JP2004016227A - Method for immobilizing lectin and tool containing immobilized lectin - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for immobilizing lectin on a support. <P>SOLUTION: The method for immobilizing the lectin comprises immobilizing an anti-tag antibody (e.g. an anti-FLAG antibody) 14 on the support 12 and binding a tag antibody (e.g. a FLAG antibody) fused with the lectin to the anti-tag antibody (e.g. the anti-FLAG antibody) 14. <P>COPYRIGHT: (C)2004,JPO

Description

【００３０】
［ファージ形成用ＶＣＳＭの作製］
ＳＢ培地（テトラサイクリン最終濃度４０μｇ／ｍｌ添加）で一晩培養しておいたＸＬ−１　Ｂｌｕｅ　２００μｌとＳＢ培地１０ｍｌを３７℃で１時間振とう培養した。ＶＣＳＭ原液を１μｌ加えてから３７℃の湯浴で３０分間温めてファージを感染させ、カナマイシン（最終濃度７０μｇ／ｍｌ）を加えてさらに３７℃で８時間振とう培養した。培養液を４℃、３５００ｒｐｍで２０分間遠心して、回収した上清を６５℃の湯浴で１５分間温めた。もう一度、４℃、３５００ｒｐｍで２０分間遠心して、回収した上清をファージ形成用ＶＣＳＭ（以下ヘルパーファージと略）とした（−８０℃保存）。
【００３１】
［ヘルパーファージのタイターチェック］
ヘルパーファージの濃度を計算するために、以下に述べる方法でタイターチェックを行った。まず、ヘルパーファージ１／１０６μｌをＸＬ−１　Ｂｌｕｅ　１００μｌに混ぜて、３７℃の湯浴で３０分間温めてファージを感染させた。一度電子レンジで加熱し溶かしてから十分に冷ましたトップアガー５ｍｌとファージを感染させたＸＬ−１　Ｂｌｕｅをよく混ぜて２×ＹＴプレートに流し込み、３７℃で一晩培養した。形成されたプラーク数からヘルパーファージの濃度を計算した。
【００３２】
［分化型Ｃａｃｏ−２細胞を用いたパニング操作］
表２に示す材料を用いて、パニングを行った。
【００３３】
【表２】

【００３４】
［パニング用ファージの調製］
乾熱滅菌済み坂口フラスコ（１ｌ）で、以下の試薬を３７℃で４０分間振とう培養した：　ＨＥＭ培地（ｐＨ６．９）（２００ｍｌ）、カルベニシリン（８０μｌ（最終濃度２０μｇ／ｍｌ））、テトラサイクリン（４００μｌ（最終濃度１０μｇ／ｍｌ））、ＣａＣｌ_２（２００μｌ（最終濃度１ｍＭ））、ＭｎＣｌ_２（２００μｌ（最終濃度１ｍＭ））、パニング用改変レクチン（１ｍｌ）。生成物に、ヘルパーファージ（１ｍｌ（約１０^１２ｐｆｕ／ｍｌ））を加えて３７℃で３０分間振とう培養し、さらにカルベニシリン（１２０μｌ（最終濃度５０μｇ／ｍｌ））、カナマイシン（５００μｌ（最終濃度２５μｇ／ｍｌ））を加えて、３７℃で１２時間振とう培養した。培養液を５０ｍｌチューブ６本に分注して、４℃、３５００ｒｐｍで３０分間遠心して、上清を回収した（計３回繰り返した）。回収した上清３０ｍｌにＰＥＧ／ＮａＣｌを６ｍｌずつ加え、低温室で氷浴に入れて一晩静置した。４℃、９５００ｒｐｍで５０分間遠心して上清を除き、再び８分間遠心して上清を完全に除いてから、１％ＢＳＡ／ＴＢＳ　１．２ｍｌで全チューブの沈殿を懸濁した。最後に４℃、１４０００ｒｐｍで１０分間遠心して、回収した上清をパニング用ファージとした（計３回繰り返した）。
【００３５】
［パニング用ファージのタイターチェック］
パニング用ファージの濃度を計算するために、以下の方法でタイターチェックを行った。まず、Ｓｕｒｅ２（２００μｌ）にパニング用ファージを加え（１／１０^８〜１／１０^６μｌ）、３７℃の湯浴で３０分間温めてから２×ＹＴＣプレートにまき、３７℃で一晩培養して形成されたコロニー数からファージの濃度を計算した。
【００３６】
［ファージ感染用Ｓｕｒｅ２の培養］
Ｓｕｒｅ２をＨＥＭ培地（ｐＨ７．０、テトラサイクリン最終濃度４０μｇ／ｍｌ添加）を用いて、３７℃で一晩培養したものをファージ感染用に使用した。
【００３７】
パニング用改変レクチンのパニングには、Ｃａｃｏ−２細胞を用いた。ここでは、その細胞の培養について述べる。同細胞の培養は、ＤＭＥＭ培地（Ｄｕｌｂｅｃｃｏ’ｓ　Ｍｏｄｉｆｉｅｄ　ＥＡＧＬＥ　ＭＥＤＩＵＭ▲２▼（日水社製＃０５９１９）４．７５ｇ／５００ｍｌ）において、オートクレーブ滅菌してから行った。その他の条件は、表３に示す。
【００３８】
【表３】

【００３９】
以下に述べるＣａｃｏ−２細胞培養に関する操作は、クリーンベンチ内で無菌的に行った。実験に用いるチップやスギウラなどは全て、オートクレーブ滅菌あるいは乾熱滅菌済みのものを使用した。Ｃａｃｏ−２用培地は、ＤＭＥＭ培地（５００ｍｌ）、３０％グルコース（１０ｍｌ）、３％Ｌ−グルタミン（７．５ｍｌ）、１Ｍ　ＨＥＰＥＳ（５ｍｌ）、１０％ＮａＨＣＯ３（１０ｍｌ）、をよく混ぜた後、５０ｍｌ分の培地を捨て、非働化ＦＣＳ　５０ｍｌを加えることにより調整した。
【００４０】
次に、細胞を１０ｃｍシャーレ（ＦＡＬＣＯＮ社製＃３５−３００３）に撒き、３７℃、５％ＣＯ_２インキュベーター内で培養した。細胞の継代はサブコンフルエントまで増えた時点で行った。まず細胞をＰＢＳで２回洗いトリプシン−ＥＤＴＡをスギウラ半分ほど加えて、３７℃で５分間処理して細胞を剥がした。剥がした細胞を培地で懸濁して４℃、１０００ｒｐｍで５分間遠心して細胞を回収し、２０倍希釈でシャーレに撒きなおした（継代間隔は約５日間）。培地の交換は継代の翌日、および１日おきに行った。
【００４１】
［Ｃａｃｏ−２細胞の分化誘導とパニング用の細胞の準備］
Ｃａｃｏ−２細胞を完全にコンフルエントになるまで培養し、最終濃度２ｍＭの酪酸ナトリウムを含む培地に交換して、さらに５日間培養を続けて分化させた。これを回収してパニング用分化型Ｃａｃｏ−２細胞として用いた。継代の時と同じ方法で分化型Ｃａｃｏ−２細胞を回収した。３回目のパニング用のみ、０．２５％グルタルアルデヒド／ＰＢＳで室温、３０分間振とうして固定した後、ＴＢＳで５回洗ったものを使用した。１回目、２回目のパニングでは非固定のまま使用した。細胞はパニング前に、約１０^６ｃｅｌｌｓを３％ＢＳＡ／ＴＢＳで懸濁し、室温で３時間ブロッキングを行った。
【００４２】
［分化型Ｃａｃｏ−２細胞を用いたパニング操作］
ブロッキングした分化型Ｃａｃｏ−２細胞約１０^６個とパニング用ファージ１０^１２ｃｆｕ（これがパニング操作におけるファージのｉｎｐｕｔ数）を１４ｍｌ（ＩＷＡＫＩ＃２３２４−０１５）チューブに入れ、１％ＢＳＡ／ＴＢＳ　１４ｍｌを加え、４℃で２時間（１、２回目のパニング）または４℃で一晩（３回目のパニング）チューブを回転させながら反応させた。細胞をＴＢＳで３回洗い未結合ファージを除き、細胞の沈殿にファージ感染用Ｓｕｒｅ２を１０ｍｌ直接加えて、３７℃の湯浴で３０分間温めてファージを感染させた。ファージを感染させたＳｕｒｅ２溶液の一部でタイターチェックを行いファージの回収率を計算した（後述）。残りはＨＥＭＣプレート（ＳＵＭＩＬＯＮプレート）に１ｍｌずつまいて３７℃で培養し、コロニーがかすかに見えてきた時点（平均８〜９時間）で４℃に移して培養を止めた。ここまでを１回のパニング操作とした。１、２回目のパニング後は、各プレートにＨＥＭ培地（ｐＨ７．０）を２０ｍｌずつ加えてコロニーを回収し、この溶液を回収ライブラリーとして再びパニング用ファージの調製操作に戻った。３回目のパニング後、形成されたコロニーをランダムに拾って、回収レクチンの塩基配列の解析に進んだ。ランダムにクローニングした６４個のクローンの解析の結果、３５個のレクチンクローンが、ストップコドンなどを含まずに完全長のレクチンをコードしている、改変部位を持つ改変ＭＡＨレクチンクローンであることがわかった（図６）。また、３５個のクローンで同一の改変部位を有するクローンは存在せず、これら３５個のクローンが３５種類の新規人工レクチンであることが明らかとなった。これら３５個の改変部位の配列は、野生型ＭＡＨの配列や、ヒト赤血球を用いたパニングにより回収された新規人工レクチンの改変部位の配列（図７）と全て異なっていた。また、この回収レクチン（「回収改変レクチン」）は、次のレクチンの固定化に用いられた。
【００４３】
［実験例２　回収改変レクチン等のタグ抗体付加と検証］
本発明の１つの実施例であるレクチン固定化法は、Ｒｅｖｅｒｓｅ　ｃｅｌｌ−ＥＬＩＳＡ法と呼ばれる方法に適用することができる。このＲｅｖｅｒｓｅ　ｃｅｌｌ−ＥＬＩＳＡ法は、ｃｅｌｌ−ＥＬＩＳＡ法を参考にしつつ、新たに開発した測定法である。ここでのレクチン固定化法は、レクチンをＥＬＴＳＡプレートに固定化するところに適用されるものである。Ｒｅｖｅｒｓｅ　ｃｅｌｌ−ＥＬＩＳＡ法において、レクチンをＥＬＩＳＡプレートに固定化するためには、まず、予めＥＬＩＳＡプレート上に抗ＦＬＡＧ抗体を固定する。次に、この抗ＦＬＡＧ抗体に結合するタグ・ペプチド（タグ抗体、フラグ抗体、フラグ配列、又は、ＦＬＡＧ抗体等）が導入されたレクチンを含むライセートをこのプレート上に加える。すると、ＦＬＡＧ配列が抗ＦＬＡＧ抗体に結合しレクチンがプレート上に固定される。この過程では、ライセート中のＦＬＡＧが付加されたレクチンのみが選択的に抗体に結合されるので、レクチンの精製と定量が可能となる。これは、抗体の特異性によるもので、結合する部位が選択的にＦＬＡＧと結合し、その量が結合部位の量によって規定されるからである。
【００４４】
［ＦＬＡＧ融合型レクチン遺伝子の作製とクローニング］
この実施例においては、上記回収改変レクチンの遺伝子および野生型ＭＡＨ遺伝子が用いられた。タグを融合するために、ベクターとして、ｐＦＬＡＧ−ＡＴＳ　Ｅｘｐｒｅｓｓｉｏｎ　Ｖｅｃｔｏｒ（ＳＩＧＭＡ社製、部品番号＃Ｅ−５７６９）が用いられた。表４のプライマーや試薬等が使用された。
【００４５】
【表４】

