferable for the stable expression of the function of the PHA layer to be coated. The particle size of the primary particles of the magnetic material constituting the structure of the present invention can be appropriately selected depending on the application, but for example, particles having a particle size in the range of 0.001 to 10 μm may be used. .
[0134]
Further, as the magnetic material constituting the structure used in the method of the present invention, those having superparamagnetism can also be preferably used. For example, when the particle diameter of ferrite is as small as about 20 nm or less, the ferrite is affected by thermal disturbance and exhibits superparamagnetism, and has no residual magnetization or coercive force. Even if it is superparamagnetic, it can be magnetically operated by applying an external magnetic field, and if it is superparamagnetic, it has no residual magnetization or coercive force, so it can be used when no external magnetic field is applied. Also, there is no possibility of magnetic aggregation due to residual magnetization.
[0135]
Further, the magnetic material used in the method of the present invention may be a composite material such as a matrix containing a metal or a metal compound, and the matrix is composed of various organic or inorganic materials. . In a method such as ferrite plating, a material in which the surface of an organic polymer is coated with a magnetic material, a material in which a magnetic material is dispersed in an organic polymer, and the like, as long as the magnetic material is partially exposed on the surface, Available.
[0136]
The magnetic material used in the method of the present invention can be used in the form of particles, a plate, or a film. It is preferable that the particles have a particle size of about 0.001 to 10 μm, assuming dispersion in a liquid sample.
[0137]
<Preparation of PHA magnetic structure>
The method for producing a PHA magnetic structure used in the method of the present invention comprises the steps of immobilizing a PHA synthase on a magnetic material, and enzymatic reaction with 3-hydroxyacyl CoA using the immobilized PHA synthase. And the step of synthesizing
[0138]
As a method for immobilizing a PHA synthase on a magnetic material, a method is generally used as long as the activity of the PHA synthase can be retained and is applicable to the target magnetic material. Any of the enzyme immobilization methods can be arbitrarily selected. For example, examples of a method for immobilizing a PHA synthase include a covalent bonding method, an ion adsorption method, a hydrophobic adsorption method, a physical adsorption method, an affinity adsorption method, a cross-linking method, and a lattice-type entrapment method. It is simple to use an immobilization method utilizing ion adsorption or hydrophobic adsorption.
[0139]
In general, an enzyme protein such as PHA synthase is a polypeptide to which a large number of amino acids are bound, and an ionic adsorbent is formed by an amino acid residue having an ionic group on a side chain such as lysine, histidine, arginine, aspartic acid, and glutamic acid. In addition, alanine, valine, leucine, isoleucine, methionine, tryptophan, phenylalanine, amino acid residues having a hydrophobic group as a side chain such as proline, or as a whole, is an organic polymer. And has properties as a hydrophobic adsorbent. Therefore, it can be adsorbed on a solid surface having ionic or hydrophobic properties, or both ionic and hydrophobic properties, though varying in degree.
[0140]
When a method of immobilizing a PHA synthase mainly by an ion adsorption method is applied, a core having an ionic functional group on its surface may be used as its nucleus. For example, kaolinite, bentonite, talc, Clay minerals such as mica, metal oxides such as alumina and titanium dioxide, insoluble inorganic salts such as silica gel, hydroxyapatite and calcium phosphate gel can be used as the core. In addition, polymers having an ionic functional group such as inorganic pigments, ion exchange resins, chitosan, and polyaminopolystyrene containing these as main components can also be used as the ion-adsorbing core.
[0141]
In the PHA magnetic structure used in the present invention, when the base material is a magnetic material, for example, in a metal oxide such as ferrite, a hydroxyl group is present on the surface thereof, and hydrogen is present on the surface of the PHA synthase with a carboxy group. An immobilization method by bonding or the like can be suitably used.
[0142]
On the other hand, when a method of immobilizing a PHA synthase mainly by hydrophobic adsorption is applied, a core having a nonpolar surface may be used as a nucleus thereof. For example, a styrene-based polymer, an acrylic-based polymer, a methacryl-based polymer may be used. Many polymers having no ionic functional group on the surface or a hydrophobic functional group on the surface, such as vinyl esters, vinyl esters, and vinyl polymers, can be used as the core. Specifically, organic pigments such as azo pigments having a plurality of aromatic rings, condensed polycyclic phthalocyanine pigments, anthraquinone pigments, etc., and carbon black are hydrophobic adsorbents. Further, the hydrophobic adsorption method can be applied to a magnetic substance subjected to a lipophilic treatment.
[0143]
The immobilization of the PHA synthase on the core by the ion adsorption method or the hydrophobic adsorption method is achieved by mixing the PHA synthase and the core in a predetermined reaction solution. At this time, it is desirable to shake or agitate the reaction vessel with appropriate strength so that the PHA synthase is evenly adsorbed on the surface of the core.
[0144]
Depending on the pH, salt concentration, and temperature of the reaction solution, the polarity of the surface charge of the core and the PHA synthase, the amount of charge, and the hydrophobicity change. It is desirable to make adjustments. For example, when the core is mainly ion-adsorbing, the amount of charge that contributes to the adsorption between the core and the PHA synthase can be increased by reducing the salt concentration. In addition, by adjusting the pH, the opposite charges can be increased. When the core is mainly hydrophobic adsorbent, the hydrophobicity of both can be increased by increasing the salt concentration. In addition, solution conditions suitable for adsorption can be set by previously measuring electrophoresis, a wetting angle, and the like, and examining the charge state and hydrophobicity of the core and the PHA synthase. Furthermore, the condition can also be determined by directly measuring the amount of adsorption between the core and the PHA synthase. The amount of adsorption is measured, for example, by adding a PHA synthase solution of a known concentration to the solution in which the core is dispersed, performing an adsorption treatment, measuring the PHA synthase concentration in the solution, and subtracting the amount of the adsorbed enzyme by a subtraction method. May be used.
[0145]
In the case of a core material in which the enzyme is difficult to be immobilized by the ion adsorption method or the hydrophobic adsorption method, the covalent bonding method may be used in consideration of the complicated operation and the possibility of deactivation of the enzyme. For example, a method of diazotizing a core material (solid particles) having an aromatic amino group and diazo-coupling an enzyme thereto, or a method of forming a peptide between a core material (solid particles) having a carboxy group and an amino group and a PHA synthase. A method of forming a bond, a method of alkylating between a core material (solid particles) having a halogen group (halogenoalkyl group) and an amino group of a PHA synthase, and a polysaccharide core particle activated with cyanogen bromide. A method of reacting an amino group of a PHA synthase, a method of cross-linking between an amino group of a core material (solid particle) and an amino group of an enzyme, a method of forming a carboxy group in the presence of a compound having an aldehyde group or a ketone group and an isocyanide compound , A method of reacting a core material (solid particles) having an amino group with a PHA synthase, a method of reacting a disulfide group (-SS-) And a method to exchange reaction with the material (solid particles) and sulfanyl group of the PHA synthesizing enzyme (-SH).
[0146]
Further, PHA synthase may be adsorbed on the surface of the core material (solid particles) by affinity adsorption. Affinity adsorption is a biological adsorption between a biopolymer and a specific substance showing a specific affinity to it, such as an enzyme and a substrate, an antibody and an antigen, a receptor and an information substance such as acetylcholine, There are mRNA and tRNA. In general, in the method of immobilizing an enzyme protein by using affinity adsorption, a substrate of an enzyme, a reaction product, a competitive inhibitor, a coenzyme, an allosteric effector, and the like are bound to a solid surface as a ligand, and this ligand is added. A method in which affinity adsorption is performed on a solid surface through binding to an enzyme protein is employed. However, for PHA synthase, for example, when 3-hydroxyacyl-CoA as a substrate is used as a ligand, the active site catalyzing PHA synthesis in the PHA synthase is blocked by binding to the ligand. This causes a problem that PHA cannot be synthesized. However, by preliminarily fusing another biopolymer to the PHA synthase and applying the affinity adsorption method using the ligand of the biopolymer, the PHA of the PHA synthase itself can be retained even after immobilization by affinity adsorption. Synthetic activity can be maintained. The fusion of the PHA synthase and the biopolymer may be performed by a genetic engineering technique, or the biopolymer may be chemically bound to the PHA synthase. As the biopolymer to be used, any one can be used as long as the ligand for the biopolymer is easily available and the ligand can be easily bound to the core.However, when a fusion is expressed by genetic recombination, The biopolymer to be fused is preferably a protein. Specifically, a transformant is used to produce a fusion protein of GST and PHA synthase using Escherichia coli in which a gene in which the gene sequence of PHA synthase is linked to the gene sequence expressing GST of the fusion partner is used, By adding this fusion protein to Sepharose to which GST ligand glutathione is bound, the fusion protein-type PHA synthase can be affinity-adsorbed onto Sepharose.
[0147]
Further, a peptide containing an amino acid sequence capable of binding to a magnetic substance is fused and presented to a PHA synthase, and the peptide portion of the amino acid sequence capable of binding to the magnetic substance is bound to the magnetic substance. Based on the above, PHA synthase can be immobilized on the surface of the magnetic substance.
[0148]
The amino acid sequence capable of binding to a magnetic substance can be determined, for example, by screening a random peptide library. In particular, for example, a phage display peptide library prepared by linking a random synthetic gene to an N-terminal gene of a surface protein (for example, geneIII protein) of M13-based phage can be suitably used. In this case, the following procedure is used to determine an amino acid sequence capable of binding to a magnetic substance. That is, the magnetic substance or at least one component constituting the magnetic substance is brought into contact by adding a phage display peptide library, and then bound and unbound phages are separated by washing. The magnetic substance-bound phage is eluted with an acid or the like, neutralized with a buffer, and then infected with Escherichia coli to amplify the magnetic substance-bound phage. When this selection is repeated a plurality of times, a plurality of clones having high binding ability to the target magnetic substance are concentrated. Here, in order to obtain a single clone, a colony is formed on a medium plate while being infected again with E. coli. After culturing each single colony in a liquid medium, the phage present in the medium supernatant is purified by precipitation with polyethylene glycol or the like, and the base sequence of the synthetic gene possessed by the clone is analyzed. The structure (amino acid sequence) of the peptide possessed can be known.
