JP2004000101A - Method for examining therapeutic agent or prophylactic for disease such as hyperlipemia - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new method for examining a therapeutic agent or a prophylactic for one or more diseases selected from hyperlipemia, arteriosclerosis and hyperglycemia. <P>SOLUTION: The method is a method for culturing a cell culture in the presence or absence of a substance to be examined, detecting the amount of mRNA of "an angiopoietin-related protein 3 or 4" gene expressed or promoter activity and selecting the substances to be examined having the detected amount of the mRNA expressed or promoter activity lower in the cell cultured in the presence of the substance to be examined than in the cell cultured in the absence of the substance to be examined. The method for examining a substances to regulate the activity of LPL and/or HTGL is a method for making "the angiopoietin-related protein 3 or 4" coexist together with the substance to be examined in a system in which LPL and/or HTGL is reacted with its substrate and then examining whether the substance to be examined controls activity inhibition of LPL and/or HTGL by the protein or not. <P>COPYRIGHT: (C)2004,JPO

Description

参考例４．　ベザフィブレート投与による影響
１１週齢の雄Ｚｕｃｋｅｒ　ｆａｔｔｙラット（日本チャールズリバーより購入）２匹に７日間、高脂血症治療剤ベザフィブレート（シグマ社製）をＦ２粉末飼料（船橋農場社製）に０．３％の濃度で混ぜて投与した。対照群のラット（２匹）にはＦ２粉末飼料のみを与えた。両方の条件のラットからそれぞれ血液サンプルを回収した後、肝臓を摘出し、直ちに液体窒素内に入れ急凍した後、−８０℃に保存した。得られた肝臓組織０．１ｇに対し５ｍｌのＴＲＩｚｏｌ試薬（ギブコ・ビーアールエル社製）を加え、超高速ホモジナイザー（ポリトロン、キネマティカ社製）を用いて氷上でホモジナイズを行った（目盛１０で２分間）。これを室温で５分間静置した後、１ｍｌのクロロホルムを加え、１５秒間激しく転倒混和した。再び室温で１０分間静置してから、１２０００×ｇ、室温で１０分間遠心分離した。遠心後、上層を回収し、２．５ｍｌのリボヌクレアーゼ不含イソプロピルアルコールを加えて混和した。これを室温で１０分間静置後、１２０００×ｇ、室温で１０分間遠心分離した後、上清を除去してリボヌクレアーゼ不含７０％エタノールを加えた。これを１２０００×ｇ、室温で５分間遠心分離し、上清を除去して、沈殿を乾燥させてから、−８０℃に保存した。得られたペレットをリボヌクレアーゼを含まない純水に溶解したものを全ＲＮＡ溶液とした。
【０４００】
一本鎖ｃＤＮＡの合成は以下の手順で行った。得られた全ＲＮＡ１μｇにリボヌクレアーゼを含まない純水を加え全量を６μｌとし、１０×ＤＮＡａｓｅ　反応緩衝液（ギブコ・ビーアールエル社製ＤＮａｓｅＩに添付）を１μｌ、ＤＮａｓｅＩ（Ａｍｐグレード、ギブコ・ビーアールエル社製）を１μｌ加え、室温で１５分間保温した後、２５ｍＭ　ＥＤＴＡ（ＤＮａｓｅＩに添付）を１μｌ加え、６５℃で１０分間保温した。この溶液に、一本鎖ｃＤＮＡ合成キット（ＳｕｐｅｒＳｃｒｉｐｔ　ｆｉｒｓｔ−ｓｔｒａｎｄ　ｓｙｎｔｈｅｓｉｓ　ｓｙｓｔｅｍ　ｆｏｒ　ＲＴ−ＰＣＲ、ギブコ・ビーアールエル社製）添付のランダムヘキサマープライマーを１μｌ、１０ｍＭ　ｄＮＴＰ　ｍｉｘを１μｌ加え、６５℃、５分間保温した後、急激に４℃まで冷却し熱変性を行った。引き続き、ｃＤＮＡ合成キット添付の１０×逆転写反応緩衝液を２μｌ、２５ｍＭ　塩化マグネシウムを４μｌ、０．１Ｍ　ジチオスレイトール溶液を２μｌ、リボヌクレアーゼインヒビターを１μｌ加え、２５℃で２分間保温し、その後、添付の逆転写酵素を１μｌ　添加した。このサンプルを２５℃で１０分保温後、４２℃で５０分保温した。さらに７０℃で１５分間保温した後、急激に４℃まで冷却し熱変性を行った。添付のリボヌクレアーゼＨを１μｌ加え、３７℃で２０分間保温してＲＮＡを分解し、−２０℃にて保存した。反応終了後、すべてのサンプルに２９μｌのリボヌクレアーゼを含まない純水を加え、さらにリボヌクレアーゼを含まない純水で５倍希釈したものを下記の定量に用いた。検量線作成用一本鎖ｃＤＮＡ溶液は、対照サンプルの１例の定量用溶液よりリボヌクレアーゼを含まない純水にて５倍ずつの希釈系列を作成することにより調製した。
【０４０１】
一方、ＴａｑＭａｎ　ＰＣＲに用いるプライマーとして、下記の配列：
５’−　ｃａａａｇｃｔｔｇａ　ａａｇｔｃｔａｃｔｇ　ｇａｇｇ　−３’　（配列表の配列番号３０）；および
５’−　ｇｇｔｔｃｔｇａａｃ　ｃａａｇｃｔｇｇｔｃ　ａ　−３’　　（配列表の配列番号３１）
を有するオリゴヌクレオチドを合成した（アマシャム・ファルマシア社に発注）。
【０４０２】
さらに、ＴａｑＭａｎ　ＰＣＲに用いるプローブとして、下記の配列：
５’−　ａａｇａｔｇｇｃｇｃ　ｔｃｃａａｃａｃａｇ　ａｇｔｃａｇ　−３’　（配列表の配列番号３２）
を有し、その５’末端にレポーター色素Ｆａｍ、３’末端にレポーター消光体のＴａｍｕｒａを結合したオリゴヌクレオチドを合成した（ＴａｑＭａｎ　フルオレセント・プローブ、アプライドバイオシステムズジャパン社製）。また「アンジオポエチン関連タンパク質３」の発現量の標準化をはかる目的で、ラット１８ｓリボゾームＲＮＡの定量を行うこととし、ＴａｑＭａｎ　リボソーマルＲＮＡコントロール・リージェント、アプライドバイオシステムズジャパン社製）を用いた。
【０４０３】
これらの材料を用いて、ＴａｑＭａｎ　ＰＣＲ法による、ラット「アンジオポエチン関連タンパク質３」遺伝子の発現量の定量を行った。ＰＣＲ反応は９６穴反応プレート（マイクロアンプ・オプチカル・９６ウェル・リアクション・プレート、アプライドバイオシステムズジャパン社製）中で行った。反応溶液の組成は１ウェル中、上記方法で得られた一本鎖ｃＤＮＡ溶液５μｌ、ＴａｑＭａｎＰＣＲ用プライマー（５０ｐｍｏｌ／μｌ）を正方向側、逆方向側ともに０．２μｌ（最終濃度２００ｎＭ）、ＴａｑＭａｎ　ＰＣＲ用プローブ（６．６μＭ）０．７５μｌ（最終濃度１００ｎＭ）、ＰＣＲ増幅用混合液（ＴａｑＭａｎ　ユニバーサル・ＰＣＲマスター・ミックス、アプライドバイオシステムズジャパン社製）２５μｌ、超純水１８．８５μｌとした。この際、検量線作成用一本鎖ｃＤＮＡ溶液は原液濃度を便宜的に「６２５」とし、以降５倍希釈を繰り返して濃度「６２５」、「１２５」、「２５」、「５」および「１」の溶液について同様に反応液を調製した。ＰＣＲ反応はＴａｑＭａｎ　ＰＣＲ専用サーマルサイクラー・検出器（ＡＢＩ７７００、アプライドバイオシステムズジャパン社製）を用いて行った。反応は、５０℃で２分間、９５℃で１０分間保温の後、９５℃で１５秒間、６０℃で１分間の反応を４０回繰り返し、１サイクルごとにレポーター色素の発光量を測定した。各サイクルごとのレポーター色素の発光量から１８Ｓリボゾーム、「アンジオポエチン関連タンパク質３」の増幅曲線を作成した。検量線作成用一本鎖ｃＤＮＡ溶液の希釈系列の増幅曲線から横軸に濃度、縦軸にサイクル数をとった検量線を作成し、各発現定量用サンプルについてはその対数増幅期において任意に設定した一定の発光量を超えたサイクル数を検量線上にプロットし、相対的な発現量を算出した。「アンジオポエチン関連タンパク質３」遺伝子の発現量は同一サンプルにおける１８Ｓリボゾームの発現量の値で補正を行った。
【０４０４】
その結果、ラット「アンジオポエチン関連タンパク質３」遺伝子の発現量は、ベザフィブレート投与群において、コントロール群に比べ７３％低下していることが示された。なお、この時用いられたベザフィブレート投与群のラットの血中中性脂肪濃度は、コントロール群に比べて５０％減少していた。
【０４０５】
参考例５．　動脈硬化および血糖値への影響
低脂血症マウスＫＫ／Ｓｎｋは配列表の配列番号２に示されるアミノ酸配列からなるポリペプチドをコードする遺伝子の変異型（以下「低脂血症変異遺伝子」という）をホモで有し、該ポリペプチドの産生量が先天的に低い。以下に記載する方法に従って動脈硬化モデルマウスまたは高血糖モデルマウスにおける低脂血症変異遺伝子の影響を検討した。
【０４０６】
１）　コンジェニックマウスの作出
ａ）交配様式
ＫＫ／Ｓｎｋマウスの雌をＣ５７ＢＬ／６Ｊマウスの雄（浜松医科大学附属動物実験施設より入手）と交雑して、戻し交雑第１世代（Ｎ１世代）を作出した。次いで、Ｎ１雌をＣ５７ＢＬ／６Ｊ雄に再度交雑してＮ２世代を得た。このＮ２個体の尾からゲノムＤＮＡを抽出し、後述ｃ）記載の方法でＰＣＲを行い、低脂血症変異遺伝子をヘテロで持つ個体を選抜した。選抜した雌をさらにＣ５７ＢＬ／６Ｊ雄と交雑して、Ｎ３世代を作出した。その後、同様の方法でＮ１０世代まで連続して選抜交雑を実施した。次いで、Ｎ１０世代のヘテロの雌雄を交雑し、得られた産児から採取したゲノムについて後述ｃ）記載の方法でＰＣＲを実施して、低脂血症変異遺伝子をホモで持つ雌雄を選抜した。その後、兄妹交配により継代し、Ｃ５７ＢＬ／６Ｊ背景の低脂血症変異コンジェニックマウス（Ｃ５７ＢＬ／６Ｊ−低脂血症）系統を得た。いずれの動物も温度２３±１℃、湿度５５±１０％、１４時間照明の動物室内で飼育し、ＣＭＦ固形飼料（オリエンタル酵母工業（株）製）と７ｐｐｍクロール水を自由摂取させた。
【０４０７】
ｂ）ゲノムＤＮＡの抽出方法
上記ａ）で選抜に用いたマウス（４週齢）より採取した尾（１．５ｃｍ）を８４０μｌの溶解液（７２０μｌの１×ＳＳＣ、８０μｌの１０％　ＳＤＳ、４０μｌの１０ｍｇ／ｍｌ　プロテイナーゼＫを含む）に浸漬し、５０℃で保温しながら一晩振盪した。次いで、１ｍｇ／ｍｌ　リボヌクレアーゼＡを２０μｌ加えて、５０℃で１時間保温した。その後、フェノール・クロロホルム抽出を２回、エタノール沈殿操作を１回行い、沈殿を１０ｍＭ　トリス−塩酸（ｐＨ７．５）、１ｍＭ　ＥＤＴＡを含む緩衝液１５０μｌに溶解した。その後、分光光度計（Ｕ−３０００、（株）日立製作所製）で２６０ｎｍ波長における吸光度を測定し、滅菌水を加えて濃度を２５ｎｇ／μｌに調整してゲノムＤＮＡ試料とした。
【０４０８】
ｃ）遺伝子型の判定
２種類のマイクロサテライトマーカー検出用プライマーセット（Ｄ４Ｍｉｔ１５およびＤ４Ｍｉｔ２１９、リサーチジェネティクス社製）を用い、サーマルサイクラー（ＰＴＣ−１００、エムジェーリサーチ社製）を使用して以下の条件でＰＣＲを行った。１．５μｌの１０×ＰＣＲバッファー［１００ｍＭ　トリス−塩酸（ｐＨ８．３）、１５ｍＭ　塩化マグネシウム、５００ｍＭ　塩化カリウムを含む。ロシュ・ダイアグノティクス社製］、２．４μｌの各１．２５ｍＭ　ｄＮＴＰｓ（宝酒造（株）社製）、各０．４５μｌの６．７μＭ　プライマー、０．３５ＵのＴａｑ　ＤＮＡポリメラーゼ（ロシュ・ダイアグノティクス社製）に滅菌水を加えて１１μｌとした後、上記ｂ）で調製したゲノムＤＮＡを４μｌ加え、反応液を調製した。この反応液を、まず９４℃で３分間加熱した後、９４℃で３０秒、５５℃で１分、７２℃で４５秒の温度サイクルを３５回繰り返し、さらに７２℃で３分保持してから、４℃で保温した。
【０４０９】
このＰＣＲ後の反応液に色素液（０．１５％　ブロモフェノールブルー、５０％グリセロール）を２μｌ加え、そのうちの５μｌを４％のアガロースゲル（ＮｕＳｉｅｖｅ　３：１アガロース（ＦＭＣバイオプロダクツ社製）で調製した）で１５０Ｖ、室温で１．５時間乃至２時間、電気泳動した。電気泳動後のゲルを臭化エチジウムで染色した後、紫外線照射下で目的のＤＮＡに相当するバンドを確認した。プライマーセットＤ４Ｍｉｔ１５で約２８０ｂｐと約３００ｂｐの２種類の断片が増幅され、かつＤ４Ｍｉｔ２１９で約１１０ｂｐと約１２０ｂｐの２種類の断片が増幅されたゲノムを有する個体を「低脂血症変異遺伝子へテロ」、Ｄ４Ｍｉｔ１５で約３００ｂｐの断片のみが増幅され、かつＤ４Ｍｉｔ２１９で約１２０ｂｐの断片のみが増幅されたゲノムを有する個体を「低脂血症変異遺伝子ホモ」とした。
【０４１０】
２）低脂血症変異遺伝子ホモ／ＡｐｏＥホモ欠損個体の作出
ａ）選抜様式
上記１）のａ）で得られたＣ５７ＢＬ／６Ｊ−低脂血症マウスとアポリポタンパク質Ｅ（以下「ＡｐｏＥ」という）ホモ欠損マウス（Ｚｈａｎｇ，　Ｓ．Ｈ．，　ｅｔ　ａｌ．　（１９９２）　Ｓｃｉｅｎｃｅ　２５８，　４６８−４７１、東京大学医学部器官病態内科学教室より入手）との交雑から交雑第１世代（Ｆ１世代）を作出した。次いで、Ｆ１世代の雌雄を交雑してＦ２世代を得た。Ｆ２世代のうち、ＡｐｏＥをホモで欠損し、低脂血症変異遺伝子をホモで持つ個体（以下「ＡｐｏＥ　ＫＯ−低脂血症マウス」という）と低脂血症変異遺伝子を持たないＡｐｏＥホモ欠損個体（以下「ＡｐｏＥ　ＫＯマウス」という）を下記の方法で遺伝子型を判定することにより選抜した。なお、いずれの動物も上記１）のａ）記載の方法で飼育した。
【０４１１】
ｂ）遺伝子型の判定方法
上記１）のｂ）記載の方法でマウス尾よりゲノムＤＮＡを抽出した。低脂血症変異遺伝子を選抜するために、変異型アレルと野生型アレルにおいてサイズ多型を検出するオリゴヌクレオチドプライマー：
５’−　ｇｇｃｔａａａｔａｇ　ｔａａａａｃｃｃｔｇ　ｇｃｇ　−３’　　（配列表の配列番号３３）；および
５’−　ｇｔｇｃｔｔｇｃｔｇ　ｔｃｔｔｔｃｃａｇｔ　ｃｔｔ　−３’　　（配列表の配列番号３４）
を合成した。このプライマーセットを用いてＰＣＲを実施すると、低脂血症変異遺伝子では約２４０ｂｐの断片が増幅され、一方変異のない正常遺伝子では、約２３０ｂｐの断片が増幅される。
【０４１２】
また、ＡｐｏＥ欠損を検出するために、欠損型アレル（ネオマイシン耐性遺伝子）を特異的に増幅するための、下記の配列からなるオリゴヌクレオチドプライマー：
５’−　ａｇｇａｔｃｔｃｇｔ　ｃｇｔｇａｃｃｃａｔ　ｇｇｃｇａ　−３’　　（配列表の配列番号３５）；および
５’−　ｇａｇｃｇｇｃｇａｔ　ａｃｃｇｔａａａｇｃ　ａｃｇａｇｇ　−３’　（配列表の配列番号３６）
（Ｇａｗ，　Ａ．，　ｅｔ　ａｌ．　（１９９５）　Ｌａｂ．　Ａｎｉｍ．　２９，　４４７−４４９参照）
をそれぞれ合成した。このプライマーセットを用いてＰＣＲを実施すると、鋳型となる遺伝子にＡｐｏＥ欠損変異がある場合のみ２００ｂｐの断片が増幅される。
【０４１３】
さらに、ＡｐｏＥ欠損のない野生型アレルを特異的に増幅するための、下記の配列からなるオリゴヌクレオチドプライマー：
５’−　ｔｃｃｃａａｇｔｃａ　ｃａｃａａｇａａｃｔ　ｇａｃ　−３’　　（配列表の配列番号３７）；および
５’−　ｃａｔｃｃａｇａａｇ　ｇｃｔａａａｇａａｇ　ｇｃａ　−３’　　（配列表の配列番号３８）
をそれぞれ合成した。このプライマーセットを用いてＰＣＲを実施すると、鋳型となる遺伝子にＡｐｏＥ欠損変異がない場合のみ１７４ｂｐの断片が増幅される。
【０４１４】
上記の３セットのプライマーを用いて、上記１）のｃ）に記載した通り、ＰＣＲと電気泳動を実施し、増幅された断片を調べた。その結果、約２４０ｂｐおよび２００ｂｐの断片が検出された個体を「ＡｐｏＥ　ＫＯ−低脂血症マウス」とし、約２３０ｂｐおよび２００ｂｐの断片が検出された個体を「ＡｐｏＥ　ＫＯマウス」として選抜した。
【０４１５】
３）低脂血症変異遺伝子ホモ／レプチン変異遺伝子ホモ個体の作出
ａ）選抜様式
上記１）のａ）で得られたＣ５７ＢＬ／６Ｊ−低脂血症マウスとレプチン変異遺伝子（Ｌｅｐ^ｏｂ）をヘテロで持つＣ５７ＢＬ／６Ｊ−Ｌｅｐ^ｏｂマウス（Ｃｏｌｅｍａｎ，　Ｄ．　Ｌ．　ａｎｄ　Ｈｕｍｍｅｌ，　Ｋ．　Ｐ．　（１９７３）　Ｄｉａｂｅｔｏｌｏｇｉａ　９　：２８７−２９３．　浜松医科大学附属動物実験施設より入手）との交雑から交雑第１世代（Ｆ１世代）を作出した。次いで、Ｆ１世代の雌雄を交雑してＦ２世代を得た。Ｆ２世代のうち、レプチン変異遺伝子と低脂血症変異遺伝子をともにホモで持つ個体（以下「ｏｂ−低脂血症マウス」という）と低脂血症変異遺伝子を持たないレプチン変異ホモ個体（以下「ｏｂマウス」という）を下記の方法で遺伝子型を判定することにより選抜した。なお、いずれの動物も上記１）のａ）記載の方法で飼育した。
【０４１６】
ｂ）遺伝子型の判定方法
上記１）のｂ）記載の方法でマウス尾よりゲノムＤＮＡを抽出した。レプチン変異を検出するために、変異型アレルを特異的に増幅するための、下記の配列からなるオリゴヌクレオチドプライマー：
５’−　ｔｇａｃｃｔｇｇａｇ　ａａｔｃｔｃｔ　−３’　（配列表の配列番号３９）；および
５’−　ｃａｔｃｃａｇｇｃｔ　ｃｔｃｔｇｇｃ　−３’　（配列表の配列番号４０）；
（Ｎａｍａｅ，　Ｍ．　ｅｔ　ａｌ．　（１９９８）　Ｌａｂ．　Ａｎｉｍ．　Ｓｃｉ．　４８，　１０３−１０４参照）
をそれぞれ合成した。このプライマーセットを用いてＰＣＲを実施すると、鋳型となる遺伝子に変異型アレルがある場合のみ約１００ｂｐの断片が増幅される。
【０４１７】
さらに、レプチン変異のない野生型アレルをを特異的に増幅するための、下記の配列からなるオリゴヌクレオチドプライマー：
５’−　ｔｇａｃｃｔｇｇａｇ　ａａｔｃｔｃｃ　−３’　（配列表の配列番号４１）；および
５’−　ｃａｔｃｃａｇｇｃｔ　ｃｔｃｔｇｇｃ　−３’　（前出、配列表の配列番号４０）
をそれぞれ合成した。このプライマーセットを用いてＰＣＲを実施すると、鋳型となる遺伝子にレプチン変異のない場合のみ約１００ｂｐの断片が増幅される。
【０４１８】
上記の２セットのプライマーおよび上記２）のｂ）に記載した低脂血症変異遺伝子を選抜するための１セットのプライマーを用いて、上記１）のｃ）に記載した通り、ＰＣＲと電気泳動を実施し、増幅された断片を調べた。その結果、約１００ｂｐ（レプチン変異型）および約２４０ｂｐの断片が検出された個体を「ｏｂ−低脂血症マウス」とし、約１００ｂｐ（レプチン変異型）および約２３０ｂｐの断片が検出された個体を「ｏｂマウス」として選抜した。
【０４１９】
４）血清脂質と動脈硬化病変面積の測定
ＡｐｏＥ　ＫＯマウス（雄３匹、２２−２３週齢）およびＡｐｏＥ　ＫＯ−低脂血症マウス（雄３匹、１９−２３週齢）を、いずれも１７時間絶食させた後にエーテル麻酔下で腹大静脈より採血した。次いで血液を３０分放置した後、２０００×ｇ、室温で１０分間遠心分離を行い、血清を回収した。血清中の中性脂肪濃度についてはトリグリセリドＥ−テストワコー（和光純薬工業（株）社製）を、血清コレステロール濃度についてはコレステロールＣ−テストワコー（和光純薬工業（株）社製）をそれぞれ用いて測定した。
【０４２０】
また、エーテル麻酔下で、各マウスの胸部を切開し、左心室より１単位／ｍｌのへパリンを含む生理食塩液で洗浄した。心臓より大動脈弓部までを取り出し、心臓と大動脈弁部の分岐点が現れるまで脂肪を除去した。脂肪除去後、大動脈直上で切断し、４％中性ホルマリン溶液中で２４時間乃至７日間浸漬固定した。固定後、常法にしたがってパラフィンブロックを作製した。このパラフィンブロックを大動脈弓部側からミクロトームで薄切して、切片に初めて弁が現れてから、５μｍの厚さで連続５枚の切片を採取し、その次の５枚を捨てた。これを１単位として、この方法で全長５００μｍまで薄切した。得られた切片には、ＥｌａｓｔｉｃａＭａｓｓｏｎ染色を施した。
【０４２１】
ＣＣＤカメラ（ＨＣ−２０００、富士写真フィルム（株）社製）を搭載した光学顕微鏡（ＢＸ−５０、オリンパス光学工業（株）社製）下において、ＥｌａｓｔｉｃａＭａｓｓｏｎ染色を施した大動脈弁部横断像をテレビモニター上に映し出し、画像解析プログラム（ＱＷｉｎ、ライカ社製）を用いて病変の境界をトレースし、病変面積を計測した。各個体とも厚さ５０μｍの間隔で合計１０枚の切片について病変面積を計測し、その総和を算出した。
【０４２２】
その結果を表２に示した。
【０４２３】
【表２】

以上の結果から、配列表の配列番号２に示されるアミノ酸配列からなるポリペプチドをコードする遺伝子の変異は、血清脂質の低下とともに動脈硬化病変の進展を抑制することが示された。
５）血糖値の測定
ＡｐｏＥ　ＫＯ（雄９匹、２４週齢）、ＡｐｏＥ　ＫＯ−低脂血症マウス（雄１０匹、２４週齢）、ｏｂマウス（雄１２匹、１７−１８週齢）、およびｏｂ−低脂血症マウス（雄１４匹、１７−１９週齢）を、いずれも１７時間絶食させた後にエーテル麻酔下で腹大静脈より採血した。次いで血液を３０分放置した後、２０００Ｇ、室温で１０分間遠心分離を行い、血清を回収した。血糖値はグルコースＢ−テストワコー（和光純薬工業（株）社製）を用いて測定した。
【０４２４】
ＡｐｏＥ　ＫＯおよびＡｐｏＥ　ＫＯ−低脂血症マウスの結果を表３に、ｏｂマウスおよびｏｂ−低脂血症マウスの結果を表４に示した。
【０４２５】
【表３】

【０４２６】
【表４】

以上の結果から、配列表の配列番号２に示されるアミノ酸配列からなるポリペプチドをコードする遺伝子の変異は、ＡｐｏＥ欠損あるいはレプチン変異による高血糖を有意に低下させることが示された。
【０４２７】
実施例７　モノクローナル抗体の調製
（ａ）　抗原蛋白質の調製
１）生産ＣＨＯ細胞株の作製
ｐＳＲαプロモーター下流（ｐＭＥ１８Ｓプラスミド）にヒトアンジオポエチン関連蛋白質３　ｃＤＮＡが位置する発現ベクターを作製した。この発現ベクターとｐＳＶ２−ｄｈｆｒ（ＡＴＣＣ）を１０％ＦＣＳ（ＧｉｂｃｏＢＲＬ社製）−ＭＥＭα（ＧｉｂｃｏＢＲＬ社製）−１０ｕｎｉｔｓ／ｍｌペニシリン−１０μｇ／ｍｌストレプトマイシン（ＧｉｂｃｏＢＲＬ社製）培地でジヒドロ葉酸還元酵素欠損ＣＨＯ細胞（ＡＴＣＣ）に共導入し、核酸除去培地において定常株を取得した。次に、本定常株をメトトレキセート０．０８μＭ濃度で生育可能な耐性株を取得し、遺伝子重複による発現量の増加が確認された株を取得した。以後、１．２８、３０、６０μＭのメトトレキセート入り核酸除去ＭＥＭα培地で生育可能な株を取得していき、最終的にコンフルエントな無血清培養の細胞培養用フラスコ上で、５乃至１０ｍｇ／ｌ／３日の生産能を有するアンジオポエチン関連蛋白質３生産ＣＨＯ細胞株を取得した。
このように作製したアンジオポエチン関連蛋白質３生産ＣＨＯ細胞株をコンフルエントまで培養後、無血清ＭＥＭα（ＧｉｂｃｏＢＲＬ社製）培地に交換し、３日間培養して無血清培養上清を得た。
２）脱塩、小分子除去
本サンプルをＳｅｐｈａｄｅｘ　Ｇ２５（アマシャム・ファルマシア・バイオテク社製）１．６リットルのゲルに対して６００ｍｌの割合で２０ｍｌ／ｍｉｎで１０回に分けて、脱塩、小分子を除去した。本サンプルをＦＰＬＣ（Ｂｉｏｐｉｌｏｔ　ｓｙｔｅｍ、アマシャム・ファルマシア・バイオテク社製）を用いたイオン交換クロマトグラフィーで分画した。
３）分画ＳｔｅｐＩ
カラム：Ｑｓｅｐｈａｒｏｓｅ　Ｆａｓｔ　Ｆｌｏｗ（アマシャム・ファルマシア・バイオテク社製）をＸＫ５０（アマシャム・ファルマシア・バイオテク社製）に１００ｍｌ充填した。
溶離緩衝液：
Ａ）２０ｍＭ　Ｔｒｉｓ−ＨＣｌ（ｐＨ７．５）−０．０１％　ＮａＮ３（ＳＩＧＭＡ社製）−０．００５％　Ｐｒｏｔｅａｓｅ　ｉｎｈｉｂｉｔｏｒ　ｃｏｃｋｔａｉｌ（ＳＩＧＭＡ社製）−０．００５％　Ｔｗｅｅｎ２０（ＳＩＧＭＡ社製）
Ｂ）２０ｍＭ　Ｔｒｉｓ−ＨＣｌ−１Ｍ　ＮａＣｌ（ｐＨ７．５）−０．０１％　ＮａＮ３（ＳＩＧＭＡ社製）−０．００５％　Ｐｒｏｔｅａｓｅ　ｉｎｈｉｂｉｔｏｒ　ｃｏｃｋｔａｉｌ（ＳＩＧＭＡ社製）−０．００５％　Ｔｗｅｅｎ２０（ＳＩＧＭＡ社製）
流速：１０ｍｌ／ｍｉｎ
フラクション溶液：１０ｍｌ／ｔｕｂｅ
温度：４℃
溶出条件：溶離液Ａ　　⇒　溶離液Ｂ（直線濃度勾配、６０分間）
得られたフラクションについて、各々０．３乃至０．４５Ｍ　ＮａＣｌ濃度で溶出されてくる画分を回収した後、一つの溶液にまとめた。まとめた溶液をサンプルとして、以下の条件で、ＦＰＬＣ（アマシャム・ファルマシア・バイオテク社製）を用いて、群特異的アフィニティークロマトグラフィーを以下の条件で実施した。
４）分画ＳｔｅｐＩＩ
カラム：Ａｆｆｉ　Ｇｅｌ　Ｂｌｕｅ（ＢｉｏＲａｄ）をＫＸ１６（アマシャム・ファルマシア・バイオテク社製）に１０ｍｌ充填した。
溶離緩衝液：
Ｃ）２０　ｍＭ　Ｔｒｉｓ−ＨＣｌ−０．５Ｍ　ＮａＣｌ（ｐＨ７．５）−０．０１％ＮａＮ３（ＳＩＧＭＡ社製）−０．００５％　Ｐｒｏｔｅａｓｅ　ｉｎｈｉｂｉｔｏｒ　ｃｏｃｋｔａｉｌ（ＳＩＧＭＡ社製）−０．００５％　Ｔｗｅｅｎ２０（ＳＩＧＭＡ社製）
Ｄ）２０　ｍＭ　Ｔｒｉｓ−ＨＣｌ−１Ｍ　ＮａＣｌ（ｐＨ７．５）−０．０１％ＮａＮ３（ＳＩＧＭＡ社製）−０．００５％　Ｐｒｏｔｅａｓｅ　ｉｎｈｉｂｉｔｏｒ　ｃｏｃｋｔａｉｌ（ＳＩＧＭＡ社製）−０．００５％　Ｔｗｅｅｎ２０（ＳＩＧＭＡ社製）
流速：１．５ｍｌ／ｍｉｎ
温度：４℃
サンプルをアプライ後、Ｃ）液を６０ｍｌを流す。
溶出条件：Ｄ）液を１００ｍｌ流し、一つの溶液として回収した。回収した溶液を等量（１００ｍｌ）のＡ）液と混濁し、作製した２００ｍｌの溶液をサンプルとして以下の条件で、ＦＰＬＣ（アマシャム・ファルマシア・バイオテク社製）を用いた群特異的アフィニティークロマトグラフィーを以下の条件で実施した。５）分画ＳｔｅｐＩＩＩ
カラム：Ｌｅｎｔｉｌ　ｌｅｃｔｉｎ　ｓｅｐｈａｒｏｓｅ^ＴＭ（アマシャム・ファルマシア・バイオテク社製）をＫＸ１６（アマシャム・ファルマシア・バイオテク社製）に１０ｍｌ充填した。
溶離緩衝液：
Ｃ）２０　ｍＭ　Ｔｒｉｓ−ＨＣｌ−０．５Ｍ　ＮａＣｌ（ｐＨ７．５）−０．０１％ＮａＮ３（ＳＩＧＭＡ社製）−０．００５％　Ｐｒｏｔｅａｓｅ　ｉｎｈｉｂｉｔｏｒ　ｃｏｃｋｔａｉｌ（ＳＩＧＭＡ社製）−０．００５％　Ｔｗｅｅｎ２０（ＳＩＧＭＡ社製））
Ｆ）２０　ｍＭ　Ｔｒｉｓ−ＨＣｌ−０．５Ｍ　ＮａＣｌ−０．３Ｍ　　Ｍｅｔｈｙｌｍａｎｎｏｐｙｒａｎｏｓｉｄｅ（ｐＨ７．５）−０．０１％　ＮａＮ３（ＳＩＧＭＡ社製）−０．００５％　Ｐｒｏｔｅａｓｅ　ｉｎｈｉｂｉｔｏｒ　ｃｏｃｋｔａｉｌ（ＳＩＧＭＡ社製）−０．００５％　Ｔｗｅｅｎ２０（ＳＩＧＭＡ社製））
流速：１ｍｌ／ｍｉｎ
温度：４℃
サンプルをアプライ後、Ｃ）液を５０ｍｌ流す。
溶出条件：Ｆ）液を５０ｍｌ流し、一つの溶液として回収した。
６）透析
回収した液をＤｉａｌｙｓｉｓ　ｔｕｂｉｎｇ−１−７／８　ｉｎ　Ｄｕａｍｅｔｅｒ（ＧｉｂｃｏＢＲＬ社製）に移し、Ｄｕｌｂｅｃｃｏ’ｓ　ＰＢＳ（−）（日水製薬社製）、０．０１％　ＮａＮ３（ＳＩＧＭＡ社製）−０．１％　Ｐｒｏｔｅａｓｅ　ｉｎｈｉｂｉｔｏｒ　ｃｏｃｋｔａｉｌ（ＳＩＧＭＡ社製）溶液２リットルで一晩、４℃で透析した。
７）サンプルの確認
透析後、回収したサンプルの濃度は、ＤＣ　ｐｒｏｔｅｉｎ　ａｓｓａｙ　ｋｉｔ（Ｂｉｏ　Ｒａｄ社）、ｗｅｓｔｅｒｎ解析、ＳＤＳ−ＰＡＧＥで測定し、「アンジオポエチン関連タンパク質３」の蛋白質量を測定した。エンドトキシン濃度はエンドトキシン測定キット（生化学工業）により測定し、２０ｍｇ／ｍｇ以下である事を確認した。このようにして精製した蛋白質を抗原として用いた。
【０４２８】
（ｂ）　免疫マウス脾細胞の調製
８週令のＢＡＬＢ／ｃ雄マウス３匹にアジュバントとしてフロインド完全アジュバント（ディフコ社）０．５ｍｌを（ａ）で精製した０．５ｍｌ（１００μｇ相当）のアンジオポエチン関連タンパク質３に加え、連結針のついた注射筒内でエマルジョンを形成させた後、皮下注射して免疫した。２回目以降の免疫ではアジュバントとしてフロインド不完全アジュバント（ディフコ社）を用いた。免疫は、約２週間おきに４回行い、２回目の免疫以降、免疫する直前に眼底静脈叢より採血し、血清中の抗「アンジオポエチン関連タンパク質３」抗体価を以下に述べる固相法による酵素免疫測定法（ＥＬＩＳＡ法）で調べた。
【０４２９】
（固相酵素免疫法）９６ウェルのＥＬＩＳＡ用プレート（マキシソープ；ヌンク社）の各ウェルに５０ｎｇの精製「アンジオポエチン関連タンパク質３」を分注し、４℃で一晩放置して抗原をプレートウェル底面にコートさせた。プレートを０．１％（ｖ／ｖ）Ｔｗｅｅｎ２０を含むＰＢＳで３回洗浄した後、ＰＢＳで１０μｇ／ｍｌに調製したＢＳＡを１００μｌ／ウェルで添加し、室温で１時間放置した。さらに０．１％（ｖ／ｖ）Ｔｗｅｅｎ２０を含むＰＢＳで３回洗浄して、これに第一抗体として段階希釈した試料（マウス血清）を３０乃至１００μｌ／ウェル分注し、室温で１時間放置した。その後、０．１％（ｖ／ｖ）Ｔｗｅｅｎ２０を含むＰＢＳで３回洗浄し、第二抗体としてペルオキシダーゼ標識ヤギ抗マウスＩｇＧ（バイオラッド社）の３０００倍希釈液を１００μｌ／ウェル分注し、室温で１乃至２時間放置する。０．１％（ｖ／ｖ）Ｔｗｅｅｎ２０を含むＰＢＳで３回洗浄後、ＴＭＢパーオキシダーゼ基質液（バイオラッド社；ＴＭＢパーオキシダーゼキット）を１００μｌ／ウェルずつ添加し、室温で３０分間保温した後、１Ｎ硫酸を１００μｌ／ウェルずつ分注し、ペルオキシダーゼ反応を停止させてから、マイクロプレートリーダー（バイオラッド社）で４５０ｎｍの吸収を測定し、抗体価を算出した。
【０４３０】
（ｃ）　　マウス骨髄腫細胞の調製
８−アザグアニン耐性マウス骨髄腫細胞Ｐ３−Ｘ６３−Ａｇ８．６５３（６５３）をＴＩＬ培地で培養し、２×１０^７以上の細胞を得た。
【０４３１】
（ｄ）　ハイブリドーマの作製
ＤＭＥＭ（日水製薬社製）でよく洗浄した免疫マウス脾細胞１．４×１０^８個とマウス骨髄腫細胞Ｐ３−Ｘ６３−Ａｇ８．６５３（６５３）　　１．５×１０^７個を混合し、８００ｒｐｍで６分間遠心分離にかけた。沈澱として得られた脾細胞、およびＰ３−Ｘ６３−Ａｇ８．６５３（６５３）を混合した細胞群をよくほぐした後、あらかじめＤＭＥＭで５０％に調製したポリエチレングリコール４０００（ＰＥＧ４０００）溶液を２ｍｌ／分となるように一滴ずつ撹拌しながら１分間添加した。次にＤＭＥＭを２ｍｌ／分で１分間、同様にして添加し、この操作をもう一度繰り返した。さらに１６ｍｌのＤＭＥＭを３分間で添加して、８００ｒｐｍで６分間遠心分離した。遠心分離後上清を捨て、３５ｍｌのＴＩＬ培地で懸濁した。
【０４３２】
（ｅ）　ハイブリドーマ群の選別
懸濁液を９６ウェルプレート（コースター社）に１００μｌ／ウェルずつ分注し、７．５％のＣＯ２インキュベーター中、３７℃で培養した。７日後、５０μｌ／ウェルでＨＡＴ培地を添加した。さらに４日後、同様にして同量のＨＡＴ培地を添加した。その３日後、コロニー状に生育してきた融合細胞のみられるウェルについて培養上清の一部を採取し、抗「アンジオポエチン関連タンパク質３」抗体価を上記（ｂ）と同様の固相酵素免疫測定法により測定し、それと同時にＨＴ培地へ置き換えた。
【０４３３】
（ｆ）　クローニング
抗体価の認められたウェルについては、限界希釈法によりクローニングを３回繰返し、安定して抗体価の認められたクローンを抗「アンジオポエチン関連タンパク質３」モノクローナル抗体産生ハイブリドーマ株として選択した。ここで得られたハイブリドーマクローンの１つは、４５Ｂ１と命名され、独立行政法人産業技術総合研究所特許生物寄託センターに２００２年３月１３日付で、寄託番号ＦＥＲＭ　ＢＰ−７９６３として寄託されている。
【０４３４】
（ｇ）　モノクローナル抗体の精製
抗「アンジオポエチン関連タンパク質３」モノクローナル抗体産生ハイブリドーマ４５Ｂ１株の培養上清を回収して、０．２２μｍのフィルター（ミリポア社）にて濾過滅菌した後、ＭＡｂＴｒａｐ　ＧＩＩ（ファルマシア社）を用いて抗体を精製した。以上の工程を経て抗「アンジオポエチン関連タンパク質３」モノクローナル抗体ＭＡｂ４５Ｂ１を得た。
【０４３５】
（ｈ）　モノクローナル抗体の抗原特異性
上記（ａ）の１）で作製した「アンジオポエチン関連タンパク質３」生産ＣＨＯ細胞株の無血清培養上清とＣＨＯ細胞の無血清培養上清とをそれぞれ吸着抗原として用いたＥＬＩＳＡにより、Ｍａｂ４５Ｂ１が「アンジオポエチン関連タンパク質３」に特異的であることを確認した。すなわち、「アンジオポエチン関連タンパク質３」生産ＣＨＯ細胞株の無血清培養上清とＣＨＯ細胞の無血清培養上清とをそれぞれ１００μｌずつ９６ウェルのＥＬＩＳＡ用プレート（マキシソープ；ヌンク社製）の各ウェルに分注し、４℃で一晩保温して抗体をプレートウェル底面に吸着させた。各ウェルをＰＢＳで３回洗浄した後、３％（ｗ／ｖ）ＢＳＡを含むＰＢＳを２００μｌ／ウェルずつ添加し、４℃で一晩保温した。その後、０．０５％（ｖ／ｖ）のＴｗｅｅｎ２０を含むＰＢＳ（以下「ＰＢＳＴ」という）で各ウェルを２回洗浄し、これに、１％（ｗ／ｖ）ＢＳＡを含むＰＢＳで段階希釈した抗体産生ハイブリドーマ４５Ｂ１培養上清を１００μｌ／ウェルずつ分注し、室温で１時間反応させた。その後、０．０５％（ｖ／ｖ）のＴｗｅｅｎ２０を含むＰＢＳで各ウェルを５回洗浄し、１％（ｗ／ｖ）ＢＳＡを含むＰＢＳで５０００分の１に希釈したＨＲＰ標識化抗マウスＩｇＧ抗体（アマシャムバイオサイエンス）を１００μｌ／ウェルずつ分注し、３７℃で１時間反応させた。さらに０．０５％（ｖ／ｖ）のＴｗｅｅｎ２０を含むＰＢＳで各ウェルを５回洗浄し、ＡＢＴＳパーオキシダーゼ基質液（ロッシュ・ダイアグノスティクス）を１００μｌ／ウェルずつ添加し、室温で３０分間保温した後、４１５ｎｍの吸収を測定した。「アンジオポエチン関連タンパク質３」生産ＣＨＯ細胞株の無血清培養上清を吸着させたウェルでは高い吸光度が見られ抗体の希釈に従って吸光度が減少したのに対し、ＣＨＯ細胞の無血清培養上清を吸着させたウェルでは抗体の希釈によらず低い吸光度しか見られなかった。
【０４３６】
（ｉ）　モノクローナル抗体の分類
マウスモノクローナル抗体アイソタイピングキット（アマシャム社）を用いて解析した結果、この抗体は、ＩｇＧ１サブクラスの抗体であると同定された。
【０４３７】
実施例８．ＭＡｂ４５Ｂ１を用いた「アンジオポエチン関連タンパク質３」定量系
（ａ）　ウサギ抗「アンジオポエチン関連タンパク質３」ポリクローナル抗体の作製
アジュバントとしてフロインド完全アジュバント（ディフコ社）０．５ｍｌを実施例７の（ａ）で精製した０．５ｍｌ（１００μｇ相当）の「アンジオポエチン関連タンパク質３」に加え、連結針のついた注射筒内でエマルジョンを形成させた後、雄ウサギの皮下に接種した。２回目以降の免疫ではアジュバントとしてフロインド不完全アジュバント（ディフコ社）を用いた。免疫は、約２週間おきに８回行った。接種後、経時的に部分採血し、抗体価の上昇を調べた。８回目の免疫後７日後に麻酔下で全採血し、５３．５ｍｌの免疫血清を得た。該血清２０ｍｌを、ＭＡｂＴｒａｐ　ＧＩＩキットを用いて精製し、１１５ｍｌの１．６４ｍｇ／ｍｌ　ＩｇＧが得られた。
【０４３８】
以上の工程を経て、ウサギ抗「アンジオポエチン関連タンパク質３」ポリクローナル抗体（以下「Ｎｏ．１抗体」という）を得た。
【０４３９】
（ｂ）　ＨＲＰ標識Ｆａｂ´化ポリクローナル抗体の作成
１）Ｆ（ａｂ´）２の調製
３０ｍｌの１．６４ｍｇ／ｍｌ　Ｎｏ．１抗体を０．１Ｍ酢酸ナトリウム緩衝液（ｐＨ４．５　０．１Ｍ　ＮａＣｌを含む）で透析したし、ＣＥＮＴＲＩＣＯＮ−３０（アミコン社）を用いて濃縮した。０．１Ｍ酢酸ナトリウム緩衝液（ｐＨ４．５　０．１Ｍ　ＮａＣｌを含む）で濃度を６ｍｇ／ｍｌに合わせた。０．１Ｍ酢酸ナトリウム緩衝液（ｐＨ４．５、０．１Ｍ　ＮａＣｌを含む）にて１０ｍｇ／ｍｌに溶解したブタ胃・ペプシン（シグマ社）２０μｌ（０．２ｍｇ）を０．５ｍｌの６ｍｇ／ｍｌ　Ｎｏ．１抗体に添加し、３７℃で６時間反応させた。反応液を０．１Ｍリン酸ナトリウム緩衝液（ｐＨ７．０）で透析した後、Ｕｌｔｒｏｇｅｌ　ＡｃＡ　４４（インビトロジェン社）カラム（１．５ｃｍ×４５ｃｍ）−０．１Ｍリン酸ナトリウム緩衝液（ｐＨ７．０）、流速０．３５ｍｌ／ｍｉｎ、１画分０．５乃至１．０ｍｌでゲル濾過した。Ｎｏ．１抗体Ｆ（ａｂ´）２Ｆ画分を２８０ｎｍの吸光度により確認し、ＣＥＮＴＲＩＣＯＮ−３０（アミコン社）を用いて濃縮した。これらの操作を繰り返し、最終的に１１．２ｍｌの２．５ｍｇ／ｍｌ　Ｎｏ．１　Ｆ（ａｂ’）２抗体が得られた。
【０４４０】
２）Ｆａｂ’の調製
１１．２ｍｌの２．５ｍｇ／ｍｌ　Ｎｏ．１　Ｆ（ａｂ´）２抗体を０．１Ｍリン酸ナトリウム緩衝液（ｐＨ６．０）で透析し、ＣＥＮＴＲＩＣＯＮ−３０（アミコン社）を用いて濃縮した。０．１Ｍリン酸ナトリウム緩衝液（ｐＨ６．０）で濃度を５ｍｇ／ｍｌに合わせた。０．４５ｍｌの５ｍｇ／ｍｌ　Ｎｏ．１　Ｆ（ａｂ´）２抗体に０．１Ｍ　２−ｍｅｒｃａｐｏｔｅｔｈｙｌａｍｉｎｅ、５０ｍＭ　ＥＤＴＡを含む０．１Ｍリン酸ナトリウム緩衝液（ｐＨ６．０）５０μｌを加え、３７℃、９０分間反応させた。反応液をＵｌｔｒｏｇｅｌ　ＡｃＡ　４４（インビトロジェン社）カラム（１．５×４５ｃｍ）−０．１Ｍリン酸ナトリウム緩衝液（ｐＨ６．０，５ｍＭ　ＥＤＴＡを含む）、流速０．３５ｍｌ／ｍｉｎ、１画分０．５乃至１．０ｍｌでゲル濾過した。Ｎｏ．１　Ｆａｂ´抗体の存在を２８０ｎｍの吸光度により確認した後、ＣＥＮＴＲＩＣＯＮ−３０（アミコン社）を用いて濃縮した。
これらの操作を繰り返し、最終的に１１．４ｍｌの１．４ｍｇ／ｍｌ　Ｎｏ．１
Ｆａｂ´抗体が得られた。
【０４４１】
３）マレイミドペルオキシダーゼの調製
西洋ワサビ・ペルオキシダーゼ（シグマ社）３６．８ｍｇを０．１Ｍリン酸ナトリウム緩衝液（ｐＨ７．０）にて６．６６ｍｇ／ｍｌに溶解した。Ｎ，Ｎ−ｄｉｍｅｔｈｙｌｆｏｒｍａｍｉｄｅにてＮ−ｓｕｃｃｉｎｉｍｉｄｙｌ−４−（Ｎ−ｍａｌｅｉｍｉｄｏｍｅｔｈｙｌ）　　ｃｙｃｌｏｈｅｘａｎｅ−１−１ｃａｒｂｏｘｙｌａｔｅ（シグマ社）を溶解して濃度を７０ｍＭに合わせた。３００μｌの６．６６ｍｇ／ｍｌ西洋ワサビ・ペルオキシダーゼに３０μｌの７０ｍＭ　Ｎ−ｓｕｃｃｉｎｉｍｉｄｙｌ−４−（Ｎ−ｍａｌｅｉｍｉｄｏｍｅｔｈｙｌ）　　ｃｙｃｌｏｈｅｘａｎｅ−１−１ｃａｒｂｏｘｙｌａｔｅを加え、３０℃、３０分間反応させた。反応液から沈殿を遠心除去した後、Ｓｅｐｈａｄｅｘ　Ｇ−２５のカラム（１×３０ｃｍ）−０．１Ｍリン酸ナトリウム緩衝液（ｐＨ６．０），流速０．４乃至０．６ｍｌ／ｍｉｎ，１画分０．５乃至１．０ｍｌでゲル濾過した。ペルオキシダーゼを含む画分を４０３ｎｍの吸光度により確認し、ＣＥＮＴＲＩＣＯＮ−３０（アミコン社）を用いて濃縮した。これらの操作を繰り返し、最終的に５．２ｍｌの６．５ｍｇ／ｍｌマレイミドペルオキシダーゼが得られた。
【０４４２】
４）マレイミド化ペルオキシダーゼとＦａｂ´の反応
０．７１ｍｌの１．４ｍｇ／ｍｌのＮｏ．１　Ｆａｂ´抗体に０．２８ｍｌの６．５ｍｇ／ｍｌマレイミド化ペルオキシダーゼを添加し、４℃、２０時間反応させた。反応後、５０ｍＭ　Ｎ−ｅｔｈｙｌｍａｌｅｉｍｉｄｅを１０μｌ添加した。Ｕｌｔｒｏｇｅｌ　ＡｃＡ４４（インビトロジェン社）カラム（１．５×４５乃至１００ｃｍ）−０．１Ｍリン酸ナトリウム緩衝液（ｐＨ６．５）、流速０．３５ｍｌ／ｍｉｎ、１画分１．０ｍｌでゲル濾過した。各画分の２８０ｎｍ及び４０３ｎｍにおける吸光度を測定し、さらに各画分のペルオキシダーゼ活性を測定した。各画分に０．２％（ｗ／ｖ）　ｔｈｉｍｅｒｏｓａｌ（シグマ社）を終濃度０．００２％になるように、１０％（ｗ／ｖ）ウシ血清アルブミンを終濃度０．１％になるように添加した後、４℃で保存した。ペルオキシダーゼ標識Ｆａｂ´の濃度はペルオキシダーゼ活性あるいは４０３ｎｍの吸光度から計算し、１０μｇ／ｍｌ以上の濃度で保存した。これらの操作を繰り返し、最終的に８．６ｍｌの０．７８ｍｇ／ｍｌのＨＲＰ標識化Ｎｏ．１　Ｆａｂ´抗体を得、以下の実験に用いた。
【０４４３】
（ｃ）「アンジオポエチン関連タンパク質３」定量系の作製
実施例７の（ｇ）で培養上清より得られた精製ＭＡｂ４５Ｂ１を０．１Ｍ炭酸緩衝液（ｐＨ９．６）で２μｇ／ｍｌに希釈し、これを１００μｌずつ９６ウェルのＥＬＩＳＡ用プレート（マキシソープ；ヌンク社製）の各ウェルに分注し、４℃で一晩保温して抗体をプレートのウェル底面に吸着させた。各ウェルをＰＢＳで３回洗浄した後、０．１％（ｗ／ｖ）ＢＳＡを含むＰＢＳを２００μｌ／ウェルずつ添加し、４℃で一晩保温した。０．０５％（ｖ／ｖ）のＴｗｅｅｎ２０を含むＰＢＳ（以下「ＰＢＳＴ」という）で各ウェルを２回洗浄し、これに、１％（ｗ／ｖ）ＢＳＡおよび０．０５％（ｖ／ｖ）のＴｗｅｅｎ２０を含むＰＢＳで段階希釈した試料（精製「アンジオポエチン関連タンパク質３」あるいはヒト血漿）を１００μｌ／ウェルずつ分注し、４℃で一晩保温した。０．０５％（ｖ／ｖ）のＴｗｅｅｎ２０を含むＰＢＳで各ウェルを７回洗浄し、１％（ｗ／ｖ）ＢＳＡおよび０．０５％（ｖ／ｖ）のＴｗｅｅｎ２０を含むＰＢＳで０．５μｇ／ｍｌに希釈したＨＲＰ標識化Ｎｏ．１　Ｆａｂ´抗体を１００μｌ／ウェルずつ分注し、３７℃で３０分間反応させた。０．０５％（ｖ／ｖ）のＴｗｅｅｎ２０を含むＰＢＳで各ウェルを９回洗浄し、ＴＭＢパーオキシダーゼ基質液（バイオラッド社　ＴＭＢパーオキシダーゼキット）を１００μｌ／ウェルずつ添加し、室温で３０分間保温した後、１Ｎ硫酸を１００μｌ／ウェルずつ分注し、ペルオキシダーゼ反応を停止させてから、マイクロプレートリーダー（バイオラッド社製）で４５０ｎｍの吸収を測定した。その結果、精製「アンジオポエチン関連タンパク質３」濃度と測定結果が正比例したことから、このＥＬＩＳＡ系により「アンジオポエチン関連タンパク質３」を直線性良く定量可能であることが判明した（図６参照）。ヒト血漿を試料とした場合も該ＥＬＩＳＡ系により検出可能であった。
【０４４４】
実施例９．　アデノウイルスベクターを用いた組換えポリペプチドの産生
（１）ＤＮＡの調製
１）コザック配列の挿入
参考例２記載の、高脂血症の治療または予防剤を試験するための方法において、遺伝子発現量を検出するためにクローニングされたｃＤＮＡに、コザックのコンセンサス配列が付加されたｃＤＮＡクローンを得るため、下記のヌクレオチド配列：
５’−　ｇｃｃａｃｃａｔｇｇｔｃａｃａａｔｔａａｇｃｔｃｃｔｔｃｔｔｔｔｔａｔｔｇ　−３’（配列表の配列番号４２）
および、制限酵素ＸｈｏＩの特異的認識配列が付加された下記のヌクレオチド配列：
５’−　ｔｃｔａｇａｇｃｃｔｃｇｔｔｃａｔｔｃａａａｇｃｔｔｔｃｔｇａａｔｃｔｇ　−３’（配列表の配列番号４３）
からなるオリゴヌクレオチドプライマーを合成した。ここで、配列番号４２に示されるプライマーの一部は配列表の配列番号５３に示される「アンジオポエチン関連タンパク質３」のｃＤＮＡのヌクレオチド番号２５乃至５５であり、配列番号４３に示されるプライマーの一部は配列表の配列番号５３のヌクレオチド番号１３８５乃至１４１５のアンチセンスに該当する。
【０４４５】
次に、参考例２記載の方法で得られた組換えファージミドｐＴｒｉｐ／ｈ５５−１に挿入されているヒトｃＤＮＡ（Ｇｅｎｂａｎｋデータベースに「アンジオポエチン関連タンパク質３」をコードするヌクレオチド配列として開示されているもの）約０．４μｇ／ｍｌを鋳型とし、上記プライマー（それぞれ０．４ｍＭ）、Ｔｂｒ　ＥＸＴ　ＤＮＡポリメラーゼ（フィンザイム社製）、１×ＰＣＲ反応緩衝液（ＤＮＡポリメラーゼに添付の１０×緩衝液を１０分の１量）、２ｍＭの塩化マグネシウム、各０．４ｍＭのｄＮＴＰｓ存在下でＰＣＲを行った。ＰＣＲは上記サンプルを９４℃で３分間加熱した後に、９４℃で３０秒、５５℃で３０秒、７２℃で１分の温度サイクル３０回、最後に６８℃で２分間保温の条件で行なった。
【０４４６】
ＰＣＲの結果得られた増幅断片（約１．４ｋｂｐ）を、ＴＡクローニングキット（インビトロジェン社製）に添付されたプラスミドベクターｐＣＲ２．１へ挿入し、挿入断片を含むプラスミドクローン（ｐＣＲ／ＫｈＡＰ５）を単離した。ＤＮＡシークエンサー（ＡＢＩ　ＰＲＩＳＭ　３７００、アプライドバイオシステムズ社製）で挿入断片の配列を解析して、ＰＣＲの際に該挿入断片内に変異が生じていないことを確認した。
【０４４７】
次に、ｐＣＲ／ＫｈＡＰ５を制限酵素ＥｃｏＲＩおよびＸｂａＩで消化して、生成した断片のうち、ヒト・「アンジオポエチン関連タンパク質３」をコードするｃＤＮＡを含む断片を、予め制限酵素ＥｃｏＲＩおよびＸｂａＩで消化した哺乳動物細胞発現ベクターｐＭＥ１８Ｓに挿入することにより、組換えプラスミドｐＭＥ１８Ｓ／ＫｈＡＰ５を得た。
【０４４８】
２）部分欠失変異体の作製
ａ）カルボキシル末端側領域を欠失した変異体
上記１）で得られた組換えプラスミドｐＭＥ１８Ｓ／ＫｈＡＰ５を制限酵素ＳｐｅＩで消化し、Ｔ４　ＤＮＡポリメラーゼで切断末端を平滑端化した後に、ＤＮＡリガーゼ反応を行って再び両端を連結することにより、ヒト・「アンジオポエチン関連タンパク質３」のシグナルペプチドを含むアミノ酸配列中のアミノ末端から２０７番目のアミノ酸残基（セリン）をコードするコドン（ａｔｃ）の後に終止コドン（ｔａｇ）が挿入された組換えプラスミドｐＭＥ１８Ｓ／ＫｈＡＰ５　ＣＣを構築した。この組換えプラスミド中の挿入断片のヌクレオチド配列は配列表の配列番号１１に示す通りである。
【０４４９】
ｂ）アミノ末端側領域を欠失した変異体
上記１）で得られた組換えプラスミドｐＭＥ１８Ｓ／ＫｈＡＰ５を制限酵素ＢｃｌＩおよびＳｐｅＩで消化し、ヒト・「アンジオポエチン関連タンパク質３」のシグナルペプチドを含むアミノ酸配列中のアミノ末端から２０７番目のアミノ酸残基（セリン）までをコードするｃＤＮＡ配列のみを含む断片を除いた後、Ｔ４　ＤＮＡポリメラーゼ切断末端を平滑端化し、ＤＮＡリガーゼ反応を行って再び両端を連結した。この操作により、ｐＭＥ１８Ｓ／ＫｈＡＰ５の中の、ヒト・「アンジオポエチン関連タンパク質３」のシグナルペプチドを含むアミノ酸配列中の２１乃至２０６番目のアミノ酸をコードするヌクレオチド配列がプロリンをコードするコドン（ｃｃｔ）で置換された組換えプラスミドｐＭＥ１８Ｓ／ＫｈＡＰ５　ＦＬＤを構築した。この組換えプラスミド中の挿入断片のヌクレオチド配列は配列表の配列番号１３に示す通りである。
【０４５０】
（２）組換えアデノウイルスの調製
上記（１）で得られた組換えプラスミドｐＭＥ１８Ｓ／ＫｈＡＰ５　ＣＣとｐＭＥ１８Ｓ／ＫｈＡＰ５　ＦＬＤ中に存在する「アンジオポエチン関連タンパク質３」遺伝子の一部領域欠失変異体をコードする遺伝子が、組込まれた組換えアデノウイルスは、アデノウイルス発現ベクターキット（宝酒造（株）社製）を用いて以下に記載する方法に従って作製した。
【０４５１】
まず、上記で（１）で得られた組換えプラスミドｐＭＥ１８Ｓ／ＫｈＡＰ５　ＣＣおよびｐＭＥ１８Ｓ／ＫｈＡＰ５　ＦＬＤを、それぞれ制限酵素ＥｃｏＲＩおよびＸｂａＩで消化して、得られたそれぞれ約１．４ｋｂおよび約０．９ｋｂのＤＮＡ断片を回収し、切断末端を平滑化した。次に、サイトメガロウイルスエンハンサーとニワトリβ−アクチンプロモーターにより挿入遺伝子が発現されるように設計されているコスミドベクターｐＡｘＣＡｗｔのＳｗａＩ切断部位にこれらの断片をそれぞれ挿入し、組換えコスミドｐＡｘＣＡ／ＫｈＡＰ５　ＣＣおよびｐＡｘＣＡ／ＫｈＡＰ５　ＦＬＤを調製した。
【０４５２】
これらの組換えコスミドＤＮＡと末端タンパク質結合ウイルスＤＮＡ（ＤＮＡ−ＴＰＣ、アデノウイルス発現ベクターキットに添付）をリン酸カルシウムトランスフェクションシステム（ライフテック社製）を用いて２９３細胞（ＡＴＣＣＣＲＬ１５７３）にコ・トランスフェクションして、組換えアデノウイルスＡｄ／ＫｈＡＰ５　ＣＣ、Ａｄ／ＫｈＡＰ５　ＦＬＤを単離し、さらに２９３細胞内で増幅させた。増幅させたウイルスの２９３細胞からの回収は、まずウイルス感染２９３細胞に３０秒×４回の超音波処理（ブランソン社製Ｂ−１２００を使用）を行うか、または凍結融解を３回繰り返し、次いで塩化セシウム密度勾配遠心による精製を２回繰り返すことによって行った。得られたウイルス液を、１０％　グリセロールを添加したＰＢＳに対して４℃で透析した後、使用するまで−７０℃以下で凍結保存した。
【０４５３】
（３）組換えアデノウイルスによる目的タンパク質の発現の確認
上記（２）記載の方法で得られた組換えアデノウイルスをＨｅＬａ細胞に感染させて、目的タンパク質の発現を確認した。
【０４５４】
まず、上記（２）記載の方法で得られた組換えアデノウイルスをそれぞれＨｅＬａ細胞（ＡＴＣＣ　ＣＣＬ２）に約５ｍ．ｏ．ｉ．（ｍｕｌｔｉｐｌｉｃｉｔｙ　ｏｆ　ｉｎｆｅｃｔｉｏｎ：多重感染度）で感染させ、無血清培地（ダルベッコ修正イーグル培地（ＤＭＥＭ））で３乃至４日培養した後の培養上清を回収した。培養上清１ｍｌを１．５ｍｌ容のエッペンドルフチューブに入れ、１００μｌのトリクロロ酢酸溶液（１ｇ／ｍｌ　トリクロロ酢酸、４ｍｇ／ｍｌ　デオキシコール酸）を添加して室温で３分間放置後、卓上遠心機にて１５０００ｒｐｍで５分遠心した。上清を除いて、氷冷したアセトンを０．５ｍｌ加えて攪拌し、卓上遠心機にて１５０００ｒｐｍで２分遠心した。上清を除いて、沈殿に再度氷冷したアセトンを０．５ｍｌ加えて攪拌し、卓上遠心機にて１５０００ｒｐｍで２分遠心して、上清を除き、沈殿物を真空乾燥した。この沈殿物をそれぞれ１０μｌの蒸留水に溶解させ、２−メルカプトエタノールを含むＳＤＳ−ＰＡＧＥ試料緩衝液（バイオラッド社製）と混合した後、９９℃で５分間加熱して、電気泳動用試料を調製した。これら試料をゲル濃度４−２０％のポリアクリルアミド密度勾配ゲルにて電気泳動した（電気泳動用緩衝液：２５ｍＭ　トリス、１９２ｍＭ　グリシン、０．１％ＳＤＳ）後、転写緩衝液（２５ｍＭ　トリス、１９２ｍＭ　グリシン、２０％メタノール）中で４℃、１時間、２００ｍＡの条件でニトロセルロースメンブレンに転写した。
【０４５５】
転写したメンブレンは０．５％　スキムミルクを添加したＰＢＳ液中に浸して４℃で一晩ブロッキングし、洗浄液（０．０５％　Ｔｗｅｅｎ２０−ＰＢＳ）で３回洗浄した。次いで、メンブレンを、約２７０ｎｇ／ｍｌの５５−１−Ｎ１抗体または約１μｇ／ｍｌの５５−１−Ｃ１抗体および５％　ウシ胎児血清を含む０．０５％　Ｔｗｅｅｎ２０−ＰＢＳ液中に入れて室温で１時間インキュベーションした後、洗浄液で３回洗浄した。なお、これら抗体はいずれも実施例２記載の方法で取得された。５５−１−Ｎ１抗体はヒト「アンジオポエチン関連タンパク質３」のアミノ末端側に特異的に結合する抗体であり、５５−１−Ｃ１抗体は同じくカルボキシル末端側に特異的に結合する抗体である。
【０４５６】
さらに、メンブレンを西洋ワサビペルオキシダーゼ標識ヤギ抗ウサギＩｇＧ（Ｈ＋Ｌ）（バイオラッド社製）を５％　ウシ胎児血清を含む０．０５％　Ｔｗｅｅｎ２０−ＰＢＳで２０００倍希釈した反応液中で室温で１時間インキュベーションした後、洗浄液で５回洗浄した。このメンブレンをラップフィルム上に置き、発光試薬溶液（ＥＣＬウエスタン検出試薬。アマシャム・ファルマシア・バイオテク社製）に１分間浸した後、１時間室温で放置してバックグラウンドを減衰させてから、Ｘ線フィルムを感光させた（１０秒間）。このＸ線フィルムを現像した結果、組換えアデノウイルスを感染させたＨｅＬａ細胞において、それぞれ目的のポリペプチドが産生されていることが確かめられた。
【０４５７】
（４）組換えアデノウイルスを接種したＫＫ／Ｓｎｋマウスの血中中性脂肪濃度の測定
上記で精製した組換えアデノウイルスＡｄ／ＫｈＡＰ５　ＣＣおよびＡｄ／ＫｈＡＰ５　ＦＬＤを１０％　グリセロール添加ＰＢＳで２．０×１０^１０ｐｆｕ／ｍｌおよび１．０×１０^１０ｐｆｕ／ｍｌに希釈し、それぞれ３匹の１９週齢の雄ＫＫ／Ｓｎｋマウスに３００μｌずつ尾静脈注射により接種した。また、陰性対照として、１０％　グリセロール添加ＰＢＳを３００μｌずつ３匹の１９週齢の雄ＫＫ／Ｓｎｋマウスに尾静脈注射により接種した。
【０４５８】
接種１日後に、各マウスの眼底よりヘマトクリット管で採血し、卓上遠心機で５２００ｒｐｍで１５分間遠心して血漿を分離し、キット（トリグリセリドＥ−テストワコー。和光製薬（株）社製）を用いて中性脂肪濃度を測定した。
【０４５９】
その結果、Ａｄ／　ＫｈＡＰ５　ＦＬＤ接種マウス群と１０％　グリセロール添加ＰＢＳ接種マウス群では、血中の中性脂肪濃度に有意な差が認められなかった（いずれも５０ｍｇ／ｄＬ以下）のに対し、Ａｄ／ＫｈＡＰ５　ＣＣ接種マウス群では血中の中性脂肪濃度の顕著な上昇が認められた（３０００ｍｇ／ｄＬ以上）。
【０４６０】
本実施例の方法において、例えばＡｄ／ＫｈＡＰ５　ＣＣを接種したマウスに、被験物質を投与し、血中中性脂肪濃度の変動を調べることにより、高脂血症、動脈硬化および高血糖から選択される一つまたは二つ以上の疾患の治療または予防剤の試験を行うことができる。
【０４６１】
実施例１０．　融合タンパク質の活性評価
（１）融合タンパク質発現形質転換大腸菌の作製
配列表の配列番号１２のアミノ酸番号１７乃至２０７、同アミノ酸番号１７乃至１９５、または同アミノ酸番号１７乃至１４７に示されるアミノ酸配列からなるポリペプチドのアミノ末端にグルタチオン・Ｓ・トランスフェラーゼ（以下「ＧＳＴ」という）が連結している融合タンパク質（以下、順番にそれぞれ「ＧＳＴ−ＣＣ」、「ＧＳＴ−ＣＣｄｅｌ」および「ＧＳＴ−Ｃ１」という。）を以下に記載する方法に従って調製した。
【０４６２】
まず、ＰＣＲ用プライマーとして、下記のヌクレオチド配列：
５’−　ｃｇｇａａｔｔｃｃｃｔｃｃａｇａａｔｔｇａｔｃａａｇ　−３’（ＧＳＴ−ＣＣ、ＧＳＴ−ＣＣｄｅｌおよびＧＳＴ−Ｃ１共用のセンスプライマー。配列表の配列番号４４）；
５’−　ｃｃｇｃｔｃｇａｇａｃｔａｇｔｃｃｔｔｃｔｇａｇｃｔｇ　−３’（ＧＳＴ−ＣＣ用アンチセンスプライマー。配列表の配列番号４５）；および
５’−　ｃｃｇｃｔｃｇａｇｔｔｇａｃｔａｔｇｃｔｇｔｔｇｇｔｔ　−３’（ＧＳＴ−ＣＣｄｅｌ用アンチセンスプライマー。配列表の配列番号４６）；および
５’−　ｃｃｇｃｔｃｇａｇｇｔｔａｇｔｔａｇｔｔｇｃｔｃｔｔｃ　−３’（ＧＳＴ−Ｃ１用アンチセンスプライマー。配列表の配列番号４７）を合成した。
【０４６３】
次に、実施例３記載のｃＤＮＡ（ｐＭＥｈ５５−１）を鋳型とし、センスプライマーおよびアンチセンスプライマー（それぞれ０．２μＭ）、Ｔｂｒ　ＥＸＴＤＮＡポリメラーゼ、１×ＰＣＲ緩衝液（Ｔｂｒ　ＥＸＴ　ＤＮＡポリメラーゼに添付の１０×緩衝液を１０倍希釈）、２ｍＭの塩化マグネシウムおよび各０．４ｍＭのｄＮＴＰｓ存在下でＰＣＲを実施した。ＰＣＲは、上記サンプルを９４℃で３分間加熱した後に、９４℃で３０秒、５５℃で３０秒、７２℃で１分の温度サイクル３０回、最後に７２℃で５分間の条件で行なった。
【０４６４】
ＰＣＲではそれぞれについて、約５８０ｂｐ（ＧＳＴ−ＣＣ用アンチセンスプライマー使用）、約５４０ｂｐ（ＧＳＴ−ＣＣｄｅｌ用アンチセンスプライマー使用）および約４００ｂｐ（ＧＳＴ−Ｃ１用アンチセンスプライマー使用）のＤＮＡ断片が増幅した。増幅したＤＮＡ断片を回収し、それぞれについて、ＥｃｏＲＩおよびＸｈｏＩで消化した。ＥｃｏＲＩおよびＸｈｏＩで消化してからウシ小腸アルカリホスファターゼ（宝酒造（株）社製）を用いて切断末端の脱リン酸化処理を行ったプラスミドベクターｐＧＥＸ−４Ｔ−２（ＧＳＴをコードする遺伝子を含む。アマシャム・ファルマシア・バイオテク社製）に、このＥｃｏＲＩおよびＸｈｏＩで消化した、ＤＮＡ断片をそれぞれ挿入し、３種の挿入断片を含む３種のプラスミドクローンを単離した。ＧＳＴ−ＣＣ用アンチセンスプライマーで増幅した断片を有するプラスミドクローンをｐＧＳＴ−ＣＣ、ＧＳＴ−ＣＣｄｅｌ用アンチセンスプライマーを使用して増幅した断片を有するプラスミドクローンをｐＧＳＴ―ＣＣｄｅｌ、ＧＳＴ−Ｃ１用アンチセンス用プライマーを使用して増幅した断片を有するプラスミドクローンをｐＧＳＴ−Ｃ１と命名した。
【０４６５】
プラスミドクローンｐＧＳＴ−ＣＣ、ｐＧＳＴ−ＣＣｄｅｌ、ｐＧＳＴ−Ｃ１または、挿入断片を含まずＧＳＴのみを発現させるためのコントロールベクターｐＧＥＸ−４Ｔ−２のそれぞれで大腸菌ＪＭ１０９株を形質転換した。形質転換は大腸菌ＪＭ１０９のコンピテントセルにプラスミドを添加し、氷上で３０分間静置した後、４２℃で３０秒間保温し、再び氷上で２分間静置してから、３７℃で１時間振盪培養をすることによって行なった。形質転換後の培養液をアンピシリン添加ＬＢ寒天プレート上に撒き、出現した組換え体のコロニーをピックアップして液体培養し、ＩＰＴＧ誘導による組換えタンパク質の発現を確認した。
【０４６６】
（２）融合タンパク質の調製
１）形質転換大腸菌の培養
プラスミドクローンｐＧＳＴ−ＣＣ、ｐＧＳＴ−ＣＣｄｅｌ、ｐＧＳＴ−Ｃ１及びコントロールベクターｐＧＥＸ−４Ｔ−２を保持する形質転換大腸菌をそれぞれ、アンピシリン入りのＬＢ培地に植菌し、３７℃で１８時間振とう培養した。培養液の一部を新しい培地に添加し、さらに３７℃にて振とう培養を行なった。６００ｎｍでの吸光度が０．５−０．７に達した時に、最終濃度１ｍＭとなるようにイソプロピル・チオガラクトピラノシド（ＩＰＴＧ）を添加しさらに４時間振とう培養を継続した。
【０４６７】
２）ｐＧＥＸ−４Ｔ−２を保持する大腸菌からの細胞破砕液の調製
コントロールベクターｐＧＥＸ−４Ｔ−２で形質転換した大腸菌の培養液を４℃、６０００ｒｐｍ、１０分間の条件で遠心処理して沈殿した菌体を回収した。回収した菌体を超音波処理用緩衝液（５０ｍＭ　トリス−塩酸（ｐＨ８．０）、５０ｍＭ　塩化ナトリウム、１ｍＭ　ＥＤＴＡ、１ｍＭ　ＤＴＴ）で懸濁し、懸濁液を４℃、６０００ｒｐｍ、１０分間の条件で遠心処理した。沈殿を−８０℃で凍結し、次いで室温にて融解して再度超音処理用緩衝液に懸濁した。懸濁液を氷浴中で超音波破砕器（トミー精工（株）社製ＵＤ−２０１）によって、出力＝５、デューティ＝５０の条件で５分間処理した。超音波処理後の破砕液に最終濃度１％となるようにトリトン　Ｘ−１００を添加し混和し、４℃、１００００ｒｐｍ、３０分間の条件で遠心処理し、上清を回収した。上清はＧＳＴｒａｐカラム（アマシャム・ファルマシア・バイオテク社製）中に流した。
【０４６８】
３）ｐＧＳＴ−ＣＣ、ｐＧＳＴ−ＣＣｄｅｌ、ｐＧＳＴ−Ｃ１を保持する大腸菌からの細胞破砕液の調製
組換えプラスミドｐＧＳＴ−ＣＣ、ｐＧＳＴ−ＣＣｄｅｌ、ｐＧＳＴ−Ｃ１で形質転換した大腸菌の培養液それぞれを、４℃、５０００ｒｐｍ、１５分間の条件で遠心処理し、沈殿した菌体を回収した。沈殿した菌体に対して、洗浄用緩衝液（０．５％　トリトン　Ｘ−１００、１ｍＭ　ＥＤＴＡ）での懸濁、４℃、５０００ｒｐｍ、１０分間の条件での遠心処理、の操作を３回繰り返し、得られた菌体を８Ｍ　尿素溶液に溶解した。この溶液を室温に１時間放置した後、４℃、１３０００ｒｐｍ、３０分間の遠心処理を行ない上清を回収した。組換えプラスミドｐＧＳＴ−ＣＣ、ｐＧＳＴ−ＣＣｄｅｌ、ｐＧＳＴ−Ｃ１で形質転換した大腸菌の由来の培養液から得られた３種の上清について透析を行なった。透析は、４Ｍ　尿素溶液に対して４℃で１時間、２Ｍ　尿素溶液に対して４℃で１時間、超音波処理用緩衝液に対して４℃で１時間、超音波処理用緩衝液に対して４℃で一晩の順で行なった。透析後、４℃、１２０００ｒｐｍ、３０分間の条件で遠心し、上清をそれぞれＧＳＴｒａｐカラム中に流した。
【０４６９】
４）融合タンパク質の精製
上記の工程を経てＧＳＴ、ＧＳＴとの融合タンパク質ＧＳＴ−ＣＣ、ＧＳＴ−Ｃ１またはＧＳＴ−ＣＣｄｅｌが吸着したＧＳＴカラムにＰＢＳを流してカラム内を洗浄して不純物を除去した。次いで、１０ｍＭ　還元型グルタチオン溶液を流して、ＧＳＴ、ＧＳＴとの融合タンパク質ＧＳＴ−ＣＣ、ＧＳＴ−Ｃ１又はＧＳＴ−ＣＣｄｅｌを溶出させた。溶出液の一部をＳＤＳ−ＰＡＧＥ電気泳動にで分離した後、ゲルをクマシー染色して、タンパク質の存在を確認した。また、溶出液をＰＢＳで４℃、一晩透析した。ここで、プラスミドｐＧＥＸ−４Ｔ−２を保持する形質転換体から精製されたタンパク質がＧＳＴであり、以下同様にｐＧＳＴ−ＣＣ、ｐＧＳＴ−Ｃ１、ｐＧＳＴ−ＣＣｄｅｌ由来のタンパク質がそれぞれ、ＧＳＴ−ＣＣ、ＧＳＴ−Ｃ１、ＧＳＴ−ＣＣｄｅｌである。
【０４７０】
（３）血中中性脂肪濃度上昇活性
上記（２）で得られたＧＳＴ、ＧＳＴ−ＣＣ、ＧＳＴ−Ｃ１、ＧＳＴ−ＣＣｄｅｌおよびＧＳＴ−Ｃ１を、ＰＢＳでタンパク質濃度約１ｍｇ／ｍｌに調整した後に、それぞれ３匹、４匹、２匹および５匹の１９週齢の雄ＫＫ／Ｓｎｋマウスに３００μｌずつ尾静脈注射により接種した。採血は摂取前と摂取３０分後に行なった。ヘマトクリット管で各マウスの眼底より採血して、卓上遠心機で５２００ｒｐｍ、１５分間遠心して血漿を分離し、接種前後における血中中性脂肪（トリアシルグリセロール）濃度を測定した。
【０４７１】
その結果を図８に示した。図８のグラフ中、縦軸は、血漿１００ｍｌ中の中性脂肪（トリアシルグリセロール）量を示している。横軸は各融合タンパク質を示す。ＧＳＴ接種マウス群（図８の「ＧＳＴ」）では接種前後で差が認められなかったのに対し、ＧＳＴ−Ｃ１接種マウス群（図８の「Ｃ１」）では若干ながら血中中性脂肪濃度の上昇が認められた。一方、ＧＳＴ−ＣＣおよびＧＳＴ−ＣＣｄｅｌ接種マウス群（図８の「ＣＣ」および「ＣＣｄｅｌ」）では血中中性脂肪濃度の顕著な上昇が認められた。
【０４７２】
本実施例の方法において、例えばＧＳＴ−ＣＣあるいはＣＣｄｅｌを接種したマウスに、被験物質を投与し、血中中性脂肪濃度の変動を調べることにより、高脂血症、動脈硬化および高血糖から選択される一つまたは二つ以上の疾患の治療または予防剤の試験を行うことができる。
【０４７３】
実施例１１．　ＬＰＬ活性測定法
（１）ラット脂肪細胞由来ＬＰＬを用いる方法
１）ＬＰＬ試料の調製
ラット白色脂肪前駆細胞（ホクドー社より購入）を増殖用培地［１０％　ウシ胎児血清（以下「ＦＣＳ」という）、１００単位／ｍｌ　ペニシリン、１００μｇ／ｍｌ　ストレプトマイシン、１７μＭ　パントテン酸、３３μＭ　（＋）−ビオチン、１００μＭ　アスコルビン酸、１μＭ　オクタン酸および５０ｎＭ　トリヨードチロニンを含むダルベッコ変法イーグル培地（以下「ＤＭＥＭ」という）］の入った細胞培養用フラスコ（培養面積２５ｃｍ^２）中で３７℃で２４時間培養した後、培地を分化誘導用培地（１０％　ＦＣＳ、１００単位／ｍｌ　ペニシリン、１００μｇ／ｍｌ　ストレプトマイシン、１７μＭ　パントテン酸、３３μＭ　（＋）−ビオチン、１００μＭ　アスコルビン酸、１μＭ　オクタン酸、５０ｎＭ　トリヨードチロニン、１０μｇ／ｍｌ　インスリンおよび２．５μＭ　デキサメタゾンを含むＤＭＥＭ）に交換し、３７℃でさらに４８時間培養した。その後、脂肪細胞維持培地（１０％　ＦＣＳ、１００Ｕ／ｍｌ　ペニシリン、１００μｇ／ｍｌ　ストレプトマイシン、１７μＭ　パントテン酸、３３μＭ　（＋）−ビオチン、１００μＭ　アスコルビン酸、１μＭオクタン酸、５０ｎＭ　トリヨードチロニンおよび１０μｇ／ｍｌ　インスリンを含むＤＭＥＭ）に交換し、２乃至３日目に１０％　ＦＣＳ、５０ｎｇ／ｍｌ　インスリンおよび１０単位／ｍｌ　ヘパリンナトリウムを含有するＤＭＥＭ培地を２ｍｌ加え、１時間培養した後、この培養上清を回収してＬＰＬ活性の測定に用いた。
【０４７４】
２）ＬＰＬ活性測定
ＬＰＬ活性の測定法は、文献記載の方法（Ｎｉｌｓｓｏｎ−Ｅｈｌｅ，　Ｐ．　ａｎｄ　Ｓｃｈｏｔｚ，　Ｍ．　Ｃ．（１９７６）　Ｊ．Ｌｉｐｉｄ　Ｒｅｓ．　１７，　５３６−５４１）に従った。なお、配列表の配列番号４のアミノ酸番号１７乃至４６０に示されるアミノ酸配列からなるポリペプチド（アンジオポエチン関連タンパク質３。）は、実施例６記載の方法に従って調製した。
【０４７５】
まず、上記１）記載の方法で調製した培養上清１００μｌを等量の基質溶液［２ｍＭ　グリセロール・トリ［９，１０（ｎ）−^３Ｈ］オレイン酸（１３１ＫＢｅｑ／μｍｏｌ、アマシャム社製）、０．１８９μｇ／ｍｌ　Ｌ　α−ホスファチジルコリン（シグマ社製）、１４ｍｇ／ｍｌ　ウシ血清アルブミン（シグマ社製）、１４０ｍＭ　トリス−塩酸（ｐＨ８．０）、１５％（ｖ／ｖ）グリセロール、１０％（ｖ／ｖ）非働化ＦＣＳ］と混合し、そこへ精製したアンジオポエチン関連タンパク質３を加え、３７℃で１２０分間保温した。その後、１．０５ｍｌの０．１Ｍ　炭酸カリウム・ほう酸緩衝液（ｐＨ１０．５）と３．２５ｍｌのメタノール：クロロホルム：ヘキサン＝１４１：１２５：１００（ｖ／ｖ）を加えて反応を停止させ、強く撹拌した後、３０００×ｇで１５分間遠心分離した。水−メタノール層の^３Ｈカウントを液体シンチレーションカウンターにて測定した。ＬＰＬ活性の１単位は、１μｍｏｌの脂肪酸を１分間に生成する活性として定義した。その結果を表５に示した。
【０４７６】
【表５】