【００４６】
［回収レクチン遺伝子のインサート側断片の作製］
ＭＡＨ由来の人工レクチン遺伝子および野生型ＭＡＨ遺伝子を鋳型にして、以下の表５の試薬・条件でＰＣＲを行い、インサート側断片を作製した。
【００４７】
【表５】

【００４８】
ＰＣＲ終了後、ＤｐｎＩを１μｌずつ添加し、３７℃で３時間反応させた。反応産物はＰＣＲ　Ｐｕｒｉｆｉｃａｔｉｏｎ　Ｋｉｔで回収し、表６に示すように制限酵素処理を行った。
【００４９】
【表６】

【００５０】
最後に再びＰＣＲ　Ｐｕｒｉｆｉｃａｔｉｏｎ　Ｋｉｔで回収して、電気泳動で確認したものをインサート側断片とした。
【００５１】
［ＦＬＡＧ発現ベクターのベクター側断片の作製］
表７に示すようにｐＦＬＡＧ−ＡＴＳ　Ｅｘｐｒｅｓｓｉｏｎ　Ｖｅｃｔｏｒを制限酵素処理した。この条件で得られたものをＰＣＲ　Ｐｕｒｉｆｉｃａｔｉｏｎ　Ｋｉｔで回収して、電気泳動でベクター側断片となることを確認した。
【００５２】
【表７】

【００５３】
［ライゲーションと大腸菌へのトランスフォーメーション］
ＴａＫａＲａ　ＤＮＡ　Ｌｉｇａｔｉｏｎ　Ｋｉｔ　Ｖｅｒ．２を用いて、表８の試薬等を用いてライゲーションを行った。即ち、１６℃で３時間ライゲーション反応を行い、このうち２μｌをＪＭ１０９にトランスフォーメーションして、２×ＹＴＣプレートに撒いた。
【００５４】
【表８】

【００５５】
［塩基配列の確認］
形成されたコロニーを拾って一晩培養した培養液からプラスミドを回収した。表４の解析用プライマーを用いて正しい塩基配列を有しているかどうかをＧｅｎｅｔｉｃ　Ａｎａ１ｙｚｅｒ　３１００で確認した。さらに、回収したプラスミドを制限酵素ＸｈｏＩ、ＢｇｌＩＩで切断し、生成するバンドの大きさが正しいかどうか電気泳動で確認した。正しくクローニングできたものは、グリセロールストックを作製して−８０℃で保存した。
【００５６】
［ＦＬＡＧ融合型レクチンのライセート作製］
ＦＬＡＧ融合型レクチンのライセート作製には、表９の材料を用いた。
【００５７】
【表９】

【００５８】
［ＦＬＡＧ融合型レクチンクローンの予備培養］
ＨＥＭ培地（カルベニシリン最終濃度５０μｇ／ｍｌ添加）４ｍｌにＦＬＡＧ融合型レクチンクローンのグリセロールストック４μｌを加え、３７℃で一晩培養した。ＨＥＭ培地（カルベニシリン最終濃度５０μｇ／ｍｌ）４ｍｌに培養液４μｌを加えて、もう一度３７℃で一晩培養した。
【００５９】
［ライセート回収用の培養とＩＰＴＧ誘導］
５０ｍｌチューブに表１０に示すような試薬を準備し、３７℃、２００ｒｐｍで３時間振とう培養した。その後、ＩＰＴＧを２００μｌ添加し（最終濃度１ｍＭ）、さらに３７℃、２００ｒｐｍで３時間振とう培養して、タンパク質の発現誘導を行った。
【００６０】
【表１０】

【００６１】
［ライセート回収］
誘導後の培養液を、９５００ｒｐｍ、４℃で２０分間遠心して大腸菌を回収した。ライセート作製用バッファー２００μｌで大腸菌を懸濁し、１．５ｍｌのエッペンドルフチューブに移した。液体窒素と３７℃の湯浴を使い、懸濁液を計５回凍結融解して、大腸菌を粉砕した。１５０００ｒｐｍ、４℃で３０分間遠心して、上清をライセートとして回収した。回収したライセートはＢＣＡ　Ｐｒｏｔｅｉｎ　Ａｓｓａｙ　Ｋｉｔでタンパク定量を行い、１ｍｇ／ｍｌの濃度に調製して分注し、回収レクチンのライセートとして−８０℃で保存した。
【００６２】
［回収したライセート中のＦＬＡＧ融合型レクチンの確認］
表１１に示すような材料を用いて回収したライセート中のＦＬＡＧ融合型レクチンの確認を行った。
【００６３】
【表１１】

【００６４】
［ＳＤＳ−ＰＡＧＥ］
ライセートに１／３量の４×Ｓａｍｐｌｅ　Ｂｕｆｆｅｒ（＋２ＭＥ）を加え、１００℃で５分間加熱して熱変成させた。各ライセートを１０μｇずつ用いて、電気泳動を行った。
【００６５】
［ウェスタンブロッティングによるＦＬＡＧ融合型レクチンの検出］
ＳＤＳ−ＰＡＧＥ後、ＰＶＤＦ膜にタンパク質をブロッティングした。一次抗体として、抗ＦＬＡＧ抗体または抗ＭＡＨ抗体を１時間反応させた。ＴＢＳＴで洗った後、二次抗体としてＡＰ標識−抗マウスＩｇＧ抗体またはＡＰ標識−抗ウサギＩｇＧ抗体を３０分間反応させた。ＴＢＳＴで洗った後、ＡｌｋａｌｉｎｅＰｈｏｓｐｈａｔａｓｅ　Ｓｕｂｓｔｒａｔｅ　Ｋｉｔで発色させて確認した。
【００６６】
作製したＦＬＡＧ融合型野生型ＭＡＨレクチン遺伝子を制限酵素ＸｈｏＩ、ＢｇｌＩＩで切断し、電気泳動で確認したところ、インサート側断片とベクター側断片に対応する、正しい大きさのバンドが確認された（図８）。この遺伝子については、ＤＮＡシークエンサーによる塩基配列解析によっても、正しくＦＬＡＧ配列を導入できたことが確認された。
【００６７】
ＦＬＡＧ融合したＭＡＨ由来の人工レクチン（Ｃ１及びＣ３５）及び野生型ＭＡＨレクチンにおいて、抗ＦＬＡＧ抗体および抗ＭＡＨ抗体を用いたウェスタンブロッティングによって、ＭＡＨの分子量である約３０ｋＤａ付近に、抗ＭＡＨ抗体と抗ＦＬＡＧ抗体の両方で共通して検出されたバンド現れることがわかった（図９）。従って、ＦＬＡＧ配列を導入した人工レクチンが正しく発現していることが、タンパク質レベルでも確認された。
【００６８】
［実験例３　回収改変レクチン等の固定化］
［人工レクチンを含むライセートの準備］
実験例２のＣａｃｏ２細胞を用いたパニングと同様に、ヒト赤血球を用いたパニングにより実験例１のパニング用改変レクチンから、回収改変レクチンとして１０種類の新規人工レクチンを回収した。この回収改変レクチンについては、実験例２と同様にして、タグ抗体を付加させた。このようなタグ抗体付きの人工レクチンと野生型ＭＡＨ、さらに人工レクチンを含まないコントロールとして、空ベクターであるｐＦＬＡＧ−ＡＴＳについて実験例２と同様の方法でライセートを調製した。以下の実験は、表１２に示す材料を用いて行った。
【００６９】
【表１２】

【００７０】
［人工レクチンの抗ＦＬＡＧ抗体への結合］
９６ｗｅｌｌ−ＥＬＩＳＡプレートに抗ＦＬＡＧ抗体を５０μｌずつ入れ（０．２５μｇ／ｗｅｌｌ）、４℃で一晩静置して抗体をｗｅｌｌに固定した。ｗｅｌｌをＴＢＳで３回洗い、３％ＢＳＡ／ＴＢＳ　１７０μｌを加え、室温で３時間ブロッキングを行った。ｗｅｌｌをＴＢＳで３回洗い、各ライセートを５０μｇずつ入れ（５０μｇ／ｗｅｌｌ）、室温で２時間揺らしてライセート中の人工レクチンを結合させた。
【００７１】
［人工レクチンの抗ＦＬＡＧ抗体への結合量の確認］
ライセート中の人工レクチンを結合させた後、ｗｅｌｌをＴＢＳＴで３回洗い、抗ＭＡＨ抗体を５０μｌずつ加え、室温で３０分間反応させた。ｗｅｌｌをＴＢＳＴで３回洗い、ＨＲＰ標識−抗ウサギＩｇＧ抗体を５０μずつ加え、室温で３０分間反応させた。ｗｅｌｌをＴＢＳＴで３回洗い、各ｗｅｌｌにＡＢＴＳ／Ｈ２０２を５０μｌずつ加え発色させ、０Ｄ４０５／４９０を測定した。
【００７２】
［抗ＦＬＡＧ抗体に結合させたレクチンの活性の確認］
ライセート中の人工レクチンを結合させた後、ｗｅｌｌをＴＢＳＴで３回洗い、ビオチン化シアリルＴを５０μｌずつ加え、室温で２時間反応させた。ｗｅｌｌをＴＢＳＴで３回洗い、ＨＲＰ標識−ストレプトアビジンを５０μｌずつ加え、室温で３０分間反応させた。ｗｅｌｌをＴＢＳＴで３回洗い、各ｗｅｌｌにＡＢＴＳ／Ｈ２０２を５０μｌずつ加え発色させ、０Ｄ４０５／４９０を測定した。
【００７３】
［ヒト赤血球等を用いたＲｅｖｅｒｓｅ　ｃｅｌｌ−ＥＬＩＳＡ法による測定］
表１３に示すような材料を用いて、Ｒｅｖｅｒｓｅ　ｃｅｌｌ−ＥＬＩＳＡ法による測定を行った。まず、実験例２と同様の方法により人工レクチンを含むライセートの準備を行った。
【００７４】
【表１３】