[0149]
The synthetic gene encoding the amino acid sequence of the peptide capable of binding to the magnetic substance, which is selected by the above-described screening method, is used by being fused to the gene of the PHA synthase using ordinary genetic engineering techniques. The peptide capable of binding to a magnetic substance can be expressed by being linked to the N-terminal or C-terminal of PHA synthase. Alternatively, expression can be carried out by inserting an appropriate spacer sequence. The spacer sequence is preferably about 3 to about 400 amino acids, and the spacer sequence may include any amino acids. Most preferably, the spacer sequence does not interfere with the function of the PHA synthase, and when the fusion protein-type PHA synthase binds to the magnetic substance via the peptide capable of binding to the magnetic substance, the spacer sequence has a binding ability. It does not interfere with the binding by the peptide possessed.
[0150]
The immobilized PHA synthase produced by the above method can be used as it is, but it should be further stored after freeze-drying and the like, and each time it should be added to the enzyme reaction medium and used. You can also.
[0151]
When the amount of PHA synthase at which the amount of CoA released in the reaction for synthesizing PHA by the polymerization of 3-hydroxyacyl CoA by the immobilized PHA synthase becomes 1 μmol per minute is defined as 1 unit (U), When the magnetic substance is the core of the capsule structure, the amount of the PHA synthase immobilized on the body is, for example, in the range of 10 units (U) to 1,000 units (U) per gram of the magnetic substance, preferably, It is good to set within the range of 50 units (U) to 500 units (U).
[0152]
The immobilized PHA synthase is added to an aqueous reaction solution containing 3-hydroxyacyl-CoA as a raw material of a desired PHA, and PHA is synthesized by the PHA synthase immobilized on the surface of the magnetic material, thereby obtaining a magnetic material. The surface of the body forms a structure coated with the synthesized PHA. At that time, the aqueous reaction solution should be constituted into a reaction system adjusted to conditions capable of exerting the activity of the immobilized PHA synthase, for example, usually in the range of pH 5.5 to pH 9.0, preferably, The pH is adjusted with a buffer so as to be in the range of pH 7.0 to pH 8.5. However, depending on the optimum pH and pH stability of the immobilized PHA synthase to be used, setting conditions other than the above pH range is not excluded. The type of the buffer can be appropriately selected and used depending on the set pH range and the like as long as it can exert the activity of the immobilized PHA synthase to be used. Buffer, specifically acetate buffer, phosphate buffer, potassium phosphate buffer, 3- (N-morpholino) propanesulfonic acid (MOPS) buffer, N-tris (hydroxymethyl) methyl-3-amino A propanesulfonic acid (TAPS) buffer, a Tris-HCl buffer, a glycine buffer, a 2- (cyclohexylamino) ethanesulfonic acid (CHES) buffer, or the like may be used. The concentration of the buffer is not particularly limited as long as it can exert the activity of the immobilized PHA synthase to be used, but is usually in the range of 5.0 mM to 1.0 M, preferably 0.1 M. A concentration of ~ 0.2 M is preferably used. The reaction temperature is appropriately set according to the characteristics of the PHA synthase to be used, but is usually set in the range of 4 ° C to 50 ° C, preferably in the range of 20 ° C to 40 ° C. However, depending on the optimum temperature and heat resistance of the PHA synthase used, setting conditions other than the above ranges is not excluded. The reaction time is to be selected according to the thickness of the PHA coating layer to be synthesized, and also depends on the stability of the PHA synthase to be used, but is usually in the range of 1 minute to 24 hours. Preferably, it is appropriately selected and set within a range of 30 minutes to 3 hours. The concentration of the substrate 3-hydroxyacyl-CoA in the reaction solution should be appropriately selected according to the composition and thickness of the PHA coating layer to be synthesized, and exhibits the activity of the immobilized PHA synthase used. It is set appropriately within the range in which it can be performed, but is usually set within the range of 0.1 mM to 1.0 M, preferably 0.2 mM to 0.2 M. In addition, when the substrate 3-hydroxyacyl-CoA concentration in the reaction solution is high, the pH of the reaction system generally tends to decrease. Therefore, when the substrate 3-hydroxyacyl-CoA concentration is set to be high, the fluctuation of pH is suppressed. Therefore, it is preferable that the buffer solution concentration is also set to be higher.
[0153]
Further, in the above step, by changing the composition such as the type and concentration of the substrate 3-hydroxyacyl-CoA in the aqueous reaction solution over time, if the shape of the formed structure is granular, it goes from the inside to the outside. The composition of the monomer unit in the thickness direction of the PHA covering the surface of the magnetic material can be changed in the direction and in the vertical direction if the structure is planar.
[0154]
As a form of the structure in which the monomer unit composition of the PHA is changed, for example, a PHA film in which the composition change of the PHA coating is continuous and a composition gradient is formed in a direction from the inside to the outside or in a vertical direction is a magnetic layer. The form which covered the body can be mentioned. As a method for producing a one-layer PHA having a composition gradient, for example, a method of adding a substrate 3-hydroxyacyl-CoA of another composition to a reaction solution while synthesizing PHA may be used.
[0155]
As another form, there can be mentioned a form in which the composition of the PHA coating is stepwise, and the PHA having a different composition covers the magnetic material in multiple layers. As a method for producing a PHA multilayer coating having a different composition, a PHA is synthesized with a certain composition of 3-hydroxyacyl-CoA, and then the structure being prepared is once recovered from the reaction solution by centrifugation or the like, and the recovered preparation is prepared. A reaction solution having a different composition of 3-hydroxyacyl-CoA may be added again to the surface of the structure, and a PHA having a different composition may be laminated.
[0156]
<Support of target component binding molecule on PHA magnetic structure>
As a method of supporting a molecule having a binding affinity for a target component (hereinafter, referred to as a target component binding molecule) on the surface of the PHA magnetic structure, the PHA coating the surface and the target component binding molecule are combined. Hydrophobicity, ionicity, physical adsorption such as van der Waals force can also be used, but in consideration of reproducibility and stability, the functional group of the PHA side chain and the functional group of the target component binding molecule It is more desirable to combine と and そ の ま ま as it is or in the presence of a conversion, modification and activation reagent to intervene an irreversible covalent bond.
[0157]
As one mode of the PHA magnetic structure used in the method of the present invention, a PHA magnetic structure having an epoxy group on a side chain of PHA covering the surface can be used. The epoxy group is an amino group (-NH ₂ ) Or a sulfanyl group (-SH). Since a covalent bond can be formed without the need for a reagent for bond formation, the target component binding molecule used is a protein such as an enzyme protein, which is liable to undergo denaturation, or an antibody (Fc) receptor such as an A protein or a G protein. It is effective for carrying proteins and the like. Further, iminodiacetic acid (IDA) is supported on the PHA covering the surface, ²⁺ By adding such a metal ion, it can be used for efficient recovery of the His-tagged target protein. The method of forming a covalent bond using the reaction with the present epoxy group is such that even when the target component binding molecule is a low molecular chemical such as a drug candidate substance, the low molecular chemical is an amino group (- NH ₂ ) Or a sulfanyl group (-SH).
[0158]
The epoxy group-containing PHA magnetic structure is reacted with an ammonium group or hexamethylenediamine hydrochloride in a molar amount of 10 to 100 times the amount of the epoxy group, and the amino group is reacted with the amino group under an alkaline condition. Can be converted to a PHA magnetic structure having the same. When the target component binding molecule is a protein or peptide, the amino group is NHS (NHS) between the main chain carboxy terminus and a side chain carboxy group of an amino acid residue of the protein or peptide such as aspartic acid or glutamic acid. An amide bond can be formed by a crosslinking agent such as N-hydroxysuccinimide). In this case, the carboxy group on the side of the target component binding molecule needs to be converted into an activated ester by NHS treatment. Therefore, it is confirmed in advance whether or not the function of the target component binding molecule is sufficiently maintained when the NHS treatment is performed. It is necessary to keep. The supporting method utilizing the amide bond formation is of course applied even when the target component binding molecule is a low molecular chemical such as a drug candidate substance, provided that the low molecular chemical has a carboxy group. be able to.
[0159]
Further, when the target component-binding molecule is a glycoprotein such as a sugar chain or lectin, or a glycolipid such as lipopolysaccharide, the amino group is located between the aldehyde structure (formyl group: -CHO) in the sugar chain. Can form a stable bond by Schiff base (-CH = N-) formation and reductive amination. A method for forming a covalent bond utilizing the reaction between the present sugar chain and an amino group is promoted by partial oxidation of a sugar chain portion by introducing an aldehyde structure into the sugar chain, and has a sugar chain in its Fc portion. It is also applicable to carrying antibody molecules such as IgG. In the method of the present amino group and aldehyde structure (formyl group: -CHO), even when the target component binding molecule is a low molecular chemical substance such as a drug candidate substance, the low molecular chemical substance has an aldehyde structure (formyl group: Of course, it can be applied as long as it has an —aldehyde structure (formyl group: —CHO) by partial oxidation.