アンジオポエチン関連タンパク質３はＬＰＬの活性を用量依存的に抑制した。この実験系において、リパーゼ反応の際に被験物質を添加すれば、該物質がＬＰＬの活性を調節する作用を有するか否かを調べることができる。
（２）　マウスヘパリン静注後血漿を用いる方法
Ｃ５７ＢＬ／６ｊマウスにヘパリンナトリウムを尾静脈投与し（１００単位／ｋｇ）、１０分後に腹大動脈より採血を行ない、遠心分離によりヘパリン静注後血漿を調製し、ＬＰＬ活性測定に使用した。ただし、血漿を用いた場合、上記（１）の２）に記載の条件で測定を行うと、血漿中に含まれる肝臓由来のＨＴＧＬとＬＰＬとの両リパーゼ活性が測定される。そこで、それらを分別して測定するために、まず、上記（１）の２）に記載の条件でリパーゼ反応を行ってリパーゼ活性を測定し、得られた結果を「総リパーゼ活性」とした。一方、別に、終濃度１Ｍとなるように塩化ナトリウムを反応液に加えることによりＬＰＬを不活化した条件でリパーゼ反応を行い、血漿中に残存するＨＴＧＬのリパーゼ活性のみを測定した。このようにして得られた総リパーゼ活性からＨＴＧＬ活性を差し引いた値をＬＰＬ活性とした。その結果を表６に示した。
【０４７７】
【表６】

アンジオポエチン関連タンパク質３は、マウスヘパリン静注後血漿中のＬＰＬおよびＨＴＧＬの両者に対してその活性をイン・ビトロで阻害することが明らかになった。この実験系において、総リパーゼ活性を測定するためのリパーゼ反応時に被験物質を添加すれば、該物質がＬＰＬまたはＨＴＧＬのいずれかの活性を調節する作用を有するか否かを調べることができる。同様に、ＨＴＧＬ活性を測定するためのリパーゼ反応時に被験物質を添加すれば、該物質がＨＴＧＬの活性を調節する作用を有するか否かを調べることができる。
【０４７８】
実施例１２．　融合タンパク質のＬＰＬ活性阻害効果
ＧＳＴ−ＣＣ、ＧＳＴ−ＣＣｄｅｌまたはＧＳＴを用いてアンジオポエチン関連タンパク質３の代わりに用いて実施例１１の（１）記載の方法でＬＰＬの活性を測定した。
ＧＳＴ（１０μｇ／ｍｌ）を添加したときのＬＰＬ活性を１００％とし、ＧＳＴ−ＣＣあるいはＧＳＴ−ＣＣｄｅｌを添加したときの相対的ＬＰＬ活性を算出した。その結果を表７に示した。
【０４７９】
【表７】

ＧＳＴ−ＣＣおよびＧＳＴ−ＣＣｄｅｌはＬＰＬの活性を用量依存的に抑制した。この実験系において、リパーゼ反応の際に被験物質を添加すれば、該物質がＬＰＬの活性を調節する作用を有するか否かを調べることができる。
【０４８０】
実施例１３．　ヒスチジンタグが付加されたポリペプチドの調製とＬＰＬ阻害活性
（１）ヒスチジンタグが付加されたポリペプチドの調製
「アンジオポエチン関連タンパク質３」のシグナルペプチドを含むアミノ酸配列中のアミノ末端から２０番目のアミノ酸残基（アスパラギン酸）をコードするコドンの直後にヒスチジンタグが挿入され、かつアミノ末端から２０８番目以降のアミノ酸配列を欠失したポリペプチドＫｈＡＰ５　ＣＣ／Ｈｉｓを、以下に記載する方法に従って調製した。
【０４８１】
１）ヒスチジンタグの挿入
「アンジオポエチン関連タンパク質３」の前駆体のアミノ酸配列のアミノ末端から２０番目（成熟体のアミノ酸配列の４番目）に相当するアミノ酸（アスパラギン酸）のカルボキシル末端側に、ヒスチジンタグ（配列表の配列番号２６のアミノ酸番号２１乃至２８に示されるアミノ酸配列）が挿入されるように、実施例９の（１）の１）で得られた組換えプラスミドｐＭＥ１８Ｓ／ＫｈＡＰ５を改変した。
【０４８２】
まず、一部が相補的な、下記のヌクレオチド配列からなるオリゴヌクレオチド：
５’−　ｇａｔｃｃｇｃａｔｃａｔｃａｃｃａｔｃａｃｃａｔ　−３’（配列表の配列番号４８）および
５’−　ｇａｔｃａｔｇｇｔｇａｔｇｇｔｇａｔｇａｔｇｃｇ　−３’（配列表の配列番号４９）
を合成し、さらに合成ＤＮＡ末端リン酸化試薬フォスファリンク（アプライドバイオシステム社製）を用いて５’末端をリン酸化した。これらをアニーリングさせたものを、ｐＭＥ１８Ｓ／ＫｈＡＰ５を制限酵素ＢｃｌＩで完全に消化して得られた約４．２ｋｂの断片に挿入した。その結果として、上記のアニーリングさせたオリゴヌクレオチドが３単位連結したＤＮＡが、ＢｃｌＩ切断部位に挿入されたプラスミドクローンが得られた。次に、このクローンをＢｃｌＩで消化したものに、ｐＭＥ１８Ｓ／ＫｈＡＰ５をＢｃｌＩで完全に消化して得られる約１８０ｂｐの断片を挿入することにより、プラスミドｐＭＥ１８Ｓ／ＫｈＡＰ５／Ｈｉｓ＋１６を得た。
【０４８３】
プラスミドｐＭＥ１８Ｓ／ＫｈＡＰ５／Ｈｉｓ＋１６には、ヒスチジンタグをコードする配列が３単位含まれており、その中の３’末端側の２単位は、目的とは逆の方向に連結された余分な配列である。この配列を省いた断片をＰＣＲにより増幅するために、センス／アンチセンス兼用プライマーとして、以下のヌクレオチド配列：
５’−ｇａａｔｔｇａｔｃａｇｃａｔｃａｔｃａｃｃａｔｃａｃｃａｃｇａｔｃ−３’（配列表の配列番号５０）
からなるオリゴヌクレオチドプライマーを合成した。次いで、ｐＭＥ１８Ｓ／ＫｈＡＰ５／Ｈｉｓ＋１６（０．４μｇ／ｍｌ）を鋳型とし、上記プライマー（０．４ｍＭ）、Ｔｂｒ　ＥＸＴ　ＤＮＡポリメラーゼ（フィンザイム社製）、１×ＰＣＲ反応緩衝液（ＤＮＡポリメラーゼに添付の１０×緩衝液を１０分の１量）、２ｍＭの塩化マグネシウム、各０．４ｍＭのｄＮＴＰｓ存在下でＰＣＲを行った。ＰＣＲは、まず９４℃で２分加熱した後に、９４℃で３０秒、６０℃で３０秒、７２℃で３０秒の温度サイクルを３０回繰り返し、最後に７２℃で７分間保温するという条件で行なった。
【０４８４】
ＰＣＲの結果得られた増幅断片（２６８ｂｐ）をＢｃｌＩで消化した後、ｐＭＥ１８Ｓ／ＫｈＡＰ５をＢｃｌＩで完全に切断して得られた約４．２ｋｂの断片に挿入した。以上の操作により、ｐＭＥ１８Ｓ／ＫｈＡＰ５に挿入されているｃＤＮＡ配列中に存在する２ヶ所のＢｃｌＩ切断部位のうち、上流側の切断部位に、ヒスチジンタグをコードするヌクレオチド配列（配列表の配列番号２５のヌクレオチド番号７８乃至１０１）が挿入されたＤＮＡを含む組換えプラスミドｐＭＥ１８Ｓ／ＫｈＡＰ５／Ｈｉｓを得た。
【０４８５】
２）カルボキシル末端側を欠損した改変体の作製
上記１）で得られたプラスミドｐＭＥ１８Ｓ／ＫｈＡＰ５／Ｈｉｓを制限酵素ＳｐｅＩで消化し、Ｔ４　ＤＮＡポリメラーゼで切断末端を平滑化した後に、ＤＮＡリガーゼ反応を行って再び両端を連結することにより、「アンジオポエチン関連タンパク質３」のシグナルペプチドを含むアミノ酸配列中のアミノ末端から２０７番目のアミノ酸残基（セリン）をコードするコドン（ａｔｃ）の後に終止コドン（ｔａｇ）が挿入され、またアミノ末端から２０番目のアミノ酸残基（アスパラギン酸）をコードするコドンの直後にヒスチジンタグをコードするヌクレオチド配列が挿入されたＤＮＡ（配列表の配列番号２５）を含む組換えプラスミドｐＭＥ１８Ｓ／ＫｈＡＰ５　ＣＣ／Ｈｉｓを構築した。
【０４８６】
３）ＣＯＳ−１細胞での発現
直径１５０ｍｍ組織培養用シャーレ（コーニング社製）に、ＣＯＳ−１細胞（０．５×１０^６細胞）を懸濁した１０％　ウシ胎児血清（以下「ＦＣＳ」という。ギブコ社製）を含むダルベッコ変法イーグル培地（以下「ＤＭＥＭ」という。ギブコ社製）３０ｍｌを入れて、３７℃、５％炭酸ガス下で３日間培養した。その後、培地を除去してから、トランスフェクション試薬（リポフェクタミン２０００１８０μｌとＯＰＴＩＭＥＭ−Ｉ溶液（いずれもギブコ社製）　３ｍｌとを混濁したもの）に３６μｇのｐＭＥ１８Ｓ／ＫｈＡＰ５　ＣＣ／Ｈｉｓを加えて混ぜたものをＣＯＳ−１細胞に加えることにより、トランスフェクションを行った。トランスフェクション後、３７℃、５％炭酸ガス下で３日間静置した。以上の操作をシャーレ１３０枚分のＣＯＳ−１細胞に対して実施し、総計約４．４Ｌの培養上清を集めた。この培養上清を、０．２２μｍセルロースアセテート製ボトルトップフィルター（コーニング社製）を用いて吸引ろ過して、ろ液を回収した。
【０４８７】
４）発現量、濃度の確認
上記３）で回収されたろ液の一部と、実施例６に記載の方法で精製された「アンジオポエチン関連タンパク質３」についてＳＤＳ−ＰＡＧＥを実施し、ゲル内のタンパク質をニトロセルロース膜（バイオラッド社製）にウェット法で転写した（０．２Ａ、９０分、４℃）。このニトロセルロース膜を、５％　スキムミルク（雪印乳業（株）社製）を含むＰＢＳ−０．０５％　ツイーン２０溶液（以下「ＰＢＳ−Ｔ」という）に一晩浸してブロッキングを行った。次いで、ニトロセルロース膜をＰＢＳ−Ｔで洗浄した後、ウエスタンブロットを行った。一次抗体には実施例２記載の５５−Ｎ１抗体を、二次抗体には西洋ワサビペルオキシダーゼ標識された抗ウサギポリクローナルＩｇＧ抗体（アマシャム社製）を使用した。５５−Ｎ１抗体が特異的に結合したバンドの検出には、ＥＣＬ試薬（アマシャム社製）を用いた。その結果、上記３）で得られたろ液中には約１．０乃至１．５ｍｇ／リットル（全ろ液中に４．４乃至６．６ｍｇ）のＫｈＡＰ５　ＣＣ／Ｈｉｓが含まれていることが明らかになった。
【０４８８】
５）ＫｈＡＰ５　ＣＣ／Ｈｉｓの精製
上記３）で得られたろ液の全量について、限外ろ過装置（ＴＩＦＦラボスケールおよび３０ＫＤａ孔径の限外ろ過膜（いずれもミリポア社製））を用いて、４４０ｍｌとなるまで限外濃縮した。さらに、得られた試料に、限外ろ過装置中で２０ｍＭ　リン酸ナトリウム、０．５Ｍ　塩化ナトリウム、０．１％　アジ化ナトリウムおよび０．０５％　プロテアーゼインヒビターカクテル（シグマ社製）を含む緩衝液（ｐＨ７．４）４リットルを徐々に加えながら限外ろ過を続行することにより、溶媒が該緩衝液に置換された試料を得た（液量６００ｍｌ）。
【０４８９】
一方、キレーティングカラム（ＨｉＴｒａｐキレーティング　５ｍｌカラム。アマシャム社製）に０．１Ｍの硫酸ニッケル　５ｍｌを約５ｍｌ／分の流速で流すことにより、担体にニッケルイオンを結合させた。このカラムに超純水を流して洗浄した後、ＨＰＬＣ送液システム（アマシャム社製）に装着して、以下に記載する条件で試料のアフィニティー精製を行った。
【０４９０】
溶媒：
Ａ液：　２０ｍＭ　リン酸ナトリウム、０．５Ｍ　塩化ナトリウム（ｐＨ７．４）、０．１％　アジ化ナトリウム、０．０５％　プロテアーゼインヒビターカクテル（シグマ社製）
Ｂ液：　２０ｍＭ　リン酸ナトリウム、０．５Ｍ　塩化ナトリウム、０．５Ｍイミダゾール（ｐＨ７．４）、０．１％　アジ化ナトリウム、０．０５％　プロテアーゼインヒビターカクテル（シグマ社製）
流速：　５ｍｌ／分
精製手順：　試料６００ｍｌをカラムに流した後、Ａ液　８０％、Ｂ液　２０％の緩衝液（０．１Ｍ　イミダゾール濃度に相当）５０ｍｌを流して非吸着物を洗浄除去した。その後、Ｂ液　５０ｍｌをカラムに流し、ヒスチジンタグを有するポリペプチドを溶出させ、溶出液を回収した。この溶出液にＡ液２００ｍｌを加えて、イミダゾール濃度０．１Ｍの溶液としたものを、第２回のアフィニティー精製用試料とした。
【０４９１】
小スケールのキレーティングカラム（ＨｉＴｒａｐキレーティング　１ｍｌカラム。アマシャム社製）に０．１Ｍの硫酸ニッケル　５ｍｌを約１ｍｌ／分の流速で流すことにより、担体にニッケルイオンを結合させ、次いで超純水を流して洗浄した後、カラムをＨＰＬＣ送液システムに装着し、試料を流してから、カラムをＨＰＬＣから取り外した。次いで、２０ｍＭ　リン酸ナトリウム、０．５Ｍ塩化ナトリウム、０．１Ｍ　イミダゾール、ｐＨ７．４の緩衝液を、１ｍｌ／分の流速で１ｍｌカラムに手動で流して、非吸着物を洗浄除去した。その後、２０ｍＭ　リン酸ナトリウム、０．５Ｍ　塩化ナトリウム、０．３Ｍ　イミダゾール、ｐＨ７．４の緩衝液を同様に１ｍｌ／分の流速で流し、溶出液を１ｍｌずつ、５分画（計５ｍｌ）回収した。
【０４９２】
回収した溶出液をそれぞれ透析チューブ（ギブコ社製）に入れて、５００ｍＬのＰＢＳ（ギブコ社製）に対して透析を行った。透析膜外液であるＰＢＳは、３回交換した。最後の透析膜外液を一部採取して、ＬＰＬ活性評価の際の陰性対照試料とした。
【０４９３】
回収した試料中のタンパク質濃度を、プロテインアッセイ（ＤＣプロテインアッセイキット（バイオラッド社製）を使用）やＳＤＳ−ＰＡＧＥ等で見積もった結果、計１．８ｍｇのＫｈＡＰ５　ＣＣ／Ｈｉｓが得られたことが判明した。そして、下記のＬＰＬ活性評価には、第２回のアフィニティー精製により得られた第２および第３の分画をまとめたものを用いた。
【０４９４】
（２）ＬＰＬ活性の測定
上記（１）の５）で得られたＫｈＡＰ５　ＣＣ／Ｈｉｓ試料を、ＧＳＴ−ＣＣの代わりに用いて、実施例１１の（１）記載の方法でＬＰＬ活性を測定した。その結果を表８に示す。
【０４９５】
【表８】

ＫｈＡＰ５　ＣＣ／Ｈｉｓは、ＬＰＬ活性を強く阻害した。この実験系において、リパーゼ反応の際に被験物質を添加すれば、該物質がＬＰＬの活性を調節する作用を有するか否かを調べることができる。
【０４９６】
（３）血中中性脂肪濃度の低下作用
実施例１０の（３）記載の方法に従って、ＫｈＡＰ５　ＣＣ／ＨｉｓをＫＫ／Ｓｎｋマウスに尾静脈投与した（１００μｇ／マウス）。投与前と投与後のマウスから末梢血を採取して中性脂肪濃度を測定した。その結果、投与前のマウスでは血中中性脂肪濃度が４０．５ｍｇ／ｄｌであったのに対し、ＫｈＡＰ５　ＣＣ／Ｈｉｓ投与３０分後では１０６．０ｍｇ／ｄｌに上昇していた。溶媒のみを投与した対照群の投与３０分後の血中中性脂肪濃度は４１．３ｍｇ投与群であった。以上の結果より、ＫｈＡＰ５　ＣＣ／Ｈｉｓは血中中性脂肪濃度を有意に上昇させることが確かめられた。
【０４９７】
実施例１４．ヘパリン結合部位に変異を導入したアンジオポエチン関連タンパク質３の血漿トリグリセリドに対する効果
（１）ヘパリン結合モチーフに変異を導入したアンギオポエチン関連蛋白−３（ＡＲＰ３）のＮ末２０７アミノ酸部分蛋白の構築
実施例１３の２）記載のプラスミドｐＭＥ１８Ｓ／ＫｈＡＰ５　ＣＣ／ＨｉｓをＥｃｏＲＩとＸｂａＩで切断して得られる約１．４ｋｂｐの断片を単離し、宝酒造製のＭｕｔａｎ−Ｓｕｐｅｒ　Ｅｘｐｒｅｓｓ　Ｋｍキットに添付のプラスミドｐＫＦ１８ｋのＥｃｏＲＩ／ＸｂａＩ切断点に挿入した。変異導入用プライマーＧＴＣＡＴＡＡＡＣＡＣＧＡＡＣＧＧＣＣＡＡＡＴＴＡＡＴＧＡＣを用い、キットの説明書に従って変異を導入し、プラスミドｐＫＦ１８／ＫｈＡＰ５　ＣＣ（−ＨＢ）／Ｈｉｓを得た。得られたプラスミドクローンの塩基配列を決定して、変異導入が行われたことおよび他の部分に変異が入っていないことを確認した。結果として、配列表の配列番号１２の６２番目のヒスチジン、６３番目リジン、６５番目のリジンがそれぞれイソロイシン、アスパラギン、アスパラギンへ置換した蛋白をコードするＤＮＡ配列が作製された。得られたプラスミドをＥｃｏＲＩとＸｂａＩで切断して約１．４ｋｂｐの断片を単離し、高発現ベクターｐＭＥ１８Ｓ（Ｈａｒａ，　Ｔ．　ｅｔ　ａｌ．（１９９２）　ＥＭＢＯ．Ｊ．　１１，　１８７５−、横田崇、新井賢一編集、バイオマニュアルシリーズ３、遺伝子クローニング実験法、羊土社、ｐ１８−２０）のＥｃｏＲＩ／ＸｂａＩ切断点に挿入した（ｐＭＥ１８Ｓ／ＫｈＡＰ５　ＣＣ（−ＨＢ））。
【０４９８】
（２）ヘパリン結合モチーフに変異を導入したアンギオポエチン関連蛋白−３（ＡＲＰ３）のＮ末２０７アミノ酸部分蛋白の活性に関する検討
１）マウス血漿中トリグリセリド濃度上昇活性
Ａ）組換えアデノウイルスの作製
ＡＲＰ３のＮ末２０７アミノ酸部分蛋白（ＡＲＰ３　ＣＣ）およびヘパリン結合モチーフに変異を導入したＡＲＰ３のＮ末２０７アミノ酸部分蛋白（ＡＲＰ３　ＣＣ（−ＨＢ））を強制発現させるため組換えアデノウイルスは、市販のキット（アデノウイルス・エクスプレッション・ベクター・キット、宝酒造（株）社製）を用いて作製した。すなわち、ｐＭＥ１８Ｓ／ＫｈＡＰ５　ＣＣ／ＨｉｓあるいはｐＫＦ１８／ＫｈＡＰ５　ＣＣ（−ＨＢ）／ＨｉｓをＥｃｏＲＩおよびＸｂａＩで消化し、得られた約１．４ｋｂのＤＮＡ断片の末端を平滑化したものを挿入ＤＮＡ断片として以下の操作に用いた。
【０４９９】
また、サイトメガロウイルスエンハンサーとニワトリβ−アクチンプロモーターにより発現されるように設計されているコスミドベクターｐＡｘＣＡｗｔ（アデノウイルス・エクスプレッション・ベクター・キットに添付）の制限酵素ＳｗａＩ認識部位にインサートＤＮＡ断片を挿入したコスミドｐＡｘＣＡ／ＫｈＡＰ５　ＣＣ／ＨｉｓおよびｐＡｘＣＡ／ＫｈＡＰ５　ＣＣ（−ＨＢ）／Ｈｉｓを作製した。ｐＡｘＣＡ／ＫｈＡＰ５　ＣＣ／Ｈｉｓ　ＤＮＡあるいはｐＡｘＣＡ／ＫｈＡＰ５　ＣＣ（−ＨＢ）／Ｈｉｓ　ＤＮＡと末端タンパク質結合ウイルスＤＮＡ（ＤＮＡ−ＴＰＣ、アデノウイルス・エクスプレッション・ベクター・キットに添付）をリン酸カルシウムトランスフェクションシステム（ライフテック社製）を用いて２９３細胞（ＡＴＣＣ　ＣＲＬ１５７３）にコ・トランスフェクションして、組換えアデノウイルスＡｄ／ＫｈＡＰ５　ＣＣ／ＨｉｓおよびＡｄ／ＫｈＡＰ５　ＣＣ（−ＨＢ）／Ｈｉｓを単離し、さらに２９３細胞中で増幅させた。増幅させたウイルスの２９３細胞からの回収は、まずウイルス感染２９３細胞に３０秒×４回の超音波処理（ブランソン社製Ｂ−１２００を使用）を行い、次いで塩化セシウム密度勾配遠心による精製を２回繰り返すことによって行った。得られたウイルス液を、１０％　グリセロールを添加したＰＢＳに対して４℃で透析した後、使用するまで−７０℃以下で凍結保存した。
【０５００】
Ｂ）組換えアデノウイルスのマウス接種後の血漿中トリグリセリド濃度
上記のようにして精製された組換えアデノウイルスＡｄ／ＫｈＡＰ５　ＣＣ／ＨｉｓおよびＡｄ／ＫｈＡＰ５　ＣＣ（−ＨＢ）／Ｈｉｓを、１０％グリセロールを含むＰＢＳで２×１０^１０ｐｆｕ／ｍｌに希釈し、それぞれ２匹（Ａｄ／ＫｈＡＰ５　ＣＣ／Ｈｉｓ）または３匹（Ａｄ／ＫｈＡＰ５　ＣＣ（−ＨＢ）／Ｈｉｓ）の２０−２１週齢の雄ＫＫ／Ｓｎｋマウスに２００μｌ（５×１０^９ｐｆｕ）ずつ尾静脈注射により接種した。接種前、接種１、２、４、７、１１、１４日後に各マウスの眼底よりヘマトクリット管で採血して、卓上遠心機で５２００ｒｐｍ１５分遠心して血漿を分離した。接種後１日後の血漿１μｌを２−メルカプトエタノールを含むＳＤＳ−ＰＡＧＥ試料緩衝液（バイオラッド社製）と混合した後、９９℃で５分間加熱して、電気泳動用試料を調製した。試料を中でゲル濃度４−２０％のポリアクリルアミド密度勾配ゲルにて電気泳動した（電気泳動用緩衝液：２５ｍＭ　トリス、１９２ｍＭ　グリシン、０．１％　ＳＤＳ）後、転写緩衝液（２５ｍＭ　トリス、１９２ｍＭ　グリシン、２０％メタノール）中で４℃、１時間、２００ｍＡの条件でニトロセルロースメンブレンに転写した。転写したメンブレンは０．５％　スキムミルクを添加したＰＢＳ液で４℃で一晩ブロッキングし、洗浄液（０．０５％　Ｔｗｅｅｎ２０−ＰＢＳ）で３回洗浄した。次いで、メンブレンを、２７０ｎｇ／ｍｌの実施例２記載の５５−１−Ｎ１抗体および５％　ウシ胎児血清を含む０．０５％　Ｔｗｅｅｎ２０−ＰＢＳ液中に入れて室温で１時間インキュベーションした後、洗浄液で３回洗浄した。さらに、メンブレンを西洋ワサビペルオキシダーゼ標識ヤギ抗ウサギＩｇＧ（Ｈ＋Ｌ）（アマシャム・バイオサイエンス社製）を５％　ウシ胎児血清を含む０．０５％　Ｔｗｅｅｎ２０−ＰＢＳで２０００倍希釈した反応液中で室温で１時間インキュベーションした後、洗浄液で５回洗浄した。このメンブレンをラップフィルム上に置き、ＥＣＬウエスタンブロッティング検出溶液に１分間浸した後、Ｘ線フィルムを感光させた。その結果、組換えアデノウイルスＡｄ／ＫｈＡＰ５　ＣＣ／Ｈｉｓを感染させたマウス血漿中およびＡｄ／ＫｈＡＰ５　ＣＣ（−ＨＢ）／Ｈｉｓを感染させたマウス血漿中に特異的なバンドが検出された（図９）。
【０５０１】
続いて中性脂肪測定用キット（トリグリセリドＥ−テストワコー、和光製薬（株）社製）を用いて中性脂肪濃度を測定した。Ａｄ／ＫｈＡＰ５　ＣＣ／Ｈｉｓ接種マウス群では血中の中性脂肪濃度が顕著に上昇したのに対し、Ａｄ／ＫｈＡＰ５　ＣＣ（−ＨＢ）／Ｈｉｓ接種マウス群では血漿中の中性脂肪濃度はほとんど上昇しなかった（図１０）。
【０５０２】
２）リポタンパク質リパーゼ（ＬＰＬ）阻害活性
Ａ）組換えタンパクの調製
ｐＭＥ１８Ｓ／ＫｈＡＰ５　ＣＣ／ＨｉｓあるいはｐＫＦ１８／ＫｈＡＰ５　ＣＣ（−ＨＢ）／ＨｉｓをＬｉｐｏｆｅｃｔＡＭＩＮＥ２０００試薬（インビトロジェン社製）を用いて、ＣＯＳ細胞（Ｇｌｕｚｍａｎ，　Ｙ．　（１９８１）　Ｃｅｌｌ　２３，　１７５−１８２、ＡＴＣＣ　ＣＲＬ−１６５０）に導入し、血清非添加のダルベッコ改変イーグル培地で４日培養した後に培養上清を回収した。培養上清をＴＩＦＦラボスケール（ミリポア社）で３０Ｋのフィルター（ミリポア社）を用いて、約１０分の１量に限外濃縮した。濃縮したサンプルは、１０倍量の５０ｍＭイミダゾール、０．５Ｍ塩化ナトリウム、０．０２Ｍリン酸ナトリウムバッファーで循環させて、本組成のバッファーに置換した。バッファー置換したサンプルは、予め０．５ｍｌの０．１Ｍ硫酸ニッケルで処理し、５０ｍＭイミダゾール、０．５Ｍ塩化ナトリウム、０．０２Ｍリン酸ナトリウムバッファーに置換したＨｉＴｒａｐ　Ｃｈｅｌａｔｉｎｇカラム（アマシャムバイオサイエンス社）に結合させた後、１００ｍＭイミダゾール、０．５Ｍ塩化ナトリウム、０．０２Ｍリン酸ナトリウムバッファーで洗浄し、３００ｍＭイミダゾール、０．５Ｍ塩化ナトリウム、０．０２Ｍリン酸ナトリウムバッファーで溶出した。得られた溶出サンプルを１，０００倍量のＰＢＳ（―）（シグマ社）で３回透析したものをそれぞれ組換えタンパクＡＲＰ３
ＣＣ、ＡＲＰ３　ＣＣ（−ＨＢ）とした。
【０５０３】
Ｂ）ＬＰＬ阻害活性
上記で得られた組換えタンパク質のＬＰＬ阻害活性を実施例１１）の（１）の２）記載の方法で検討した。ＡＲＰ３　ＣＣ（―ＨＢ）のＬＰＬ阻害活性はＡＲＰ３　ＣＣのＬＰＬ阻害活性に比べて著しく低かった（図１１）。
【０５０４】
以上より、ＡＲＰ３　ＣＣ（―ＨＢ）はＡＲＰ３の機能阻害剤のスクリーニングの際の陰性対照として利用できる。
【０５０５】
実施例１５．ヒスチジンタグ付加蛋白発現アデノウイルスベクターを接種したマウスの血漿の調製
（１）組換えアデノウイルスの調製
アデノウイルス発現ベクターキット（宝酒造（株）社製）を用いて、以下の方法にしたがって、組換えアデノウイルスを作製した。実施例１３の（１）の１）で得られた、組換えプラスミドｐＭＥ１８Ｓ／ＫｈＡＰ５／Ｈｉｓを、制限酵素ＥｃｏＲＩおよびＸｂａＩで消化して、約１．４ｋｂのＤＮＡ断片を回収し、切断末端を平滑化した。次に、サイトメガロウイルスエンハンサーとニワトリβ−アクチンプロモーターにより挿入遺伝子が発現されるように設計されているコスミドベクターｐＡｘＣＡｗｔのＳｗａＩ切断部位にこれらの断片をそれぞれ挿入し、組換えコスミドｐＡｘＣＡ／ＫｈＡＰ５／Ｈｉｓを調製した。
【０５０６】
この組換えコスミドＤＮＡと末端タンパク質結合ウイルスＤＮＡ（ＤＮＡ−ＴＰＣ、アデノウイルス発現ベクターキットに添付）をリン酸カルシウムトランスフェクションシステム（ライフテック社製）を用いて２９３細胞（ＡＴＣＣ　ＣＲＬ１５７３）にコ・トランスフェクションして、組換えアデノウイルスＡｄ／ＫｈＡＰ５／Ｈｉｓを単離し、さらに２９３細胞内で増幅させた。増幅させたウイルスの２９３細胞からの回収は、まずウイルス感染２９３細胞に３０秒×４回の超音波処理（ブランソン社製Ｂ−１２００を使用）を行うか、または凍結融解を３回繰り返し、次いで塩化セシウム密度勾配遠心による精製を２回繰り返すことによって行った。得られたウイルス液を、１０％　グリセロールを添加したＰＢＳに対して４℃で透析した後、使用するまで−７０℃以下で凍結保存した。
【０５０７】
（２）組換えアデノウイルスによる目的タンパク質の発現の確認
上記（１）記載の方法で得られた組換えアデノウイルスをＨｅＬａ細胞に感染させて、目的タンパク質の発現を確認した。
【０５０８】
まず、上記（１）記載の方法で得られた組換えアデノウイルスをそれぞれＨｅＬａ細胞（ＡＴＣＣ　ＣＣＬ２）に約５ｍ．ｏ．ｉ．（ｍｕｌｔｉｐｌｉｃｉｔｙ　ｏｆ　ｉｎｆｅｃｔｉｏｎ：多重感染度）で感染させ、無血清培地（ダルベッコ修正イーグル培地（ＤＭＥＭ））で３乃至４日培養した後の培養上清を回収した。培養上清１ｍｌを１．５ｍｌ容のエッペンドルフチューブに入れ、１００μｌのトリクロロ酢酸溶液（１ｇ／ｍｌ　トリクロロ酢酸、４ｍｇ／ｍｌ　デオキシコール酸）を添加して室温で３分間放置後、卓上遠心機にて１５０００ｒｐｍで５分遠心した。上清を除いて、氷冷したアセトンを０．５ｍｌ加えて攪拌し、卓上遠心機にて１５０００ｒｐｍで２分遠心した。上清を除いて、沈殿に再度氷冷したアセトンを０．５ｍｌ加えて攪拌し、卓上遠心機にて１５０００ｒｐｍで２分遠心して、上清を除き、沈殿物を真空乾燥した。この沈殿物をそれぞれ１０μｌの蒸留水に溶解させ、２−メルカプトエタノールを含むＳＤＳ−ＰＡＧＥ試料緩衝液（バイオラッド社製）と混合した後、９９℃で５分間加熱して、電気泳動用試料を調製した。これら試料をゲル濃度４−２０％のポリアクリルアミド密度勾配ゲルにて電気泳動した（電気泳動用緩衝液：２５ｍＭ　トリス、１９２ｍＭ　グリシン、０．１％ＳＤＳ）後、転写緩衝液（２５ｍＭ　トリス、１９２ｍＭ　グリシン、２０％メタノール）中で４℃、１時間、２００ｍＡの条件でニトロセルロースメンブレンに転写した。
【０５０９】
転写したメンブレンは０．５％　スキムミルクを添加したＰＢＳ液中に浸して４℃で一晩ブロッキングし、洗浄液（０．０５％　Ｔｗｅｅｎ２０−ＰＢＳ）で３回洗浄した。次いで、メンブレンを、約２７０ｎｇ／ｍｌの実施例２記載の５５−１−Ｎ１抗体および５％　ウシ胎児血清を含む０．０５％　Ｔｗｅｅｎ２０−ＰＢＳ液中に入れて室温で１時間インキュベーションした後、洗浄液で３回洗浄した。
【０５１０】
さらに、メンブレンを西洋ワサビペルオキシダーゼ標識ヤギ抗ウサギＩｇＧ（Ｈ＋Ｌ）（バイオラッド社製）を５％　ウシ胎児血清を含む０．０５％　Ｔｗｅｅｎ２０−ＰＢＳで２０００倍希釈した反応液中で室温で１時間インキュベーションした後、洗浄液で５回洗浄した。このメンブレンをラップフィルム上に置き、発光試薬溶液（ＥＣＬウエスタン検出試薬。アマシャム・ファルマシア・バイオテク社製）に１分間浸した後、１時間室温で放置してバックグラウンドを減衰させてから、Ｘ線フィルムを感光させた（１０秒間）。このＸ線フィルムを現像した結果、組換えアデノウイルスを感染させたＨｅＬａ細胞において、目的のポリペプチドが産生されていることが確かめられた。
【０５１１】
（３）組換えアデノウイルスを接種したＫＫ／Ｓｎｋマウス血漿の調製
上記（１）で精製した組換えアデノウイルスＡｄ／ＫｈＡＰ５／Ｈｉｓを１０％グリセロール添加ＰＢＳで７．２×１０^９ｐｆｕ／ｍｌに希釈し、５匹の１８週齢の雄ＫＫ／Ｓｎｋマウスに３００μｌずつ尾静脈注射により接種した。
【０５１２】
接種２日後に、各マウスの腹大動脈より６０μｌの１％ＥＤＴＡを予め入れた注射筒（テルモ社製の２６ゲージの注射針および１ｍｌのシリンジ）を用いて採血し、１．５ｍｌのエッペンドルフチューブに移し、転倒混和後に、卓上遠心機で５２００ｒｐｍで１５分間遠心して血漿を分離した。
【０５１３】
（４）ＫＫ／Ｓｎｋマウス血漿中目的タンパク質の精製
得られた血漿を１０ｍＭイミダゾール／ＰＢＳ、４℃で４日間透析後、ＨｉＴｒａｐ　Ｃｈｅｌａｔｉｎｇ　ＨＰカラム（Ａｍｅｒｓｈａｍ社）へ添加し、２０ｍＭイミダゾール／ＰＢＳおよび１００ｍＭイミダゾール／ＰＢＳ洗浄を行った。溶出は３００ｍＭイミダゾール／ＰＢＳで行い、各フラクションはゲル濃度４−２０％のポリアクリルアミド密度勾配ゲルにて電気泳動（電気泳動用緩衝液：２５ｍＭ　トリス、１９２ｍＭ　グリシン、０．１％　ＳＤＳ）後、ＧｅｌＣｏｄｅ染色（Ｐｉｅｒｃｅ社）を行い目的タンパク質の有無を確認した。
【０５１４】
（５）ＫＫ／Ｓｎｋマウス血漿中目的タンパク質切断点の同定
Ｈｉｓ−ｔａｇアフィニティー精製で得たヒト・「アンジオポエチン関連タンパク質３」画分に終濃度１０　ｍＭ　ｄｉｔｈｉｏｔｈｒｅｉｔｏｌ　（Ｓｉｇｍａ，　Ｓｔ．　ｌｏｕｉｓ，　ＭＯ）を加えて還元した後、終濃度５５　ｍＭ　ｉｏｄｏａｃｅｔａｍｉｄｅ　（Ｗａｋｏ，　Ｏｓａｋａ，　Ｊａｐａｎ）を加えアルキル化反応を行った。その後Ｌｙｓｙｌ　Ｅｎｄｏｐｅｐｔｉｄａｓｅ　（Ｗａｋｏ，　Ｏｓａｋａ，　Ｊａｐａｎ）を加えて３７　℃で一晩消化、またはＰＮＧａｓｅ　Ｆ　（Ｎｅｗ　Ｅｎｇｌａｎｄ　Ｂｉｏｌａｂｓ，　Ｂｅｖｅｒｌｙ，　ＭＡ）で３７　℃、２時間糖鎖除去処理を行った後、Ｓｅｑｕｅｎｃｉｎｇ　Ｇｒａｄｅ　Ｅｎｄｏｐｒｏｔｅｉｎａｓｅ　Ｇｌｕ−Ｃ　（Ｐｒｏｍｅｇａ，　Ｍａｄｉｓｏｎ，　ＷＩ）を加えて３７　℃で一晩消化した。消化反応液に０．０５　％　ｆｏｒｍｉｃ　ａｃｉｄ　／　Ｈ２Ｏを加えＬＣ／ＥＳＩ　ＭＳ／ＭＳ　（ｌｉｑｕｉｄ　ｃｈｒｏｍａｔｏｇｒａｐｈｙ　ｅｌｅｃｔｒｏｓｐｒａｙ　ｉｏｎｉｚａｔｉｏｎｔａｎｄｅｍ　ｍａｓｓ　ｓｐｅｃｔｒｏｍｅｔｒｙ）測定の試料とした。ＬＣには逆相クロマトグラフィー担体Ｉｎｅｒｔｓｉｌ　ＯＤＳ−２　（ＧＬ　Ｓｃｉｅｎｃｅ，　Ｔｏｋｙｏ，　Ｊａｐａｎ）を詰めた自作のエレクトロスプレーニードルをカラムとして用い、ＣａｐＬＣ　Ｓｙｓｔｅｍ　（Ｗａｔｅｒｓ，　Ｍｉｌｆｏｒｄ，　ＭＡ）で送液した。溶出溶媒としてＡ液：０．０５　％　ｆｏｒｍｉｃ　ａｃｉｄ　／　Ｈ２Ｏ　、Ｂ液：０．０５　％　ｆｏｒｍｉｃａｃｉｄ　／　ａｃｅｔｏｎｉｔｒｉｌｅ　を使用し、１時間で０　％から３０　％　までＢ液濃度を直線的に上げるグラジエント溶出を行った。溶出液は直接Ｑ−Ｔｏｆ２　（Ｍｉｃｒｏｍａｓｓ，　Ｍａｎｃｈｅｓｔｅｒ，　ＵＫ）に導入してＥＳＩ　ＭＳ／ＭＳ　測定を行った。
【０５１５】
得られたＭＳスペクトルからヒト・「アンジオポエチン関連タンパク質３」のペプチドマッピングを行ったところ、Ｌｙｓｙｌ　Ｅｎｄｏｐｅｐｔｉｄａｓｅで消化したサンプルにはヒト・「アンジオポエチン関連タンパク質３」に由来するＥＩＥＮＱＬＲＲＴＳＩＱＥＰＴＥＩＳＬＳＳＫＰＲ（アミノ酸残基１９８−２２１）、ＥＩＥＮＱＬＲＲＴＳＩＱＥＰＴＥＩＳＬＳＳＫＰＲＡＰＲ（１９８−２２４）という２本のペプチドが、ｅｎｄｏｐｒｏｔｅｉｎａｓｅ　Ｇｌｕ−Ｃで消化したサンプルにはＩＳＬＳＳＫＰＲ（２１４−２２１）、ＩＳＬＳＳＫＰＲＡＰＲ（２１４−２２４）という２本のペプチドが存在した。これらのペプチドのカルボキシル末端はこの２種の酵素の基質認識部位ではない。従ってヒト・「アンジオポエチン関連タンパク質３」の血中切断部位としてＡｒｇ２２１とＡｌａ２２２の間およびＡｒｇ２２４とＴｈｒ２２５の間の２ヶ所を確認した。
【０５１６】
実施例１６．　融合アンジオポエチン関連タンパク質４の活性評価
（１）融合タンパク質の調製
配列表の配列番号１６のアミノ酸番号２６乃至１４３に示されるアミノ酸配列からなるポリペプチドのアミノ末端にグルタチオン・Ｓ・トランスフェラーゼ（以下「ＧＳＴ」という）が連結している融合タンパク質（以下、「ＧＳＴ−ＡＲＰ４Ｎ」という）を以下に記載する方法に従って調製した。
【０５１７】
まず、ＰＣＲ用プライマーとして、下記のヌクレオチド配列：
５’−　ｔｃｃｃｃｃｇｇｇｇｇａｃｃｃｇｔｇｃａｇｔｃｃａａｇ−３’（ＧＳＴ−ＡＲＰ４Ｎ用のセンスプライマー。配列表の配列番号５１）；および
５’−　ｃｃｇｃｔｃｇａｇｃｔｇｇｃｔｔｔｇｃａｇａｔｇｃｔｇ−３’（ＧＳＴ−ＡＲＰ４Ｎ用アンチセンスプライマー。配列表の配列番号５２）を合成した。
【０５１８】
次に、Ｉ．Ｍ．Ａ．Ｇ．Ｅ　Ｃｏｎｓｏｒｔｉｕｍより購入したｃＤＮＡ　ｃｌｏｎｅ（ｃｌｏｎｅ　４１４９０３９）を鋳型とし、センスプライマーおよびアンチセンスプライマー（それぞれ０．２μＭ）、Ｔｂｒ　ＥＸＴ　ＤＮＡポリメラーゼ、１×ＰＣＲ緩衝液（Ｔｂｒ　ＥＸＴ　ＤＮＡポリメラーゼに添付の１０×緩衝液を１０倍希釈）、２ｍＭの塩化マグネシウムおよび各０．４ｍＭのｄＮＴＰｓ存在下でＰＣＲを実施した。ＰＣＲは、上記サンプルを９４℃で３分加熱した後に、９４℃で３０秒、５５℃で３０秒、７２℃で３０秒の温度サイクルを３０回繰り返した後、最後に７２℃で７分間の条件で行なった。
【０５１９】
ＰＣＲで増幅した約３７０ｂｐのＤＮＡ断片を回収し、ＳｍａＩおよびＸｈｏＩで消化した。ＳｍａＩおよびＸｈｏＩで消化してからウシ小腸アルカリホスファターゼ（宝酒造（株）社製）を用いて切断末端の脱リン酸化処理を行ったプラスミドベクターｐＧＥＸ−４Ｔ−２（ＧＳＴをコードする遺伝子を含む。アマシャム・ファルマシア・バイオテク社製）にこの断片を挿入し、挿入断片を含むプラスミドクローンを単離した。このプラスミドクローンをｐＧＳＴ−ＡＲＰ４Ｎと命名した。
【０５２０】
組換えプラスミドクローンｐＧＳＴ−ＡＲＰ４Ｎ、または、挿入断片を含まずＧＳＴのみを発現させるためのコントロールベクターｐＧＥＸ−４Ｔ−２で大腸菌ＪＭ１０９株をそれぞれ形質転換した。形質転換は大腸菌ＪＭ１０９株のコンピテントセルにプラスミドを添加し、氷上で３０分間静置した後、４２℃で３０秒間保温し、再び氷上で２分間静置してから、３７℃で１時間振盪培養をすることによって行なった。形質転換後の培養液をアンピシリン添加ＬＢ寒天プレート上に撒き、出現した組換え体のコロニーをピックアップして液体培養し、ＩＰＴＧ誘導による組換えタンパク質の発現を確認した。
【０５２１】
（２）融合タンパク質の調製
１）形質転換大腸菌の培養
プラスミドクローンｐＧＳＴ−ＡＲＰ４Ｎ、コントロールベクターｐＧＥＸ−４Ｔ−２を保持する形質転換大腸菌をそれぞれ、アンピシリン入りのＬＢ培地に植菌し、３７℃で１８時間振とう培養した。培養液の一部を新しい培地に添加し、さらに３７℃にて振とう培養を行なった。６００ｎｍでの吸光度が０．５−０．７に達した時に、最終濃度１ｍＭとなるようにイソプロピル・チオガラクトピラノシド（ＩＰＴＧ）を添加しさらに４時間振とう培養を継続した。
【０５２２】
２）ｐＧＥＸ−４Ｔ−２を保持した大腸菌からの細胞破砕液の調製
コントロールベクターｐＧＥＸ−４Ｔ−２で形質転換した大腸菌の培養液を４℃、６０００ｒｐｍ、１０分間の条件で遠心処理して沈殿した菌体を回収した。回収した菌体を超音波処理用緩衝液（５０ｍＭ　トリス−塩酸（ｐＨ８．０）、５０ｍＭ　塩化ナトリウム、１ｍＭ　ＥＤＴＡ、１ｍＭ　ＤＴＴ）で懸濁し、懸濁液を４℃、６０００ｒｐｍ、１０分間の条件で遠心処理した。沈殿を−８０℃で凍結し、次いで室温にて融解して再度超音処理用緩衝液に懸濁した。懸濁液を氷浴中で超音波破砕器（トミー精工（株）社製ＵＤ−２０１）によって、出力＝５、デューティ＝５０の条件で５分間処理した。超音波処理後の破砕液に最終濃度１％となるようにトリトン　Ｘ−１００を添加し混和し、４℃、１００００ｒｐｍ、３０分間の条件で遠心処理し、上清を回収した。上清はＧＳＴｒａｐカラム（アマシャム・ファルマシア・バイオテク社製）中に流した。
【０５２３】
３）ｐＧＳＴ−ＡＲＰ４Ｎを保持する大腸菌からの細胞破砕液の調製
組換えプラスミドｐＧＳＴ−ＡＲＰ４Ｎで形質転換した大腸菌の培養液それぞれを、４℃、５０００ｒｐｍ、１５分間の条件で遠心処理し、沈殿した菌体を回収した。沈殿した菌体に対して、洗浄用緩衝液（０．５％　トリトン　Ｘ−１００、１ｍＭ　ＥＤＴＡ）での懸濁、４℃、５０００ｒｐｍ、１０分間の条件での遠心処理、の操作を３回繰り返し、得られた菌体を８Ｍ　尿素溶液に溶解した。この溶液を室温に１時間放置した後、４℃、１３０００ｒｐｍ、３０分間の遠心処理を行ない上清を回収した。得られた上清を透析した。透析は、４Ｍ　尿素溶液に対して４℃で１時間、２Ｍ　尿素溶液に対して４℃で１時間、超音波処理用緩衝液に対して４℃で１時間、超音波処理用緩衝液に対して４℃で一晩の順で行なった。透析後、４℃、１２０００ｒｐｍ、３０分間の条件で遠心し、ＧＳＴｒａｐカラム中に流した。
【０５２４】
４）融合タンパク質の精製
上記の工程を経てＧＳＴ、ＧＳＴとの融合タンパク質（以下、「ＧＳＴ−ＡＲＰ４Ｎ」という）が吸着したＧＳＴカラムにＰＢＳを流してカラム内を洗浄して不純物を除去した。次いで、１０ｍＭ　還元型グルタチオン溶液を流して、ＧＳＴ、ＧＳＴ−ＡＲＰ４Ｎを溶出させた。溶出液の一部をＳＤＳ−ＰＡＧＥ電気泳動にで分離した後、ゲルをクマシー染色して、タンパク質の存在を確認した。また、溶出液をＰＢＳで４℃、一晩透析した。
【０５２５】
（２）血中中性脂肪濃度上昇活性
上記（１）で精製したＧＳＴおよびＧＳＴ−ＡＲＰ４Ｎを、ＰＢＳでタンパク質濃度約１ｍｇ／ｍｌに調整し、それぞれ２匹の１９週齢の雄ＫＫ／Ｓｎｋマウスに約３００μｌずつ尾静脈注射により接種した。採血は摂取前と接種３０分後に行なった。ヘマトクリット管で各マウスの眼底より採血して、卓上遠心機で５２００ｒｐｍ１５分遠心して血漿を分離し、接種前後における血中中性脂肪濃度を測定した。結果を表９に示した。
【０５２６】
【表９】

ＧＳＴ接種マウス群では接種前後で差が認められなかったのに対し、ＧＳＴ−ＡＲＰ４Ｎ接種マウス群では有意に血中中性脂肪濃度の上昇が認められた。
【０５２７】
本実施例の方法において、例えばＧＳＴ−ＡＲＰ４Ｎを接種したマウスに、被験物質を投与し、血中中性脂肪濃度の変動を調べることにより、高脂血症、動脈硬化および高血糖から選択される一つまたは二つ以上の疾患の治療または予防剤の試験を行うことができる。
【０５２８】
実施例１７．　ＬＰＬ活性阻害効果
（１）ＬＰＬ試料の調製
ラット白色脂肪前駆細胞（ホクドー社より購入）を増殖用培地［１０％　ウシ胎児血清（以下「ＦＣＳ」という）、１００単位／ｍｌ　ペニシリン、１００μｇ／ｍｌ　ストレプトマイシン、１７μＭ　パントテン酸、３３μＭ　（＋）−ビオチン、１００μＭ　アスコルビン酸、１μＭ　オクタン酸および５０ｎＭ　トリヨードチロニンを含むダルベッコ変法イーグル培地（以下「ＤＭＥＭ」という）］の入った細胞培養用フラスコ（培養面積２５ｃｍ^２）中で３７℃で２４時間培養した後、培地を分化誘導用培地（１０％　ＦＣＳ、１００単位／ｍｌ　ペニシリン、１００μｇ／ｍｌ　ストレプトマイシン、１７μＭ　パントテン酸、３３μＭ　（＋）−ビオチン、１００μＭ　アスコルビン酸、１μＭ　オクタン酸、５０ｎＭ　トリヨードチロニン、１０μｇ／ｍｌ　インスリンおよび２．５μＭ　デキサメタゾンを含むＤＭＥＭ）に交換し、３７℃でさらに４８時間培養した。その後、脂肪細胞維持培地（１０％　ＦＣＳ、１００Ｕ／ｍｌ　ペニシリン、１００μｇ／ｍｌ　ストレプトマイシン、１７μＭ　パントテン酸、３３μＭ（＋）−ビオチン、１００μＭ　アスコルビン酸、１μＭオクタン酸、５０ｎＭ　トリヨードチロニンおよび１０μｇ／ｍｌ　インスリンを含むＤＭＥＭ）に交換した。２乃至３日後に１０％　ＦＣＳ、５０ｎｇ／ｍｌ　インスリンおよび１０単位／ｍｌ　ヘパリンナトリウムを含有するＤＭＥＭ培地を２ｍｌ加え、１時間培養した後、この培養上清を回収してＬＰＬ活性の測定に用いた。
【０５２９】
（２）ＬＰＬ活性測定
ＬＰＬ活性の測定法は、文献記載の方法（Ｎｉｌｓｓｏｎ−Ｅｈｌｅ，　Ｐ．　ａｎｄ　Ｓｃｈｏｔｚ，　Ｍ．　Ｃ．（１９７６）　Ｊ．Ｌｉｐｉｄ　Ｒｅｓ．　１７，　５３６−５４１）に従った。
【０５３０】
まず、上記１）記載の方法で調製した培養上清１００μｌを等量の基質溶液［２ｍＭ　グリセロール・トリ［９，１０（ｎ）−^３Ｈ］オレイン酸（１３１ＫＢｅｑ／μｍｏｌ、アマシャム社製）、０．１８９μｇ／ｍｌ　Ｌ　α−ホスファチジルコリン（シグマ社製）、１４ｍｇ／ｍｌ　ウシ血清アルブミン（シグマ社製）、１４０ｍＭ　トリス−塩酸（ｐＨ８．０）、１５％（ｖ／ｖ）グリセロール、１０％（ｖ／ｖ）非働化ＦＣＳ］と混合し、そこへ実施例１５で得られたＧＳＴまたはＧＳＴ−ＡＲＰ４Ｎを加え、３７℃で１２０分間保温した。その後、１．０５ｍｌの０．１Ｍ　炭酸カリウム・ほう酸緩衝液（ｐＨ１０．５）と３．２５ｍｌのメタノール：クロロホルム：ヘキサン＝１４１：１２５：１００（ｖ／ｖ）を加えて反応を停止させ、強く撹拌した後、３０００×ｇで１５分間遠心分離した。水−メタノール層の^３Ｈカウントを液体シンチレーションカウンターにて測定した。ＬＰＬ活性の１単位は、１μｍｏｌの脂肪酸を１分間に生成する活性として定義した。さらに、ＧＳＴ（１０μｇ／ｍｌ）を添加したときのＬＰＬ活性を１００％とし、ＧＳＴ−ＡＲＰ４Ｎを添加したときの相対的ＬＰＬ活性を算出した。その結果を表１０に示した。
【０５３１】
【表１０】

ＧＳＴ−ＡＲＰ４ＮはＬＰＬの活性を用量依存的に抑制した。この実験系において、リパーゼ反応の際に被験物質を添加すれば、該物質がＬＰＬの活性を調節する作用を有するか否かを調べることができる。
【０５３２】
実施例１８．アンジオポエチン関連タンパク質４とグルタチオン・Ｓ・トランスフェラーゼとの融合タンパク質の活性評価
（１）融合タンパク質発現形質転換大腸菌の作製
配列表の配列番号１６のアミノ酸番号２６乃至４０６に示されるアミノ酸配列からなるポリペプチドのアミノ末端にグルタチオン・Ｓ・トランスフェラーゼ（以下「ＧＳＴ」という）が連結している融合タンパク質（以下、「ＧＳＴ−ＡＲＰ４Ｆｕｌｌ」という。）を以下に記載する方法に従って調製した。
【０５３３】
まず、ＰＣＲ用プライマーとして、下記のヌクレオチド配列：
５’−　ｔｃｃｃｃｃｇｇｇｇｇａｃｃｃｇｔｇｃａｇｔｃｃａａｇ−３’（ＧＳＴ−ＡＲＰ４Ｆｕｌｌ用のセンスプライマー。配列表の配列番号５５）；および
５’−　ｃｃｇｃｔｃｇａｇｇｇａｇｇｃｔｇｃｃｔｃｔｇｃｔｇｃ−３’（ＧＳＴ−ＡＲＰ４Ｆｕｌｌ用アンチセンスプライマー。配列表の配列番号５６）を合成した。
【０５３４】
次に、Ｉ．Ｍ．Ａ．Ｇ．Ｅ．　Ｃｏｎｓｏｒｔｉｕｍより購入のｃＤＮＡ（Ｉ．Ｄ．　４１４９０３９）１０ｎｇを鋳型とし、センスプライマーおよびアンチセンスプライマー（それぞれ０．２μＭ）、Ｔｂｒ　ＥＸＴ　ＤＮＡポリメラーゼ、１×ＰＣＲ緩衝液（Ｔｂｒ　ＥＸＴ　ＤＮＡポリメラーゼに添付の１０×緩衝液を１０倍希釈）、２ｍＭの塩化マグネシウムおよび各０．４ｍＭのｄＮＴＰｓ存在下でＰＣＲを実施した。ＰＣＲは、上記サンプルを９４℃で３分間加熱した後に、９４℃で３０秒、５５℃で３０秒、７２℃で１分３０秒の温度サイクル３５回、最後に７２℃で５分間の条件で行なった。
【０５３５】
ＰＣＲでは約１１４０ｂｐのＤＮＡ断片が増幅した。増幅したＤＮＡ断片を回収し、ＳｍａＩおよびＸｈｏＩで消化した。ＳｍａＩおよびＸｈｏＩで消化してからウシ小腸アルカリホスファターゼ（宝酒造（株）社製）を用いて切断末端の脱リン酸化処理を行ったプラスミドベクターｐＧＥＸ−４Ｔ−２（ＧＳＴをコードする遺伝子を含む。アマシャム・ファルマシア・バイオテク社製）に、このＳｍａＩおよびＸｈｏＩで消化した、ＤＮＡ断片を挿入し、挿入断片を含むプラスミドクローンを単離した。このプラスミドクローンをｐＧＳＴ−ＡＲＰ４Ｆｕｌｌと命名した。
【０５３６】
プラスミドクローンｐＧＳＴ−ＡＲＰ４Ｆｕｌｌで大腸菌ＥｐｉｃｕｒｉａｎＣｏｌｉ　ＢＬ２１　ＣｏｄｏｎＰｌｕｓ　（ＤＥ３）−ＲＩＬ株（Ｓｔｒａｔａｇｅｎｅ社製）を、または、挿入断片を含まずＧＳＴのみを発現させるためのコントロールベクターｐＧＥＸ−４Ｔ−２で大腸菌ＪＭ１０９株をそれぞれ形質転換した。
【０５３７】
形質転換は１０倍希釈したＸＬ１０−Ｇｏｌｄ　β−ｍｅｒｃａｐｔｏｅｔｈａｎｏｌ　ｍｉｘ　２ｍｌを大腸菌Ｅｐｉｃｕｒｉａｎ　Ｃｏｌｉ　ＢＬ２１　ＣｏｄｏｎＰｌｕｓ　（ＤＥ３）−ＲＩＬのコンピテントセル　１００ｍｌに予め添加し、氷上で２分置きに攪拌しながら１０分間冷却後にプラスミドを添加し、氷上で３０分間静置後、４２℃で２０秒間保温し、再び氷上で２分間静置してから、ＳＯＣ培養液中で３７℃、１時間振盪培養をすることによって行なった。形質転換後の培養液をアンピシリンおよびクロラムフェニコール添加ＬＢ寒天プレート上に撒き、出現した組換え体のコロニーをピックアップして液体培養し、ＩＰＴＧ誘導による組換えタンパク質の発現を確認した。
【０５３８】
一方、大腸菌ＪＭ１０９の形質転換はコンピテントセルにプラスミドを添加し、氷上で３０分間静置した後、４２℃で３０秒間保温し、再び氷上で２分間静置してから、ＳＯＣ培養液中で３７℃、１時間振盪培養をすることによって行なった。形質転換後の培養液をアンピシリン添加ＬＢ寒天プレート上に撒き、出現した組換え体のコロニーをピックアップして液体培養し、ＩＰＴＧ誘導による組換えタンパク質の発現を確認した。
【０５３９】
（２）融合タンパク質の調製
１）形質転換大腸菌の培養
プラスミドクローンｐＧＳＴ−ＡＲＰ４ＦｕｌｌおよびコントロールベクターｐＧＥＸ−４Ｔ−２を保持する形質転換大腸菌をそれぞれ、アンピシリン／クロラムフェニコールおよびアンピシリン入りのＬＢ培地に植菌し、３７℃で１８時間振とう培養した。培養液の一部を新しい培地に添加し、さらに３７℃にて振とう培養を行なった。６００ｎｍでの吸光度が０．５−０．７に達した時に、最終濃度１ｍＭとなるようにイソプロピル・チオガラクトピラノシド（ＩＰＴＧ）を添加しさらに４時間振とう培養を継続した。
【０５４０】
２）ｐＧＥＸ−４Ｔ−２を保持する大腸菌からの細胞破砕液の調製
コントロールベクターｐＧＥＸ−４Ｔ−２で形質転換した大腸菌の培養液を４℃、６０００ｒｐｍ、１０分間の条件で遠心処理して沈殿した菌体を回収した。回収した菌体を超音波処理用緩衝液（５０ｍＭ　トリス−塩酸（ｐＨ８．０）、５０ｍＭ　塩化ナトリウム、１ｍＭ　ＥＤＴＡ、１ｍＭ　ＤＴＴ）で懸濁し、懸濁液を４℃、６０００ｒｐｍ、１０分間の条件で遠心処理した。沈殿を−８０℃で凍結し、次いで室温にて融解して再度超音処理用緩衝液に懸濁した。懸濁液を氷浴中で超音波破砕器（トミー精工（株）社製ＵＤ−２０１）によって、出力＝５、デューティ＝５０の条件で５分間処理した。超音波処理後の破砕液に最終濃度１％となるようにトリトン　Ｘ−１００を添加し混和し、４℃、１００００ｒｐｍ、３０分間の条件で遠心処理し、上清を回収した。上清はＧＳＴｒａｐカラム（アマシャム・ファルマシア・バイオテク社製）中に流した。
【０５４１】
３）ｐＧＳＴ−ＡＲＰ４Ｆｕｌｌを保持する大腸菌からの細胞破砕液の調製
組換えプラスミドｐＧＳＴ−ＡＲＰ４Ｆｕｌｌで形質転換した大腸菌の培養液を、４℃、５０００ｒｐｍ、１５分間の条件で遠心処理し、沈殿した菌体を回収した。沈殿した菌体に対して、洗浄用緩衝液（０．５％　トリトン　Ｘ−１００、１ｍＭ　ＥＤＴＡ）での懸濁、４℃、５０００ｒｐｍ、１０分間の条件での遠心処理、の操作を３回繰り返し、得られた菌体を８Ｍ　尿素溶液に溶解した。この溶液を室温に１時間放置した後、４℃、１３０００ｒｐｍ、３０分間の遠心処理を行ない上清を回収した。組換えプラスミドｐＧＳＴ−ＡＲＰ４Ｆｕｌｌで形質転換した大腸菌由来の培養液から得られた上清について透析を行なった。透析は、４Ｍ　尿素溶液に対して４℃で１時間、２Ｍ　尿素溶液に対して４℃で１時間、超音波処理用緩衝液に対して４℃で１時間、超音波処理用緩衝液に対して４℃で一晩の順で行なった。透析後、４℃、１２０００ｒｐｍ、３０分間の条件で遠心し、上清をＧＳＴｒａｐカラム中に流した。
【０５４２】
４）融合タンパク質の精製
上記の工程を経てＧＳＴ又はＧＳＴとの融合タンパク質ＧＳＴ−ＡＲＰ４Ｆｕｌｌが吸着したＧＳＴカラムにＰＢＳを流してカラム内を洗浄して不純物を除去した。次いで、１０ｍＭ　還元型グルタチオン溶液（ｐＨ８．０）を流して、ＧＳＴ、ＧＳＴとの融合タンパク質ＧＳＴ−ＡＲＰ４Ｆｕｌｌを溶出させた。溶出液の一部をＳＤＳ−ＰＡＧＥ電気泳動にて分離した後、ゲルをクマシー染色して、タンパク質の存在を確認した。また、溶出液をＰＢＳで４℃、一晩透析した。ここで、プラスミドｐＧＥＸ−４Ｔ−２を保持する形質転換体から精製されたタンパク質がＧＳＴであり、ｐＧＳＴ−ＡＲＰ４Ｆｕｌｌを保持する形質転換体から精製されたタンパク質が、ＧＳＴ−ＡＲＰ４Ｆｕｌｌである。
【０５４３】
（３）血中中性脂肪濃度上昇活性
上記（２）で得られたＧＳＴおよびＧＳＴ−ＡＲＰ４Ｆｕｌｌを、ＰＢＳでタンパク質濃度約０．５ｍｇ／ｍｌに調整した後に、それぞれ２匹の１６週齢の雄ＫＫ／Ｓｎｋマウスに１００μｌずつ尾静脈注射により接種した。採血は摂取前と摂取３０分後に行なった。ヘマトクリット管で各マウスの眼底より採血して、卓上遠心機で５２００ｒｐｍ、１５分間遠心して血漿を分離し、接種前後における血中中性脂肪（トリアシルグリセロール）濃度を測定した。
【０５４４】
その結果を表１１に示した。ＧＳＴ接種マウス群（表１１の「ＧＳＴ」）では接種前後で差が認められなかったのに対し、ＧＳＴ−ＡＲＰ４Ｆｕｌｌ接種マウス群（表１１の「ＧＳＴ−ＡＲＰ４Ｆｕｌｌ」）では顕著な血中中性脂肪濃度の上昇が認められた。
【０５４５】
本実施例の方法において、例えばＧＳＴ−ＡＲＰ４Ｆｕｌｌを接種したマウスに、被験物質を投与し、血中中性脂肪濃度の変動を調べることにより、高脂血症、動脈硬化および高血糖から選択される一つまたは二つ以上の疾患の治療または予防剤の試験を行うことができる。
【０５４６】
【表１１】