【００７５】
［赤血球の準備］
ヒト赤血球はヒトＡ型末梢血、又は、４種類の異なる動物赤血球（ニワトリ、ウシ、ウマ、若しくは、ウサギ）を１０００ｇ、４℃で５分間遠心して赤血球画分を沈殿させ上清を除き、ＰＢＳで懸濁して再び遠心を行う洗いの操作を５回繰り返したものを使用した。測定に用いる際は、５×１０^６個／５０μｌになるよう１％ＢＳＡ／ＴＢＳＴで希釈した。
【００７６】
［赤血球を用いたＲｅｖｅｒｓｅ　ｃｅｌｌ−ＥＬＩＳＡ法による測定］
上述と同様の方法により、ライセート中の人工レクチンを抗ＦＬＡＧ抗体に結合させた。結合後、ｗｅｌｌをＴＢＳＴで３回洗い、赤血球を５０μｌずつ加え（５×１０^６個／ｗｅｌｌ）、室温で２時間結合させた。ｗｅｌｌをＴＢＳＴで３回洗い、各ｗｅｌｌにＡＢＴＳ／Ｈ_２Ｏ_２を５０μｌずつ加え発色させ、０Ｄ_{４０５／４９０}を測定した。
【００７７】
まず各人工レクチンをできるだけ多く固相化できるように、各ｗｅｌｌに最大量固定できる抗ＦＬＡＧ抗体の量と、人工レクチンの抗体への結合量を最大化できるライセート量について条件検討を行った。そして、抗ＦＬＡＧ抗体を０．２５μｇ／ｗｅｌｌずつ４℃で一晩固定し、各ライセートを総タンパク質量で５０μｇ分、室温で２時間結合させるという条件を確定した。この条件のもと人工レクチンの結合を行うと、各人工レクチンごとの結合量がほぼ一定になることが明らかとなった。またｐＦＬＡＧ−ＡＴＳをトランスフォーメーションした大腸菌から回収したレクチンを含まないライセートでは、シグナルがほとんど検出されないことより、ライセート中の人工レクチンが特異的に結合していることが明らかとなった（図１０）。また、それぞれ親和性は異なるものの、各レクチンがそれぞれ結合することが分かっているビオチン化シアリルＴ糖鎖を用いた活性の確認では、各レクチンのシアリルＴに対する結合が確認されたことから、活性も保持されていることが確認された（図１１）。以上の結果より、Ｒｅｖｅｒｓｅ　ｃｅｌｌ−ＥＬＩＳＡ法を採用することで、ライセート中に含まれる各人工レクチンの活性を保持しつつ、精製および結合量の標準化が達成されることが明らかとなり、多数の人工レクチンライセートを用いても定量的な測定が行えることがわかる。
【００７８】
ヒト赤血球を用いたＲｅｖｅｒｓｅ　ｃｅｌｌ−ＥＬＩＳＡ法による測定を行ない、各人工レクチンについてヒト赤血球の結合が確認された（図１２）。ヒト赤血球表面に存在するシアリルＴに対する各レクチンの結合性と、ヒト赤血球に対する結合性が完全には相関しなかったが、これは各レクチンが認識できるシアリルＴ以外の糖鎖が、赤血球表面に発現していることなどが理由として考えられる。このような比較が行えるのも、Ｒｅｖｅｒｓｅ　ｃｅｌｌ−ＥＬＩＳＡ法においてはライセートを用いても定量的な議論が可能なためであり、今後Ｒｅｖｅｒｓｅ　ｃｅｌｌ−ＥＬＩＳＡ法が実際の細胞の分別や同定に有用であることが期待される。
【００７９】
ヒト赤血球と同様の方法で、４種類の異なる動物赤血球についてもＲｅｖｅｒｓｅ　ｃｅｌｌ−ＥＬＩＳＡ法による測定を行った。測定の結果、各動物赤血球に対応する結合パターンを描くことに成功した（図１３）。この測定では、ヒトＡ型、ニワトリ、ウマ、ウサギの赤血球は、ある程度似通った結合パターンを示したが、ウシの赤血球は明らかに異なる結合パターンを示した。これらの赤血球がこのような方法により見分けられることがわかる。特に、ウシの赤血球は明確に見分けられる。
【００８０】
［実験例４　回収改変レクチン等のタグ付加］
この実験例では、ＭＡＨのループＤのアミノ酸を改変して、作った人工レクチンを生成し、その人工レクチンにタグ抗体を付けたタグ付き人工レクチンを作った。このタグ付き人工レクチンは、上記実験例３の方法と同様にして、固定化されることができる。
【００８１】
［ループＤの伸長］
テンプレートとしてＭＡＨのｃＤＮＡを使用してＰＣＲ法によりループＤが延長された（図１４）。具体的には、野生型ＭＡＨ遺伝子（２μｌ）、インサート断片作製用プライマー（２種）（１μｌずつ（各１００ｐｍｏｌ））、１０×ｄＮＴＰ（１０μｌ）、１０×ＰＣＲ　Ｂｕｆｆｅｒ（１０μｌ）、ミリＱ（７５μｌ）、ａｍｐｌｉ　Ｔａｑ　Ｇｏｌｄ（１μｌ）の混合物を、９５℃×９分に保ち、（９４℃×１分、５４℃×２分、そして、７２℃×２分）のサイクルを３０サイクル、そして、７２℃×１０分保持の後、４℃に冷やした。　ＰＣＲ終了後、ＤｐｎＩを１μｌずつ添加し、３７℃で２時間反応させた。反応産物はＰＣＲ　Ｐｕｒｉｆｉｃａｔｉｏｎ　Ｋｉｔ（ＱＩＡＧＥ社製）で回収された。生成物は、制限酵素ＸｈｏＩ．ｃｏｎｃ及びＢｇｌＩＩ．ｃｏｎｃで処理された。そして再び、反応産物はＰＣＲ　Ｐｕｒｉｆｉｃａｔｉｏｎ　Ｋｉｔ（ＱＩＡＧＥ社製）で回収された（Ｄ改変レクチン）。
【００８２】
【表１４】

【００８３】
［タグ用のベクター」
ここでは、上述の方法により、ループＤが改変されたレクチンの塩基配列のＮ末端にタグを付加するために、ｐＦＬＡＧ−ＡＴＳを用いることにした。このベクターのマルチクローニングサイトにはＭＡＨを組み込むのに都合の良い制限酵素部位がなかったので、Ｓｉｔｅ−Ｄｉｒｅｃｔｅｄ　Ｍｕｔａｇｅｎｅｓｉｓのプロトコールに従い、ＢｇｌＩＩｓｉｔｅをＳｐｅＩｓｉｔｅに作り変えた。まず、改変したい部位を含むプライマーをｓｅｎｓｅ側、ａｎｔｉ側でまったく相補的になるように設計した。次にＰＣＲを行い、ＤｐｎＩ　１ｍｌを加え３７℃で１時間インキュベートした。このＤＮＡ溶液をそのまま用いてＸＬ１−Ｂｌｕｅにトランスフォーメーションした。最後に、得られたコロニーをショートカルチャーしてプラスミドを回収し、ＢｉｇＤｙｅ　Ｔｅｒｍｉｎａｔｏｒ　Ｃｙｃｌｅ　ＳｅｑｕｅｎｃｉｎｇによりそのＤＮＡ配列を読んで、改ｐＦＬＡＧ−ＡＴＳを同定した。このとき、ＰＣＲ反応液の組成は、［ｔｅｍｐｌａｔｅ（ｐＦＬＡＧ−ＡＴＳ）１μｌ、ｐｒｉｍｅｒ（ｐＦＬＡＧ−ＳｐｅＩ−ｓｅｎｓｅ１００ｎｇ／ｍｌ，ｐＦＬＡＧ−ＳｐｅＩ−ａｎｔｉ　１００ｎｇ／ｍｌ）各１．２５ｍｌ、１０×ＰＣＲ　ｂｕｆｆｅｒ　５ｍｌ、ｄＮＴＰ　１ｍｌ、ＭｉｌｌｉＱ　４０．５ｍｌ、ｐｆｕ　ｔｕｒｂｏ　１ｍｌ］であった。また、ＰＣＲの反応条件は［９５℃３０ｓｅｃ，１２ｃｙｃｌｅｓ　ｏｆ（９５℃３０ｓｅｃ，５５℃１ｍｉｎ，６８℃１０ｍｉｎ）］であった。また、プライマーの配列は、表１５に示す。
【００８４】
【表１５】

【００８５】
［ベクターの移しかえ］
上記Ｄ改変レクチンと野生型ＭＡＨのｃＤＮＡをｐＦＬＡＧ−ＣＴＳからＰＣＲで増幅して制限消化（ＸｈｏＩｃｏｎｃ．とＳｐｅＩｃｏｎｃ．（ロッシュ））し、改ｐＦＬＡＧ−ＡＴＳに組み込んだ。これをＪＭ１０９にトランスフォーメーションし、得られたクローンをＢｉｇＤｙｅ　Ｔｅｒｍｉｎａｔｏｒ　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇにより同定した。このとき、ＰＣＲ反応液の組成は、［ｔｅｍｐｌａｔｅ　０．５ｍｌ、ｐｒｉｍｅｒ（Ｎ−Ｆｌａｇ−ＸｈｏＩ　１００ｎｇ／ｍｌ，ＭＡＨ−ＳｐｅＩ−ａｎｔｉ　１００ｎｇ／ｍｌ）各０．５ｍｌ、ｄＮＴＰ　４ｍｌ、１０×ＰＣＲ　ｂｕｆｆｅｒ　５ｍｌ、ＴａｑＧｏｌｄ　１ｍｌ、ＭｉｌｌｉＱ　３８．５ｍｌ］であった。また、ＰＣＲの反応条件は一般的なもの［９６℃５ｍｉｎ，３０ｃｙｃｌｅｓ　ｏｆ（９６℃１ｍｉｎ，５５℃１ｍｉｎ，７２℃２ｍｉｎ），７２℃５ｍｉｎ］であった。また、プライマーの配列は、表１６に示す。
【００８６】
【表１６】