[0160]
Further, the amino group can be bound to a target component binding molecule having a sulfanyl group (-SH) in the presence of a maleimide derivative, a pyridyldithio compound, or an iodine / bromine acetyl compound. When a maleimide derivative is used, the conditions for the reaction between the amino group and the sulfanyl group (—SH) are as follows: 0.1 M sodium phosphate (pH 6.5-7.5) at 4 ° C. to room temperature For about 15 to 20 hours at room temperature in a PBS buffer (pH 7.5) when a pyridyldithio compound is used, and in a 0.05 M sodium borate solution (pH 8) when using an iodine / bromine acetyl compound. In 3), a reaction for 1 hour at room temperature under light shielding is a basic reaction condition, and the reaction condition can be appropriately changed depending on the type of the target component binding molecule and the subsequent purpose. It is possible.
[0161]
As another mode of loading on the PHA magnetic structure used in the method of the present invention, a method utilizing a PHA magnetic structure having a carboxy group in a side chain of the PHA can be mentioned. When the target component binding molecule is a protein or peptide, the carboxy group is NHS between the main chain amino terminal and an amino group on an amino acid residue side chain such as lysine or arginine of the protein or peptide. An amide bond can be formed by a crosslinking agent such as (N-hydroxysuccinimide). In this amide bond formation reaction, the carboxy group of PHA is converted into an activated ester by NHS treatment in advance, thereby improving the rate and frequency of amide bond formation. In addition, when the target component binding molecule is a DNA or an oligonucleotide, a method for forming an amide bond using the carboxy group of the present PHA side chain may be performed by a known method using a DNA or an oligonucleotide whose terminal is aminated. By using a nucleotide, it can be carried on PHA by the above reaction method. In the method of forming an amide bond using the carboxy group of the PHA side chain, even when the target component binding molecule is a low molecular chemical such as a drug candidate substance, the low molecular chemical has an amino group. Then, of course, it can be applied.
[0162]
As another mode of loading on the PHA magnetic structure used in the method of the present invention, the PHA has a halogen such as a chloro group (-Cl), a bromo group (-Br), or a fluoro group (-F) in a side chain. Examples using a PHA magnetic structure can be given. When the halogen is a chloro group or a bromo group, a sulfide bond (-S-) can be formed under mild conditions with a target component binding molecule having a sulfanyl group (-SH).
[0163]
In addition, a substance that specifically binds to a “target component binding molecule” that is desired to be supported on a PHA-coated structure using the above active functional group (for example, when the “target component binding molecule” is an antibody, , Protein A, protein G, etc.) on the surface, and then specifically bind the target “target component binding molecule” to a substance that specifically binds to the “target component binding molecule”. Thus, a method of carrying the compound on a structure may be mentioned, and the “target component binding molecule” may be further modified and then carried by the same method. As an example of the latter, biotin is supported on the surface of a carboxy-type PHA magnetic structure using a reagent NHS-iminobiotin (manufactured by Pierce), while a target component binding molecule modified with avidin or streptavidin is specific to biotin. A method in which the polymer is supported on the structure by the specific bonding. Other examples of available affinity binding pairs include lectin and sugar, hapten and antibody, protein A or G and antibody Fc, other specific binding protein pairs, phenylboronic acid and salicylhydroxamic acid, or reaction with each other. However, in general, there are other pairs of chemical moieties that do not react with the protein.
[0164]
In order to prevent non-specific adsorption on the structure, it is preferable to coat the surface of the structure that is not supported with a “blocking agent” that does not lose the activity of the supported target component binding molecule. As a blocking agent suitable for this "blocking treatment", there are serum proteins such as collagen, gelatin (particularly, cold-water fish skin gelatin), skim milk, BSA, and many other substances including a hydrophobic portion and a hydrophilic portion which do not react with the protein. Compounds.
[0165]
<Contact and binding between PHA magnetic structure carrying target component binding molecule and target component>
In the method of the present invention, the contact between the target component binding molecule supported on the PHA magnetic structure and the target component is usually performed in an aqueous medium, but the target component is difficult to contact with a certain drug candidate substance. When the compound is a water-soluble molecule, a polar solvent such as alcohol, acetone, DMSO (dimethylsulfoxide) or DMF (dimethylformamide), or a surfactant such as Tween, Triton, or SDS is added, and further, toluene, xylene, or hexane is added. A non-polar solvent such as described above may be added to make contact in an emulsion system to promote the binding reaction. However, when these solvents and surfactants are used, their addition concentrations need to be selected within a range that does not impair the affinity binding function of the supported target component binding molecule.
[0166]
In the method of the present invention, in order to promote the contact and binding between the target component binding molecule and the target component carried on the PHA magnetic structure, the heating means is used to the extent that the affinity function of the target component binding molecule is not impaired. Alternatively, it is possible to use a stirring means, and in that case, it is also possible to use means such as ultrasonic waves.
[0167]
In addition, since the structure used in the method of the present invention is a magnetic structure in particular, as a means for promoting contact and binding between the target component-binding molecule and the target component carried on the surface thereof, a magnetic force is used. It is also possible to use. In the case of performing an operation by a magnetic force, a structure that exerts a magnetic force (a permanent magnet, an electromagnet, or the like: sometimes collectively referred to as a magnet hereinafter) can be used to repeatedly perform the operation of releasing and releasing the magnetic force. When an electromagnet is used, energization and cutoff of the electromagnet are repeated by switching to capture and release the magnetic structure. As an example of an operation using a magnet, a probe-shaped electromagnet is inserted into a reaction vessel, and the energization and cutoff of the electromagnet are repeatedly performed by switching a switch, turning on / off a power supply, etc., to capture and release a magnetic structure. There is a way to repeat. As another example, a magnet is arranged outside the container side, and the applied magnetic field strength is repeatedly changed by switching a switch, turning on / off a power supply, or adjusting the distance of the magnet itself to the reaction container. To repeat the capture and release of the magnetic structure.
[0168]
In the method of the present invention, "binding" between a target component and a target component-binding molecule is an element of a binding pair, that is, one molecule specifically binds to the other molecule by chemical or physical action. It is. In addition to binding between antigen and antibody by well-known antigen-antibody reaction, biotin and avidin, carbohydrates and lectins, between complementary sequences of nucleic acids and nucleotides, agonists and receptor molecules, enzyme cofactors and enzymes, enzyme inhibitors Antibodies specific to enzymes, peptide sequences and their sequences or whole proteins, polymer acids and bases, dyes and protein binders, peptides and specific protein binders (ribonucleases, S-peptides and ribonuclease S-proteins), sugars and boric acid, and Examples include, but are not limited to, binding between similar pairs of molecules with an affinity that allows for molecular association in a binding assay. In addition, binding pairs can include elements that are analogs of the original binding element, such as analyte analogs or binding elements produced by recombinant techniques or molecular engineering. If the binding element is an immunoreactant, for example, an antibody, an antigen, a hapten or a complex thereof can be used, and when an `` antibody '' is used, a monoclonal antibody or a polyclonal antibody, a recombinant protein antibody or a natural antibody, It may be a chimeric antibody, a mixture (one or more), a single chain antibody-displaying phage antibody (including whole phage), a fragment (one or more) thereof displaying a single chain antibody, and a mixture of antibody and protein binding elements.
[0169]
In recent years, with the development of evolutionary molecular engineering, a technique for screening a nucleic acid molecule having high affinity for a target molecule such as a protein from a random oligonucleotide library, that is, an aptamer (sometimes called a nucleic acid antibody) ( “Systematic evolution of ligands by exponential enrichment”; SELEX or in vitro selection) was developed. A number of quick and easy preparations of high-affinity ligands have been reported by using the aptamer-based screening method (for example, Nature, 355: 564 (1992), International Patent Application WO 92/14843, JP-A-8-252100, JP-A-9-216895, etc.).
[0170]
Alternatively, regarding the binding between a transcription factor of a nucleic acid that is a protein and a nucleic acid containing a specific base sequence, it is expected that the cause of disease occurrence is investigated, and further, application to effective diagnosis and treatment is expected.
[0171]
The term “binding” to which the method of the present invention is directed includes, of course, such affinity binding between nucleic acids and proteins.
[0172]
The term "bonding" to which the method of the present invention is applied includes any physical or chemical attachment, whether permanent or primary, or intimate specific / selective association. Generally, ionic bond interactions, hydrogen bonds, hydrophobic forces, van der Waals forces, etc., allow physical attachment between the ligand molecule of interest and the receptor. The interaction of "binding" can be as short as when a chemical change occurs due to the binding. This is common when the binding component is an enzyme and the analyte "target component binding molecule" is a substrate for the enzyme. Further, the chemical linkage may be permanent or reversible. Binding may be specific, especially under different conditions.
[0173]
In fact, in the method of the present invention, as an example of the step of contacting and binding the target component with the target component-binding molecule carried on the PHA magnetic structure, the PHA magnetic structure carrying the target component-binding molecule is obtained by using a tissue This is a step of contacting with a biological sample containing a natural protein such as a fluid such as a cell homogenate or serum, and specifically adsorbing and binding the natural protein to a target component binding molecule carried on the surface of the PHA magnetic structure.
[0174]
As another example of the step, as used in the well-known phage display antibody selection method, a suitable bacteriophage surface is prepared by using a magnetic structure carrying a protein as a target component binding molecule. Can be selectively bound.
[0175]
Further, as another example of the step, a receptor contained in the biological sample is contacted with a biological sample containing the receptor, such as a supernatant of a hybridoma or a fluid such as a phage display, to specifically recognize the receptor contained in the biological sample. Alternatively, a step of adsorbing the target component binding molecule carried on the PHA magnetic structure may be considered.
[0176]
<Magnetic separation and washing of target component-bound magnetic composite>
In a method of separating and washing a target component-bound magnetic complex after binding a target component to a magnetic complex (a magnetic structure supporting a target component-binding molecule), a structure (permanent magnet) that exerts a magnetic force is used. And electromagnets, etc.) to repeatedly perform the release and release of the magnetic force to perform the operation of magnetic separation. When an electromagnet is used, the switch is switched to energize and de-energize the electromagnet to capture and release the target component-bound magnetic complex.