実施例１９．　ＧＳＴ−ＡＲＰ４ＦｕｌｌのＬＰＬ活性阻害効果
実施例１１の（１）の１）記載の方法にしたがって調製した、ラット白色脂肪前駆細胞培養上清１００μｌを等量の基質溶液（２ｍＭ　グリセロール・トリ［９，１０（ｎ）−^３Ｈ］オレイン酸（１３１ＫＢｅｑ／μｍｏｌ、アマシャム社製）、０．１８９μｇ／ｍｌ　Ｌ　α−ホスファチジルコリン（シグマ社製）、１４ｍｇ／ｍｌ　ウシ血清アルブミン（シグマ社製）、１４０ｍＭ　トリス−塩酸（ｐＨ８．０）、１５％（ｖ／ｖ）グリセロール、１０％（ｖ／ｖ）非働化ＦＣＳ）と混合し、そこへ実施例１８で得られたＧＳＴ−ＡＲＰ４ＦｕｌｌまたはＧＳＴを加え、３７℃で１２０分間保温した。その後、１．０５ｍｌの０．１Ｍ炭酸カリウム・ほう酸緩衝液（ｐＨ１０．５）と３．２５ｍｌのメタノール：クロロホルム：ヘキサン＝１４１：１２５：１００（ｖ／ｖ）を加えて反応を停止させ、強く撹拌した後、３０００×ｇで１５分間遠心分離した。水−メタノール層の^３Ｈカウントを液体シンチレーションカウンター（ベックマン社製）にて測定した。ＬＰＬ活性の１単位は、１μｍｏｌの脂肪酸を１分間に生成する活性として定義した。さらに、ＧＳＴ（１０μｇ／ｍｌ）を添加したときのＬＰＬ活性を１００％とし、ＧＳＴ−ＡＲＰ４Ｆｕｌｌを添加したときの相対的ＬＰＬ活性を算出した。その結果を表１２に示した。
【０５４７】
【表１２】

ＧＳＴ−ＡＲＰ４ＦｕｌｌはＬＰＬの活性を用量依存的に抑制した。この実験系において、リパーゼ反応の際に被験物質を添加すれば、該物質がＬＰＬの活性を調節する作用を有するか否かを調べることができる。
【０５４８】
実施例２０．　マウス「アンジオポエチン関連タンパク質３」遺伝子の解析
ａ）ＢＡＣ（Ｂａｃｔｅｒｉａｌ　ａｒｔｉｆｉｃｉａｌ　ｃｈｒｏｍｏｓｏｍｅ）ＤＮＡの調製
マウスＢＡＣクローン３５５Ｌ−１（リサーチ・ジェネティクス社より購入）を保持する大腸菌を１２．５μｇ／ｍｌのクロラムフェニコールを含む１００ｍｌのＬＢ培地で、３７℃で一晩培養した後、培養液を４ｍｌずつ４個のチューブユニットに分注し、ＤＮＡ自動抽出装置（ＰＩ−５０、クラボウ（株）社製）を用いてＤＮＡを抽出した。さらにこのサンプルを塩化セシウム法によって精製した。
【０５４９】
ｂ）　ＤＮＡの断片化
３０ｎｇ／μｌのＢＡＣ　ＤＮＡ　３００μｌを１．５ｍｌのエッペンドルフチューブに移し、ソニケーター（ソニファイアーＩＩ　２５０、ブランソン社製）、およびスペシャルマイクロチップ（ブランソン社製）を用いて、ソニケーターの出力調整を１、デューティー・サイクルを「コンスタント」の条件にそれぞれ設定して、ＤＮＡを３秒間、２回ソニケーションし断片化した。ＤＮＡの断片化の度合いは、アガロースゲル電気泳動を行って確認した。
【０５５０】
ｃ）　ＤＮＡ断片の末端修復
上記で断片化されたＤＮＡに、終濃度５ｍＭの塩化マグネシウム、０．０５ｍＭのｄＮＴＰ（宝酒造（株）社製）存在下で１０単位のＴ４　ＤＮＡポリメラーゼ（宝酒造（株）社製）を加え、２５℃で１５分間保温した。さらに、５単位のクレノウ断片（宝酒造（株）社製）を加え、２５℃で１５分間保温して、断片化ＤＮＡの末端を平滑化した。ＤＮＡを精製後、終濃度２ｍＭのＡＴＰ（宝酒造（株）社製）、１単位のポリヌクレオチドキナーゼ（宝酒造（株）社製）およびポリヌクレオチドキナーゼ反応緩衝液（ポリヌクレオチドキナーゼに添付、終濃度：５０ｍＭ　トリス−塩酸（ｐＨ８．０）、１０ｍＭ　塩化マグネシウム、５ｍＭ　ジチオスレイトール）を加え、３７℃で３０分間保温してＤＮＡ末端をリン酸化した。反応終了後、ＴＥ飽和フェノール／クロロホルムにてポリヌクレオチドキナーゼを失活させた。
【０５５１】
ｄ）　ショ糖密度勾配遠心
１０％ショ糖溶液は、１０ｍｌの５０％ショ糖溶液、０．２５ｍｌの２０×ＳＳＣおよび０．１ｍｌの０．５Ｍ　エチレンジアミン四酢酸（以下「ＥＤＴＡ」という）を混合し、滅菌水で全量５０ｍｌに調整することにより作製した。３８％ショ糖溶液は、３８ｍｌの５０％ショ糖溶液、０．２５ｍｌの２０×ＳＳＣおよび０．１ｍｌの０．５Ｍ　ＥＤＴＡを混合して、滅菌水で全量５０ｍｌに調整することにより作製した。１３ＰＡチューブ（日立工機（株）社製）に３８％ショ糖溶液を５ｍｌ加え、次いで１０％ショ糖溶液を５ｍｌ静かに加えた後、グラジエントフォーマー（グラジエント・メイト、コスモバイオ（株）社製）を用いて速度（ＳＰＥＥＤ）１０、時間（ＴＩＭＥ）２分３０秒および角度（ＡＮＧＬＥ）８°の条件に設定してショ糖密度勾配を作製した。得られたショ糖密度勾配溶液に上記ｃ）で得られたＤＮＡを加え、１５００００×ｇ、２０℃で１６時間遠心分離した。その後、チューブの下部より注射針（１８Ｇ）を用いて穴を開け、チューブ内液を下層から３００μｌずつ分画して回収した。０．８％　アガロースゲルにて各画分の一部を電気泳動し、約２．０ｋｂのＤＮＡ断片を含んでいた画分を選択し、エタノール沈殿後に１０μｌのＴＥに溶解した。
【０５５２】
ｅ）　ベクター調製およびライブラリーの作製
上記で得られたＢＡＣ　ＤＮＡ由来の約２．０ｋｂ断片と０．１μｇのｐＵＣ１９　ＤＮＡ／ＳｍａＩ（ファーメンタス社製）をＤＮＡライゲーションキット（バージョン２、宝酒造（株）社製）を用いて連結した。反応液をエレクトロポレーション用コンピテント大腸菌（エレクトロ・マックスＤＨ１０Ｂセル、ギブコ・ビーアールエル社製）に加え、エレクトロポレーション用キュベット（ジーンパルサーキュベット　０．１ｃｍ、バイオラッド社製）に移し、エレクトロポレーション装置（ジーンパルサーＩＩ、バイオラッド社製）を用いて、電圧１．８ｋＶ、電気容量２５μＦ、抵抗２００Ωの条件でエレクトロポレーションした。その後１ｍｌのＳＯＣ培地（ギブコ・ビーアールエル社製）を加え、３７℃で１時間振とうし、終濃度５０μｇ／ｍｌのアンピシリン、終濃度５０μｇ／ｍｌのＸ−ｇａｌ（ギブコ・ビーアールエル社製）および終濃度２５μｇ／ｍｌのＩＰＴＧ（ギブコ・ビーアールエル社製）を含むＬＢ固形培地（１．２％　バクトアガー含有）上に塗り広げ、３７℃で一晩培養した。
【０５５３】
ｆ）ＤＮＡの調製
上記ｅ）の結果、白色を呈したコロニーを選択し、終濃度５０μｇ／ｍｌのアンピシリンを含むＴＢＧ培地（１リットル当たり、トリプトン　１２ｇ、イーストエキストラクト　２４ｇ、リン酸一カリウム　２．３ｇ、リン酸二カリウム　１２．５ｇ、グリセロール　４ｍｌおよび１Ｍ　グルコース　２０ｍｌを含む）１．２ｍｌ／ウェルを入れた培養用マイクロウェルプレート（２．２ｍｌ　ディープ・ウェル・プレート、日本ジェネティクス社製）に１コロニー／ウェルで植菌した。プレートの上面を通気性粘着シート（日本ジェネティクス社製）にてシールした後、プレートミキサーを用いて緩やかに撹拌しながら、３７℃で１６．５時間培養した。その後、培養液を６００μｌずつとって、新しい培養用マイクロウェルプレートに分注し、マイクロプレート用遠心機（４Ｋ１５Ｃ、シグマ社製）を用いて、６０００ｒｐｍ、２０℃にて５分間遠心した。上清を除き、沈殿した菌体からＤＮＡ抽出キット（ＱＩＡｐｒｅｐ　　９６・ターボ・バイオロボット・キット、Ｑｉａｇｅｎ社製）およびＤＮＡ抽出装置（バイオロボット９６００、Ｑｉａｇｅｎ社製）を用いてＤＮＡを抽出した。
【０５５４】
ｇ）ヌクレオチド配列解析
シークエンシング用プライマーとして、下記のヌクレオチド配列：
５’−　ｔｇｔａａａａｃｇａ　ｃｇｇｃｃａｇｔ　−３’（−２１Ｍ１３ｆｏｒｗａｒｄ、配列表の配列番号５７）；および
５’−　ｃａｇｇａａａｃａｇ　ｃｔａｔｇａｃｃ　−３’（Ｍ１３ｒｅｖｅｒｓｅ、配列表の配列番号５８）
からなるオリゴヌクレオチドをＤＮＡ合成装置にて化学合成した。
【０５５５】
上記ｆ）で得られた各ＤＮＡ　１０μｌに対して、プライマー（−２１Ｍ１３ｆｏｒｗａｒｄまたはＭ１３ｒｅｖｅｒｓｅ）を３．２ｐｍｏｌ、シークエンシング試薬（ビッグダイ・ターミネーター・サイクル・シークエンスＦＳレディ・リアクション・キット、アプライドバイオシステムズ社製）を８μｌ加え、滅菌水にて全量を２０μｌとした。サーマルサイクラー（ジーンアンプ・ＰＣＲシステム９７００、アプライドバイオシステムズ社製）にてサイクルシークエンス反応を実施（９６℃で１０秒、５０℃で５秒、６０℃で４分を１サイクルとして２５サイクル）して、得られたサンプルをＤＮＡシークエンサー（ＡＢＩ３７００、アプライドバイオシステムズ社製）に供してヌクレオチド配列を解析した。得られた配列データは、ＤＮＡシークエンスソフト（Ｓｅｑｕｅｎｃｈｅｒ、Ｇｅｎｅ　Ｃｏｄｅｓ社製）を用いて編集、整列化された。解析の結果、配列表の配列番号２７に示されるヌクレオチド配列を含むＤＮＡが目的のプロモーターＤＮＡとして選択された。マウス「アンジオポエチン関連タンパク質３」をコードする遺伝子の転写開始点（エクソン１の５’末端境界部位）はＧｅｎＢａｎｋデータベースに登録されている最も５’末端側が長いＥＳＴ（Ｅｘｐｒｅｓｓｅｄ　Ｓｅｑｕｅｎｃｅ　Ｔａｇ）である「ＡＩ１９５５２４」を参照して決定した。すなわち、ＡＩ１９５５２４のヌクレオチド配列のうち、ベクター部分を除いた最初のヌクレオチドを転写開始点と判断し、その位置を「１」と定義した。このようにして、マウス「アンジオポエチン関連タンパク質３」遺伝子の転写開始点より５’末端側４１８ｂｐから３’末端側１３２ｂｐまでの領域に相当するヌクレオチド配列からなるＤＮＡが同定された。
【０５５６】
実施例２１．　ルシフェラーゼ発現プラスミドベクターの構築
ａ）プラスミドｐＧＬ８−３の構築
実施例２０の結果を基に、マウス「アンジオポエチン関連タンパク質３」をコードする遺伝子の５’末端側上流域を特異的に増幅するためのＰＣＲプライマーとして、下記のヌクレオチド配列：
５’−　ａａｇｇｔａｃｃｇｃ　ｔｇｔｔｔｃｃａｇａ　ｔａａａｃａａａ　−３’　（プライマー１、配列表の配列番号５９）；および
５’−　ｔｃａａａｔｇａｔｇ　ａａａｇｇｔｃｔｇｇ　ａｔｃｃａｃｔｃｔｇ　ｇａｔｇｃ　−３’　（プライマー２、配列表の配列番号６０）
からなるオリゴヌクレオチドをそれぞれ化学合成した。
【０５５７】
上記プライマー（終濃度各０．２μＭ）と、ＬＡ　ＰＣＲキット（宝酒造（株）社製）添付の１０×ＬＡバッファー（Ｍｇ＋）を５μｌ、同じく添付ｄＮＴＰを８μｌ、実施例２０のａ）に記載した方法で精製したＢＡＣクローン３５５Ｌ−１　ＤＮＡ　１μｌおよび滅菌水を加えて４９．５μｌとし、キット添付のＬＡ　Ｔａｑポリメラーゼを０．５μｌ加えて全量を５０μｌとした。サーマルサイクラー（ジーンアンプＰＣＲシステム９６００、アプライドバイオシステムズ社製）を使用してＰＣＲを実施した（温度条件：９４℃で２分間加熱してから、次に９８℃で２０秒、６８℃で３分の温度サイクルを３０回繰り返し、４℃に冷却、保存した）。
【０５５８】
このＰＣＲ後の反応液中のＤＮＡをエタノール沈殿で回収した後、滅菌水８０μｌに溶解した。このＤＮＡを制限酵素ＢａｍＨＩとＫｐｎＩで順次消化した。一方、ルシフェラーゼ発現ベクターｐＧＬ３−Ｂａｓｉｃ（プロメガ社製）を同様にＫｐｎＩとＢｇｌＩＩで消化し、アルカリホスファターゼ（宝酒造（株）社製）を用いて脱リン酸化した後、上記ＤＮＡ断片とＤＮＡライゲーションキット（バージョン２、宝酒造（株）社製）を用いて連結した。このＤＮＡで大腸菌ＤＨ１０Ｂのコンピテント細胞（ギブコビーアールエル社製）を形質転換し、アンピシリン耐性のコロニーを得た。
【０５５９】
いくつかのコロニーを少量培養して、培養菌体からプラスミドを抽出し、ＤＮＡシークエンサー（モデル３７００、（（株）アプライドバイオシステムズ事業部製）を用いて挿入断片のヌクレオチド配列を解析し、配列表の配列番号２７のヌクレオチド番号１乃至５２６に示されるヌクレオチド配列からなるＤＮＡが挿入されたプラスミドをｐＧＬ８−３と命名した。プラスミドｐＧＬ８−３は、２００１（平成１３）年６月７日付けで独立行政法人産業技術総合研究所・特許生物寄託センターに国際寄託され、受託番号ＦＥＲＭ　ＢＰ−７６２７が付された。このプラスミドを保有する形質転換大腸菌を５０μｇ／ｍｌのアンピシリンを含む１００ｍｌの液体ＬＢ培地中で、３７℃で一晩培養し、この培養液から、プラスミド抽出精製キット（ウィザード・ピュアフェクション・キット、プロメガ社製））を用いてＤＮＡを回収し、精製した。
【０５６０】
ｂ）プラスミドｐＧＬ１３−１の構築
下記のヌクレオチド配列：
５’−　ａａｇｇｔａｃｃａａ　ｔｔｇｃａｔｃｃａｇ　ａｇｔｇｇａｔｃｃａ　ａ　−３’　（配列表の配列番号６１）；および
５’−　ｔｔｇｇａｔｃｃａｃ　ｔｃｔｇｇａｔｇｃａ　ａｔｔｇｇｔａｃｃｔ　ｔ　−３’　（配列表の配列番号６２）
を有するオリゴヌクレオチドを合成した。
【０５６１】
上記オリゴヌクレオチド各５μｇを混合してアニーリングさせてから、制限酵素ＢａｍＨＩとＫｐｎＩで順次消化した。一方、ルシフェラーゼ発現ベクターｐＧＬ３−Ｂａｓｉｃ（プロメガ社製）を同様にＫｐｎＩとＢｇｌＩＩで消化し、アルカリホスファターゼ（宝酒造（株）社製）を用いて脱リン酸化した後、上記ＤＮＡ断片とＤＮＡライゲーションキット（バージョン２、宝酒造（株）社製）を用いて連結した。このＤＮＡで大腸菌ＤＨ１０Ｂのコンピテント細胞（ギブコビーアールエル社製）を形質転換し、アンピシリン耐性のコロニーを得た。
【０５６２】
いくつかのコロニーの培養菌体からプラスミドを抽出してＤＮＡシークエンサー（モデル３７００、（株）パーキンエルマージャパン・アプライドバイオシステムズ事業部製）を用いてヌクレオチド配列を解析し、マウス「アンジオポエチン関連タンパク質３」遺伝子の＋９３乃至＋１０８領域に相当するＤＮＡ（配列表の配列番号２７のヌクレオチド番号５１１乃至５２６）が挿入されたプラスミドｐＧＬ１３−１を取得した。このプラスミドを保有する形質転換大腸菌を５０μｇ／ｍｌのアンピシリンを含む１００ｍｌの液体ＬＢ培地中で、３７℃で一晩培養し、この培養液から、プラスミド抽出精製キット（プロメガ社製）を用いてＤＮＡを回収し、精製した。
【０５６３】
ルシフェラーゼ発現ベクターｐＧＬ３−Ｂａｓｉｃ（プロメガ社製）ＤＮＡでも同様にして大腸菌ＤＨ１０Ｂのコンピテント細胞（ギブコビーアールエル社製）を形質転換し、アンピシリン耐性のコロニーを培養し、プラスミド抽出精製キット（プロメガ社製）を用いてＤＮＡを回収し、精製した。このプラスミドＤＮＡをネガティブコントロールとして使用した。
【０５６４】
実施例２２．　アンジオポエチン関連タンパク質３遺伝子のプロモーター活性実施例２１で単離したＤＮＡのプロモーター活性について評価するため、レポーターアッセイを実施した。すなわち、実施例２１で構築したホタルルシフェラーゼ発現プラスミド（ｐＧＬ８−３またはｐＧＬ１３−１）を、ヒト肝臓由来細胞株ＨｅｐＧ２にトランスフェクションして、ホタルルシフェラーゼ活性の変化を調べた。なお、細胞へのトランスフェクション効率についての各プラスミド間の実験ロット差を補正するための内部標準として、ウミシイタケルシフェラーゼ発現プラスミドｐＲＬ−ＣＭＶ（プロメガ社製）を用いた。
【０５６５】
まず、ＨｅｐＧ２細胞（大日本製薬（株）社より購入）を、１０％ウシ胎児血清（モアゲート社製）、ペニシリン／ストレプトマイシン、２ｍＭのＬ−グルタミン、および１ｍＭのピルビン酸を含むＭＥＭ培地（ギブコ・ビーアールエル社製）中で、３７℃で５％炭酸ガスのインキュベーター内で培養した。セミコンフルエントになるまで増殖させた大角フラスコ（住友ベークライト社製）に、ＰＢＳ（−）（日水製薬（株）社製）を加えて、セルスクレーパー（住友ベークライト（株）社製）を用いて細胞を掻きとって回収した。その細胞を５×１０^５個／ウェルで６穴培養プレート（コーニング社製）に蒔き、一晩培養した。１μｇのｐＧＬ３−Ｂａｓｉｃ、ｐＧＬ８−３、あるいはｐＧＬ１３−１　ＤＮＡと０．０４ｎｇのｐＲＬ−ＣＭＶ　ＤＮＡを混合して、滅菌水で全量を５μｌとしてＤＮＡ溶液とした。一方、１００μｌのウシ胎児血清を含まないＭＥＭ培地（その他の成分は上記と同様）に６μｌのトランスフェクション試薬（ＴｒａｎｓＩＴ−ＬＴ１、宝酒造（株）社製）を添加し室温で５分間保温した。この溶液に先に調製したＤＮＡサンプルを添加し、室温で５分間保温した。細胞は血清を含まないＭＥＭ培地（その他の成分は上記と同じ）２ｍｌで２回洗浄し、２ｍｌの同培地に置き換えた。ＤＮＡ溶液を３０μｌ／ウェルずつ３ウェルへ添加して、４時間培養後に２ｍｌ／ウェルの１０％ウシ胎児血清を含むＭＥＭ培地（その他の成分は上記と同じ）へ置き換え３７℃で一晩培養した。培養後、培地を除去して２００μｌ／ウェルの細胞溶解剤（Ｐａｓｓｉｖｅ　Ｌｙｓｉｓ　Ｂｕｆｆｅｒ（プロメガ社製））に溶解して、細胞抽出液を調製した。そして、そのうちの２０μｌを用いて、デュアル−ルシフェラーゼ・レポーター・アッセイ・システム（プロメガ社製）を、添付のプロトコールに従って用いることにより、ホタルおよびウミシイタケルシフェラーゼ活性を測定した（ルミナスＣＴ−９０００Ｄ、ダイアヤトロン社製を使用）。実験結果については、内部標準のウミシイタケルシフェラーゼ活性で補正したホタルルシフェラーゼ活性として表した。
【０５６６】
その結果、ｐＧＬ８−３を導入したＨｅｐＧ２細胞では、ｐＧＬ３−Ｂａｓｉｃを導入した細胞に比べて約１９倍のルシフェラーゼ活性が観察されたことから、ｐＧＬ８−３に組み込まれた配列表の配列番号２７に示されるヌクレオチド配列からなるＤＮＡにはプロモーターとしての活性が存在することが明らかとなった。一方、ｐＧＬ１３−１を導入した細胞では、ルシフェラーゼ活性がｐＧＬ８−３を導入した細胞の１／１０に低下した。以上の結果より、配列表の配列番号２７のヌクレオチド番号１乃至５１０に示されるＤＮＡ、すなわちマウス「アンジオポエチン関連タンパク質３」遺伝子の５’末端側上流−４１８乃至＋９２に相当する領域にプロモーター活性が存在することが明らかとなった。
【０５６７】
本実施例の方法において、例えばｐＧＬ８−３で形質転換したＨｅｐＧ２細胞を被験物質存在下で培養したときのルシフェラーゼ活性を測定することにより、本発明のＤＮＡのプロモーター活性を阻害する物質の試験を行うことができる。このプロモーター活性を阻害する物質は、高脂血症、動脈硬化および高血糖から選択される一つまたは二つ以上の疾患の治療または予防剤となりうる。
【０５６８】
実施例２３．　「アンジオポエチン関連タンパク質４」プロモーター領域の解析
ａ）マウス「アンジオポエチン関連タンパク質４」プロモーター領域の単離
マウス「アンジオポエチン関連タンパク質４」をコードする遺伝子の５’末端側上流域を特異的に増幅するためのＰＣＲプライマーとして、下記のヌクレオチド配列：
５’−　ｃｇｇｇｇｔａｃｃｇａｇｔｇｇｇｔｇｃｔｇｇｇａａｇｃａａ　−３’（プライマー１、配列表の配列番号６３）；および
５’−　ｃｃｃａａｇｃｔｔｇｃｃｔｔｇｇｇｔｇｃａｇｃａａｃｇｃｔ　−３’（プライマー２、配列表の配列番号６４）
からなるオリゴヌクレオチドをＤＮＡ合成装置（アマシャム　バイオサイエンス株式会社）にて化学合成した。
【０５６９】
マウス１２９Ｓｖ（三共株式会社安全性研究所で飼育）由来のゲノムＤＮＡ１００ｎｇを鋳型とし、上記プライマー（それぞれ３００ｎＭ）、ＫＯＤ　ＤＮＡポリメラーゼ（東洋紡社製）を１μｌ、１×ＰＣＲ反応緩衝液（ＤＮＡポリメラーゼに添付の１０×緩衝液＃１を１０分の１量）、１ｍＭの塩化マグネシウム、各０．２ｍＭのｄＮＴＰおよび滅菌水を加えて全量を５０μｌとしＰＣＲを行った。ＰＣＲはサーマルサイクラー（ジーンアンプＰＣＲシステム９７００、アプライドバイオシステムズ社製）を使用し、上記サンプルを９４℃で２分間加熱した後に、９４℃で１５秒、６０℃で３０秒、６８℃で３分の温度サイクル３５回、最後に６８℃で３分間保温の条件で行なった。得られたＰＣＲ産物はＱＩＡｑｕｉｃｋ　ＰＣＲ　Ｐｕｒｉｆｉｃａｔｉｏｎ　Ｋｉｔ（キアゲン社製）で精製後、ＫｐｎＩおよびＨｉｎｄＩＩＩによる切断を行い、１．０％　１×ＴＡＥアガロース電気泳動による目的バンドの分離後、ＳＵＰＲＥＣ−０１（宝酒造（株）社製）、エタノール沈殿によりＤＮＡの回収を行った。
【０５７０】
一方、ルシフェラーゼ発現ベクターｐＧＬ３−Ｂａｓｉｃ（プロメガ社製）を同様にＫｐｎＩおよびＨｉｎｄＩＩＩで消化し、アルカリホスファターゼ（宝酒造（株）社製）を用いて脱リン酸化した後、上記ＤＮＡ断片とＤＮＡライゲーションキット（バージョン２、宝酒造（株）社製）を用いて連結した。このＤＮＡで大腸菌ＤＨ１０Ｂのコンピテント細胞（ギブコビーアールエル社製）を形質転換し、アンピシリン耐性のコロニーを得た。
いくつかのコロニーを少量培養して、培養菌体からプラスミドを抽出し、ＤＮＡシークエンサー（モデル３７００、（アプライドバイオシステムズジャパン社製）を用いて挿入断片のヌクレオチド配列を解析し、配列表の配列番号２８のヌクレオチド番号１乃至２６０に示されるヌクレオチド配列からなるＤＮＡを含む約２．９ｋｂの断片が挿入されたプラスミドを得た（ｐＧＬ２−４）。
このプラスミドを保有する形質転換大腸菌を５０μｇ／ｍｌのアンピシリンを含む１００ｍｌの液体ＬＢ培地中で、３７℃で一晩培養し、この培養液から、プラスミド抽出精製キット（ＥｎｄｏＦｒｅｅ　Ｐｌａｓｍｉｄ　Ｍａｘｉ　Ｋｉｔ、キアゲン社製）を用いてＤＮＡを回収し、精製した。
【０５７１】
ｂ）ルシフェラーゼ発現プラスミドベクターｐＧＬ１１−４およびｐＧＬ１２−４の構築
マウス「アンジオポエチン関連タンパク質４」をコードする遺伝子の５’末端側上流域を特異的に増幅するためのＰＣＲプライマーとして、下記のヌクレオチド配列：
５’−　ｃｇｇｇｇｔａｃｃａｃａａａｇｃｃｔｇｔｇｇｃａｔｔｇｃａ　−３’（プライマー３、配列表の配列番号６５）；および
５’−　ｃｇｇｇｇｔａｃｃｃｔｃｃｃｃｃａｇａａｃｔｃｃａｇｃｔｇ　−３’（プライマー４、配列表の配列番号６６）
からなるオリゴヌクレオチドをＤＮＡ合成装置にて化学合成した。
【０５７２】
プラスミドｐＧＬ２−４を鋳型とし、上記プライマー（それぞれ３００ｎＭ）、ＫＯＤ　ＤＮＡポリメラーゼ（東洋紡社製）を１μｌ、１×ＰＣＲ反応緩衝液（ＤＮＡポリメラーゼに添付の１０×緩衝液＃１を１０分の１量）、１ｍＭの塩化マグネシウム、各０．２ｍＭのｄＮＴＰｓおよび滅菌水を加えて全量を５０μｌとしＰＣＲを行った。ＰＣＲはサーマルサイクラー（ジーンアンプＰＣＲシステム９７００、アプライドバイオシステムズ社製）を使用し、上記サンプルを９４℃で２分間加熱した後に、９４℃で１５秒、６０℃で１５秒の温度サイクル３０回、最後に６８℃で３分間保温の条件で行なった。得られたＰＣＲ産物はＱＩＡｑｕｉｃｋ　ＰＣＲ　Ｐｕｒｉｆｉｃａｔｉｏｎ　Ｋｉｔ（キアゲン社製）で精製後、ＫｐｎＩおよびＨｉｎｄＩＩＩによる切断を行い、１．０％　１×ＴＡＥアガロース電気泳動による目的バンドの分離後、ＳＵＰＲＥＣ−０１（宝酒造（株）社製）、エタノール沈殿によりＤＮＡの回収を行った。
【０５７３】
一方、ルシフェラーゼ発現ベクターｐＧＬ３−Ｂａｓｉｃ（プロメガ社製）を同様にＫｐｎＩおよびＨｉｎｄＩＩＩで消化し、アルカリホスファターゼ（宝酒造（株）社製）を用いて脱リン酸化した後、上記ＤＮＡ断片とＤＮＡライゲーションキット（バージョン２、宝酒造（株）社製）を用いて連結した。このＤＮＡで大腸菌ＤＨ１０Ｂのコンピテント細胞（ギブコビーアールエル社製）を形質転換し、アンピシリン耐性のコロニーを得た。いくつかのコロニーを少量培養して、培養菌体からプラスミドを抽出し、ＤＮＡシークエンサー（モデル３７００、（アプライドバイオシステムズジャパン社製）を用いて挿入断片のヌクレオチド配列を解析し、配列表の配列番号２８のヌクレオチド番号１乃至２６０に示されるヌクレオチド配列からなるＤＮＡが挿入されたプラスミドをｐＧＬ１１−４と命名し、配列表の配列番号２８のヌクレオチド番号１８９乃至２６０に示されるヌクレオチド配列からなるＤＮＡが挿入されたプラスミドをｐＧＬ１２−４と命名した。
【０５７４】
このプラスミドを保有する形質転換大腸菌を５０μｇ／ｍｌのアンピシリンを含む１００ｍｌの液体ＬＢ培地中で、３７℃で一晩培養し、この培養液から、プラスミド抽出精製キット（ＥｎｄｏＦｒｅｅ　Ｐｌａｓｍｉｄ　Ｍａｘｉ　Ｋｉｔ、キアゲン社製）を用いてＤＮＡを回収し、精製した。
【０５７５】
マウス「アンジオポエチン関連タンパク質４」をコードする遺伝子の転写開始点（エクソン１の５’末端境界部位）はＧｅｎＢａｎｋデータベースに登録されている最も５’末端側が長いＥＳＴ（Ｅｘｐｒｅｓｓｅｄ　Ｓｅｑｕｅｎｃｅ　Ｔａｇ）である「ＡＫ０１４５６４」を参照して決定した。すなわち、ＡＫ０１４５６４のヌクレオチド配列のうち、ベクター部分を除いた最初のヌクレオチドを転写開始点と判断し、その位置を「１」と定義した。このようにして、マウス「アンジオポエチン関連タンパク質４」遺伝子の転写開始点より５’末端側９０ｂｐから３’末端側１７０ｂｐまでの領域に相当するヌクレオチド配列からなるＤＮＡの単離を行った。
【０５７６】
ルシフェラーゼ発現ベクターｐＧＬ３−Ｂａｓｉｃ（プロメガ社製）ＤＮＡも同様にして大腸菌ＤＨ１０Ｂのコンピテント細胞（ギブコビーアールエル社製）を形質転換し、アンピシリン耐性のコロニーを培養し、プラスミド抽出精製キット（ＥｎｄｏＦｒｅｅ　Ｐｌａｓｍｉｄ　Ｍａｘｉ　Ｋｉｔ、キアゲン社製）を用いてＤＮＡを回収し、精製した。このプラスミドＤＮＡをネガティブコントロールとして使用した。
【０５７７】
実施例２４．　アンジオポエチン関連タンパク質４遺伝子のプロモーター活性実施例２３で単離したＤＮＡのプロモーター活性について評価するため、レポーターアッセイを実施した。すなわち、実施例２３で構築したホタルルシフェラーゼ発現プラスミド（ｐＧＬ１１−４またはｐＧＬ１２−４）を、サル腎臓由来細胞株ＣＯＳ１にトランスフェクションして、ホタルルシフェラーゼ活性の変化を調べた。なお、細胞へのトランスフェクション効率についての各プラスミド間の実験ロット差を補正するための内部標準として、ウミシイタケルシフェラーゼ発現プラスミドｐＲＬ−ＴＫ（プロメガ社製）を用いた。
【０５７８】
まず、ＣＯＳ１細胞（アメリカン・タイプ・カルチャー・コレクション（Ａｍｅｒｉｃａｎ　Ｔｙｐｅ　Ｃｕｌｔｕｒｅ　Ｃｏｌｌｅｃｔｉｏｎ：ＡＴＣＣ）より購入）を、１０％ウシ胎児血清（ギブコ・ビーアールエル社製）およびペニシリン／ストレプトマイシンを含むＤ−ＭＥＭ培地（ギブコ・ビーアールエル社製、Ｃａｔｌｏｇ　Ｎｕｍｂｅｒ　１１９６５−０９２）中で、３７℃で５％炭酸ガスのインキュベーター内で培養した。２４穴培養プレート（コーニング社製）でセミコンフルエント（ｓｅｍｉ−ｃｏｎｆｌｕｅｎｔ）になるまで増殖させ、０．２μｇのｐＧＬ３−Ｂａｓｉｃ、ｐＧＬ１１−４、あるいはｐＧＬ１２−４　ＤＮＡと６ｎｇのｐＲＬ−ＴＫ　ＤＮＡを混合して、滅菌水で全量を５μｌとしてＤＮＡ溶液とした。一方、２０μｌのウシ胎児血清を含まないＤ−ＭＥＭ培地（その他の成分は上記と同様）に１μｌのトランスフェクション試薬（ＦｕＧＥＮＥ６、ロシュ社製）を添加し軽く攪拌した後、この溶液に先に調製したＤＮＡサンプルを添加し、室温で１５分間保温した。ＤＮＡ溶液を３ウエルへ添加して、３７℃で二晩培養した。培養後、培地を除去し、２５０μｌ／ウエルの細胞溶解剤（Ｐａｓｓｉｖｅ　Ｌｙｓｉｓ　Ｂｕｆｆｅｒ（プロメガ社製））に溶解して、細胞抽出液を調製した。そして、そのうちの２０μｌを用いて、デュアル−ルシフェラーゼ・レポーター・アッセイ・システム（プロメガ社製）を、添付のプロトコールに従って用いることにより、ホタルおよびウミシイタケルシフェラーゼ活性を測定した（ルミナスＣＴ−９０００Ｄ、ダイアヤトロン社製を使用）。実験結果については、内部標準のウミシイタケルシフェラーゼ活性で補正したホタルルシフェラーゼ活性とし、ｐＧＬ３−Ｂａｓｉｃコントロールを１とした時の相対値で表した。
【０５７９】
その結果、ｐＧＬ１１−４を導入したＣＯＳ１細胞では、ｐＧＬ３−Ｂａｓｉｃを導入した細胞に比べて約３０倍のルシフェラーゼ活性が観察されたことから、ｐＧＬ１１−４に組み込まれた配列表の配列番号２８に示されるヌクレオチド配列からなるＤＮＡにはプロモーターとしての活性が存在することが明らかとなった。一方、ｐＧＬ１２−４を導入した細胞では、ｐＧＬ３−Ｂａｓｉｃを導入した細胞に対して約１．７倍のルシフェラーゼ活性を示すにとどまった。その結果を表１３に示した。以上の結果より、配列表の配列番号２８のヌクレオチド番号１乃至２６０に示されるＤＮＡ、すなわちマウス「アンジオポエチン関連タンパク質４」遺伝子の５’末端側上流−９０乃至＋１７０に相当する領域にプロモーター活性が存在することが明らかとなった。なお、表中（Ｍｅａｎ±ＳＤ）はルシフェラーゼ活性の平均値と標準偏差を意味する。
【０５８０】
【表１３】

本実施例の方法において、例えばｐＧＬ１１−４で形質転換したＣＯＳ１細胞を被験物質存在下で培養したときのルシフェラーゼ活性を測定することにより、本発明のＤＮＡのプロモーター活性を阻害する物質の試験を行うことができる。このプロモーター活性を阻害する物質は、高脂血症、動脈硬化および高血糖から選択される一つまたは二つ以上の疾患の治療または予防剤となりうる。
【０５８１】
【発明の効果】以上述べたように、本発明により、配列表の配列番号１のヌクレオチド番号４７から１４１１に示されるヌクレオチド配列または配列表の配列番号３のヌクレオチド番号７８から１４５７に示されるヌクレオチド配列からなる遺伝子が、高脂血症、動脈硬化および高血糖から選択される一つまたは二つ以上の疾患に関与する遺伝的素因の一つであり、従って該遺伝子の発現量を抑制する物質が高脂血症、動脈硬化および高血糖から選択される一つまたは二つ以上の疾患の治療または予防剤となり得ることが確かめられた。すなわち、本発明の方法、該方法のうち核酸の検出を行う態様においてプローブまたはプライマーとして使用されるポリヌクレオチドおよび同じくポリペプチドの検出を行う態様において使用される抗体は、高脂血症、動脈硬化および高血糖から選択される一つまたは二つ以上の疾患の治療または予防剤の探索に有用である。また、本発明のポリペプチドが血中中性脂肪濃度を上昇させる活性を有すること、該活性は融合タンパク質の形状でも保持され得ること、さらに本発明のポリペプチドがＬＰＬ活性の阻害効果を有すること、および本発明のポリペプチドを用いることにより高脂血症、動脈硬化および高血糖から選択される一つまたは二つ以上の疾患の治療または予防剤の試験をイン・ビボ、イン・ビトロのいずれにおいても実施できることが示された。
【０５８２】
さらに、本発明により、プロモーター活性を有する新規ＤＮＡが提供された。本発明のＤＮＡは、「アンジオポエチン関連タンパク質３」または「アンジオポエチン関連タンパク質４」の発現量を抑制する物質を試験するための方法に用いることができるので、新しい作用機作を有する高脂血症、動脈硬化および高血糖から選択される一つまたは二つ以上の疾患の治療または予防剤の開発に有用である。
【配列表フリーテキスト】
配列番号９：　ポリクローナル抗体を取得するための抗原として使用された合成オリゴペプチド
配列番号１１：　ヒト・「アンジオポエチン関連タンパク質３」のアミノ末端側領域をコードするＤＮＡ
配列番号１２：　ヒト・「アンジオポエチン関連タンパク質３」のアミノ末端側領域をコードするポリペプチドのアミノ酸配列
配列番号１３：　ヒト・「アンジオポエチン関連タンパク質３」のカルボキシル末端側領域をコードするペプチドのポリペプチドのＤＮＡ配列
配列番号１４：　ヒト・「アンジオポエチン関連タンパク質３」のカルボキシル末端側領域をコードするポリペプチドのアミノ酸配列
配列番号２５：　ＫｈＡＰ５　ＣＣ／ＨｉｓをコードするＤＮＡ
配列番号２６：　ＫｈＡＰ５　ＣＣ／Ｈｉｓのアミノ酸配列
配列番号４２：　コザックのコンセンサス配列が付加されたＰＣＲプライマー
配列番号４３：　制限酵素ＸｈｏＩの特異的配列が付加されたＰＣＲプライマー配列番号４４：　ＧＳＴ−ＣＣ、ＧＳＴ−ＣＣｄｅｌ及びＧＳＴ−Ｃ１の一部をコードするＤＮＡを増幅するためのＰＣＲセンスプライマー
配列番号４５：　ＧＳＴ−ＣＣの一部をコードするＤＮＡを増幅するためのＰＣＲアンチセンスプライマー
配列番号４６：　ＧＳＴ−ＣＣｄｅｌの一部をコードするＤＮＡを増幅するためのＰＣＲアンチセンスプライマー
配列番号４７：　ＧＳＴ−Ｃ１の一部をコードするＤＮＡを増幅するためのＰＣＲアンチセンスプライマー
配列番号４８：　ヒスチジンタグをコードする配列を挿入するためのオリゴヌクレオチド
配列番号４９：　ヒスチジンタグをコードする配列を挿入するためのオリゴヌクレオチド
配列番号５０：　プラスミドｐＭＥ１８Ｓ／ＫｈＡＰ５／Ｈｉｓの部分断片を増幅するためのセンス／アンチセンスプライマー
配列番号５１：　ＧＳＴ−ＡＲＰ４Ｎの一部をコードするＤＮＡを増幅するためのＰＣＲセンスプライマー
配列番号５２：　ＧＳＴ−ＡＲＰ４Ｎの一部をコードするＤＮＡを増幅するためのＰＣＲアンチセンスプライマー
配列番号５５：　ＧＳＴ−ＡＲＰ４Ｆｕｌｌの一部をコードするＤＮＡを増幅するためのＰＣＲセンスプライマー
配列番号５６：　ＧＳＴ−ＡＲＰ４Ｆｕｌｌの一部をコードするＤＮＡを増幅するためのＰＣＲアンチセンスプライマー
配列番号５７：　シークエンシングプライマー　−２１Ｍ１３ｆｏｒｗａｒｄ
配列番号５８：　シークエンシングプライマー　Ｍ１３ｒｅｖｅｒｓｅ
配列番号５９：　ＰＣＲプライマー１
配列番号６０：　ＰＣＲプライマー２
配列番号６１：　プラスミドｐＧＬ１３−１の挿入断片を調製するためのオリゴヌクレオチド
配列番号６２：　プラスミドｐＧＬ１３−１の挿入断片を調製するためのオリゴヌクレオチド
配列番号６３：　制限酵素ＫｐｎＩに特異的な配列を付加したＰＣＲセンスプライマー。
配列番号６４：　制限酵素ＨｉｎｄＩＩＩに特異的な配列を付加したＰＣＲアンチセンスプライマー。
配列番号６５：　制限酵素ＫｐｎＩに特異的な配列を付加したＰＣＲセンスプライマー。
配列番号６６：　制限酵素ＨｉｎｄＩＩＩに特異的な配列を付加したＰＣＲアンチセンスプライマー。
【０５８３】
【配列表】