【００８７】
［ＢｉｇＤｙｅ　Ｔｅｒｍｉｎａｔｏｒ　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇプロトコールの変更点］
ここでは既存のプロトコールを変更して行った。ＰＣＲ反応液の組成は、５ｘＳｅｑｕｅｎｃｉｎｇ　Ｂｕｆｆｅｒ　２ｍｌから１０ｘ　ＰＣＲ　Ｂｕｆｆｅｒ　２ｍｌへと、また、エタノール沈殿により調整後のシーケンスサンプルを溶かす溶媒は、Ｈｉ−Ｄｉ　Ｆｏｒｍａｍｉｄｅ　２０ｍｌからＭｉｌｌｉＱ　２０ｍｌへと変更した。
【００８８】
上記により得られた大腸菌を、カルベニシリンを入れたＨＥＭ（ＨｉｇｈｌｙＥｎｒｉｃｈｅｄ　Ｍｅｄｉｕｍ）で単離して、ｓｍａｌｌ−ｃｕｌｔｕｒｅ（ｏｖｅｒ　ｎｉｇｈｔ）した。翌日、ＨＥＭ２０ｍｌ，ＣａＣｌ_２（１Ｍ）２０ｍｌ，ＭｎＣｌ_２（１Ｍ）２０ｍｌ，ＭｇＣｌ_２（４．９Ｍ）８１．７ｍｌからなる培地にｓｍａｌｌ−ｃｕｌｔｕｒｅした大腸菌液を２００ｍｌ加えて３時間ｐｒｅ−ｃｕｌｔｕｒｅした。ここへ１００ｍＭ　ＩＰＴＧを２００ｍｌ加えて３時間培養し誘導をかけた。次に９，５００ｒｐｍで１０分間遠心して大腸菌を回収し、ＴＢＳ２００ｍｌに懸濁して−８０℃に保存した。後日、大腸菌の凍結（液体窒素）と融解（３７℃湯浴）を５回繰り返し、１５，０００ｒｐｍで２０分間遠心して上清をライセートとして回収した。このライセートはタンパク定量を行った後に−８０℃保存し、これを使用する準備が整ってからタンパク濃度１ｍｇ／ｍｌに希釈して４℃保存した。
【００８９】
［ＳＤＳ−ＰＡＧＥ＆Ｗｅｓｔｅｒｎ　Ｂｌｏｔｔｉｎｇ］
上記ライセート中にＤ改変レクチンのフラッグ付き（改変ＦＬＡＧ−ＭＡＨ）が存在しているのか調べるために、ＳＤＳ−ＰＡＧＥ、およびＷｅｓｔｅｒｎ　Ｂｌｏｔｔｉｎｇを行った。抗体染色では、１次抗体としてａｎｔｉ−ＭＡＨ　ｒａｂｂｉｔ　ｐｏｌｙｃｌｏｎａｌ抗体（当室で準備）またはａｎｔｉ−ＦＬＡＧ　Ｍ２　ｍｏｎｏｃｌｏｎａｌ抗体を用い、２次抗体にはそれぞれＡＰ−ｇｏａｔ　ａｎｔｉ−ｒａｂｂｉｔ　ＩｇＧ抗体とＡＰ−ｇｏａｔ　ａｎｔｉ−ｍｏｕｓｅ　ＩｇＧ抗体を用いてＡＢＣ　Ｋｉｔで発色させて、確認した。このようにタグ付きのＤ改変レクチンが得られ、上述と同様な方法により、固定化することができる。
【００９０】
［タグ付きＤ改変レクチンの固定化］
ＳＵＭＩＬＯＮ　Ｈ　タイププレートに希釈したａｎｔｉ−ＦＬＡＧ　Ｍ２　ｍｏｎｏｃｌｏｎａｌ抗体を５０ｍｌずつ入れて（０．２５ｍｇ／ｗｅｌｌ）、４℃で一晩静置した。翌日ＴＢＳで３回洗ったあとに３％ＢＳＡ／ＴＢＳ　１７０ｍｌで３時間ブロッキングした。これをＴＢＳで３回洗い、希釈分注してある上述のライセートを５０ｍｌずつ加えて２時間振とうした。このことにより、タグ付きのＤ改変レクチンがこのタイププレートに固定されることになる。次にＴＢＳＴで洗ってから、ビオチン化糖ポリマーを希釈して５０ｍｌずつ（０．２ｍｇ／ｗｅｌｌ）加えて２時間振とうした。ＴＢＳＴで洗った後に、１０００倍希釈したＳｔｒｅｐｔａｖｉｄｉｎ−ＨＲＰ　Ｃｏｎｊｕｇａｔｅを５０ｍｌずつ加えて３０分間振とうし、ＡＢＴＳ／Ｈ_２Ｏ_２　５０ｍｌで発色させてＯＤ_４０５／４９０^{を測定し、Ｄ改変レクチンが固定されていることを確認した。}
【００９１】
［ＩｇＡグライコフォームの識別への適用］
以下、本発明のレクチン固定化法を適用した例を示す。改変レクチンライブラリをＩｇＡグライコフォーム識別法に適用には、（１）改変レクチンライブラリーを作成する。（２）ＩｇＡ腎症患者ＩｇＡに親和性の高いレクチンをパニング法により選択し、レクチンサブライブラリーを作製する。（３）上記の（２）で得られたレクチンサブライブラリーの中から必要に応じＩｇＡ腎症患者と健常人のＩｇＡの違いをよく反映する改変レクチンを選び、タグを付ける。（４）このタグ付き改変レクチンをマイクロタイタープレートに本発明の方法で固定し、レクチンプレートを作製する（図１５）。（５）ＩｇＡのグライコフォームのレクチンライブラリーによる識別ＩｇＡ腎症患者と健常人の血清ＩｇＡのレクチンプレートへの結合パターンの比較・解析を行う。（６）血清診断アッセイ条件の検討市販健常人ＩｇＡに糖鎖を酵素的・化学的に付加し、人工ＩｇＡを作製する。健常人血清に人工ＩｇＡを混合し、レクチンプレートを用いたパターン解析を行う。血清中に存在する他の血清蛋白質の影響を検討し、アッセイ条件の最適化を行う。
【００９２】
［骨芽細胞亜集団の識別方法への適用］
まず、（１）改変レクチンライブラリーを作成する。次に（２）レクチンライブラリーの作製骨芽細胞に親和性の高いレクチンをパニング法等により選択し、レクチンサブライブラリーを作製する。このとき、タグの選別機能を使うこともできる。（３）上記の（２）で得られたレクチンサブライブラリーの中から必要に応じ分化のステージをよく反映するレクチンを選び、タグを付けてマイクロタイタープレートに本発明の方法で固定化し、レクチンプレートを作製する。（４）骨芽細胞の分化誘導とレクチンライブラリーによる識別培養した間葉系幹細胞を培養・分離する。より具体的には、間葉系幹細胞を培養後、骨芽細胞に分化させ、分化開始から５日目、１０日目、１５日目、２０日目の細胞を分離する。（５）分化過程の各時点で分離した細胞をレクチンプレートにて分析し、細胞表面糖鎖構造と骨形成能との相関を検討する。骨形性能の測定には骨型アルカリフォスファターゼ活性の測定およびオステオカルシン含有量の測定を行う（標準の作成）。即ち、分離した細胞を骨芽細胞を足場となるｂ−ＴＣＰブロックとの複合体の形でラット背部皮下に移植し、移植後４週、８週において摘出後、（ｉ）オステオカルシン含有量と（ｉｉ）骨型アルカリファスファターゼ活性を測定する。（６）分化過程が不明の細胞をレクチンプレートで分析し、標準と比較する（図１６参照）。
【００９３】
ここで、間葉系幹細胞とは、組織や臓器に成長する元となる細胞である幹細胞のうち、骨髄の中に存在するものをいい、間葉系幹細胞は骨、軟骨、脂肪、心臓、神経、肝臓などの細胞に分化することが確認されており、ほとんどすべての組織にも分化することのできる胚性幹細胞（ＥＳ細胞）に近い能力を秘めている。間葉系幹細胞は、β−ＴＣＰ（β−ｔｒｉｃａｌｃｉｕｍ　ｐｈｏｓｐｈａｔｅ）というリン酸カルシウムを主成分として生体内に優れた親和性、吸収性および骨伝導性を有する人工材料を培養する足場として用いる。
【００９４】
【発明の効果】
以上のような本発明によれば、糖鎖解析に主要な機能を発揮する糖鎖認識部位を阻害することなく、レクチンを支持体に固定することができ、糖鎖解析の効率が向上されるばかりでなく、糖鎖解析条件と異なる条件でレクチンが固定できるという利点がある。また、糖鎖解析用のレクチンの濃度や糖鎖解析部位の濃度が均一にすることができる。更に、特にレクチン及びその他の物質を含むライセート等から直接レクチンを固定できるという利点がある。
【００９５】
【配列表】