[0177]
As an example of the embodiment, a probe-type magnet is used to capture the target component-bound magnetic complex in the container, and then remove the remaining liquid from the container. A washing solution is introduced into the container to wash the target component-bound magnetic complex still captured on the probe. Alternatively, the probe-type magnet in which the target component-bound magnetic complex has been captured can be removed from the reaction solution, moved to a washing solution, and subjected to a washing operation.
[0178]
Alternatively, the magnet may be located outside the container side to keep the target component-bound magnetic complex adhered to the container inside during liquid exchange. When the external magnet is removed, the target component-bound magnetic complex is released, and is washed by mixing with the exchanged liquid (washing solution).
[0179]
For the operation of the present magnetic separation, it is possible to use a number of magnetic separators that are commercially available for the purpose of operating magnetic fine particles. Examples of commercially available magnetic separators include DYNAL MCP manufactured by DYNAL, MAIA Magnetic Separator manufactured by Serono Diagnostics, Magnetite Sapport Standard manufactured by Takara Shugou, Inc.
[0180]
<Elute and release target components from magnetic structures>
In the method of the present invention, if necessary, after isolating the target component-bound magnetic complex, the bound target component can be eluted and released from the target component-binding molecule. When the target component-binding molecule and the target component are a protein-protein combination, they can be released under normal dissociation conditions (pH 2, 4M guanidine, 2M ammonium thiocyanate, 1% SDS, etc.). At this time, the released target component protein is often denatured. Even if the released target component protein is denatured, there is no particular problem when performing purification by electrophoresis or HPLC, but if you want to verify the original three-dimensional structure of the target component protein, a dialysis or other restoration step May be required.
[0181]
<Detection of target component>
As a method for detecting a target component used in the method of the present invention, any method that can be used in an immunoassay, a hybridization assay, such as a normal colorimetric method, a fluorescent method, a chemiluminescent method, a radioisotope method, etc., may be employed. Is possible. Furthermore, the target component eluted / released from the target component binding molecule by the above-described method can be analyzed using the same method. Further, when the target component is DNA, the target component can be analyzed by a technique such as PCR. After amplification, it is also possible to verify the base sequence using a sequencer or the like.If the target component is a protein, after enzymatic digestion, the enzyme-digested fragment is separated by two-dimensional electrophoresis or HPLC. , Electrospray ionization-MS analysis, and matrix-assisted laser desorption / ionization-time-of-flight mass spectrometry (MALDI ToF-) (MS).
[0182]
Of course, the detection of the target component by other spectral analysis such as nuclear magnetic resonance spectroscopy (NMR), infrared absorption spectroscopy (IR), and ultraviolet absorption spectroscopy (UV) alone or in combination. Is also possible.
[0183]
【Example】
Hereinafter, the present invention will be described more specifically with reference to examples. However, the examples described below are examples of the best embodiment according to the present invention, but the technical scope of the present invention is not limited to these examples.
[0184]
Reference Example 1. Preparation of transformant having PHA synthase producing ability
YN2 strain was placed in a 100 ml LB medium (1% polypeptone (Nippon Pharmaceutical Co., Ltd.), 0.5% yeast extract (Difco), 0.5% sodium chloride, pH 7.4) at 30 ° C. After overnight culture, chromosomal DNA was separated and recovered by the method of Marmer et al. The chromosomal DNA of the obtained YN2 strain was completely digested with a restriction enzyme Hind III. As a cloning vector, pUC18 was used and cut with a restriction enzyme HindIII. After the terminal dephosphorylation treatment (Molecular Cloning, 1, 572, (1989); Cold Spring Harbor Laboratory publication), the DNA ligation kit Ver. II (manufactured by Takara Shuzo Co., Ltd.) was used to ligate the vector cleavage site (cloning site) to the completely digested HindIII fragment of chromosomal DNA. Escherichia coli HB101 strain was transformed using the plasmid vector into which the chromosomal DNA fragment was incorporated, and a DNA library of YN2 strain was prepared.
[0185]
Next, in order to select a DNA fragment containing the PHA synthase gene of the YN2 strain, a probe for colony hybridization was prepared. Oligonucleotides consisting of the nucleotide sequences of SEQ ID NO: 2 and SEQ ID NO: 3 were synthesized (Amersham Pharmacia Biotech Co., Ltd.), and PCR was performed using the oligonucleotides as primers and chromosomal DNA as a template. The PCR-amplified DNA fragment was used as a probe for colony hybridization. The labeling of the probe was performed using a commercially available labeling enzyme system AlkPhos Direct (manufactured by Amersham Pharmacia Biotech Co., Ltd.). Using the obtained labeled probe, an Escherichia coli strain having a recombinant plasmid containing a PHA synthase gene was selected from a chromosomal DNA library of the YN2 strain by colony hybridization.
[0186]
By recovering the plasmid from the selected strains by the alkaline method, a DNA fragment containing the PHA synthase gene derived from the YN2 strain could be obtained. The gene DNA fragment obtained here was recombined into a vector pBBR122 (Mo BiTec) containing a broad host range replication region that does not belong to any of the incompatibility groups IncP, IncQ or IncW. This recombinant plasmid was inserted into a Pseudomonas chicory eye YN2ml strain (a strain lacking PHA synthesizing ability) by electroporation and transformed. As a result, the PHA synthesizing ability of the YN2ml strain was restored and showed complementarity. Therefore, it was confirmed that the selected gene DNA fragment contained a PHA synthase gene region translatable into PHA synthase in Pseudomonas chicoryi YN2ml strain.
[0187]
The nucleotide sequence of this gene DNA fragment was determined by the Sanger method. As a result, it was confirmed that the determined nucleotide sequences contained the nucleotide sequences represented by SEQ ID NO: 4 and SEQ ID NO: 5, each encoding a peptide chain. For these PHA synthase genes, PCR was performed using chromosomal DNA as a template to re-prepare the full length of the PHA synthase gene. That is, an upstream primer (SEQ ID NO: 6) and a downstream primer (SEQ ID NO: 7) for the PHA synthase gene having the nucleotide sequence represented by SEQ ID NO: 4, and a PHA having the nucleotide sequence represented by SEQ ID NO: 5 An upstream primer (SEQ ID NO: 8) and a downstream primer (SEQ ID NO: 9) for the synthase gene were synthesized respectively (Amersham Pharmacia Biotech Co., Ltd.).
[0188]
Using these primer pairs, PCR amplification was performed for each of the nucleotide sequences represented by SEQ ID NO: 4 and SEQ ID NO: 5 to amplify the full length of the PHA synthase gene (LA-PCR kit; Takara Shuzo Co., Ltd. )). Next, the obtained PCR-amplified fragment and the expression vector pTrc99A were digested with a restriction enzyme HindIII, dephosphorylated (Molecular Cloning, Vol. 1, p. 572, 1989; Cold Spring Harbor Laboratory), and then expressed. A DNA fragment containing the full length of the PHA synthase gene excluding unnecessary nucleotide sequences at both ends at the cleavage site of the vector pTrc99A was obtained by using a DNA ligation kit Ver. II (manufactured by Takara Shuzo Co., Ltd.).
[0189]
Escherichia coli (Escherichia coli HB101: Takara Shuzo) was transformed with the obtained recombinant plasmid by the calcium chloride method. The obtained recombinants were cultured, the recombinant plasmids were amplified, and the respective recombinant plasmids were collected. The recombinant plasmid retaining the gene DNA of SEQ ID NO: 4 was designated as pYN2-C1, and the recombinant plasmid retaining the gene DNA of SEQ ID NO: 5 was designated as pYN2-C2. Escherichia coli (Escherichia coli HB101 fB fadB deficient strain) was transformed by the calcium chloride method with pYN2-C1 and pYN2-C2, and a recombinant Escherichia coli strain, a pYN2-C1 recombinant strain, and a pYN2-C2 strain, which retained the respective recombinant plasmids, were transformed. A recombinant strain was obtained.
[0190]
Reference example 2. Production of PHA synthase 1
An oligonucleotide (SEQ ID NO: 10) serving as an upstream primer and an oligonucleotide (SEQ ID NO: 11) serving as a downstream primer were designed and synthesized for pYN2-C1 (Amersham Pharmacia Biotech Co., Ltd.). )). PCR was performed using this oligonucleotide as a primer and pYN2-C1 as a template to amplify the full length of a PHA synthase gene having a BamHI restriction site upstream and an XhoI restriction site downstream (LA-PCR kit; Takara Shuzo Co., Ltd.) Made).
[0191]
Similarly, an oligonucleotide (SEQ ID NO: 12) serving as an upstream primer and an oligonucleotide (SEQ ID NO: 13) serving as a downstream primer were designed and synthesized for pYN2-C2 (Amersham Pharmacia. Biotech Co., Ltd.). Using this oligonucleotide as a primer, PCR was performed using pYN2-C2 as a template to amplify the full length of the PHA synthase gene having a BamHI restriction site upstream and an XhoI restriction site downstream (LA-PCR kit; Takara Shuzo Co., Ltd.) Made).
[0192]
Each of the purified PCR amplification products was digested with BamHI and XhoI, and inserted into the corresponding sites of plasmid pGEX-6P-1 (Amersham Pharmacia Biotech). Escherichia coli (JM109) was transformed with these vectors to obtain an expression strain. The expression strain was confirmed using a DNA fragment obtained by treating a large amount of plasmid DNA with BamHI and XhoI using Miniprep (Wizard Minipreps DNA Purification Systems, manufactured by PROMEGA). After the obtained strain was precultured overnight in 10 ml of LB-Amp medium, 0.1 ml of the culture was added to 10 ml of LB-Amp medium and shaken at 37 ° C. and 170 rpm for 3 hours. Culture was continued. Thereafter, IPTG was added (final concentration: 1 mM), and culture was continued at 37 ° C for 4 to 12 hours.