【図面の簡単な説明】
【図１】ＫＫマウス臓器由来の試料についてのノーザンブロット解析の結果を表す図。
【図２】遺伝子導入ＣＯＳ−１細胞培養上清を試料としたウエスタンブロット解析の結果を表す図。
【図３】遺伝子導入ＨｅＬａ細胞培養上清を試料としたウエスタンブロット解析の結果を表す図。
【図４】組換えアデノウイルスを感染させたＫＫ／Ｓｎｋマウスの末梢血中の中性脂肪濃度測定結果を表す図。
【図５】ＫＫマウスおよび組換えアデノウイルスを感染させたＫＫ／Ｓｎｋマウスの肝臓由来の試料についてのノーザンブロット解析の結果を表す図。
【図６】精製「アンジオポエチン関連タンパク質３」のＥＬＩＳＡによる検量線を表す図。
【図７】「アンジオポエチン関連タンパク質１乃至５」の構造。左側がアミノ末端、右側がカルボキシル末端である。アンジオポエチン関連タンパク質は、アミノ末端から、シグナルペプチド、ヘリックス領域、フィブリノーゲン様領域からなる。
【図８】融合タンパク質のＫＫ／Ｓｎｋマウスにおける血中中性脂肪濃度に及ぼす影響を示した図。
【図９】組換えアデノウイルスを感染させたマウスの血漿を試料としたウエスタンブロット解析の結果を表す図。
【図１０】組換えアデノウイルスを感染させたマウスの末梢血中の中性脂肪濃度測定結果を表す図。
【図１１】ヘパリン結合部位に変異を導入したアンジオポエチン関連タンパク質３の血漿トリグリセリドに対する効果を表す図。[Claims]
1. A continuous amino acid sequence contained in the amino acid sequence represented by SEQ ID NO: 12 in the sequence listing, wherein the amino acid at amino acid number 17 is an amino terminal and any one of amino acids 147 to 207 is selected. A polypeptide comprising an amino acid sequence having a carboxyl terminus
2. The polypeptide according to claim 1, comprising an amino acid sequence represented by amino acids 17 to 207 of SEQ ID NO: 12 in the sequence listing.
3. The polypeptide according to claim 1, comprising an amino acid sequence represented by amino acids 17 to 195 of SEQ ID NO: 12 in the sequence listing.
4. The polypeptide according to claim 1, comprising an amino acid sequence represented by amino acids 17 to 147 of SEQ ID NO: 12 in the sequence listing.
5. A continuous amino acid sequence contained in the amino acid sequence shown in SEQ ID NO: 16 in the sequence listing, which comprises an amino acid sequence having amino acid number 26 as an amino terminal and amino acid number 143 as a carboxyl terminal. peptide.
6. A continuous amino acid sequence contained in the amino acid sequence shown in SEQ ID NO: 16 in the sequence listing, wherein amino acid number 26 is an amino terminal and any one of amino acid numbers 144 to 183 is a carboxyl terminal. A polypeptide comprising an amino acid sequence represented by the formula:
7. A polypeptide comprising an amino acid sequence represented by SEQ ID NO: 26 in the sequence listing.
Claim 81Or7An activity of increasing the neutral fat concentration in the blood of a mammal, wherein one or several amino acid residues are deleted, inserted, added or substituted at one or several sites in the amino acid sequence described above; An equivalent of a polypeptide having the formula:
Claim 91Or8A DNA encoding the polypeptide of any one of the above.
10. A continuous sequence contained in the nucleotide sequence shown in SEQ ID NO: 11 in the sequence listing, wherein the nucleotide at nucleotide number 18 is the 5 'end and any one of nucleotide numbers 458 to 638 is used. Consisting of a nucleotide sequence having a 3 'end.
11. A continuous sequence contained in the nucleotide sequence shown in SEQ ID NO: 11 in the sequence listing, wherein the nucleotide of nucleotide No. 18 is the 5 ′ end and any one of nucleotides 458 to 602 is used. Consisting of a nucleotide sequence having a 3 'end.
12. A continuous sequence contained in the nucleotide sequence shown in SEQ ID NO: 15 in the sequence listing, wherein the nucleotide at nucleotide 173 is at the 5 ′ end and the nucleotide at nucleotide 601 is at the 3 ′ end. DNA consisting of
13. A DNA comprising a nucleotide sequence represented by nucleotide numbers 18 to 662 of SEQ ID NO: 25 in the sequence listing.
Claim 149Or13A recombinant vector comprising the DNA of any one of the above.
Claim 1514A host cell transformed with the recombinant vector as described above.
Claim 16FifteenCulturing the host cell as described above, and then recovering a polypeptide having an activity of increasing the concentration of neutral fat in the blood of the mammal from the culture, A method for producing a polypeptide having an activity to increase fat concentration.
17. A method for testing an agent for treating or preventing one or more diseases selected from hyperlipidemia, arteriosclerosis and hyperglycemia, comprising the following steps:
1) For non-human mammals,A polypeptide comprising the amino acid sequence of amino acids 17 to 460 of SEQ ID NO: 4 in the Sequence Listing, a polypeptide lacking 1 to 253 residues at the carboxyl terminal thereof, and a polypeptide having amino acids 26 to 406 of SEQ ID NO: 16 in the Sequence Listing A polypeptide consisting of the amino acid sequence shown, a polypeptide lacking the carboxyl-terminal 1-263 residues, orClaim1Or8Administering one or more polypeptides selected from the polypeptides described in 1 and a test substance; and
2) Measure the neutral fat concentration in the blood of the animal of 1) above.
18. A method for testing an agent for treating or preventing one or more diseases selected from hyperlipidemia, arteriosclerosis and hyperglycemia, comprising the following steps:
1) For non-human mammals,The nucleotide sequence according to any one of claims 9 to 13, wherein the nucleotide sequence is represented by nucleotide numbers 78 to 1457 of SEQ ID NO: 3 in the sequence listing, the nucleotide sequence represented by nucleotide numbers 173 to 1393 of SEQ ID NO: 15 in the sequencing listing, or Contains DNAInfecting an adenovirus carrying the recombinant adenovirus vector;
2) {administering a test substance to the animal of the above step 1);
3) Measure the neutral fat concentration in the blood of the animal in step 2) above.
19. A method for testing an agent for treating or preventing one or more diseases selected from hyperlipidemia, arteriosclerosis and hyperglycemia, which comprises lipoprotein lipase (hereinafter referred to as "LPL") and And / or a system comprising reacting hepatic triacylglycerol lipase (hereinafter referred to as "HTGL") with a substrate, together with a test substance, a polypeptide comprising an amino acid sequence represented by amino acids 17 to 460 of SEQ ID NO: 4 in the sequence listing. A polypeptide lacking 1 to 253 residues at its carboxyl terminus;A polypeptide consisting of the amino acid sequences of amino acids 26 to 406 of SEQ ID NO: 16 in the sequence listing, a polypeptide lacking carboxyl terminal 1 to 263 residues thereof,Or claims1Or8A method comprising coexisting the described polypeptide, and then examining the inhibitory effect of the test substance on the inhibitory effect of the polypeptide on LPL and / or HTGL activity.
20. A method for testing a substance that modulates LPL activity, comprising the following steps:
1) The following[1]-A through[3]Prepare a mixture of the described materials and the test substance:
[1]A material comprising -a @ LPL;
[2]-A material comprising a substrate of LPL;
[3]ポ リ a polypeptide consisting of the amino acid sequence of amino acids 17 to 460 of SEQ ID NO: 4 in the sequence listing, a polypeptide lacking 1 to 253 residues at the carboxyl terminal thereof,A polypeptide consisting of the amino acid sequences of amino acids 26 to 406 of SEQ ID NO: 16 in the sequence listing, a polypeptide lacking carboxyl terminal 1 to 263 residues thereof,Or claims1Or8The described polypeptide;
2) 1) above[1]-A through[3]A mixture consisting only of the materials of 1) is prepared separately from 1);
3) In the mixture of 1) and 2) above, measure the amount of fatty acid released from the substrate, respectively, and compare the measured values.
21. The LPL-containing material is a culture supernatant of a cultured adipocyte derived from a mammal.20The described method.
22. The LPL-containing material is a culture supernatant of a cultured adipocyte derived from a rodent or a primate.20Or21The method described in.
23. The material comprising an LPL substrate, wherein the material is glycerol trioleic acid.20Or22The method according to any one of the preceding claims.
24. A method for testing a substance that regulates LPL and / or HTGL activity, comprising the following steps:
1) The following[1]-B through[3]Prepare a mixture of the described materials and the test substance:
[1]A material comprising -b @ LPL and / or HTGL;
[2]-B material comprising a substrate of LPL and / or HTGL;
[3](4) a polypeptide comprising the amino acid sequence of amino acids 17 to 460 of SEQ ID NO: 4 in the sequence listing, or a polypeptide lacking carboxyl terminal 1 to 253 residuesA polypeptide consisting of the amino acid sequences of amino acid numbers 26 to 406 of SEQ ID NO: 16 in the sequence listing, a polypeptide lacking carboxyl terminal 1 to 263 residues thereof,Or claims1Or8The described polypeptide;
2) 1) above[1]-B through[3]A mixture consisting only of the materials of 1) is prepared separately from 1);
3) In the mixture of 1) and 2) above, measure the amount of fatty acid released from the substrate and compare the measured values.
25. The method according to claim 25, wherein the material containing LPL and / or HTGL is plasma from a mammal to which heparin has been intravenously administered.24The described method.
(26) the material containing LPL and / or HTGL is a culture supernatant of cultured adipocytes derived from mammals;24The described method.
27. The material containing LPL and / or HTGL is a culture supernatant of cultured adipocytes derived from rodents or primates.24Or26The described method.
28. The material comprising a substrate for LPL and / or HTGL, wherein the material is glycerol trioleic acid.24 to 27The method according to any one of the preceding claims.
DETAILED DESCRIPTION OF THE INVENTION
[0001]
[0001] The present invention relates to a novel method for testing an agent for treating or preventing one or more diseases selected from hyperlipidemia, arteriosclerosis and hyperglycemia.
[0002]
In addition, the present invention provides a novel polypeptide useful for searching or testing an agent for treating or preventing one or more diseases selected from hyperlipidemia, arteriosclerosis and hyperglycemia, and the polypeptide using the polypeptide. It relates to a new test method.
[0003]
The present invention also tests lipid-lowering agents, particularly substances that modulate lipase activity, which are useful as agents for treating or preventing one or more diseases selected from hyperlipidemia, arteriosclerosis and hyperglycemia. A new method for doing so.
[0004]
The present invention also relates to a novel DNA having a promoter activity, which is useful for testing an agent for treating or preventing one or more diseases selected from hyperlipidemia, arteriosclerosis, and hyperglycemia.
[0005]
BACKGROUND OF THE INVENTION Cardiovascular disease is still the leading cause of death in most developed countries of the world. Atherosclerosis is the main etiology of acute coronary syndrome (unstable angina, acute myocardial infarction, sudden ischemic death), cerebral thrombosis and cerebral infarction. It is estimated that in the United States each year, 550,000 people die from myocardial infarction caused by atherosclerosis. According to statistics in 1991, cardiovascular disease due to atherosclerosis is the leading cause of death in Japan. Epidemiological studies have shown that atherosclerosis is caused by many risk factors, such as hypercholesterolemia, hyperlipidemia, hypertension, and smoking.
[0006]
An abnormal increase in serum lipid levels is called hyperlipidemia or hyperlipidemia. Serum lipids include cholesterol (cholesterol esters, free cholesterol), phospholipids (lecithin, sphingomyelin, etc.), triglycerides (neutral fats), free fatty acids, and other sterols. Is an increase in neutral fats and cholesterol in the blood (COMMON DISASEASE SERIES No. 19 hyperlipidemia edited by Haruo Nakamura, published October 10, 1991, Nankodo, Austin et al., Circulation 101, 2777-2782 (2000). )). The causal relationship between hypercholesterolemia and the onset of ischemic heart disease has been clarified, and its position as an independent risk factor for ischemic heart disease has been established. Is being done. In recent years, although not as epidemiologically as serum cholesterol levels, hypertriglyceridemia has been re-recognized as a risk factor for various diseases related to arteriosclerosis (Rubins et al., N Engl J Med). 341, {410-418} (1999)). Therefore, appropriate control of blood lipid levels is extremely important for prevention or treatment of various diseases related to arteriosclerosis, such as ischemic heart disease and cerebral infarction (Brewer et al., Am J Cardiol. 83, {3F-12F} (1999)).
As a drug for lowering the neutral fat, fibric acid-based compounds, for example, clofibrate, fenofibrate, bezafibrate, gemfibrozil and the like are provided as medicaments, and lowers the plasma neutral fat, and cholesterol levels. It is known to be useful for the prevention of ischemic heart disease in individuals with elevated cholesterol levels (Havel, R. J. and Kane, J. P. (1973) 13 Ann. Rev. Pharmac. 287- 308, Circulation 102, 21-27 (2000), Frick, M. H. et al. (1987) N. Engl. J. Med. 317, 1237-1245).
[0007]
On the other hand, drugs that lower blood cholesterol level include those that capture and inhibit the absorption of bile acids such as cholestyramine and colestipol (for example, US Pat. No. 4,027,009), melinamide (French Patent No. 1,476,569), and the like. In addition to those that inhibit the intestinal absorption of cholesterol by inhibiting the acyl coenzyme A cholesterol acyltransferase (ACAT), drugs that inhibit cholesterol biosynthesis have attracted attention. As cholesterol biosynthesis inhibitors, lovastatin (US Pat. No. 4,231,938), simvastatin (US Pat. No. 4,444,784), and pravastatin (US), which particularly inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, are used. Japanese Patent No. 4346227) is provided as a medicine.
[0008]
Obtaining a safer and more effective lipid-lowering agent exhibiting a neutral lipid lowering effect is a drug useful for the prevention or treatment of hyperlipidemia or the treatment or prevention of arteriosclerosis, and thus more useful as a medicament. Will be provided.
[0009]
Diabetes, on the other hand, is a disease process derived from a number of causative factors, characterized by elevated plasma glucose levels or hyperglycemia. Uncontrolled hyperglycemia is associated with increased premature mortality due to increased risk for microvascular and macrovascular disease including nephropathy, neuropathy, retinopathy, hypertension, stroke and heart disease I do. Therefore, control of glucose homeostasis is critically important as a treatment for hyperglycemia and, consequently, diabetes.
[0010]
Among the genes used in the method of the present invention, the cDNA sequence described in SEQ ID NO: 1 in the Sequence Listing is disclosed as a nucleotide sequence encoding “angiopoietin-related protein 3” on the Genbank database, International Patent Application Publication No. WO 99/55869 discloses the same nucleotide sequence as encoding "zalpha5". However, the gene having these nucleotide sequences is not associated with hyperlipidemia, arteriosclerosis and hyperglycemia. No association is known, and no DNA sequence is known for its transcriptional regulation. Therefore, a part of the nucleotide sequence encoding “angiopoietin-related protein 3” to “zalpha5” or a promoter thereof is useful for testing an agent for treating or preventing hyperlipidemia, arteriosclerosis and hyperglycemia. Not even known.
[0011]
Six families of “angiopoietin-related proteins” are known (“angiopoietin-related proteins 1, 2, 3, 4, 5, and 6”). "Angiopoietin-related protein 1, 2, 3, 4, 5, and 6" is a secretory protein characterized by a coil structure on the amino terminal side and a fibrinogen-like domain on the carboxyl terminal side, like angiopoietin (FIG. 7). For example, in “angiopoietin-related protein 3”, out of 460 amino acids, the signal peptide extends from the amino terminus to the 16th amino acid, and the helical region extends from the 17th amino acid to the 207th amino acid, and includes a coil structure. It is said that the region from the 241st to the 241st amino acid is a linker region, and the region from the 242nd to 460th amino acid is a fibrinogen binding region.
[0012]
"Angiopoietin-related protein 1" and "Angiopoietin-related protein 2" were identified from human heart by PCR based on homology of angiopoietin (Kim, I., et al. (1999) FEBS Lett. 443: 353-356). {Kim, {I.,} et al (1999) {J. Biol. Chem. 274: 26523-26528). "Angiopoietin-related protein 1" and "angiopoietin-related protein 2" show 59% homology. The homology of “angiopoietin-related proteins 1 and 2” to angiopoietin 1 is 26-29%. The homology of “angiopoietin-related proteins 1 and 2” to angiopoietin 2 is 34%.
[0013]
Although "angiopoietin-related protein 1" is expressed in a wide range of organs, it does not show angiopoietin-like endothelial cell stimulating activity. On the other hand, the expression of “angiopoietin-related protein 2” in the heart, small intestine, spleen and stomach was observed, and although it showed a stimulating activity on vascular endothelial cells, the expression of “angiopoietin 1 or angiopoietin 2 receptor“ TIE2 ” No binding was observed.
[0014]
On the other hand, “angiopoietin-related protein 3” was found by homology search using EST and was shown to be expressed only in liver and kidney (Conklin, D., et al. (1999) Genomics). 62, 477-482). Regarding “angiopoietin-related protein 3”, the function of the C-terminal region has been reported (Conklin, D., et al. (1999) Genomics 62, 477-482), but the N-terminal region has any function. It was not known about. Furthermore, it is not known at all that a mutant having only the N-terminal region lacking the C-terminal region has a function. Further, a partner factor of “angiopoietin-related protein 3” is not known, and a method of in vitro testing a drug that regulates the concentration of triglyceride in blood using angiopoietin-related protein 3 and its partner factor is also known. unknown.
[0015]
“Angiopoietin-related protein 4” is a protein involved in lipid metabolism or glucose homeostasis under the control of PPARγ (Yoon, J C, et al. (2000) Molec. Cell. Biol 20, 5343-5349), a PPARα target gene , A novel "angiopoietin-related protein" derived from the liver that induces apoptosis in endothelial cells (Kersten, S., et al. (2000) J. Biol. Chem. 275, 28488-28493) (Kim, I., et al. (2000) Biochem. J. 346, 603-610). Expression of “angiopoietin-related protein 4” in white adipocytes and placenta has been reported, but no report has been made regarding the presence or absence of angiopoietin-like activity.
In addition, a specific DNA region (promoter / enhancer region) involved in the regulation of the expression of “angiopoietin-related protein 4” has not been identified, and the promoter of the gene is not used for treatment or prevention of hyperlipidemia, arteriosclerosis, and hyperglycemia. It was also not known to be useful for testing agents.
[0016]
“Angiopoietin-related protein 5” has only a registered gene, and its function is unknown.
[0017]
“Angiopoietin-related protein 6” has been identified as a cornea-specifically expressed gene, cornea-translated transscript 6 (CDT6) (Peek, R., et al. (1998) Invest. Ophthalmol. Vis. Sci. 39, 1782-). 1788). "Angiopoietin-related protein 6" showed tumor growth inhibition in a mouse transplanted cancer model (Peek, R., et al. (2001) J. Biol. Chem. Epub ahead of print).
[0018]
Thus, it is conceivable that the “angiopoietin-related protein” plays a function different from the vascular endothelial growth factor-like activity possessed by angiopoietin.
[0019]
The carboxyl-terminal fibrinogen-like domain common to the “angiopoietin-related protein” family has not been reported to be a region involved in binding to the “TIE receptor”. It is unknown what function the terminal coil structure has.
[0020]
Furthermore, it was not known at all that the deletion of only the amino-terminal region lacking the fibrinogen-like region on the carboxyl terminal side of the “angiopoietin-related protein” family has a function.
[0021]
An object of the present invention is to provide a novel method for testing an agent for treating or preventing one or more diseases selected from hyperlipidemia, arteriosclerosis and hyperglycemia. Is to provide.
[0022]
Further, an object of the present invention is to provide a novel polypeptide and the polypeptide useful for searching or testing an agent for treating or preventing one or more diseases selected from hyperlipidemia, arteriosclerosis and hyperglycemia. It is to provide a new test method used.
[0023]
Another object of the present invention is to increase the blood lipid concentration by increasing the activity of LPL and / or HTGL, which is useful as a substance that regulates lipase activity, in particular, an agent for preventing or treating hyperlipidemia and / or arterial effect. It is an object of the present invention to provide a new method for testing a substance that reduces the odor.
[0024]
Another object of the present invention is to specify a promoter region involved in the transcriptional control of the “angiopoietin-related protein 3” gene and the “angiopoietin-related protein 4” gene, and to provide a method for screening a compound that regulates the activity of the promoter. is there. More specifically, a novel method for testing a therapeutic or prophylactic agent for hyperlipidemia, arteriosclerosis or hyperglycemia and a DNA used in the method are provided.
[0025]
[Means for Solving the Problems]
The present invention
(1) A method for testing the effect of a test substance on treating or preventing one or more diseases selected from hyperlipidemia, arteriosclerosis, and hyperglycemia, comprising the following steps:
1) culturing cultured cells derived from a mammal in the presence or absence of a test substance;
2) detecting the expression level of mRNA having the nucleotide sequence described in any one of the following a) to e) (where t in the sequence is replaced by u) in the cultured cells obtained in 1) above: Do:
a) a nucleotide sequence represented by nucleotide numbers 47 to 1411 of SEQ ID NO: 1 in the sequence listing;
b) a nucleotide sequence represented by nucleotide numbers 78 to 1457 of SEQ ID NO: 3 in the sequence listing;
c) Transformed E. coli. a nucleotide sequence of a DNA inserted into a phagemid retained by E. coli pBK / m55-1 SANK 72199 (FERM BP-6940);
d) Transformed E. coli. a nucleotide sequence of a DNA inserted into a phagemid carried by E. coli {pTrip / h55-1} SANK # 72299 (FERM @ BP-6941);
e) a polynucleotide having an activity of hybridizing under stringent conditions with a polynucleotide comprising an antisense sequence of the nucleotide sequence according to any one of the above a) to d) to increase the concentration of neutral fat in blood. A nucleotide sequence encoding a peptide; and
3) As a result of the above step 2), the detected mRNA expression level is compared between the cells cultured in the absence of the test substance and the cells cultured in the presence of the test substance.
[0026]
(2) The method according to (1), wherein the cultured cell derived from a mammal is derived from a liver.
[0027]
(3) The method according to (1) or (2), wherein the cultured cell derived from a mammal is derived from a primate or a rodent.
[0028]
(4) The method according to any one of (1) to (3), wherein the cultured cells derived from mammals are derived from humans or mice.
[0029]
(5) The method according to any one of (1) to (4), wherein the means for detecting the expression level of mRNA is a Northern blot, a dot blot or a slot blot.
[0030]
(6) The method according to any one of (1) to (4), wherein the means for detecting the expression level of mRNA is RT-PCR.
[0031]
(7) The method according to any one of (1) to (4), wherein the means for detecting the mRNA expression level is a ribonuclease protection assay.
[0032]
(8) The method according to any one of (1) to (4), wherein the means for detecting the expression level of mRNA is a run-on assay.
[0033]
(9) In the method according to any one of (1) to (4), the means for detecting the expression level of mRNA is prepared using a complementary DNA group derived from animal tissues or cells or a partial sequence of the DNA. A method using a gene chip.
[0034]
(10) A method for testing the effect of a test substance on treating or preventing one or more diseases selected from hyperlipidemia, arteriosclerosis and hyperglycemia, comprising the following steps:
1) culturing the cultured cells in the presence or absence of a test substance;
2) The production amount of the amino acid sequence encoded by the nucleotide sequence described in any one of the following a) to e) or a polypeptide comprising a part thereof in the cultured cell supernatant obtained in 1) above: , Using an antibody that specifically binds to the polypeptide:
a) a nucleotide sequence represented by nucleotide numbers 47 to 1411 of SEQ ID NO: 1 in the sequence listing;
b) a nucleotide sequence represented by nucleotide numbers 78 to 1457 of SEQ ID NO: 3 in the sequence listing;
c) Transformed E. coli. a nucleotide sequence of a DNA inserted into a phagemid retained by E. coli pBK / m55-1 SANK 72199 (FERM BP-6940);
d) Transformed E. coli. a nucleotide sequence of a DNA inserted into a phagemid carried by E. coli {pTrip / h55-1} SANK # 72299 (FERM @ BP-6941);
e) a polynucleotide having an activity of hybridizing under stringent conditions with a polynucleotide comprising an antisense sequence of the nucleotide sequence according to any one of the above a) to d) to increase the concentration of neutral fat in blood. A nucleotide sequence encoding a peptide; and
3) As a result of the above step 2), the amount of the detected polypeptide is compared between the cells cultured in the absence of the test substance and the cells cultured in the presence of the test substance.
[0035]
(11) The method according to (10), wherein the antibody that specifically binds to the amino acid sequence encoded by the nucleotide sequence according to any one of a) to e) or a polypeptide comprising a part thereof is Polypeptide consisting of the amino acid sequence represented by amino acid Nos. 17-455 of SEQ ID NO: 2 or a part thereof, polypeptide consisting of the amino acid sequence represented by amino acid No. 19-455 or a part thereof, or amino acid of SEQ ID NO: 4 A method which specifically binds to a polypeptide consisting of the amino acid sequence represented by Nos. 17-460 or a part thereof.
[0036]
(12) 方法 In the method according to (10) or (11), specifically binds to a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence according to any one of a) to e) or a part thereof Wherein the antibody specifically recognizes the amino acid sequence represented by amino acid numbers 1 to 13 of SEQ ID NO: 9 or the amino acid sequence represented by amino acid numbers 1 to 14 of SEQ ID NO: 10 in the sequence listing. Method.
[0037]
(13) The polypeptide according to any one of (10) to (12), which comprises the amino acid sequence encoded by the nucleotide sequence according to any one of (a) to (e) or a part thereof A method wherein the operation of detecting the production amount of E. coli using an antibody that specifically binds to the polypeptide is a Western blot, a dot blot, or a slot blot.
[0038]
(14) The polypeptide according to any one of (10) to (12), which comprises the amino acid sequence encoded by the nucleotide sequence according to any one of (a) to (e) or a part thereof Wherein the operation of detecting the amount of the produced using an antibody that specifically binds to the polypeptide is an enzyme-linked immunosorbent assay (ELISA) or a radioisotope immunoassay (RIA). Method.
[0039]
(15) A method comprising using the monoclonal antibody Mab45B1 produced by the mouse hybridoma 45B1 as the antibody immobilized on a plate in the ELISA method according to (14). The hybridoma 45B1 has been deposited with the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary on March 13, 2002 under the deposit number FERM @ BP-7963.
[0040]
(16) An agent for treating or preventing one or more diseases selected from hyperlipidemia, arteriosclerosis and hyperglycemia, which comprises the antibody described in i) and / or ii) below. Kit for testing:
i) an antibody which specifically binds to a polypeptide consisting of an amino acid sequence encoded by the nucleotide sequence according to any one of the following a) to e) or a part thereof:
a) a nucleotide sequence represented by nucleotide numbers 47 to 1411 of SEQ ID NO: 1 in the sequence listing;
b) a nucleotide sequence represented by nucleotide numbers 78 to 1457 of SEQ ID NO: 3 in the sequence listing;
c) Transformed E. coli. a nucleotide sequence of a DNA inserted into a phagemid retained by E. coli pBK / m55-1 SANK 72199 (FERM BP-6940);
d) Transformed E. coli. a nucleotide sequence of a DNA inserted into a phagemid carried by E. coli {pTrip / h55-1} SANK # 72299 (FERM @ BP-6941);
e) a polynucleotide having an activity of hybridizing under stringent conditions with a polynucleotide comprising an antisense sequence of the nucleotide sequence according to any one of the above a) to d) to increase the concentration of neutral fat in blood. A nucleotide sequence encoding a peptide;
ii) An antibody which specifically binds to an amino acid sequence represented by amino acid numbers 1 to 13 of SEQ ID NO: 9 or an amino acid sequence represented by amino acid numbers 1 to 14 of SEQ ID NO: 10 in the sequence listing.
[0041]
(17) Contains, as an active ingredient, DNA or RNA having a nucleotide sequence containing an antisense sequence of a nucleotide sequence consisting of 15 to 30 consecutive nucleotides in the nucleotide sequence of nucleotide numbers 78 to 1457 of SEQ ID NO: 3 in the sequence listing. A composition for gene therapy of one or more diseases selected from hyperlipidemia, arteriosclerosis and hyperglycemia.
[0042]
(18) A single continuous amino acid sequence contained in the amino acid sequence represented by SEQ ID NO: 12 in the sequence listing, wherein the amino acid at amino acid number 17 is the amino terminal and the amino acid at any one of amino acids 147 to 207 is carboxyl. A polypeptide comprising an amino acid sequence to be terminated.
[0043]
(19) The polypeptide according to (18), comprising the amino acid sequence represented by amino acid numbers 17 to 207 of SEQ ID NO: 12 in the sequence listing.
[0044]
(20) The polypeptide according to (18), comprising an amino acid sequence represented by amino acid numbers 17 to 195 of SEQ ID NO: 12 in the sequence listing.
[0045]
(21) The polypeptide according to (18), which comprises the amino acid sequence represented by amino acid numbers 17 to 147 of SEQ ID NO: 12 in the sequence listing.
[0046]
(22) A polypeptide comprising a continuous amino acid sequence contained in the amino acid sequence represented by SEQ ID NO: 16 in the sequence listing, the amino acid sequence having amino acid number 26 as an amino terminal and amino acid number 143 as a carboxyl terminal.
[0047]
(23) A single continuous amino acid sequence contained in the amino acid sequence represented by SEQ ID NO: 16 in the sequence listing, wherein amino acid number 26 is the amino terminal and one of amino acid numbers 144 to 183 is the carboxyl terminal A polypeptide consisting of an amino acid sequence.
[0048]
(24) A continuous amino acid sequence contained in the amino acid sequence of SEQ ID NO: 18 in the sequence listing, the amino acid sequence having amino acid number 22 as an amino terminal and any one of amino acid numbers 202 to 276 as a carboxyl terminal A polypeptide consisting of
[0049]
(25) A single continuous amino acid sequence contained in the amino acid sequence of SEQ ID NO: 20 in the sequence listing, wherein the amino acid number is 23 as an amino terminal and any one of amino acid numbers 206 to 275 is a carboxyl terminal. A polypeptide consisting of
[0050]
(26) a single continuous amino acid sequence contained in the amino acid sequence of SEQ ID NO: 22 in the sequence listing, wherein the amino acid number is 21 as an amino terminal and any one of amino acids 185 to 258 is a carboxyl terminal; A polypeptide consisting of
[0051]
(27) A single continuous amino acid sequence contained in the amino acid sequence shown in SEQ ID NO: 24 in the sequence listing, wherein amino acid number 27 is the amino terminal and one of amino acid numbers 122 to 129 is the carboxyl terminal. A polypeptide consisting of an amino acid sequence.
[0052]
(28) A polypeptide consisting of an amino acid sequence having amino acid number 26 in SEQ ID NO: 16 as the amino terminal and 406 as the carboxyl terminal in the sequence listing.
[0053]
(29) A polypeptide consisting of an amino acid sequence having amino acid No. 22 of SEQ ID No. 18 in the sequence listing as an amino terminal and 491 as a carboxyl terminal.
[0054]
(30) A polypeptide having an amino acid sequence having amino acid number 23 of SEQ ID NO: 20 as an amino terminal and 493 as a carboxyl terminal in the sequence listing.
[0055]
(31) A polypeptide consisting of an amino acid sequence having amino acid number 21 in SEQ ID NO: 22 as the amino terminal and 470 as the carboxyl terminal in the sequence listing.
[0056]
(32) A polypeptide consisting of an amino acid sequence having amino acid number 27 in SEQ ID NO: 24 and amino acid number 346 in the sequence listing.
[0057]
(33) A polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 26 in the sequence listing.
[0058]
(34) Mammalian blood in which one or several amino acid residues are deleted, inserted, added or substituted at one or several sites of the amino acid sequence according to (18) to (33). An equivalent of a polypeptide having an activity to increase the concentration of neutral fat in the same.
[0059]
(35) DNA encoding the polypeptide according to any one of (18) to (34).
[0060]
(36) A continuous sequence contained in the nucleotide sequence shown in SEQ ID NO: 11 in the sequence listing, in which the nucleotide at nucleotide number 18 is the 5 'end and any one of nucleotide numbers 458 to 638 is 3 'A DNA consisting of a nucleotide sequence to be a terminus.
[0061]
(37) A continuous sequence contained in the nucleotide sequence shown in SEQ ID NO: 11 in the sequence listing, in which the nucleotide at nucleotide number 18 is the 5 ′ end and any one nucleotide from nucleotide numbers 458 to 602 is 3 'A DNA consisting of a nucleotide sequence to be a terminus.
[0062]
(38) A single continuous sequence contained in the nucleotide sequence shown in SEQ ID NO: 15 in the sequence listing, consisting of a nucleotide sequence having the nucleotide of nucleotide number 173 as the 5 'end and the nucleotide number 601 as the 3' end. DNA.
[0063]
(39) A single continuous sequence contained in the nucleotide sequence shown in SEQ ID NO: 15 in the sequence listing, consisting of a nucleotide sequence having the nucleotide of nucleotide number 173 as the 5 'end and the nucleotide number 1393 as the 3' end. DNA.
[0064]
(40) A single continuous sequence contained in the nucleotide sequence shown in SEQ ID NO: 17 in the sequence listing, wherein the nucleotide at nucleotide number 434 is the 5 'end, and any one nucleotide from nucleotide numbers 1039 to 1261 is 3 'A DNA consisting of a nucleotide sequence to be a terminus.
[0065]
(41) A single continuous sequence contained in the nucleotide sequence shown in SEQ ID NO: 17 in the sequence listing, comprising a nucleotide sequence having the nucleotide of nucleotide number 434 as the 5 'end and nucleotide number 1909 as the 3' end. DNA.
[0066]
(42) A single continuous sequence contained in the nucleotide sequence shown in SEQ ID NO: 19 in the sequence listing, wherein the nucleotide at nucleotide number 22 is the 5 'end, and any one of nucleotide numbers 639 to 846 is 3 'A DNA consisting of a nucleotide sequence to be a terminus.
[0067]
(43) A single continuous sequence contained in the nucleotide sequence shown in SEQ ID NO: 19 in the sequence listing, comprising a nucleotide sequence having nucleotide 5 at the 5 ′ end and nucleotide 3150 at the 3 ′ end. DNA.
[0068]
(44) A single continuous sequence contained in the nucleotide sequence shown in SEQ ID NO: 21 in the sequence listing, in which the nucleotide at nucleotide number 242 is the 5 'end and any one nucleotide from nucleotide numbers 796 to 1015 is 3 'A DNA consisting of a nucleotide sequence to be a terminus.
[0069]
(45) A single continuous sequence contained in the nucleotide sequence shown in SEQ ID NO: 21 in the sequence listing, comprising a nucleotide sequence having the nucleotide of nucleotide number 242 as the 5 'end and nucleotide number 1654 as the 3' end. DNA.
[0070]
(46) A single continuous sequence contained in the nucleotide sequence shown in SEQ ID NO: 23 of the sequence listing, wherein the nucleotide at nucleotide number 86 is the 5 'end, and any one of nucleotide numbers 373 to 394 is 3 'A DNA consisting of a nucleotide sequence to be a terminus.
[0071]
(47) A single continuous sequence contained in the nucleotide sequence shown in SEQ ID NO: 23 of the sequence listing, consisting of a nucleotide sequence having nucleotide No. 86 as the 5 ′ end and nucleotide No. 1048 as the 3 ′ end. DNA.
[0072]
(48) DNA comprising a nucleotide sequence represented by nucleotide numbers 18 to 662 of SEQ ID NO: 25 in the sequence listing.
[0073]
(49) A recombinant vector containing the DNA according to any one of (35) to (48).
[0074]
(50) The recombinant vector according to (49), which is an adenovirus vector.
[0075]
(51) A host cell transformed with the recombinant vector according to (49) or (50).
[0076]
(52) A mammal, which comprises culturing the host cell according to (51), and then recovering, from the culture, a polypeptide having an activity of increasing the concentration of neutral fat in the blood of the mammal. A method for producing a polypeptide having an activity of increasing the concentration of neutral fat in blood.
[0077]
(53) A method for testing an agent for treating or preventing one or more diseases selected from hyperlipidemia, arteriosclerosis and hyperglycemia, comprising the following steps:
1) administering to the non-human mammal one or more polypeptides selected from the polypeptides according to (18) to (34), and a test substance; and
2) Measure the neutral fat concentration in the blood of the animal of 1) above.
[0078]
(54) A method for testing an agent for treating or preventing one or more diseases selected from hyperlipidemia, arteriosclerosis and hyperglycemia, comprising the following steps:
1) Infect a non-human mammal with an adenovirus carrying the recombinant adenovirus vector according to (50);
2) {administering a test substance to the animal of the above step 1);
3) Measure the neutral fat concentration in the blood of the animal in step 2) above.
[0079]
(55) A method for testing an agent for treating or preventing one or more diseases selected from hyperlipidemia, arteriosclerosis, and hyperglycemia, which comprises lipoprotein lipase (hereinafter, referred to as “LPL”) and / or In a system for reacting hepatic triacylglycerol lipase (hereinafter referred to as "HTGL") with its substrate, a polypeptide comprising an amino acid sequence represented by amino acid numbers 17 to 460 of SEQ ID NO: 4 together with a test substance, A polypeptide lacking the carboxyl terminal 1 to 253 residues or the polypeptide according to (18) to (34) is allowed to coexist, and then the test substance is inhibited from inhibiting the LPL and / or HTGL activity by the polypeptide. A method characterized by examining the effect.
[0080]
(56) {A method for testing a substance that modulates LPL activity, comprising the following steps:
1) The following[1]-A through[3]Prepare a mixture of the described materials and the test substance:
[1]A material comprising -a @ LPL;
[2]-A material comprising a substrate of LPL;
[3]ポ リ a polypeptide consisting of the amino acid sequence of amino acids 17 to 460 of SEQ ID NO: 4 in the sequence listing, a polypeptide lacking carboxyl terminal 1 to 253 residues, or a polypeptide according to (18) to (34);
2) 1) above[1]-A through[3]A mixture consisting only of the materials of 1) is prepared separately from 1);
3) In the mixture of 1) and 2) above, measure the amount of fatty acid released from the substrate, respectively, and compare the measured values.
[0081]
(57) The method according to (56), wherein the material containing LPL is a culture supernatant of a cultured adipocyte derived from a mammal.
[0082]
(58) The method according to (56) or (57), wherein the material containing LPL is a culture supernatant of a cultured adipocyte derived from a rodent or a primate.
[0083]
(59) The method according to any one of (56) to (58), wherein the material containing the substrate of LPL is glycerol trioleic acid.
[0084]
(60) A method for testing a substance that modulates LPL and / or HTGL activity, comprising the following steps:
1) The following[1]-B through[3]Prepare a mixture of the described materials and the test substance:
[1]A material comprising -b @ LPL and / or HTGL;
[2]-B material comprising a substrate of LPL and / or HTGL;
[3]ポ リ a polypeptide consisting of the amino acid sequence of amino acids 17 to 460 of SEQ ID NO: 4 in the sequence listing, a polypeptide lacking 1 to 253 residues at the carboxyl terminus or a polypeptide according to (18) to (34);
2) 1) above[1]-B through[3]A mixture consisting only of the materials of 1) is prepared separately from 1);
3) In the mixture of 1) and 2) above, measure the amount of fatty acid released from the substrate and compare the measured values.
[0085]
(61) The method according to (60), wherein the material containing LPL and / or HTGL is plasma from a mammal to which heparin has been intravenously administered.
[0086]
(62) The method according to (60) or (61), wherein the material containing the substrate of ΔLPL and / or HTGL is glycerol trioleate.
[0087]
(63) The material comprising LPL and / or HTGL is a culture supernatant of a cultured adipocyte derived from a mammal or plasma derived from a mammal to which heparin has been intravenously administered (59) ) Described method.
[0088]
(64) The method according to (59) or (63), wherein the material containing LPL and / or HTGL is a culture supernatant of a cultured adipocyte derived from a rodent or a primate.
[0089]
(65) The method according to any one of (59), (63) or (64), wherein the material containing the substrate of ΔLPL and / or HTGL is glycerol trioleate.
[0090]
(66) DNA represented by any one of the following 1) to 3):
1) In the nucleotide sequence shown in SEQ ID NO: 27 in the sequence listing, a nucleotide sequence having the nucleotide shown by nucleotide No. 1 as the 5 ′ end and the nucleotide shown by any one of nucleotide numbers 510 to 526 as the 3 ′ end DNA consisting of:
2) DNA that hybridizes with the DNA of 1) above under stringent conditions and has promoter activity in mammalian liver-derived cells;
3) A DNA comprising a nucleotide sequence having 95% or more nucleotide sequence identity to the DNA described in 1) above and having promoter activity in mammalian liver-derived cells.
[0091]
(67) A DNA comprising the nucleotide sequence represented by nucleotide numbers 1 to 510 of SEQ ID NO: 27 in the sequence listing.
[0092]
(68) DNA comprising a nucleotide sequence represented by nucleotide numbers 1 to 510 of SEQ ID NO: 27 in the sequence listing.
[0093]
(69) A DNA having a nucleotide sequence represented by nucleotide numbers 1 to 526 of SEQ ID NO: 27 in the sequence listing.
[0094]
(70) DNA represented by any one of the following 1) to 3):
1) a nucleotide sequence having a nucleotide represented by nucleotide No. 1 as a 5 ′ end and a nucleotide represented by any one of nucleotides 188 to 260 as a 3 ′ end in the nucleotide sequence represented by SEQ ID NO: 28 in the sequence listing DNA consisting of:
2) DNA that hybridizes with the DNA of 1) above under stringent conditions and has promoter activity in mammalian cells;
3) A DNA comprising a nucleotide sequence having 95% or more nucleotide sequence identity with the DNA described in 1) above and having promoter activity in mammalian cells.
[0095]
(71) A DNA comprising a nucleotide sequence represented by nucleotide numbers 1 to 188 of SEQ ID NO: 28 in the sequence listing, and having promoter activity in mammalian cells.
[0096]
(72) A DNA having a nucleotide sequence represented by nucleotide numbers 1 to 188 of SEQ ID NO: 28 in the sequence listing.
[0097]
(73) An expression vector containing the DNA according to any one of (66) to (72).
[0098]
(74) The DNA according to any one of (66) to (72) is linked to the upstream side of the foreign gene in the same transcriptional direction as the sequence of the foreign gene, and allows the foreign gene to be expressed in mammalian cells. Expression vector to be obtained.
[0099]
(75) The expression according to (74), wherein the foreign gene sequence is a gene sequence encoding a protein selected from the group consisting of luciferase, horseradish peroxidase, alkaline phosphatase, β-galactosidase and green fluorescent protein. vector.
[0100]
(76) The expression vector according to (74), wherein the foreign gene sequence is a gene sequence encoding luciferase.
[0101]
(77) Recombinant plasmid pGL8-3 (FERM BP-7627).
[0102]
(78) Expression vector pGL11-4.
[0103]
(79) A host cell transformed with the expression vector according to any one of (73) to (78).
[0104]
(80) A method for testing the effect of a test substance on treating or preventing one or more diseases selected from hyperlipidemia, arteriosclerosis and hyperglycemia, comprising the following steps i) to iii):
i) a step of culturing a host cell transformed with the expression vector according to any one of (73) to (78) in the presence or absence of a test substance;
ii) a step of detecting or measuring the production amount of a protein encoded by a foreign gene in the expression vector according to any one of (73) to (78) in the host cell of i);
iii) a step of comparing the results of the above ii) between detection and measurement of cells cultured in the presence of the test substance and cells cultured in the absence of the test substance.
[0105]
(81) Identifying a cDNA encoding a protein having a therapeutic or preventive effect on one or more diseases selected from hyperlipidemia, arteriosclerosis and hyperglycemia, comprising the following steps i) and ii): how to:
i)[1](73) a host cell transformed with the expression vector according to any one of (78) (hereinafter, referred to as "cell A");[2]A cell that transiently or stably expresses the test cDNA by transforming cell A with a recombinant plasmid in which the test cDNA has been incorporated into a mammalian expression vector (hereinafter referred to as “cell B”);[3]A cell which has been transformed with another recombinant plasmid but does not express the test cDNA (hereinafter referred to as "cell C").[1]Or[3]Individually culturing cells selected from the group consisting of:
ii) measuring the expression level of the protein encoded by the foreign gene in the expression vector according to any one of (73) to (78) in the cell A, the cell B, and the cell C in the above i). The process of comparing.
[0106]
(82) A method for detecting a protein that regulates the “angiopoietin-related protein 3” gene promoter activity, comprising contacting a sample containing the protein with the DNA according to any one of (66) to (69), Then, detecting the protein bound to the DNA.
[0107]
(83) A method for testing a substance having an activity of regulating the promoter activity of the “angiopoietin-related protein 3” gene, comprising the following steps i) and ii):
i) A radiolabeled DNA of any one of (66) to (69) to which a nuclear extract of mouse cells and a test substance were added, and only a nuclear extract of mouse cells was added. Prepare each one;
ii) Electrophoresis of each mixture sample of the above i), and the mobility is compared between a band found in a sample to which only a nuclear extract of mouse cells is added and a band found in a sample to which a test substance is added.
[0108]
(84) A DNA consisting of at least 10 contiguous nucleotides, a nucleic acid consisting of a nucleotide sequence containing its antisense sequence, or a derivative thereof in the nucleotide sequence represented by nucleotide numbers 1 to 526 of SEQ ID NO: 27 in the sequence listing.
[0109]
(85) A method for isolating a protein that regulates the “angiopoietin-related protein 4” gene promoter activity, comprising contacting a DNA-containing sample with the DNA according to any one of (70) to (72), Then, isolating the protein bound to the DNA.
[0110]
(86) A method for testing a substance having an activity of regulating the “angiopoietin-related protein 4” gene promoter activity, comprising the following steps i) and ii):
i) A radiolabeled DNA of any one of (70) to (72), to which a nuclear extract of mouse cells and a test substance were added, and only a nuclear extract of mouse cells was added. Prepare each one;
ii) Electrophoresis of each mixture sample of the above i), and the mobility is compared between a band found in a sample to which only a nuclear extract of mouse cells is added and a band found in a sample to which a test substance is added.
[0111]
(87) A DNA consisting of at least 10 contiguous nucleotides in the nucleotide sequence represented by nucleotide numbers 1 to 260 of SEQ ID NO: 28 in the sequence listing, and a nucleotide sequence containing its antisense sequence, (70) to (72) A nucleic acid which promotes or inhibits the promoter activity of the DNA according to any one of the above, or a derivative thereof.
About.
[0112]
That is, the present invention relates to the expression of genes involved in the regulation of neutral fat concentration in mammalian blood, as shown in SEQ ID NOs: 1, 3, 11, 15, 17, 19, 21, 23 and 25 in the sequence listing. The present invention provides a method for testing the effect of a test substance as an agent for treating or preventing hyperlipidemia, arteriosclerosis, and hyperglycemia, and nucleic acid probes, primers, and antibodies used in the method.
[0113]
The present inventors compare the gene of the congenital hypolipidemic mouse with the gene of the hyperlipidemic mouse for the purpose of searching for a target gene for a therapeutic or preventive agent for hyperlipidemia, As a result of narrowing down the location of the causative gene on the chromosome, we succeeded in specifying a gene that is highly expressed in hyperlipidemic mice. As a result of homology search, cDNA derived from this gene was disclosed as a nucleotide sequence encoding “angiopoietin-related protein 3” in the Genbank database. However, the present inventors have determined that a gene having this nucleotide sequence can be hypolipidemic. It has been found that administration of a human-type recombinant protein expressed in mice or expressed in animal cells increases the concentration of triglyceride in the blood, and a novel gene whose gene has not been reported so far. We confirmed that we had function.
[0114]
Furthermore, in mice obtained by crossing innately a congenic mouse that does not normally express the “angiopoietin-related protein 3” gene with an apolipoprotein E-deficient mouse that develops arteriosclerosis, the development of arteriosclerosis is suppressed. It was confirmed that it was done. Thus, a new test method for a therapeutic or preventive agent for arteriosclerosis using a gene expression detection system using the nucleotide sequence or a part thereof as a probe or primer was successfully constructed. Further, a novel test method for a therapeutic or prophylactic agent for arteriosclerosis using an experimental system for preparing an antibody specific to the polypeptide encoded by the nucleotide sequence and detecting the production amount of the polypeptide using the antibody Successfully built.
[0115]
Furthermore, in a mouse obtained by crossing a congenic mouse that does not normally express the "angiopoietin-related protein 3" gene and an apolipoprotein E-deficient mouse or a mouse having a leptin mutant gene that develops atherosclerosis, It was confirmed that the hyperglycemia status was improved. Thus, it has been found that a novel test method for a therapeutic or preventive agent for hyperglycemia can be constructed by using a gene expression detection system using the nucleotide sequence or a part thereof as a probe or primer. Further, a novel test method for a therapeutic or preventive agent for hyperglycemia using an experimental system for preparing an antibody specific to the polypeptide encoded by the nucleotide sequence and detecting the production amount of the polypeptide using the antibody is provided. Succeeded in building.
[0116]
In addition, the present inventors focused on the amino-terminal coil structure commonly possessed by “angiopoietin-related proteins” and lacked the carboxyl-terminal fibrinogen-like region of “angiopoietin-related proteins” and “angiopoietin-related proteins”. It has been found that the defective polypeptide having only the amino-terminal region has a function of increasing blood neutral fat concentration in mammals. Furthermore, when the present inventors administer “angiopoietin-related protein” to blood, the fibrinogen-like region on the carboxyl terminal side is cleaved, and a polypeptide having a helical region on the amino terminal side is generated. It was also found to be involved in increasing fat concentration. In addition, the present inventors have used in vivo and in vitro a polypeptide with only the amino-terminal region lacking the carboxyl-terminal fibrinogen-like region of "angiopoietin-related protein" or "angiopoietin-related protein". A method for testing an agent for treating or preventing one or more diseases selected from hyperlipidemia, arteriosclerosis and hyperglycemia was established.
[0117]
The helical region, linker region, and fibrinogen-like region in the “angiopoietin-related protein” can be determined by, for example, three-dimensional crystal structure analysis, but can also be predicted using a computer program. Such a program includes GCG \ Package \ Ver. 10.2 (COIL @ SCAN) (Lupas, et al., Science 252: 1162-1164.1991.). A coil structure exists in the helical region.
[0118]
According to the above-mentioned computer program or experimental results, in "angiopoietin-related protein 1", amino acid numbers 1 to 21 of SEQ ID NO: 18 in the sequence listing are signal peptides, and 22 to 202 are helical regions containing a coil structure. 277 to 491 are fibrinogen-like regions, and in “angiopoietin-related protein 2”, a helix containing amino acid numbers 1 to 22 of SEQ ID NO: 20 is a signal peptide and 23 to 206 is a coil structure Regions 276 to 493 are fibrinogen-like regions. In “angiopoietin-related protein 3”, amino acid numbers 1 to 16 of SEQ ID NO: 4 in the sequence listing include a signal peptide, and 17 to 207 include a coil structure. Helix territory 242 to 460 are fibrinogen-like regions, and in “angiopoietin-related protein 4”, a helix containing amino acid numbers 1 to 25 of SEQ ID NO: 16 in the sequence listing is a signal peptide and 26 to 144 is a coil structure The region 184 to 406 is a fibrinogen-like region. In the “angiopoietin-related protein 5,” amino acids 1 to 20 of SEQ ID NO: 22 in the sequence listing include a signal peptide, and 21 to 185 include a coil structure. The helical region, # 259 to 470, is a fibrinogen-like region. In "angiopoietin-related protein 6," amino acid numbers 1 to 26 of SEQ ID NO: 24 in the sequence listing include a signal peptide, and 27 to 122 include a coil structure. Herrick Area, is the fibrinogen-like area up to 130 or 346.
[0119]
In addition, the present inventors conducted a search for a partner factor of “angiopoietin-related protein 3”, which is responsible for increasing the blood neutral fat concentration in vivo. As a result, the lipase activity of LPL and / or It was found to be inhibited by protein 3. Then, as a method for testing in vitro a substance that regulates the blood neutral fat concentration increasing effect of angiopoietin-related protein 3, a method utilizing this lipase activity inhibitory effect of LPL and / or HTGL was developed, The present invention has been completed.
[0120]
Since angiopoietin-related protein 3 inhibits LPL activity, if a substance that substantially increases LPL activity by suppressing the interaction between the protein and LPL is found, a hyperlipidemia having a new mechanism of action can be obtained. Alternatively, an agent useful for treating or preventing arteriosclerosis will be provided.
[0121]
In addition, the present inventors set the site (nucleotide number 419 of SEQ ID NO: 27 in the sequence listing) predicted to be the transcription start site of the “angiopoietin-related protein 3” gene in the mouse genome as “1”, and From the site of 418 nucleotides upstream, a DNA consisting of a nucleotide sequence of 108 nucleotides downstream of the 3 ′ end (hereinafter referred to as “−418 to +108”; nucleotide numbers 1 to 526 of SEQ ID NO: 27 in the sequence listing) is added to luciferase. Was introduced into human HepG2 cells and the amount of luciferase produced was measured to find that the "-418 to +108" region of the "angiopoietin-related protein 3" gene had promoter activity. Was. Further, the present inventors shortened the 5 'terminal region of the mouse "angiopoietin-related protein 3" gene linked to the luciferase gene to "+93 to +108" (nucleotide numbers 511 to 526 of SEQ ID NO: 27 in the sequence listing). We also found that promoter activity was lost. The present inventors have further found that by using a DNA having the above promoter activity, a compound that regulates the promoter activity can be screened.
[0122]
Further, the present inventors set the site (nucleotide number 91 of SEQ ID NO: 28 in the sequence listing) predicted as the transcription start site of the “angiopoietin-related protein 4” gene in the mouse genome as “1”, and DNA consisting of a nucleotide sequence of 169 nucleotides downstream from the site of 90 nucleotides upstream (hereinafter referred to as “−90 to +170”; corresponding to nucleotide numbers 1 to 260 of SEQ ID NO: 28 in the sequence listing) A reporter gene linked to DNA encoding luciferase is introduced into monkey kidney cells COS1, and the amount of luciferase produced is measured, whereby the "-90 to +170" region of the "angiopoietin-related protein 4" gene has promoter activity. I found that. The present inventors also shortened the 5'-terminal region of the mouse "angiopoietin-related protein 4" gene linked to the luciferase gene to "+99 to +170" (corresponding to nucleotide numbers 189 to 260 of SEQ ID NO: 28 in the sequence listing). Then, they found that the promoter activity was lost.
The present inventors have further found that by using a DNA having the above promoter activity, a compound that regulates the promoter activity can be screened.
[0123]
The DNA of the present invention may contain any nucleotide sequence in the 5'-terminal upstream region of the mouse "angiopoietin-related protein 3" gene as long as it has promoter activity in mammalian liver-derived cells. Preferable examples include a DNA consisting of the nucleotide sequence shown in nucleotide numbers 1 to 526 of SEQ ID NO: 27 in the sequence listing, but the present invention is not limited thereto.
[0124]
On the other hand, DNA consisting of a nucleotide sequence corresponding to +93 to +108 on the 5 ′ end side of the mouse “angiopoietin-related protein 3” gene (nucleotide numbers 511 to 526 of SEQ ID NO: 27 in the sequence listing) has substantially no promoter activity. Absent. Therefore, the nucleotide sequence (nucleotide numbers 1 to 510 of SEQ ID NO: 27 in the sequence listing) corresponding to positions −418 to +92 in the 5′-end upstream region of the mouse “angiopoietin-related protein 3” gene is essential for the promoter activity. Since the region is considered to be included, the promoter DNA of the present invention preferably contains at least a part of the nucleotide sequence shown by nucleotide numbers 1 to 510 of SEQ ID NO: 27 in the sequence listing.
[0125]
The DNA of the present invention may contain any nucleotide sequence in the 5'-terminal upstream region of the mouse "angiopoietin-related protein 4" gene as long as it has promoter activity in mammalian cells. Preferably, a DNA comprising the nucleotide sequence shown in nucleotide numbers 1 to 260 of SEQ ID NO: 28 in the sequence listing can be mentioned, but the present invention is not limited thereto.
[0126]
On the other hand, DNA consisting of a nucleotide sequence corresponding to +99 to +170 at the 5 ′ end of the mouse “angiopoietin-related protein 4” gene (corresponding to nucleotide numbers 189 to 260 in SEQ ID NO: 28 in the sequence listing) substantially has a promoter activity. I do not have. Therefore, a nucleotide sequence corresponding to positions −90 to +98 (corresponding to nucleotide numbers 1 to 188 of SEQ ID NO: 28 in the sequence listing) in the 5′-terminal upstream region of the mouse “angiopoietin-related protein 4” gene has a promoter activity. Since the essential region is considered to be included, the promoter DNA of the present invention more preferably at least partially includes the nucleotide sequence represented by nucleotide numbers 1 to 188 of SEQ ID NO: 28 in the sequence listing.
[0127]
In the present invention, “hybridize under stringent conditions” means to hybridize at 68 ° C. in a commercially available hybridization solution ExpressHyb Hybridization Solution (manufactured by Clontech) or to hybridize under the same conditions. Say.
[0128]
BEST MODE FOR CARRYING OUT THE INVENTION
I.方法 Method using gene expression as an index
One method of the present invention specifically includes, for example, a nucleotide sequence represented by nucleotide numbers 47 to 1411 of SEQ ID NO: 1 in the sequence listing, a nucleotide sequence represented by nucleotide numbers 78 to 1457 of SEQ ID NO: 3 in the sequence listing, Nucleotide sequence represented by nucleotide numbers 173 to 1390 of SEQ ID NO: 15 in the Sequence Listing, nucleotide sequence represented by nucleotide numbers 434 to 1909 of SEQ ID NO: 17 in the Sequence Listing, nucleotide sequence represented by nucleotide numbers 22 to 1500 of SEQ ID NO: 19 of Sequence Listing Having the nucleotide sequence shown in SEQ ID NO: 21 from nucleotide numbers 242 to 1652 in the sequence listing or the nucleotide sequence shown in nucleotide numbers 8 to 1045 in SEQ ID NO: 23, or the polypeptide encoded by them. The expression of a gene encoding a polypeptide having the activity of increasing the concentration of a substance in the blood is measured by the specific detection of the nucleic acid (mRNA) or the polypeptide, and the test is performed to decrease the expression level of the gene. The substance is selected as a candidate substance for an agent for treating or preventing one or more diseases selected from hyperlipidemia, arteriosclerosis, and hyperglycemia. Hereinafter, an embodiment for detecting a nucleic acid and an embodiment for detecting a polypeptide will be described separately.
[0129]
(A) Detection of nucleic acid
1) Probe
Among the embodiments for detecting nucleic acid, the probe used in the method using nucleic acid hybridization is DNA or RNA, and the nucleotide sequence thereof is as follows a) to e):
a) a nucleotide sequence represented by nucleotide numbers 47 to 1411 of SEQ ID NO: 1 in the sequence listing;
b) a nucleotide sequence represented by nucleotide numbers 78 to 1457 of SEQ ID NO: 3 in the sequence listing;
c) Transformed E. coli. a nucleotide sequence of a DNA fragment inserted into a phagemid carried by E. coli \ pBK / m55-1 \ SANK \ 72199 (FERM \ BP-6940);
d) Transformed E. coli. a nucleotide sequence of a DNA fragment inserted into a phagemid carried by E. coli pTrip / h55-1 SANK 72299 (FERM BP-6941);
e) a polynucleotide having an activity of hybridizing under stringent conditions with a polynucleotide having the antisense sequence of the nucleotide sequence according to any one of the above a) to d) to increase the concentration of neutral fat in blood. A nucleotide sequence encoding a peptide;
(Where t in the sequence is read as u), and may be, for example, a polynucleotide having the antisense sequence of the nucleotide sequence described in any one of the above a) to e). Having the nucleotide sequence according to any one of the above a) to e), such as a nucleotide, a polynucleotide having a partial sequence consisting of at least 15 consecutive nucleotides in the antisense sequence, or a variant of the antisense sequence; Any substance capable of specifically detecting the polyribonucleotide by hybridizing with the polyribonucleotide under stringent conditions can be used in the method of the present invention. Among them, a polynucleotide having an antisense sequence of the nucleotide sequence described in a) above can be obtained by a well-known method, for example, based on the nucleotide sequence information shown in SEQ ID NO: 1 from a mouse liver-derived cDNA library, for example, A cDNA clone cloned by a plaque hybridization method, a colony hybridization method, a PCR method, or the like, is directly labeled by a well-known method, or is labeled in a replication or transcription reaction by a polymerase reaction using the cDNA clone as a template. It can be obtained as a labeled probe. The polynucleotide having the antisense sequence of the nucleotide sequence described in b) can be obtained by performing the same operation based on the nucleotide sequence information shown in SEQ ID NO: 3 in the sequence listing from a cDNA library derived from human liver, for example. A labeled probe can be obtained from the cloned cDNA clone. On the other hand, a polynucleotide having an antisense sequence of the nucleotide sequence described in c) or d) above was submitted to the National Institute of Advanced Industrial Science and Technology (currently the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary) by the National Institute of Advanced Industrial Science and Technology. E. coli strain E. coli deposited internationally on November 19, 2014 and having the accession number FERM @ BP-6940 or FERM @ BP-6941. coliBK / m55-1 {SANK} 72199 strain or E. coli E. coli {pTrip / h55-1} SANK # 72299 can be obtained from the recombinant phagemid.
[0130]
Further, the polynucleotide having the antisense sequence of the nucleotide sequence described in e) above can be used as a probe, for example, using the polynucleotide having the antisense sequence of the nucleotide sequence described in a) to d) obtained as described above as a probe. Can be obtained from a cDNA clone isolated by cloning a cDNA library derived from a mammal (preferably liver) by plaque hybridization or colony hybridization.
[0131]
Furthermore, a polynucleotide having a partial sequence consisting of at least 15 contiguous nucleotides in the antisense sequence of the nucleotide sequence described in any one of the above a) to e) may be chemically modified if it has about several tens of nucleotides. It can be obtained by synthesis. Alternatively, an arbitrary partial sequence in a cDNA clone having the nucleotide sequence according to any one of the above a) to e) obtained as described above is subcloned by, for example, PCR or the like, and then the same as above. Can also be prepared as a probe having an antisense sequence by the method described in
[0132]
For example, a polynucleotide consisting of a nucleotide sequence in which several nucleotides are added, deleted and / or added in a partial sequence consisting of several tens of nucleotides in the antisense sequence of the nucleotide sequence described in SEQ ID NO: 1 in the sequence listing Even if it hybridizes with the polyribonucleotide described in any one of the above a) to e) under stringent conditions, it can be used in the method of the present invention. Such a polynucleotide can be produced using a chemical synthesis method or a well-known mutagenesis method in the technical field to which the present invention belongs using an enzymatic reaction such as a polymerase chain reaction (hereinafter, referred to as “PCR”). is there.
[0133]
Further, the probe used in the method of the present invention is not limited to a probe consisting of a single nucleotide sequence. That is, in the method of the present invention, for example, a mixture of a plurality of types of nucleotide sequences satisfying the above requirements may be used as a probe, or multiplex detection may be performed using each of the plurality of types of nucleotide sequences individually.
[0134]
2) Primers for RT-PCR
In another embodiment of the present invention for detecting a nucleic acid, a reverse transcriptase reaction using mRNA as a template is first performed, and then PCR is performed to specifically amplify a DNA fragment, that is, so-called RT-PCR is performed. Is the way. In this method, in order to specifically amplify the nucleotide sequence of interest, an antisense primer complementary to a specific partial sequence of the mRNA of interest and a sequence of cDNA generated by reverse transcriptase from the antisense primer are used. A sense primer complementary to a specific partial sequence therein is used.
[0135]
The antisense primer used for both the reverse transcriptase reaction and the PCR is substantially a nucleotide sequence of at least 18 nucleotides, preferably at least 23 nucleotides, in the antisense sequence of the nucleotide sequence described in a) to e) above. Consists of
[0136]
On the other hand, the sequence of the sense primer used in the PCR is the nucleotide sequence described in the above a) to e), which is a region further 5 'end than the most 5' end position in the sequence corresponding to the complementary strand of the antisense primer. Consists of any subsequence of at least 18 contiguous nucleotides, preferably at least 21 nucleotides, in the sequence present at However, if the sense primer and the antisense primer have mutually complementary sequences, annealing of the primers will amplify non-specific sequences, which may hinder specific gene detection. It is preferable to design primers that avoid various combinations.
[0137]
In each of these antisense primers and sense primers, a nucleotide sequence unrelated to the nucleotide sequences described in a) to e) above may be added as a linker to the 5 ′ end of the nucleotide sequence defined above. . However, it is preferable that the linker does not cause non-specific annealing with the nucleic acid in the reaction solution during the reaction so as not to hinder the specific gene detection.
[0138]
3) Cells or animals whose gene expression is to be detected
Next, the cultured cell used in the method of the present invention may be a mammalian cell that expresses the gene having the nucleotide sequence described in any one of the above a) to e). Preferably, a cultured cell derived from a mammalian liver (preferably, a primary cultured hepatocyte), for example, a cell into which a gene having the nucleotide sequence described in any one of the above a) to e) is introduced together with its promoter region. For example, artificially transformed cells (eg, CHO cells) can also be used. As the mammalian species, humans, mice, rats or hamsters are preferable, humans or mice are more preferable, and more preferably, the primary cultured liver of KK mouse (available from CLEA Japan), which is a hyperlipidemic mouse, is preferable. Cells include, but are not limited to. When it is judged that it is more preferable than using cultured cells, a test substance is administered to a mammal individual, and then any of the above a) to e) in the organ or tissue cell extracted from the animal individual A method for detecting the expression of a gene having the nucleotide sequence described in any one of the above may also be employed. At this time, the organ or tissue to be detected for gene expression may be any organ or tissue that expresses the gene having the nucleotide sequence described in any one of the above a) to e), but is preferably liver. Preferred mammalian species in this embodiment are preferably humans, mice, rats or hamsters, more preferably humans or mice. For example, as the mouse, the KK mouse is preferably used, but the present invention is not limited to this.
[0139]
The cultured cells used in the method of the present invention may be prepared under any conditions capable of expressing the gene having the nucleotide sequence according to any one of the above a) to e) (provided that no test substance is added). May be cultured. For example, when the culture conditions established for the cultured cells are known, and the cells express the gene having the nucleotide sequence according to any one of the above a) to e) under the conditions, Culture may be performed under conditions.
[0140]
4) Addition of test substance
During the culture of the cells, a test substance is added to the culture medium and cultured for a certain period. The test substance includes compounds, metabolites of microorganisms, extracts of plant and animal tissues, derivatives thereof, and mixtures thereof. In addition, a nucleic acid or a derivative thereof (including antisense nucleotide, ribozyme, RNAi, etc.) designed to suppress the expression of a gene having the nucleotide sequence according to any one of the above a) to e) is tested. It is also possible to use it as a substance. The dose and concentration of the test substance may be set as appropriate, or a plurality of doses may be set, for example, by preparing a dilution series. The period for culturing in the presence of the test substance may be appropriately set, but is preferably 30 minutes to 24 hours. When a test substance is administered to a mammal individual, administration forms such as oral administration, intravenous injection, intraperitoneal injection, transdermal administration, and subcutaneous injection are used depending on the properties of the test substance.
[0141]
5) Sample preparation
When extracting RNA from the cells cultured as described above, immediately after the culture is completed, the cells are directly dissolved in a solvent for RNA extraction (for example, one containing a component having a function of inactivating ribonuclease such as phenol). Is preferred. Alternatively, the cells may be collected carefully by a method such as carefully scraping with a scraper or gently separating from the culture substrate using a proteolytic enzyme such as trypsin so as not to destroy the cells. Move on to the extraction process.
[0142]
RNA extraction methods include guanidine thiocyanate / cesium chloride ultracentrifugation method, guanidine thiocyanate / hot phenol method, guanidine hydrochloride method, guanidine thiocyanate / phenol / chloroform method (Chomczynski, P. and Sacchi, N., ( 1987) {Anal.} Biochem., {162, 156-159), etc., but the guanidine acid thiocyanate / phenol / chloroform method is preferred.
[0143]
The method for further purifying mRNA from the obtained RNA is as described below. That is, since it is known that many mRNAs present in the cytoplasm of eukaryotic cells have a poly (A) sequence at the 3 'end, for example, an oligo (dT) probe biotinylated using this feature MRNA is adsorbed on the paramagnetic particles, and the paramagnetic particles on which streptavidin is immobilized capture the mRNA using the binding between biotin and streptavidin, and after the washing operation, the mRNA is eluted to purify the mRNA. Can be. Alternatively, a method in which mRNA is adsorbed on an oligo (dT) cellulose column and then eluted and purified may be employed. Further, mRNA can be further fractionated by sucrose density gradient centrifugation or the like. However, for the method of the present invention, these mRNA purification steps are not essential, as long as the expression of the gene having the nucleotide sequence described in any one of the above a) to e) can be detected. Alternatively, total RNA can be used in subsequent steps.
[0144]
6) Sample immobilization
In the case of performing detection by nucleic acid hybridization, in order to specifically detect a gene in the RNA sample obtained as described above, the RNA sample is subjected to a hybridization experiment membrane (hereinafter simply referred to as “membrane”) through agarose electrophoresis. (Northern blot method) or by directly infiltrating the sample into the membrane, so-called dot blot method or slot blot method, and immobilized on the membrane. Examples of the membrane include a nitrocellulose membrane (for example, High Bond-C Pure (manufactured by Amersham Pharmacia)), a positive charge nylon membrane (for example, High Bond-N + Amersham Pharmacia), or a hydrophilic nylon membrane (for example). For example, High Bond-N / NX (manufactured by Amersham Pharmacia) or the like is used.
[0145]
Examples of agarose electrophoresis methods for Northern blot include agarose formamide gel electrophoresis, a method in which a sample is treated with glyoxal and dimethyl sulfoxide, denatured, and then electrophoresed on an agarose gel prepared with a phosphate buffer, and agarose gel methylation. Mercury electrophoresis (Maniatis, T. et al., (1982) in, "Molecular Cloning, A Laboratory, Manual", Cold Spring Harbor, Laboratory, NY), and the like, but are not limited thereto.
[0146]
As a so-called blotting method for transferring RNA from the gel after the electrophoresis to the membrane, a capillary transfer method (Maniatis, T. et al. (1982) in "Molecular Cloning A Laboratories Manual" Cold Spring Harbor Laboratories, Laboratories, California). Method, electrophoresis (Maniatis, T. et al., (1989) in Molecular Cloning A Laboratory Manual, 2nd ed, Cold Spring Harbor Laboratory, NY). Equipment for dot blotting and slot blotting is also commercially available (for example, Biodot (manufactured by Bio-Rad)).
[0147]
After the blotting is completed, an operation of fixing the RNA transferred to the membrane to the membrane is performed (this operation differs depending on the material of the membrane, and depending on the product, the fixing operation may not be necessary).
[0148]
7) Probe labeling
In the detection by nucleic acid hybridization, a labeling method and a detection method of a probe for detecting a specific mRNA in the RNA sample immobilized as described above are described below:
i) Radioisotope labeling
A nick translation method (for example, using a nick translation kit (manufactured by Amersham Pharmacia) or the like) or a random prime method (for example, a multiprime DNA labeling system (eg, a multi-prime DNA labeling system) Amersham Pharmacia Co., Ltd.) and an end labeling method (eg, using a megalabel (Takara Shuzo Co., Ltd.), 3'-end labeling kit (Amersham Pharmacia Co., Ltd.), etc.) to prepare a labeled DNA probe. Alternatively, a labeled RNA probe is prepared by an in vitro transcription method using an SP6 promoter or T7 promoter in a vector into which a template DNA has been subcloned. Detection of these probes can be performed by autoradiography using an X-ray film or an imaging plate. In the case of an X-ray film, densitometry (for example, GS-700 imaging densitometer (manufactured by Bio-Rad)) Is used, and in the case of an imaging plate, quantification using a BAS2000II (manufactured by Fuji Film) system is also possible.
[0149]
ii) Enzyme label
A DNA or RNA fragment is directly labeled with an enzyme. Enzymes used for labeling include, for example, alkaline phosphatase (AlkPhos \ Direct \ system \ for \ chemiluminescence (manufactured by Amersham-Pharmacia)), horseradish peroxidase (Horseradish \ Peroxydase, ECL and A.D.D.C. System (Amersham Pharmacia) or the like). The probe is detected by immersing the membrane in an enzyme reaction buffer containing a substrate capable of detecting the catalytic reaction of the labeled enzyme, for example, a chromogenic substance or a substrate that emits light by the catalytic reaction. . When a chromogenic substrate is used, it can be visually detected.When a luminescent substrate is used, autoradiography using an X-ray film or an imaging plate or a photograph using an instant camera as in the case of radioisotope labeling It can be detected by photographing. Furthermore, when a luminescent substrate is used, quantification using densitometry or a BAS2000II system is also possible.
[0150]
iii) Labeling with other molecules
Fluorescein labeling: The DNA fragment is labeled by a nick translation method, a random prime method, or a 3 'end labeling method (such as an ECL 3'-oligolabeling system commercially available from Amersham Pharmacia). Alternatively, the RNA is labeled by in vitro transcription with the SP6, T7 promoter;
Biotin labeling: Label the 5 'end of the DNA (using an oligonucleotide biotin labeling kit commercially available from Amersham Pharmacia) or label the DNA fragment with the nick translation method or random prime method.
[0151]
Digoxigenin-modified dUTP labeling: DNA fragments are labeled by a nick translation method, a random prime method or the like.
[0152]
In each case, detection of these labeled molecules includes an operation of binding a molecule specifically binding to the labeled molecule to a probe by labeling the molecule with a radioisotope or an enzyme. Specific binding molecules include, for example, an anti-fluorescein antibody or an anti-digoxigenin antibody in the case of fluorescein or digoxigenin, and avidin or streptavidin in the case of biotin. After these are bound to the probe, detection can be carried out with the labeled radioisotope or enzyme according to the same method as described in i) or ii) above.
[0153]
8) Hybridization
Hybridization can be performed by a method well known in the art to which the present invention belongs. The relationship between the composition of the hybridization solution or washing solution and the hybridization temperature or washing temperature in the present invention can be according to, for example, the description in the literature (Bio-Experiment Illustrated 4, p148, published by Shujunsha). Is as follows:
Hybridization solution: {ExpressHyb} Hybridization \ Solution (Clontech);
Final concentration of probe (for radiolabeled probe): 1-2 × 10⁶cpm / ml (preferably 2 × 10⁶cpm / ml);
Hybridization temperature and time: @ 68 ° C, 1 to 24 hours.
[0154]
Cleaning conditions for the membrane:
i) in 0.1 to 5 × SSC (most preferably 2 × SSC), 0.05 to 0.1% in SDS (most preferably 0.05%) sodium dodecyl sulfate (hereinafter “SDS”) at room temperature to Shaking at 42 ° C. (most preferably room temperature) for 20 to 60 minutes (most preferably 20 minutes) is performed 2 to 6 times (most preferably 3 times) with the exchange of the washing solution;
ii) After the above-mentioned i), the operation of shaking in 0.1 × SSC, 0.1% SDS at 50 to 65 ° C. for 20 to 60 minutes is performed 2 to 6 times by exchanging the washing solution. Alternatively, the operation of shaking in 0.2 to 0.5 × SSC, 0.1% SDS at 62 to 65 ° C. for 20 to 60 minutes is performed 2 to 6 times by changing the washing solution. Most preferably, the operation of shaking in 0.1 × SSC, 0.1% ０．１SDS at 50 ° C. for 20 minutes is performed three times by changing the washing solution.
[0155]
After the washing is completed, detection and quantification according to the probe labeling method are performed as described in 7) above. Genes known to have stable expression levels per cell (eg, 23 kDa highly basic protein, α-tubulin, glyceraldehyde-3-dehydrogenase, hypoxanthine guanine phosphoribosyltransferase, Probes for detecting gene expression of phospholipase A2, ribosomal protein S9, ubiquitin, and the like are commercially available)), and the expression level of each sample is corrected for variations due to differences in RNA levels between the samples. The relative value of the expression level of the gene to be detected, based on the expression level of the stable expression gene, was compared between the cell group to which the test substance was administered and the cell group to which the test substance was not administered. By doing so, a more precise evaluation can be performed.
[0156]
As a result, the test substance that decreases the expression level of the gene having the nucleotide sequence according to any one of the above a) to e) is one or two selected from hyperlipidemia, arteriosclerosis, and hyperglycemia. It can be a therapeutic or prophylactic agent for one or more diseases.
[0157]
9) RT-PCR reaction
In the embodiment in which the nucleic acid is detected by RT-PCR, the conditions of each reaction are as described below. The sample for detection by RT-PCR is usually poly (A)⁺It need not be purified to RNA.
[0158]
i) Reverse transcriptase reaction
Example of reaction solution composition (total volume 20 μl):
Total RNA as appropriate;
Magnesium chloride @ 2.5-5 mM (preferably 5 mM);
1 × RNA PCR buffer (10 mM Tris-HCl (pH 8.3 to 9.0 (preferably 8.3) at 25 ° C., 50 mM potassium chloride));
dNTPs @ 0.5-1 mM (preferably 1 mM);
Antisense primer {1 μM} (2.5 μM of a commercially available random primer or oligo (dT) primer (12-20 nucleotides) can be added as a substitute for the antisense primer);
Reverse transcriptase 0.25 to 1 unit / μl (preferably 0.25 unit / μl);
Adjust to 20 μl with sterile water.
[0159]
Reaction temperature conditions:
After incubating at 30 ° C for 10 minutes (only when using a random primer), incubating at 42 to 60 ° C (preferably 42 ° C) for 15 to 30 minutes (preferably 30 minutes), and further heating at 99 ° C for 5 minutes, the enzyme And then cooled at 4-5 ° C (preferably 5 ° C) for 5 minutes.
[0160]
ii) PCR
Example of reaction solution composition:
Magnesium chloride 2 to 2.5 mM (preferably 2.5 mM);
1 × PCR buffer (10 mM Tris-HCl (pH 8.3 to 9.0 at 25 ° C. (preferably 8.3)), 50 mM potassium chloride;
dNTPs 0.2 to 0.25 mM (preferably 0.25 mM);
Antisense and sense primers 0.2-0.5 μM (preferably 0.2 μM);
Taq polymerase @ 1 to 2.5 units (preferably 2.5 units);
The total volume is adjusted to 80 μl by adding sterile water, and the total volume is added to the total volume of the reaction solution after the completion of the reverse transcription reaction before PCR is started.
[0161]
Reaction temperature conditions: First, heating at 94 ° C. for 2 minutes, and then at 90 to 95 ° C. (preferably 94 ° C.) for 30 seconds, 40 to 60 ° C. (preferably, the dissociation temperature (Tm) calculated from the characteristics of the primer) A temperature cycle of 70 to 75 ° C. (preferably 72 ° C.) for 1.5 seconds at 28 to 50 cycles (preferably 28 cycles) at a temperature lower than 20 ° C. for 30 seconds, and then cooling to 4 ° C. I do.
[0162]
After completion of the PCR, the reaction solution is subjected to electrophoresis to detect whether or not a band of a desired size has been amplified. In order to perform quantitative detection, PCR is carried out under the same conditions using a serially diluted cDNA clone as a standard template DNA, and the number of temperature cycles at which quantitative detection is possible is determined. A part of the reaction solution is sampled and subjected to electrophoresis. Also, for example, by using radiolabeled dCTP during the PCR reaction, quantification can be performed using the amount of radioactivity incorporated in the band as an index.
[0163]
The detection results are compared between a sample derived from cells cultured in the presence of the test substance and a sample derived from cells cultured in the absence of the test substance, and the nucleotide according to any one of the above a) to e) A test substance having a reduced expression level of a gene having a sequence can be a therapeutic or preventive agent for one or more diseases selected from hyperlipidemia, arteriosclerosis, and hyperglycemia.
[0164]
10) Other methods
As another method for measuring the expression level of the gene having the nucleotide sequence described in any one of the above a) to e), the following methods can be exemplified.
[0165]
i) Ribonuclease protection assay (RNase protection assay): a labeled probe in an RNA sample having only the mRNA having the nucleotide sequence according to any one of the above a) to e) (where t in the sequence is replaced with u) After hybridizing to form a double-stranded polynucleotide and then incubating the sample with ribonuclease, the probe-hybridized mRNA is not digested by ribonuclease due to its double-stranded form. Since the other RNAs are digested, only the double-stranded polynucleotide remains (if the probe is shorter than the mRNA to be detected, the double-stranded polynucleotide corresponding to the length of the probe remains). By quantifying the double-stranded polynucleotide, the expression level of the target gene is measured. Specifically, for example, the method described below is used.
[0166]
To ensure that the extra labeled probe that does not form a double strand is separated from the double-stranded polynucleotide to facilitate quantification, the extra labeled probe is preferably digested with ribonuclease. The labeling probe may be DNA or RNA as long as it is capable of digesting the main-strand DNA. The method for preparing the labeled probe is as described in the above 1) and 7), but the length of the probe used in this method is preferably about 50 to 500 nucleotides.
[0167]
The RNA probe is prepared, for example, according to the following method. First, the template DNA is inserted into a plasmid vector (for example, pGEM-T (promega)) having a bacteriophage promoter (T7, SP6, T3 promoter, etc.). Next, this recombinant plasmid vector is digested with a restriction enzyme so that it is cleaved only one point immediately downstream of the inserted fragment. Using the obtained linear DNA as a template, an in vitro transcription reaction is carried out in the presence of radiolabeled ribonucleotides. In this reaction, an enzyme such as T7, SP6, or T3 polymerase is used in accordance with the promoter in the vector. The above operation can be performed using, for example, Riboprobe System-T7, -SP6 or -T3 (all manufactured by Promega).
[0168]
The steps up to preparing the RNA sample are as described in the above 3) to 5). 10 to 20 μg of the prepared total RNA sample and 5 × 10⁵A ribonuclease protection assay is performed using an excess of labeled probe equivalent to cpm. This operation can be performed using a commercially available kit (HybSpeed @ RPA @ Kit, manufactured by Ambion). The obtained sample after ribonuclease digestion is electrophoresed on a 4 to 12% polyacrylamide gel containing 8M urea, the gel is dried, and autoradiography is performed with an X-ray film. By the above operation, the band of the double-stranded polynucleotide which escaped the ribonuclease digestion can be detected, and the quantification can be carried out according to the method described in 7) i) above). Furthermore, as in the case of Northern blot analysis, if the expression level of the β-actin gene is simultaneously measured for the purpose of correcting variations caused by a difference in RNA amount between each sample, more precise evaluation will be performed. be able to.
[0169]
In this way, the detection results are compared between a sample derived from cells cultured in the presence of the test substance and a sample derived from cells cultured in the absence of the test substance, and any one of the above a) to e) is detected. The test substance in which the expression level of the gene having the nucleotide sequence described in (1) is reduced can be a therapeutic or preventive agent for one or more diseases selected from hyperlipidemia, arteriosclerosis and hyperglycemia.
[0170]
ii) Run-on assay (Run-on assay, Greenberg, M. E. and Ziff, E. B. (1984) Nature 311, 433-438, and Groudine, M. et al. (1981) Mol. CellBol. This method is a method for measuring the transcriptional activity of a target gene by isolating the nucleus from a cell, and is not a method for detecting mRNA in a cell as described above. In the present invention, it is included in the “method for detecting the expression level of a gene”. When a transcription reaction is performed in a test tube using the isolated cell nucleus, only the reaction in which transcription is already started before the nucleus is isolated and the one where the mRNA chain is being generated elongates proceeds. I do. At the time of isolating the nucleus by adding a radiolabeled ribonucleotide during this reaction to label the elongating mRNA and detecting the mRNA contained therein that hybridizes to the unlabeled probe The transcription activity of the target gene can be measured. In order to use the data of the time when the influence of the test substance appears most remarkably, the time from the addition of the test substance to the cultured cells to the isolation of the nucleus is, for example, 30 minutes after addition, 1 hour after, 2 hours , 4 hours, 8 hours, and 24 hours later, nuclei can be isolated from the cells and assayed, respectively. The specific operation method is the same as that described in the above-mentioned reference except that a probe that is not labeled is prepared according to the description in the above 1). In this way, the detection results are compared between a sample derived from cells cultured in the presence of the test substance and a sample derived from cells cultured in the absence of the test substance, and any one of the above a) to e) is detected. A test substance having a reduced transcriptional activity of a gene having the nucleotide sequence described in (1) can be a therapeutic or preventive agent for one or more diseases selected from hyperlipidemia, arteriosclerosis, and hyperglycemia.
[0171]
iii) DNA chip analysis, DNA microarray analysis
[1] Preparation of sample for probe acquisition
First, mRNA of a sample derived from cells cultured in the presence of the test substance and mRNA of a sample derived from cells cultured in the absence of the test substance are extracted and purified. The steps up to preparing the RNA sample are as described in the above 3) to 5).
[0172]
[2] Labeling of probe
As a starting material for preparing a probe for DNA chip analysis or DNA microarray analysis, unpurified total RNA can be used, but poly (A) purified by the method described in the above 5) can be used.⁺More preferably, it is RNA.
[0173]
In performing detection by nucleic acid hybridization, a labeling method and a detection method of a probe will be described below.
[0174]
Probe for analysis using Affymetrix DNA chip: に 従 い A biotin-labeled cRNA probe is used according to the protocol attached to the Affymetrix DNA chip.
[0175]
Probe for analysis using DNA microarray: (1) Poly (A) by reverse transcriptase reaction⁺When preparing cDNA from RNA, the cDNA is fluorescently labeled by adding d-UTP or the like labeled with a fluorescent dye (for example, Cy3 or Cy5). At this time, if the sample derived from the cells cultured in the presence of the test substance and the sample derived from the cells cultured in the absence of the test substance are labeled with different dyes, both are mixed and used at the time of subsequent hybridization. be able to.プ ロ ー ブ Probe for analysis using a membrane filter: ポ リ Poly (A) by reverse transcriptase reaction⁺When producing cDNA from RNA, radioisotopes (eg,³²P,³³The probe is labeled by adding d-CTP or the like labeled with P).
[0176]
[3] Immobilized sample
Examples of the immobilized sample to be hybridized with the labeled probe obtained in the above step [2] include the following.
[0177]
A gene chip (manufactured by Affymetrix) on which an antisense oligonucleotide synthesized based on an EST (expressed sequence tag) sequence or an mRNA sequence on a database is immobilized (Lipshutz, R. J. et al. (1999) Nature) genet. 21, suppliment, 20-24):
Most preferably, the EST sequence or mRNA sequence is derived from an animal of the same species as the cell used for preparing the probe, but is not limited thereto. However, a gene having the nucleotide sequence according to any one of the above a) to e), a gene published as a nucleotide sequence encoding “angiopoietin-related protein 3” on the Genbank database, or any one of them A sequence on which a sequence containing two partial sequences is immobilized is used.
[0178]
CDNA or RT-PCR product prepared from mRNA obtained from an organ tissue (preferably liver) of an animal of the same species or a closely related animal as the cell used for preparing the probe, or a cell isolated or established from the organ tissue DNA microarray or membrane filter on which is immobilized:
These cDNA or RT-PCR products are cloned by performing a reverse transcriptase reaction or PCR with primers prepared based on sequence information such as an EST database of animals used as mRNA materials. . As a material for preparing a sample, a gene having the nucleotide sequence according to any one of the above a) to e), a gene disclosed as a nucleotide sequence encoding “angiopoietin-related protein 3” on the Genbank database Is used (preferably from the liver). As the cDNA or the RT-PCR product, mRNA having different expression levels is determined in advance by a subtraction method (Diatchenko, L. et al. (1996) Proc. Natl. Acad. Sci. USA USA 93, 6025-6030). ), Differential display method (Kato, K. (1995) Nucleic Acids Res. 23, 3685-3690) and the like. As the DNA microarray or the filter, a commercially available product containing a gene to be detected, that is, a gene published as a nucleotide sequence encoding “angiopoietin-related protein 3” on the Genbank database may be used. Can be used to prepare a DNA microarray or a filter on which a gene having the nucleotide sequence according to any one of the above a) to e) is immobilized (for example, GMS417 Alayer manufactured by Takara Shuzo Co., Ltd.). ).
[0179]
The probe prepared in the above [2] is hybridized to this solid-phased sample separately or mixed under the same conditions simultaneously (Brown, {P.O.} et. Al. (1999) @ Nature @ gene. 21, @suppliment, 33-37).
[0180]
[4] Analysis
In the case of analysis using a DNA chip manufactured by Affymetrix: Hybridization and analysis are performed according to the protocol attached to the DNA chip manufactured by Affymetrix.
[0181]
In the case of analysis using a DNA microarray: For example, when a commercial DNA microarray manufactured by Takara Shuzo Co., Ltd. is used, hybridization and washing are performed according to the company's protocol, and a fluorescent signal detector (eg, GMS418 Array Scanner (Takara Shuzo Co., Ltd.) Analysis) after detecting the fluorescent signal.
[0182]
In the case of analysis using a filter: Hybridization can be performed by a method well known in the art to which the present invention belongs. For example, after performing hybridization and washing, analysis is performed using an analyzer (for example, Atlas Image (manufactured by Clontech)).
[0183]
In any of the above cases, a probe derived from a cell-derived sample cultured in the presence of the test substance and a probe derived from a cell-derived sample cultured in the absence of the test substance are respectively hybridized to the solid-phased sample of the same lot. . At this time, the hybridization conditions other than the probe used are the same. As described in the above [2], when each probe is labeled with a different fluorescent dye, a mixture of both probes can be hybridized to one immobilized sample (Brown, P. O. et al). (1999) Nature genet. 21, suppliment, 33-37).
[0184]
As a result of the analysis, a probe derived from a cell-derived sample cultured in the presence of the test substance and a probe derived from a cell-derived sample cultured in the absence of the test substance, the target gene of the solid-phased sample, The amount of the probe hybridized to the gene having the nucleotide sequence described in any one of a) to e) or the gene having the nucleotide sequence encoding “angiopoietin-related protein 3” is measured. As a result, the amount of the probe hybridized to the target gene was compared, and the probe derived from the cell-derived sample cultured in the presence of the test substance was better than the probe derived from the cell-derived sample cultured in the absence of the test substance. If the amount is small, the test substance is a substance that suppresses the expression of the gene having the nucleotide sequence according to any one of the above a) to e), and is selected from hyperlipidemia, arteriosclerosis, and hyperglycemia. It can be an agent for treating or preventing one or more diseases.
[0185]
iv) Other
In addition to the above, a sample derived from cells cultured in the presence of a test substance has the nucleotide sequence according to any one of the above a) to e) as compared to a sample derived from cells cultured in the absence of the test substance The following methods can be mentioned as a method for detecting the identification of a test substance that reduces the expression level of a gene.
[0186]
That is, in a single reaction, a technique known as “Taqman” combining PCR with hybridization probing (hereinafter “probing”) (Holland, P. M. et al. (1991) Proc. Natl. Acad. Sci. USA 88, 7276-7280) or a method in which PCR is combined with probing in a single reaction (Higuchi et al. Biotechnology, 10, 413-417 (1992)). In the latter method, ethidium bromide, a nucleic acid detection reagent that emits fluorescence when excited by ultraviolet light, is added to a PCR reaction solution. Since the fluorescence of ethidium bromide increases in the presence of double-stranded DNA, the increase in fluorescence detected upon irradiation with excitation light may correlate with the accumulation of double-stranded PCR product.
[0187]
Furthermore, as another method combining PCR amplification and probing, the method described in EP-A-0601889 can be used. Furthermore, it is also possible to quantify the amount of mRNA using a light cycler system (manufactured by Roche Diagnostics Inc., see Japanese Patent Publication No. 2000-312600).
[0188]
(B) Detection of polypeptide
Next, as another embodiment of the method of the present invention, there is a method for detecting a polypeptide encoded by a gene to be detected in the above-described embodiment for detecting gene expression. In this embodiment, after the polypeptide in the sample is immobilized on the bottom surface in the well of a 96-well plate, a membrane, or the like, detection is performed using an antibody that specifically recognizes the target polypeptide. Among them, the method using a 96-well plate is generally called an enzyme-linked immunosorbent assay (ELISA) or a radioisotope immunoassay (RIA). On the other hand, as a method for immobilizing a sample on a membrane, a method in which a polypeptide is transferred to a membrane via polyacrylamide electrophoresis of a sample (Western blotting), or a method in which a sample or a dilution thereof is directly impregnated into a membrane, is called a dot. Blot method and slot blot method are mentioned.
[0189]
1) Sample preparation
The conditions regarding the type of cultured cells used in the embodiment for detecting the polypeptide as described above are the same as those in the embodiment for detecting the gene expression described above. Alternatively, a method in which a test substance is administered to a mammal individual, and then serum or the like collected from the animal individual is used as a sample may be employed. Preferred mammalian species in this case are humans, mice, rats or hamsters, more preferably humans or mice. For example, as a mouse, a KK mouse which is a hyperlipidemic mouse is preferably used, but the present invention is not limited thereto. The culture conditions of the cultured cells and the method of administering the test substance are the same as those in the embodiment in which gene expression is detected. The test substance to be tested for its activity as a therapeutic or prophylactic agent for hyperlipidemia includes compounds, metabolites of microorganisms, extracts of plant and animal tissues, derivatives thereof, and mixtures thereof. Can be.
[0190]
As a material for preparing a sample for the present embodiment, a culture supernatant or a cytoplasmic fraction of a cell culture cultured in the presence or absence of a test substance can be used, and the culture supernatant is preferable. . The culture supernatant is collected after the completion of the culture, and if necessary, subjected to a filtration sterilization treatment to be subjected to a process for preparing a sample for ELISA / RIA or a sample for Western blot.
[0191]
As a sample for ELISA / RIA, for example, the collected culture supernatant is used as it is, or a sample appropriately diluted with a buffer is used.
[0192]
The preparation method of the sample for Western blot (for electrophoresis) is as follows. First, for example, the culture supernatant is treated with trichloroacetic acid to precipitate the protein, and the precipitate is obtained by centrifugation. The precipitate is washed with ice-cooled acetone, air-dried, and then dissolved in a sample buffer (eg, Bio-Rad) containing 2-mercatorethanol for SDS-polyacrylamide electrophoresis.
[0193]
In the case of dot / slot blot, for example, the collected culture supernatant itself or one appropriately diluted with a buffer is directly adsorbed to the membrane by using a blotting device or the like.
[0194]
2) Sample immobilization
In order to specifically detect the polypeptide in the sample obtained as described above, the sample is immobilized. Examples of the membrane used for the Western blotting, dot blotting or slot blotting include nitrocellulose membrane (for example, manufactured by Bio-Rad), nylon membrane (for example, Hybond-ECL (Amersham-Pharmacia)), cotton Examples thereof include a membrane (for example, a blot absorbent filter (manufactured by Bio-Rad) and the like) and a polyvinylidene difluoride (PVDF) membrane (for example, manufactured by Bio-Rad) and the like.
[0195]
As a so-called blotting method for transferring the polypeptide from the gel after electrophoresis to the membrane, a wet blotting method (CURRENT PROTOCOLS IN IMMUNOLOGY volume 2 ed by J. E. Coligan, A.M. M. Shevach, W. Strober), semi-dry blotting method (see CURRENT PROTOCOLS IN IMMUNOLOGY volume2) and the like. Equipment for dot blotting and slot blotting is also commercially available (for example, Biodot (Biorad)).
[0196]
On the other hand, in order to perform detection / quantification by the ELISA method / RIA method, a sample or a diluted solution thereof (for example, 0.05% Add phosphate buffered saline containing sodium chloride (diluted with PBS) and let stand at 4 ° C. to room temperature overnight or at 37 ° C. for 1 to 3 hours. Is immobilized on a solid phase.
[0197]
3) Antibody
The antibody used in this embodiment is a polypeptide produced by expressing a gene containing the nucleotide sequence described in a) to e) of the above (A) in animal cells, preferably a polypeptide of SEQ ID NO: 2 in the sequence listing. A polypeptide consisting of the amino acid sequence represented by amino acid Nos. 17-455 (more preferably 19-455) or a portion thereof, or a polypeptide consisting of the amino acid sequence represented by amino acid Nos. 17-460 of SEQ ID NO: 4 or one thereof The part is specifically recognized. Suitable examples of such an antibody include, for example, a polypeptide having the amino acid sequence of amino acid Nos. 17-455 of SEQ ID NO: 2 (more preferably, 19-455) and amino acid No. 17 of SEQ ID NO: 4 -460, which binds to any of the polypeptides consisting of the amino acid sequence but does not bind to any other mouse or human protein.
[0198]
Antibodies for this embodiment can be selected from protein proteins that serve as antigens, or amino acid sequences thereof, using conventional methods (for example, Shinsei Kagaku Kenkyusho 1, Protein 1, p. 389-397, 1992). Can be obtained by immunizing an animal with the above polypeptide and collecting and purifying antibodies produced in the living body. In addition, according to known methods (eg, Kohler and Milstein, Nature 256, 495-497, 1975, Kennet, Red, Monoclonal Antibody p. 365-367, 1980, Prenum Press, Invention of NY, in accordance with the present invention). A hybridoma can be established by fusing an antibody-producing cell producing an antibody against the protein with a myeloma cell to obtain a monoclonal antibody.
[0199]
In general, the production of monoclonal antibodies involves the following working steps. Preparation of antibody-producing cells by (a) purifying a biopolymer used as an antigen, (b) immunizing a mouse by injection of the antigen, and collecting and assaying blood to determine the timing of splenectomy. (C) preparation of myeloma cells (myeloma), (d) removal of spleen, and in the case of spleen cells and myeloma, (e) selection of hybridoma group producing the desired antibody, (f) single cell clone (G) cultivation of hybridomas to produce monoclonal antibodies in large quantities, or breeding of mice transplanted with hybridomas, (h) bioactivity of monoclonal antibodies thus produced , Or through a working process such as a test of the characteristics as a labeling reagent.
[0200]
The monoclonal antibody of the present invention can also be produced according to the above steps, but is not limited thereto.For example, antibody-producing cells other than splenocytes, myeloma, antibody-producing cells of other mammals, and myeloma can be used. Needless to say.
[0201]
(A) Purification of antigen
The antigen may be any polypeptide as long as it is the polypeptide according to claims 10 to 12, and is a fusion protein obtained by introducing the DNA of the present invention into Escherichia coli, expressing and purifying the same, and COS in the DNA of the present invention. Proteins purified from serum-free culture supernatant obtained by transfecting -1 cells or CHO cells are effective as antigens.
[0202]
(B) Preparation of antibody-producing cells
A protein purified from a serum-free culture supernatant obtained by transfecting the DNA of the present invention into CHO cells is mixed with an adjuvant such as Freund's complete or incomplete adjuvant or alum, and used as an immunogen. Immunize experimental animals. As an experimental animal, it is desirable to use a BALB / c mouse, since all myelomas derived from a frequently used mouse originate from a BALB / c mouse, and its properties have been studied relatively in detail. If both antibody-producing cells and myeloma are derived from BALB / c mice, the resulting hybridomas can grow in the intraperitoneal cavity of BALB / c mice, so that there is an advantage that monoclonal antibodies can be obtained from ascites without complicated procedures. . However, as described above, the present invention is not limited to this.
[0203]
The method of administering the immunogen upon immunization may be any of subcutaneous injection, intraperitoneal injection, intravenous injection, intradermal injection, and intramuscular injection, but subcutaneous injection or intraperitoneal injection is preferred.
[0204]
Immunization may be repeated once or at appropriate intervals, preferably multiple times at one to five week intervals. The antibody titer against the antigen in the serum of the immunized animal is measured, and if the animal with a sufficiently high antibody titer is used as a source of the antibody-producing cells, the effect of the subsequent operation can be improved. For the fusion, it is preferable to use animal-derived antibody-producing cells 3 to 5 days after the final immunization.
[0205]
Examples of the method for measuring the antibody titer used here include various known techniques such as radioisotope immunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), fluorescent antibody method, and passive hemagglutination method. However, the RIA method and the ELISA method are preferable from the viewpoints of sensitivity, speed, accuracy, and possibility of automation.
[0206]
The measurement of the antibody titer according to the present invention can be performed, for example, by the following procedure according to the ELISA method. That is, the antigen is adsorbed on the solid phase, and the surface of the solid phase on which the antigen is not adsorbed is covered with a protein irrelevant to the antigen, for example, bovine serum albumin (BSA). A sample (for example, mouse serum) is brought into contact with the antibody, the antibody in the sample is allowed to bind to the above antigen, and an antibody against an enzyme-labeled mouse antibody is added as a second antibody, and the antibody is bound to the mouse antibody. In addition, the antibody titer can be calculated by measuring the color development based on the decomposition of the substrate.
[0207]
(C) 工程 Preparation process of myeloma cells
As the bone marrow cells, cell lines generally obtained from mice, for example, an 8-azaguanine-resistant mouse (derived from BALB / c) myeloma cell line P3-x63 @ Ag8-U1 (P3-U1) (Current @ Topics @ in @ Microbiology) and {Immology, 81, 1-7 (1978)), P3-NSI / 1-Ag4.1 (NS-1) (European {J. Immunology, 6, 511-519 (1976)}), SP2 / O-Ag14 (SP) -2) (Nature, 276, 269-270 (1978)), P3-x63-Ag8.653 (653) (J. Immunology, 123, 1548-1550 (1979)), P3-x63-Ag8 (x63) ( Nature, 256, 495-497 1975)) or the like is preferably used.
[0208]
These cell lines can be cultured in a suitable medium, for example, 8-azaguanine medium (glutamine (1.5 mM), 2-mercaptoethanol (5 × 10 5 in RPMI-1640 medium).^-5M), gentamicin (10 μg / ml)}, and a medium supplemented with 8-azaguanine in a medium supplemented with fetal calf serum (FCS 10%), Iscover's Modified Dulbecco's Medium (IMDM), or Dulbec´s Modified. The cells are subcultured in Eagle Medium (DMEM). Three to four days before cell fusion, TIL medium (TIL Media I (500 ml), 5 ml PENICILLIN-STREPTOMYCIN SOLUTION (Sigma), and bovine fetal serum ( Subcultured in a medium supplemented with FCS (10%) and 2 × 10⁷Ensure the number of cells above.
[0209]
(D) Cell fusion
Antibody-producing cells are plasma cells and their precursor lymphocytes, which may be obtained from any site in an individual, generally from spleen, lymph nodes, peripheral blood, or any suitable combination thereof. However, spleen cells are most commonly used.
[0210]
After the final immunization, a site where antibody-producing cells are present, for example, a spleen is removed from a mouse having a predetermined antibody titer, and spleen cells as antibody-producing cells are prepared. The most commonly used means for fusing the splenocytes with the myeloma cells obtained in step (c) at present is a method using polyethylene glycol, which has relatively low cytotoxicity and easy fusion operation. . This method includes the following steps.
[0211]
The spleen cells and myeloma cells are thoroughly washed with a medium or a phosphate buffer (PBS), mixed so that the ratio of spleen cells to myeloma cells is about 5 to 10: 1, and centrifuged. The supernatant is discarded, the precipitated cell group is loosened, and a mixed solution of polyethylene glycol (PEG, molecular weight: 1,000 to 4,000) and a medium is added with stirring, followed by centrifugation several minutes later. The supernatant is discarded again, and the precipitated cells are suspended in a TIL medium and dispensed into wells on a culture plate. When cell growth was confirmed in each well, HAT medium (hypoxanthine 10 was added to TIL medium).^-6To 10^-3M, aminopterin 10^-8To 10^-7M, thymidine 10^-6To 10^-4M-containing medium).
[0212]
(E) Selection of hybridoma group
The culture plate is cultured at 35 to 40 ° C. in a CO 2 incubator. Thereafter, a HAT medium equivalent to half of the medium is added every 1 to 3 days, and the cells are cultured in a CO2 incubator at 35 to 40 ° C for 10 to 14 days.
[0213]
The myeloma cells are 8-azaguanine-resistant strains, and the myeloma cells and hybridomas between myeloma cells cannot survive in HAT medium. However, since antibody-producing cells or hybridomas between antibody-producing cells and myeloma cells can survive, and hybridomas between antibody-producing cells have a long life, hybridomas can be selected by culturing in HAT medium. It becomes.
[0214]
The holes in which the hybridomas growing in colonies are observed are converted to HT medium (medium obtained by removing aminopterin from HAT medium). Thereafter, a part of the culture supernatant is taken, and the antibody titer is measured by, for example, ELISA.
The 8-azaguanine-resistant cell line was used as described above, but other cells can be used according to the selection of the hybridoma, and in that case, the composition of the medium to be used naturally changes.
[0215]
(F) Cloning
The hybridoma which has been found to produce a specific antibody by the measurement of the antibody titer is transferred to another plate and cloned. Examples of the cloning method include a method in which one hybridoma is diluted and contained in one well by limiting dilution, a method in which a colony is collected in a soft agar medium, and a method in which one cell is taken out by a micromanipulator. The method includes a "sorter clone" in which one cell is separated by a cell sorter, and the limiting dilution method is simple and often used.
[0216]
Cloning is repeated 2 to 4 times, for example, by the limiting dilution method with respect to the wells in which the antibody titer is recognized, and those having a stable antibody titer are selected as hybridoma strains producing the monoclonal antibody of the present invention.
[0219]
(G) Preparation of monoclonal antibody by hybridoma culture
After cloning, the hybridoma is cultured by replacing the HT medium with a normal medium. Large-scale culture is performed by rotary culture using a large culture bottle or spinner culture. The monoclonal antibody of the present invention can be obtained by collecting and purifying the IgG fraction by performing gel filtration or the like on the supernatant in this large-scale culture. In addition, it can be propagated in the abdominal cavity of mice of the same strain (for example, BALB / c described above) or Nu / Nu mice.
[0218]
As a simple method, a monoclonal antibody purification kit (for example, MAbTrap @ GII manufactured by Pharmacia) can also be used.
[0219]
(H) Identification of monoclonal antibody
The isotype and subclass of the monoclonal antibody thus obtained are determined as follows. As an identification method, there is an Ouchterlony method, an ELISA method, or an RIA method. The Otterlony method is simple but requires concentration when the concentration of monoclonal antibody is low. If the ELISA method or the RIA method is used, it is possible to react the culture supernatant with the antigen-adsorbed solid phase as it is and to use antibodies corresponding to various IgG subclasses as secondary antibodies. Further, a kit for isotyping (eg, a mouse monoclonal antibody isotyping kit manufactured by Amersham) can be used as a simpler method.
[0220]
Furthermore, protein quantification can be calculated by the Foreign Lowry method and the absorbance at 280 nm (1.4 (OD280) = immunoglobulin 1 mg / ml).
[0221]
The hybridoma clone that stably produces the monoclonal antibody of the present invention at a high concentration is named 45B1, and is deposited with the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary on March 13, 2002, under the deposit number FERM. Deposited as BP-7963.
[0222]
The monoclonal antibody thus obtained has high specificity for the protein of the present invention and can be obtained uniformly by culturing the hybridoma, and thus can be used for isolation and purification of the protein of the present invention. For example, the protein of the present invention can be isolated and purified with the monoclonal antibody by immunoprecipitation using an antigen-antibody reaction. That is, the monoclonal antibody is added to a solution containing the protein of the present invention, stored at room temperature, mixed with Protein G Sepharose (Pharmacia), and further stored at room temperature. The precipitate is obtained by centrifugation, washed several times with PBS containing 0.1% Tween 20, etc., and centrifuged again. The precipitate obtained is a complex of the protein, the monoclonal antibody, and protein G Sepharose. . The precipitate is suspended in an appropriate buffer, and then heat-treated to dissociate the complex into each component, followed by centrifugation to collect the supernatant. From the supernatant, the protein can be easily isolated and purified by molecular sieve chromatography (gel filtration) or the like.
[0223]
In addition, the monoclonal antibody can be used as a ligand in an affinity chromatography method for isolating and purifying the protein. Affinity chromatography is an extremely effective method for isolating and purifying only substances that specifically bind to a carrier from a mixture, and greatly reduces the required purification process compared to gel filtration of the mixture. be able to. The preparation of the immobilized monoclonal antibody useful in the affinity chromatography can be performed according to the immobilization method of various enzymes, for example, a method using a CNBr-activated carrier can be generally used, and such a carrier can be used. Are various materials generally used for chromatography, for example, cellulose, agarose, cross-linked dextran, polyacrylamide, porous glass, or those obtained by introducing a spacer into these carriers, particularly Sepharose 4B (Pharmacia), Affigel 10 (manufactured by Bio-Rad) is a preferred example. In order to actually purify the protein using the immobilized monoclonal antibody, the protein is packed in a column and then passed through a solution containing the protein. By this operation, the protein is adsorbed on the column. Elution with a solvent such as glycine-hydrochloride buffer (pH 2.5), sodium chloride solution, propion solution, dioxane, ethylene glycol, chaotropic salt, guanidine hydrochloride, or urea allows the adsorbed protein to be purified with high purity. Elute.
[0224]
The monoclonal antibody of the present invention is not limited to a mouse-derived antibody, and may be any mammal-derived antibody as long as it has a binding property to the protein of the present invention. Further, the monoclonal antibody of the present invention is not limited to one produced by a hybridoma, and may be one produced by a genetic engineering technique. For example, a chimeric antibody, a humanized antibody and the like are also included in the category of the monoclonal antibody of the present invention as long as they have a binding property to the protein of the present invention. Such an antibody can be prepared, for example, according to the method of Man @ Sung @ Co et al. (Man @ Sung @ Co, @ et @ al. @ (1992) @J. Immunol. @ 148, 1149-1154).
[0225]
As an antigen for preparing the antibody used in this embodiment, a polypeptide consisting of the amino acid sequence represented by amino acid Nos. 17-455 (preferably 19-455) of SEQ ID NO: 2 in the sequence listing, or at least 6 Or a polypeptide consisting of the amino acid sequence represented by amino acid Nos. 17-460 of SEQ ID NO: 4 or a polypeptide consisting of at least six consecutive partial amino acid sequences thereof, or SEQ ID NO: A polypeptide comprising the amino acid sequence represented by amino acid Nos. 26-406 of 16 or a polypeptide comprising at least six consecutive partial amino acid sequences thereof, or a polypeptide comprising the amino acid sequence represented by amino acid Nos. 22-491 of SEQ ID NO: 18 Peptides or less A polypeptide consisting of six consecutive partial amino acid sequences, or a polypeptide consisting of the amino acid sequence represented by amino acid numbers 23-493 of SEQ ID NO: 20, or a polypeptide consisting of at least six consecutive partial amino acid sequences thereof, or A polypeptide consisting of the amino acid sequence represented by amino acid numbers 21-470 of SEQ ID NO: 22 or a polypeptide consisting of at least six consecutive partial amino acid sequences thereof, or the amino acid sequence represented by amino acid numbers 27-346 of SEQ ID NO: 24 Or a polypeptide comprising at least six consecutive partial amino acid sequences thereof, or a derivative in which an arbitrary amino acid sequence or a carrier is added to these polypeptides. Number 9 A polypeptide comprising the amino acid sequence represented by amino acid No. 1-14 of SEQ ID NO: 10 or the N-terminus of a polypeptide consisting of amino acid No. 1-14, wherein keyhole limpet hemocyanin is bound as a carrier. is there.
[0226]
The polypeptide consisting of the amino acid sequence represented by amino acid numbers 17-455 (preferably 19-455) of SEQ ID NO: 2 or the polypeptide consisting of the amino acid sequence represented by amino acids 17-460 of SEQ ID NO: 4 is For example, a polypeptide encoded by the nucleotide sequence represented by nucleotide numbers 47-1411 of SEQ ID NO: 1 in the sequence listing or the nucleotide sequence represented by nucleotide numbers 78-1457 of SEQ ID NO: 3 in the sequence listing is transformed into a host cell by genetic manipulation. Can be obtained. Specifically, other prokaryotic or eukaryotic host cells can be transformed by incorporating the DNA having the above nucleotide sequence into an appropriate vector DNA. Furthermore, by introducing an appropriate promoter and a sequence involved in expression into these vectors, the gene can be expressed in each host.
[0227]
Examples of prokaryotic cell hosts include Escherichia coli and Bacillus subtilis. To transform a gene of interest into these host cells, the host cells are transformed with a plasmid vector that contains replicons or origins of replication from species compatible with the host and regulatory sequences. Further, the vector is preferably a vector having a sequence capable of imparting phenotypic (phenotypic) selectivity to transformed cells.
[0228]
For example, K12 strain or the like is often used as Escherichia coli, and pBR322 or a pUC-type plasmid is generally used as a vector, but is not limited thereto, and any known various strains and vectors can be used.
[0229]
Examples of the promoter include, in Escherichia coli, tryptophan (trp) promoter, lactose (lac) promoter, tryptophan lactose (tac) promoter, lipoprotein (lpp) promoter, polypeptide chain elongation factor Tu (tufB) promoter, and the like. Any promoter can be used to produce the polypeptide of interest.
[0230]
As the Bacillus subtilis, for example, strain 207-25 is preferable, and as a vector, pTUB228 (Ohmura, K. et al. (1984) J. Biochem. is not. By ligating a DNA sequence encoding the signal peptide sequence of Bacillus subtilis α-amylase, extracellular secretory expression becomes possible.
[0231]
Eukaryotic host cells include cells such as vertebrates, insects, and yeast. Examples of vertebrate cells include monkey COS cells (Gluzman, {Y.} (1981) {Cell} 23, {175-). 182, ATCC @ CRL-1650) and Chinese hamster ovary cells (CHO cells, ATCC @ CCL-61) deficient in dihydrofolate reductase (Urlaub, G. and ａｓChasin, L.A. (1980) Proc. Natl. Acad. Sci. (USA # 77, # 4126-4220) and the like are often used, but are not limited thereto.
[0232]
As a vertebrate cell expression promoter, a promoter having an upstream site of a gene to be normally expressed, an RNA splice site, a polyadenylation site, a transcription termination sequence and the like can be used. May be provided. Examples of the expression vector include, but are not limited to, pSV2dhfr (Subramani, S. et al. (1981) Mol. Cell. Biol. 1, 854-864) having the SV40 early promoter.
[0233]
As an example, when a COS cell is used as a host cell, the expression vector has an SV40 origin of replication, is capable of autonomous growth in COS cells, and further has a transcription promoter, a transcription termination signal, and an RNA splice site. Can be used. The expression vector was prepared by the diethylaminoethyl (DEAE) -dextran method (Luthman, H. and Magnesson, G. (1983) Nucleic Acids Res, 11, 1295-1308), and calcium phosphate-DNA coprecipitation method (Graham, F. L.). and van der Eb, A. J. (1973) Virology 52, 456-457), and electric pulse perforation (Neummann, E. et al. (1982) EMBO J. 1, 1, 841-845). Thus, the desired transformed cells can be obtained. When a CHO cell is used as a host cell, a vector capable of expressing a neo gene functioning as an antibiotic G418 resistance marker together with an expression vector, for example, pRSVneo (Sambrook, {J. et al. (1989)}: {"Molecular Cloning"] A Laboratory Laboratory Manual, Cold Spring Harbor Laboratory, NY, pSV2-neo (Southern, PJ J. and Berg, P. (1982) J. Mol. Appl. By selecting a G418-resistant colony, a transformed cell stably producing the desired polypeptide can be obtained. .
[0234]
When an insect cell is used as a host cell, a cell line derived from the ovary cell (Sf-9 or Sf-21) of Spodoptera frugiperda of Lepidoptera and Noctuidae or a High Five cell derived from an egg cell of Trichoplusia ni (Wickham, {T.}. al, (1992) ｔｅBiotechnol. Prog. I: 391-396) and the like are often used as host cells, and the baculovirus transfer vector used is a polyhedrin protein protein promoter of autographa nucleopolyhedrovirus (AcNPV). pVL1392 / 1393 is often used (Kidd, I. M. and VC. Emery (1993) The use of baculovirus. s as expression vectors. Applied Biochemistry and Biotechnology 42, 137-159). In addition, vectors using promoters of baculovirus P10 and homobasic protein can also be used. Furthermore, by connecting the secretion signal sequence of the envelope protein protein GP67 of AcNPV to the N-terminal side of the protein of interest, the recombinant protein can be expressed as a secreted protein (Zhe-mei @ Wang, @ et @ al.
(1998) @Biol. {Chem. , $ 379, $ 167-174).
[0235]
As an expression system using a eukaryotic microorganism as a host cell, yeast is generally well known. Among them, yeast of the genus Saccharomyces, for example, baker's yeast Saccharomyces cerevisiae and petroleum yeast Pichia pastoris are preferable. Examples of expression vectors for eukaryotic microorganisms such as the yeast include, for example, a promoter for an alcohol dehydrogenase gene (Bennetzen, J. L. and Hall, B. D. (1982) J. Biol. Chem. 257, 3018-3025) ) Or a promoter of an acid phosphatase gene (Miyanohara, A. et al. (1983) Proc. Natl. Acad. Sci. USA 80, 1-5). When expressed as a secretory protein, it can be expressed as a recombinant having a secretory signal sequence and a cleavage site of an endogenous protease or a known protease possessed by a host cell at the N-terminal side. For example, in a system in which human mast cell tryptase of a trypsin-type serine protease is expressed in petroleum yeast, the activity is achieved by connecting the secretory signal sequence of α-factor of yeast and the cleavage site of KEX2 protease possessed by petroleum yeast to the N-terminal side and expressing it. It is known that type tryptase is secreted into the medium (Andrew, L. Niles, et al. (1998) Biotechnol. Appl. Biochem. 28, 125-131).
[0236]
The transformant obtained as described above can be cultured according to a conventional method, and the culture produces the target polypeptide inside or outside the cell. As the medium used for the culture, various types commonly used depending on the host cell used can be appropriately selected. For example, in the case of the above-mentioned COS cells, RPMI1640 medium or Dulbecco's modified Eagle medium (hereinafter referred to as “DMEM”) A medium obtained by adding a serum component such as fetal bovine serum to a medium such as the above can be used as necessary.
[0237]
Recombinant protein produced in or outside the cells of the transformant by the above culture can be separated and purified by various known separation procedures utilizing the physical and chemical properties of the protein. it can. As the method, specifically, for example, treatment with a normal protein precipitant, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high-performance liquid chromatography ( Examples thereof include various liquid chromatography such as HPLC), dialysis, and combinations thereof. In addition, by connecting histidine consisting of 6 residues to the recombinant protein to be expressed, the protein can be efficiently purified by a nickel affinity column. By combining the above methods, the polypeptide of the present invention can be easily produced in large quantities with high yield and high purity.
[0238]
The antibody obtained as described above can be used for various immunological measurement methods such as RIA, ELISA, fluorescent antibody method, passive hemagglutination, immunohistological staining, and the like.
[0239]
4) Detection
The antibody obtained by the method described in the above 3) may be directly labeled, or may be used as a primary antibody to specifically recognize the antibody (recognize an antibody derived from the animal in which the antibody was produced). Used for detection in cooperation with the next antibody.
[0240]
Preferred examples of the type of label include, but are not limited to, an enzyme (alkaline phosphatase or horseradish peroxidase) or biotin (however, an operation of further binding enzyme-labeled streptavidin to biotin of the secondary antibody is added). Various pre-labeled antibodies (or streptavidin) are commercially available for methods that use labeled secondary antibodies (or labeled streptavidin). I for RIA¹²⁵The measurement is carried out using a liquid scintillation counter or the like using an antibody labeled with a radioisotope such as
[0241]
By detecting the activity of these labeled enzymes, the amount of the polypeptide that is the antigen is measured. In the case of alkaline phosphatase or horseradish peroxidase, substrates that emit color or emit light by the catalyst of those enzymes are commercially available.
[0242]
When a color-developing substrate is used, it can be visually detected by Western blotting or dot / slot blotting. In the ELISA method, quantification is preferably carried out by measuring the absorbance (measurement wavelength varies depending on the substrate) of each well using a commercially available microplate reader. Also, preferably, a standard curve prepared by preparing a dilution series of the antigen used for the production of the antibody in the above 3), performing a detection operation simultaneously with other samples using this as a standard antigen sample, and plotting the standard antigen concentration and the measured value It is possible to quantify the antigen concentration in other samples by preparing
[0243]
On the other hand, when a substrate that emits light is used, it can be detected by Western radiography or dot / slot blotting by autoradiography using an X-ray film or an imaging plate, or photography using an instant camera, Quantitation using densitometry or the BAS2000II system is also possible. When a luminescent substrate is used in the ELISA method, the enzyme activity is measured using a luminescent microplate reader (for example, manufactured by Bio-Rad).
[0244]
5) Measurement operation
i) Western blot, dot blot or slot blot
First, in order to prevent non-specific adsorption of antibodies, the membrane is immersed in a buffer containing a substance that inhibits such non-specific adsorption (skim milk, bovine serum albumin, gelatin, polyvinylpyrrolidone, etc.) for a certain time in advance. Perform an operation (blocking). The composition of the blocking solution is, for example, phosphate buffered saline (PBS) or Tris buffered saline (TBS) containing 5% {skim milk, 0.05 to 0.1%} Tween 20. Instead of skim milk, 1 to 10% bovine serum albumin, 0.5 to 3% gelatin or 1% polyvinylpyrrolidone may be used. The blocking time is 16 to 24 hours at 4 ° C. or 1 to 3 hours at room temperature.
[0245]
Next, the membrane was washed with PBS or TBS containing 0.05 to 0.1% Tween 20 (hereinafter referred to as “washing solution”) to remove an excess blocking solution, and then the membrane was manufactured by the method described in 3) above. The antibody is immersed in a solution appropriately diluted with a washing solution for a certain period of time to bind the antibody to the antigen on the membrane. The dilution ratio of the antibody at this time can be determined, for example, by performing a preliminary Western blotting experiment using a sample obtained by serially diluting the recombinant antigen described in 3) above. This antibody reaction operation is preferably performed at room temperature for 1 hour. After completion of the antibody reaction operation, the membrane is washed with a washing solution. Here, when the used antibody is labeled, the detection operation can be performed immediately. When an unlabeled antibody is used, a secondary antibody reaction is subsequently performed. For example, when a commercially available product is used, the labeled secondary antibody is diluted 2,000 to 20,000 times with a washing solution and used (if a suitable dilution ratio is described in the attached instruction, follow the description). After washing and removing the primary antibody, the membrane is immersed in a secondary antibody solution at room temperature for 1 to 3 hours, washed with a washing solution, and then subjected to a detection operation according to the labeling method. The washing operation is performed, for example, by first shaking the membrane in the washing solution for 15 minutes, exchanging the washing solution for a new one, shaking for 5 minutes, exchanging the washing solution again, and shaking for 5 minutes. If necessary, the washing solution may be further exchanged for washing.
[0246]
ii) ELISA / RIA
First, as in the case of Western blotting, blocking is performed in advance to prevent nonspecific adsorption of the antibody to the bottom surface in the well of the plate on which the sample is immobilized by the method 2). The blocking conditions are as described in the section of Western blot.
[0247]
Next, the wells are washed with PBS or TBS containing 0.05 to 0.1% Tween 20 (hereinafter, referred to as “washing solution”) to remove an excess blocking solution, and then manufactured by the method described in 3) above. A solution obtained by appropriately diluting the antibody with a washing solution is dispensed and incubated for a certain period of time to bind the antibody to the antigen. The dilution ratio of the antibody at this time can be determined, for example, by performing a preliminary ELISA experiment using a serially diluted recombinant antigen described in 3) above as a sample. This antibody reaction operation is preferably performed at room temperature for about 1 hour. After completion of the antibody reaction operation, the inside of the well is washed with a washing solution. Here, when the used antibody is labeled, the detection operation can be performed immediately. When an unlabeled antibody is used, a secondary antibody reaction is subsequently performed. For example, when a commercially available product is used, the labeled secondary antibody is diluted 2,000 to 20,000 times with a washing solution and used (if a suitable dilution ratio is described in the attached instruction, follow the description). After washing and removing the primary antibody, a secondary antibody solution is dispensed into the wells, incubated at room temperature for 1 to 3 hours, washed with a washing solution, and then subjected to a detection operation according to the labeling method. The washing operation is performed by, for example, dispensing a washing solution into a well and shaking for 5 minutes, replacing the washing solution with a new one, shaking for 5 minutes, exchanging the washing solution again and shaking for 5 minutes. If necessary, the washing solution may be further exchanged for washing.