【図面の簡単な説明】
【図１】種々のレクチンのアミノ酸配列。
【図２】ＭＡＨの塩基配列と予想されるアミノ酸配列を示した図である。
【図３】ＭＡＨの立体構造の予測図である。
【図４】本発明の１つの実施例となるレクチンを固定する方法を用いたツールを示す概念図である。
【図５】ＭＡＨレクチンの遺伝子改変方法を表した図である。
【図６】Ｃａｃｏ２によるパニングにより回収された改変ＭＡＨレクチンのアミノ酸配列（Ｃループ）を示した図である。
【図７】ヒト赤血球を用いたパニングにより回収されてきたレクチンの改変部位のアミノ酸配列を示す図である。
【図８】ＦＬＡＧ融合型レクチン遺伝子の制限酵素処理による確認の例を示した図である。
【図９】ウェスタンブロッティングによるＦＬＡＧ融合型レクチンの発現の確認を示す図である。
【図１０】抗ＦＬＡＧ抗体への各人工レクチンの結合量に対応する発色状態を示す図である。
【図１１】抗ＦＬＡＧ抗体に結合した各レクチンのシアリルＴへの結合活性に対応する発色状態を示す図である。
【図１２】ヒト赤血球に対するＲｅｖｅｒｓｅ　Ｃｅｌｌ−ＥＬＩＳＡ法による測定結果を示す図である。
【図１３】種々の赤血球に対するＲｅｖｅｒｓｅ　Ｃｅｌｌ−ＥＬＩＳＡ法による測定結果を示す図である。
【図１４】ＭＡＨレクチンのループＤへのアミノ酸挿入位置を示す図である。
【図１５】レクチンチップの一例およびその使用方法（ＩｇＡについて）を示す図である。
【図１６】レクチンチップの一例およびその使用方法（骨芽細胞の分化度プロファイリングについて）を示す図である。
【符号の説明】
１０　糖鎖解析ツールの一例
１２　ＥＬＩＳＡプレート
１４　抗ＦＬＡＧ抗体
１６　ＦＬＡＧ融合型人工レクチン
１８　糖鎖認識部位
２０　解析対象の細胞
２２　細胞表面糖鎖[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for immobilizing a lectin, in particular, a method for immobilizing a genetically modified lectin, and a sugar chain analysis tool including a lectin immobilized using the immobilization method.
[0002]
[Prior art]
Recent advances in glycotechnology have been remarkable, including proteoglycans in the cell wall of plant cells that contribute to cell stabilization, glycolipids that affect cell differentiation, proliferation, adhesion, migration, etc., and cell-cell interactions and cells. The mechanism by which high-molecular sugar chains such as glycoproteins involved in recognition substitute, assist, amplify, regulate, or inhibit each other's functions while controlling sophisticated and precise biological reactions is being clarified. . In addition, active research has been made on the occurrence of diseases due to abnormalities of sugar chains on the cell surface and the interaction between sugar chains and receptors, and the role of sugar chains in the infection of viruses such as AIDS.
[0003]
In particular, sugar chains on the surface of cells can be used to distinguish and identify certain cells from other cells, such as normal cells and cancer cells, or cells at different stages of differentiation. There have been many reports that stem cells change according to their differentiation stage in response to changes in the cell line. As described above, analysis based on sugar chains (for example, classification and identification) is considered to be very significant.
[0004]
Lectins are considered to be very useful as a means of such sugar chain analysis.However, it is important to consider whether the types of naturally occurring lectins are limited and whether the lectins are easily accessible. . That is, in order to identify many types of sugar chains, lectins having different properties corresponding to the number of sugar chains are theoretically necessary. On the other hand, for easy use, it is desirable that a specific type of lectin is isolated and fixed on a carrier (or support).
[0005]
Conventionally, when immobilizing lectin, methods such as simply adsorbing lectin to the substrate or covalently bonding it to the substrate have been used. It is difficult to immobilize, and covalent bonding requires a complicated chemical reaction. Further, it has been proposed that a sugar chain polymer having a sugar chain that specifically binds to a lectin to be immobilized is adsorbed on a substrate, and the lectin is immobilized via the sugar chain polymer (Japanese Patent Application Laid-Open No. Hei 8 -319300). In this method, the lectin is immobilized by utilizing the sugar chain specificity of the lectin originally required for the analysis, and it is difficult to say that the so-called active site for the sugar chain is sufficiently used for the sugar chain analysis.
[0006]
[Problems to be solved by the invention]
Therefore, an object of the present invention is to provide a method for immobilizing a predetermined lectin efficiently and stably, and to provide a method for immobilizing a lectin capable of sufficiently utilizing a sugar chain analysis site of the lectin for the analysis. is there.
[0007]
[Means for Solving the Invention]
In view of the above problems, in the present invention, a first recognition part having a recognition ability for a predetermined first substance or a part thereof and a predetermined second substance or a part thereof are recognized. A lectin having a second recognition portion having a recognition ability, wherein the lectin is fixed to a predetermined support by the first recognition portion.
[0008]
Here, the first substance may include a substance that is recognized by an ability other than the sugar chain recognition ability of lectin and can be directly or indirectly bound. For example, an anti-tag antibody is included in the first substance. And a tag antibody can be included in the first recognition moiety. Further, the second substance may include a sugar chain, and the second recognition portion may include a sugar chain recognition site of the lectin. In addition, the lectin may include a lectin produced by genetic modification, and may include a non-naturally occurring artificial lectin.
[0009]
More specifically, the present invention provides the following.
[0010]
(1) An immobilization method for immobilizing a lectin on a support, comprising the steps of encoding a lectin and a tag peptide into a vector DNA, and producing an LT complex protein using the encoded DNA. And a step of bringing the LT protein into contact with a support on which an anti-tag antibody having binding or associating properties with the tag peptide is immobilized.
[0011]
(2) The method for immobilizing a lectin according to the above (1), wherein the lectin is a legume lectin.
[0012]
(3) The lectin according to (1) or (2) above, wherein a modified lectin in which one or more amino acids of loop C or loop D is modified is encoded in the vector DNA. Lectin immobilization method.
[0013]
(4) The immobilization of the lectin according to any one of (1) to (3), wherein an amino acid sequence obtained by adding the tag peptide to a C-terminal region or an N-terminal region is encoded in the vector DNA. Method.
[0014]
(5) A sugar chain analysis tool including a support on which a lectin is immobilized by the lectin immobilization method according to any one of (1) to (4).
[0015]
(6) A lectin-immobilized support comprising a lectin to which a tag / peptide has been added by a genetic modification operation, and a solid support containing an anti-tag antibody.
[0016]
(7) A sugar chain analysis tool including the lectin-immobilized support of (6).
[0017]
(8) A tag antibody vector used for adding the tag protein described in any of (1) to (7) above to the lectin.
[0018]
Here, the support may include a solid support.For example, the solid support may be a test paper using a paper such as a filter paper, or an organic substance such as nitrocellulose, latex, or a plastic material. May be included. Further, the solid support may include an inorganic substance such as glass and mica, a metal such as platinum, silver, copper, gold, aluminum and silicon, and a nonmetallic material such as CC composite and carbon. In addition, if a gel-like porous body having a large specific surface area is used, a large number of binding sites can be provided, which can be considered to be advantageous. Examples of such a support include agarose gel, allyl dextran gel and the like. Further, the shape of the solid support may include any shape such as a sheet, a plate, a sphere, a bead, a cup, and a rectangle, and is made by combining various substances and materials as described above. Any shape may be included. Tag peptides may include flag peptides, which may also be referred to as flags, and may include flag antibodies. The protein thus tagged can bind or associate with the anti-tag antibody. Contacting the protein may include placing it in a state that allows it to bind or associate with the anti-tag antibody.
[0019]
Lectins may include various types of lectins, but preferably include lectins derived from plants. More preferably, it comprises a rich variety of legume lectins. For example, other lectins as shown in FIG. 1 may be included. More preferably, it includes Maackia @ amurensis @ hemagglutinin (hereinafter abbreviated as MAH). FIG. 2 shows the predicted amino acid sequence of MAH lectin.
[0020]
Generally, legume lectins have a structure in which sugar chains are sandwiched between loops C and D (FIG. 3), but it is preferable to modify such sites that sandwich sugar chains. Therefore, altering the amino acid of loop C or loop D may mean altering the amino acid of such a sugar chain recognition site. For example, in FIG. 2, 466 to 498 (127 to 137 in the amino acid sequence equivalent from the N-terminal serine of the wild-type MAH) are 721 to 780 (wild-type MAH 212 to 231) corresponding to the amino acid sequence counted from the N-terminal serine is considered to be a base sequence corresponding to the amino acid of loop D, and such a region may be included as a target region for modification. Further, in loop D, more preferably, a region of 742 to 756 (219 to 223 in the amino acid sequence equivalent from the N-terminal serine of wild-type MAH) may be included as an object of modification. Here, the nucleotide sequence was counted from the start codon, and the amino acid sequence was counted from the N-terminal amino acid group. Modifying these amino acids may include altering the order, number, type, etc. of the aligned amino acids, and may include inserting one or more amino acids.
[0021]
The tag peptide may be added to the C-terminal region or the N-terminal region, but may be added to both the C-terminal region and the N-terminal region. The C-terminal region may include the C-terminal and its peripheral region, and the N-terminal region may include the N-terminal and its peripheral region. Since it is preferable that the tag peptide does not significantly affect the site having the sugar chain recognition function, the tag peptide may be added to a region having little effect.
[0022]
Lectins immobilized by the method described above can be used in various fields. That is, in a means (including a tool) for sugar chain analysis utilizing the sugar chain discriminating ability. For example, when cells can be identified by sugar chain analysis using sugar chain identification ability, it is used in a cell identification tool or kit. Examples of the cell identification tool include a pad and a pallet for cell identification. The concept of such a tool is schematically shown in FIG. 4 taking one example. The main element 10 of the tool is, for example, an ELISA plate 12 (an example that can be included in the support, the support is not limited to this), and an anti-tag antibody 14 immobilized thereon. And the tag-fused artificial lectin 16 bound to the anti-tag antibody and the tag antibody. The tag-fused artificial lectin 16 is fixed to the plate 12 in such a manner that a site 18 capable of recognizing a sugar chain can be brought into contact with the sugar chain so that the sugar chain to be analyzed can be sandwiched therebetween. Here, the ELISA plate 12 is used as a support, but various substances and materials can be applied to such a support as described above. When a strong material is selected, or when a chemical reaction occurs and analysis is difficult, various selections should be made according to the purpose, such as selecting a substance having low reactivity. The cell 20 desired to be identified has cell surface sugar chains 22 on its surface, and some of them are sandwiched between the sites 18 capable of recognizing sugar chains, whereby the sugar chains are recognized and sugar chain analysis becomes possible. . As described above, the anti-tag antibody performs a binding function with the tag antibody incorporated into the lectin. Therefore, without being limited to a tag antibody and an anti-tag antibody, one of two proteins or other substances having a combination binding property is fixed to a support, and the other is incorporated into a lectin, thereby immobilizing the lectin. Can be
[0023]
Here, “sugar chain analysis” refers to the identification of cells having sugar chains, the presence of proteins in all types of liquids and / or solids produced from living organisms containing proteins (urine proteins, salivary proteins, The “sugar chain analysis method” may include a method for performing the above, including diagnosis of sugar modification (glycoform) of a runny nose protein (including serum proteins, etc.), disease diagnosis, and other analyses. When the structure (or state) of the sugar chain of a cell or the glycosylation of a glycoprotein is different depending on the disease (or health) state, the disease (or health) state can be determined by sugar chain analysis. Tools (diagnostics, diagnostic devices, etc.) may be included. For example, if the wells or spots containing the lectins selected for cell identification are arranged in a permutation appropriate for cell identification, the pattern is displayed so that the pattern can be easily recognized visually. For this purpose, the position may be arranged at a predetermined position. Here, "display" may be either direct or indirect. Even in the case of visual pattern information, if the number is enormous, visual inspection with the naked eye or other methods is often insufficient, and it is also possible to perform cell identification by cluster analysis using a computer. It is possible. In cluster analysis, a computer may be used to check which part of a part to be compared (for example, a standard and a test object, or a control and a comparative substance) has a large difference. Software can also be used.
[0024]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in more detail with reference to the drawings with reference to specific examples. However, only one specific example is a material, and the present invention is not limited to these examples. It is not limited.
[0025]
【Example】
[Experimental example 1: Preparation of modified lectin]
Maacki amurensis hemagglutinin (MAH), which is a legume lectin specific to sugar chains containing sialic acid, was used. This lectin can recognize a hydrocarbon sequence in which the carbohydrate chain is composed of sialic acid residues, that is, Neu5Acα2-3Galβ1-4GlcNAc (5). At least a part of the part that seems to be related to this sugar binding site is randomly modified by genetic engineering techniques, and an artificial lectin library that can distinguish multiple types of different sugar chain variations from the randomly modified lectin is created did. MAH has a relative molecular weight of 29,000 and consists of subunit dimers.
[0026]
In the nucleotide sequence of the cDNA encoded by MAH and the amino acid sequence derived therefrom, MAH consists of 287 amino acids and contains a 30 amino acid single peptide. FIGS. 5A and 5B are diagrams showing the generation of a mutation in the MAH sugar chain recognition domain. A is a schematic diagram showing a partial overlap extension method for introducing a mutation (overlap @ extension @ method). B is a diagram showing the generation of a mutation in the sugar chain recognition domain of MAH. That is, of the 285 amino acids of the MAH lectin, the nucleotide sequence from the 127th to the 137th portion, which is a sugar binding site, was randomized by overlap extension PCR using a synthetic oligonucleotide as a primer. However, aspartic acid 135, which is considered essential for interaction with sialic acid, and aspartic acid 127, which is considered essential for coordination with metal ions, and histidine 132 were immobilized (FIG. 5). Since the types of lectins that can be produced are enormous, lectins and phages were selected and recovered by the following panning.
[0027]
Here, panning refers to recovering a phage by utilizing the ability of a protein expressed on the phage to bind to a substance to which the protein binds (eg, an antigen and an antibody, a lectin and a sugar chain, a receptor and a ligand, etc.). Refers to a technique, for example, by adding a partner substance that binds to the protein expressed by the phage, bind the expressed protein, phage, and other substances, and after flowing out the phage solution, express the bound expression. This is a method for recovering proteins, phages, and other substances. The recovered phage can be infected with Escherichia coli, amplified, and panned again (described above) to recover phages with high binding. The panning used herein is a technique for enriching and selecting a specific clone from a library, based on the binding property. Therefore, if the number of times of panning is small, there is a concern that artificial lectins having low binding properties have not yet been sufficiently eliminated.If the number is too large, enrichment proceeds by panning, and only a limited type of lectins with strong binding can be recovered. Become. Therefore, it is considered that there is an optimal number of times of the panning operation. In this embodiment, three times of panning, which is considered to be just right, were employed.
[0028]
[Preparation of VCSM (helper phage) for phage formation]
VCSM for phage formation (helper phage) was prepared using the materials shown in Table 1.
[0029]
[Table 1]