[0193]
IPTG-induced Escherichia coli was collected (8,000 × g, 2 minutes, 4 ° C.), and 1/10 volume of 4 ° C. phosphate buffered saline (PBS; 8 g NaCl, 1.44 g Na). ₂ HPO ₄ , 0.24 g KH ₂ PO ₄ , 0.2 g KCl, 1,000 ml purified water). The cells were disrupted by freeze-thaw and sonication, and centrifuged (8,000 × g, 10 minutes, 4 ° C.) to remove solid contaminants. After confirming the presence of the target expressed protein in the supernatant by SDS-PAGE, the induced and expressed GST fusion protein was purified by glutathione Sepharose 4B (manufactured by Amersham Pharmacia Biotech). Glutathione Sepharose to be used was previously subjected to a treatment for suppressing nonspecific adsorption. That is, Glutathione Sepharose was washed three times with the same amount of PBS (8,000 × g, 1 minute, 4 ° C.), and the same amount of PBS containing 4% bovine serum albumin was added, followed by treatment at 4 ° C. for 1 hour. . After the treatment, the cells were washed twice with the same volume of PBS, and resuspended in 1/2 volume of PBS. 40 μl of pretreated glutathione sepharose was added to 1 ml of the cell-free extract, and the mixture was gently stirred at 4 ° C. In the above procedure, the fusion protein GST-YN2-C1 and the fusion protein GST-YN2-C2 were adsorbed on glutathione sepharose. After adsorption, glutathione sepharose was collected by centrifugation (8,000 × g, 1 minute, 4 ° C.), and washed three times with 400 μl of PBS. Thereafter, 40 μl of 10 mM glutathione was added, and the mixture was stirred at 4 ° C. for 1 hour to elute the adsorbed fusion protein. The supernatant was collected by centrifugation (8,000 × g, 2 minutes, 4 ° C.), and then dialyzed against PBS to purify the GST fusion protein. A single band derived from the GST fusion protein was confirmed by SDS-PAGE.
[0194]
After 500 μg of each GST fusion protein was digested with PreScission protease (Amersham Pharmacia Biotech Co., Ltd., 5 U), the protease and GST were removed by passing through Glutathione Sepharose. The flow-through fraction was further applied to a Sephadex G200 column equilibrated with PBS to obtain final purified expression proteins YN2-C1 and YN2-C2. SDS-PAGE confirmed a single band at 60.8 kDa and a single band at 61.5 kDa, respectively.
[0195]
The recombinant enzyme was concentrated using a biological solution sample concentrating agent (Mizubutori-kun AB-1100, manufactured by ATTO Co., Ltd.) to obtain a 10 U / ml purified enzyme solution. The activity of each purified enzyme was measured by the method described above. The protein concentration in the sample was measured using a micro BCA protein quantitative reagent kit (Pierce Chemical). Table 1 shows the results of the activity measurement of each purified enzyme.
[0196]
[Table 1]

[0197]
Reference example 3. Production of PHA synthase 2
The P91 strain, the H45 strain, the YN2 strain, or the P161 strain was inoculated into 200 ml of an M9 medium containing 0.5% of yeast extract (manufactured by Difco) and 0.1% of octanoic acid, at 30 ° C. and 125 strokes / min. With shaking. After 24 hours, the cells are collected by centrifugation (10,000 × g, 4 ° C., 10 minutes), resuspended in 200 ml of 0.1 M Tris-HCl buffer (pH 8.0), and centrifuged again. Washes by. The washed cells were resuspended in 2.0 ml of 0.1 M Tris-HCl buffer (pH 8.0), crushed by an ultrasonic crusher, and centrifuged (12,000 × g, 4 ° C., 10 ° C.). After that, the supernatant was recovered to obtain a crude enzyme.
Each purified enzyme activity was measured by the method described above, and Table 2 shows the measurement results.
[0198]
[Table 2]

[0199]
Reference example 4. Preparation of magnetic material
In an aqueous ferrous sulfate solution, l. An aqueous solution containing ferrous hydroxide was prepared by mixing 0 to 1.1 equivalents of a sodium hydroxide solution. While maintaining the pH of the aqueous solution at about 8, air was blown therein to perform an oxidation reaction at 80 to 90 ° C. to prepare a slurry liquid for generating seed crystals.
[0200]
Next, an aqueous ferrous sulfate solution was added to this slurry liquid so as to have an equivalent amount of 0.9 to 1.2 equivalents to the initial alkali amount (sodium component of caustic soda), and the slurry liquid was maintained at pH around 8. To promote the oxidation reaction while blowing air. After the oxidation reaction, the generated magnetic iron oxide particles were washed, filtered, and dried, and the aggregated particles were crushed to obtain a granular magnetic body 1 having an average particle diameter of 0.1 μm.
[0201]
Reference example 5. Preparation of epoxy-type PHA magnetic structure fine particles 1
To 10 parts by mass of a PHA synthase solution (10 U / ml) derived from the pYN2-C1 recombinant strain, 1 part by mass of the magnetic substance prepared in Reference Example 4 and 39 parts by mass of PBS were added, and the mixture was added at 30 ° C. for 30 minutes. By gentle shaking, the PHA synthase was adsorbed on the surface of the magnetic material. This was centrifuged (10,000 × g, 4 ° C., 10 minutes), the precipitate was suspended in a PBS solution, and centrifuged again (10,000 × g, 4 ° C., 10 minutes) to immobilize the immobilized PHA synthase. Got.
[0202]
The immobilized PHA synthase was suspended in 48 parts by mass of 0.1 M phosphate buffer (pH 7.0), and (R, S) -3-hydroxy-5-phenoxyvaleryl-CoA (3-phenoxypropanal and bromo After hydrolyzing 3-hydroxy-5-phenoxyvaleric acid ester obtained by a Reformatsky reaction with ethyl acetate to obtain 3-hydroxy-5-phenoxyvaleric acid, Eur. J. Biochem., 250, 432- 439 (prepared by the method described in (1997)) 0.8 parts by mass, (R, S) -3-hydroxy-7,8-epoxyoctanoyl CoA (Int. J. Biol. Macromol., 12, 85-91) (1990) Epoxidation of the unsaturated moiety of 3-hydroxy-7-octenoic acid with 3-chlorobenzoic acid Thereafter, 0.2 part by mass of Eur. J. Biochem., 250, 432-439 (1997)) and 0.1 part by mass of bovine serum albumin (manufactured by Sigma) were added, and 30 ° C. The mixture was gently shaken for 2 hours to obtain PHA magnetic structure fine particles.
[0203]
10 μl of the above sample was collected on a slide glass, 10 μl of a 1% Nile Blue A aqueous solution was added, and the mixture was mixed on the slide glass. Then, a cover glass was placed, and a fluorescence microscope (330 to 380 nm excitation filter, 420 nm long-pass absorption) was used. (Filter, manufactured by Nikon Corporation). As a result, it was confirmed that the surface of the fine particles of the PHA magnetic structure emitted fluorescence in each of the samples. Therefore, it was shown that the PHA magnetic structure fine particles were surely coated on the surface with PHA.
[0204]
Further, a part of the sample was collected by centrifugation (10,000 × g, 4 ° C., 10 minutes), dried in vacuo, suspended in chloroform, and stirred at 60 ° C. for 20 hours to form PHA forming an envelope. Was extracted. About this extract, ¹ H-NMR analysis was performed (equipment used: FT-NMR: Bruker DPX400, measurement nuclide: ¹ H, solvent used: deuterated chloroform (containing TMS)). As a result, the composition and unit% of the sheath PHA of the PHA magnetic structure fine particles were: 3-hydroxy-5-phenoxyvaleric acid unit: 75%, 3-hydroxy-7,8-epoxyoctanoic acid unit: 25%. It was confirmed that there was.
[0205]
Reference example 6. Preparation of vinylphenyl type PHA magnetic structure fine particles
1 mass of 0.3 μm-diameter magnetite fine particles “magnetite EPT500” (manufactured by Toda Kogyo Co., Ltd.) synthesized by a wet method in 10 mass parts of a PHA synthase solution (10 U / ml) derived from the pYN2-C1 recombinant strain And 39 parts by mass of PBS were added, and the mixture was gently shaken at 30 ° C. for 30 minutes to adsorb PHA synthase on the surface of the fine particles. This was centrifuged (10,000 × g, 4 ° C., 10 minutes), the precipitate was suspended in a PBS solution, and centrifuged again (10,000 × g, 4 ° C., 10 minutes) to synthesize immobilized PHA. The enzyme was obtained.
[0206]
The immobilized PHA synthase was suspended in 48 parts by mass of a 0.1 M phosphate buffer (pH 7.0), and (R) -3-hydroxy-5-phenylvaleryl-CoA (Eur. J. Biochem., 250, 432-439 (prepared by the method described in (1997)) 0.8 parts by mass, (R) -3-hydroxy-5- (4-vinylphenyl) valeryl CoA (Eur. J. Biochem., 250, 432-439). (Prepared by the method described in (1997)) 0.2 parts by mass and 0.1 parts by mass of bovine serum albumin (manufactured by Sigma) were added, and the mixture was gently shaken at 30 ° C. for 2 hours to remove the PHA magnetic structure fine particles. Obtained.