[0248]
In the present invention, the so-called sandwich ELISA can be carried out, for example, by the method described below. First, in one of the amino acid sequence represented by amino acid numbers 17-455 (preferably 19-455) of SEQ ID NO: 2 and the amino acid sequence represented by amino acid numbers 17-460 of SEQ ID NO: 4, Two regions having high hydrophilicity are selected, a partial peptide consisting of 6 or more amino acids in each region is synthesized, and two types of antibodies using the partial peptides as antigens are obtained. One of the antibodies is labeled as described in 4) above. The unlabeled antibody is immobilized on the bottom surface in the well of a 96-well ELISA plate according to the method described in 2) above. After blocking, the sample solution is placed in a well and incubated at room temperature for 1 hour. After washing the wells, the labeled antibody diluent is dispensed into each well and incubated. After washing the inside of the well again, a detection operation according to the labeling method is performed.
[0249]
6) Evaluation
According to the method described above, the detection results are compared between a sample derived from cells cultured in the presence of the test substance and a sample derived from cells cultured in the absence of the test substance. The test substance, which has reduced the production of a polypeptide to which the antibody specifically produced in step (1) binds, is used for treating or preventing one or more diseases selected from hyperlipidemia, arteriosclerosis, and hyperglycemia. It can be an agent. In addition, by combining the antibody prepared by the method described in 3) above and other reagents used in the above series of methods, one or two or more selected from hyperlipidemia, arteriosclerosis, and hyperglycemia A kit for testing an agent for treating or preventing a disease of the present invention is provided.
[0250]
The polypeptide to be detected in the method of the present invention can be obtained in accordance with the above description of the method for obtaining an antigen for producing an antibody, or can have the nucleotide sequence or nucleotide sequence shown in nucleotide numbers 47 to 1411 of SEQ ID NO: 1 in the sequence listing. It can also be produced in animal cells using an adenovirus vector into which DNA having the nucleotide sequence shown in nucleotide numbers 78-1457 of SEQ ID NO: 3 in the column list has been incorporated. Examples of a method for constructing such a recombinant adenovirus vector include a method using a commercially available kit (for example, adenovirus expression vector kit, manufactured by Takara Shuzo Co., Ltd.). The fact that the gene to be detected in the method of the present invention is closely correlated with the blood triglyceride concentration of a mammal means that, for example, a recombinant adenovirus having a recombinant adenovirus vector obtained as described above is used. When a mammal, for example, a mouse is infected to express a gene carried by the recombinant adenovirus vector, an increase in blood triglyceride concentration is observed, and the recombinant protein obtained as described above is expressed in a mouse. Increase in blood triglyceride concentration when administered to mice, and in a congenital hypolipidemic mouse, the expression level of a gene having a nucleotide sequence represented by nucleotide numbers 47 to 1411 of SEQ ID NO: 1 in the sequence listing is reduced. It is evidenced by less than hyperlipidemic mice.
[0251]
In addition, a non-human animal into which a foreign gene containing the nucleotide sequence according to any one of the above a) to e) obtained by genetic manipulation has been introduced so as to be able to highly express the gene is used. The present invention also includes a method for testing an agent for treating or preventing one or more diseases selected from hyperlipidemia, arteriosclerosis and hyperglycemia. Animals used in this method include, for example, KK /SnkA mouse or a transgenic mouse in which a DNA having the nucleotide sequence shown in nucleotide No. 47-1411 of SEQ ID No. 1 in the Sequence Listing or the nucleotide sequence shown in nucleotide No. 78-1457 of SEQ ID No. 3 in the Sequence Listing is introduced into the mouse; However, the present invention is not limited to these.
[0252]
The transgenic animal can be obtained by obtaining a fertilized egg from the animal, transfecting the animal, and then transplanting the embryo into a pseudopregnant animal and generating the transgenic animal. Engineering Experiment Manual (edited by Tatsuji Nomura, Motoya Katsuki, ed. 1987), Operation Manual for Mouse Embryos (“Manipulating the Mouse Embryo, A Laboratory Manual”, B. Hogan, F., Costinini and L. E. L., L. E., L. E. L.) (Translated by Yutaka Toyoda, Youtsumori Mori and Yoichiro Iwakura, 1989), and Japanese Patent Publication No. 5-48093). Specifically, for example, in the case of a mouse, first, a female mouse (a mouse whose blood triglyceride concentration is lower than normal, for example, KK /SnkAfter administration of an ovulation-inducing agent to a mouse (preferably, but not limited to), it is mated with a male of the same strain, and the pronuclear fertilized egg is collected from the oviduct of the female mouse the next day. Next, the DNA fragment solution to be introduced is injected into the pronucleus of a fertilized egg using a micro glass tube. In addition, as a regulatory gene such as a promoter or an enhancer for expressing a gene to be introduced in animal cells, any regulatory gene that functions in the cell of the introduced animal can be used. The fertilized egg into which the DNA has been injected is transplanted into the oviduct of a pseudopregnant foster female mouse (Slc: ICR, etc.), and is born about 20 days later by spontaneous delivery or cesarean section. As a method for confirming that the thus obtained animal has the introduced gene, DNA is extracted from the tail of the animal or the like, and the DNA is used as a template to detect the sense and antisense specific for the introduced gene. A method of performing PCR using sense primers, digesting the DNA with several types of restriction enzymes, gel electrophoresis, and blotting the DNA in the gel onto a nitrocellulose membrane or nylon membrane, etc. And a method of performing Southern blot analysis using a probe labeled as a probe. Whether or not the introduced gene is actually expressed in the animal body can be confirmed by measuring the concentration of neutral fat in peripheral blood. When the introduced gene is actually expressed in the animal, the blood triglyceride concentration is higher than in an animal into which the gene has not been introduced.
[0253]
A test substance is administered to the transgenic animal thus obtained, and a substance that reduces the concentration of triglyceride in blood is selected (preferably, the test substance is administered to an animal to which no gene is introduced, and the results are compared. Then, a substance that significantly lowers the blood triglyceride concentration in the transgenic animal is selected). In this method, not only the substance that suppresses or inhibits the expression of the introduced gene, but also the function of the polypeptide itself encoded by the gene, and interacts with the polypeptide to contribute to the functional expression of the polypeptide Substances such as factors (for example, receptors) that inhibit any of the biological reactions related to an increase in blood triglyceride concentration due to the action of the polypeptide can be found. Such a substance can also be an agent for treating or preventing one or more diseases selected from hyperlipidemia, arteriosclerosis, and hyperglycemia.
[0254]
The present invention also relates to a polypeptide other than an antibody, that is, a receptor for a polypeptide to be detected, which specifically binds to a polypeptide to be detected in the method of the present invention (hereinafter, simply referred to as “polypeptide to be detected”). When the receptor is a protein present on the cell membrane, the receptor can be cloned, for example, according to the method described below. First, a recombinant consisting of a nucleotide sequence represented by nucleotide numbers 47 to 1411 of SEQ ID NO: 1 in the Sequence Listing or a nucleotide sequence represented by nucleotide numbers 78 to 1457 of SEQ ID NO: 3 in the Sequence Listing can be expressed in a mammalian cell. A vector is constructed, introduced into COS-1, the culture supernatant is collected, and the recombinant polypeptide of the polypeptide to be detected is purified. The obtained recombinant polypeptide is labeled with fluorescein isothiocyanate (hereinafter referred to as "FITC"). Next, a labeled recombinant polypeptide is added to cultured cells derived from various mammalian tissues to identify cells to which the labeled recombinant polypeptide specifically binds on the cell membrane, that is, cells that express the receptor. A cDNA library derived from the identified cells is incorporated into a vector that can be expressed in mammalian cells, and expressed in cells that do not express the receptor (preferably COS-1 or CHO cells, more preferably CHO cells). The above-mentioned FITC-labeled recombinant polypeptide is added to the cells in which the cDNA library has been expressed, and after culturing for a certain period of time, the cells are collected and the cells to which the FITC-labeled recombinant polypeptides are bound are selected using a cell sorter. I do. The cDNA introduced from the obtained cells is cloned by PCR or the like. If necessary, the above operations are repeated to finally clone a cDNA encoding a receptor that specifically binds to the FITC-labeled recombinant polypeptide. Also, the receptor itself can be recovered from the cells thus cloned.
[0255]
If cDNA encoding the receptor of the polypeptide to be detected is obtained in this way, the interaction between the polypeptide to be detected and the receptor is further inhibited by using the cells used as the material of the cDNA library. Used for screening for substances, substances that exert the same function as the polypeptide to be detected by binding to the receptor, or substances that inhibit signal transduction initiated by the interaction between the polypeptide to be detected and the receptor. be able to. Specifically, for example, mouse genomic DNA is appropriately fragmented by restriction enzyme digestion or the like, and pEGFP-1 (manufactured by Clontech) is commercially available as a vector for a green-fluorescent protein: reporter assay at one end thereof. ) Is introduced into the cells used as the material of the cDNA library. During the culturing of the cells, a recombinant polypeptide (not labeled) of the polypeptide to be detected is added, and the cells producing the green fluorescent protein are selected with a cell sorter. The sorted cells produce green fluorescent protein in the presence of the recombinant polypeptide. The cells were cultured in a 96-well culture plate such that the number of cells per well was substantially the same, and only the test substance was added, or the recombinant polypeptide and the test substance were added simultaneously and cultured for a certain period of time. Thereafter, the amount of green fluorescent protein produced is measured using a fluorescent plate reader or the like. When the test substance alone produces green fluorescent protein when cultured, the substance is considered to be an agonist of the polypeptide to be detected. On the other hand, when the amount of green fluorescent protein produced in the well to which the recombinant polypeptide and the test substance are simultaneously added is smaller than that in the well to which the recombinant polypeptide alone is added, the test substance is an antagonist of the polypeptide to be detected or the polypeptide. It is a signal transduction inhibitor of a peptide and is considered to be useful as an agent for treating or preventing one or more diseases selected from hyperlipidemia, arteriosclerosis and hyperglycemia.
[0256]
When the thus obtained antagonist is a protein or peptide, the polynucleotide having a nucleotide sequence encoding the protein or peptide is one or two selected from hyperlipidemia, arteriosclerosis, and hyperglycemia. It can be used for gene therapy for the above diseases. Such polynucleotides can be obtained, for example, by analyzing the amino acid sequence of the identified antagonist protein or polypeptide, synthesizing an oligonucleotide probe consisting of a nucleotide sequence encoding the amino acid sequence, and preparing various cDNA libraries or genomic libraries. It can be obtained by performing screening. When the peptide having antagonist activity is derived from a randomly synthesized artificial peptide library, a DNA consisting of a nucleotide sequence encoding the amino acid sequence of the peptide is chemically synthesized. In gene therapy, the thus-obtained polynucleotide encoding the antagonist is incorporated into, for example, a viral vector to infect a patient with the virus (detoxified) having the recombinant viral vector. Since an antagonist is produced in the patient's body and inhibits the function of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing, one or more selected from hyperlipidemia, arteriosclerosis and hyperglycemia Disease progress can be suppressed.
[0257]
As a method for introducing a gene therapy agent into cells, a gene transfer method using a viral vector or a non-viral gene transfer method (Nikkei Science, April 1994, pp. 20-45, Experimental Medicine Special Edition, 12 (15) (1994), Experimental Medicine Separate Volume, “Basic Technology for Gene Therapy”, Youtosha (1996)).
[0258]
As a method for gene transfer using a virus vector, for example, TR4 or mutant TR4 is encoded in a DNA virus or RNA virus such as retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, pox virus, polio virus, and simbis virus. And a method of incorporating and introducing DNA to be incorporated. Among them, a method using a retrovirus, an adenovirus, an adeno-associated virus, and a vaccinia virus is particularly preferable. Examples of non-viral gene transfer methods include a method of directly administering an expression plasmid intramuscularly (DNA vaccine method), a liposome method, a lipofectin method, a microinjection method, a calcium phosphate method, and an electroporation method. The vaccine method and the liposome method are preferred.
[0259]
Further, in order for the gene therapy agent to actually act as a medicine, an in vivo (in vivo) method in which DNA is directly introduced into the body or a certain cell is taken out from a human, DNA is introduced outside the body into the cell, and the cell is transferred into the body. Ex-vivo method (Nikkei Science, April 1994, pp. 20-45, Monthly Pharmaceutical Affairs, 36 (1), 23-48 (1994), Experimental Medicine Special Edition, {12} (15) (1994) )).
[0260]
For example, when the gene therapy agent is administered by an in vivo method, it is administered by an appropriate administration route, such as vein, artery, subcutaneous, intradermal, intramuscular, etc., depending on the disease, condition and the like. When administered by an in vivo method, the gene therapy agent is generally used as an injection or the like, but if necessary, a conventional carrier may be added. In the case of a liposome or a membrane-fused liposome (Sendai virus-liposome or the like), a liposome preparation such as a suspension, a freezing agent, a centrifugal concentrated frozen agent and the like can be obtained.
[0261]
A nucleotide sequence complementary to the partial sequence of the nucleotide sequence shown in nucleotide numbers 78-1457 of SEQ ID NO: 3 in the sequence listing can be used for so-called antisense therapy. The antisense molecule is a stable DNA such as a DNA consisting of usually 15 to 30 mer, or a phosphorothioate, methylphosphonate or morpholino derivative thereof, which is complementary to a part of the nucleotide sequence represented by nucleotide numbers 78 to 1457 of SEQ ID NO: 3 in the sequence listing. It can be used as a stable DNA derivative such as a DNA derivative or 2'-O-alkyl RNA. Such antisense molecules can be introduced into cells by methods well known in the art of the present invention, such as by microinjection, liposome encapsulation, or expression using a vector having an antisense sequence. Such antisense therapy is effective in reducing the activity of the protein encoded by the nucleotide sequence shown by nucleotide numbers 78 to 1457 of SEQ ID NO: 3 in the sequence listing, particularly hyperlipidemia, arteriosclerosis and It is useful for treating one or more diseases selected from hyperglycemia.
[0262]
A composition useful as a medicine containing the antisense oligonucleotide can be produced by a known method such as mixing a pharmaceutically acceptable carrier. Examples of such carriers and manufacturing methods are described in Applied Antisense Oligonucleotide Technology (1998, Wiley-Liss, Inc.). Then, one or two selected from hyperlipidemia, arteriosclerosis, and hyperglycemia in which the expression of the gene containing the nucleotide sequence represented by nucleotide numbers 78 to 1457 of SEQ ID NO: 3 and the activity of the gene product are abnormal. Each person is administered an amount sufficient to treat one or more diseases. The effective amount can vary depending on various factors such as the condition, weight, sex, and age of each individual, and the administration method such as subcutaneous, topical, oral, and intramuscular. For example, for intravenous injection, 0.02 to 0.2 mg / kg / hour for 2 hours, and for subcutaneous administration, 1 to 200 mg / kg / hour.²/ Day.
[0263]
II.試 験 Test method using polypeptide
The polypeptide of the present invention is, for example, (a) one continuous sequence contained in the nucleotide sequence shown in SEQ ID NO: 11 in the sequence listing, in which nucleotide No. 18 has a 5 ′ terminal and nucleotides 458 to 638 DNA containing a nucleotide sequence having any one at the 3 ′ end, (b) a continuous sequence contained in the nucleotide sequence shown in SEQ ID NO: 11 in the sequence listing, wherein nucleotide No. 18 is a 5 ′ end A DNA containing a nucleotide sequence having any one of nucleotide numbers 458 to 602 as a 3 'end, (c) a continuous sequence contained in the nucleotide sequence shown in SEQ ID NO: 15 in the sequence listing, and DNA containing a nucleotide sequence having 173 at the 5 ′ end and nucleotide number 601 at the 3 ′ end, (d (E) a DNA comprising a continuous sequence contained in the nucleotide sequence shown in SEQ ID NO: 15 in the sequence listing and having a nucleotide sequence having nucleotide number 173 as a 5 'end and nucleotide number 1393 as a 3' end; One continuous sequence contained in the nucleotide sequence shown in SEQ ID NO: 17 in the sequence listing, having nucleotide number 434 as its 5 'end and any one of nucleotide numbers 1039 to 1261 as its 3' end And (f) a nucleotide sequence comprising one continuous sequence contained in the nucleotide sequence shown in SEQ ID NO: 17 in the sequence listing, having nucleotide number 434 as a 5 'end and nucleotide number 1909 as a 3' end. And (g) a nucleotide sequence shown in SEQ ID NO: 19 in the sequence listing. A DNA comprising a nucleotide sequence having nucleotide number 22 at the 5 'end and any one of nucleotide numbers 639 to 846 at the 3' end, (h) a sequence in the sequence listing A DNA comprising a nucleotide sequence having a nucleotide sequence having nucleotide number 22 at the 5 'end and nucleotide number 1503 at the 3' end, which is one continuous sequence contained in the nucleotide sequence represented by No. 19; A DNA comprising a continuous sequence contained in the nucleotide sequence represented by No. 21 and having a nucleotide sequence having nucleotide number 242 as a 5 ′ end and any one of nucleotide numbers 796 to 1015 as a 3 ′ end, ( j) one continuous sequence contained in the nucleotide sequence shown in SEQ ID NO: 21 in the sequence listing Thus, DNA containing a nucleotide sequence having nucleotide number 242 as the 5 'end and nucleotide number 1654 as the 3' end, (k) a continuous sequence contained in the nucleotide sequence shown in SEQ ID NO: 23 in the sequence listing. A DNA comprising a nucleotide sequence having nucleotide number 86 as the 5 'end and any one of nucleotide numbers 373 to 394 as the 3' end, (l) contained in the nucleotide sequence shown in SEQ ID NO: 23 in the sequence listing (A) to (l) of a DNA comprising a nucleotide sequence having a nucleotide sequence with nucleotide number 86 at the 5 ′ end and nucleotide number 1048 at the 3 ′ end, Producing a polypeptide encoded by the DNA in a host cell by using a conventional gene recombination technique. Ri may be obtained.
[0264]
In addition, in the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 11 in the sequence listing, that is, in the amino acid sequence of SEQ ID NO: 12 in the sequence listing, the amino acid sequence shown in amino acid numbers 1 to 16 is translated after translation in mammalian cells. Since it is removed as a signal peptide (Conklin, D. et al. (1999) Genomics 62, 477-482), in the above production method, when the host is derived from a mammalian cell, the amino acid of SEQ ID NO: 12 in the sequence listing is used. A polypeptide having No. 17 as the amino terminus can be obtained. Similarly, in the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 15 of the sequence listing, that is, in the amino acid sequence of SEQ ID NO: 16 in the sequence listing, the amino acid sequence shown by amino acid numbers 1 to 25 is translated after translation in mammalian cells. Since it is removed as a signal peptide, in the above-mentioned production method, when the host is derived from a mammalian cell, a polypeptide having amino acid number 26 of SEQ ID NO: 16 in the sequence listing can be obtained. Similarly, in the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 17 in the sequence listing, that is, in the amino acid sequence of SEQ ID NO: 18 in the sequence listing, the amino acid sequence shown in amino acid Nos. 1-21 is translated after translation in mammalian cells. Since it is removed as a signal peptide, in the above production method, when the host is derived from a mammalian cell, a polypeptide having the amino terminal at amino acid number 22 of SEQ ID NO: 18 in the sequence listing can be obtained. Similarly, in the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 19 in the sequence listing, that is, in the amino acid sequence of SEQ ID NO: 20 in the sequence listing, the amino acid sequence shown in amino acid numbers 1-22 is translated after translation in mammalian cells. Since it is removed as a signal peptide, in the above-mentioned production method, when the host is derived from a mammalian cell, a polypeptide having amino acid number 23 of SEQ ID NO: 20 in the sequence listing as an amino terminal can be obtained. Similarly, in the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 21 in the sequence listing, that is, in the amino acid sequence of SEQ ID NO: 22 in the sequence listing, the amino acid sequence shown in amino acid numbers 1 to 20 is translated after translation in mammalian cells. Since it is removed as a signal peptide, in the above production method, when the host is derived from a mammalian cell, a polypeptide having amino acid number 21 of SEQ ID NO: 22 in the sequence listing as an amino terminal can be obtained. Similarly, in the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 23 of the sequence listing, that is, in the amino acid sequence of SEQ ID NO: 24 in the sequence listing, the amino acid sequence shown by amino acid numbers 1-26 is translated after translation in mammalian cells. Since it is removed as a signal peptide, in the above production method, when the host is derived from a mammalian cell, a polypeptide having amino acid number 27 of SEQ ID NO: 24 in the sequence listing as an amino terminal can be obtained.
[0265]
The method for producing the polypeptide of the present invention using a host having no function of cleaving a signal peptide of a mammalian protein, such as a prokaryotic cell, includes, for example, (a) SEQ ID NO: 12 in the sequence listing. A continuous amino acid sequence in the amino acid sequence shown in the above, the amino acid sequence having amino acid number 17 as the amino terminal and any one of amino acid numbers 147 to 207 or 147 to 195 as the carboxyl terminal, b) one continuous amino acid sequence in the amino acid sequence represented by SEQ ID NO: 16 in the sequence listing, wherein amino acid number 26 is an amino terminal and amino acid number 143 is a carboxyl terminal; A single continuous amino acid sequence contained in the amino acid sequence shown in SEQ ID NO: 16, (D) a continuous amino acid sequence in the amino acid sequence of SEQ ID NO: 18 in the sequence listing, wherein the amino acid sequence has an amino terminal at No. 26 and a carboxyl terminal at one of amino acid numbers 144 to 183; Is the amino terminal, and any one of amino acid numbers 202 to 276 is the carboxyl terminal. (E) A continuous amino acid sequence in the amino acid sequence of SEQ ID NO: 20 in the sequence listing, wherein amino acid number 23 is the amino acid sequence. An amino acid sequence having any one of amino acid numbers 206 to 275 as a carboxyl terminal; (f) a continuous amino acid sequence in the amino acid sequence of SEQ ID NO: 22 in the sequence listing, wherein amino acid number 21 is an amino terminal An amino acid sequence having any one of amino acids 185 to 258 as a carboxyl terminal; (G) one continuous amino acid sequence contained in the amino acid sequence shown in SEQ ID NO: 24 in the sequence listing, wherein amino acid number 27 is an amino terminal, and one of amino acid numbers 122 to 129 is a carboxyl terminal; (H) an amino acid sequence having amino acid number 26 of SEQ ID NO: 16 in the sequence listing as the amino terminus and a carboxyl terminus of 406; (i) amino acid number 22 of SEQ ID NO. 18 in the sequence listing as the amino terminus; (J) an amino acid sequence having amino acid number 23 of SEQ ID NO: 20 in the sequence listing as an amino terminus and an amino acid sequence having 493 as a carboxyl terminus; and (k) an amino acid sequence of SEQ ID NO. An amino acid sequence having 470 as a carboxyl terminal, and (l) an amino acid sequence of SEQ ID NO: 24 in the sequence listing. (A) to (l) of the amino acid sequence having amino acid No. 27 as an amino terminus and 346 as a carboxyl terminus, specifically cleaved by a protease which does not cleave the amino acid sequence. Escherichia coli to produce a fusion protein consisting of an amino acid sequence in which the amino acid sequence to be linked is further linked, and the amino terminal side of the amino acid sequence is linked to the amino acid sequence of a protein having specific affinity for a specific compound. After purifying the protein using affinity with a specific compound, a method of removing the amino acid sequence of the protein having affinity with the specific compound on the amino terminal side by the protease can be used. As an example of purification utilizing the affinity for such a specific compound, for example, a fusion protein to which an amino acid sequence of glutathione S-transferase (hereinafter, referred to as “GST”) is linked is produced by Escherichia coli. After purifying the fusion protein using the affinity between glutathione and GST, a method of removing the GST sequence with a protease can be mentioned. However, as shown in the examples of the present specification, the function of the polypeptide of the present invention is not impaired even in the state of a fusion protein in which the GST sequence is not removed.
[0266]
In addition, a polypeptide in which a histidine tag for facilitating purification is inserted into the amino terminus of the “angiopoietin-related protein” (within the signal peptide) and the fibrinogen binding region at the carboxyl terminus is also deleted. A histidine for facilitating purification is provided on the amino terminal side (in the signal peptide) of “angiopoietin-related protein 3” as shown in SEQ ID NO: 24 in the sequence listing without losing the function of increasing the concentration of triglyceride. The polypeptide in which the tag is inserted and the 253 residues at the carboxyl terminal side are deleted also retains its function.
[0267]
That is, the present invention relates to (a) one continuous amino acid sequence in the amino acid sequence shown in SEQ ID NO: 12 in the sequence listing, wherein amino acid number 17 is an amino terminal and any one of amino acid numbers 147 to 207 is used. (B) an amino acid sequence having amino acid number 17 of SEQ ID NO: 12 in the sequence listing as an amino terminus and having one of amino acid numbers 147 to 195 as a carboxyl terminus; An amino acid sequence having one continuous amino acid sequence in the amino acid sequence represented by SEQ ID NO: 16, wherein amino acid number 26 is an amino terminal and amino acid number 143 is a carboxyl terminal, (d) amino acid sequence of SEQ ID NO: 18 in the sequence listing A single continuous amino acid sequence in the sequence, wherein amino acid number 22 is the amino terminal, (E) an amino acid sequence having a carboxyl terminal at any one of 202 to 276, (e) a continuous amino acid sequence in the amino acid sequence of SEQ ID NO: 20 in the sequence listing, wherein amino acid number 23 is an amino terminal, (F) a continuous amino acid sequence in the amino acid sequence of SEQ ID NO: 22 in the sequence listing, wherein amino acid number 21 is the amino terminal, and amino acid numbers 185 to 258 (G) a continuous amino acid sequence in the amino acid sequence of SEQ ID NO: 24 in the sequence listing, wherein any one of the amino acids has amino terminal 27 and any of amino acids 122 to 129; An amino acid sequence having one carboxyl terminal; (h) an amino acid sequence of SEQ ID NO: 16 in the sequence listing; An amino acid sequence having acid number 26 as the amino terminal and 406 as the carboxyl terminal, (i) an amino acid sequence having amino acid number 22 of SEQ ID NO: 18 as the amino terminal and 491 as the carboxyl terminal, and (j) an amino acid sequence having the carboxyl terminal. An amino acid sequence having amino acid number 23 of SEQ ID NO: 20 as an amino terminal and 493 as a carboxyl terminal; (k) an amino acid sequence having amino acid number 21 of SEQ ID NO. 22 in the sequence listing as an amino terminal and 470 as a carboxyl terminal; A) an amino acid sequence in which amino acid number 27 of SEQ ID NO: 24 in the sequence listing is an amino terminal and 346 is a carboxyl terminal, and any one of (a) to (l) is linked to an arbitrary amino acid sequence; And polypeptides having an activity to increase blood triglyceride concentration in mammals It is. In this case, the amino acid sequence to be linked does not include an amino acid sequence of 18 or more consecutive residues derived from the amino acid sequence of each human “angiopoietin-related protein”.
[0268]
The nucleotide sequence information of the cDNA of “angiopoietin-related protein” can be obtained from the National Institute of Genetics database (GeneBank). “Angiopoietin-related protein 1” has accession number XM_001720, “Angiopoietin-related protein 2” has accession number NM — 012098, “Angiopoietin-related protein 3” has accession number AF152562, and “Angiopoietin-related protein 4” has accession number AF202636. “Angiopoietin-related protein 5” is registered with accession number NM_039171, and “Angiopoietin-related protein 6” is registered with accession number Y16132.
[0269]
Nucleotides having nucleotide number 18 in SEQ ID NO: 11 as the 5 'end and any one of nucleotide numbers 458 to 638 as the 3' end in the sequence listing may be, for example, a human / angiopoietin-related protein cloned from a human liver cDNA library. Or a recombinant E. coli strain E. coli deposited internationally at the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary. A polymerase chain reaction (hereinafter, referred to as “the following) using a recombinant phagemid held by colitrip / h55-1 SANK 72299 (FERM BP-6941) as a template and designed to amplify DNA having a desired nucleotide sequence. PCR ”). Alternatively, the recombinant E. coli strain E. E. coli @ pTrip / h55-1 @ SANK72299 (FERM @ BP-6941) at the desired position in the nucleotide sequence of the cDNA inserted into the recombinant phagemid pTrip / h55-1 carried by FERM @ BP-6941, identical to the open reading frame of the cDNA It can also be obtained by performing site-specific artificial mutation such that a nucleotide sequence serving as a stop codon is inserted on the frame.
(A) One continuous sequence contained in the nucleotide sequence shown in SEQ ID NO: 17 in the sequence listing, wherein nucleotide number 434 is the 5 'end and any one of nucleotide numbers 1039 to 1261 is the 3' end A nucleotide consisting of a nucleotide sequence, (b) one continuous sequence contained in the nucleotide sequence shown in SEQ ID NO: 17 in the sequence listing, wherein the nucleotide of nucleotide number 434 is the 5 'end and the nucleotide number 1909 is the 3' end (C) a nucleotide consisting of a nucleotide sequence having nucleotide number 22 shown in SEQ ID NO: 19 in the sequence listing at the 5 ′ end and having any one of nucleotide numbers 639 to 846 at the 3 ′ end, (D) the nucleotidic sequence represented by SEQ ID NO: 19 in the sequence listing (E) a nucleotide consisting of a nucleotide sequence having a nucleotide at nucleotide number 22 at its 5 ′ end and nucleotide number 1503 at its 3 ′ end, (e) a nucleotide of SEQ ID NO: 21 in the sequence listing, No. 242 as a 5 'end and a nucleotide containing a nucleotide sequence having any one of nucleotide numbers 796 to 1015 as a 3' end, (f) one continuous nucleotide contained in the nucleotide sequence shown in SEQ ID NO: 21 in the sequence listing A nucleotide sequence consisting of a nucleotide sequence having a nucleotide at nucleotide number 242 as the 5 'end and nucleotide number 1654 at the 3' end, and (g) a continuous nucleotide contained in the nucleotide sequence shown in SEQ ID NO: 23 in the sequence listing. A sequence comprising nucleotide number 86 A nucleotide consisting of a nucleotide sequence having a nucleotide at the 5 ′ end and having any one of nucleotide numbers 373 to 394 as the 3 ′ end; (h) a continuous 1 contained in the nucleotide sequence shown in SEQ ID NO: 23 in the sequence listing; (A) to (h), each of which is a nucleotide sequence having a nucleotide sequence having nucleotide 5 at the 5′-terminus and nucleotide 3 at the 3′-terminus, all of which are derived from an appropriate human organ CDNA encoding human "angiopoietin-related protein 1, 2, 4, 5, 6" or a commercially available cDNA clone (available from, for example, IMAGE GE Consortium) From the desired nucleotide sequence using The DNA can be obtained by performing PCR using oligonucleotide primers designed to amplify the DNA.
[0270]
However, the DNA encoding the polypeptide of the present invention is not limited thereto, and may be, for example, those which have been appropriately modified in consideration of the frequency of codon usage of the host used in the above-described gene recombination technique, or those which have been artificially synthesized DNA, for example. In a host cell, (a) a single continuous amino acid sequence in the amino acid sequence shown in SEQ ID NO: 12 in the sequence listing, wherein amino acid number 17 is the amino terminal, and amino acid number is 147 to 207 or a polypeptide having an amino acid sequence having any one of amino acid numbers 147 to 195 as a carboxyl terminal; and (b) a continuous amino acid sequence in the amino acid sequence shown in SEQ ID NO: 16 in the sequence listing. Amino acid number 26 as the amino terminal and amino acid number 143 as the carboxyl terminal (C) a continuous amino acid sequence contained in the amino acid sequence shown in SEQ ID NO: 16 in the sequence listing, wherein the amino acid sequence is amino acid terminal 26, and any of amino acid sequences 144 to 183 (D) a continuous amino acid sequence in the amino acid sequence of SEQ ID NO: 18 in the sequence listing, wherein amino acid number 22 is the amino terminal and amino acids 202 to 276 (E) a continuous amino acid sequence in the amino acid sequence of SEQ ID NO: 20 in the sequence listing, wherein amino acid number 23 is the amino terminal and amino acid number 206 is Consisting of an amino acid sequence having a carboxyl terminal at any one of Peptide, (f) a continuous amino acid sequence in the amino acid sequence of SEQ ID NO: 22 in the sequence listing, wherein the amino acid sequence is amino acid number 21 as the amino terminal and amino acid number 185 to 258 as the carboxyl terminal. (G) a continuous amino acid sequence contained in the amino acid sequence shown in SEQ ID NO: 24 in the sequence listing, wherein amino acid number 27 is the amino terminal, and any one of amino acid numbers 122 to 129 is A polypeptide comprising an amino acid sequence having a carboxyl terminus; (h) a polypeptide comprising an amino acid sequence having amino acid number 26 of SEQ ID NO: 16 as an amino terminus and 406 as a carboxyl terminus; Amino acid No. 18 at the amino terminal and 491 at the carboxyl terminal A polypeptide comprising an amino acid sequence; (j) a polypeptide comprising an amino acid sequence having amino acid number 23 of SEQ ID NO: 20 as an amino terminal and 493 as a carboxyl terminal; and (k) an amino acid sequence of SEQ ID NO: 22 in the sequence table. A polypeptide comprising an amino acid sequence having amino acid No. 21 as the amino terminal and 470 as a carboxyl terminal, and (1) a polypeptide comprising an amino acid sequence having amino acid 27 as the amino terminal of SEQ ID NO: 24 and 346 as the carboxyl terminal in the sequence listing Any of the above (a) to (1) is included in the present invention as long as it has a nucleotide sequence capable of producing the polypeptide.
[0271]
A recombinant vector obtained by incorporating such a DNA having a nucleotide sequence encoding the polypeptide of the present invention into an appropriate vector DNA can transform a prokaryotic or eukaryotic host cell. it can. Furthermore, by introducing an appropriate promoter and a sequence involved in expression into these vectors, the introduced gene can be expressed in each host.
[0272]
Examples of prokaryotic cell hosts include Escherichia coli and Bacillus subtilis. To transform a gene of interest into these host cells, the host cells are transformed with a plasmid vector that contains replicons or origins of replication from species compatible with the host and regulatory sequences. Further, the vector is preferably a vector having a sequence capable of imparting phenotypic (phenotypic) selectivity to transformed cells.
[0273]
For example, K12 strain or JM109 strain is often used as Escherichia coli, and pBR322 or a pUC-based plasmid is generally used as a vector, but is not limited thereto, and any known various strains and vectors can be used. .
[0274]
When expressing proteins derived from other organisms in Escherichia coli, translation efficiency may be limited by differences in codon usage between species, which may result in a bias in the amount of tRNA in Escherichia coli cells. It is considered that the cause is that several types of tRNA are depleted. In such a case, expression using a strain that expresses a relatively large amount of a relatively small amount of tRNA in Escherichia coli may be successful. An example of such E. coli is BL21 {CodonPlus} (DE3) -RIL of E. coli. This strain is a strain in which the copy number of a relatively small amount of a tRNA gene (arginine, isoleucine, leucine) is increased in Escherichia coli.
[0275]
Examples of the promoter include, in Escherichia coli, tryptophan (trp) promoter, lactose (lac) promoter, tryptophan lactose (tac) promoter, lipoprotein (lpp) promoter, polypeptide chain elongation factor Tu (tufB) promoter, and the like. Any promoter can be used to produce the polypeptide of interest.
[0276]
As the Bacillus subtilis, for example, strain 207-25 is preferable, and as a vector, pTUB228 (Ohmura, K. et al. (1984) J. Biochem. is not. By ligating a DNA sequence encoding the signal peptide sequence of Bacillus subtilis α-amylase, extracellular secretory expression becomes possible.
[0277]
Eukaryotic host cells include cells such as vertebrates, insects, and yeast. Examples of vertebrate cells include monkey COS cells (Gluzman, {Y.} (1981) {Cell} 23, {175-). 182, ATCC @ CRL-1650) and Chinese hamster ovary cells (CHO cells, ATCC @ CCL-61) deficient in dihydrofolate reductase (Urlaub, G. and ａｓChasin, L.A. (1980) Proc. Natl. Acad. Sci. (USA # 77, # 4126-4220) and the like are often used, but are not limited thereto.
[0278]
As a vertebrate cell expression promoter, a promoter having an upstream site of a gene to be normally expressed, an RNA splice site, a polyadenylation site, a transcription termination sequence and the like can be used. May be provided. Examples of the expression vector include, but are not limited to, pSV2dhfr (Subramani, S. et al. (1981) Mol. Cell. Biol. 1, 854-864) having the SV40 early promoter.
[0279]
As an example, when a COS cell is used as a host cell, the expression vector has an SV40 origin of replication, is capable of autonomous growth in COS cells, and further has a transcription promoter, a transcription termination signal, and an RNA splice site. Can be used. The expression vector was prepared by the diethylaminoethyl (DEAE) -dextran method (Luthman, H. and Magnesson, G. (1983) Nucleic Acids Res, 11, 1295-1308), and calcium phosphate-DNA coprecipitation method (Graham, F. L.). and van der Eb, A. J. (1973) Virology 52, 456-457), and electric pulse perforation (Neummann, E. et al. (1982) EMBO J. 1, 1, 841-845). Thus, the desired transformed cells can be obtained. When CHO cells are used as host cells, a vector capable of expressing a neo gene functioning as an antibiotic G418 resistance marker together with an expression vector, for example, pRSVneo (Sambrook, {J. et al. (1989)}: {"Molecular Cloning"] A Laboratory Laboratory Manual, Cold Spring Harbor Laboratory, NY, pSV2-neo (Southern, PJ J. and Berg, P. (1982) J. Mol. Appl. By selecting a G418-resistant colony, a transformed cell stably producing the desired polypeptide can be obtained.
[0280]
When an insect cell is used as a host cell, a cell line derived from an ovary cell (Sf-9 or Sf-21) of Spodoptera frugiperda of Lepidoptera and Noctuidae, and a High Five cell derived from an egg cell of Trichoplusia ni (Wickham, {T.}. al, (1992) ｔｅBiotechnol. Prog. I: 391-396) and the like, and as a baculovirus transfer vector, pVL1392 using a polyhedrin protein promoter of autographer nucleopolyhedrovirus (AcNPV) as a baculovirus transfer vector. / 1393 (Kidd, I. M. and V. C. C. Emery (1993) The These of Baculoviruses as expression vectors. Applied Biochemistry and Biotechnology 42, 137-159). In addition to this, a vector using a promoter of baculovirus P10 or an isobasic protein can also be used. Furthermore, by linking the secretion signal sequence of the envelope surface protein GP67 of AcNPV to the amino terminus of the target protein, it is possible to express the recombinant protein as a secretory protein (Zhe-mei Wang, et al. (1998)). Biol. Chem., 379, 167-174).
[0281]
As an expression system using a eukaryotic microorganism as a host cell, yeast is generally well known. Among them, yeast of the genus Saccharomyces, for example, baker's yeast Saccharomyces cerevisiae and petroleum yeast Pichia pastoris are preferable. Examples of expression vectors for eukaryotic microorganisms such as the yeast include, for example, a promoter for an alcohol dehydrogenase gene (Bennetzen, J. L. and Hall, B. D. (1982) J. Biol. Chem. 257, 3018-3025) ) Or a promoter of an acid phosphatase gene (Miyanohara, A. et al. (1983) Proc. Natl. Acad. Sci. USA 80, 1-5). When expressed as a secretory protein, it can be expressed as a recombinant having a secretory signal sequence and a cleavage site of an endogenous protease or a known protease possessed by the host cell at the amino terminal side. For example, in a system in which human mast cell tryptase of a trypsin-type serine protease is expressed in petroleum yeast, the activity is achieved by connecting the secretion signal sequence of α-factor of yeast and the cleavage site of KEX2 protease possessed by petroleum yeast to the amino terminus side. It is known that type tryptase is secreted into the medium (Andrew, L. Niles, et al. (1998) ｔｅBiotechnol. Appl. Biochem.） 28, 125-131).
[0282]
The transformant obtained as described above can be cultured according to a conventional method, and the culture produces the target polypeptide inside or outside the cell. As the medium used for the culture, various types commonly used depending on the host cell used can be appropriately selected. For example, in the case of the above-mentioned COS cells, RPMI1640 medium or Dulbecco's modified Eagle medium (hereinafter referred to as “DMEM”) A medium obtained by adding a serum component such as fetal bovine serum to a medium such as the above can be used as necessary.
[0283]
Recombinant protein produced in or outside the cells of the transformant by the above culture can be separated and purified by various known separation procedures utilizing the physical and chemical properties of the protein. it can. As the method, specifically, for example, treatment with a normal protein precipitant, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high-performance liquid chromatography ( Examples thereof include various liquid chromatography such as HPLC, dialysis, and combinations thereof. In addition, by connecting histidine consisting of 6 residues to the recombinant protein to be expressed, the protein can be efficiently purified by a nickel affinity column. By combining the above methods, the polypeptide of the present invention can be easily produced in large quantities with high yield and high purity.
[0284]
The polypeptide of the present invention can be produced by animal cells using an adenovirus vector into which a DNA having a nucleotide sequence encoding the polypeptide has been incorporated, in addition to being obtained according to the above method. Examples of a method for constructing such a recombinant adenovirus vector include a method using a commercially available kit (for example, adenovirus expression vector kit, manufactured by Takara Shuzo Co., Ltd.). The close correlation of the polypeptide of the present invention with the blood triglyceride concentration of a mammal indicates that a recombinant adenovirus having a recombinant adenovirus vector obtained as described above can be used in a mammal, such as a mouse. Infecting E. coli and expressing the gene carried by the recombinant adenovirus vector is evidenced by the observed increase in blood triglyceride (triglyceride) concentration.
[0285]
When the “angiopoietin-related protein” is administered to mouse blood, a peptide in which the carboxyl terminal peptide has been removed is generated. For example, when “angiopoietin-related protein 3” is administered to the blood, two types of polypeptides, cleaved between amino acids 221 and 222 of amino acid SEQ ID NO: 52 and peptides cleaved between amino acids 224 and 225 Can be obtained. That is, to increase the blood lipid concentration, `` angiopoietin-related protein '' may be administered, or a mutant polypeptide modified to include only the helical region of `` angiopoietin-related protein '' may be administered. .
[0286]
The polypeptide of the present invention obtained by the above method may be used in a method for testing an agent for treating or preventing one or more diseases selected from, for example, hyperlipidemia, arteriosclerosis, and hyperglycemia as described below. Can be used.
[0287]
First, the non-human animal used in this method may be, for example, a KK / mutant having a mutant gene that does not normally produce “angiopoietin-related protein” (eg, “angiopoietin-related protein 3” and / or “angiopoietin-related protein 4”).SnkPreferred are, but not limited to, mice and knockout mice that have been genetically engineered to not express the “angiopoietin-related protein” (eg, “angiopoietin-related protein 3” and / or “angiopoietin-related protein 4”) gene.
[0288]
As the polypeptide to be administered,[1]KK / which has a mutant gene that does not normally produce “angiopoietin-related protein 3”SnkFor a mouse, for example, one continuous amino acid sequence in the amino acid sequence shown in SEQ ID NO: 12 in the sequence listing, amino acid number 17 is an amino terminal, and any one of amino acid numbers 147 to 207 is Using a polypeptide having an amino acid sequence to be a carboxyl terminus,[2]KK / with a mutated gene that does not normally produce “angiopoietin-related protein 4”SnkFor a mouse, for example, a continuous amino acid sequence in the amino acid sequence shown in SEQ ID NO: 16 in the sequence listing, wherein amino acid number 26 is the amino terminal, and one of amino acid numbers 143 to 183 is carboxyl. A polypeptide having an amino acid sequence at the end can be used, but is not limited thereto as long as an increase in blood neutral fat concentration can be confirmed. The polypeptide of the present invention may be used alone or in combination of two or more.
[0289]
Examples of a method for administering the polypeptide of the present invention to the above animals include administration to blood vessels, oral administration, and the like. However, the administration method is not limited as long as an increase in blood neutral fat concentration can be confirmed. The dose of the polypeptide of the present invention to the above animal is not limited as long as an increase in blood fat concentration can be confirmed. For example, when administered to the tail vein of a mouse, the dose is 0.01 μg to 1 μg per mouse body weight. It can be administered in the range of 10 mg.
[0290]
When the polypeptide of the present invention is administered to the above animals, an increase in blood neutral fat (triglyceride) concentration is observed.
[0291]
In this experimental system, the polypeptide of the present invention and a test substance are administered to a test animal, and a substance that reduces the concentration of neutral fat in blood is selected. More preferably,[1]An animal to which only the polypeptide of the present invention has been administered,[2]Animals to which only the test substance has been administered, and[3]Compare the results between animals that received both,[2]Does not affect blood triglyceride levels in animals of[3]In animals[1]Are selected as substances that specifically suppress the function of the polypeptide of the present invention. In this method, the function of the polypeptide of the present invention itself or a factor (for example, a receptor) that interacts with the polypeptide and contributes to the expression of the function of the polypeptide, such as neutral lipids in blood due to the action of the polypeptide, Substances that inhibit any of the biological reactions associated with increasing concentrations can be found. Such a substance can be a therapeutic or prophylactic agent for one or more diseases selected from hyperlipidemia, arteriosclerosis and hyperglycemia.
[0292]
On the other hand, since the polypeptide of the present invention has an effect of inhibiting the activity of LPL and / or HTGL in vitro, it can be used in a test method for a substance that regulates LPL and / or HTGL activity as described below. it can.
[0293]
This test method is a method for measuring the activity of LPL and / or HTGL in the LPL and / or HTGL reaction and the substance of the present invention to examine the effect of regulating the activity of LPL and / or HTGL. (Referred to as “test substance”). More specifically, described below[1]-A through[3]Or[1]-B through[3]In a system for examining LPL activity in the presence of the above materials, a change in LPL activity when a test substance is further added is examined.
[0294]
[1]-Material containing LPL
As a material containing LPL, a culture supernatant of a cell line derived from a mammalian adipocyte or a precursor cell of an adipocyte that produces LPL can be used. If the cell line is undifferentiated, it is preferable to prepare the material after differentiation into adipocytes. Examples of cell lines producing LPL include mouse cell line 3T3-L1 (purchasable from Dainippon Pharmaceutical Co., Ltd.), 3T3-F442A, and Ob17. Alternatively, cells obtained by differentiating rat white preadipocytes (primary cultured cells) into adipocytes can also be suitably used, but the present invention is not limited thereto.
[0295]
Further, as a material containing LPL, purified LPL or recombinant LPL (see Methods {Enzymol.} (1996) # 263, # 319-26) can also be used. For example, LPL from milk (commercially available from Sigma) can be used in the method of the present invention.
[0296]
[1]Materials containing -b @ LPL and HTGL
As the material containing LPL and HTGL, plasma derived from peripheral blood collected from a mammal 10 to 15 minutes after intravenous injection of 10 to 100 units of heparin per kg of body weight is preferable.
[0297]
[2]Or[2]Material comprising a substrate of LPL and / or HTGL
Next, the material containing the substrate of LPL and / or HTGL contains a substance known to release fatty acids by the catalysis of these lipases, and contains a component that inhibits or inactivates the activity of these lipases. It does not matter if it is not glycerol tri [9,10 (n)-³Particularly preferred are compounds in which the fatty acid released by the LPL catalyst, such as [H] oleic acid, is labeled with a radioisotope or similarly labeled with a substance that emits fluorescence at a specific excitation wavelength. When the amount of free fatty acid is measured by a biochemical method, an unlabeled substrate (for example, glycerol trioleic acid) may be used.
[0298]
[3]The polypeptide of the present invention
As the polypeptide of the present invention, a polypeptide consisting of the amino acid sequence represented by amino acid Nos. 17 to 460 of SEQ ID NO: 4 in the sequence listing, a polypeptide lacking 1 to 253 residues at the carboxyl terminal thereof, or a polypeptide of claims 18 to 34 The described polypeptides can be used, but are not limited thereto.
[0299]
The test substance in the method of the present invention is not particularly limited, and extracts from cultures of microorganisms and tissues of animals and plants, extracts from cultures of cultured cells, artificially synthesized inorganic or organic compounds, and recombinants Any of a protein, an antibody or a fragment thereof, a composition in which a plurality of types of substances are mixed, and the like can be used.
[0300]
In addition, a buffer solution for pH adjustment or a protein component such as albumin may be added to the reaction solution as needed.
[0301]
These materials are mixed and subjected to an LPL reaction under the conditions described below:
Temperature conditions: $ 27-37 ° C, preferably 37 ° C;
PH of the reaction solution: $ 8.0 to 8.5, preferably 8.0;
Sodium chloride concentration in the reaction solution: 0.1 to 0.15M, preferably 0.15M;
Reaction time: 15 to 120 minutes, preferably 120 minutes.
[0302]
When a material containing both LPL and HTGL is used, such as plasma from a mammal to which heparin has been administered, the lipase activity of LPL and HTGL can be measured under the above conditions. When only HTGL activity is measured, the lipase reaction is allowed to proceed under the condition where LPL is inactivated by changing the concentration of sodium chloride to 1 M without changing other conditions. The LPL activity is a value obtained by subtracting the HTGL activity from the total lipase activity.
[0303]
After completion of the reaction, an organic solvent is added to the reaction solution and mixed, and then the layer containing free fatty acids and the layer containing unreacted substrate are separated, and the amount of free fatty acids collected in the former is measured.
[0304]
An example of a test method using an unlabeled substrate is shown below (Clin. Chem. # 30, 748 (1984)).
[0305]
0.5 ml of a substrate solution (7.5 μmol / glycerol / trioleic acid, 22.5 mg / gum arabic, 100 μmol / tris-hydrochloric acid (pH 8.2), 50 μmol / sodium chloride, 25 mg / albumin, 140 μl / human plasma) was incubated at 37 ° C. for 80 minutes. Thereafter, the adipocyte culture supernatant is added, and simultaneously the polypeptide of the present invention and a test substance are added, and the mixture is reacted at 28 ° C. for 60 minutes. Next, 2.5 ml of isopropanol: heptane: sulfuric acid (2.5 mol / L) = 40: 10: 2 (v / v) was added to the reaction solution, mixed for 10 minutes, and left at room temperature for 40 minutes. Collect 1 ml and dry with nitrogen. The obtained product is dissolved in a 5% {Triton} X-100 solution, and the amount of fatty acid is measured using a commercially available fatty acid measurement kit (for example, NEFA-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.)).
[0306]
As a result of the test conducted by the above method, the test substance which inhibits the LPL and / or HTGL activity inhibition by the polypeptide of the present invention and consequently increases the LPL and / or HTGL activity is a neutral lipid in blood. It is selected as a candidate for a therapeutic or prophylactic agent for one or more diseases selected from new hyperlipidemia, arteriosclerosis and hyperglycemia, which decrease the concentration.
[0307]
III. Test method using promoter DNA
The DNA of the present invention can be obtained, for example, from the nucleotide library of mouse “angiopoietin-related protein 3” cDNA (Conklin, {D. et al. Genomics 62, 477-482 (1999)) or mouse “ Angiopoietin-related protein 4 "nucleotide sequence of cDNA (Kersten, S., et al., (2000) J. Biol. Chem. 275, 28488-28493; Yoon, J. C., et al. (2000) Mol. Cell. Biol. ..20, 5343-5349) as a probe. Alternatively, the DNA of the present invention can be directly amplified by the polymerase chain reaction (hereinafter, referred to as “PCR”) using mouse genomic DNA as a template without passing through a library screening step based on this sequence information. .
[0308]
The recombinant plasmid pGL8-3 into which the DNA of the present invention has been inserted was internationally deposited with the National Institute of Advanced Industrial Science and Technology and the Patent Organism Depositary on June 7, 2001, under the accession number. FERM @ BP-7627 is attached. Therefore, the DNA of the present invention can also be obtained from the plasmid.
[0309]
Anyone having ordinary knowledge in the technical field to which the present invention pertains may substitute a part of the nucleotide sequence of the natural promoter DNA with another nucleotide, or delete or add a nucleotide, to modify the natural sequence. It is possible to prepare a DNA having a promoter activity equivalent to that of the type of promoter DNA. Thus, a DNA having a nucleotide sequence in which a nucleotide is substituted, deleted or added in a natural nucleotide sequence and having a promoter activity equivalent to that of the natural promoter DNA is also included in the DNA of the present invention. Modification of the nucleotide sequence includes, for example, introduction of a deletion using a restriction enzyme or a DNA exonuclease, introduction of a mutation by a site-directed mutagenesis method, modification of a promoter sequence by a PCR method using a mutation primer, direct introduction of a synthetic mutant DNA, etc. It can be done by a method. Preferred among these are DNAs comprising the nucleotide sequence represented by nucleotide numbers 1 to 510 of SEQ ID NO: 27 in the Sequence Listing or the nucleotide sequences represented by nucleotide numbers 1 to 260 of SEQ ID NO: 2 in the Sequence Listing, Preferably, it is a DNA comprising the nucleotide sequence represented by nucleotide numbers 1 to 526 of SEQ ID NO: 27 in the sequence listing or the nucleotide sequence represented by nucleotide numbers 1 to 188 of SEQ ID NO: 28 in the sequence listing.
[0310]
Further, the DNA of the present invention is a stringent DNA with the nucleotide sequence represented by nucleotide numbers 1 to 526 of SEQ ID NO: 27 or the nucleotide sequence represented by nucleotide numbers 1 to 260 of SEQ ID NO: 28 in the sequence listing. Those containing DNA that can hybridize under the conditions are mentioned. The DNA capable of hybridizing in this manner may be any DNA having promoter activity. Such a DNA is usually 70% or more, preferably 80% or more, of the nucleotide sequence represented by nucleotide numbers 1 to 526 of SEQ ID NO: 27 or the nucleotide sequence represented by nucleotide numbers 1 to 260 of SEQ ID NO: 28 in the sequence listing. As described above, more preferably 95% or more nucleotide sequence identity. Such DNAs include mutant genes found in nature, artificially modified mutant genes, homologous genes derived from heterologous organisms, and the like.
[0311]
In the present invention, hybridization under stringent conditions is performed under ordinary stringent conditions (roast stringent conditions) by using 5 × SSC (0.75 M sodium chloride, 0.075 M sodium citrate). ) Or in a hybridization solution having a salt concentration equivalent to this, for about 12 hours under a temperature condition of 37-42 ° C, and pre-washing as necessary with a solution such as 5xSSC or a solution having a salt concentration equivalent thereto. After that, washing can be carried out in 1 × SSC or a solution having a salt concentration equivalent thereto. Further, under conditions having higher stringency (high stringency conditions), the washing can be carried out in the above manner by performing the washing in a solution having a salt concentration of 0.1 × SSC or an equivalent thereof.
[0312]
Whether or not the thus obtained DNA of the present invention has a promoter activity is determined by ligating a marker gene such as luciferase downstream thereof (hereinafter referred to as “reporter gene”) as described below. It can be confirmed by transforming an animal cell and examining whether or not the expression of the marker gene in the transformed cell is detected.
[0313]
The expression vector used in the present invention has the DNA of the present invention linked to the upstream side of the foreign gene in the same transcriptional direction as the sequence of the foreign gene, and is capable of expressing the foreign gene in mammalian cells. In such an expression vector, since the DNA of the present invention is linked upstream of the foreign gene, the foreign gene can be expressed under the control of the promoter in the DNA of the present invention. The expression vector of the present invention may be in any form that can be replicated by introduction into a mammalian cell, and may be, for example, either circular or linear having an appropriate replicon.
[0314]
Examples of the method of gene transfer using a viral vector include a method of incorporating cDNA into a retrovirus, an adenovirus, an adeno-associated virus, a herpes virus, a vaccinia virus, a pox virus, a polio virus, or another DNA virus, or an RNA virus to introduce cDNA. . Among them, a method using a retrovirus, an adenovirus, an adeno-associated virus, and a vaccinia virus is preferable.
[0315]
Examples of non-viral gene transfer methods include a method of directly administering an expression plasmid intramuscularly (DNA vaccine method), a liposome method, a lipofection method, a microinjection method, a calcium phosphate method, an electroporation method, and the like. The DNA vaccine method and the liposome method are preferred.
[0316]
The expression vector is prepared by the diethylaminoethyl (DEAE) -dextran method (Luthman, H. and Magnusson, G. (1983) Nucleic Acids Res, 11, 1295-1308), and the calcium phosphate-DNA coprecipitation method (Graham, F. L.). and van der Eb, A. J. (1973) Virology 52, 456-457), and electric pulse perforation (Neummann, E. et al. (1982) EMBO J. 1, 841-845) and lipofection (Lopata). et al. (1984) Nucl. Acids Res. 12, 5707-5717, Sussman and Milman (198). ) Mol. Cell. Biol. 4, 1641-1643) can be incorporated into COS cells by such, thus it is possible to obtain the desired transformed cells. However, when the cultured cell line is a so-called floating cell, it is preferable to use a method other than the calcium phosphate-DNA coprecipitation method. In each case, optimized transfection conditions are used according to the cells used.
[0317]
Alternatively, a transgenic animal obtained by obtaining a fertilized egg from an animal, transferring the gene, transplanting the animal into a pseudopregnant animal, and developing the animal can be used, and the procedure is a known method [Developmental Engineering Experiment Manual (Nomura Tatsuji {supervision, edited by Katsuki Motoya}, 1987), JP-A-5-48093, etc.]. Specifically, for example, in the case of a mouse, first, an ovulation-inducing agent is administered to a female mouse, then mated with a male of the same strain, and the pronuclear fertilized egg is collected from the oviduct of the female mouse the next day. Next, the DNA fragment solution to be introduced is injected into the pronucleus of a fertilized egg using a micro glass tube. The regulatory gene such as an enhancer is not particularly limited as long as it has the DNA of the present invention as a promoter for expressing the gene to be introduced in animal cells and functions as an enhancer or the like in the cell of the introduced animal. The fertilized egg into which the DNA has been injected is transplanted into a fallopian tube of a pseudopregnant foster female mouse (Slc: ICR, etc.), and is born by spontaneous delivery or cesarean section 20 days later.
[0318]
The method for confirming that the thus obtained animal carries the transgene includes, for example: extracting DNA from the animal's tail or the like and using the DNA with specific sense and antisense primers. After digesting the DNA with a restriction enzyme, gel electrophoresis, blotting the DNA in the gel onto a nylon membrane or the like, and performing Southern blot analysis using all or a part of the labeled transgene as a probe Methods and the like can be mentioned.
[0319]
Examples of the foreign gene include a gene encoding a protein for treating a target disease, a marker gene, and the like.
[0320]
Hereinafter, a method for using the DNA of the present invention will be described.
[0321]
1) Use as an inducible expression vector
When the DNA of the present invention is found to exhibit promoter activity by a specific stimulus, a vector or the like in which the DNA of the present invention is inserted upstream of a desired gene is prepared and introduced into somatic cells. By adding, the desired gene can be inducibly expressed.
[0322]
2) Regulation of promoter activity of DNA using competitive inhibition by DNA
The present invention relates to a DNA containing at least a part of the sequence of the above promoter DNA (including a derivative thereof; hereinafter, referred to as “partial sequence”). A method for competitively inhibiting the binding between the above promoter DNA and a protein capable of binding to the promoter DNA (for example, a transcription factor) irrespective of whether or not the partial sequence itself has promoter activity, by using such a partial sequence. Can be implemented. For example, when the partial sequence corresponds to a binding site of a protein that inhibits the promoter activity on the DNA sequence of the present invention, the promoter activity can be promoted by this method, and conversely, the promoter activity can be promoted. When it corresponds to the binding site of a protein (including a transcription factor), the promoter activity can be inhibited. The partial sequence DNA used for such competitive inhibition usually has a chain length of at least 6 nucleotides, preferably at least 10 nucleotides. Examples of the type of partial sequence selected for use in competitive inhibition include, for example, a sequence containing a binding consensus site of a transcription factor on a promoter. Derivatives of such partial sequence DNA include those obtained by linking several partial sequences, those obtained by incorporating them into an adenovirus vector or the like to enable gene transfer into animal cells, and the 3′-terminal of DNA. Alternatively, one in which biotin is linked to the 5 ′ end can be used.
[0323]
As is clear from the examples below, when "angiopoietin-related protein 3" and "angiopoietin-related protein 4" are forcibly expressed in hypolipidemic mice, the concentration of neutral fat in the blood increases. Due to the effect of the mutant gene that does not normally produce “angiopoietin-related protein 3”, a marked decrease in blood lipid concentration, suppression of progression of arteriosclerosis and improvement of hyperglycemia were observed, and the same applies to “angiopoietin-related protein 4”. Since a phenomenon is considered to be observed, the partial sequence having a function of suppressing the promoter activity of the DNA of the present invention or a derivative thereof is one or more selected from hyperlipidemia, arteriosclerosis, and hyperglycemia. Useful as an active ingredient of a pharmaceutical composition for treating or preventing the above diseases.
[0324]
3) Screening for proteins that regulate promoter activity
In order to regulate the promoter activity of the DNA of the present invention, in addition to the above-described competitive inhibition using the partial sequence or the like, a method using a protein that regulates the promoter activity of the DNA of the present invention can also be used. The DNA of the present invention can be used for screening a protein that regulates its promoter activity. Therefore, the present invention relates to a method for screening a protein that regulates the promoter activity of the DNA as described below. Such proteins include proteins directly binding to the DNA of the present invention to promote or inhibit its promoter activity, and indirectly acting on cell membrane receptors or intracellular proteins to promote the promoter activity of the DNA of the present invention. And proteins that promote or inhibit the activity.
[0325]
3-1) Screening for protein binding to promoter DNA
This method includes a step of bringing a test protein sample into contact with the DNA of the present invention and selecting a protein that binds to the DNA of the present invention. Such a method includes, for example, affinity purification of a protein binding thereto using the promoter DNA of the present invention. As an example of a specific method, the promoter DNA of the present invention is biotinylated and bound to streptavidin-bound magnetic beads to prepare DNA affinity beads. This is then incubated with the nuclear extract of the cells to purify the proteins in the nuclear extract that specifically bind to the DNA of the invention, and to determine its structure. As a result, a protein that directly binds to the DNA of the present invention and a protein that does not have DNA binding activity but forms a complex with the protein as a subunit and binds to the DNA of the present invention can be purified (Gabrielsen O. S et al. , {Nucleic acid Research 17, 17, 6253-6267 (1989), Savoysky E et al., Oncogene 9, 1839-1846 (1994)).
[0326]
3-2) Screening of protein using promoter activity as an index
In this method, a test DNA is introduced into cells that carry a marker gene [described in detail in 5-2] below linked to the downstream of the DNA of the present invention, and an expression product that regulates the expression of the marker gene is selected. Process. Such a method includes, for example, a one-hybrid method using yeast or animal cells. Specifically, a reporter gene into which the DNA of the present invention and a marker gene have been inserted is stably introduced into a cell, and then a gene library is introduced thereinto to obtain a clone exhibiting promotion or inhibition of expression of the reporter gene. Select and select a protein that binds to the DNA of the invention. According to this method, in addition to the protein directly binding to the DNA of the present invention, a protein that acts on an endogenous protein in a cell and indirectly regulates the promoter activity of the DNA of the present invention can be obtained. In addition, the one-hybrid method of yeast (Li JJ and Herskowitz I., Science 262, 1870-1873) (1993), Wang MM and Reed RR, Nature 364, 121-126 (1993) ), And “Matchmaker @ system (Clontech)” and the like are sold as kits.
[0327]
Still another embodiment includes a step of contacting a test sample with cells holding a marker gene linked downstream of the DNA of the present invention, and selecting a protein that regulates the expression of the marker gene. As an example of a specific method, a cell into which a reporter gene having the DNA of the present invention and a marker gene inserted therein is stably introduced is incubated with a test sample (for example, a culture supernatant of a cell into which a gene library has been introduced). Then, a protein that promotes or inhibits the expression of the marker gene is selected. In this method, a protein that indirectly affects the promoter activity of the DNA of the present invention via a cell membrane receptor or the like is obtained.
[0328]
4) Method for screening cDNA encoding a protein that regulates promoter activity
Using the DNA of the present invention, it is also possible to directly isolate a DNA encoding a protein that regulates its promoter activity. The present invention also relates to a method for screening a cDNA encoding a protein that regulates the promoter activity of the DNA of the present invention. One embodiment of this screening method includes a step of contacting an expression product of a test DNA with the DNA of the present invention and selecting a cDNA encoding a protein that binds to the DNA of the present invention. Such methods include, for example, the Southwestern method. Specifically, each protein is expressed in Escherichia coli into which a gene library has been introduced, and these proteins are transferred to a filter membrane, and then directly blotted using the DNA of the present invention as a probe, and clones expressing proteins that bind to the DNA probe are used. To isolate the gene encoding it. According to this method, a gene encoding a protein having an activity of binding to the DNA of the present invention can be obtained (see Experimental Medicine Separate Volume, Bio Manual Series, “Transcription Factor Research Methods”, published by Yodosha, pages 177-188). .
[0329]
Another embodiment includes a step of introducing a test cDNA into cells having a marker gene linked downstream of the DNA of the present invention, and selecting a cDNA encoding an expression product that regulates the expression of the marker gene. Such methods include, for example, the above-mentioned one-hybrid method using yeast or animal cells.
[0330]
That is, the DNA of the present invention and a reporter gene into which a marker gene has been inserted are stably introduced into cultured cells derived from mammals (hereinafter referred to as "cell A"), and further, the cells are tested for expression vectors for mammalian cells. Cells into which the recombinant plasmid into which the cDNA has been inserted (hereinafter referred to as "cell B") are cultured. As a control, cells A were cultured as they were, or an expression vector for mammalian cells containing no test cDNA in cells A, or a cell into which a recombinant plasmid containing test cDNA but not expressing it was introduced ( (Hereinafter referred to as “cell C”). The expression level of the marker gene is compared between cell B and cell A or cell C, and a cDNA clone that promotes or inhibits the expression of the marker gene is selected. In this method, not only cDNA encoding a protein that directly binds to the DNA of the present invention, but also cDNA encoding a protein that acts on an endogenous protein in a cell and indirectly regulates the promoter activity of the DNA of the present invention. Obtainable.
[0331]
In still another embodiment, the expression product of a test cDNA is brought into contact with a cell retaining a marker gene linked downstream of the DNA of the present invention, and a cDNA encoding an expression product that regulates the expression of the marker gene is selected. Process. An example of a specific method is as follows. A cell into which a reporter gene into which a DNA and a marker gene of the present invention have been inserted is stably introduced, and an expression product of a test cDNA (for example, a culture supernatant of a cell into which a cDNA library has been introduced). ) Is isolated to isolate a protein or cDNA encoding the protein that promotes or inhibits the expression of the marker gene. According to this method, a cDNA encoding a protein that acts on the promoter activity of the DNA of the present invention indirectly via a cell membrane receptor or the like can be obtained.
[0332]
5) Screening for compounds that regulate promoter activity
Using the DNA of the present invention, a compound that regulates its promoter activity can be screened. That is, the present invention relates to a method for screening a compound that regulates the promoter activity of the DNA as described below.
[0333]
5-1) Screening using binding between promoter and protein as an index
This method includes a step of contacting a DNA of the present invention with a test sample in the presence of a test compound, and selecting a compound that promotes or inhibits the binding between the DNA of the present invention and a protein in the test sample. Specifically, for example, a nuclear extract of a cell is bound to a probe obtained by labeling the DNA of the present invention with a radioisotope or the like, and the protein in the nuclear extract and the DNA of the present invention are subjected to polyacrylamide gel electrophoresis. The band of the complex is detected by the gel shift method (see Experimental Medical Supplement, Bio Manual Series, “Transcription Factor Research Method”, Yodosha Co., Ltd., pp. 107-112). When a DNA probe is added, a test compound is also added, and a compound that promotes or inhibits the formation of a band of a complex between the protein in the nuclear extract and the DNA of the present invention is selected. In this method, a compound that directly acts on the DNA of the present invention and a compound that acts on a protein that binds to the DNA of the present invention are obtained. For example, when a protein that binds to the DNA of the present invention inhibits the promoter activity of the DNA of the present invention in vivo, a compound that inhibits the binding of the protein to the DNA of the present invention is a DNA of the present invention. Is considered to be able to promote the promoter activity. If a protein that binds to the DNA of the present invention has already been isolated, a recombinant protein of the protein can be used instead of the cell nuclear extract.
[0334]
5-2) Screening using promoter activity as an index
This method includes a step of bringing a test compound into contact with cells retaining a marker gene linked to the 3 'end of the DNA of the present invention, and selecting a compound that regulates the expression of the marker gene. In this method, a compound that directly or indirectly regulates the promoter activity of the DNA of the present invention is obtained.
[0335]
In this method, the marker protein encoded by the marker gene linked to the 3 ′ end of the DNA of the present invention is different from any other protein that the host cell can produce in a series of steps of the method of the present invention. Any cell that can be distinguished (preferably, such that the cell before transformation does not have a gene encoding the same or similar protein as the marker protein) may be used. For example, even if the marker protein is toxic to the cell or if the cell confers resistance to sensitive antibiotics, the presence or absence of the marker gene will determine the viability of the cell. Can be determined. However, a more preferable marker gene used in the present invention can specifically and quantitatively detect the expression level (for example, when a specific antibody against the protein encoded by the marker gene has been obtained). ) It is a structural gene. More preferably, it is a gene encoding an enzyme or the like which specifically reacts with an exogenous substrate to generate a metabolite which can be easily measured quantitatively. Such may include, for example, the genes encoding the proteins listed below, but the invention is not limited thereto:
Chloramphenicol acetyltransferase: Adds an acetyl group to chloramphenicol. Detectable by so-called CAT assay. A pCAT3-Basic vector (promega) is commercially available as a vector that can be used to prepare a vector for a reporter assay simply by incorporating a promoter;
Firefly luciferase: quantified by measuring bioluminescence generated when metabolizing persiferin. Similarly, a pGL3-Basic vector (promega) is commercially available as a vector for a reporter assay;
β-galactosidase: が あ る There are substrates that can be measured by color reaction, fluorescence or chemiluminescence, respectively. Pβgal-Basic (Promega) is commercially available as a vector for reporter assays;
Secreted alkaline phosphatase: (1) There are substrates that can be measured by color reaction, bioluminescence or chemiluminescence, respectively. PSEAP2-Basic (Clontech) is commercially available as a vector for reporter assays;
Green-fluorescent protein: Although not an enzyme, it can quantify directly because it emits fluorescence. Similarly, pEGFP-1 (manufactured by Clontech) is commercially available as a vector for a reporter assay.
[0336]
Examples of a method for introducing an expression plasmid into a cultured cell line include a DEAE-dextran method (Luthman, H. and Magnesson, G. (1983) Nucleic Acids） Res. 11, 1295-1308), a calcium phosphate-DNA coprecipitation method (Graham, F. L. and van der Eb, AJ (1973) Virology 52, 456-457), electric pulse perforation (Neummann, E. et al. (1982) EMBO J. 1, 841-845), lipofection. (Lopata et al. (1984) Nucl. Acids Res. 12, 12, 5707-5717, Sussman and Milman (1984)). Mol. Cell. Biol.4, can be exemplified 1641-1643) or the like, not limited to these, other methods commonly used in the art to which this invention belongs may also be employed. However, when the cultured cell line is a so-called floating cell, it is preferable to use a method other than the calcium phosphate-DNA coprecipitation method. In each case, optimized transfection conditions are used according to the cells used.
[0337]
By culturing cells transfected with an expression vector having a marker gene linked to the 3 'end of the DNA of the present invention, transcription of the marker gene is promoted. In culturing under conditions that allow the expression of the marker gene, after culturing under conditions where any test substance is added or not in the medium, the expression level of the marker gene is measured, and by adding the test substance, It is tested whether or not a change occurs in the expression level of the marker gene. The "conditions for expressing the marker gene" may be any conditions under which the cells can survive and produce the protein. Preferably, the medium is a medium (serum such as fetal bovine serum) suitable for the cell line used. Components may be added) at 36-38 ° C. (most preferably 37 ° C.) for 2-3 days in the presence of air containing 4-6% (most preferably 5%) carbon dioxide. Incubate (most preferably for 2 days).
[0338]
In such a system, a test substance that suppresses the induction of reporter gene expression upon introduction of an expression vector having a marker gene linked to the 3 ′ end of the DNA of the present invention is hyperlipidemia, arteriosclerosis, etc. And a candidate substance useful as an agent for treating or preventing one or more diseases selected from hyperglycemia.
[0339]
If a protein that regulates the promoter activity of the DNA of the present invention has already been obtained, the protein (or a derivative thereof) is brought into contact with the DNA of the present invention in the presence of a test compound, and the protein (or Compounds that promote or inhibit the binding of the derivative of the present invention to the DNA of the present invention can be selected to screen for compounds that regulate the promoter activity of the DNA of the present invention. Specifically, for example, a protein that binds to the DNA of the present invention fused with glutathione S-transferase (or only the DNA binding domain) may be purified and bound to a microplate covered with an anti-glutathione S-transferase antibody. Thereafter, the DNA of the present invention, which has been biotinylated, is brought into contact with this protein, and the binding between the protein and the DNA of the present invention is detected with streptavidinated alkaline phosphatase. When the DNA of the present invention is added, a test compound is also added, and a compound that promotes or inhibits the binding between the protein and the DNA of the present invention is selected. In this way, compounds that act directly on the DNA of the present invention and compounds that act on proteins that bind to the present invention are obtained. For example, when a protein that binds to the DNA of the present invention inhibits the promoter activity of the DNA of the present invention in vivo, a compound that inhibits the binding of the protein to the DNA of the present invention is used as a compound that inhibits the promoter activity of the DNA of the present invention. It is thought that it can promote.
[0340]
As is clear from the following Reference Examples, when "angiopoietin-related protein 3" and "angiopoietin-related protein 4" are forcibly expressed in hypolipidemic mice, the concentration of neutral fat in the blood increases. Due to the effect of the mutant gene that does not normally produce “angiopoietin-related protein 3”, a marked decrease in blood lipid concentration, suppression of progression of arteriosclerosis and improvement of hyperglycemia were observed, and the same applies to “angiopoietin-related protein 4”. Since a phenomenon is considered to be observed, the partial sequence having a function of suppressing the promoter activity of the DNA of the present invention or a derivative thereof is one or more selected from hyperlipidemia, arteriosclerosis, and hyperglycemia. Can be a therapeutic or prophylactic agent for the disease of
[0341]
EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the present invention is limited thereto. In addition, in the following examples, unless otherwise specified, each operation relating to gene manipulation is referred to as "Molecular Cloning" [Sambrook, @J. Fritsch, {E. F. And Maniatis, T. (Published by Cold Spring Laboratories Press, 1989), or in the case of using a commercially available reagent or kit, in accordance with the instructions of the commercially available product.
[0342]
Reference Example 1. Cloning of cDNA
Using the mouse liver as a material, a cDNA having the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing was obtained according to the following method.
[0343]
a) Extraction of mRNA from mouse liver
Two 9-week-old KK mice (male, obtained from an animal experimental facility attached to Hamamatsu Medical University) were dissected and their livers were excised, immediately placed in liquid nitrogen, and rapidly frozen. The weight was measured, and 3.1 g was ground in a mortar in the presence of liquid nitrogen. 5.5 M guanidine thiocyanate (hereinafter referred to as GT) buffer (5.5 M guanidine thiocyanate, 25 mM sodium citrate (pH 7.0), 0.5% sarkosyl, 0.2 M β-mercaptoethanol) (30 ml) was added, and the mixture was pestled. The mixture was crushed, a new GT buffer (10 ml) was added, the mixture was crushed with a pestle, and the lysate was recovered. The mortar was washed with a GT buffer (20 ml), and this solution was also collected. After 36 ml of the collected solution was centrifuged at 3000 rpm and 10 ° C. for 10 minutes, the supernatant was transferred to a new tube, and suction and discharge were repeated 20 times with an 18-gauge injection needle. Next, total RNA was separated by density gradient centrifugation using cesium trifluoroacetic acid (CsTFA). The CsTFA stock solution (19 ml) was diluted with ribonuclease-free double-distilled water (18.924 ml), and the diluted solution (6.18 ml) was placed in six 13PA (Beckman) tubes, and the previously collected sample (1 tube) Per ml). This was centrifuged at 30,000 rpm (approximately 125000 g) at 20 ° C. for 20.5 hours using a swing rotor (Hitachi Koki Co., Ltd., P40ST) at 20 ° C., and the supernatant was removed. The obtained pellet was suspended in 3.3 mL of extraction buffer attached to an mRNA purification kit (Quick-prep mRNA purification kit manufactured by Amersham Pharmacia). The mRNA was purified by using the purification kit according to the attached protocol. Using 5 μg of the thus obtained mRNA as a template and a cDNA library preparation kit (ZAP Express cDNA Gigapack III Gold Cloning Kit manufactured by Stratagene) according to the attached protocol, a λ phage cDNA library was prepared. Was prepared.
[0344]
b) Primary screening of cDNA library
Escherichia coli infected with the λ phage cDNA library obtained by the method described in a) above was plated on an agar medium plate (NZY medium: 0.5% sodium chloride, 0.2% magnesium sulfate) prepared in a culture dish having a diameter of 9 cm. Heptahydrate, 0.5% yeast extract, 1% {casein hydrolyzate, 1.5% @ agar), 1.8 x 10 per plate⁵The cells were dispersed so that individual plaques were formed, and cultured at 37 ° C. for 8 hours. At 14 places of the agar medium on which the plaque was formed, the agar medium was extracted together with the plaque using the bottom of a 250 μl wide diameter pipette tip (manufactured by RAININ), and the agar medium pieces were each 100 μl of SM buffer solution (0. Put into a plastic centrifuge tube containing 1M sodium chloride, 8mM magnesium sulfate, 50mM Tris-HCl (pH 7.5), 0.01% gelatin), vortex vigorously using a vortex mixer, and then 1 to 2 hours at 4 ° C. After standing, the mixture was centrifuged at 12,000 × g for 5 minutes to recover the supernatant, which was used as a phage suspension.
[0345]
On the other hand, the following nucleotide sequences were used as primers for PCR:
Primer 1: 5 '-{gactgatcaa @ atgttgag @ cttt} -3' (SEQ ID NO: 5 in the Sequence Listing); and Primer 2: 5 '-{tgcatccaga @ gtggatccag @ a} -3' (SEQ ID NO: 6 in the Sequence Listing)
Using an automatic DNA synthesizer (Model 394: manufactured by PerkinElmer Japan Applied Biosystems Division) using a phosphoramidite method (Mateucci, M.D., andCarturers, M.H.). 1981) {J. Am. Chem. Soc. 103, 3185-3191).
[0346]
5 μl of the phage suspension obtained as described above was mixed with 2.5 μl of 10 × PCR buffer (attached to Taq polymerase manufactured by Takara Shuzo Co., Ltd.) and 4 μl of dNTP mixed solution (2.5 mM each, Takara Shuzo) Primer 1 and 2, adjusted to 1 μl each 7.5 μM, 0.25 μl Taq polymerase (Takara Shuzo Co., Ltd.), 11.25 μl sterilized water After mixing and heating at 94 ° C. for 5 minutes, a temperature cycle of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds and 72 ° C. for 30 seconds was repeated 30 times, and finally, the temperature was kept at 72 ° C. for 7 minutes. And stored at 4 ° C. The reaction was electrophoresed on a 4% agarose gel (prepared on NuSieve # 3: 1 agarose (FMC Bioproducts)) to analyze the amplification of specific fragments. As a result of screening 14 phage suspensions in this way, two positive samples in which the target cDNA fragment was specifically amplified were obtained.
[0347]
c) Secondary screening
Using 100 ng of mouse genomic DNA (manufactured by Clontech) as a template, PCR was performed under the conditions described in b) above, and agarose electrophoresis was performed to recover the amplified DNA fragment. Using this DNA fragment as a template, a multi-prime DNA labeling system (Amersham Pharmacia) was used.³²After performing a reaction for generating a DNA fragment labeled with P, the reaction solution was injected into a nick column (manufactured by Amersham Pharmacia). The column was washed once by flowing 400 μl of TE (10 mM Tris-hydrochloric acid (pH 7.5), 1 mM EDTA), and further washed with 400 μl of TE to collect an eluate. The whole amount of the eluted fraction was used as a labeled probe in the following secondary screening.
[0348]
On the other hand, the phage suspension that was determined to be positive in b) was diluted 100-fold with SM buffer, and 2 μl of the phage-infected Escherichia coli was dispersed on an agar medium plate prepared in a 9 cm-diameter culture dish. And incubated at 37 ° C. for 8 hours. A circular nylon membrane (Hybond N +, manufactured by Amersham Pharmacia Co., Ltd.) adjusted to the inside diameter of the petri dish was placed on the agar medium on which the plaque had been formed, and the plaque was transferred by leaving it at 4 ° C. for 5 minutes. After penetrating three places of the membrane to the agar medium using an 18G injection needle, marks for alignment are provided, and the membrane is peeled off and placed in an alkaline solution (1.5 M sodium chloride, 0.5 M sodium hydroxide). 2 minutes, then into a neutralization solution (1.5 M sodium chloride, 0.5 M Tris-HCl (pH 8.0)) for 5 minutes, and further into a solution containing 2 × SSC, 0.2 M Tris-HCl (pH 7.5) After soaking for 30 seconds, it was completely air-dried at room temperature.
[0349]
This membrane was incubated (prehybridization) at 68 ° C. for 1 hour in 20 ml of a hybridization solution (ExpressHyb Hybridization Solution, manufactured by Clontech), and then replaced with an 8 ml hybridization solution containing a labeled probe at 68 ° C., 6 μm. Incubated for hours. Subsequently, the operation of washing the membrane with a solution containing 2 × SSC, 0.05% SDS while gently shaking at room temperature for 15 minutes is repeated three times, and then 0.1 × SSC, 0.1% The operation of washing with a solution containing SDS at 50 ° C. for 30 minutes was repeated three times.
[0350]
Autoradiography was performed on the washed membrane, and the original plaque at a position recognized as positive was recovered from the agar medium, and the phage suspension was subjected to PCR under the conditions described in b) above. Specific amplification of the DNA fragment was observed in 6 out of 10 plaque samples.
[0351]
The phage suspension of the sample in which amplification was observed most strongly was analyzed using the helper phage and host bacteria attached to the ZAP Express cDNA Gigapack III Gold Cloning Kit (Stratagene). In vivo excision (In Vivo Excision) was performed according to the attached protocol, and E. coli colonies containing phagemid were formed on agar medium. These colonies were isolated, phagemids were respectively extracted, and PCR was carried out by the method described in b) above. As a result, colonies showing specific amplification of the DNA fragment were selected and cultured, and a 1.6 kbp cDNA insert was obtained. Transformed E. coli carrying phagemid # 55-1 having E. coli. coli @ pBK / m55-1 {SANK} 72199 was isolated.
[0352]
As a result of analyzing the entire nucleotide sequence of the cDNA inserted into the obtained phagemid # 55-1, it was found to be the sequence shown in SEQ ID NO: 1 in the Sequence Listing (however, the nucleotide number of SEQ ID NO: 1 in the Sequence Listing). 1-8 is a vector-derived adapter sequence). This sequence was the same as the sequence registered in the GenBank database as mouse “angiopoietin-related protein 3” (accession number: AF162224). The transformed E. coli E. coli harboring this phagemid # 55-1. coli @ pBK / m55-1 @ SANK @ 72199 was internationally deposited on November 19, 1999 at the National Institute of Advanced Industrial Science and Technology (currently the National Institute of Advanced Industrial Science and Technology @ Patent Depositary). Accession number FERM @ BP-6940.
[0353]
Embodiment 1 FIG. Northern blot analysis
a) Extraction of total RNA from mouse organs
Northern blot analysis was performed for the purpose of examining the tissue expressing the cDNA obtained in Reference Example 1. First, KK mice (hyperlipidemic mice) and KK /SnkTestis, spleen, kidney, kidney, small intestine, liver of mice (hypolipidemic mutant mice; Shiraki et al., The 7th Diabetes Study Group (1993)) And extraction of total RNA from brain. 18 week old KK mice and KK /SnkThe mouse was dissected, and each organ was excised, immediately put into liquid nitrogen, rapidly frozen, and stored at -80 ° C. About 15 ml of TRIzol reagent (manufactured by Gibco BRL) was added to 0.5 g of the organ, and the mixture was homogenized on ice using an ultra-high-speed homogenizer Polytron (manufactured by Kinematica) (at a scale of 6 minutes for 2 minutes). After allowing this to stand at room temperature for 5 minutes, 3 ml of chloroform was added, and the mixture was mixed by overturning vigorously by hand for 15 seconds. After leaving still at room temperature for 3 minutes, the mixture was centrifuged at 12000 × g and 4 ° C. for 15 minutes. After centrifugation, the upper layer was recovered and mixed with 0.8 volume of ribonuclease-free isopropyl alcohol. This was allowed to stand at room temperature for 10 minutes, and then centrifuged at 12,000 × g at 4 ° C. for 10 minutes. Then, the supernatant was removed, and ribonuclease-free 70% ethanol was added. This was centrifuged at 12000 × g at 4 ° C. for 10 minutes, the supernatant was removed, and the precipitate was dried and stored at −80 ° C.
[0354]
b) Electrophoresis and blotting of total RNA
After adjusting the total RNA of each collected organ to 4 μg / μl with ribonuclease-free double distilled water, 5 μl of this RNA solution and RNA sample buffer (1.15 × MOPS buffer (1 × MOPS buffer is 20 mM MOPS, 5 mM sodium acetate, 1 mM ethylenediaminetetraacetic acid (hereinafter referred to as “EDTA”), 2.4 M formaldehyde, 57% formamide, 7% glycerol, 18 μg / ml bromophenol blue, 18 μg / ml xylene cyanol, 0.18 mM EDTA ) 16 μl were mixed and kept at 65 ° C. for 10 minutes, and then left on ice for 5 minutes. The whole amount of the sample solution was subjected to an agarose gel for electrophoresis containing 1.17% formalin (1 × MOPS buffer, 1.17% @ agarose (high strength, for analysis, manufactured by Bio-Rad), 0.66M @ formaldehyde). Injected into one well and electrophoresed. The electrophoresis was performed by energizing at 50 V for about 1 hour and then energizing at 100 V for about 1.5 hours in a submarine electrophoresis tank containing 1 × MOPS buffer containing 500 ng / ml ethidium bromide. . After completion of the electrophoresis, the RNA in the agarose gel was subjected to a capillary transfer method (Maniatis, T. et al., (1982) in, "Molecular Cloning, A Laboratory, Manual", Cold Spring Harbor, Laboratories, NY, NY, NY, NY). (Transfer solution was 20 × SSC). The membrane was washed with 2 × SSC for 5 minutes, air-dried, and irradiated with ultraviolet rays (1200 J / cm) using an ultraviolet irradiation device for crosslinks (Spectrolinker XL-1000, manufactured by Tommy Seiko Co., Ltd.).²) To fix the RNA.
[0355]
c) Preparation of probe
Using the primers synthesized in b) of Reference Example 1, PCR was performed under the following conditions using a thermal cycler (GeneAmp PCR System 9600, manufactured by Perkin Elmer Japan Applied Biosystems Division). First, sterile water was added to primers (0.5 μM each in final concentration) and Tween 20 (manufactured by Sigma, 0.1% in final concentration) to make 7.5 μl, and then 2 × PCR Solution Premix Taq (Takara Shuzo Co., Ltd.) ): 7.5 μl of 0.05 units / μl (including Taq polymerase, 0.4 mM dNTPs, 20 mM tris-hydrochloric acid (pH 8.3), 100 mM potassium chloride, and 3 mM magnesium chloride). Further, 1 μl (corresponding to 100 ng) of mouse genomic DNA (manufactured by Clontech) was added to prepare a reaction solution. The reaction solution was first heated at 94 ° C. for 3 minutes, and then subjected to a temperature cycle of 94 ° C. for 30 seconds, 55 ° C. for 1 minute, and 72 ° C. for 45 seconds 35 times, and then kept at 4 ° C.
[0356]
1 μl of the reaction solution after the PCR was taken, and the amplified DNA fragment was cloned into a plasmid vector using a TA cloning kit (dual promoter version A, manufactured by Invitrogen) according to the attached protocol. Competent Escherichia coli was transformed with this recombinant plasmid vector and cultured on LB agar medium containing 50 μg / ml ampicillin. As a result, Escherichia coli colonies which grew showing ampicillin resistance were selected and cultured overnight at 37 ° C. in 4 ml of a liquid LB medium containing 50 μg / ml of ampicillin. Plasmid DNA was recovered from 3.5 ml of the culture solution using a plasmid automatic extraction device (PI-50, manufactured by Kurabo Industries, Ltd.). Nucleotide sequence analysis was performed on the obtained plasmid DNA, and the plasmid into which the desired PCR product had been inserted was used in the following procedure.
[0357]
After 8 μg of the selected plasmid DNA was digested with a restriction enzyme EcoRI, phenol / chloroform extraction and ethanol precipitation were performed, and the obtained precipitate was dissolved in 10 μl of sterilized water. To this solution, 2 μl of a dye solution (0.25% bromophenol blue, 0.25% xylene cyanol, 15% ficoll (type 400)) was added, and the whole amount was subjected to polyacrylamide gel electrophoresis (gel concentration 8%, 100 V, (Room temperature, 3 hours). After the gel after the electrophoresis is stained with ethidium bromide, a piece of the gel corresponding to the target DNA (approximately 200 bp, SEQ ID NO: 29 in the sequence listing) is cut off with a razor blade under ultraviolet irradiation, and placed in a microcentrifuge tube. Transferred and crushed. To this, 300 μl of an elution buffer (0.5 M ammonium acetate, 10 mM EDTA (pH 8.0), 0.1% SDS) was added, and the mixture was incubated at 37 ° C. overnight, and extracted twice with phenol / chloroform. Precipitation was performed once and the precipitate was dissolved in 20 μl of sterile water.
[0358]
About 5 μl of the obtained DNA solution, by the method described in c) of Reference Example 1,³²A probe labeled with P (400 μl) was prepared.
[0359]
d) Hybridization
The membrane prepared in the above b) was placed in 20 ml of a hybridization solution (ExpressHyb Hybridization Solution, manufactured by Clontech) and incubated at 68 ° C. for 1 hour (prehybridization).³²Incubated overnight at 68 ° C. in 20 ml of hybridization solution containing P-labeled probe. Thereafter, the membrane is washed three times in a solution containing 2 × SSC and 0.05% SDS at room temperature for 20 minutes, and further in a solution containing 0.1 × SSC and 0.1% ΔSDS at 50 ° C. for 20 minutes. After washing three times, autoradiography was performed.
[0360]
As a result, the expression of the detected gene was found only in the liver, and its expression level was higher than that of KK mice (hyperlipidemic mice).SnkIn mice (hypolipidemic mice), it was found to be significantly reduced (FIG. 1).
[0361]
In the above-described experimental system, for example, an RNA sample is prepared from primary cultured hepatocytes of KK mice cultured in the presence or absence of a test substance, and the same operation is performed in the following to thereby obtain hyperlipidemia, arteriosclerosis, The effect as an agent for treating or preventing one or more diseases selected from hyperglycemia can be examined. A test substance that decreases the expression level of a gene detected in this experiment can be a therapeutic or preventive agent for one or more diseases selected from hyperlipidemia, arteriosclerosis, and hyperglycemia. When performing multi-sample processing, dot blot or slot blot can be performed without electrophoresis.
[0362]
Reference example 2. Cloning of human cDNA
1) Preparation of probe
To obtain a human cDNA corresponding to the mouse cDNA shown in SEQ ID NO: 1 in the sequence listing, the following nucleotide sequence was used:
5 '-{tcctcttagtt @ atttcctcca @ g} -3' (SEQ ID NO: 7 in Sequence Listing); and
5 '-{tggttttgcca @ gcgatagatac @ -3' (SEQ ID NO: 8 in Sequence Listing)
Was synthesized.
[0363]
Next, 1 μl of human genomic DNA (manufactured by Boehringer Mannheim, 200 mg / ml), 1 μl of Taq polymerase (rTaq, manufactured by Takara Shuzo Co., Ltd., 5 units / μl), 10 × PCR buffer (manufactured by Takara Shuzo Co., Ltd.) 10 μl, 16 μl of a dNTP mixture (2.5 mM each), 2 μl of each 20 μM primer, and 68 μl of sterilized water were mixed. After heating the reaction solution at 94 ° C. for 5 minutes, a temperature cycle of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 30 seconds was repeated 30 times, and finally, the temperature was maintained at 72 ° C. for 10 minutes. And stored at 4 ° C. After electrophoresis of this reaction solution on a 2% agarose gel, the gel of the amplified DNA band was cut out and purified. The DNA thus obtained was labeled with a DNA labeling kit (BcaBest labeling kit, manufactured by Takara Shuzo Co., Ltd.).³²Those labeled with P were used as probes in 2) below.
[0364]
2) Primary screening of cDNA library
Of a commercially available human liver-derived cDNA library (Human Liver 5'-STRETCH cDNA Library, manufactured by Clontech), 1 × 10⁶Plaque DNA was immobilized on a nylon membrane. That is, E. coli infected with the cDNA library was placed on 20 agar plates prepared in a culture dish having a diameter of 9 cm, and 5 × 10⁴The cells were dispersed so that individual plaques were formed, and cultured at 37 ° C. for 8 hours. A circular nylon membrane (Hybond N +, manufactured by Amersham Pharmacia Co., Ltd.) adjusted to the inside diameter of the petri dish was placed on the agar medium on which the plaque had been formed, and the plaque was transferred by leaving it at 4 ° C. for 5 minutes. After penetrating three places of the membrane to the agar medium using an 18G injection needle, marks for alignment are provided, and the membrane is peeled off and placed in an alkaline solution (1.5 M sodium chloride, 0.5 M sodium hydroxide). 2 minutes, then into a neutralization solution (1.5 M sodium chloride, 0.5 M Tris-HCl (pH 8.0)) for 5 minutes, and further into a solution containing 2 × SSC, 0.2 M Tris-HCl (pH 7.5) After soaking for 30 seconds, it was completely air-dried at room temperature. Next, ultraviolet rays were irradiated (1200 J / cm) with an ultraviolet irradiation device for crosslinks (Spectrolinker XL-1000, manufactured by Tommy Seiko Co., Ltd.).²) To fix the DNA.
[0365]
The membrane thus prepared was incubated overnight at 65 ° C. in a hybridization solution (ExpressHyb \ solution, Clontech) containing the labeled probe obtained in 1) above. Thereafter, the membrane is washed with a solution containing 2 × SSC and 0.05% ΔSDS three times for 15 minutes each at room temperature, and then further washed with a solution containing 0.1 × SSC and 0.1% ΔSDS for 30 minutes. After washing twice at 50 ° C., autoradiography was performed.
[0366]
The plaque at the position of the positive signal found as a result was collected together with the medium from the agar medium plate, and each 100 μl of SM buffer solution (0.1 M sodium chloride, 8 mM magnesium sulfate, 50 mM Tris-HCl (pH 7.5) was used. ), 0.01% gelatin), suspended well at 4 ° C. for 2 hours, and centrifuged at 12,000 × g for 5 minutes to collect the supernatant.
[0367]
Escherichia coli was again infected with the thus obtained primary positive phage solution so that 500 plaques per petri dish were formed on an agar medium prepared in a culture dish having a diameter of 9 cm. After culturing, secondary screening was performed by repeating the above procedure. Escherichia coli strain BM25.8 (manufactured by Clontech) was infected with the obtained secondary positive clone phage and cultured at 37 ° C. on an agar medium to form an E. coli colony containing phagemid. A colony was isolated, cultured in a small amount in a liquid medium, phagemid was extracted, and the inserted fragment was analyzed by restriction enzyme digestion. As a result, E. coli E. coli harboring clone # h5-1 having an inserted fragment of 1.6 kbp was analyzed. E. coli pTrip / h55-1 SANK 72299 was isolated. As a result of analyzing the nucleotide sequence of the inserted fragment of this clone, the nucleotide sequence was consistent with the cDNA sequence (registration number: AF152562) registered as a human "angiopoietin-related protein 3" in GenBank (SEQ ID NO: 3 in the sequence listing; however, Nucleotide numbers 1 to 14 in SEQ ID NO: 3 in the sequence listing are vector-derived adapter sequences). In addition, transformed E. coli E. coli harboring this phagemid # h5-1. coli @ pTrip / h55-1 @ SANK @ 72299 was internationally deposited on November 19, 1999 with the National Institute of Advanced Industrial Science and Technology (now the National Institute of Advanced Industrial Science and Technology @ Patent Organism Depositary), and the accession number FERM. BP-6941 was attached.
[0368]
Embodiment 2. FIG.の Preparation of polyclonal antibody
Recognizes both the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 1 and SEQ ID NO: 3 in the sequence listing, that is, the polypeptide having the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing For the purpose of producing antibodies, two amino acid sequences selected from the regions conserved between the mouse and human polypeptides as antigens:
Glu-Pro-Lys-Ser-Arg-Phe-Ala-Met-Leu-Asp-Asp-Val-Lys-Cys (55-1-N1, SEQ ID NO: 9 in Sequence Listing);
Cys-Gly-Glu-Asn-Asn-Leu-Asn-Gly-Lys-Tyr-Asn-Lys-Pro-Arg (55-1-C1, SEQ ID NO: 10 in Sequence Listing)
Was synthesized by a chemical synthesis method (device used: Model 433, manufactured by PerkinElmer Japan). However, the amino acid sequence of 55-1-N1 has a cysteine residue added to the C-terminal of the original amino acid sequence in order to bind keyhole limpet hemocyanin (hereinafter referred to as “KLH”) as a carrier. . On the other hand, the N-terminal cysteine to which KLH is bound at 55-1-C1 is derived from the original amino acid sequence.
[0369]
Next, synthetic peptide 55-1-N1 11.1 mg and KLH 21.5 mg, or 55-1-C1 10.2 mg and KLH 21.2 mg were respectively added to N- (6-maleimidocaproyloxy) succinimide (EMCS, Condensed using a reagent (manufactured by Dojindo Laboratories, Inc.) (using a 0.02 M phosphate buffer (pH 7.5) containing 8 M urea and 0.9% sodium chloride as a condensation reaction solvent) at room temperature. Incubated for 15 hours). This reaction solution was placed in an 8M urea solution, dialyzed against running water, further dialyzed against pure water, and then lyophilized to obtain a KLH-bound peptide antigen. 1 ml of physiological saline was added to about 10 mg of these peptide antigens to form a fine suspension using an ultrasonic oscillator (sonicator), vortex mixer, glass rod, or the like. Thereafter, the total volume was adjusted to 7.5 ml each with physiological saline, and further 1 ml was subdivided into vials and stored frozen.
[0370]
At the time of immunization, the antigen solution for one vial was melted, mixed with the same amount of adjuvant, and injected subcutaneously or intradermally into the back of two rabbits, respectively. The adjuvant was Freund's complete adjuvant, and Freund's incomplete adjuvant was used for the second and subsequent immunizations. Immunization was performed four times every two weeks, and after the second immunization, test blood was collected and the antibody titer in the serum was examined by enzyme immunoassay (ELISA) using a solid phase method. A 96-well ELISA plate (96-well H type, manufactured by Sumitomo Bakelite Co., Ltd.) was coated with each antigen peptide, and horseradish peroxidase-labeled anti-rabbit IgG antibody was used as a secondary antibody. Thirteen days after the fourth immunization, whole blood was collected.
[0371]
After blood collection, the antibody was purified using an affinity column. That is, the carrier EMC-activated by reacting N- (6-maleimidocaproyloxy) succinimide (EMCS, manufactured by Dojindo Laboratories, Inc.) with aminoalkyl agarose (Affigel 102, manufactured by Bio-Rad). The peptide (55-1-N1: 7.82 mg, 55-1-C1: 8.07 mg) was bound to agarose (about 5 ml). Unreacted EMC groups were inactivated by treatment with 0.1 M mercaptoethylamine hydrochloride (containing 5 mM EDTA). 85 ml of the antiserum was diluted 2-fold with PBS (containing 0.02 M phosphate buffer (pH 7.0), 0.9% sodium chloride), and a precipitate was obtained by ammonium sulfate precipitation (final concentration: 40%). The substance was dissolved in PBS, desalted and dialyzed against PBS, and the dialysate was used as a crude IgG fraction. The chromatographic operation was performed three times on the affinity column. That is, the operation of charging the affinity column with 1/3 of the crude IgG fraction and re-injecting the fraction into the column was repeated three times. A total of 40 ml of the flow-through portion and the washing was collected as a non-adsorbed fraction. After thoroughly washing with 1 M sodium chloride-containing PBS to remove non-specifically bound to the column, 4 M magnesium chloride solution, 3.5 M potassium thiocyanate solution and 0.1 M glycine-hydrochloride buffer (pH 2. 3) was sequentially passed through the column, and the antibody specifically bound to the peptide bound on the column carrier was eluted as an affinity-purified antibody. Since the target antibody was contained in the eluates of the 4M magnesium chloride solution and the 3.5M potassium thiocyanate solution, the respective eluates dialyzed against PBS were used as antibodies in the following procedure.
[0372]
Embodiment 3 FIG.発 現 Expression in COS-1 cells and Western blot analysis
The # h5-1 phagemid DNA obtained in Reference Example 2 was digested with restriction enzymes EcoRI and XbaI, subjected to 8% polyacrylamide gel electrophoresis, and subjected to the method described in Example 1 c). A 1.6 kb fragment was isolated and purified. On the other hand, a high expression vector pME18S (Hara, T. et al. (1992) EMBO. J. 11, 11875-, Takashi Yokota, Kenichi Arai, edited by Bio Manual Series 3, Gene Cloning Experiment, Yodosha, p18-20 ) Was similarly digested with EcoRI and XbaI, the ends were dephosphorylated, and ligated with the above cDNA fragment using a DNA ligation kit (version 2, Takara Shuzo Co., Ltd.). Escherichia coli was transformed with this DNA, and the resulting transformant was analyzed for the plasmid DNA carried by the restriction enzyme, and a strain having a 1.6 kb DNA fragment was selected and named pMEh55-1.
[0373]
Next, the transformed E. coli harboring pMEh55-1 was cultured overnight at 37 ° C. in 100 ml of a liquid LB medium containing 50 μg / ml of ampicillin. From this culture solution, pMEh55-1 DNA was recovered using a plasmid purification kit (Wizard Purefection Plasmid DNA Purification System, manufactured by Promega) and purified by the cesium chloride method.
[0374]
COS-1 cells were transfected with the thus obtained plasmid pMEh55-1. Transfection into COS-1 cells was performed by an electroporation method using a gene transfer device GTE-1 manufactured by Shimadzu Corporation. That is, first, cells were recovered by trypsin-EDTA treatment from flasks in which COS-1 cells were grown to semi-confluence, and washed with PBS (-) buffer (Takara Shuzo Co., Ltd.). Next, the cells were washed with PBS (−) buffer at 6 × 10 5⁷Resuspended at cells / ml. The plasmid DNA (pMEh55-1) recovered by the above method was adjusted to 200 μg / ml with a PBS (−) buffer. The cell suspension and the DNA solution were mixed in an amount of 20 μl each, placed in a chamber with an electrode spacing of 2 mm, and pulsed at 600 V for 30 μsec twice at 1 second intervals. After the chamber was cooled at 4 ° C. for 5 minutes, the cell-DNA mixture in the chamber was added to 10 ml of 10% {DMEM containing fetal bovine serum}, transferred to a Petri dish, and cultured at 37 ° C. under 5% of CO 2 overnight. Thereafter, the culture supernatant was removed, and the cells were washed with a serum-free medium (DMEM).
10 ml was added and the cells were cultured for 3 days.
[0375]
The culture supernatant was recovered from the cell culture thus obtained. 0.3 ml of a serum-free culture supernatant of COS-1 cells obtained by transfecting with a negative control plasmid pME18S or pMEh55-1 containing no cDNA was treated with trichloroacetic acid (hereinafter referred to as “TCA”) to precipitate proteins. A precipitate was obtained by centrifugation. The precipitate is washed with ice-cooled acetone, air-dried, and then dissolved in a sample buffer (manufactured by Bio-Rad) containing 2-mercaptoethanol for SDS-polyacrylamide electrophoresis (SDS-PAGE), 4-20% SDS-PAGE was performed under reducing conditions using a polyacrylamide density gradient gel (Multigel 4/20, manufactured by Daiichi Pure Chemicals Co., Ltd.).
[0376]
After the electrophoresis, the band was transferred from the polyacrylamide gel in a transfer buffer (192 mM glycine, 20% methanol, 25 mM Tris) using a gel membrane transfer device (manufactured by Marisol, NP7513) at 4 ° C. for 90 minutes at 200 mA. It was transferred to a nitrocellulose membrane (manufactured by Bio-Rad).
[0377]
The nitrocellulose membrane after transfer was subjected to Western blot analysis using the antibody obtained in Example 2 (hereinafter, referred to as “55-1-N1 antibody” or “55-1-C1 antibody”). That is, first, the nitrocellulose membrane was washed with PBS containing 0.05% Tween 20 (hereinafter, referred to as “0.05%@Tween 20-PBS”) (at room temperature once for 15 minutes, then twice for 5 minutes), and then In a plastic bag (trade name: Hybridvac, manufactured by Cosmo Bio Co., Ltd.), add 20 ml of 0.05% Tween 20-PBS containing 5% skim milk (manufactured by Snow Brand Milk Products Co., Ltd.) and shake for 1 hour at room temperature I'm sorry. After one hour, the membrane was taken out and washed in 0.05% Tween 20-PBS for 15 minutes × 1 time and then for 5 minutes × 2 times. After washing, the membrane was transferred to a new plastic bag, and containing a 55-1-N1 antibody or a 55-1-C1 antibody (100-fold dilution), 1% bovine serum albumin (hereinafter referred to as “BSA”, manufactured by Sigma). Twenty ml of 05% Tween 20-PBS was added and shaken at room temperature for 1 hour. One hour later, the membrane was taken out and washed with a 0.05% Tween 20-PBS solution for 15 minutes × 1 time and 5 minutes × 2 times. Thereafter, the membrane was transferred to a new plastic bag, and 20 ml of a solution of horseradish peroxidase-labeled anti-rabbit IgG antibody (manufactured by Amersham Pharmacia) diluted 2000-fold with 0.05% Tween 20-PBS containing 1% BSA was added. Shake at room temperature for 1 hour. One hour later, the membrane was taken out and washed with 0.05% @ Tween 20-PBS for 15 minutes x 1 and then for 5 minutes x 4 times. After washing, the membrane was placed on a wrap film, and the band to which the 55-1-N1 antibody or 55-1-C1 antibody was bound was detected using an ECL western blotting detection solution (manufactured by Amersham Pharmacia) ( The membrane was placed on a wrap film, immersed in an ECL western blotting detection solution for 1 minute, left at room temperature for 1 hour to attenuate the background, and then exposed to X-ray film (3 seconds). As a result, both antibodies detected specific bands in the COS-1 culture supernatant obtained by introducing the pMEh55-1 plasmid DNA (FIG. 2).
[0378]
A similar experiment can be performed on the COS-1 cell culture supernatant expressing the mouse cDNA obtained in Reference Example 1. That is, a clone (pME55-1) obtained by digesting the phagemid # 55-1 DNA obtained in Reference Example 1 with restriction enzymes EcoRI and XbaI and incorporating an inserted DNA fragment of about 1.6 kb into pME18S (pME55-1). Is introduced into COS-1 cells in the same manner as described above, and the culture supernatant is collected. When a sample prepared from this culture supernatant was subjected to Western blot analysis using the 55-1-N1 antibody and the 55-1-C1 antibody, the pME55-1 plasmid DNA was introduced when any of the antibodies was detected. A specific band is detected in the obtained COS-1 culture supernatant.
[0379]
In the above-described experimental system, for example, a sample is prepared from a culture supernatant of primary cultured hepatocytes of a KK mouse cultured in the presence or absence of a test substance, and by performing the same operation, hyperlipidemia of the test substance, The effect as an agent for treating or preventing one or more diseases selected from arteriosclerosis and hyperglycemia can be examined. A test substance that reduces the amount of antigen detected in this experiment can be a therapeutic or prophylactic agent for one or more diseases selected from hyperlipidemia, arteriosclerosis, and hyperglycemia. When performing multi-sample processing, dot blot or slot blot can be performed without electrophoresis.
[0380]
Embodiment 4. FIG.作 製 Preparation of recombinant adenovirus and expression in cultured cells
A recombinant adenovirus for forcibly expressing the mouse-derived cDNA obtained in Reference Example 1 was prepared using a commercially available kit (Adenovirus Expression Vector Kit, manufactured by Takara Shuzo Co., Ltd.). That is, the phagemid clone # 55-1 obtained in Reference Example 1 was digested with restriction enzymes EcoRI and NotI, and the obtained DNA fragment of about 1.7 kb was blunt-ended at the end to obtain an inserted DNA fragment. Used for the operation.
[0381]
In addition, an insert DNA fragment was inserted into a restriction enzyme SwaI recognition site of a cosmid vector pAxCAwt (attached to an adenovirus expression vector kit) designed to be expressed by a cytomegalovirus enhancer and a chicken β-actin promoter. Cosmid pAxCA / mAP5 was prepared. pAxCA / mAP5 DNA and terminal protein-linked virus DNA (DNA-TPC, attached to the adenovirus expression vector kit) were co-transfected into 293 cells (ATCC @ CRL1573) using a calcium phosphate transfection system (manufactured by Lifetech). The recombinant adenovirus Ad / mAP-5 was isolated and further amplified in 293 cells. In addition, an adenovirus Ad / LacZ having a recombinant adenovirus vector into which the LacZ gene excised from the control cosmid pAxCAiLacZ was incorporated was similarly prepared and amplified in 293 cells. To recover the amplified virus from 293 cells, the virus-infected 293 cells were first subjected to sonication for 30 seconds × 4 times (using B-1200 manufactured by Branson), and then purified by cesium chloride density gradient centrifugation. This was done by repeating the procedure several times. The obtained virus solution was dialyzed at 4 ° C. against PBS supplemented with 10% Δglycerol, and then stored frozen at −70 ° C. or lower until use.
[0382]
About 5 m. Of the recombinant adenovirus thus obtained was introduced into HeLa cells (ATCC @ CCL2). o. i. (Multiplicity of infection: multiplicity of infection), and cultured for 3 to 4 days in a serum-free medium (Dulbecco's modified Eagle's medium (DMEM)) to collect the culture supernatant. 1 ml of this supernatant was placed in a 1.5 ml Eppendorf tube, 100 μl of trichloroacetic acid was added, the mixture was allowed to stand at room temperature for 3 minutes, and then centrifuged at 15000 rpm for 5 minutes using a tabletop centrifuge. The supernatant was removed, 0.5 ml of ice-cooled acetone was added, and the mixture was stirred well and centrifuged again at 15,000 rpm for 2 minutes using a tabletop centrifuge. The supernatant was removed, 0.5 ml of ice-cold acetone was added again, and the mixture was stirred well. The mixture was centrifuged at 15000 rpm for 2 minutes using a desktop centrifuge to remove the supernatant, and the precipitate was dried under vacuum. This precipitate was dissolved in 10 μl of distilled water, mixed with SDS-PAGE sample buffer containing 2-mercaptoethanol (manufactured by Bio-Rad), and heated at 99 ° C. for 5 minutes to prepare a sample for electrophoresis. did. The sample was subjected to electrophoresis on a polyacrylamide density gradient gel having a gel concentration of 4 to 20% (electrophoresis buffer: 25 mM @ Tris, 192 mM @ glycine, 0.1% @ SDS), and then at 4 ° C in a transfer buffer. It was transferred to a nitrocellulose membrane for 1 hour at 200 mA. The transferred membrane was blocked overnight at 4 ° C. with a PBS solution containing 0.5% skim milk, and washed three times with a washing solution (0.05% Tween 20-PBS). Then, the membrane was mixed with a mixture of the antiserum before purification of the 55-1-N1 antibody and the antiserum before purification of the 55-1-C1 antibody in 5% {0.05% containing fetal bovine serum} Tween 20-PBS. After incubating for 1 hour at room temperature in a reaction solution diluted 10000-fold, it was washed three times with a washing solution. Furthermore, the membrane was incubated at room temperature for 1 hour at room temperature in a reaction solution in which horseradish peroxidase-labeled goat anti-rabbit IgG (H + L) (manufactured by Bio-Rad) was diluted 10,000 times with 5% {0.05% fetal bovine serum} Tween 20-PBS. After that, it was washed 5 times with a washing solution. The membrane was placed on a wrap film, immersed in an ECL western blotting detection solution for 1 minute, left at room temperature for 1 hour to attenuate the background, and then exposed to X-ray film (3 seconds). As a result, a specific band was detected in the lane of the sample derived from the culture supernatant of the HeLa cell infected with the recombinant adenovirus Ad / mAP-5 (FIG. 3).
[0383]
On the other hand, a recombinant adenovirus (Ad / hAP5) having the cDNA retained by the phagemid clone # h5-1 incorporated in an adenovirus vector was prepared, and the same experiment was carried out. A specific band was similarly detected as a result of Western blot analysis of Qing (FIG. 3).
[0384]
Embodiment 5 FIG. In vivo expression using recombinant adenovirus
Recombinant adenovirus Ad / mAP-5 or Ad / LacZ purified as described above was added to PBS containing 10% glycerol in 2 × 10 5¹⁰pfu (plaqueforming @ unit) / ml, 3 (Ad / mAP-5) or 2 (Ad / LacZ) 13-14 week old male KK /Snk100 μl (2 × 10⁹pfu) by tail vein injection. One day after inoculation, blood was collected from the fundus of each mouse using a hematocrit tube, centrifuged at 5200 rpm for 15 minutes with a tabletop centrifuge to separate plasma, and a neutral fat measurement kit (Triglyceride E-Test Wako, Wako Pharmaceutical Co., Ltd.) Was used to measure the neutral fat concentration. No significant difference in blood triglyceride concentration was observed in the Ad / LacZ-vaccinated mouse group, whereas the blood triglyceride concentration was significantly different between the Ad / mAP-5-vaccinated mouse group and the other two groups. A significant difference was observed in the concentration (FIG. 4).
[0385]
On the other hand, a similar experiment was performed using a recombinant adenovirus (Ad / hAP5) having a cDNA retained by the phagemid clone # h5-1 incorporated into an adenovirus vector, and male KK /SnkAs a result of expression in mice, a significant increase in the concentration of neutral fat in the blood was observed, and it was found that the human-type molecule also functions in the mouse body (FIG. 4).
[0386]
In addition, total RNA was recovered from the livers of the group of adenovirus-infected mice in which a significant difference was detected in the neutral fat concentration in the blood, and Northern blot analysis was performed by the method described in Example 1. It was confirmed that the expression was actually high (FIG. 5). Adenovirus infection KK /SnkThe band detected in the group of mice is larger than the band detected in the KK mouse that has not been genetically engineered, but this is due to the fact that the effective transcription initiation site or poly (A) added signal in the recombinant adenovirus vector is not used. It is thought to be caused by a difference from that on the gene. In any case, immediately after the first translation initiation codon in the cDNA incorporated in the recombinant adenovirus vector, a termination codon is present on the same reading frame as the translation initiation codon, so that this mRNA has The difference in size does not affect the translated amino acid sequence.
[0387]
Embodiment 6 FIG.精製 Purification of recombinant protein and determination of N-terminal amino acid sequence
According to the method described below, an animal cell is transformed with the expression vector pMEh55-1 into which a human cDNA having the nucleotide sequence shown in SEQ ID NO: 3 in the sequence listing prepared in Example 3 is inserted, and the transformation is performed. The recombinant protein secreted into the cell culture supernatant was purified and its N-terminal amino acid sequence was determined.
[0388]
(1) Obtaining a transformant and preparing a culture supernatant
Dihydrofolate reductase in an αMEM (Gibco BRL) medium containing 10% FCS (Gibco BRL), 10 units / ml penicillin and 10 μg / ml streptomycin (Gibco BRL) After growing deficient CHO cells (ATCC @ CRL-9096), the pMEh55-1 plasmid was added at 1 μg / 10% using a transfection reagent (FuGENE6: manufactured by Roche Diagnostics).⁶Transfected at the percentage of cells. Specifically, the cells were placed in a 200-cell culture dish (150 mmφ, manufactured by Corning Incorporated) at 1.5 × 10⁷The cells were cultured in a Petri dish, and 15 μg of the pMEh55-1 plasmid per Petri dish was used for transfection. After the cells after the transfection operation were cultured in the above-mentioned medium containing FCS for 24 hours, the medium was replaced with a serum-free αMEM medium (30 ml / dish), and the cells were further cultured for 3 days. Was recovered.
[0389]
(2) Purification of recombinant protein
1) A 1.6-liter gel of Sephadex G25 (manufactured by Amersham Pharmacia Biotech) packed in a column (Streamline C-100: manufactured by Amersham Pharmacia Biotech) was obtained in the above (1). 600 ml of the culture supernatant was injected (flow rate: 20 ml / min). Next, an elution buffer (20 mM Tris-HCl (pH 7.5), 0.01% sodium azide (Sigma), 0.05% protease inhibitor mixture (Sigma), 0.05% Tween 20 (Sigma) (Hereinafter referred to as "solution A") was passed through the column at a flow rate of 50 ml / min, and the first 1500 ml of eluate was collected. This operation was performed 10 times to desalt and remove low molecules of the 6 liter culture supernatant obtained in the above (1).
[0390]
2) Next, the eluate obtained in the above 1) was fractionated by ion exchange chromatography using an FPLC apparatus (BioPilot System, manufactured by Amersham-Pharmacia Biotech) under the following conditions.
Column: 100 ml of Q Sepharose Fast Flow (manufactured by Amersham Pharmacia Biotech) was packed in an XK50 / 100 column (manufactured by Amersham Pharmacia Biotech).
Elution buffer composition:
[Solution A] See above
[Solution B] 20 mM Tris-HCl, 1 M sodium chloride (pH 7.5), 0.01% sodium azide, 0.05% protease inhibitor mixed solution, 0.05% Tween 20
Flow rate: $ 10ml / min
Fractionation: 10ml / tube each
Temperature: 4 ℃
Elution conditions: A fraction eluted with a linear concentration gradient from {100%} solution A to 100% {solution B (for 60 minutes) at a sodium chloride concentration of 0.3 to 0.4 M was collected.
[0391]
3) {Further, the fraction collected by the ion exchange chromatography in the above 2) was subjected to group-specific affinity chromatography under the following conditions using an FPLC apparatus.
Column: Affigel Blue (manufactured by Bio-Rad) (10 ml) was packed in an XK16 / 40 column (manufactured by Amersham Pharmacia Biotech).
Elution buffer composition:
[Solution C] 20 mM Tris-HCl, 0.5 M sodium chloride (pH 7.5), 0.01% sodium azide, 0.05% protease inhibitor mixture, 0.05% Tween20
[Solution D] 20 mM Tris-HCl, 1 M sodium chloride (pH 7.5), 0.01% sodium azide, 0.05% protease inhibitor mixture, 0.05% Tween20
Flow rate: $ 1.5ml / min
Temperature: 4 ℃
After the fraction collected in 2) above was passed through a column, 60 ml of solution C was washed, followed by 100 ml of solution D, and an eluate of solution D was collected.
[0392]
4) An equal amount (100 ml) of the solution A was added to the eluate obtained in the above 3). This was subjected to group-specific affinity chromatography under the conditions described below using an FPLC apparatus.
Column: 10 ml of Lentill Lectin Sepharose 4B (manufactured by Amersham Pharmacia Biotech) was packed in an XK16 / 40 column.
Elution buffer composition:
[Liquid C] See above
[Solution F] 20 mM Tris-HCl, 0.5 M sodium chloride, 0.3 M methyl mannopyranoside (pH 7.5), 0.01% sodium azide, 0.05% protease inhibitor mixed solution, 0.05 % @ Tween20
Flow rate: $ 1ml / min
Temperature: 4 ℃
After the sample was passed through the column, 50 ml of the solution C was washed to wash, and then 50 ml of the solution F was flown to collect an eluate of the solution F.
[0393]
The eluate was transferred to a dialysis tube (exclusion limit molecular weight: 10 KDa: manufactured by Gibco BRL), and Dulbecco PBS (-) (Nissui Pharmaceutical) containing a mixed solution of 0.01% sodium azide and 0.1% protease inhibitor was used. Dialysis was performed overnight at 4 ° C. against 2 liters. Thereafter, the solution in the dialysis tube was collected, and to 4 ml of the solution, 1/10 volume (0.4 ml) of TCA containing 4 mg / ml sodium deoxycholate was added, and the obtained precipitate was collected. The resulting precipitate was collected and dissolved in 50 μl of sterile ultrapure water.
[0394]
(3) Determination of N-terminal amino acid sequence
PNGaseF (manufactured by New England Biolabs) was added to the sample purified in the above (2) to cleave N-linked sugar chains. Specifically, first, 6 μl of 10 × denaturing buffer (attached to PNGase F reagent) was added to 50 μl of the sample, stirred, and heated in a boiling water bath for 10 minutes. After the temperature was returned to room temperature, 6 μl of 10% @nonidet P-40 and 6 μl of 10 × G7 buffer (attached to the PNGase F reagent) were sequentially added and stirred. 3 μl of PNGaseF was added thereto, and the mixture was stirred and kept in a water bath at 37 ° C. for 2 hours or more.
[0395]
For the reaction solution after the N-linked sugar chain cleavage reaction, a 4 to 20% concentration gradient acrylamide gel (Multigel 4/20, manufactured by Daiichi Pure Chemicals Co., Ltd.) and a mini-slab electrophoresis apparatus (Nippon Aido Co., Ltd.) SDS-PAGE under reducing conditions using the following method. After the electrophoresis, the proteins separated in the gel were transferred to a polyvinylidene difluoride (PVDF) membrane (manufactured by Bio-Rad) having a pore size of 0.2 μm using a gel membrane transfer device (manufactured by Marisol) (2 mA / cm).², 4 hours at 4 ° C). After the transfer, the membrane was wetted with 100% methanol (Wako Pure Chemical Industries, Ltd.) for 10 seconds, washed with ultrapure water for 2 minutes, and then stained with Coomassie staining solution (Biorad) for 5 minutes. Further, as a result of immersing the membrane in methanol and decoloring for 2 minutes, a band corresponding to about 50 KDa was observed. The membrane of this band was cut out, and the N-terminal sequence was analyzed using a protein sequencer (PPSQ-10: manufactured by Shimadzu Corporation).
[0396]
As a result, the N-terminal amino acid sequence of the band of about 50 KDa is
Ser-Arg-Ile-Asp-Gln-Asp-Asn-Ser-Ser-Phe-Asp (amino acids 17 to 27 of SEQ ID NO: 4 in the sequence listing)
Met. From this, the protein encoded by the nucleotide sequence represented by nucleotide numbers 78 to 1457 of SEQ ID NO: 3 in the sequence listing is composed of 16 N-terminal peptides (amino acid numbers of SEQ ID NO: 4 in the sequence listing) in mammalian cells. 1 to 16) were cut off, and it was revealed that they were secreted as a mature form having a serine residue (amino acid No. 17 of SEQ ID NO: 4 in the sequence listing) immediately following the N-terminus.
[0397]
Reference example 3.精製 Purification of recombinant protein and administration to mice
300 μg of the recombinant protein purified in Example 6 was used for 15-week-old KK /SnkThe tail vein was administered to four male mice. As a control, another four animals received PBS (manufactured by Gibco BRL) (0.5 ml / mouse). At 1, 3, 6, and 24 hours after the administration, blood was collected from each mouse using a 75 μl heparin-treated capillary blood collection tube (Funakoshi) and centrifuged at 5200 rpm at 4 ° C. for 15 minutes (THM manufactured by Tommy Seiko Co., Ltd.). -1 rotor was used), and the plasma was separated and collected. Similarly, plasma was separated by fundus oculi blood sampling before administration of the purified protein, and collected as a sample of the pre-administration value. The triglyceride E test Wako (manufactured by Wako Pure Chemical Industries, Ltd.) was used for measuring the neutral fat concentration in blood. That is, 2 μl of the collected plasma was added to a 96-well plate (manufactured by Corning Incorporated), 200 μl of a coloring reagent was added thereto, and the mixture was stirred and allowed to stand at room temperature for 10 minutes. After the operation was completed, the absorbance at a main wavelength of 600 nm and the auxiliary wavelength of 700 nm was measured with a microplate reader, and the neutral fat concentration was calculated from the value obtained by subtracting the measured value of the auxiliary wavelength from the measured value of the main wavelength.
[0398]
The results are shown in Table 1. Administration of the recombinant protein was shown to increase blood neutral fat levels.
[0399]
[Table 1] (Unit: mg / dl)