[0030]
[Preparation of VCSM for phage formation]
200 μl of XL-1 {Blue} cultured overnight in SB medium (addition of a final concentration of 40 μg / ml of tetracycline) and 10 ml of SB medium were shake-cultured at 37 ° C. for 1 hour. After adding 1 μl of the VCSM stock solution, the mixture was warmed in a 37 ° C. water bath for 30 minutes to infect the phage, and kanamycin (final concentration: 70 μg / ml) was added, followed by shaking culture at 37 ° C. for 8 hours. The culture was centrifuged at 4 ° C., 3500 rpm for 20 minutes, and the collected supernatant was warmed in a 65 ° C. water bath for 15 minutes. Again, the mixture was centrifuged again at 4 ° C. and 3500 rpm for 20 minutes, and the collected supernatant was used as VCSM for phage formation (hereinafter abbreviated as helper phage) (stored at −80 ° C.).
[0031]
[Helper phage titer check]
To calculate the concentration of helper phage, a titer check was performed by the method described below. First, 1/106 μl of helper phage was mixed with 100 μl of XL-1 {Blue}, and the mixture was warmed in a 37 ° C. water bath for 30 minutes to infect the phage. Once heated and melted in a microwave oven, 5 ml of sufficiently cooled top agar and XL-1 @ Blue infected with phage were mixed well, poured into a 2 × YT plate, and cultured at 37 ° C. overnight. The concentration of helper phage was calculated from the number of plaques formed.
[0032]
[Panning operation using differentiated Caco-2 cells]
Panning was performed using the materials shown in Table 2.
[0033]
[Table 2]

[0034]
[Preparation of panning phage]
The following reagents were cultured with shaking in a dry-heat sterilized Sakaguchi flask (1 L) at 37 ° C. for 40 minutes: @HEM medium (pH 6.9) (200 ml), carbenicillin (80 μl (final concentration 20 μg / ml)), tetracycline ( 400 μl (final concentration 10 μg / ml), CaCl₂(200 μl (final concentration 1 mM)), MnCl₂(200 μl (final concentration 1 mM)), modified lectin for panning (1 ml). The product contains helper phage (1 ml (about 10¹²pfu / ml)) and shake culture at 37 ° C. for 30 minutes. Carbenicillin (120 μl (final concentration: 50 μg / ml)) and kanamycin (500 μl (final concentration: 25 μg / ml)) are added. The cells were cultured with shaking for an hour. The culture solution was dispensed into six 50 ml tubes and centrifuged at 4 ° C. and 3,500 rpm for 30 minutes to collect the supernatant (repeated three times in total). 6 ml of PEG / NaCl was added to 30 ml of the collected supernatant, and the mixture was placed in an ice bath in a low-temperature room and allowed to stand overnight. The supernatant was removed by centrifugation at 9500 rpm for 50 minutes at 4 ° C., and the supernatant was completely removed by centrifugation again for 8 minutes. Then, the precipitate in all tubes was suspended in 1.2 ml of 1% BSA / TBS. Finally, the mixture was centrifuged at 4 ° C. and 14000 rpm for 10 minutes, and the collected supernatant was used as panning phage (repeated three times in total).
[0035]
[Titer check of panning phage]
To calculate the concentration of panning phage, a titer check was performed by the following method. First, panning phage was added to Sure2 (200 μl) (1/10⁸~ 1/10⁶μl), warmed in a 37 ° C. water bath for 30 minutes, spread on a 2 × YTC plate, cultured at 37 ° C. overnight, and calculated the phage concentration from the number of colonies formed.
[0036]
[Culture of Sure2 for phage infection]
Sure2 was cultured overnight at 37 ° C. using a HEM medium (pH 7.0, added with a final tetracycline concentration of 40 μg / ml) and used for phage infection.
[0037]
Caco-2 cells were used for panning of the modified lectin for panning. Here, the culture of the cells will be described. The cells were cultured after autoclaving in a DMEM medium (Dulbecco's Modified EAGLE MEDIUM 2 (# 05919, manufactured by Nissui) 4.75 g / 500 ml). Other conditions are shown in Table 3.
[0038]
[Table 3]

[0039]
The following operations related to Caco-2 cell culture were performed aseptically in a clean bench. All the chips and cedars used in the experiments were autoclaved or dry-heat sterilized. The medium for Caco-2 was prepared by thoroughly mixing a DMEM medium (500 ml), 30% glucose (10 ml), 3% L-glutamine (7.5 ml), 1M @ HEPES (5 ml), and 10% NaHCO3 (10 ml). The medium for 50 ml was discarded and adjusted by adding 50 ml of inactivated FCS.
[0040]
Next, the cells were seeded on a 10-cm Petri dish (# 35-3003, manufactured by FALCON), and were cultivated at 37 ° C and 5%₂The cells were cultured in an incubator. The cells were passaged when they had increased to subconfluence. First, the cells were washed twice with PBS, and trypsin-EDTA was added to about half of Sugiura, and treated at 37 ° C. for 5 minutes to detach the cells. The detached cells were suspended in a medium and centrifuged at 4 ° C. and 1000 rpm for 5 minutes to collect the cells, which were then re-spread on a Petri dish at a 20-fold dilution (the passage interval was about 5 days). The medium was changed the next day after the passage and every other day.
[0041]
[Induction of Caco-2 cell differentiation and preparation of cells for panning]
Caco-2 cells were cultured until they were completely confluent, replaced with a medium containing a final concentration of 2 mM sodium butyrate, and further cultured for 5 days to differentiate. This was recovered and used as differentiated Caco-2 cells for panning. Differentiated Caco-2 cells were collected in the same manner as in the passage. For the third panning only, the one that was shaken and fixed with 0.25% glutaraldehyde / PBS at room temperature for 30 minutes and then washed five times with TBS was used. The first and second pannings were used without fixing. Cells should be ca.⁶The cells were suspended in 3% BSA / TBS, and blocking was performed at room temperature for 3 hours.
[0042]
[Panning operation using differentiated Caco-2 cells]
Approximately 10 blocked differentiated Caco-2 cells⁶Individuals and 10 phages for panning¹²Put cfu (this is the phage input number in the panning operation) in a 14 ml (IWAKI # 2324-015) tube, add 1% BSA / TBS @ 14 ml, and add 2 hours at 4 ° C. (first and second panning) or 4 ° C. The reaction was performed overnight (third panning) while rotating the tube. The cells were washed three times with TBS to remove unbound phage, 10 ml of Sure2 for phage infection was directly added to the cell precipitate, and the phage was infected by warming in a 37 ° C. water bath for 30 minutes. A titer check was performed on a part of the Sure2 solution infected with the phage, and the phage recovery was calculated (described later). The remainder was spread on a HEMC plate (SUMILON plate) in an amount of 1 ml each and cultured at 37 ° C. When the colonies became faint (8 to 9 hours on average), the colonies were transferred to 4 ° C to stop the culture. Up to this point, one panning operation was performed. After the first and second rounds of panning, 20 ml of HEM medium (pH 7.0) was added to each plate to collect colonies, and this solution was used as a recovery library to return to the operation of preparing phage for panning again. After the third round of panning, the formed colonies were randomly picked, and the analysis of the base sequence of the recovered lectin proceeded. Analysis of the 64 randomly cloned clones revealed that 35 lectin clones were modified MAH lectin clones with modified sites, encoding full-length lectins without stop codons and the like. (FIG. 6). In addition, there was no clone having the same modification site in 35 clones, and it was revealed that these 35 clones were 35 kinds of novel artificial lectins. The sequences of these 35 modified sites were all different from the sequence of the wild-type MAH and the sequence of the modified site of the novel artificial lectin recovered by panning using human erythrocytes (FIG. 7). This recovered lectin ("recovered modified lectin") was used for immobilizing the next lectin.
[0043]
[Experimental Example 2 Addition and verification of tag antibody such as recovered modified lectin]
The lectin immobilization method as one embodiment of the present invention can be applied to a method called Reverse @ cell-ELISA method. This Reverse @ cell-ELISA method is a measurement method newly developed with reference to the cell-ELISA method. The lectin immobilization method here is applied to immobilizing a lectin on an ELTSA plate. In the Reverse Cell-ELISA method, in order to immobilize the lectin on the ELISA plate, first, an anti-FLAG antibody is immobilized on the ELISA plate in advance. Next, a lysate containing a lectin into which a tag peptide (a tag antibody, a flag antibody, a flag sequence, a FLAG antibody, or the like) that binds to the anti-FLAG antibody is added is added to the plate. Then, the FLAG sequence binds to the anti-FLAG antibody, and the lectin is fixed on the plate. In this process, only the lectin to which FLAG is added in the lysate is selectively bound to the antibody, so that the lectin can be purified and quantified. This is due to the specificity of the antibody, because the binding site selectively binds to FLAG, and the amount is determined by the amount of the binding site.
[0044]
[Preparation and cloning of FLAG fusion lectin gene]
In this example, the gene for the recovered and modified lectin and the wild-type MAH gene were used. In order to fuse the tag, pFLAG-ATS \ Expression \ Vector (manufactured by SIGMA, part number # E-5768) was used as a vector. The primers and reagents in Table 4 were used.
[0045]
[Table 4]

[0046]
[Preparation of insert-side fragment of recovered lectin gene]
Using the artificial lectin gene derived from MAH and the wild-type MAH gene as templates, PCR was performed under the reagents and conditions shown in Table 5 below to prepare an insert-side fragment.
[0047]
[Table 5]

[0048]
After completion of the PCR, 1 μl of DpnI was added thereto, and the mixture was reacted at 37 ° C. for 3 hours. The reaction product was collected by PCR {Purification Kit} and treated with restriction enzymes as shown in Table 6.
[0049]
[Table 6]

[0050]
Finally, the DNA was recovered again by PCR \ Purification \ Kit and confirmed by electrophoresis was used as an insert-side fragment.
[0051]
[Preparation of vector-side fragment of FLAG expression vector]
As shown in Table 7, pFLAG-ATS \ Expression \ Vector was treated with a restriction enzyme. The product obtained under these conditions was recovered by PCR \ Purification \ Kit, and it was confirmed by electrophoresis that it became a vector fragment.
[0052]
[Table 7]

[0053]
[Ligation and transformation into E. coli]
TaKaRa DNA Ligation Kit Ver. 2 was ligated using the reagents and the like shown in Table 8. That is, a ligation reaction was performed at 16 ° C. for 3 hours, and 2 μl of the reaction was transformed into JM109 and spread on a 2 × YTC plate.
[0054]
[Table 8]

[0055]
[Confirmation of base sequence]
The formed colonies were picked up, and the plasmid was recovered from a culture solution cultured overnight. Using the primers for analysis in Table 4, it was confirmed by Genetic @ Analyzer @ 3100 whether the base sequence was correct. Furthermore, the recovered plasmid was digested with restriction enzymes XhoI and BglII, and the size of the generated band was confirmed by electrophoresis to determine whether the size was correct. Those that could be cloned correctly were prepared as glycerol stocks and stored at -80 ° C.
[0056]
[Preparation of lysate of FLAG fusion type lectin]
The materials shown in Table 9 were used for preparing a lysate of the FLAG fusion lectin.
[0057]
[Table 9]