[0207]
10 μl of the above sample was collected on a slide glass, 10 μl of a 1% Nile Blue A aqueous solution was added, and the mixture was mixed on the slide glass. Then, a cover glass was placed, and a fluorescence microscope (330 to 380 nm excitation filter, 420 nm long-pass absorption) was used. (Filter, manufactured by Nikon Corporation). As a result, it was confirmed that the surface of the PHA magnetic structure fine particles emits fluorescence, and it was found that the PHA magnetic structure fine particles were surely covered with PHA.
[0208]
Further, a part of the sample was collected by centrifugation (10,000 × g, 4 ° C., 10 minutes), dried in vacuo, suspended in chloroform, and stirred at 60 ° C. for 20 hours to remove PHA forming an envelope. Extracted. About this extract, ¹ H-NMR analysis was performed (equipment used: FT-NMR: Bruker DPX400, measurement nuclide: ¹ H, solvent used: deuterated chloroform (containing TMS)). As a result, the composition and unit% of the sheath PHA of the PHA magnetic structure fine particles were: 3-hydroxy-5-phenylvaleric acid unit: 83%, 3-hydroxy-5- (4-vinylphenyl) valeric acid unit: It was confirmed to be 17%.
[0209]
Reference example 7. Preparation of fine particles of epoxy-type PHA magnetic structure 2
The vinyl group-type PHA magnetic structure fine particles produced in Reference Example 6 were subjected to an epoxidation reaction of the vinyl group. One part by mass of the vinylphenyl-type PHA magnetic structure fine particles was added to a four-necked round bottom flask, and 6 parts by mass of distilled water was added thereto and stirred. The inside of the flask was heated to 40 ° C., and 1 part by mass of a hexane solution containing 30% of peracetic acid was continuously added dropwise thereto and reacted at 40 ° C. for 5 hours with stirring. The reaction proceeded without aggregation of the PHA magnetic structure fine particles. After completion of the reaction, the mixture was cooled to room temperature, and the reaction solution was filtered to collect PHA magnetic structure fine particles. The collected PHA magnetic structure fine particles were resuspended in distilled water and then centrifuged (3000 × g, 4 ° C., 30 minutes). After the separation, the PHA magnetic structure fine particles were resuspended in distilled water and centrifuged again to wash. Further, this washing operation was repeated three times. Thereafter, by vacuum drying, PHA magnetic structure fine particles having the following composition were obtained.
[0210]
Further, a part of the sample was collected by centrifugation (10,000 × g, 4 ° C., 10 minutes), dried in vacuo, suspended in chloroform, and stirred at 60 ° C. for 20 hours to form PHA forming an envelope. Was extracted. About this extract, ¹ H-NMR analysis was performed (equipment used: FT-NMR: Bruker DPX400, measurement nuclide: ¹ H, solvent used: deuterated chloroform (containing TMS)). The unit% of each side chain unit contained in the sheath PHA calculated from the measurement results is: 3-hydroxy-5-phenylvaleric acid unit: 85%, 3-hydroxy-5- (4-vinylphenyl) valeric acid Unit: 11%, 3-hydroxy-5- (4- (1,2-epoxyethyl) phenyl) valeric acid unit: 4%. Since this reaction system is a heterogeneous reaction, it is considered that PHA on the surface of the fine particles is epoxidized and remains unreacted PHA inside the coating.
[0211]
Reference Example 8. Preparation of carboxy-type PHA magnetic structure fine particles
The vinyl group-type PHA magnetic structure fine particles produced in Reference Example 6 were subjected to an oxidative cleavage reaction of the vinyl group. 10 parts by mass of the vinylphenyl-type PHA magnetic structure fine particles were transferred to a three-necked flask, and 300 parts by weight of distilled water to which 50 ppm of hydrogen peroxide had been added was added. Ozone was blown in at a rate of 1 part by weight / hour, followed by stirring at room temperature for 3 hours. The reaction proceeded without aggregation of the magnetic structure fine particles. After completion of the reaction, the PHA magnetic structure fine particles were recovered by filtering the reaction solution. The PHA magnetic structure fine particles were resuspended in distilled water, and then centrifuged (3000 × g, 4 ° C., 30 minutes) to wash the residual hydrogen peroxide solution. Further, this washing operation was repeated twice. Thereafter, by vacuum drying, PHA magnetic fine particles having the following composition were obtained.
[0212]
Further, a part of the sample was collected by centrifugation (10,000 × g, 4 ° C., 10 minutes), dried in vacuo, suspended in chloroform, and stirred at 60 ° C. for 20 hours to remove PHA forming an envelope. Extracted. About this extract, ¹ H-NMR analysis was performed (equipment used: FT-NMR: Bruker DPX400, measurement nuclide: ¹ H, solvent used: deuterated chloroform (containing TMS)). The unit% of each side chain unit contained in the sheath PHA calculated from the measurement results is as follows: 3-hydroxy-5-phenylvaleric acid unit: 84%, 3-hydroxy-5- (4-vinylphenyl) valeric acid unit : 11%, 3-hydroxy-5- (4-carboxyphenyl) valeric acid unit: 5%. Since this reaction system is a heterogeneous reaction, it is considered that PHA on the surface of the fine particles is epoxidized and remains unreacted PHA inside the coating.
[0213]
Reference example 9. Preparation of bromo-type PHA magnetic structure fine particles
To 10 parts by mass of a PHA synthase solution (10 U / ml) derived from the YN2-C1 recombinant strain, 1 part by mass of the magnetic substance prepared in Reference Example 4 and 39 parts by mass of PBS were added, and the mixture was added at 30 ° C. for 30 minutes. By gentle shaking, the PHA synthase was adsorbed on the surface of the magnetic substance. This was centrifuged (10,000 × g, 4 ° C., 10 minutes), the precipitate was suspended in a PBS solution, and centrifuged again (10,000 × g, 4 ° C., 10 minutes) to synthesize immobilized PHA. The enzyme was obtained.
[0214]
The immobilized PHA synthase was suspended in 48 parts by mass of a 0.1 M phosphate buffer (pH 7.0), and (R, S) -3-hydroxy-5-phenoxyvaleryl CoA (3-phenoxypropanal and bromo After hydrolyzing 3-hydroxy-5-phenoxyvaleric acid ester obtained by a Reformatsky reaction with ethyl acetate to obtain 3-hydroxy-5-phenoxyvaleric acid, Eur. J. Biochem., 250, 432- 439 (prepared by the method described in (1997)) 0.8 parts by mass, (R) -3-hydroxy-8-bromooctanoyl-CoA (Eur. J. Biochem., 250, 432-439 (1997)) 0.2 parts by mass) and 0.1 part by mass of bovine serum albumin (manufactured by Sigma) were added thereto, and the mixture was slowly heated at 30 ° C. for 2 hours. Shaking to obtain a PHA magnetic structure particles.
[0215]
10 μl of the above PHA magnetic structure fine particles were collected on a slide glass, 10 μl of a 1% Nile Blue A aqueous solution was added, mixed on the slide glass, a cover glass was placed, and a fluorescence microscope (330 to 380 nm excitation filter) was placed. , 420 nm long-pass absorption filter, manufactured by Nikon Corporation). As a result, it was confirmed that the particle surface of the magnetic structure emitted fluorescence. Therefore, it was shown that the PHA magnetic structure fine particles were coated on the surface with PHA.
[0216]
Further, a part of the PHA magnetic structure fine particles was collected by centrifugation (10,000 × g, 4 ° C., 10 minutes), dried in vacuum, suspended in chloroform, and stirred at 60 ° C. for 20 hours. The PHA forming the envelope was extracted. About this extract, ¹ H-NMR analysis was performed (equipment used: FT-NMR: Bruker DPX400, measurement nuclide: ¹ H, solvent used: deuterated chloroform (containing TMS)). The unit% of each side chain unit contained in the envelope PHA calculated from the measurement results was 3-hydroxy-5-phenoxyvaleric acid unit: 89% and 3-hydroxy-8-bromooctanoic acid unit: 11%. Was.
[0219]
Embodiment 1 FIG. AFP immunodetection with anti-AFP antibody supported on epoxy-type PHA magnetic structure microparticles
1. Loading of Antibody on Epoxy-Type PHA Magnetic Structure Particles
The epoxy-type PHA magnetic structure fine particles prepared in Reference Example 5 were uniformly dispersed in a phosphate buffer (0.1 M; pH 7.4) (theoretical concentration: 5-10 × 10 ⁸ ) / Anti-AFP (α-fetoprotein) antibody dissolved in the same phosphate buffer, and the final ratio of the PHA magnetic structure fine particles to the antibody was 5-10 μg / 10 ⁷ And gently stirred by pipetting.
[0218]
After reacting at 30 ° C. for 1 hour on a rotary shaker, bovine serum albumin (BSA) was added to a final concentration of 0.3% for blocking, and the reaction was further performed for 15 hours to obtain an epoxy-type PHA magnetic material. An antibody supported on the surface of the fine particles of the structure was obtained.
[0219]
2. Reaction with target component (antigen: AFP)
In a 5 mL Eppendorf tube (BSA treatment), 20 μL of a 1 μg / mL AFP solution was mixed with 500 μL of the dispersion of the anti-AFP antibody-supported epoxy-type PHA magnetic structure fine particles prepared in 1 and allowed to react at 37 ° C. for 30 minutes. Was. The tube was brought into contact with a magnet to capture the fine particles of the magnetic structure, and the supernatant was removed by decantation. Thereafter, 2 mL of a 0.04% NaCl solution was added to the tube and stirred. As in the above, the magnetic structure fine particles were captured by a magnet, and the supernatant was removed by decantation. This washing operation was repeated three times.