Reference example 4.影響 Effect of bezafibrate administration
Two 11-week-old male Zucker fatty rats (purchased from Charles River Japan) were treated with bezafibrate (manufactured by Sigma) for treatment of hyperlipidemia at a concentration of 0.3% in F2 powder feed (manufactured by Funabashi Farm) for 7 days. And mixed. Rats in the control group (2 rats) received only F2 powder diet. After collecting a blood sample from each of the rats under both conditions, the liver was excised, immediately placed in liquid nitrogen, rapidly frozen, and stored at -80 ° C. 5 g of TRIzol reagent (manufactured by Gibco BRL) was added to 0.1 g of the obtained liver tissue, and homogenized on ice using an ultra-high-speed homogenizer (Polytron, manufactured by Kinematica) (2 on a scale of 10). Minutes). After allowing this to stand at room temperature for 5 minutes, 1 ml of chloroform was added, and the mixture was mixed by inverting vigorously for 15 seconds. It was left still at room temperature for 10 minutes, and then centrifuged at 12,000 × g and room temperature for 10 minutes. After centrifugation, the upper layer was recovered, and 2.5 ml of ribonuclease-free isopropyl alcohol was added and mixed. This was allowed to stand at room temperature for 10 minutes, and then centrifuged at 12,000 × g and room temperature for 10 minutes. The supernatant was removed, and ribonuclease-free 70% ethanol was added. This was centrifuged at 12,000 × g at room temperature for 5 minutes, the supernatant was removed, and the precipitate was dried and stored at −80 ° C. A solution obtained by dissolving the obtained pellet in pure water containing no ribonuclease was used as a total RNA solution.
[0400]
The synthesis of single-stranded cDNA was performed according to the following procedure. To 1 μg of the obtained total RNA, pure water containing no ribonuclease was added to make the total volume 6 μl, 1 μl of 10 × DNAase II reaction buffer (attached to DNase I manufactured by Gibco BRL), DNase I (Amp grade, Gibco BRL) 1 μl) and incubated at room temperature for 15 minutes, then 1 μl of 25 mM ΔEDTA (attached to DNase I) was added, and the mixture was incubated at 65 ° C. for 10 minutes. To this solution was added 1 μl of a random hexamer primer attached to a single-stranded cDNA synthesis kit (SuperScript first-strand synthesis system for RT-PCR, manufactured by Gibco BRL), and 1 μl of 10 mM dNTP mix was added. After keeping the temperature for 4 minutes, the mixture was rapidly cooled to 4 ° C. to perform heat denaturation. Subsequently, 2 μl of the 10 × reverse transcription reaction buffer attached to the cDNA synthesis kit, 4 μl of 25 mM magnesium chloride, 2 μl of 0.1 M dithiothreitol solution, and 1 μl of ribonuclease inhibitor were added, and the mixture was incubated at 25 ° C. for 2 minutes. Of reverse transcriptase was added in an amount of 1 μl. This sample was kept at 25 ° C. for 10 minutes, and then kept at 42 ° C. for 50 minutes. After further keeping the temperature at 70 ° C. for 15 minutes, it was rapidly cooled to 4 ° C. to perform heat denaturation. 1 μl of the attached ribonuclease H was added, and the mixture was kept at 37 ° C. for 20 minutes to decompose RNA, and stored at −20 ° C. After the completion of the reaction, 29 μl of pure water containing no ribonuclease was added to all the samples, and further diluted 5-fold with pure water containing no ribonuclease was used for the following quantification. The single-stranded cDNA solution for preparing the calibration curve was prepared by preparing a five-fold dilution series with pure water containing no ribonuclease from the quantification solution of one example of the control sample.
[0401]
On the other hand, the following sequences were used as primers for TaqMan @ PCR:
5 '-{caagctttga {aagtctactg {gagg} -3'} (SEQ ID NO: 30 in the sequence listing); and
5 '-{ggttctgaac {caagctggtc} a} -3'} (SEQ ID NO: 31 in Sequence Listing)
Was synthesized (ordered from Amersham Pharmacia).
[0402]
Further, as a probe used in TaqMan @ PCR, the following sequence:
5 '-{agatagggcgc} tccacaacag {agttag} -3'} (SEQ ID NO: 32 in Sequence Listing)
An oligonucleotide having a 5'-terminal reporter dye Fam and a 3'-terminal bound to a reporter quencher Tamura was synthesized (TaqMan @ fluorescent probe, manufactured by Applied Biosystems Japan). For the purpose of standardizing the expression level of “angiopoietin-related protein 3”, rat 18s ribosomal RNA was quantified, and TaqMan (ribosomal RNA Control Regent, manufactured by Applied Biosystems Japan) was used.
[0403]
Using these materials, the expression level of the rat “angiopoietin-related protein 3” gene was quantified by TaqManＭPCR. The PCR reaction was performed in a 96-well reaction plate (MicroAmp Optical 96-well Reaction Plate, manufactured by Applied Biosystems Japan). The composition of the reaction solution was as follows: in one well, 5 μl of the single-stranded cDNA solution obtained by the above method, 0.2 μl of the TaqMan PCR primer (50 pmol / μl) in both the forward and reverse directions (final concentration: 200 nM), TaqMan @ PCR 0.75 μl (final concentration: 100 nM) of a probe for use (6.6 μM), 25 μl of a PCR amplification mixture (TaqMan @ Universal PCR Master Mix, manufactured by Applied Biosystems Japan), and 18.85 μl of ultrapure water. At this time, the concentration of the stock solution of the single-stranded cDNA solution for preparing the calibration curve was set to “625” for convenience, and the concentration was changed to “625”, “125”, “25”, “5” and “1” by repeating 5-fold dilution. , A reaction solution was prepared in the same manner. The PCR reaction was performed using a TaqMan @ PCR dedicated thermal cycler / detector (ABI7700, manufactured by Applied Biosystems Japan). The reaction was carried out at 50 ° C. for 2 minutes and at 95 ° C. for 10 minutes, followed by repeating the reaction at 95 ° C. for 15 seconds and 60 ° C. for 1 minute 40 times, and the luminescence of the reporter dye was measured every cycle. An amplification curve of 18S ribosome and “angiopoietin-related protein 3” was prepared from the emission amount of the reporter dye in each cycle. From the amplification curve of the single-stranded cDNA solution for the preparation of a calibration curve, a calibration curve was prepared with the concentration on the horizontal axis and the number of cycles on the vertical axis, and each expression quantification sample was arbitrarily set in the logarithmic amplification phase. The number of cycles exceeding the given luminescence amount was plotted on a calibration curve, and the relative expression amount was calculated. The expression level of the “angiopoietin-related protein 3” gene was corrected with the value of the 18S ribosome expression level in the same sample.
[0404]
As a result, it was shown that the expression level of the rat “angiopoietin-related protein 3” gene was 73% lower in the bezafibrate-administered group than in the control group. The neutral lipid concentration in the blood of the rats in the bezafibrate-administered group used at this time was reduced by 50% as compared with the control group.
[0405]
Reference example 5. Effects on arteriosclerosis and blood glucose
Hypolipidemic mouse KK /SnkHas a homologous variant of a gene encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 (hereinafter referred to as "hypolipidemic mutant gene"), and the amount of production of the polypeptide is innate. Low. The effects of hypolipidemic mutant genes in atherosclerosis model mice or hyperglycemia model mice were examined according to the method described below.
[0406]
1) Creation of a congenic mouse
a) Mating style
KK /SnkFemale mice were crossed with male C57BL / 6J mice (obtained from Hamamatsu Medical University Animal Research Facility) to produce the first generation of backcross (N1 generation). The N1 female was then recrossed to a C57BL / 6J male to obtain the N2 generation. Genomic DNA was extracted from the tail of this N2 individual, and PCR was performed by the method described in c) below to select individuals heterozygously containing the hypolipidemic mutant gene. The selected females were further crossed with C57BL / 6J males to create the N3 generation. Thereafter, selection hybridization was continuously performed in the same manner up to N10 generations. Next, heterozygous males and females of the N10 generation were crossed, and PCR was carried out on a genome collected from the obtained baby by the method described in c) described below to select males and females homozygous for the hypolipidemic mutant gene. Thereafter, the cells were passaged by sibling mating to obtain a hypolipidemic mutant congenic mouse (C57BL / 6J-hypolipidemia) strain with a C57BL / 6J background. All animals were bred in an animal room at a temperature of 23 ± 1 ° C., a humidity of 55 ± 10%, and illuminated for 14 hours, and were allowed to freely ingest CMF solid feed (manufactured by Oriental Yeast Co., Ltd.) and 7 ppm crawl water.
[0407]
b) Method for extracting genomic DNA
The tail (1.5 cm) collected from the mouse (4 weeks old) used for selection in a) above contains 840 μl of lysate (720 μl of 1 × SSC, 80 μl of 10% SDS, 40 μl of 10 mg / ml proteinase K) ) And shaken overnight while keeping the temperature at 50 ° C. Next, 20 μl of 1 mg / ml @ ribonuclease A was added, and the mixture was kept at 50 ° C. for 1 hour. Thereafter, phenol / chloroform extraction was performed twice and ethanol precipitation operation was performed once, and the precipitate was dissolved in 150 μl of a buffer solution containing 10 mM Tris-HCl (pH 7.5) and 1 mM EDTA. Thereafter, the absorbance at a wavelength of 260 nm was measured with a spectrophotometer (U-3000, manufactured by Hitachi, Ltd.), and the concentration was adjusted to 25 ng / μl by adding sterilized water to obtain a genomic DNA sample.
[0408]
c) Determination of genotype
Using two types of primer sets for detecting microsatellite markers (D4Mit15 and D4Mit219, manufactured by Research Genetics), PCR was performed under the following conditions using a thermal cycler (PTC-100, manufactured by MJ Research). 1.5 μl of 10 × PCR buffer [containing 100 mM Tris-HCl (pH 8.3), 15 mM magnesium chloride, 500 mM potassium chloride. Roche Diagnostics, Inc.], 2.4 μl of each 1.25 mM {dNTPs (manufactured by Takara Shuzo), 0.45 μl of each 6.7 μM primer, 0.35 U of Taq DNA polymerase (Roche Diagnostics) Was added to sterilized water to make 11 μl, and 4 μl of the genomic DNA prepared in b) was added to prepare a reaction solution. This reaction solution was first heated at 94 ° C. for 3 minutes, and then subjected to a temperature cycle of 94 ° C. for 30 seconds, 55 ° C. for 1 minute, 72 ° C. for 45 seconds 35 times, and further kept at 72 ° C. for 3 minutes. And kept at 4 ° C.
[0409]
To the reaction solution after the PCR, 2 µl of a dye solution (0.15% @ bromophenol blue, 50% glycerol) was added, and 5 µl of the solution was prepared on a 4% agarose gel (NuSieve @ 3: 1 agarose (manufactured by FMC Bioproducts)). Was electrophoresed at 150 V at room temperature for 1.5 to 2 hours. After staining the gel after electrophoresis with ethidium bromide, a band corresponding to the target DNA was confirmed under ultraviolet irradiation. An individual having a genome in which two kinds of fragments of about 280 bp and about 300 bp are amplified by the primer set D4Mit15 and two kinds of fragments of about 110 bp and about 120 bp are amplified by the D4Mit219 is referred to as “heterolipidemia mutant gene”. An individual having a genome in which only a fragment of about 300 bp was amplified by D4Mit15 and only a fragment of about 120 bp was amplified by D4Mit219 was defined as "hypolipidemic mutant gene homozygous".
[0410]
2) Creation of hypolipidemic mutant gene homo / ApoE homo deficient individuals
a) Selection style
The C57BL / 6J-hypolipidemic mouse and apolipoprotein E (hereinafter referred to as "ApoE") homo-deficient mouse (Zhang, SH, et al. (1992) Science 258, obtained in 1) a) above. 468-471, obtained from the Department of Internal Medicine, School of Medicine, The University of Tokyo). Next, F1 generation males and females were crossed to obtain F2 generation. Of the F2 generation, individuals homozygously deficient in ApoE and homozygous for the hypolipidemic mutant gene (hereinafter referred to as "ApoE @ KO-hypolipidemic mice") and homozygous ApoE deficient lacking the hypolipidemic mutant gene Individuals (hereinafter referred to as “ApoE @ KO mice”) were selected by determining their genotypes by the following method. All animals were bred by the method described in 1) a) above.
[0411]
b) Method for determining genotype
Genomic DNA was extracted from the mouse tail by the method described in 1) b) above. Oligonucleotide primers to detect size polymorphisms in mutant and wild-type alleles to select hypolipidemic mutant genes:
5 '-{ggctaaatag {taaaaccctg {gcg} -3'} (SEQ ID NO: 33 in the sequence listing); and
5 '-{gtgctttgctg {tctttccagt {ctt} -3'} (SEQ ID NO: 34 in Sequence Listing)
Was synthesized. When PCR is performed using this primer set, a fragment of about 240 bp is amplified for a hypolipidemic mutant gene, while a fragment of about 230 bp is amplified for a normal gene without mutation.
[0412]
In addition, an oligonucleotide primer having the following sequence for specifically amplifying a defective allele (neomycin resistance gene) in order to detect ApoE deficiency:
5 '-{agtagctcgt {cgtgacccat {ggcga} -3'} (SEQ ID NO: 35 in the sequence listing); and
5 '-{gagcggcgat} accgtaaaagc {acgagg} -3'} (SEQ ID NO: 36 in Sequence Listing)
(See Gaw, A., et al. (1995) Lab. Anim. 29, 447-449)
Were respectively synthesized. When PCR is performed using this primer set, a 200 bp fragment is amplified only when an ApoE-deficient mutation is present in the gene serving as a template.
[0413]
Furthermore, an oligonucleotide primer having the following sequence for specifically amplifying a wild-type allele without ApoE deletion:
5 '-{tcccaagtca {cacaagaact {gac} -3'} (SEQ ID NO: 37 in the sequence listing); and
5 '-{catccagaaggcctaaaagaaggca-3-3} (SEQ ID NO: 38 in Sequence Listing)
Were respectively synthesized. When PCR is performed using this primer set, a 174 bp fragment is amplified only when there is no ApoE-deficient mutation in the template gene.
[0414]
Using the three sets of primers described above, PCR and electrophoresis were performed as described in 1) c) above, and the amplified fragments were examined. As a result, individuals in which fragments of about 240 bp and 200 bp were detected were selected as "ApoE @ KO-hypolipidemic mice", and individuals in which fragments of about 230 bp and 200 bp were detected were selected as "ApoE @ KO mice".
[0415]
3) Production of homozygous hypolipidemic mutant / leptin mutant gene
a) Selection style
C57BL / 6J-hypolipidemic mouse obtained in 1) a) above and a leptin mutant gene (Lep^ob) With heterologous C57BL / 6J-Lep^obThe first generation (F1 generation) was produced from crosses with mice (Coleman, D. L. and Hummel, K. P. (1973) Diabetologia 9: 287-293, obtained from Hamamatsu Medical University Animal Research Facility). . Next, F1 generation males and females were crossed to obtain F2 generation. Of the F2 generation, an individual homozygous for both the leptin mutant gene and the hypolipidemic mutant gene (hereinafter referred to as “ob-hypolipidemic mouse”) and a homozygous leptin mutant gene not having the hypolipidemic mutant gene (hereinafter, “ob-hypolipidemic mouse”) "Ob mice") were selected by genotyping in the following manner. All animals were bred by the method described in 1) a) above.
[0416]
b) Method for determining genotype
Genomic DNA was extracted from the mouse tail by the method described in 1) b) above. An oligonucleotide primer having the following sequence for specifically amplifying a mutant allele to detect a leptin mutation:
5 '-{tgacctggag {aattctct} -3'} (SEQ ID NO: 39 in the sequence listing); and
5 '-{catccaggct {ctctggc} -3'} (SEQ ID NO: 40 in the sequence listing);
(See Namae, M. et al. (1998) Lab. Anim. Sci. 48, 103-104)
Were respectively synthesized. When PCR is performed using this primer set, a fragment of about 100 bp is amplified only when a gene serving as a template has a mutant allele.
[0417]
Furthermore, an oligonucleotide primer having the following sequence for specifically amplifying a wild-type allele without leptin mutation:
5 '-{tgacctggag {aattctcc} -3'} (SEQ ID NO: 41 in the sequence listing);
5 '-{catccaggct {ctctggc} -3'} (supra, SEQ ID NO: 40 in the sequence listing)
Were respectively synthesized. When PCR is performed using this primer set, a fragment of about 100 bp is amplified only when there is no leptin mutation in the template gene.
[0418]
PCR and electrophoresis as described in 1) c) above, using the above two sets of primers and one set of primers for selecting the hypolipidemic mutant gene described in 2) b) above Was performed to examine the amplified fragment. As a result, an individual in which a fragment of about 100 bp (leptin mutant) and a fragment of about 240 bp were detected was referred to as an “ob-hypolipidemic mouse”, and an individual in which a fragment of about 100 bp (leptin mutant) and a fragment of about 230 bp were detected was used. Selected as "ob mouse".
[0419]
4) Measurement of serum lipid and atherosclerotic lesion area
ApoE @ KO mice (3 males, 22-23 weeks old) and ApoE @ KO-hypolipidemic mice (3 males, 19-23 weeks old) were all starved under ether anesthesia after fasting for 17 hours. Blood was collected from the vein. Next, the blood was left for 30 minutes, and then centrifuged at 2000 × g and room temperature for 10 minutes to collect serum. Triglyceride E-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.) was used for serum neutral fat concentration, and cholesterol C-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.) was used for serum cholesterol concentration. It measured using.
[0420]
Also, under ether anesthesia, the chest of each mouse was incised, and the left ventricle was washed with a physiological saline solution containing 1 unit / ml of heparin. From the heart to the aortic arch, the fat was removed until the junction between the heart and the aortic valve appeared. After fat removal, they were cut just above the aorta and immersed and fixed in a 4% neutral formalin solution for 24 hours to 7 days. After fixation, a paraffin block was prepared according to a conventional method. The paraffin block was sliced with a microtome from the side of the aortic arch, and after the valve appeared on the section for the first time, five consecutive sections were collected at a thickness of 5 μm, and the next five sections were discarded. Using this as one unit, this method was sliced to a total length of 500 μm. The obtained sections were subjected to Elastica Masson staining.
[0421]
Under an optical microscope (BX-50, manufactured by Olympus Optical Co., Ltd.) equipped with a CCD camera (HC-2000, manufactured by Fuji Photo Film Co., Ltd.), a cross section of the aortic valve section subjected to Elastica Masson staining is displayed on a television. The image was displayed on a monitor, and the boundary of the lesion was traced using an image analysis program (QWin, manufactured by Leica), and the lesion area was measured. For each individual, the lesion area was measured for a total of 10 sections at intervals of 50 μm in thickness, and the total was calculated.
[0422]
The results are shown in Table 2.
[0423]
[Table 2]