[0058]
[Preliminary culture of FLAG fusion type lectin clone]
To 4 ml of HEM medium (adding a final concentration of 50 μg / ml of carbenicillin), 4 μl of a glycerol stock of a FLAG-fused lectin clone was added, and cultured at 37 ° C. overnight. 4 μl of the culture solution was added to 4 ml of HEM medium (carbenicillin final concentration 50 μg / ml), and the cells were cultured again at 37 ° C. overnight.
[0059]
[Culture for lysate recovery and IPTG induction]
The reagents shown in Table 10 were prepared in a 50 ml tube, and cultured with shaking at 37 ° C. and 200 rpm for 3 hours. Thereafter, 200 μl of IPTG was added (final concentration: 1 mM), and the cells were cultured with shaking at 37 ° C. and 200 rpm for 3 hours to induce protein expression.
[0060]
[Table 10]

[0061]
[Lysate collection]
The culture solution after the induction was centrifuged at 9500 rpm at 4 ° C. for 20 minutes to collect E. coli. Escherichia coli was suspended in 200 μl of the lysate preparation buffer and transferred to a 1.5 ml Eppendorf tube. The suspension was freeze-thawed a total of 5 times using liquid nitrogen and a hot water bath at 37 ° C. to pulverize Escherichia coli. After centrifugation at 15000 rpm at 4 ° C. for 30 minutes, the supernatant was collected as a lysate. The recovered lysate was quantified for protein with BCA \ Protein \ Assay \ Kit, adjusted to a concentration of 1 mg / ml, dispensed, and stored at -80 ° C as a lysate of the recovered lectin.
[0062]
[Confirmation of FLAG fusion lectin in recovered lysate]
The FLAG-fused lectin in the lysate recovered using the materials shown in Table 11 was confirmed.
[0063]
[Table 11]

[0064]
[SDS-PAGE]
One third of 4 × Sample Buffer (+ 2ME) was added to the lysate, and the mixture was heated at 100 ° C. for 5 minutes for thermal denaturation. Electrophoresis was performed using 10 μg of each lysate.
[0065]
[Detection of FLAG fusion lectin by Western blotting]
After SDS-PAGE, proteins were blotted on a PVDF membrane. As a primary antibody, an anti-FLAG antibody or an anti-MAH antibody was reacted for 1 hour. After washing with TBST, an AP-labeled anti-mouse IgG antibody or an AP-labeled anti-rabbit IgG antibody was reacted as a secondary antibody for 30 minutes. After washing with TBST, color was confirmed with Alkaline Phosphatases \ Substrate \ Kit and confirmed.
[0066]
The prepared FLAG-fused wild-type MAH lectin gene was digested with restriction enzymes XhoI and BglII and confirmed by electrophoresis. As a result, bands of the correct size corresponding to the insert-side fragment and the vector-side fragment were confirmed (FIG. 8). ). It was also confirmed that the FLAG sequence could be correctly introduced into this gene by nucleotide sequence analysis using a DNA sequencer.
[0067]
In the FLAG-fused MAH-derived artificial lectins (C1 and C35) and wild-type MAH lectin, anti-MAH antibody and anti-FLAG were obtained by Western blotting using an anti-FLAG antibody and an anti-MAH antibody at around the molecular weight of about 30 kDa of MAH. It was found that a band commonly detected by both antibodies appeared (FIG. 9). Therefore, it was confirmed at the protein level that the artificial lectin into which the FLAG sequence was introduced was correctly expressed.
[0068]
[Experimental Example 3 Immobilization of recovered and modified lectin]
[Preparation of lysate containing artificial lectin]
Similar to the panning using Caco2 cells in Experimental Example 2, 10 types of novel artificial lectins were recovered as modified lectins from the modified lectin for panning in Experimental Example 1 by panning using human erythrocytes. A tag antibody was added to the recovered and modified lectin in the same manner as in Experimental Example 2. A lysate was prepared in the same manner as in Experimental Example 2 for pFLAG-ATS, an empty vector, as a control containing no such artificial lectin with a tag antibody, wild-type MAH, and an artificial lectin. The following experiment was performed using the materials shown in Table 12.
[0069]
[Table 12]

[0070]
[Binding of artificial lectin to anti-FLAG antibody]
The anti-FLAG antibody was placed in a 96-well ELISA plate in an amount of 50 μl each (0.25 μg / well), and allowed to stand at 4 ° C. overnight to fix the antibody to the well. The well was washed three times with TBS, and 170 μl of 3% BSA / TBS was added, and blocking was performed at room temperature for 3 hours. The wells were washed three times with TBS, 50 μg of each lysate was added (50 μg / well), and shaken at room temperature for 2 hours to bind the artificial lectin in the lysate.
[0071]
[Confirmation of binding amount of artificial lectin to anti-FLAG antibody]
After binding the artificial lectin in the lysate, the well was washed three times with TBST, and 50 μl of anti-MAH antibody was added thereto, followed by reaction at room temperature for 30 minutes. The wells were washed three times with TBST, HRP-labeled anti-rabbit IgG antibodies were added in 50 μl / well, and reacted at room temperature for 30 minutes. The wells were washed three times with TBST, and 50 μl of ABTS / H202 was added to each well to develop color, and 0D405 / 490 was measured.
[0072]
[Confirmation of activity of lectin bound to anti-FLAG antibody]
After binding the artificial lectin in the lysate, the well was washed three times with TBST, and 50 μl of biotinylated sialyl T was added thereto, followed by reaction at room temperature for 2 hours. The wells were washed three times with TBST, HRP-labeled-streptavidin was added in an amount of 50 μl each, and reacted at room temperature for 30 minutes. The wells were washed three times with TBST, and 50 μl of ABTS / H202 was added to each well to develop color, and 0D405 / 490 was measured.
[0073]
[Measurement by Reverse @ cell-ELISA using human erythrocytes etc.]
Using the materials shown in Table 13, measurement was performed by the Reverse @ cell-ELISA method. First, a lysate containing an artificial lectin was prepared in the same manner as in Experimental Example 2.
[0074]
[Table 13]

[0075]
[Preparation of red blood cells]
Human erythrocytes were obtained by centrifuging human type A peripheral blood or four different animal erythrocytes (chicken, cow, horse, or rabbit) at 1000 g at 4 ° C. for 5 minutes to precipitate the erythrocyte fraction, removing the supernatant, and removing PBS. The washing operation of suspending and centrifuging again was repeated 5 times. 5 × 10 when used for measurement⁶Each sample was diluted with 1% BSA / TBST to 50 μl.
[0076]
[Measurement by Reverse @ cell-ELISA using red blood cells]
The artificial lectin in the lysate was bound to the anti-FLAG antibody by the same method as described above. After binding, the wells were washed three times with TBST, and 50 μl of red blood cells were added thereto (5 × 10 5⁶Per well) and allowed to bind for 2 hours at room temperature. Wash the wells three times with TBST, and add ABTS / H to each well.₂O₂Was added to the mixture at 50 μl to develop color, and 0D_405/490Was measured.
[0077]
First, conditions were examined for the amount of anti-FLAG antibody that can be immobilized to each well in the maximum amount and the amount of lysate that can maximize the amount of artificial lectin bound to the antibody so that each artificial lectin can be immobilized as much as possible. Then, conditions were determined in which the anti-FLAG antibody was fixed at 0.25 μg / well at 4 ° C. overnight and each lysate was allowed to bind for 50 μg in total protein amount for 2 hours at room temperature. When binding of artificial lectins was performed under these conditions, it became clear that the amount of binding of each artificial lectin was almost constant. In the lysate containing no lectin recovered from E. coli transformed with pFLAG-ATS, almost no signal was detected, indicating that the artificial lectin in the lysate was specifically bound (FIG. 10). . In addition, the activity was confirmed using a biotinylated sialyl-T sugar chain, which is known to bind to each lectin, although the affinity differs, and the activity was confirmed because the binding of each lectin to sialyl-T was confirmed. It was confirmed that it was retained (FIG. 11). From the above results, it is clear that by employing the Reverse @ cell-ELISA method, purification and standardization of the binding amount can be achieved while maintaining the activity of each artificial lectin contained in the lysate. It can be seen that quantitative measurement can be performed using lysate.
[0078]
The measurement was performed by the Reverse @ cell-ELISA method using human erythrocytes, and the binding of human erythrocytes to each artificial lectin was confirmed (FIG. 12). Although the binding of each lectin to sialyl T present on the surface of human erythrocytes and the binding to human erythrocytes did not completely correlate, this is because sugar chains other than sialyl T that can be recognized by each lectin are expressed on the erythrocyte surface. This is probably because you are doing it. Such comparison can be performed because the Reverse @ cell-ELISA method can be used for quantitative discussion even using a lysate, and the Reverse @ cell-ELISA method will be useful for actual cell sorting and identification in the future. It is expected.
[0079]
In the same manner as for human erythrocytes, measurement was also performed on the four different animal erythrocytes by the Reverse @ cell-ELISA method. As a result of the measurement, a binding pattern corresponding to each animal red blood cell was successfully drawn (FIG. 13). In this measurement, red blood cells of human type A, chicken, horse, and rabbit showed a somewhat similar binding pattern, while bovine red blood cells showed a distinctly different binding pattern. It can be seen that these red blood cells can be identified by such a method. In particular, bovine erythrocytes are clearly discernable.
[0080]
[Experimental Example 4 Addition of tag such as recovered and modified lectin]
In this experimental example, an artificial lectin was produced by modifying the amino acid of loop D of MAH, and an artificial lectin with a tag was attached to the artificial lectin. This tagged artificial lectin can be immobilized in the same manner as in the method of Experimental Example 3 above.
[0081]
[Elongation of loop D]
Loop D was extended by PCR using MAH cDNA as a template (FIG. 14). Specifically, wild-type MAH gene (2 μl), insert fragment preparation primers (2 types) (1 μl each (100 pmol each)), 10 × dNTP (10 μl), 10 × PCR @ Buffer (10 μl), Milli-Q (75 μl) ), Keep the mixture of ampli @ Taq Gold (1 μl) at 95 ° C. × 9 minutes, 30 cycles of (94 ° C. × 1 minute, 54 ° C. × 2 minutes, and 72 ° C. × 2 minutes) and 72 After holding at 10 ° C for 10 minutes, it was cooled to 4 ° C. (4) After completion of PCR, DpnI was added in an amount of 1 μl each, and reacted at 37 ° C. for 2 hours. The reaction product was collected by PCR @ Purification @ Kit (manufactured by QIAGE). The product was obtained from the restriction enzyme XhoI. conc and BglII. conc. Then, the reaction product was recovered again by PCR @ Purification @ Kit (manufactured by QIAGE) (D-modified lectin).
[0082]
[Table 14]

[0083]
[Tag Vectors]
Here, pFLAG-ATS was used to add a tag to the N-terminus of the base sequence of the lectin in which loop D was modified by the method described above. Since the multicloning site of this vector did not have a convenient restriction enzyme site for incorporating MAH, BglIIsite was changed to SpeIsite according to the protocol of Site-Directed Mutagenesis. First, a primer containing a site to be modified was designed to be completely complementary on the sense side and the anti side. Next, PCR was performed, 1 ml of DpnI was added, and the mixture was incubated at 37 ° C. for 1 hour. Using this DNA solution as it was, it was transformed into XL1-Blue. Finally, the resulting colonies were short-cultured to recover the plasmid, and the DNA sequence was read using BigDye Terminator Cycle Sequencing to identify the modified pFLAG-ATS. At this time, the composition of the PCR reaction solution was [template (pFLAG-ATS) 1 μl, primer (pFLAG-SpeI-sense 100 ng / ml, pFLAG-SpeI-anti @ 100 ng / ml) 1.25 ml each, 10 × PCR @ buffer @ 5 ml, dNTP 1 ml, MilliQ 40.5 ml, pfu turbo 1 ml]. The reaction conditions of the PCR were [95 ° C. 30 sec, 12 cycles @ of (95 ° C. 30 sec, 55 ° C. 1 min, 68 ° C. 10 min)]. Table 15 shows the sequences of the primers.
[0084]
[Table 15]