[0220]
3. Reaction with enzyme-labeled secondary antibody
J. According to the method described in Immunoassay, 4, 209 (1983) and Biochemistry, 11 (12), 2291 (1972), an alkaline phosphatase-labeled anti-AFP antibody Fab ′ was prepared to a final concentration of 0.1 μg / mL. Into a 0.1 M Tris-HCl buffer (2% BSA, 1 mM MgCl ₂ , 0.1 mM ZnCl ₂ Containing; pH 7.5), and dissolve 500 μL of it in 3. Was added to the antigen-AFP-bound magnetic complex fine particles obtained in the above, and reacted at 37 ° C. for 10 minutes. Then, 2. The antigen-AFP-bound magnetic complex fine particles reacted with the enzyme-labeled secondary antibody were washed in the same manner as described above.
[0221]
4. detection
3. 500 μL of glycine-NaOH buffer solution (0.1 M: pH 10.3: MgCl 2) was placed in the tube containing the antigen-AFP-bound magnetic complex fine particles reacted with the enzyme-labeled secondary antibody obtained in ₂ (1 mM) and ovalbumin (250 mg / L), and reacted at 37 ° C. for 5 minutes. Then, 500 μl of the above buffer containing 4-nitrophenyl phosphate (5.5 mM final concentration) was added, and The reaction was performed at 60 ° C. for 60 minutes. After completion of the reaction, 500 μL of a 1 M aqueous NaOH solution was added to stop the reaction, and ultraviolet / visible absorbance was measured. As a result, absorption at 405 nm derived from the reaction product of the labeling enzyme alkaline phosphatase was detected, confirming that the antigen AFP was actually recovered.
[0222]
Embodiment 2. FIG. Recovery of Concanavalin A Using Cellopentaose-Supported Aminated PHA Magnetic Structure Fine Particles
1. Amination of epoxy-type PHA magnetic structure fine particles
2,2 ′-(Ethylenedioxy) -dimethylamine was added to the epoxy-type PHA magnetic structure fine particles produced in Reference Example 7, and the mixture was reacted at 30 ° C. for 15 hours to carry out amination. After completion of the reaction, the resultant was washed five times with bis-2-methoxyethyl ether to remove residual amine, and further washed three times with distilled water to obtain aminated PHA magnetic structure fine particles.
[0223]
2. Support of cellopentaose on aminated PHA magnetic structure fine particles
Step 1. The fine particles of the aminated PHA magnetic structure obtained in the above were uniformly dispersed in a phosphate buffer solution as in Example 1, and cellopentaose (D-(+)-Cellopentaose: manufactured by Sigma) was previously contained in periodic acid. Oxidation of the non-reducing end with sodium acid to form an aldehyde structure (—CHO: formyl group) and stirring at 30 ° C. for 30 hours to carry cellopentaose on the aminated PHA magnetic structure fine particles Was done. Thereafter, in order to protect excess amino groups remaining on the surface of the fine particles, butanedienoic anhydride was added and reacted, followed by washing with distilled water three times to obtain fine particles of aminated PHA magnetic structure carrying cellopentaose.
[0224]
3. Recovery of Concanavalin A
1. Dissolve Concanavalin A (Concanavalin A: Sigma) and BSA in the phosphate buffer; After adding the cellopentaose-supported aminated PHA magnetic structure fine particles obtained in 1) and reacting at 30 ° C. for 15 hours, the fine particles were collected by magnetic force. The collected microparticles were treated with sodium dodecyl sulfate (SDS), and SDS-PAGE analysis of the eluate showed a substantially single band at 104 kDa, indicating that concanavalin A was recovered.
[0225]
Embodiment 3 FIG. Screening of DNA fragment using carboxy-type PHA magnetic structure fine particles
1. Synthesis and amination of model nucleic acid M13p18ssDNA
A 20-mer oligonucleotide having a base sequence complementary to the Escherichia coli M13 phage mp18 single-stranded DNA shown in SEQ ID NO: 14 was synthesized using a DNA automatic synthesizer (381A, manufactured by ABI).
SEQ ID NO: 14: 5′-GTTGTAAAACGACGGCCAGT-3 ′
Then, instead of a normal amidite reagent, an amino group (-NH) is added to the 5 'side of the 20-mer oligonucleotide using a deoxyuridylic acid derivative monomer (compound [19]) into which an amino group has been introduced. ₂ ) Was introduced (compound [20]). Cleavage from the CPG support, deprotection, and purification by high performance liquid chromatography (HPLC) were performed by a conventional method.
[0226]
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[0227]
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[0228]
2. Loading of aminated probe oligonucleotide on carboxy-type PHA magnetic structure fine particles
The carboxy-type PHA magnetic structure fine particles obtained in Reference Example 8 were washed in advance with a 0.01 M sodium hydroxide solution. -3- (3-diethylaminopropyl) -carbodiimide-hydrochloric acid) and an aminated oligonucleotide represented by the formula [20], and reacted at 4 ° C. for 18 hours to obtain probe-oligonucleotide-supported PHA magnetic structure fine particles. Got.
[0229]
3. Synthesis and labeling of target model oligonucleotides
A 20-mer oligonucleotide (SEQ ID NO: 15) that is completely complementary to the oligonucleotide of SEQ ID NO: 14 described above, a 1-base mismatch (SEQ ID NO: 16), and a 2-base mismatch (SEQ ID NO: 17) oligo Each nucleotide was synthesized by an automatic synthesizer and purified by a conventional method.
SEQ ID NO: 15: 5′-ACTGGCCGTCGTTTTTACAAC-3 ′
SEQ ID NO: 16: 5′-ACTGGCCGTCCTTTTTACAAC-3 ′
SEQ ID NO: 17: 5′-ACTGGCGGGTCGTTATACAAC-3 ′
The three types of the purified model oligonucleotides were aminated at the 5 ′ end using a deoxyuridylic acid derivative monomer (compound [19]) in the same manner as in step 1.
[0230]
Separately, 170 mg of a cyanine dye (compound [21]) was dissolved in 5 ml of dry DMF according to the method disclosed in Japanese Patent No. 03368011, and 50 μl of dry pyridine was added thereto. Further, after 128 mg of DSC (disucciimidyl carbonate) was added, the mixture was stirred in a dark place at room temperature for 20 hours. 150 ml of diethyl ether was added to the reaction mixture, and the deposited precipitate was collected, washed with diethyl ether and dried. The obtained active ester (compound [22]) was used as it was for labeling a target model oligonucleotide (complete complement: SEQ ID NO: 15).
[0231]
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[0232]
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[0233]
In the same manner, the aminated product of the oligonucleotide having a single base mismatch (SEQ ID NO: 16) and the double base mismatch (SEQ ID NO: 17) was labeled with compound [23] which is an azulene dye active ester ( Compound [24]).
[0234]
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[0235]
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[0236]
4. Screening for perfectly complementary oligonucleotides
The probe-oligonucleotide-supported PHA magnetic structure fine particles obtained in the step 2 and the labeled target oligonucleotide obtained in the step 3 are adjusted so that the probe / target amount ratio becomes 1/10 theoretically, After holding at 2 ° C. for 2 minutes, the temperature was returned to room temperature over time (hybridization conditions).
[0237]
The fine particles were separated by magnetic force, excited with a 780 nm laser, and measured with a fluorometer. As a result, fluorescence having a peak near 820 nm derived from the compound of the chemical formula [21] was observed. It was confirmed that DNA was selectively recovered.
[0238]
Embodiment 4. FIG. Recovery of nonylphenol using carboxy-type PHA magnetic structure particles carrying anti-alkylphenol antibody
1. Carrying anti-alkylphenol antibody on carboxy-type PHA magnetic structure fine particles
The carboxy group on the PHA side chain of the carboxy-type PHA magnetic structure fine particles prepared in Reference Example 8 was converted into N-ethyl-N ′-(dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) according to a conventional method. ) And reacted with a theoretically twice the amount of an anti-alkylphenol antibody (manufactured by Wako Pure Chemical Industries, Ltd.) to obtain PHA magnetic structure fine particles carrying the anti-alkylphenol antibody.
[0239]
2. Recovery and detection of nonylphenol
According to the method of Wako Pure Chemical's "Alkylphenol ELISA Kit", mix the nonylphenol standard stock solution and the solution of the antigen-enzyme complex, add the anti-alkylphenol antibody-supporting PHA magnetic structure fine particles obtained in Step 1, and add the outside of the container. The reaction was promoted by switching the electromagnet which was brought into close contact with the switch. After performing this operation at 30 ° C. for 30 minutes, the fine particles were captured on the inner wall surface of the container while the electromagnet was turned on, and the reaction solution was removed. Next, a cleaning solution was added to the container, and the electromagnet was switched to promote cleaning. After performing this washing step three times for 5 minutes each time with replacing the washing solution, a chromogenic substrate solution was added, and when the absorbance was measured, strong absorption was observed at 450 nm. According to this method, nonylphenol was recovered. Can be performed.
[0240]
Embodiment 5 FIG. Separation and recovery of liposomes using long-chain alkane-supported bromo-type PHA magnetic structure particles
1. Dodecane loading on bromo-type PHA magnetic structure fine particles using dodecanethiol
1-Dodecanethiol is dissolved in hexane, sodium iodide, potassium carbonate and diethylamine are added, mixed with the bromo-type PHA magnetic structure fine particles prepared in Reference Example 9, and stirred at room temperature for 20 hours to form a sulfide bond. Dodecane-supported PHA magnetic structure fine particles were obtained via (-S-).
[0241]
2. Recovery and detection of fluorescent liposomes
Using “Lipsome Kit” manufactured and sold by Sigma, liposomes containing FITC therein were prepared according to the protocol, and stirred and mixed with the dodecane-supported PHA magnetic structure fine particles obtained in step 1. After 15 minutes of mixing, the probe-type electromagnet is inserted into the container, the power switch for the probe-type electromagnet is turned on, the fine particles are captured on the probe-type electromagnet, and then the reaction solution is transferred from the reaction container to the container containing the washing liquid. The power switch was turned off to release the fine particles, and the mixture was stirred for 15 minutes. After this washing operation was repeated three times, measurement with a fluorimeter revealed that the fluorescence had a maximum at 520 nm based on FITC, indicating that the liposome was efficiently collected and detected by this method.