From the above results, it was shown that the mutation of the gene encoding the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing suppresses the development of arteriosclerotic lesions as well as the decrease in serum lipids.
5) Measurement of blood sugar level
ApoE @ KO (9 males, 24 weeks old), ApoE @ KO-hypolipidemic mice (10 males, 24 weeks old), ob mice (12 males, 17-18 weeks old), and ob-hypolipidemic Each of the affected mice (14 males, 17-19 weeks old) was fasted for 17 hours and then blood was collected from the abdominal vena cava under ether anesthesia. Next, the blood was left for 30 minutes, and then centrifuged at 2000 G and room temperature for 10 minutes to collect serum. The blood glucose level was measured using glucose B-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.).
[0424]
The results of ApoE @ KO and ApoE @ KO-hypolipidemic mice are shown in Table 3, and the results of ob mice and ob-hypolipidemic mice are shown in Table 4.
[0425]
[Table 3]

[0426]
[Table 4]

From the above results, it was shown that the mutation of the gene encoding the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing significantly reduced hyperglycemia caused by ApoE deficiency or leptin mutation.
[0427]
Example 7 Preparation of Monoclonal Antibody
(A) Preparation of antigen protein
1) Preparation of production CHO cell line
An expression vector in which the human angiopoietin-related protein 3 cDNA was located downstream of the pSRα promoter (pME18S plasmid) was prepared. This expression vector and pSV2-dhfr (ATCC) were transformed with 10% FCS (manufactured by GibcoBRL) -MEMα (manufactured by GibcoBRL) -10 units / ml penicillin-10 μg / ml streptomycin (manufactured by GibcoBRL) CHO cells lacking dihydrofolate reductase (ATCC) and a constant strain was obtained in a nucleic acid-free medium. Next, a resistant strain capable of growing the present constant strain at a concentration of 0.08 μM of methotrexate was obtained, and a strain in which an increase in the expression level due to gene duplication was confirmed. Thereafter, strains capable of growing on a MEMα medium with nucleic acid containing 1.28, 30, and 60 μM of methotrexate were obtained, and finally 5 to 10 mg / l / 3 on a confluent serum-free cell culture flask. An angiopoietin-related protein 3 producing CHO cell line having daily productivity was obtained.
The angiopoietin-related protein 3 producing CHO cell line thus prepared was cultured to confluence, then replaced with a serum-free MEMα (manufactured by GibcoBRL) medium, and cultured for 3 days to obtain a serum-free culture supernatant.
2) Desalting, small molecule removal
This sample was desalted at 1.6 ml of Sephadex G25 (manufactured by Amersham Pharmacia Biotech) at a rate of 600 ml at 20 ml / min for 10 times to remove salts and remove small molecules. This sample was fractionated by ion exchange chromatography using FPLC (Biopilot @ system, manufactured by Amersham Pharmacia Biotech).
3) Fractionation StepI
Column: 100 ml of Qsepharose Fast Flow (manufactured by Amersham Pharmacia Biotech) was packed in XK50 (manufactured by Amersham Pharmacia Biotech).
Elution buffer:
A) 20 mM Tris-HCl (pH 7.5) -0.01% NaN3 (Sigma) -0.005% Proteinase inhibitor cocktail (Sigma) -0.005% Tween20 (Sigma)
B) 20 mM Tris-HCl-1M NaCl (pH 7.5) -0.01% NaN3 (manufactured by SIGMA)-0.005% Protease inhibitor inhibitor cocktail (manufactured by SIGMA)-0.005% @ Tween20 (manufactured by SIGMA)
Flow rate: 10 ml / min
Fraction solution: 10 ml / tube
Temperature: 4 ° C
Elution conditions: Eluent A ⇒ Eluent B (linear concentration gradient, 60 minutes)
From the obtained fractions, fractions eluted at a concentration of 0.3 to 0.45 M @NaCl were collected and combined into one solution. Using the combined solution as a sample, group-specific affinity chromatography was performed under the following conditions using FPLC (Amersham Pharmacia Biotech) under the following conditions.
4) Fractionation Step II
Column: 10 ml of Affi \ Gel \ Blue (BioRad) was packed in KX16 (manufactured by Amersham Pharmacia Biotech).
Elution buffer:
C) 20 mM Tris-HCl-0.5 M NaCl (pH 7.5) -0.01% NaN3 (manufactured by SIGMA) -0.005% Protease inhibitor inhibitor cocktail (manufactured by SIGMA) -0.005% Tween 20 (manufactured by SIGMA) Made)
D) 20 mM Tris-HCl-1M NaCl (pH 7.5) -0.01% NaN3 (manufactured by SIGMA) -0.005% Protease inhibitor inhibitor cocktail (manufactured by SIGMA) -0.005% Tween 20 (manufactured by SIGMA)
Flow rate: 1.5 ml / min
Temperature: 4 ° C
After applying the sample, pour 60 ml of the solution C).
Elution conditions: D) 100 ml of the solution was flowed and collected as one solution. The collected solution was turbid with an equal volume (100 ml) of the solution A), and a group-specific affinity chromatography using FPLC (manufactured by Amersham Pharmacia Biotech) under the following conditions was performed using the prepared 200 ml solution as a sample. The test was performed under the following conditions. 5) Fractionation Step III
Column: Lentil @ lectin @ sepharose^TM(Amersham Pharmacia Biotech) was filled into KX16 (Amersham Pharmacia Biotech) in an amount of 10 ml.
Elution buffer:
C) 20 mM Tris-HCl-0.5 M NaCl (pH 7.5) -0.01% NaN3 (manufactured by SIGMA) -0.005% Protease inhibitor inhibitor cocktail (manufactured by SIGMA) -0.005% Tween 20 (manufactured by SIGMA) Made))
F) 20 mM Tris-HCl-0.5 M NaCl-0.3 M Methylmannopyranoside (pH 7.5) -0.01% NaN3 (manufactured by SIGMA) -0.005% Protease inhibitor inhibitor cocktail (manufactured by SIGMA) -0.005 % @ Tween20 (manufactured by SIGMA))
Flow rate: 1 ml / min
Temperature: 4 ° C
After applying the sample, flow 50 ml of solution C).
Elution conditions: F) 50 ml of the solution was flowed and collected as one solution.
6) Dialysis
The collected liquid was transferred to Dialysis Tubing-1-7 / 8 in Duamer (manufactured by Gibco BRL), and Dulbecco's PBS (-) (manufactured by Nissui Pharmaceutical), 0.01% NaN3 (manufactured by SIGMA) -0. It was dialyzed overnight at 4 ° C. against 2 liters of a 1% proteinase inhibitor cocktail (manufactured by SIGMA) solution.
7) Confirmation of sample
After dialysis, the concentration of the collected sample was measured by DC protein assay kit (Bio Rad), western analysis, and SDS-PAGE, and the protein content of “angiopoietin-related protein 3” was measured. The endotoxin concentration was measured with an endotoxin measurement kit (Seikagaku Corporation) and confirmed to be 20 mg / mg or less. The protein thus purified was used as an antigen.
[0428]
(B) Preparation of immunized mouse splenocytes
0.5 ml of Freund's complete adjuvant (Difco) was added as an adjuvant to three 8-week-old BALB / c male mice, and 0.5 ml (corresponding to 100 μg) of angiopoietin-related protein 3 purified in (a) was added thereto. After an emulsion was formed in the injection syringe, immunization was performed by subcutaneous injection. In the second and subsequent immunizations, Freund's incomplete adjuvant (Difco) was used as an adjuvant. Immunization was performed four times approximately every two weeks. After the second immunization, blood was collected from the fundus venous plexus immediately before the immunization, and the anti- "angiopoietin-related protein 3" antibody titer in the serum was determined by the solid phase method described below. It was examined by an immunoassay (ELISA method).
[0429]
(Solid-phase enzyme immunoassay) Dispense 50 ng of purified “angiopoietin-related protein 3” into each well of a 96-well ELISA plate (Maxisorp; Nunc), and let stand at 4 ° C. overnight to allow the antigen to plate well. The bottom was coated. After the plate was washed three times with PBS containing 0.1% (v / v) Tween 20, BSA adjusted to 10 μg / ml with PBS was added at 100 μl / well and left at room temperature for 1 hour. After further washing three times with PBS containing 0.1% (v / v) Tween 20, 30 to 100 μl / well of a serially diluted sample (mouse serum) as the first antibody was dispensed and left at room temperature for 1 hour. did. Thereafter, the plate was washed three times with PBS containing 0.1% (v / v) Tween 20, and a 3000-fold diluted solution of peroxidase-labeled goat anti-mouse IgG (Bio-Rad) was dispensed as a second antibody at 100 μl / well, and the mixture was added at room temperature. And leave for 1-2 hours. After washing three times with PBS containing 0.1% (v / v) Tween 20, a TMB peroxidase substrate solution (Bio-Rad; TMB peroxidase kit) was added at 100 μl / well, and the mixture was incubated at room temperature for 30 minutes. 100 μl / well of 1N sulfuric acid was dispensed to stop the peroxidase reaction, and the absorbance at 450 nm was measured with a microplate reader (Bio-Rad) to calculate the antibody titer.
[0430]
(C) Preparation of mouse myeloma cells
8-azaguanine-resistant mouse myeloma cells P3-X63-Ag8.653 (653) were cultured in TIL medium and 2 × 10⁷The above cells were obtained.
[0431]
(D) Preparation of hybridoma
Immune mouse spleen cells 1.4 × 10 10 well washed with DMEM (Nissui Pharmaceutical)⁸Individual and mouse myeloma cell P3-X63-Ag8.653 (653) 1.5 × 10⁷Individuals were mixed and centrifuged at 800 rpm for 6 minutes. After splenocytes obtained as a precipitate and a cell group in which P3-X63-Ag8.653 (653) were mixed well, a polyethylene glycol 4000 (PEG4000) solution previously adjusted to 50% with DMEM was added at 2 ml / min. The mixture was added dropwise for 1 minute while stirring. Next, DMEM was added at 2 ml / min for 1 minute in the same manner, and this operation was repeated once. An additional 16 ml of DMEM was added over 3 minutes and centrifuged at 800 rpm for 6 minutes. After centrifugation, the supernatant was discarded and suspended in 35 ml of TIL medium.
[0432]
(E) Selection of hybridoma group
The suspension was dispensed at 100 μl / well into a 96-well plate (Coaster) and cultured at 37 ° C. in a 7.5% CO 2 incubator. Seven days later, HAT medium was added at 50 μl / well. Four days later, the same amount of HAT medium was added in the same manner. Three days later, a part of the culture supernatant was collected from the wells in which the fused cells that had grown in a colony were formed, and the anti- "angiopoietin-related protein 3" antibody titer was determined by the same enzyme-linked immunosorbent assay as in (b) above. It was measured and at the same time replaced with HT medium.
[0433]
(F) Cloning
Cloning was repeated three times in the wells in which the antibody titer was recognized by the limiting dilution method, and the clone in which the antibody titer was stably recognized was selected as the anti- "angiopoietin-related protein 3" monoclonal antibody-producing hybridoma strain. One of the hybridoma clones obtained here was named 45B1 and deposited with the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary on March 13, 2002, under the deposit number FERM @ BP-7963.
[0434]
(G) Purification of monoclonal antibody
The culture supernatant of the hybridoma 45B1 strain producing anti- "angiopoietin-related protein 3" monoclonal antibody is collected, sterilized by filtration through a 0.22 μm filter (Millipore), and then purified using MAbTrap @ GII (Pharmacia). did. Through the above steps, an anti- "angiopoietin-related protein 3" monoclonal antibody MAb45B1 was obtained.
[0435]
(H) 抗原 Antigen specificity of monoclonal antibody
Mab45B1 was identified as “angiopoietin” by ELISA using the serum-free culture supernatant of the “Angiopoietin-related protein 3” -producing CHO cell line and the serum-free culture supernatant of CHO cells prepared in 1) of (a) above as adsorbed antigens, respectively. Specific protein 3 ". That is, 100 μl of the serum-free culture supernatant of the CHO cell line producing “angiopoietin-related protein 3” and 100 μl of the serum-free culture supernatant of the CHO cells were added to each well of a 96-well ELISA plate (Maxisorp; Nunc). After dispensing, the mixture was kept at 4 ° C. overnight, and the antibody was adsorbed to the bottom of the plate well. After washing each well three times with PBS, 200 μl / well of PBS containing 3% (w / v) BSA was added, and the mixture was incubated at 4 ° C. overnight. Thereafter, each well was washed twice with PBS containing 0.05% (v / v) Tween 20 (hereinafter referred to as “PBST”), and serially diluted with PBS containing 1% (w / v) BSA. The antibody-producing hybridoma 45B1 culture supernatant was dispensed at 100 μl / well and allowed to react at room temperature for 1 hour. Thereafter, each well was washed 5 times with PBS containing 0.05% (v / v) Tween 20, and HRP-labeled anti-mouse IgG diluted 1/5000 with PBS containing 1% (w / v) BSA. An antibody (Amersham Bioscience) was dispensed at 100 μl / well and allowed to react at 37 ° C. for 1 hour. Further, each well was washed five times with PBS containing 0.05% (v / v) Tween 20, ABTS peroxidase substrate solution (Roche Diagnostics) was added at 100 μl / well, and incubated at room temperature for 30 minutes. Thereafter, the absorption at 415 nm was measured. High absorbance was observed in the wells to which the serum-free culture supernatant of the “Angiopoietin-related protein 3” -producing CHO cell line was adsorbed, and the absorbance decreased with dilution of the antibody, whereas the serum-free culture supernatant of CHO cells was adsorbed. The wells showed low absorbance regardless of antibody dilution.
[0436]
(I) Classification of monoclonal antibodies
As a result of analysis using a mouse monoclonal antibody isotyping kit (Amersham), this antibody was identified as an antibody of the IgG1 subclass.
[0437]
Embodiment 8 FIG. "Angiopoietin-related protein 3" quantification system using MAb45B1
(A) Preparation of rabbit anti- "angiopoietin-related protein 3" polyclonal antibody
As an adjuvant, 0.5 ml of Freund's complete adjuvant (Difco) was added to 0.5 ml (corresponding to 100 μg) of “angiopoietin-related protein 3” purified in (a) of Example 7, and the emulsion was prepared in a syringe with a connecting needle. After inoculation, male rabbits were inoculated subcutaneously. In the second and subsequent immunizations, Freund's incomplete adjuvant (Difco) was used as an adjuvant. Immunization was performed eight times approximately every two weeks. After the inoculation, blood was partially collected over time to check for an increase in antibody titer. Seven days after the eighth immunization, whole blood was collected under anesthesia to obtain 53.5 ml of immune serum. 20 ml of the serum was purified using the MAbTrap @ GII kit, yielding 115 ml of 1.64 mg / ml @ IgG.
[0438]
Through the above steps, a rabbit anti- "angiopoietin-related protein 3" polyclonal antibody (hereinafter referred to as "No. 1 antibody") was obtained.
[0439]
(B) Preparation of HRP-labeled Fab′-modified polyclonal antibody
1) Preparation of F (ab ') 2
30 ml of 1.64 mg / ml @ No. One antibody was dialyzed against a 0.1 M sodium acetate buffer (containing pH 4.5４0.1 M NaCl) and concentrated using CENTRICON-30 (Amicon). The concentration was adjusted to 6 mg / ml with a 0.1 M sodium acetate buffer (containing pH 4.5 / 0.1 M / NaCl). 20 μl (0.2 mg) of porcine stomach and pepsin (Sigma) dissolved in 10 mg / ml with a 0.1 M sodium acetate buffer (pH 4.5, containing 0.1 M NaCl) was added to 0.5 ml of 6 mg / ml No. . One antibody was added and reacted at 37 ° C. for 6 hours. The reaction solution was dialyzed against a 0.1 M sodium phosphate buffer (pH 7.0), and then an Ultragel AcAc 44 (Invitrogen) column (1.5 cm x 45 cm)-0.1 M sodium phosphate buffer (pH 7.0). Gel filtration was performed at a flow rate of 0.35 ml / min and a fraction of 0.5 to 1.0 ml. No. 1 Antibody F (ab ') 2F fraction was confirmed by absorbance at 280 nm and concentrated using CENTRICON-30 (Amicon). These operations were repeated until 11.2 ml of 2.5 mg / ml @ No. 1'F (ab ') 2 antibody was obtained.
[0440]
2) Preparation of Fab '
11.2 ml of 2.5 mg / ml @ No. The 1F (ab ') 2 antibody was dialyzed against a 0.1 M sodium phosphate buffer (pH 6.0) and concentrated using CENTRICON-30 (Amicon). The concentration was adjusted to 5 mg / ml with a 0.1 M sodium phosphate buffer (pH 6.0). 0.45 ml of 5 mg / ml @ No. 50 μl of 0.1 M sodium phosphate buffer (pH 6.0) containing 0.1 M of 2-mercaptoethylamine and 50 mM of EDTA was added to the 1F (ab ′) 2 antibody, and reacted at 37 ° C. for 90 minutes. The reaction solution was an Ultragel AcA 44 (Invitrogen) column (1.5 x 45 cm)-0.1 M sodium phosphate buffer (pH 6.0, containing 5 mM EDTA), flow rate 0.35 ml / min, fraction 0.1. Gel filtration was performed on 5 to 1.0 ml. No. After confirming the presence of 1 'Fab' antibody by absorbance at 280 nm, it was concentrated using CENTRICON-30 (Amicon).
These operations were repeated, and finally 11.4 ml of 1.4 mg / ml @ No. 1
Fab 'antibody was obtained.
[0441]
3) Preparation of maleimide peroxidase
36.8 mg of horseradish peroxidase (Sigma) was dissolved at 6.66 mg / ml in a 0.1 M sodium phosphate buffer (pH 7.0). In N, N-dimethylformamide, N-succinimidyl-4- (N-maleimidomethyl) @ cyclohexane-1-1-1carboxylate (Sigma) was dissolved to adjust the concentration to 70 mM. To 300 μl of 6.66 mg / ml horseradish peroxidase was added 30 μl of 70 mM {N-succinimidyl-4- (N-maleimidomethyl)} cyclohexane-1-1-carboxylate, and reacted at 30 ° C. for 30 minutes. After removing the precipitate from the reaction solution by centrifugation, Sephadex® G-25 column (1 × 30 cm) -0.1 M sodium phosphate buffer (pH 6.0), flow rate of 0.4 to 0.6 ml / min, one fraction Gel filtration was performed between 0.5 and 1.0 ml. The fraction containing peroxidase was confirmed by absorbance at 403 nm, and concentrated using CENTRICON-30 (Amicon). These operations were repeated to finally obtain 5.2 ml of 6.5 mg / ml maleimide peroxidase.
[0442]
4) Reaction of maleimidated peroxidase with Fab '
0.71 ml of 1.4 mg / ml No. 0.28 ml of 6.5 mg / ml maleimidated peroxidase was added to 1 Fab ′ antibody, and the mixture was reacted at 4 ° C. for 20 hours. After the reaction, 10 μl of 50 mM ΔN-ethylmaleimide was added. Gel filtration was performed using an Ultragel @ AcA44 (Invitrogen) column (1.5 × 45 to 100 cm) -0.1 M sodium phosphate buffer (pH 6.5), a flow rate of 0.35 ml / min, and a fraction of 1.0 ml. The absorbance at 280 nm and 403 nm of each fraction was measured, and the peroxidase activity of each fraction was measured. In each fraction, 0.2% (w / v) @thimerosal (Sigma) was added to a final concentration of 0.002%, and 10% (w / v) bovine serum albumin was added to a final concentration of 0.1%. And stored at 4 ° C. The concentration of peroxidase-labeled Fab ′ was calculated from the peroxidase activity or the absorbance at 403 nm, and stored at a concentration of 10 μg / ml or more. These operations were repeated until 8.6 ml of 0.78 mg / ml HRP-labeled No. 1 Fab ′ antibody was obtained and used in the following experiments.
[0443]
(C) Preparation of "Angiopoietin-related protein 3" quantitative system
The purified MAb45B1 obtained from the culture supernatant in (g) of Example 7 was diluted to 2 μg / ml with 0.1 M carbonate buffer (pH 9.6), and 100 μl of each was diluted to a 96-well ELISA plate (Maxisorp). ; Nunc), and incubated at 4 ° C. overnight to adsorb the antibody to the bottom of the wells of the plate. After washing each well three times with PBS, 200 μl / well of PBS containing 0.1% (w / v) BSA was added, and the mixture was incubated at 4 ° C. overnight. Each well was washed twice with PBS containing 0.05% (v / v) Tween 20 (hereinafter referred to as “PBST”), to which 1% (w / v) BSA and 0.05% (v / v) were added. ), A sample (purified “angiopoietin-related protein 3” or human plasma) serially diluted with PBS containing Tween 20 was dispensed at 100 μl / well and incubated at 4 ° C. overnight. Wash each well seven times with PBS containing 0.05% (v / v) Tween 20, and 0.5 μg with PBS containing 1% (w / v) BSA and 0.05% (v / v) Tween 20. / Ml diluted in HRP-labeled No. 1 Fab ′ antibody was dispensed at 100 μl / well and allowed to react at 37 ° C. for 30 minutes. Wash each well 9 times with PBS containing 0.05% (v / v) Tween 20, add 100 μl / well of TMB peroxidase substrate solution (Bio-Rad @ TMB peroxidase kit), and keep at room temperature for 30 minutes After that, 1N sulfuric acid was dispensed at 100 μl / well at a time to stop the peroxidase reaction, and the absorbance at 450 nm was measured with a microplate reader (manufactured by Bio-Rad). As a result, since the concentration of the purified “angiopoietin-related protein 3” was directly proportional to the measurement result, it was found that “angiopoietin-related protein 3” could be quantitatively determined with high linearity by this ELISA system (see FIG. 6). Even when human plasma was used as a sample, detection was possible by the ELISA system.
[0444]
Embodiment 9 FIG.産生 Production of recombinant polypeptide using adenovirus vector
(1) Preparation of DNA
1) Insertion of Kozak sequence
In the method for testing a therapeutic or prophylactic agent for hyperlipidemia described in Reference Example 2, a method for obtaining a cDNA clone obtained by adding a Kozak consensus sequence to a cDNA cloned for detecting a gene expression level. , The following nucleotide sequence:
5 '-{gccaccatggtcacaatttagctccttctttttttattg} -3' (SEQ ID NO: 42 in Sequence Listing)
And the following nucleotide sequence to which a specific recognition sequence of the restriction enzyme XhoI has been added:
5 '-{tctagagcctcgttcattcaaaagttttctgaatctg} -3' (SEQ ID NO: 43 in Sequence Listing)
Was synthesized. Here, a part of the primer shown in SEQ ID NO: 42 is nucleotide numbers 25 to 55 of the cDNA of “angiopoietin-related protein 3” shown in SEQ ID NO: 53 in the sequence listing, and a part of the primer shown in SEQ ID NO: 43 Corresponds to the antisense of nucleotide numbers 1385 to 1415 of SEQ ID NO: 53 in the sequence listing.
[0445]
Next, a human cDNA inserted into the recombinant phagemid pTrip / h55-1 obtained by the method described in Reference Example 2 (disclosed as a nucleotide sequence encoding “angiopoietin-related protein 3” in the Genbank database) Using about 0.4 μg / ml as a template, the above primers (0.4 mM each), Tbr EXT DNA polymerase (manufactured by Finzyme), 1 × PCR reaction buffer (10 × buffer attached to the DNA polymerase was reduced to 1/10 Amount) PCR was performed in the presence of 2 mM magnesium chloride and 0.4 mM dNTPs each. PCR was performed by heating the sample at 94 ° C. for 3 minutes, and then performing a temperature cycle of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, 72 ° C. for 1 minute 30 times, and finally a temperature of 68 ° C. for 2 minutes. .
[0446]
The amplified fragment (approximately 1.4 kbp) obtained as a result of the PCR was inserted into a plasmid vector pCR2.1 attached to a TA cloning kit (manufactured by Invitrogen), and a plasmid clone (pCR / KhAP5) containing the inserted fragment was simply isolated. Released. The sequence of the insert was analyzed using a DNA sequencer (ABI PRISM 3700, manufactured by Applied Biosystems), and it was confirmed that no mutation occurred in the insert during PCR.
[0447]
Next, pCR / KhAP5 was digested with restriction enzymes EcoRI and XbaI, and a fragment containing cDNA encoding human "angiopoietin-related protein 3" was digested with restriction enzymes EcoRI and XbaI. Recombinant plasmid pME18S / KhAP5 was obtained by inserting into animal cell expression vector pME18S.
[0448]
2) Preparation of partially deleted mutant
a) Mutants lacking the carboxyl terminal region
The recombinant plasmid pME18S / KhAP5 obtained in the above 1) is digested with the restriction enzyme SpeI, the cut ends are blunt-ended with T4 DNA polymerase, and then DNA ligase reaction is performed to ligate both ends again. A recombinant plasmid pME18S / in which a stop codon (tag) was inserted after a codon (atc) encoding an amino acid residue at position 207 from the amino terminus (serine) in the amino acid sequence containing the signal peptide of "angiopoietin-related protein 3" KhAP5 @ CC was constructed. The nucleotide sequence of the insert in this recombinant plasmid is as shown in SEQ ID NO: 11 in the sequence listing.
[0449]
b) Mutants lacking the amino-terminal region
The recombinant plasmid pME18S / KhAP5 obtained in 1) above is digested with restriction enzymes BclI and SpeI, and the amino acid residue at position 207 from the amino terminus in the amino acid sequence containing the signal peptide of human "angiopoietin-related protein 3" ( After removal of the fragment containing only the cDNA sequence coding for serine), the cut end of the T4 DNA polymerase was blunt-ended, and a DNA ligase reaction was performed to ligate both ends again. By this operation, in pME18S / KhAP5, the nucleotide sequence encoding the 21st to 206th amino acids in the amino acid sequence containing the signal peptide of human “angiopoietin-related protein 3” is replaced with a codon (cct) encoding proline. The constructed recombinant plasmid pME18S / KhAP5ΔFLD was constructed. The nucleotide sequence of the insert in this recombinant plasmid is as shown in SEQ ID NO: 13 of the Sequence Listing.
[0450]
(2) Preparation of recombinant adenovirus
A recombinant encoding a partial region deletion mutant of the “angiopoietin-related protein 3” gene present in the recombinant plasmids pME18S / KhAP5ΔCC and pME18S / KhAP5ΔFLD obtained in the above (1) was integrated. The adenovirus was prepared using an adenovirus expression vector kit (Takara Shuzo Co., Ltd.) according to the method described below.
[0451]
First, the recombinant plasmids pME18S / KhAP5ΔCC and pME18S / KhAP5ΔFLD obtained in (1) above were digested with restriction enzymes EcoRI and XbaI, respectively, to obtain about 1.4 kb and about 0.9 kb, respectively. The DNA fragment was recovered and the cut ends were blunted. Next, these fragments were respectively inserted into the SwaI cleavage site of the cosmid vector pAxCAwt designed to express the inserted gene by the cytomegalovirus enhancer and the chicken β-actin promoter, and the recombinant cosmid pAxCA / KhAP5 CC and pAxCA / KhAP5 @ FLD was prepared.
[0452]
These recombinant cosmid DNA and terminal protein-coupled virus DNA (DNA-TPC, attached to the adenovirus expression vector kit) were co-transfected into 293 cells (ATCC CRL1573) using a calcium phosphate transfection system (manufactured by Lifetech). Thus, the recombinant adenoviruses Ad / KhAP5ΔCC and Ad / KhAP5ΔFLD were isolated and further amplified in 293 cells. Recovery of the amplified virus from the 293 cells was performed by first sonicating the virus-infected 293 cells for 30 seconds × 4 times (using B-1200 manufactured by Branson) or repeating freeze-thawing three times, and then Purification by cesium chloride density gradient centrifugation was repeated twice. The obtained virus solution was dialyzed at 4 ° C. against PBS supplemented with 10% Δglycerol, and then stored frozen at −70 ° C. or lower until use.
[0453]
(3) Confirmation of expression of target protein by recombinant adenovirus
HeLa cells were infected with the recombinant adenovirus obtained by the method described in (2) above, and the expression of the target protein was confirmed.
[0454]
First, about 5 m of the recombinant adenovirus obtained by the method described in the above (2) was introduced into HeLa cells (ATCC @ CCL2). o. i. (Multiplicity of infection: multiplicity of infection), and cultured for 3 to 4 days in a serum-free medium (Dulbecco's modified Eagle's medium (DMEM)) to collect the culture supernatant. 1 ml of the culture supernatant is placed in a 1.5 ml Eppendorf tube, 100 μl of a trichloroacetic acid solution (1 g / ml trichloroacetic acid, 4 mg / ml deoxycholic acid) is added, and the mixture is left at room temperature for 3 minutes. Centrifuged at 15000 rpm for 5 minutes. After removing the supernatant, 0.5 ml of ice-cooled acetone was added and stirred, and the mixture was centrifuged at 15000 rpm for 2 minutes using a desktop centrifuge. The supernatant was removed, 0.5 ml of ice-cooled acetone was added to the precipitate again, and the mixture was stirred. The mixture was centrifuged at 15000 rpm for 2 minutes using a tabletop centrifuge, the supernatant was removed, and the precipitate was dried under vacuum. Each of the precipitates was dissolved in 10 μl of distilled water, mixed with an SDS-PAGE sample buffer solution (manufactured by Bio-Rad) containing 2-mercaptoethanol, and heated at 99 ° C. for 5 minutes to prepare a sample for electrophoresis. Prepared. These samples were subjected to electrophoresis on a polyacrylamide density gradient gel having a gel concentration of 4 to 20% (electrophoresis buffer: 25 mM Tris, 192 mM glycine, 0.1% SDS), and then a transfer buffer (25 mM Tris, 192 mM glycine). , 20% methanol) at 4 ° C. for 1 hour at 200 mA on a nitrocellulose membrane.
[0455]
The transferred membrane was immersed in a PBS solution containing 0.5% skim milk, blocked at 4 ° C overnight, and washed three times with a washing solution (0.05% Tween 20-PBS). The membrane is then placed in a solution of about 270 ng / ml of 55-1-N1 antibody or about 1 μg / ml of 55-1-C1 antibody and 5% {fetal bovine serum in 0.05%} Tween 20-PBS at room temperature. After incubation for 1 hour, the plate was washed three times with a washing solution. These antibodies were all obtained by the method described in Example 2. The 55-1-N1 antibody is an antibody that specifically binds to the amino terminal side of human “angiopoietin-related protein 3”, and the 55-1-C1 antibody is an antibody that specifically binds to the carboxyl terminal side.
[0456]
Further, the membrane was incubated at room temperature for 1 hour at room temperature in a reaction solution obtained by diluting a horseradish peroxidase-labeled goat anti-rabbit IgG (H + L) (manufactured by Bio-Rad) with 5% {0.05% containing fetal bovine serum} 2000 times in Tween 20-PBS. After that, it was washed 5 times with a washing solution. This membrane is placed on a wrap film, immersed in a luminescence reagent solution (ECL Western detection reagent, manufactured by Amersham Pharmacia Biotech) for 1 minute, and left at room temperature for 1 hour to attenuate the background. The film was exposed (10 seconds). As a result of developing this X-ray film, it was confirmed that each of the target polypeptides was produced in HeLa cells infected with the recombinant adenovirus.
[0457]
(4) KK / inoculated with recombinant adenovirusSnkMeasurement of blood triglyceride concentration in mice
The recombinant adenoviruses Ad / KhAP5 @ CC and Ad / KhAP5 @ FLD purified above were mixed with PBS containing 10% glycerol at 2.0 × 10 5¹⁰pfu / ml and 1.0 × 10¹⁰pfu / ml, each of three 19-week-old male KK /SnkMice were inoculated by tail vein injection of 300 μl each. As a negative control, three 19-week-old male KK /SnkMice were inoculated by tail vein injection.
[0458]
One day after the inoculation, blood was collected from the fundus of each mouse using a hematocrit tube, centrifuged at 5200 rpm for 15 minutes using a tabletop centrifuge to separate plasma, and a kit (Triglyceride E-Test Wako, manufactured by Wako Pharmaceutical Co., Ltd.) was used. Neutral fat concentration was measured.
[0459]
As a result, no significant difference was observed in the neutral fat concentration in the blood between the group of mice inoculated with Ad / {KhAP5} FLD and the group of mice inoculated with PBS supplemented with 10% glycerol (all 50 mg / dL or less). In the group of mice inoculated with / KhAP5 顕 著 な CC, a remarkable increase in the concentration of neutral fat in blood was observed (3000 mg / dL or more).
[0460]
In the method of this example, a test substance is administered to a mouse inoculated with, for example, Ad / KhAP5ΔCC, and the change in blood triglyceride concentration is examined to select from hyperlipidemia, arteriosclerosis, and hyperglycemia. Testing of therapeutic or prophylactic agents for one or more diseases can be performed.
[0461]
Embodiment 10 FIG.評 価 Evaluation of fusion protein activity
(1) Production of transformed E. coli expressing fusion protein
Glutathione S. transferase (hereinafter “GST”) is added to the amino terminus of the polypeptide consisting of amino acid Nos. 17 to 207, 17 to 195, or 17 to 147 of SEQ ID NO: 12 in the sequence listing. (Hereinafter referred to as “GST-CC”, “GST-CCdel” and “GST-C1”, respectively, in that order) were prepared according to the method described below.
[0462]
First, the following nucleotide sequence was used as a primer for PCR:
5 '-{cggaattccctccagaatttatcaag} -3' (sense primer shared by GST-CC, GST-CCdel and GST-C1; SEQ ID NO: 44 in Sequence Listing);
5 '-{ccgctcgagactagtcccttctgagctg} -3' (antisense primer for GST-CC; SEQ ID NO: 45 in the sequence listing); and
5 '-{ccgctcgagttgactatgctgttggtt} -3' (antisense primer for GST-CCdel; SEQ ID NO: 46 in the sequence listing); and
5 '-{ccgctcgaggttagtagtagtgctctttc} -3' (antisense primer for GST-C1; SEQ ID NO: 47 in Sequence Listing) was synthesized.
[0463]
Next, using the cDNA (pMEh55-1) described in Example 3 as a template, a sense primer and an antisense primer (0.2 μM each), Tbr EXT DNA polymerase, 1 × PCR buffer (10 × attached to Tbr EXT DNA polymerase) PCR was performed in the presence of 2 mM magnesium chloride and 0.4 mM dNTPs each. PCR was performed by heating the sample at 94 ° C. for 3 minutes, followed by 30 temperature cycles of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, 72 ° C. for 1 minute, and finally 72 ° C. for 5 minutes. .
[0464]
In the PCR, DNA fragments of about 580 bp (using antisense primers for GST-CC), about 540 bp (using antisense primers for GST-CCdel) and about 400 bp (using antisense primers for GST-C1) were amplified. The amplified DNA fragments were recovered and each was digested with EcoRI and XhoI. Plasmid vector pGEX-4T-2 (containing the gene encoding GST, digested with EcoRI and XhoI, and then dephosphorylated at the cut ends using bovine intestinal alkaline phosphatase (Takara Shuzo Co., Ltd.). DNA fragments digested with EcoRI and XhoI were respectively inserted into the DNA fragments into a plasmid (Pharmacia Biotech), and three plasmid clones containing the three inserts were isolated. A plasmid clone having a fragment amplified with the antisense primer for GST-CC was used for pGST-CC, and a plasmid clone having a fragment amplified using the antisense primer for GST-CCdel was used for pGST-CCdel and antisense for GST-C1. The plasmid clone having the fragment amplified using the primer was designated as pGST-C1.
[0465]
Escherichia coli strain JM109 was transformed with each of the plasmid clones pGST-CC, pGST-CCdel, pGST-C1, or a control vector pGEX-4T-2 for expressing only GST without an insert fragment. For transformation, the plasmid was added to competent cells of Escherichia coli JM109, allowed to stand on ice for 30 minutes, kept at 42 ° C for 30 seconds, again allowed to stand on ice for 2 minutes, and shake-cultured at 37 ° C for 1 hour. This was done by doing The transformed culture was spread on an LB agar plate supplemented with ampicillin, and colonies of the resulting recombinants were picked up and cultured in liquid to confirm the expression of the recombinant protein induced by IPTG.
[0466]
(2) Preparation of fusion protein
1) Culture of transformed Escherichia coli
Transformed E. coli cells harboring plasmid clones pGST-CC, pGST-CCdel, pGST-C1 and control vector pGEX-4T-2 were each inoculated into LB medium containing ampicillin, and cultured at 37 ° C. for 18 hours with shaking. A part of the culture solution was added to a fresh medium, and shaking culture was further performed at 37 ° C. When the absorbance at 600 nm reached 0.5-0.7, isopropyl thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM, and shaking culture was continued for another 4 hours.
[0467]
2) Preparation of cell lysate from E. coli carrying pGEX-4T-2
A culture solution of Escherichia coli transformed with the control vector pGEX-4T-2 was centrifuged at 4 ° C. and 6000 rpm for 10 minutes to collect the precipitated cells. The collected cells were suspended in a buffer for sonication (50 mM Tris-hydrochloric acid (pH 8.0), 50 mM sodium chloride, 1 mM EDTA, 1 mM DTT), and the suspension was incubated at 4 ° C. and 6000 rpm for 10 minutes. Centrifuged. The precipitate was frozen at -80 ° C, then thawed at room temperature and resuspended in sonication buffer. The suspension was treated in an ice bath with an ultrasonic crusher (UD-201 manufactured by Tommy Seiko Co., Ltd.) for 5 minutes under conditions of output = 5 and duty = 50. Triton @ X-100 was added to the crushed liquid after the ultrasonic treatment so as to have a final concentration of 1%, mixed, and centrifuged at 4 ° C. and 10,000 rpm for 30 minutes to collect the supernatant. The supernatant was passed through a GSTrap column (Amersham Pharmacia Biotech).
[0468]
3) Preparation of cell lysate from E. coli carrying pGST-CC, pGST-CCdel, and pGST-C1
Each culture of Escherichia coli transformed with the recombinant plasmids pGST-CC, pGST-CCdel, and pGST-C1 was centrifuged at 4 ° C. and 5000 rpm for 15 minutes, and the precipitated cells were collected. The operation of suspending the precipitated cells in a washing buffer (0.5% Triton X-100, 1 mM EDTA) and centrifuging at 5000 rpm for 10 minutes at 4 ° C was repeated three times. The obtained cells were dissolved in an 8M urea solution. After leaving this solution at room temperature for 1 hour, centrifugation was performed at 4 ° C. and 13000 rpm for 30 minutes to collect the supernatant. Three types of supernatants obtained from cultures derived from Escherichia coli transformed with the recombinant plasmids pGST-CC, pGST-CCdel and pGST-C1 were dialyzed. Dialysis was performed at 4 ° C. for 1 hour against a 4M urea solution, 1 hour at 4 ° C. against a 2M urea solution, 1 hour at 4 ° C. against a sonication buffer, and At 4 ° C. overnight. After the dialysis, the mixture was centrifuged at 4 ° C. and 12,000 rpm for 30 minutes, and the supernatant was passed through a GSTrap column.
[0469]
4) Purification of fusion protein
Through the above steps, GST, a fusion protein with GST, GST-CC, GST-C1, or GST-CCdel, PBS was flowed through the GST column to which the GST was adsorbed, and the inside of the column was washed to remove impurities. Next, GST, a fusion protein with GST, GST-CC, GST-C1, or GST-CCdel was eluted by flowing a 10 mM reduced glutathione solution. After a part of the eluate was separated by SDS-PAGE electrophoresis, the gel was subjected to Coomassie staining to confirm the presence of the protein. The eluate was dialyzed against PBS at 4 ° C. overnight. Here, the protein purified from the transformant carrying the plasmid pGEX-4T-2 is GST, and similarly, proteins derived from pGST-CC, pGST-C1, and pGST-CCdel are GST-CC and GST, respectively. -C1, GST-CCdel.
[0470]
(3) Blood neutral fat concentration increasing activity
GST, GST-CC, GST-C1, GST-CCdel and GST-C1 obtained in the above (2) were adjusted to a protein concentration of about 1 mg / ml with PBS, and then 3, 4, 2, and Five 19-week-old male KK /SnkMice were inoculated by tail vein injection of 300 μl each. Blood was collected before and 30 minutes after ingestion. Blood was collected from the fundus of each mouse with a hematocrit tube, centrifuged at 5200 rpm for 15 minutes with a tabletop centrifuge to separate plasma, and the blood neutral fat (triacylglycerol) concentration before and after inoculation was measured.
[0471]
The result is shown in FIG. In the graph of FIG. 8, the vertical axis indicates the amount of neutral fat (triacylglycerol) in 100 ml of plasma. The horizontal axis indicates each fusion protein. In the GST-inoculated mouse group ("GST" in FIG. 8), no difference was observed before and after inoculation, whereas in the GST-C1-inoculated mouse group ("C1" in FIG. 8), the blood neutral fat concentration was slightly increased. A rise was noted. On the other hand, in the group of mice inoculated with GST-CC and GST-CCdel (“CC” and “CCdel” in FIG. 8), a remarkable increase in blood neutral fat concentration was observed.
[0472]
In the method of the present example, for example, a test substance is administered to a mouse inoculated with GST-CC or CCdel, and a change in blood triglyceride concentration is examined to select from hyperlipidemia, arteriosclerosis, and hyperglycemia. Testing of a therapeutic or prophylactic agent for one or more diseases to be performed can be performed.
[0473]
Embodiment 11 FIG. LPL activity measurement method
(1) Method using rat adipocyte-derived LPL
1) Preparation of LPL sample
Rat white adipose precursor cells (purchased from Hokudo) were cultured in a growth medium [10% fetal calf serum (hereinafter referred to as “FCS”), 100 units / ml penicillin, 100 μg / ml streptomycin, 17 μM pantothenic acid, 33 μM} (+)-biotin. Dulbecco's modified Eagle's medium containing 100 μM ascorbic acid, 1 μM octanoic acid and 50 nM triiodothyronine (hereinafter referred to as “DMEM”)].²) At 37 ° C. for 24 hours, and then the medium was cultured for differentiation induction (10% FCS, 100 units / ml penicillin, 100 μg / ml streptomycin, 17 μM pantothenic acid, 33 μM (+)-biotin, 100 μM ascorbic acid, 1 μM The solution was replaced with octanoic acid, 50 nM {triiodothyronine, 10 μg / ml in insulin and 2.5 μM in dexamethasone), and the cells were further cultured at 37 ° C. for 48 hours. Then, the adipocyte maintenance medium (10% FCS, 100 U / ml penicillin, 100 μg / ml streptomycin, 17 μM pantothenic acid, 33 μM (+)-biotin, 100 μM ascorbic acid, 1 μM octanoic acid, 50 nM triiodothyronine and 10 μg / ml insulin And DMEM containing 10% {FCS, 50 ng / ml} insulin and 10 units / ml sodium heparin was added on the 2nd and 3rd days, and after culturing for 1 hour, the culture supernatant was collected. And used for measuring LPL activity.
[0474]
2) LPL activity measurement
The LPL activity was measured according to the method described in the literature (Nilsson-Ehle, P. and Schottz, M. C. (1976) J. Lipid Res. 17, 536-541). In addition, the polypeptide (angiopoietin-related protein 3) consisting of the amino acid sequence represented by amino acid numbers 17 to 460 of SEQ ID NO: 4 in the sequence listing was prepared according to the method described in Example 6.
[0475]
First, 100 μl of the culture supernatant prepared by the method described in 1) above was added to an equal volume of a substrate solution [2 mM @ glycerol tri [9,10 (n)-³H] Oleic acid (131 KBeq / μmol, manufactured by Amersham), 0.189 μg / ml {L} α-phosphatidylcholine (Sigma), 14 mg / ml {bovine serum albumin (Sigma), 140 mM} Tris-HCl (pH 8.0) , 15% (v / v) glycerol, 10% (v / v) inactivated FCS], and purified Angiopoietin-related protein 3 was added thereto, followed by incubation at 37 ° C for 120 minutes. Thereafter, 1.05 ml of 0.1 M potassium carbonate / borate buffer (pH 10.5) and 3.25 ml of methanol: chloroform: hexane = 141: 125: 100 (v / v) were added to stop the reaction. After stirring, the mixture was centrifuged at 3000 × g for 15 minutes. Water-methanol layer³H count was measured with a liquid scintillation counter. One unit of LPL activity was defined as the activity of producing 1 μmol of fatty acid in one minute. Table 5 shows the results.
[0476]
[Table 5]

Angiopoietin-related protein 3 suppressed the activity of LPL in a dose-dependent manner. In this experimental system, if a test substance is added during the lipase reaction, it can be determined whether or not the substance has an effect of regulating LPL activity.
(2) Method using plasma after intravenous injection of mouse heparin
Heparin sodium was administered to C57BL / 6j mice via the tail vein (100 units / kg), blood was collected from the abdominal aorta 10 minutes later, and heparin was intravenously injected by centrifugation to prepare plasma, which was used for measuring LPL activity. However, in the case where plasma is used, when the measurement is performed under the conditions described in (1) -2) above, both HTGL and LPL lipase activities derived from liver contained in plasma are measured. Then, in order to separate and measure them, first, a lipase reaction was performed under the conditions described in (1) -2) above, and lipase activity was measured. The obtained result was defined as “total lipase activity”. On the other hand, a lipase reaction was separately performed under conditions in which LPL was inactivated by adding sodium chloride to the reaction solution to a final concentration of 1 M, and only the lipase activity of HTGL remaining in plasma was measured. The value obtained by subtracting the HTGL activity from the total lipase activity thus obtained was defined as LPL activity. Table 6 shows the results.
[0477]
[Table 6]

Angiopoietin-related protein 3 was found to inhibit the activity of both LPL and HTGL in plasma in vitro after intravenous injection of mouse heparin. In this experimental system, if a test substance is added during the lipase reaction for measuring the total lipase activity, it can be examined whether or not the substance has an effect of regulating either LPL or HTGL activity. Similarly, if a test substance is added during a lipase reaction for measuring HTGL activity, it can be determined whether or not the substance has an effect of regulating HTGL activity.
[0478]
Embodiment 12 FIG. ＬLPL activity inhibitory effect of fusion protein
Using GST-CC, GST-CCdel or GST instead of angiopoietin-related protein 3, LPL activity was measured by the method described in Example 11, (1).
The LPL activity when GST (10 μg / ml) was added was defined as 100%, and the relative LPL activity when GST-CC or GST-CCdel was added was calculated. Table 7 shows the results.
[0479]
[Table 7]