[0085]
[Transfer vector]
The D-modified lectin and wild-type MAH cDNA were amplified by PCR from pFLAG-CTS, subjected to restriction digestion (XhoIconc. And SpeIconc. (Roche)), and incorporated into a modified pFLAG-ATS. This was transformed into JM109, and the resulting clone was identified by BigDye \ Terminator \ Cycle \ Sequencing. At this time, the composition of the PCR reaction solution was as follows: [template = 0.5 ml, primer (N-Flag-XhoI 100 ng / ml, MAH-SpeI-anti 100 ng / ml) each 0.5 ml, dNTP 4 ml, 10 × PCR buffer 5 ml, TaqGold 1 ml, MilliQ 38.5 ml]. The reaction conditions of the PCR were general conditions [96 ° C. for 5 min, 30 cycles @ of (96 ° C. for 1 min, 55 ° C. for 1 min, 72 ° C. for 2 min), 72 ° C. for 5 min]. Table 16 shows the sequences of the primers.
[0086]
[Table 16]

[0087]
[Changes of BigDye \ Terminator \ Cycle \ Sequencing Protocol]
Here, the existing protocol was modified. The composition of the PCR reaction solution was changed from 5 × Sequencing Buffer 2 ml to 10 × PCR Buffer 2 ml, and the solvent for dissolving the sequence sample prepared by ethanol precipitation was changed from Hi-Di {formamide} 20 ml to MilliQ 20 ml.
[0088]
The Escherichia coli thus obtained was isolated by HEM (Highly Enriched Medium) containing carbenicillin and subjected to small-culture (over @ night). The next day, 20 ml of HEM, CaCl₂(1M) 20ml, MnCl₂(1M) 20ml, MgCl₂200 ml of a small-cultured E. coli solution was added to a medium consisting of 81.7 ml of (4.9 M) and pre-cultured for 3 hours. 200 ml of 100 mM @IPTG was added thereto, and the mixture was cultured for 3 hours to induce induction. Next, E. coli was recovered by centrifugation at 9,500 rpm for 10 minutes, suspended in 200 ml of TBS, and stored at -80 ° C. At a later date, E. coli was repeatedly frozen (liquid nitrogen) and thawed (bath at 37 ° C.) five times, centrifuged at 15,000 rpm for 20 minutes, and the supernatant was collected as a lysate. The lysate was stored at −80 ° C. after protein quantification, and after preparation for use, diluted to a protein concentration of 1 mg / ml and stored at 4 ° C.
[0089]
[SDS-PAGE & Western Blotting]
SDS-PAGE and Western @ Blotting were performed to examine whether a flag with D-modified lectin (modified FLAG-MAH) was present in the lysate. In the antibody staining, anti-MAH rabbit polyclonal antibody (prepared in our room) or anti-FLAG M2 monoclonal antibody was used as a primary antibody, and AP-goat anti-rabbit IgG antibody and AP-goat anti-antibody were used as secondary antibodies, respectively. Color was developed using ABC @ Kit using a mouse @ IgG antibody and confirmed. Thus, a tagged D-modified lectin is obtained, and can be immobilized by the same method as described above.
[0090]
[Immobilization of tagged D-modified lectin]
50 ml of the diluted anti-FLAG M2 monoclonal antibody was added to a SUMILON H type plate (0.25 mg / well) and allowed to stand at 4 ° C overnight. The next day, the plate was washed three times with TBS and then blocked with 3% BSA / TBS @ 170 ml for 3 hours. This was washed three times with TBS, and the diluted lysate was added in an amount of 50 ml each and shaken for 2 hours. This results in the immobilization of the tagged D-modified lectin on this type plate. Next, after washing with TBST, the biotinylated saccharide polymer was diluted, added in 50 ml portions (0.2 mg / well), and shaken for 2 hours. After washing with TBST, Streptavidin-HRP @ Conjugate diluted 1000-fold was added in 50 ml portions and shaken for 30 minutes, and ABTS / H₂O₂$ 50ml for color development and OD₄₀5/490^{Was measured, and it was confirmed that the D-modified lectin was fixed.}
[0091]
[Application to identification of IgA glycoform]
Hereinafter, an example in which the lectin immobilization method of the present invention is applied will be described. To apply the modified lectin library to the IgA glycoform identification method, (1) create a modified lectin library. (2) A lectin having a high affinity for IgA nephropathy patient IgA is selected by a panning method, and a lectin sub-library is prepared. (3) From the lectin sub-library obtained in the above (2), a modified lectin which reflects the difference between IgA of IgA nephropathy patients and healthy individuals is selected and tagged as required. (4) This modified lectin with a tag is immobilized on a microtiter plate by the method of the present invention to prepare a lectin plate (FIG. 15). (5) Discrimination of IgA glycoform by lectin library Comparison and analysis of binding patterns of serum IgA to lectin plate between IgA nephropathy patients and healthy persons are performed. (6) Examination of serum diagnostic assay conditions A commercially available healthy human IgA is enzymatically and chemically added with a sugar chain to produce an artificial IgA. Artificial IgA is mixed with healthy human serum, and pattern analysis is performed using a lectin plate. Investigate the effects of other serum proteins present in serum and optimize assay conditions.
[0092]
[Application to identification method of osteoblast subpopulation]
First, (1) a modified lectin library is prepared. Next, (2) Preparation of lectin library A lectin having high affinity for osteoblasts is selected by a panning method or the like, and a lectin sublibrary is prepared. At this time, a tag sorting function can be used. (3) From the lectin sub-library obtained in (2) above, select a lectin that reflects the stage of differentiation well if necessary, attach it to a tag and immobilize it on a microtiter plate by the method of the present invention. Make a plate. (4) Induction of differentiation of osteoblasts and discrimination by lectin library Cultured and separated mesenchymal stem cells are cultured. More specifically, after culturing the mesenchymal stem cells, the cells are differentiated into osteoblasts, and the cells on the fifth, tenth, fifteenth, and twenty days from the start of the differentiation are separated. (5) The cells separated at each point in the differentiation process are analyzed on a lectin plate, and the correlation between the cell surface sugar chain structure and the bone forming ability is examined. For the measurement of bone shape performance, bone type alkaline phosphatase activity is measured and osteocalcin content is measured (preparation of standard). That is, the isolated cells were implanted subcutaneously at the back of the rat in the form of a complex of osteoblasts with a b-TCP block serving as a scaffold, and excised at 4 weeks and 8 weeks after implantation. ii) Bone alkaline phosphatase activity is measured. (6) The cells whose differentiation process is unknown are analyzed on a lectin plate and compared with a standard (see FIG. 16).
[0093]
Here, the mesenchymal stem cells are the stem cells that are cells that grow into tissues and organs and are present in the bone marrow, and the mesenchymal stem cells are bone, cartilage, fat, heart, and nerve. It has been confirmed to differentiate into cells such as the liver and the like, and has a capability close to that of embryonic stem cells (ES cells) that can also differentiate into almost all tissues. The mesenchymal stem cells are used as a scaffold for culturing an artificial material having excellent affinity, absorbability and osteoconductivity in a living body, which is mainly composed of calcium phosphate called β-triccalium phosphate (β-TCP).
[0094]
【The invention's effect】
According to the present invention as described above, a lectin can be immobilized on a support without inhibiting a sugar chain recognition site that exerts a main function in sugar chain analysis, and the efficiency of sugar chain analysis is improved. In addition, there is an advantage that the lectin can be immobilized under conditions different from the sugar chain analysis conditions. In addition, the concentration of the lectin for sugar chain analysis and the concentration of the sugar chain analysis site can be made uniform. Furthermore, there is an advantage that lectin can be directly fixed, particularly from a lysate containing lectin and other substances.
[0095]
[Sequence list]

[Brief description of the drawings]
FIG. 1. Amino acid sequences of various lectins.
FIG. 2 is a diagram showing a nucleotide sequence and a predicted amino acid sequence of MAH.
FIG. 3 is a prediction diagram of a three-dimensional structure of MAH.
FIG. 4 is a conceptual diagram showing a tool using a lectin fixing method according to one embodiment of the present invention.
FIG. 5 is a diagram showing a method for genetically modifying MAH lectin.
FIG. 6 is a view showing an amino acid sequence (C loop) of a modified MAH lectin recovered by panning with Caco2.
FIG. 7 is a view showing an amino acid sequence of a lectin modification site recovered by panning using human erythrocytes.
FIG. 8 is a diagram showing an example of confirmation of a FLAG fusion lectin gene by treatment with a restriction enzyme.
FIG. 9 shows the confirmation of FLAG fusion lectin expression by Western blotting.
FIG. 10 is a diagram showing a color development state corresponding to the amount of each artificial lectin bound to an anti-FLAG antibody.
FIG. 11 is a diagram showing a color development state corresponding to the binding activity of each lectin bound to an anti-FLAG antibody to sialyl-T.
FIG. 12 is a view showing the results of measurement of human erythrocytes by Reverse Cell-ELISA.
FIG. 13 is a diagram showing the results of measurement of various erythrocytes by the Reverse Cell-ELISA method.
FIG. 14 is a view showing the position of amino acid insertion into loop D of MAH lectin.
FIG. 15 is a diagram showing an example of a lectin chip and a method of using the same (for IgA).
FIG. 16 is a diagram showing an example of a lectin chip and a method of using the same (for osteoblast differentiation profiling).
[Explanation of symbols]
Example of 10 sugar chain analysis tool
12 ELISA plate
14 anti-FLAG antibody
16 FLAG fused artificial lectin
18 sugar chain recognition site
20 Cells to be analyzed
22 cell surface sugar chains

Claims (8)

An immobilization method for immobilizing a lectin on a support,
Encoding the lectin and tag peptide into the vector DNA;
Using the encoded DNA to make an LT complex protein;
Contacting the LT protein with a support on which an anti-tag antibody having binding or association with the tag peptide is immobilized,
A method for immobilizing a lectin, comprising: The lectin immobilization method according to claim 1, wherein the lectin is a legume lectin. The method for immobilizing a lectin according to claim 1 or 2, wherein a modified lectin in which one or more amino acids of loop C or loop D is modified in the lectin is encoded in the vector DNA. The method for immobilizing a lectin according to any one of claims 1 to 3, wherein an amino acid sequence obtained by adding the tag peptide to a C-terminal region or an N-terminal region is encoded in the vector DNA. A sugar chain analysis tool comprising a lectin-immobilized support by the lectin immobilization method according to claim 1. A lectin to which a tag / peptide has been added by a genetic modification operation,
And a solid support containing an anti-tag antibody. A sugar chain analysis tool comprising the lectin-immobilized support according to claim 6. A tag antibody vector used for adding the tag peptide according to any one of claims 1 to 7 to a lectin.

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