[0242]
Embodiment 6 FIG. Separation and recovery of transcription factor proteins using carboxy-type PHA magnetic structure microparticles carrying a consensus binding sequence gene fragment
The separation and recovery of ATF-2 was attempted by utilizing the affinity between ATF-2, a transcription factor having a basic leucine zipper, and its consensus binding sequence.
[0243]
1. Synthesis of terminal thiol type ATF-2 consensus binding sequence DNA fragment
Using an automatic DNA synthesizer, a single-stranded nucleic acid of SEQ ID NO: 18 was synthesized. The 5 ′ end of the single-stranded DNA of SEQ ID NO: 18 was synthesized with a thiol-modifier (GlenResearch) at the time of synthesis using an automatic DNA synthesizer to obtain a sulfanyl. A group (-SH) was introduced (compound [25]). Subsequently, the DNA was recovered by ordinary deprotection, purified by high performance liquid chromatography, and used in the following experiments.
SEQ ID NO: 18: 5′-TGACATCA-3 ′
[0244]
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[0245]
2. Carrying terminal thiolated DNA fragments on carboxy-type PHA magnetic structure fine particles
The carboxy group on the PHA side chain of the carboxy-type PHA magnetic structure fine particles prepared in Reference Example 8 was converted into N-ethyl-N ′-(dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) according to a conventional method. ) And activated against thiols with 2- (2-pyridinyldithio) ethanamine (PDEA) dissolved in borate buffer (pH 8.5 adjusted with NaOH), obtained in step 1. A terminal thiolated DNA fragment was added for loading. After the reaction was completed, the active groups remaining on the PHA magnetic structure fine particles were inactivated by cysteine-NaCl.
[0246]
3. Binding reaction, labeling
According to the protocol of Clontech, the DNA fragment-carrying PHA magnetic structure fine particles obtained in step 2 were introduced into a PBS solution of AFT-2, and the DNA fragment and AFT-2 were separated by a magnet in the same manner as in Example 4. It promoted the binding reaction due to affinity. After completion of the reaction, the plate was washed in the same manner as in Example 4, and an antigen-antibody reaction was performed in a FITC-labeled anti-AFT-2 antibody (polyclonal) solution by a conventional method.
[0247]
4. Washing and detection
After the antigen-antibody reaction was completed, washing was performed by a procedure using a magnet according to the method of Example 4, and fluorescence was detected. As a result, fluorescence having a maximum at 520 nm based on labeled FITC was confirmed. Recovery based on affinity binding of the factor protein-consensus sequence DNA fragment was shown to be detectable.
[0248]
【The invention's effect】
In the present invention, as the polymer material layer covering the carrier surface, a polyhydroxyalkanoate, which is a polymer material having high biocompatibility, for example, by using a polyhydroxyalkanoate containing a 3-hydroxyalkanoic acid unit, It is possible to efficiently separate and detect a target substance in a specimen under conditions closer to biological conditions.
[0249]
[Sequence list]

[Brief description of the drawings]
FIG. 1 schematically shows the structure of a target component-binding molecule-supporting PHA structure in which a molecule having a binding affinity for a target component is supported on a PHA covering a carrier (magnetic material) surface used in the present invention. FIG.
FIG. 2 is a diagram schematically illustrating a process of forming a selective bond between a target component and a target component binding molecule on a PHA structure carrying a target component binding molecule in the present invention.
FIG. 3 is a diagram schematically showing a magnetic separation process of a PHA magnetic structure supporting a target component-binding molecule, which has formed a selective bond with a target component, using a structure exerting a magnetic force in the present invention. is there.
[Explanation of symbols]
1 Carrier substrate (magnetic material)
2 Polyhydroxyalkanoate coating layer
3. Sites selectively supporting molecules having a binding affinity for a target component in polyhydroxyalkanoate
4. Molecules with binding affinity for target components
5 Target components
6, 7 Mixed components other than target component
8 Structures that exert magnetic force (permanent magnets, electromagnets, etc.)

Claims (21)

A method for separating a target component contained in a sample,
A step in which a molecule having a binding affinity for the target component prepares a carrier immobilized on its surface,
Mixing the carrier and the sample,
The step of binding the target component contained in the sample and the molecule having the binding affinity immobilized on the carrier surface, which is mixed in the mixing step,
The binding step, through binding with the molecule having the binding affinity, the target component immobilized on the carrier, a step of separating from the sample together with the carrier from the sample,
A method for separating a target component, wherein the carrier is at least partially coated with a polyhydroxyalkanoate. The method according to claim 1, wherein the carrier is a carrier comprising a magnetic material. The method according to claim 1, wherein the polyhydroxyalkanoate is a polyhydroxyalkanoate containing a 3-hydroxyalkanoic acid unit. In the step of separating the target component immobilized on the carrier from the sample together with the carrier through binding with the molecule having the binding affinity,
The method according to claim 2, further comprising a step of applying a magnetic field to the carrier containing the magnetic material to magnetically separate the carrier from the sample. Immobilization of a molecule having a binding affinity for the target component on the surface of the carrier,
As the polyhydroxyalkanoate, which is coated on at least a part of the surface of the carrier, an epoxy group, an amino group, a carboxy group, and a polyhydroxyalkanoate having at least one functional group selected from the group consisting of halogen molecules Using
The method according to claim 1 or 2, wherein a molecule having a binding affinity for the target component is immobilized using at least one of the functional groups of the polyhydroxyalkanoate. A molecule having a binding affinity for the target component, which is immobilized on the carrier surface,
The molecule according to claim 1 or 2, wherein the molecule comprises at least one selected from the group consisting of nucleic acids, proteins, peptides, sugar chains, lipids, low molecular weight compounds, and complexes thereof. Method. The target component separated from the sample,
The component according to claim 1 or 2, wherein the component comprises at least one selected from the group consisting of nucleic acids, proteins, peptides, sugar chains, lipids, low molecular weight compounds, and complexes thereof. Method. The method according to claim 2, wherein the shape of the carrier comprising the magnetic material is granular. The method according to claim 2, wherein the shape of the carrier containing the magnetic material is a flat plate or a film. 3. The method according to claim 2, wherein the magnetic substance in the carrier containing the magnetic substance is a magnetic substance made of a metal or a metal compound having magnetism. A method for detecting a target component contained in a sample,
A step in which a molecule having a binding affinity for the target component prepares a carrier immobilized on its surface,
Mixing the carrier and the sample,
The step of binding the target component contained in the sample and the molecule having the binding affinity immobilized on the carrier surface, which is mixed in the mixing step,
The binding step, through the binding with the molecule having the binding affinity, the step of selectively detecting the target component immobilized on the carrier,
A method for detecting a target component, wherein the carrier is at least partially coated with a polyhydroxyalkanoate. By the binding step, through the binding to the molecule having the binding affinity, the step of selectively detecting the target component immobilized on the carrier,
By the binding step, through the binding with the molecule having the binding affinity, the target component immobilized on the carrier, the step of separating from the sample together with the carrier from the sample,
Selectively separating the target component immobilized on the carrier through binding with the molecule having the binding affinity, which is separated from the sample together with the carrier. The method according to claim 11. The method according to claim 12, wherein the carrier is a carrier containing a magnetic material. 14. The method according to claim 12, wherein the carrier is a carrier at least partially coated with a polyhydroxyalkanoate containing a 3-hydroxyalkanoic acid unit. In the step of separating the target component immobilized on the carrier from the sample together with the carrier through binding with the molecule having the binding affinity,
The method according to claim 13, further comprising a step of applying a magnetic field to the carrier containing the magnetic material to magnetically separate the sample from the sample. In the step of selectively detecting the target component immobilized on the carrier,
11. The method according to claim 11, wherein the detection method is one detection method selected from the group consisting of a colorimetry method, a fluorescence method, a chemiluminescence method, and a radioisotope method, or a combination of two or more detection methods. A method according to any one of claims 13 to 13. In the step of selectively detecting the target component immobilized on the carrier,
As a detection method, one detection method selected from the group consisting of nuclear magnetic resonance spectroscopy, mass spectroscopy, infrared absorption spectroscopy, and ultraviolet absorption spectroscopy, or using a combination of two or more detection methods. 14. The method according to any one of claims 11 to 13, characterized in that: A method for screening a target component contained in a medium,
The screening is directed to a mixed sample in which a mixture containing the target component is contained in the medium,
A step in which a molecule having a binding affinity for the target component prepares a carrier immobilized on its surface,
Mixing the carrier and the mixed sample,
The step of binding the target component contained in the mixed sample and the molecule having the binding affinity immobilized on the carrier surface, which is mixed in the mixing step,
The binding step, through the binding with the molecule having the binding affinity, the target component immobilized on the carrier, the step of separating from the mixed sample together with the carrier,
A method for screening a target component, wherein the carrier is at least partially coated with a polyhydroxyalkanoate. The method according to claim 18, wherein the carrier is a carrier containing a magnetic material. The method according to claim 18 or 19, wherein the polyhydroxyalkanoate is a polyhydroxyalkanoate containing a 3-hydroxyalkanoic acid unit. In the step of separating the target component immobilized on the carrier from the mixed sample together with the carrier through binding with the molecule having the binding affinity,
20. The method according to claim 19, further comprising a step of applying a magnetic field to the carrier containing the magnetic material to magnetically separate the mixed sample from the mixed sample.

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