GST-CC and GST-CCdel suppressed the activity of LPL in a dose-dependent manner. In this experimental system, if a test substance is added at the time of the lipase reaction, it can be examined whether or not the substance has an effect of regulating LPL activity.
[0480]
Embodiment 13 FIG. Preparation of histidine-tagged polypeptide and LPL inhibitory activity
(1) Preparation of histidine-tagged polypeptide
A histidine tag is inserted immediately after the codon encoding the 20th amino acid residue (aspartic acid) from the amino terminus in the amino acid sequence containing the signal peptide of “angiopoietin-related protein 3,” and the amino acids 208 and beyond from the amino terminus The sequence-deleted polypeptide KhAP5ΔCC / His was prepared according to the method described below.
[0481]
1) Insertion of histidine tag
A histidine tag (SEQ ID NO: 2 in the sequence listing) is attached at the carboxyl-terminal side of the amino acid (aspartic acid) corresponding to the 20th amino acid (aspartic acid) from the amino terminal of the amino acid sequence of the precursor of “angiopoietin-related protein 3” (the fourth amino acid sequence of the mature form). The recombinant plasmid pME18S / KhAP5 obtained in Example 9 (1) -1) was modified so that the amino acid sequence represented by amino acid Nos. 21 to 28 (26) was inserted.
[0482]
First, an oligonucleotide partially complementary is composed of the following nucleotide sequence:
5 '-{gatccgcatcatcaccatcaccat} -3' (SEQ ID NO: 48 in the sequence listing) and
5 '-{gatcatggtgatggtgatgatgcg} -3' (SEQ ID NO: 49 in Sequence Listing)
Was synthesized and phosphorylated at the 5 'end using a synthetic DNA terminal phosphorylation reagent Phosphalink (manufactured by Applied Biosystems). The annealed product was inserted into a fragment of about 4.2 kb obtained by completely digesting pME18S / KhAP5 with the restriction enzyme BclI. As a result, a plasmid clone was obtained in which the DNA in which the above-described annealed oligonucleotide was linked in 3 units was inserted into the BclI cleavage site. Next, a plasmid pME18S / KhAP5 / His + 16 was obtained by inserting a fragment of about 180 bp obtained by completely digesting pME18S / KhAP5 with BclI into one obtained by digesting this clone with BclI.
[0483]
Plasmid pME18S / KhAP5 / His + 16 contains 3 units of the sequence encoding the histidine tag, of which 2 units at the 3 'end are extra sequences linked in the opposite direction to the intended purpose. . The following nucleotide sequence was used as a sense / antisense primer in order to amplify the fragment without this sequence by PCR:
5'-gaattgatcagcatcatcaccataccacgatc-3 '(SEQ ID NO: 50 in Sequence Listing)
Was synthesized. Next, using pME18S / KhAP5 / His + 16 (0.4 μg / ml) as a template, the above primer (0.4 mM), Tbr EXT DNA polymerase (Finzyme), 1 × PCR reaction buffer (10 × PCR was performed in the presence of 2 mM magnesium chloride and 0.4 mM dNTPs each. The PCR was carried out under the following conditions: first, heating at 94 ° C. for 2 minutes, then repeating a temperature cycle of 94 ° C. for 30 seconds, 60 ° C. for 30 seconds, 72 ° C. for 30 seconds 30 times, and finally keeping the temperature at 72 ° C. for 7 minutes. Done.
[0484]
The amplified fragment (268 bp) obtained as a result of PCR was digested with BclI, and then inserted into a fragment of about 4.2 kb obtained by completely digesting pME18S / KhAP5 with BclI. By the above operation, a nucleotide sequence encoding a histidine tag (SEQ ID NO: 25 in the sequence listing) was placed at the upstream cleavage site among the two BclI cleavage sites present in the cDNA sequence inserted into pME18S / KhAP5. Thus, a recombinant plasmid pME18S / KhAP5 / His containing DNA into which nucleotides 78 to 101 were inserted was obtained.
[0485]
2) Preparation of a variant lacking the carboxyl terminal
The plasmid pME18S / KhAP5 / His obtained in the above 1) is digested with a restriction enzyme SpeI, the cut ends are blunted with T4 DNA polymerase, and a DNA ligase reaction is carried out to ligate both ends again. A stop codon (tag) is inserted after the codon (atc) encoding the 207th amino acid residue (serine) from the amino terminus in the amino acid sequence containing the signal peptide of "protein 3", and the 20th amino acid from the amino terminus A recombinant plasmid pME18S / KhAP5ΔCC / His containing DNA (SEQ ID NO: 25 in the sequence listing) in which a nucleotide sequence encoding a histidine tag was inserted immediately after a codon encoding a residue (aspartic acid).
[0486]
3) Expression in COS-1 cells
COS-1 cells (0.5 × 10 5) were placed in a 150 mm diameter tissue culture dish (manufactured by Corning).⁶The cells were suspended in 30 ml of Dulbecco's modified Eagle's medium (hereinafter, referred to as "DMEM", manufactured by Gibco) containing 10% fetal bovine serum (hereinafter, referred to as "FCS", manufactured by Gibco), and 37 ° C, 5% The cells were cultured under carbon dioxide for 3 days. Thereafter, after removing the medium, 36 μg of pME18S / KhAP5 @ CC / His was added to a transfection reagent (a turbidity of lipofectamine 2000180 μl and OPTIMEM-I solution (all manufactured by Gibco) @ 3 ml) and mixed. Transfection was performed by adding to COS-1 cells. After transfection, the cells were allowed to stand at 37 ° C. under 5% carbon dioxide for 3 days. The above operation was performed on 130 Petri dishes of COS-1 cells, and a total of about 4.4 L of culture supernatant was collected. This culture supernatant was subjected to suction filtration using a bottle top filter (manufactured by Corning) made of 0.22 μm cellulose acetate, and the filtrate was collected.
[0487]
4) Confirmation of expression level and concentration
A part of the filtrate collected in the above 3) and “Angiopoietin-related protein 3” purified by the method described in Example 6 were subjected to SDS-PAGE, and the protein in the gel was converted to a nitrocellulose membrane (Bio-Rad, Inc.). Was transferred by a wet method (0.2 A, 90 minutes, 4 ° C.). The nitrocellulose membrane was immersed in a PBS-0.05% Tween 20 solution (hereinafter referred to as "PBS-T") containing 5% skim milk (manufactured by Snow Brand Milk Products Co., Ltd.) overnight to perform blocking. Next, after washing the nitrocellulose membrane with PBS-T, Western blot was performed. The 55-N1 antibody described in Example 2 was used as the primary antibody, and a horseradish peroxidase-labeled anti-rabbit polyclonal IgG antibody (manufactured by Amersham) was used as the secondary antibody. An ECL reagent (manufactured by Amersham) was used to detect a band to which the 55-N1 antibody specifically bound. As a result, the filtrate obtained in the above 3) may contain about 1.0 to 1.5 mg / liter (4.4 to 6.6 mg in the whole filtrate) of KhAP5 @ CC / His. It was revealed.
[0488]
5) Purification of KhAP5 @ CC / His
The total amount of the filtrate obtained in the above 3) was ultraconcentrated to 440 ml using an ultrafiltration apparatus (TIFF lab scale and an ultrafiltration membrane having a pore size of 30 KDa (both manufactured by Millipore)). Further, a buffer solution containing 20 mM sodium phosphate, 0.5 M sodium chloride, 0.1% sodium azide and 0.05% protease inhibitor cocktail (manufactured by Sigma) was added to the obtained sample in an ultrafiltration apparatus. Ultrafiltration was continued while gradually adding 4 liters (pH 7.4) to obtain a sample in which the solvent was replaced with the buffer solution (liquid volume: 600 ml).
[0489]
On the other hand, nickel ions were bound to the carrier by flowing 0.1 M nickel sulfate (5 ml) at a flow rate of about 5 ml / min through a chelating column (HiTrap chelating # 5 ml column; manufactured by Amersham). After the column was washed by flowing ultrapure water, the sample was attached to an HPLC liquid sending system (manufactured by Amersham), and affinity purification of the sample was performed under the following conditions.
[0490]
solvent:
Solution A: 20 mM sodium phosphate, 0.5 M sodium chloride (pH 7.4), 0.1% sodium azide, 0.05% protease inhibitor cocktail (manufactured by Sigma)
Solution B: {20 mM sodium phosphate, 0.5 M sodium chloride, 0.5 M imidazole (pH 7.4), 0.1% sodium azide, 0.05%} protease inhibitor cocktail (manufactured by Sigma)
Flow rate: $ 5ml / min
Purification procedure: After flowing 600 ml of the sample through the column, 50 ml of a buffer solution (solution A: 80%, solution B: 20% (corresponding to 0.1 M concentration of imidazole)) was flowed to wash and remove nonadsorbed substances. Thereafter, 50 ml of solution B was passed through the column to elute the polypeptide having a histidine tag, and the eluate was collected. A solution having an imidazole concentration of 0.1 M by adding 200 ml of Solution A to this eluate was used as a second sample for affinity purification.
[0490]
Nickel ions were bound to the carrier by flowing 0.1 M nickel sulfate 5 ml at a flow rate of about 1 ml / min through a small-scale chelating column (HiTrap chelating 1 ml column, manufactured by Amersham), and then ultrapure water was added. After flushing and washing, the column was mounted on an HPLC solution delivery system, the sample was flushed, and the column was removed from the HPLC. Subsequently, a buffer of 20 mM sodium phosphate, 0.5 M sodium chloride, 0.1 M imidazole, pH 7.4 was manually passed through a 1 ml column at a flow rate of 1 ml / min to wash and remove non-adsorbed substances. Thereafter, a buffer solution of 20 mM sodium phosphate, 0.5 M sodium chloride, 0.3 M imidazole, and pH 7.4 was similarly passed at a flow rate of 1 ml / min, and the eluate was collected in 5 fractions (1 ml each) for 5 fractions (total 5 ml). .
[0492]
Each of the collected eluates was placed in a dialysis tube (manufactured by Gibco), and dialyzed against 500 mL of PBS (manufactured by Gibco). The dialysis membrane external solution, PBS, was replaced three times. A part of the last extradialysis membrane solution was collected and used as a negative control sample for evaluation of LPL activity.
[0493]
As a result of estimating the protein concentration in the collected sample using a protein assay (using a DC protein assay kit (manufactured by Bio-Rad)) or SDS-PAGE, a total of 1.8 mg of KhAP5ΔCC / His was obtained. found. In the following LPL activity evaluation, a summary of the second and third fractions obtained by the second round of affinity purification was used.
[0494]
(2) Measurement of LPL activity
Using the KhAP5ΔCC / His sample obtained in (1) 5) above instead of GST-CC, LPL activity was measured by the method described in Example 11 (1). Table 8 shows the results.
[0495]
[Table 8]

KhAP5ΔCC / His strongly inhibited LPL activity. In this experimental system, if a test substance is added during the lipase reaction, it can be determined whether or not the substance has an effect of regulating LPL activity.
[0496]
(3) Lowering effect of blood triglyceride concentration
According to the method described in Example 10, (3), KhAP5ΔCC / His wasSnkThe mice were administered tail vein (100 μg / mouse). Peripheral blood was collected from mice before and after administration and the neutral fat concentration was measured. As a result, in the mice before administration, the blood triglyceride concentration was 40.5 mg / dl, whereas it increased to 106.0 mg / dl 30 minutes after administration of KhAP5ΔCC / His. The blood neutral fat concentration of the control group to which only the solvent was administered 30 minutes after administration was 41.3 mg. From the above results, it was confirmed that KhAP5ΔCC / His significantly increased blood neutral fat concentration.
[0497]
Embodiment 14 FIG. Effect of angiopoietin-related protein 3 with a mutation in the heparin binding site on plasma triglycerides
(1) Construction of N-terminal 207 amino acid partial protein of angiopoietin-related protein-3 (ARP3) in which a mutation is introduced into a heparin-binding motif
An approximately 1.4 kbp fragment obtained by cutting the plasmid pME18S / KhAP5 @ CC / His described in Example 13-2) with EcoRI and XbaI was isolated, and the plasmid pKF18k attached to the Mutan-Super @ Express @ Km kit manufactured by Takara Shuzo was isolated. It was inserted at the EcoRI / XbaI break. Using the mutation introduction primer GTCATAAAACACGAACGGCCAAATTAATGAC, a mutation was introduced according to the instructions of the kit to obtain plasmid pKF18 / KhAP5ΔCC (-HB) / His. The nucleotide sequence of the obtained plasmid clone was determined, and it was confirmed that the mutation was introduced and that no mutation was contained in other portions. As a result, a DNA sequence encoding a protein in which histidine at position 62, lysine at position 63, and lysine at position 65 of SEQ ID NO: 12 in the sequence listing were substituted with isoleucine, asparagine, and asparagine, respectively. The resulting plasmid was digested with EcoRI and XbaI to isolate a fragment of about 1.4 kbp, and the high expression vector pME18S (Hara, T. et al. (1992) {EMBO.J. 11, 11, 1875-, Takashi Yokota, Arai) Inserted at the EcoRI / XbaI cleavage point of Kenichi Edited, Bio Manual Series 3, Gene Cloning Experimental Method, Yodosha, p18-20) (pME18S / KhAP5 @ CC (-HB)).
[0498]
(2) Study on activity of N-terminal 207 amino acid partial protein of angiopoietin-related protein-3 (ARP3) having mutation introduced into heparin binding motif
1) Triglyceride concentration increasing activity in mouse plasma
A) Preparation of recombinant adenovirus
In order to forcibly express the N-terminal 207 amino acid partial protein of ARP3 (ARP3 @ CC) and the N-terminal 207 amino acid partial protein of ARP3 (ARP3 @ CC (-HB)) having a mutation introduced into the heparin-binding motif, a recombinant adenovirus is commercially available. It was prepared using a kit (Adenovirus Expression Vector Kit, manufactured by Takara Shuzo Co., Ltd.). That is, pME18S / KhAP5ΔCC / His or pKF18 / KhAP5ΔCC (-HB) / His was digested with EcoRI and XbaI, and the obtained DNA fragment of about 1.4 kb was blunt-ended to obtain an inserted DNA fragment. Used for the operation.
[0499]
In addition, an insert DNA fragment was inserted into a restriction enzyme SwaI recognition site of a cosmid vector pAxCAwt (attached to an adenovirus expression vector kit) designed to be expressed by a cytomegalovirus enhancer and a chicken β-actin promoter. Cosmids pAxCA / KhAP5 @ CC / His and pAxCA / KhAP5 @ CC (-HB) / His were made. pAxCA / KhAP5 CC / His DNA or pAxCA / KhAP5 CC (-HB) / His DNA and terminal protein-bound virus DNA (DNA-TPC, attached to the adenovirus expression vector kit) were added to a calcium phosphate transfection system (Lifetech) 293 cells (ATCC @ CRL1573) were used to isolate recombinant adenoviruses Ad / KhAP5 @ CC / His and Ad / KhAP5 @ CC (-HB) / His and further amplified in 293 cells I let it. To recover the amplified virus from 293 cells, the virus-infected 293 cells were first subjected to sonication for 30 seconds × 4 times (using B-1200 manufactured by Branson), and then purified by cesium chloride density gradient centrifugation. This was done by repeating the procedure several times. The obtained virus solution was dialyzed at 4 ° C. against PBS supplemented with 10% Δglycerol, and then stored frozen at −70 ° C. or lower until use.
[0500]
B) Plasma triglyceride concentration after inoculation of mice with recombinant adenovirus
Recombinant recombinant adenoviruses Ad / KhAP5ΔCC / His and Ad / KhAP5ΔCC (-HB) / His purified as described above were 2 × 10 6 in PBS containing 10% glycerol.¹⁰pfu / ml, and 2 (Ad / KhAP5 @ CC / His) or 3 (Ad / KhAP5 @ CC (-HB) / His) male KK / 20-21 weeks old, respectively.Snk200 μl (5 × 10⁹pfu) by tail vein injection. Before inoculation, 1, 2, 4, 7, 11, and 14 days after inoculation, blood was collected from the fundus of each mouse using a hematocrit tube, and centrifuged at 5200 rpm for 15 minutes using a tabletop centrifuge to separate plasma. One day after the inoculation, 1 μl of plasma was mixed with an SDS-PAGE sample buffer (manufactured by Bio-Rad) containing 2-mercaptoethanol, and heated at 99 ° C. for 5 minutes to prepare a sample for electrophoresis. The sample was subjected to electrophoresis in a polyacrylamide density gradient gel having a gel concentration of 4 to 20% (electrophoresis buffer: 25 mM @ Tris, 192 mM @ glycine, 0.1% @ SDS), and then a transfer buffer (25 mM @ Tris, 192 mM). (Glycine, 20% methanol) and transferred to a nitrocellulose membrane at 200C for 1 hour at 4 ° C. The transferred membrane was blocked overnight at 4 ° C. with a PBS solution containing 0.5% skim milk, and washed three times with a washing solution (0.05% Tween 20-PBS). Next, the membrane was incubated in a 0.05% Tween 20-PBS solution containing 270 ng / ml of the 55-1-N1 antibody described in Example 2 and 5% {fetal bovine serum} for 1 hour at room temperature, and then washed with a washing solution. Washed three times. Furthermore, the membrane was diluted with horseradish peroxidase-labeled goat anti-rabbit IgG (H + L) (manufactured by Amersham Biosciences) 5% 0.05% containing fetal bovine serum 2,000 times in Tween20-PBS in a reaction solution obtained by 2,000-fold dilution at room temperature to obtain a solution. After incubation for an hour, the plate was washed five times with a washing solution. This membrane was placed on a wrap film, immersed in an ECL western blotting detection solution for 1 minute, and exposed to an X-ray film. As a result, specific bands were detected in the plasma of mice infected with the recombinant adenovirus Ad / KhAP5ΔCC / His and in the plasma of mice infected with Ad / KhAP5ΔCC (-HB) / His (FIG. 9). ).
[0501]
Subsequently, the neutral fat concentration was measured using a neutral fat measurement kit (Triglyceride E-Test Wako, manufactured by Wako Pharmaceutical Co., Ltd.). In the group of mice inoculated with Ad / KhAP5 CC / His, the triglyceride concentration in the blood increased remarkably, whereas in the group of mice inoculated with Ad / KhAP5 CC (-HB) / His, the level of triglyceride in plasma almost increased Not done (FIG. 10).
[0502]
2) Lipoprotein lipase (LPL) inhibitory activity
A) Preparation of recombinant protein
Using pME18S / KhAP5 @ CC / His or pKF18 / KhAP5 @ CC (-HB) / His as a LipofectAMINE2000 reagent (manufactured by Invitrogen), COS cells (Gluzman, Y. (1981) Cell 23, 175-182, ATCC CRL-16). ) And cultured for 4 days in Dulbecco's modified Eagle's medium without serum, and the culture supernatant was recovered. The culture supernatant was ultraconcentrated to about 1/10 volume using a 30K filter (Millipore) at TIFF Lab Scale (Millipore). The concentrated sample was circulated with a 10-fold volume of 50 mM imidazole, 0.5 M sodium chloride, and 0.02 M sodium phosphate buffer, and replaced with a buffer of the present composition. The buffer-replaced sample was treated in advance with 0.5 ml of 0.1 M nickel sulfate and bound to a HiTrap @ Chelating column (Amersham Biosciences), which was replaced with 50 mM imidazole, 0.5 M sodium chloride, and 0.02 M sodium phosphate buffer. After that, the mixture was washed with 100 mM imidazole, 0.5 M sodium chloride, and 0.02 M sodium phosphate buffer, and eluted with 300 mM imidazole, 0.5 M sodium chloride, and 0.02 M sodium phosphate buffer. The obtained eluted sample was dialyzed three times with 1,000 times the volume of PBS (-) (Sigma), and the recombinant protein ARP3 was obtained.
CC, ARP3 @ CC (-HB).
[0503]
B) LPL inhibitory activity
The LPL inhibitory activity of the recombinant protein obtained above was examined by the method described in Example 11) (1) -2). The LPL inhibitory activity of ARP3ΔCC (—HB) was significantly lower than the LPL inhibitory activity of ARP3ΔCC (FIG. 11).
[0504]
As described above, ARP3ΔCC (-HB) can be used as a negative control in screening for an ARP3 function inhibitor.
[0505]
Embodiment 15 FIG. Preparation of plasma of mice inoculated with adenovirus vector expressing histidine-tagged protein
(1) Preparation of recombinant adenovirus
Using an adenovirus expression vector kit (Takara Shuzo Co., Ltd.), a recombinant adenovirus was prepared according to the following method. The recombinant plasmid pME18S / KhAP5 / His obtained in Example 13 (1) -1) was digested with restriction enzymes EcoRI and XbaI to recover a DNA fragment of about 1.4 kb, and the cut end was blunted. It has become. Next, these fragments were respectively inserted into the SwaI cleavage site of the cosmid vector pAxCAwt designed to express the inserted gene by the cytomegalovirus enhancer and the chicken β-actin promoter, and the recombinant cosmid pAxCA / KhAP5 / His was inserted. Was prepared.
[0506]
This recombinant cosmid DNA and terminal protein-linked virus DNA (DNA-TPC, attached to the adenovirus expression vector kit) were co-transfected into 293 cells (ATCC @ CRL1573) using a calcium phosphate transfection system (manufactured by Lifetech). Thus, the recombinant adenovirus Ad / KhAP5 / His was isolated and further amplified in 293 cells. Recovery of the amplified virus from the 293 cells was performed by first sonicating the virus-infected 293 cells for 30 seconds × 4 times (using B-1200 manufactured by Branson) or repeating freeze-thawing three times, and then Purification by cesium chloride density gradient centrifugation was repeated twice. The obtained virus solution was dialyzed at 4 ° C. against PBS supplemented with 10% Δglycerol, and then stored frozen at −70 ° C. or lower until use.
[0507]
(2) Confirmation of expression of target protein by recombinant adenovirus
HeLa cells were infected with the recombinant adenovirus obtained by the method described in (1) above, and the expression of the target protein was confirmed.
[0508]
First, each recombinant adenovirus obtained by the method described in the above (1) was injected into HeLa cells (ATCC @ CCL2) for about 5 m. o. i. (Multiplicity of infection: multiplicity of infection), and cultured for 3 to 4 days in a serum-free medium (Dulbecco's modified Eagle's medium (DMEM)) to collect the culture supernatant. 1 ml of the culture supernatant is placed in a 1.5 ml Eppendorf tube, 100 μl of a trichloroacetic acid solution (1 g / ml trichloroacetic acid, 4 mg / ml deoxycholic acid) is added, and the mixture is left at room temperature for 3 minutes. Centrifuged at 15000 rpm for 5 minutes. After removing the supernatant, 0.5 ml of ice-cooled acetone was added and stirred, and the mixture was centrifuged at 15000 rpm for 2 minutes using a desktop centrifuge. The supernatant was removed, 0.5 ml of ice-cooled acetone was added to the precipitate again, and the mixture was stirred. The mixture was centrifuged at 15000 rpm for 2 minutes using a tabletop centrifuge, the supernatant was removed, and the precipitate was dried under vacuum. Each of the precipitates was dissolved in 10 μl of distilled water, mixed with an SDS-PAGE sample buffer solution (manufactured by Bio-Rad) containing 2-mercaptoethanol, and heated at 99 ° C. for 5 minutes to prepare a sample for electrophoresis. Prepared. These samples were subjected to electrophoresis on a polyacrylamide density gradient gel having a gel concentration of 4 to 20% (electrophoresis buffer: 25 mM Tris, 192 mM glycine, 0.1% SDS), and then a transfer buffer (25 mM Tris, 192 mM glycine). , 20% methanol) at 4 ° C. for 1 hour at 200 mA on a nitrocellulose membrane.
[0509]
The transferred membrane was immersed in a PBS solution containing 0.5% skim milk, blocked at 4 ° C overnight, and washed three times with a washing solution (0.05% Tween 20-PBS). Next, the membrane was incubated in a 0.05% Tween 20-PBS solution containing about 270 ng / ml of the 55-1-N1 antibody described in Example 2 and 5% {fetal bovine serum} for 1 hour at room temperature, and then washed. And washed three times.
[0510]
Further, the membrane was incubated at room temperature for 1 hour at room temperature in a reaction solution obtained by diluting a horseradish peroxidase-labeled goat anti-rabbit IgG (H + L) (manufactured by Bio-Rad) with 5% {0.05% containing fetal bovine serum} 2000 times in Tween 20-PBS. After that, it was washed 5 times with a washing solution. This membrane is placed on a wrap film, immersed in a luminescence reagent solution (ECL Western detection reagent, manufactured by Amersham Pharmacia Biotech) for 1 minute, and left at room temperature for 1 hour to attenuate the background. The film was exposed (10 seconds). As a result of developing this X-ray film, it was confirmed that the target polypeptide was produced in HeLa cells infected with the recombinant adenovirus.
[0511]
(3) KK / inoculated with recombinant adenovirusSnkPreparation of mouse plasma
The recombinant adenovirus Ad / KhAP5 / His purified in the above (1) was added to PBS containing 10% glycerol at 7.2 × 10 5⁹pfu / ml and 5 18-week-old male KK /SnkMice were inoculated by tail vein injection of 300 μl each.
[0512]
Two days after the inoculation, blood was collected from the abdominal aorta of each mouse using a syringe (a 26-gauge syringe needle and a 1 ml syringe manufactured by Terumo Corporation) previously filled with 60 μl of 1% EDTA, and placed in a 1.5 ml Eppendorf tube. After transferring and mixing by inversion, plasma was separated by centrifugation at 5200 rpm for 15 minutes using a tabletop centrifuge.
[0513]
(4) KK /SnkPurification of target protein in mouse plasma
The obtained plasma was dialyzed against 10 mM imidazole / PBS at 4 ° C. for 4 days, added to a HiTrap®Chelating®HP column (Amersham), and washed with 20 mM imidazole / PBS and 100 mM imidazole / PBS. Elution was performed with 300 mM imidazole / PBS. Each fraction was subjected to electrophoresis (electrophoresis buffer: 25 mM Tris, 192 mM glycine, 0.1% SDS) on a 4-20% polyacrylamide density gradient gel, and then GelCode. Staining (Pierce) was performed to confirm the presence or absence of the target protein.
[0514]
(5) KK /SnkIdentification of target protein cleavage point in mouse plasma
After reducing the human / “angiopoietin-related protein 3” fraction obtained by His-tag affinity purification by adding a final concentration of 10 mM mM dithiothreitol (Sigma, St. Louis, MO), a final concentration of 55 mM mM iodoacetamide (Wako, Osaka, Japan) Japan) was added to carry out an alkylation reaction. Thereafter, Lysyl Endopeptidase (Wako, Osaka, Japan) was added and digested at 37 ° C. overnight, or PNGase F was treated with PNGase F (New England Biolabs, Beverly, Mass.), Followed by removal of the glycan from the enzyme after 2 hours of removal of the sugar chain. Glu-C (Promega, Madison, WI) was added and digested at 37 ° C overnight. To the digestion reaction solution was added 0.05% {formic acid} / ＯH2O to prepare a sample for LC / ESI MS / MS (liquid chromatography electrospray ionization tandem mass spectrometry). For LC, a self-produced electrospray needle packed with a reverse phase chromatography carrier InertsilｓODS-2 (GL Science, Tokyo, Japan) was used as a column, and liquid was sent by CapLC System (Waters, Milford, MA). Solution A: 0.05% {formic acid / H2O} and Solution B: 0.05% {formicacid / acetonitrile} are used as elution solvents, and gradient elution is performed to linearly increase the concentration of solution B from 0% to 30% in one hour. Was. The eluate was directly introduced into Q-Tof2 (Micromass, Manchester, UK) to perform ESI MS / MS measurement.
[0515]
When peptide mapping of human "angiopoietin-related protein 3" was performed from the obtained MS spectrum, the sample digested with Lysyl @ Endopeptidase showed EIENQLRRRTSIQEPTEISLSSKPR derived from human "angiopoietin-related protein 3" (amino acid residues 198-221). EIENQLRRRTSIQEPTEISLSSKPRAPR (198-224), and two peptides of ISLSSKPR (214-221) and ISLSSKPRAPR (214-224) were present in the sample digested with endoproteinase @ Glu-C. The carboxyl terminus of these peptides is not a substrate recognition site for the two enzymes. Therefore, two sites between Arg221 and Ala222 and between Arg224 and Thr225 were confirmed as cleavage sites in the blood of human "angiopoietin-related protein 3".
[0516]
Embodiment 16 FIG.活性 Evaluation of activity of fusion angiopoietin-related protein 4
(1) Preparation of fusion protein
A fusion protein in which glutathione S-transferase (hereinafter, referred to as “GST”) is linked to the amino terminus of a polypeptide comprising the amino acid sequence of amino acids 26 to 143 of SEQ ID NO: 16 in the sequence listing (hereinafter, “GST- ARP4N ") was prepared according to the method described below.
[0517]
First, the following nucleotide sequence was used as a primer for PCR:
5 '-{tccccccggggggaccccgtgcagtcccaag-3' (sense primer for GST-ARP4N; SEQ ID NO: 51 in Sequence Listing);
5 '-@ ccgctcgagctgggctttgcagatgctg-3' (Antisense primer for GST-ARP4N; SEQ ID NO: 52 in the sequence listing) was synthesized.
[0518]
Next, I. M. A. G. FIG. E Using a cDNA clone (clone 4149039) purchased from Consortium as a template, a sense primer and an antisense primer (0.2 μM each), Tbr EXT DNA polymerase, 1 × PCR buffer (10 × buffer attached to Tbr EXT DNA polymerase) Was diluted 10-fold), and PCR was performed in the presence of 2 mM magnesium chloride and 0.4 mM dNTPs each. PCR was performed by heating the sample at 94 ° C. for 3 minutes, repeating a temperature cycle of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 30 seconds 30 times, and finally for 7 minutes at 72 ° C. Performed under conditions.
[0519]
A DNA fragment of about 370 bp amplified by PCR was recovered and digested with SmaI and XhoI. Plasmid vector pGEX-4T-2 (including the gene encoding GST, which has been digested with SmaI and XhoI and then dephosphorylated at the cut ends using bovine intestinal alkaline phosphatase (Takara Shuzo Co., Ltd.). -Pharmacia Biotech Co., Ltd.), and a plasmid clone containing the inserted fragment was isolated. This plasmid clone was named pGST-ARP4N.
[0520]
Escherichia coli JM109 strain was transformed with the recombinant plasmid clone pGST-ARP4N or the control vector pGEX-4T-2 for expressing only GST without an insert fragment. For transformation, the plasmid was added to competent cells of Escherichia coli JM109 strain, allowed to stand on ice for 30 minutes, kept at 42 ° C for 30 seconds, again allowed to stand on ice for 2 minutes, and then shaken at 37 ° C for 1 hour. This was performed by culturing. The transformed culture was spread on an LB agar plate supplemented with ampicillin, and colonies of the resulting recombinants were picked up and cultured in liquid to confirm the expression of the recombinant protein induced by IPTG.
[0521]
(2) Preparation of fusion protein
1) Culture of transformed Escherichia coli
The transformed E. coli cells harboring the plasmid clone pGST-ARP4N and the control vector pGEX-4T-2 were each inoculated into an LB medium containing ampicillin and cultured with shaking at 37 ° C. for 18 hours. A part of the culture solution was added to a fresh medium, and shaking culture was further performed at 37 ° C. When the absorbance at 600 nm reached 0.5-0.7, isopropyl thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM, and shaking culture was continued for another 4 hours.
[0522]
2) Preparation of cell lysate from E. coli carrying pGEX-4T-2
A culture solution of Escherichia coli transformed with the control vector pGEX-4T-2 was centrifuged at 4 ° C. and 6000 rpm for 10 minutes to collect the precipitated cells. The collected cells were suspended in a buffer for sonication (50 mM Tris-hydrochloric acid (pH 8.0), 50 mM sodium chloride, 1 mM EDTA, 1 mM DTT), and the suspension was incubated at 4 ° C. and 6000 rpm for 10 minutes. Centrifuged. The precipitate was frozen at -80 ° C, then thawed at room temperature and resuspended in sonication buffer. The suspension was treated in an ice bath with an ultrasonic crusher (UD-201 manufactured by Tommy Seiko Co., Ltd.) for 5 minutes under conditions of output = 5 and duty = 50. Triton @ X-100 was added to the crushed liquid after the ultrasonic treatment so as to have a final concentration of 1%, mixed, and centrifuged at 4 ° C. and 10,000 rpm for 30 minutes to collect the supernatant. The supernatant was passed through a GSTrap column (Amersham Pharmacia Biotech).
[0523]
3) Preparation of cell lysate from E. coli carrying pGST-ARP4N
Each culture of Escherichia coli transformed with the recombinant plasmid pGST-ARP4N was centrifuged at 4 ° C. and 5000 rpm for 15 minutes, and the precipitated cells were collected. The operation of suspending the precipitated cells in a washing buffer (0.5% Triton X-100, 1 mM EDTA) and centrifuging at 5000 rpm for 10 minutes at 4 ° C was repeated three times. The obtained cells were dissolved in an 8M urea solution. After leaving this solution at room temperature for 1 hour, centrifugation was performed at 4 ° C. and 13000 rpm for 30 minutes to collect the supernatant. The resulting supernatant was dialyzed. Dialysis was performed at 4 ° C. for 1 hour against a 4M urea solution, 1 hour at 4 ° C. against a 2M urea solution, 1 hour at 4 ° C. against a sonication buffer, and At 4 ° C. overnight. After the dialysis, the mixture was centrifuged at 4 ° C. and 12000 rpm for 30 minutes, and then passed through a GSTrap column.
[0524]
4) Purification of fusion protein
Through the above steps, GST and a fusion protein with GST (hereinafter, referred to as “GST-ARP4N”) were adsorbed on a GST column to which PBS was adsorbed, and the inside of the column was washed to remove impurities. Next, GST and GST-ARP4N were eluted by flowing a 10 mM reduced glutathione solution. After a part of the eluate was separated by SDS-PAGE electrophoresis, the gel was subjected to Coomassie staining to confirm the presence of the protein. The eluate was dialyzed against PBS at 4 ° C. overnight.
[0525]
(2) Blood neutral fat concentration increasing activity
GST and GST-ARP4N purified in the above (1) were adjusted to a protein concentration of about 1 mg / ml with PBS, and two 19-week-old male KK /SnkMice were inoculated by tail vein injection in approximately 300 μl. Blood was collected before ingestion and 30 minutes after inoculation. Blood was collected from the fundus of each mouse with a hematocrit tube, centrifuged at 5200 rpm for 15 minutes with a tabletop centrifuge to separate plasma, and the blood neutral fat concentration before and after inoculation was measured. The results are shown in Table 9.
[0526]
[Table 9]

In the GST-inoculated mouse group, no difference was observed before and after the inoculation, whereas in the GST-ARP4N-inoculated mouse group, a significant increase in blood neutral fat concentration was observed.
[0527]
In the method of the present example, for example, a test substance is administered to a mouse inoculated with GST-ARP4N, and a change in blood triglyceride concentration is examined to select from hyperlipidemia, arteriosclerosis, and hyperglycemia. Testing of therapeutic or prophylactic agents for one or more diseases can be performed.
[0528]
Embodiment 17 FIG. LPL activity inhibitory effect
(1) Preparation of LPL sample
Rat white adipose precursor cells (purchased from Hokudo) were cultured in a growth medium [10% fetal calf serum (hereinafter referred to as “FCS”), 100 units / ml penicillin, 100 μg / ml streptomycin, 17 μM pantothenic acid, 33 μM} (+)-biotin. Dulbecco's modified Eagle's medium containing 100 μM ascorbic acid, 1 μM octanoic acid and 50 nM triiodothyronine (hereinafter referred to as “DMEM”)].²) At 37 ° C. for 24 hours, and then the medium was cultured for differentiation induction (10% FCS, 100 units / ml penicillin, 100 μg / ml streptomycin, 17 μM pantothenic acid, 33 μM (+)-biotin, 100 μM ascorbic acid, 1 μM The solution was replaced with octanoic acid, 50 nM {triiodothyronine, 10 μg / ml in insulin and 2.5 μM in dexamethasone), and the cells were further cultured at 37 ° C. for 48 hours. Then, the adipocyte maintenance medium (10% FCS, 100 U / ml penicillin, 100 μg / ml streptomycin, 17 μM pantothenic acid, 33 μM (+)-biotin, 100 μM ascorbic acid, 1 μM octanoic acid, 50 nM triiodothyronine and 10 μg / ml insulin) With DMEM). After 2 to 3 days, 2 ml of a DMEM medium containing 10% {FCS, 50 ng / ml} insulin and 10 units / ml sodium heparin was added, and the mixture was cultured for 1 hour. The culture supernatant was recovered and used for measurement of LPL activity. .
[0529]
(2) LPL activity measurement
The LPL activity was measured according to the method described in the literature (Nilsson-Ehle, P. and Schottz, M. C. (1976) J. Lipid Res. 17, 536-541).
[0530]
First, 100 μl of the culture supernatant prepared by the method described in 1) above was added to an equal volume of a substrate solution [2 mM @ glycerol tri [9,10 (n)-³H] Oleic acid (131 KBeq / μmol, manufactured by Amersham), 0.189 μg / ml {L} α-phosphatidylcholine (Sigma), 14 mg / ml {bovine serum albumin (Sigma), 140 mM} Tris-HCl (pH 8.0) , 15% (v / v) glycerol, 10% (v / v) inactivated FCS], and the GST or GST-ARP4N obtained in Example 15 was added thereto, and the mixture was kept at 37 ° C for 120 minutes. Thereafter, 1.05 ml of 0.1 M potassium carbonate / borate buffer (pH 10.5) and 3.25 ml of methanol: chloroform: hexane = 141: 125: 100 (v / v) were added to stop the reaction. After stirring, the mixture was centrifuged at 3000 × g for 15 minutes. Water-methanol layer³H count was measured with a liquid scintillation counter. One unit of LPL activity was defined as the activity of producing 1 μmol of fatty acid in one minute. Furthermore, the LPL activity when GST (10 μg / ml) was added was defined as 100%, and the relative LPL activity when GST-ARP4N was added was calculated. Table 10 shows the results.
[0531]
[Table 10]

GST-ARP4N suppressed LPL activity in a dose-dependent manner. In this experimental system, if a test substance is added during the lipase reaction, it can be determined whether or not the substance has an effect of regulating LPL activity.
[0532]
Embodiment 18 FIG. Activity evaluation of fusion protein of angiopoietin-related protein 4 and glutathione S-transferase
(1) Production of transformed E. coli expressing fusion protein
A fusion protein in which glutathione S. transferase (hereinafter, referred to as “GST”) is linked to the amino terminus of a polypeptide comprising the amino acid sequence of amino acids 26 to 406 of SEQ ID NO: 16 in the sequence listing (hereinafter, “GST- ARP4Full ") was prepared according to the method described below.
[0533]
First, the following nucleotide sequence was used as a primer for PCR:
5 '-{tccccccggggggacccgtgcagtccaag-3' (sense primer for GST-ARP4Full; SEQ ID NO: 55 in Sequence Listing);
5′- ccgctcgaggggggctgcctctgctgc-3 ′ (Antisense primer for GST-ARP4Full; SEQ ID NO: 56 in the sequence listing) was synthesized.
[0534]
Next, I. M. A. G. FIG. E. FIG. Using 10 ng of cDNA (ID @ 4149039) purchased from Consortium as a template, sense primer and antisense primer (0.2 μM each), Tbr @ EXT DNA polymerase, 1 × PCR buffer (10 × attached to Tbr @ EXT DNA polymerase) PCR was performed in the presence of 2 mM magnesium chloride and 0.4 mM dNTPs each. PCR was performed by heating the sample at 94 ° C. for 3 minutes, and then performing 35 temperature cycles of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, 72 ° C. for 1 minute and 30 seconds, and finally 72 ° C. for 5 minutes. Done.
[0535]
In the PCR, a DNA fragment of about 1140 bp was amplified. The amplified DNA fragment was recovered and digested with SmaI and XhoI. Plasmid vector pGEX-4T-2 (including the gene encoding GST, which has been digested with SmaI and XhoI and then dephosphorylated at the cut ends using bovine intestinal alkaline phosphatase (Takara Shuzo Co., Ltd.). -Pharmacia Biotech Co., Ltd.), the DNA fragment digested with SmaI and XhoI was inserted, and a plasmid clone containing the inserted fragment was isolated. This plasmid clone was named pGST-ARP4Full.
[0536]
Escherichia coli EpicurianColi {BL21} CodonPlus (DE3) -RIL strain (Stratagene) was used for plasmid clone pGST-ARP4Full, or Escherichia coli JM109 strain was used for control vector pGEX-4T-2 for expressing only GST without an insert fragment. Transformed.
[0537]
For transformation, 2 ml of 10-fold diluted XL10-Gold β-mercaptoethanol {mix} was previously added to 100 ml of competent cells of Escherichia coli Epicurian Coli BL21 CodonPlus (DE3) -RIL, and cooled on ice for 10 minutes with stirring for 2 minutes. Was added, and the mixture was allowed to stand on ice for 30 minutes, kept at 42 ° C. for 20 seconds, again allowed to stand on ice for 2 minutes, and cultured in an SOC culture solution at 37 ° C. for 1 hour with shaking. The transformed culture solution was spread on an LB agar plate supplemented with ampicillin and chloramphenicol, and the resulting recombinant colonies were picked up and subjected to liquid culture to confirm the expression of the recombinant protein induced by IPTG.
[0538]
On the other hand, for transformation of E. coli JM109, the plasmid was added to competent cells, left on ice for 30 minutes, kept at 42 ° C for 30 seconds, again left on ice for 2 minutes, and then placed in SOC culture medium. This was performed by shaking culture at 37 ° C. for 1 hour. The transformed culture was spread on an LB agar plate supplemented with ampicillin, and colonies of the resulting recombinants were picked up and cultured in liquid to confirm the expression of the recombinant protein induced by IPTG.
[0539]
(2) Preparation of fusion protein
1) Culture of transformed Escherichia coli
The transformed E. coli cells harboring the plasmid clone pGST-ARP4Full and the control vector pGEX-4T-2 were each inoculated into LB medium containing ampicillin / chloramphenicol and ampicillin, and cultured with shaking at 37 ° C for 18 hours. A part of the culture solution was added to a fresh medium, and shaking culture was further performed at 37 ° C. When the absorbance at 600 nm reached 0.5-0.7, isopropyl thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM, and shaking culture was continued for another 4 hours.
[0540]
2) Preparation of cell lysate from E. coli carrying pGEX-4T-2
A culture solution of Escherichia coli transformed with the control vector pGEX-4T-2 was centrifuged at 4 ° C. and 6000 rpm for 10 minutes to collect the precipitated cells. The collected cells were suspended in a buffer for sonication (50 mM Tris-hydrochloric acid (pH 8.0), 50 mM sodium chloride, 1 mM EDTA, 1 mM DTT), and the suspension was incubated at 4 ° C. and 6000 rpm for 10 minutes. Centrifuged. The precipitate was frozen at -80 ° C, then thawed at room temperature and resuspended in sonication buffer. The suspension was treated in an ice bath with an ultrasonic crusher (UD-201 manufactured by Tommy Seiko Co., Ltd.) for 5 minutes under conditions of output = 5 and duty = 50. Triton @ X-100 was added to the crushed liquid after the ultrasonic treatment so as to have a final concentration of 1%, mixed, and centrifuged at 4 ° C. and 10,000 rpm for 30 minutes to collect the supernatant. The supernatant was passed through a GSTrap column (Amersham Pharmacia Biotech).
[0541]
3) Preparation of cell lysate from E. coli carrying pGST-ARP4Full
A culture solution of Escherichia coli transformed with the recombinant plasmid pGST-ARP4Full was centrifuged at 4 ° C. and 5000 rpm for 15 minutes, and the precipitated cells were collected. The operation of suspending the precipitated cells in a washing buffer (0.5% Triton X-100, 1 mM EDTA) and centrifuging at 5000 rpm for 10 minutes at 4 ° C was repeated three times. The obtained cells were dissolved in an 8M urea solution. After leaving this solution at room temperature for 1 hour, centrifugation was performed at 4 ° C. and 13000 rpm for 30 minutes to collect the supernatant. The supernatant obtained from the culture solution derived from Escherichia coli transformed with the recombinant plasmid pGST-ARP4Full was dialyzed. Dialysis was performed at 4 ° C. for 1 hour against a 4M urea solution, 1 hour at 4 ° C. against a 2M urea solution, 1 hour at 4 ° C. against a sonication buffer, and At 4 ° C. overnight. After the dialysis, the mixture was centrifuged at 4 ° C. and 12,000 rpm for 30 minutes, and the supernatant was passed through a GSTrap column.
[0542]
4) Purification of fusion protein
Through the above steps, GST or a fusion protein with GST, GST-ARP4 Full, was adsorbed on a GST column to which PBS was adsorbed, and the column was washed to remove impurities. Next, a 10 mM -reduced glutathione solution (pH 8.0) was allowed to flow to elute GST and a fusion protein GST-ARP4Full with GST. After a part of the eluate was separated by SDS-PAGE electrophoresis, the gel was stained with Coomassie to confirm the presence of the protein. The eluate was dialyzed against PBS at 4 ° C. overnight. Here, the protein purified from the transformant carrying the plasmid pGEX-4T-2 is GST, and the protein purified from the transformant carrying pGST-ARP4Full is GST-ARP4Full.
[0543]
(3) Blood neutral fat concentration increasing activity
After adjusting GST and GST-ARP4 Full obtained in the above (2) to a protein concentration of about 0.5 mg / ml with PBS, each of the two 16-week-old male KK /SnkMice were inoculated by tail vein injection of 100 μl each. Blood was collected before and 30 minutes after ingestion. Blood was collected from the fundus of each mouse with a hematocrit tube, centrifuged at 5200 rpm for 15 minutes with a tabletop centrifuge to separate plasma, and the blood neutral fat (triacylglycerol) concentration before and after inoculation was measured.
[0544]
Table 11 shows the results. In the GST-inoculated mouse group ("GST" in Table 11), no difference was observed before and after inoculation, whereas in the GST-ARP4 Full-inoculated mouse group ("GST-ARP4 Full" in Table 11), remarkable blood triglyceride was observed. An increase in concentration was observed.
[0545]
In the method of the present example, for example, a test substance is administered to a mouse inoculated with GST-ARP4Full, and a change in blood triglyceride concentration is examined to select from hyperlipidemia, arteriosclerosis, and hyperglycemia. Testing of therapeutic or prophylactic agents for one or more diseases can be performed.
[0546]
[Table 11]

Embodiment 19 FIG. Ｌ LST activity inhibitory effect of GST-ARP4Full
100 μl of rat white adipose precursor cell culture supernatant prepared according to the method described in 1) of Example 11 (1) was added to an equal volume of a substrate solution (2 mM @ glycerol tri [9,10 (n)-³H] Oleic acid (131 KBeq / μmol, manufactured by Amersham), 0.189 μg / ml {L} α-phosphatidylcholine (Sigma), 14 mg / ml {bovine serum albumin (Sigma), 140 mM} Tris-HCl (pH 8.0) , 15% (v / v) glycerol and 10% (v / v) inactivated FCS), and the GST-ARP4Full or GST obtained in Example 18 was added thereto, followed by incubation at 37 ° C for 120 minutes. Thereafter, 1.05 ml of 0.1 M potassium carbonate / borate buffer (pH 10.5) and 3.25 ml of methanol: chloroform: hexane = 141: 125: 100 (v / v) were added to stop the reaction. After stirring, the mixture was centrifuged at 3000 × g for 15 minutes. Water-methanol layer³The H count was measured with a liquid scintillation counter (Beckman). One unit of LPL activity was defined as the activity of producing 1 μmol of fatty acid in one minute. Further, the LPL activity when GST (10 μg / ml) was added was defined as 100%, and the relative LPL activity when GST-ARP4Full was added was calculated. Table 12 shows the results.
[0547]
[Table 12]

GST-ARP4Full suppressed the activity of LPL in a dose-dependent manner. In this experimental system, if a test substance is added during the lipase reaction, it can be determined whether or not the substance has an effect of regulating LPL activity.
[0548]
Embodiment 20 FIG.解析 Analysis of mouse “angiopoietin-related protein 3” gene
a) Preparation of BAC (Bacterial Artificial Chromosome) DNA
After culturing E. coli carrying mouse BAC clone 355L-1 (purchased from Research Genetics) in 100 ml of LB medium containing 12.5 μg / ml chloramphenicol at 37 ° C. overnight, Each 4 ml was dispensed into four tube units, and DNA was extracted using an automatic DNA extraction device (PI-50, manufactured by Kurabo Industries, Ltd.). This sample was further purified by the cesium chloride method.
[0549]
b) DNA fragmentation
Transfer 300 ng of 30 ng / μl of BAC DNA to a 1.5 ml Eppendorf tube, and adjust the output of the sonicator to 1 using a sonicator (Sonifier II 250, manufactured by Branson) and a special microchip (manufactured by Branson). -The DNA was sonicated and fragmented twice for 3 seconds, with the cycle set to each "constant" condition. The degree of DNA fragmentation was confirmed by agarose gel electrophoresis.
[0550]
c) Terminal repair of DNA fragment
To the DNA fragmented above, 10 units of T4 DNA polymerase (manufactured by Takara Shuzo Co., Ltd.) was added in the presence of magnesium chloride at a final concentration of 5 mM and dNTP (manufactured by Takara Shuzo Co., Ltd.) at a final concentration of 5 mM. Incubated at 150C for 15 minutes. Further, 5 units of Klenow fragment (manufactured by Takara Shuzo Co., Ltd.) were added, and the mixture was kept at 25 ° C. for 15 minutes to blunt the ends of the fragmented DNA. After purifying the DNA, ATP (manufactured by Takara Shuzo) having a final concentration of 2 mM, 1 unit of polynucleotide kinase (manufactured by Takara Shuzo) and a polynucleotide kinase reaction buffer (attached to the polynucleotide kinase, final concentration: 50 mM tris-hydrochloric acid (pH 8.0), 10 mM magnesium chloride, 5 mM dithiothreitol were added, and the mixture was incubated at 37 ° C. for 30 minutes to phosphorylate the DNA termini. After completion of the reaction, the polynucleotide kinase was inactivated with TE-saturated phenol / chloroform.
[0551]
d) Sucrose density gradient centrifugation
A 10% sucrose solution is prepared by mixing 10 ml of a 50% sucrose solution, 0.25 ml of 20 × SSC and 0.1 ml of 0.5 M ethylenediaminetetraacetic acid (hereinafter referred to as “EDTA”) to a total volume of 50 ml with sterile water. It was produced by adjusting. A 38% sucrose solution was prepared by mixing 38 ml of a 50% sucrose solution, 0.25 ml of 20 × SSC and 0.1 ml of 0.5 M @EDTA, and adjusting the total volume to 50 ml with sterile water. 5 ml of a 38% sucrose solution was added to a 13PA tube (manufactured by Hitachi Koki Co., Ltd.), and 5 ml of a 10% sucrose solution was gently added. Then, a gradient former (Gradient Mate, Cosmo Bio Co., Ltd.) was used. ), The speed (SPEED) was set to 10, the time (TIME) was set to 2 minutes and 30 seconds, and the angle (ANGLE) was set to 8 ° to produce a sucrose density gradient. The DNA obtained in the above c) was added to the obtained sucrose density gradient solution, and centrifuged at 150,000 × g at 20 ° C. for 16 hours. Thereafter, a hole was made from the lower part of the tube using an injection needle (18G), and the liquid in the tube was fractionated from the lower layer by 300 μl and collected. A portion of each fraction was electrophoresed on a 0.8% agarose gel, a fraction containing a DNA fragment of about 2.0 kb was selected, and after ethanol precipitation, dissolved in 10 μl of TE.
[0552]
e) Vector preparation and library construction
The about 2.0 kb fragment derived from BAC DNA obtained above and 0.1 μg of pUC19 DNA / SmaI (manufactured by Fermentas) were ligated using a DNA ligation kit (version 2, manufactured by Takara Shuzo Co., Ltd.). The reaction solution was added to competent E. coli for electroporation (Electro-Max DH10B cell, manufactured by Gibco BRL), and transferred to a cuvette for electroporation (Gene Pulser cuvette @ 0.1 cm, manufactured by Bio-Rad). Electroporation was performed using a poration device (Gene Pulser II, manufactured by Bio-Rad) under the conditions of a voltage of 1.8 kV, an electric capacity of 25 μF, and a resistance of 200Ω. Thereafter, 1 ml of SOC medium (manufactured by Gibco BRL) was added, and the mixture was shaken at 37 ° C. for 1 hour, and ampicillin having a final concentration of 50 μg / ml and X-gal having a final concentration of 50 μg / ml (Gibco BRL) were added. And IPTG (manufactured by Gibco BRL) having a final concentration of 25 μg / ml, spread on an LB solid medium (containing 1.2% Bacto-Agar), and cultured at 37 ° C. overnight.
[0553]
f) Preparation of DNA
As a result of the above e), a white colony was selected, and a TBG medium containing ampicillin having a final concentration of 50 μg / ml (tryptone 12 g, yeast extract 24 g, monopotassium phosphate 2.3 g, dibasic phosphate per liter) was used. 1 colony / well in a culture microwell plate (2.2 ml / deep well plate, manufactured by Nippon Genetics) containing 1.2 ml / well of potassium (12.5 g, glycerol (4 ml) and 1 M glucose (20 ml)) Bacteria. After sealing the upper surface of the plate with a gas-permeable pressure-sensitive adhesive sheet (manufactured by Nippon Genetics), the plate was cultured at 37 ° C. for 16.5 hours with gentle stirring using a plate mixer. Thereafter, 600 μl of the culture solution was taken and dispensed into a new microwell plate for culture, and centrifuged at 6000 rpm, 20 ° C. for 5 minutes using a microplate centrifuge (4K15C, manufactured by Sigma). The supernatant was removed, and DNA was extracted from the precipitated cells using a DNA extraction kit (QIAprep # 96 turbo biorobot kit, manufactured by Qiagen) and a DNA extraction device (BioRobot 9600, manufactured by Qiagen).
[0554]
g) nucleotide sequence analysis
The following nucleotide sequences are used as sequencing primers:
5 '-{tgtaaaacga {cggccagt} -3' (-21M13forward, SEQ ID NO: 57 in Sequence Listing);
5 '-{caggaaaacag @ ctatgacc} -3' (M13 reverse, SEQ ID NO: 58 in Sequence Listing)
Was chemically synthesized using a DNA synthesizer.
[0555]
For each DNA (10 μl) obtained in the above f), 3.2 pmol of a primer (-21 M13 forward or M13 reverse) and a sequencing reagent (Big Dye Terminator Cycle Sequence FS Ready Reaction Kit, manufactured by Applied Biosystems) Was added, and the total volume was adjusted to 20 μl with sterilized water. A cycle sequence reaction was carried out using a thermal cycler (GeneAmp PCR System 9700, manufactured by Applied Biosystems) (25 cycles at 96 ° C for 10 seconds, 50 ° C for 5 seconds, and 60 ° C for 4 minutes as one cycle). The obtained sample was subjected to a DNA sequencer (ABI3700, manufactured by Applied Biosystems) to analyze the nucleotide sequence. The obtained sequence data was edited and aligned using DNA sequencing software (Sequencher, Gene @ Codes). As a result of the analysis, a DNA containing the nucleotide sequence shown in SEQ ID NO: 27 in the sequence listing was selected as the target promoter DNA. The transcriptional start point of the gene encoding the mouse “angiopoietin-related protein 3” (5 ′ terminal boundary site of exon 1) is “AI195524” which is the longest EST (Expressed Sequence Tag) registered in the GenBank database. Was determined with reference to FIG. That is, in the nucleotide sequence of AI195524, the first nucleotide excluding the vector portion was determined as the transcription start point, and the position was defined as "1". Thus, a DNA consisting of a nucleotide sequence corresponding to a region from 418 bp on the 5 ′ end to 132 bp on the 3 ′ end from the transcription start point of the mouse “angiopoietin-related protein 3” gene was identified.
[0556]
Embodiment 21 FIG. Construction of luciferase expression plasmid vector
a) Construction of plasmid pGL8-3
Based on the results of Example 20, the following nucleotide sequence as a PCR primer for specifically amplifying the 5'-terminal upstream region of the gene encoding mouse "angiopoietin-related protein 3":
5 '-{aaggtaccgc} tgtttccaga {taaaaaaa} -3'} (primer 1, SEQ ID NO: 59 in sequence listing); and
5 '-{tcaatgatg {aaaggtctgg} atccactctg {gatgc} -3'} (primer 2, SEQ ID NO: 60 in Sequence Listing)
Were chemically synthesized.
[0557]
5 μl of 10 × LA buffer (Mg +) attached to the above-mentioned primer (final concentration: 0.2 μM each) and LAΔPCR kit (manufactured by Takara Shuzo Co., Ltd.), and 8 μl of dNTP attached, described in Example 20a) 1 μl of the BAC clone 355L-1 DNA purified by the method and sterilized water were added to make 49.5 μl, and 0.5 μl of LA Taq polymerase attached to the kit was added to make the total volume 50 μl. PCR was performed using a thermal cycler (GeneAmp PCR System 9600, manufactured by Applied Biosystems) (temperature conditions: heated at 94 ° C for 2 minutes, then at 98 ° C for 20 seconds, at 68 ° C for 3 minutes). Was repeated 30 times and cooled to 4 ° C. and stored).
[0558]
DNA in the reaction solution after the PCR was recovered by ethanol precipitation, and then dissolved in 80 μl of sterilized water. This DNA was sequentially digested with restriction enzymes BamHI and KpnI. On the other hand, the luciferase expression vector pGL3-Basic (manufactured by Promega) is similarly digested with KpnI and BglII, dephosphorylated using alkaline phosphatase (manufactured by Takara Shuzo Co., Ltd.), and then the above DNA fragment and DNA ligation kit (manufactured by Takara Shuzo). Version 2, Takara Shuzo Co., Ltd.). Escherichia coli DH10B competent cells (manufactured by Gibco BRL) were transformed with this DNA, and ampicillin-resistant colonies were obtained.
[0559]
Some colonies were cultured in a small amount, a plasmid was extracted from the cultured cells, and the nucleotide sequence of the inserted fragment was analyzed using a DNA sequencer (Model 3700, manufactured by Applied Biosystems Co., Ltd.). The plasmid into which the DNA consisting of the nucleotide sequence shown by nucleotide numbers 1 to 526 of SEQ ID NO: 27 was inserted was named pGL8-3.The plasmid pGL8-3 became independent on June 7, 2001 (Heisei 13). It was deposited internationally with the National Institute of Advanced Industrial Science and Technology and the Patent Organism Depositary, under the accession number FERM @ BP-7627. The transformed E. coli harboring this plasmid was transformed into 100 ml of liquid LB medium containing 50 μg / ml of ampicillin. And cultured overnight at 37 ° C. (Wizard Pure transfection kit, manufactured by Promega) a DNA with) was recovered and purified.
[0560]
b) Construction of plasmid pGL13-1
The following nucleotide sequence:
5 '-{aaggtccaa {ttgcatccag} agtggatcca {a} -3'} (SEQ ID NO: 61 in the sequence listing); and
5 '-{ttggatccac {tctggatgca} attggtacct {t} -3'} (SEQ ID NO: 62 in Sequence Listing)
Was synthesized.
[0561]
After 5 μg of each of the above oligonucleotides were mixed and annealed, they were sequentially digested with restriction enzymes BamHI and KpnI. On the other hand, the luciferase expression vector pGL3-Basic (manufactured by Promega) is similarly digested with KpnI and BglII, dephosphorylated using alkaline phosphatase (manufactured by Takara Shuzo Co., Ltd.), and then the above DNA fragment and DNA ligation kit (manufactured by Takara Shuzo). Version 2, Takara Shuzo Co., Ltd.). Escherichia coli DH10B competent cells (manufactured by Gibco BRL) were transformed with this DNA, and ampicillin-resistant colonies were obtained.
[0562]
Plasmids were extracted from cultured cells of several colonies, and the nucleotide sequence was analyzed using a DNA sequencer (Model 3700, manufactured by PerkinElmer Japan Applied Biosystems Division), and the mouse "Angiopoietin-related protein 3" was analyzed. A plasmid pGL13-1 into which DNA corresponding to the +93 to +108 region of the gene (nucleotide numbers 511 to 526 of SEQ ID NO: 27 in the sequence listing) was inserted was obtained. The transformed E. coli harboring this plasmid was cultured overnight at 37 ° C. in 100 ml of a liquid LB medium containing 50 μg / ml of ampicillin, and DNA was purified from the culture using a plasmid extraction and purification kit (Promega). Was collected and purified.
[0563]
Escherichia coli DH10B competent cells (manufactured by Gibco BRL) are similarly transformed with luciferase expression vector pGL3-Basic (manufactured by Promega) DNA, ampicillin-resistant colonies are cultured, and a plasmid extraction and purification kit (Promega) Was recovered and purified. This plasmid DNA was used as a negative control.
[0564]
Embodiment 22 FIG.プ ロ モ ー タ ー Promoter activity of angiopoietin-related protein 3 gene In order to evaluate the promoter activity of the DNA isolated in Example 21, a reporter assay was performed. That is, the firefly luciferase expression plasmid (pGL8-3 or pGL13-1) constructed in Example 21 was transfected into a human liver-derived cell line HepG2, and changes in firefly luciferase activity were examined. In addition, Renilla luciferase expression plasmid pRL-CMV (promega) was used as an internal standard for correcting experimental lot differences between plasmids in transfection efficiency into cells.
[0565]
First, HepG2 cells (purchased from Dainippon Pharmaceutical Co., Ltd.) were placed in a MEM medium (Gibco.com) containing 10% fetal bovine serum (manufactured by Moregate), penicillin / streptomycin, 2 mM L-glutamine, and 1 mM pyruvate. (Manufactured by BRL) at 37 ° C. in a 5% carbon dioxide gas incubator. PBS (-) (manufactured by Nissui Pharmaceutical Co., Ltd.) was added to a large-angle flask (manufactured by Sumitomo Bakelite Co., Ltd.) grown to semi-confluence, and a cell scraper (manufactured by Sumitomo Bakelite Co., Ltd.) was used. Cells were scraped and collected. 5 x 10 cells⁵Cells / well were seeded on a 6-well culture plate (manufactured by Corning) and cultured overnight. 1 μg of pGL3-Basic, pGL8-3, or pGL13-1 DNA was mixed with 0.04 ng of pRL-CMV DNA, and the total volume was adjusted to 5 μl with sterile water to prepare a DNA solution. On the other hand, 6 μl of transfection reagent (TransIT-LT1, manufactured by Takara Shuzo Co., Ltd.) was added to 100 μl of MEM medium without fetal calf serum (the other components were the same as described above), and the mixture was incubated at room temperature for 5 minutes. The DNA sample prepared above was added to this solution, and the solution was kept at room temperature for 5 minutes. Cells were washed twice with 2 ml of serum-free MEM medium (other components were the same as above) and replaced with 2 ml of the same medium. The DNA solution was added to 3 wells at 30 μl / well, and after 4 hours of culture, replaced with 2 ml / well of MEM medium containing 10% fetal bovine serum (other components were the same as above) and cultured overnight at 37 ° C. After the culture, the medium was removed, and the cells were dissolved in 200 μl / well of a cell lysing agent (Passive \ Lysis \ Buffer (manufactured by Promega)) to prepare a cell extract. The firefly and Renilla luciferase activities were measured by using a dual-luciferase reporter assay system (promega) using 20 μl thereof according to the attached protocol (Luminas CT-9000D, Diatron). Used by the company). The experimental results were expressed as firefly luciferase activities corrected for the Renilla luciferase activity of the internal standard.
[0566]
As a result, in HepG2 cells into which pGL8-3 had been introduced, about 19-fold luciferase activity was observed as compared with cells into which pGL3-Basic had been introduced. Thus, SEQ ID NO: 27 of the sequence listing incorporated into pGL8-3 It was revealed that DNA having the nucleotide sequence shown had activity as a promoter. On the other hand, in the cells into which pGL13-1 was introduced, the luciferase activity was reduced to 1/10 of the cells into which pGL8-3 was introduced. From the above results, the promoter activity is present in the DNA represented by nucleotide numbers 1 to 510 of SEQ ID NO: 27 in the sequence listing, that is, the region corresponding to -418 to +92 at the 5 'end upstream of the mouse "angiopoietin-related protein 3" gene. It became clear to do.
[0567]
In the method of the present example, for example, a substance that inhibits the promoter activity of the DNA of the present invention is measured by measuring luciferase activity when HepG2 cells transformed with pGL8-3 are cultured in the presence of a test substance. be able to. The substance that inhibits this promoter activity can be a therapeutic or preventive agent for one or more diseases selected from hyperlipidemia, arteriosclerosis and hyperglycemia.
[0568]
Embodiment 23 FIG.解析 Analysis of “angiopoietin-related protein 4” promoter region
a) Isolation of mouse “angiopoietin-related protein 4” promoter region
The following nucleotide sequence as a PCR primer for specifically amplifying the 5'-terminal upstream region of the gene encoding mouse "angiopoietin-related protein 4":
5 '-{cgggggtaccgagtggggtgctgggagaagcaa-3' (primer 1, SEQ ID NO: 63 in Sequence Listing);
5 '-{cccaagcttgcctttgggtgcagcaacgct} -3' (primer 2, SEQ ID NO: 64 in Sequence Listing)
Was chemically synthesized using a DNA synthesizer (Amersham Bioscience Co., Ltd.).
[0569]
Using 100 ng of genomic DNA derived from mouse 129Sv (bred at Safety Laboratory, Sankyo Co., Ltd.) as a template, 1 μl of the above primers (300 nM each) and KOD 、 DNA polymerase (manufactured by Toyobo), 1 × PCR reaction buffer (attached to DNA polymerase) (1/10 volume of 10 × buffer solution # 1)), 1 mM magnesium chloride, 0.2 mM dNTP and sterilized water were added to make the total volume 50 μl, and PCR was performed. PCR was performed using a thermal cycler (GeneAmp PCR System 9700, manufactured by Applied Biosystems), and the sample was heated at 94 ° C for 2 minutes, then at 94 ° C for 15 seconds, at 60 ° C for 30 seconds, and at 68 ° C for 3 minutes. The temperature cycle was repeated 35 times, and finally, the temperature was kept at 68 ° C. for 3 minutes. The resulting PCR product was purified by QIAquick PCR Purification Kit (manufactured by Qiagen), cut with KpnI and HindIII, and the target band was separated by 1.0% 1 × TAE agarose electrophoresis. Then, SUPREC-01 (Takara Shuzo) DNA) was recovered by ethanol precipitation.
[0570]
On the other hand, the luciferase expression vector pGL3-Basic (manufactured by Promega) is similarly digested with KpnI and HindIII, dephosphorylated using alkaline phosphatase (manufactured by Takara Shuzo Co., Ltd.), and then the above DNA fragment and DNA ligation kit (manufactured by Takara Shuzo). Version 2, Takara Shuzo Co., Ltd.). Escherichia coli DH10B competent cells (manufactured by Gibco BRL) were transformed with this DNA, and ampicillin-resistant colonies were obtained.
Some colonies were cultured in a small amount, a plasmid was extracted from the cultured cells, and the nucleotide sequence of the inserted fragment was analyzed using a DNA sequencer (Model 3700, manufactured by Applied Biosystems Japan). A plasmid into which an approximately 2.9 kb fragment containing a DNA consisting of the nucleotide sequence shown by nucleotide numbers 1 to 260 of 28 was inserted was obtained (pGL2-4).
The transformed Escherichia coli carrying this plasmid was cultured overnight at 37 ° C. in 100 ml of a liquid LB medium containing 50 μg / ml of ampicillin, and a plasmid extraction and purification kit (EndoFree Plasmid Maxi Kit, manufactured by Qiagen) ) Was recovered and purified.
[0571]
b) Construction of luciferase expression plasmid vectors pGL11-4 and pGL12-4
The following nucleotide sequence as a PCR primer for specifically amplifying the 5'-terminal upstream region of the gene encoding mouse "angiopoietin-related protein 4":
5 '-{cggggtaccacaaaagcctgtgggcattgca} -3' (primer 3, SEQ ID NO: 65 in the sequence listing); and
5 '-{cgggggtacccctccccccagaactccagctg} -3' (primer 4, SEQ ID NO: 66 in Sequence Listing)
Was chemically synthesized using a DNA synthesizer.
[0572]
Using the plasmid pGL2-4 as a template, the above primer (300 nM each), 1 μl of KOD DNA polymerase (manufactured by Toyobo), 1 × PCR reaction buffer (10 × buffer # 1 attached to the DNA polymerase in 1/10 volume) 1) Magnesium chloride (1 mM), dNTPs (0.2 mM each) and sterilized water were added to make the total volume 50 μl, and PCR was performed. PCR was performed using a thermal cycler (GeneAmp PCR System 9700, manufactured by Applied Biosystems), and after heating the sample at 94 ° C for 2 minutes, 30 temperature cycles of 94 ° C for 15 seconds and 60 ° C for 15 seconds were performed 30 times. Finally, the heat treatment was performed at 68 ° C. for 3 minutes. The resulting PCR product was purified by QIAquick PCR Purification Kit (manufactured by Qiagen), cut with KpnI and HindIII, and the target band was separated by 1.0% 1 × TAE agarose electrophoresis. Then, SUPREC-01 (Takara Shuzo) DNA) was recovered by ethanol precipitation.
[0573]
On the other hand, the luciferase expression vector pGL3-Basic (manufactured by Promega) is similarly digested with KpnI and HindIII, dephosphorylated using alkaline phosphatase (manufactured by Takara Shuzo Co., Ltd.), and then the above DNA fragment and DNA ligation kit (manufactured by Takara Shuzo). Version 2, Takara Shuzo Co., Ltd.). Escherichia coli DH10B competent cells (manufactured by Gibco BRL) were transformed with this DNA, and ampicillin-resistant colonies were obtained. Some colonies were cultured in a small amount, a plasmid was extracted from the cultured cells, and the nucleotide sequence of the inserted fragment was analyzed using a DNA sequencer (Model 3700, manufactured by Applied Biosystems Japan). The plasmid into which the DNA consisting of the nucleotide sequence represented by nucleotide numbers 1 to 260 of 28 was inserted was named pGL11-4, and the DNA consisting of the nucleotide sequence represented by nucleotide numbers 189 to 260 of SEQ ID NO: 28 in the sequence listing was inserted. The resulting plasmid was designated as pGL12-4.
[0574]
The transformed Escherichia coli carrying this plasmid was cultured overnight at 37 ° C. in 100 ml of a liquid LB medium containing 50 μg / ml of ampicillin, and a plasmid extraction and purification kit (EndoFree Plasmid Maxi Kit, manufactured by Qiagen) ) Was recovered and purified.
[0575]
The transcriptional start point of the gene encoding the mouse “angiopoietin-related protein 4” (5 ′ terminal boundary site of exon 1) is “AK014564”, which is an EST (Expressed Sequence Tag) with the longest 5 ′ end registered in the GenBank database. Was determined with reference to FIG. That is, in the nucleotide sequence of AK014564, the first nucleotide excluding the vector portion was determined as the transcription start point, and the position was defined as "1". In this manner, DNA consisting of a nucleotide sequence corresponding to a region from 90 bp at the 5 'end to 170 bp at the 3' end from the transcription start point of the mouse "angiopoietin-related protein 4" gene was isolated.
[0576]
Similarly, the luciferase expression vector pGL3-Basic (Promega) DNA is used to transform competent cells of Escherichia coli DH10B (Gibco BRL), culture ampicillin-resistant colonies, and use a plasmid extraction and purification kit (EndoFree @ Plasmid). DNA was recovered and purified using Maxi Kit (manufactured by Qiagen). This plasmid DNA was used as a negative control.
[0577]
Embodiment 24 FIG.プ ロ モ ー タ ー Promoter activity of angiopoietin-related protein 4 gene In order to evaluate the promoter activity of the DNA isolated in Example 23, a reporter assay was performed. That is, the firefly luciferase expression plasmid (pGL11-4 or pGL12-4) constructed in Example 23 was transfected into a monkey kidney-derived cell line COS1, and the change in firefly luciferase activity was examined. Renilla luciferase expression plasmid pRL-TK (promega) was used as an internal standard for correcting experimental lot differences between plasmids in transfection efficiency into cells.
[0578]
First, COS1 cells (purchased from American Type Culture Collection (ATCC)) were obtained from a D-MEM medium containing 10% fetal bovine serum (manufactured by Gibco BRL) and penicillin / streptomycin. The cells were cultured in a 5% carbon dioxide incubator at 37 ° C in Gibco BRL Co., Ltd. (Catlog Number 11965-092). The cells were grown on a 24-well culture plate (manufactured by Corning) until they reached semi-confluence, and 0.2 μg of pGL3-Basic, pGL11-4, or pGL12-4 あ る い は DNA was mixed with 6 ng of pRL-TK DNA. Then, the total volume was adjusted to 5 μl with sterile water to prepare a DNA solution. On the other hand, 1 μl of transfection reagent (FuGENE6, manufactured by Roche) was added to 20 μl of D-MEM medium containing no fetal bovine serum (other components were the same as described above), and the mixture was gently stirred. The added DNA sample was added, and the mixture was kept at room temperature for 15 minutes. The DNA solution was added to 3 wells and cultured at 37 ° C. for 2 nights. After the culture, the medium was removed, and the cells were dissolved in 250 μl / well of a cell lysing agent (Passive Lysis Buffer (Promega)) to prepare a cell extract. The firefly and Renilla luciferase activities were measured by using a dual-luciferase reporter assay system (promega) using 20 μl thereof according to the attached protocol (Luminas CT-9000D, Diatron). Used by the company). The experimental results were expressed as firefly luciferase activity corrected with the Renilla luciferase activity of the internal standard, and expressed as a relative value when the pGL3-Basic control was set to 1.
[0579]
As a result, in COS1 cells into which pGL11-4 had been introduced, about 30-fold luciferase activity was observed as compared with cells into which pGL3-Basic had been introduced. It was revealed that DNA having the nucleotide sequence shown had activity as a promoter. On the other hand, cells into which pGL12-4 had been introduced only showed about 1.7 times the luciferase activity as compared to cells into which pGL3-Basic had been introduced. Table 13 shows the results. From the above results, the promoter activity is present in the DNA represented by nucleotide numbers 1 to 260 of SEQ ID NO: 28 in the sequence listing, that is, in the region corresponding to -90 to +170 at the 5′-end upstream of the mouse “angiopoietin-related protein 4” gene. It became clear to do. In the table, (Mean ± SD) means the average value and the standard deviation of the luciferase activity.
[0580]
[Table 13]

In the method of the present example, a substance that inhibits the promoter activity of the DNA of the present invention is measured by measuring luciferase activity when, for example, COS1 cells transformed with pGL11-4 are cultured in the presence of a test substance. be able to. The substance that inhibits this promoter activity can be a therapeutic or preventive agent for one or more diseases selected from hyperlipidemia, arteriosclerosis and hyperglycemia.
[0581]
As described above, according to the present invention, the nucleotide sequence represented by nucleotide numbers 47 to 1411 of SEQ ID NO: 1 in the sequence listing or the nucleotide sequence represented by nucleotide numbers 78 to 1457 of SEQ ID NO: 3 in the sequence listing is obtained. Is one of the genetic predispositions involved in one or more diseases selected from hyperlipidemia, arteriosclerosis and hyperglycemia, and therefore a substance that suppresses the expression level of the gene is It has been confirmed that it can be a therapeutic or preventive agent for one or more diseases selected from hyperlipidemia, arteriosclerosis and hyperglycemia. That is, the method of the present invention, the polynucleotide used as a probe or a primer in the method for detecting a nucleic acid in the method and the antibody used in the method for detecting a polypeptide in the method for detecting a nucleic acid are hyperlipidemia, arteriosclerosis, etc. It is also useful for searching for an agent for treating or preventing one or more diseases selected from hyperglycemia. In addition, the polypeptide of the present invention has an activity of increasing the concentration of triglyceride in blood, the activity can be retained in the form of a fusion protein, and the polypeptide of the present invention has an inhibitory effect on LPL activity. , And the use of the polypeptide of the present invention to test a therapeutic or prophylactic agent for one or more diseases selected from hyperlipidemia, arteriosclerosis and hyperglycemia, either in vivo or in vitro. It was also shown that the method can be carried out.
[0582]
Furthermore, the present invention provides a novel DNA having a promoter activity. Since the DNA of the present invention can be used in a method for testing a substance that suppresses the expression level of “angiopoietin-related protein 3” or “angiopoietin-related protein 4”, hyperlipidemia having a new mechanism of action, It is useful for developing an agent for treating or preventing one or more diseases selected from arteriosclerosis and hyperglycemia.
[Sequence List Free Text]
SEQ ID NO: 9: Synthetic oligopeptide used as antigen for obtaining polyclonal antibody
SEQ ID NO: 11: DNA encoding amino-terminal region of human “angiopoietin-related protein 3”
SEQ ID NO: 12: amino acid sequence of polypeptide encoding amino-terminal region of human “angiopoietin-related protein 3”
SEQ ID NO: 13: DNA sequence of a polypeptide of a peptide encoding the carboxyl terminal region of human "angiopoietin-related protein 3"
SEQ ID NO: 14: Amino acid sequence of a polypeptide encoding the carboxyl terminal region of human “angiopoietin-related protein 3”
SEQ ID NO: 25: DNA encoding {KhAP5} CC / His
SEQ ID NO: 26: Amino acid sequence of {KhAP5} CC / His
SEQ ID NO: 42: PCR primer to which a Kozak consensus sequence has been added
SEQ ID NO: 43: ＰＣＲ PCR primer to which a specific sequence of restriction enzyme XhoI is added SEQ ID NO: 44: ＰＣＲ PCR sense primer for amplifying DNA encoding GST-CC, GST-CCdel and a part of GST-C1
SEQ ID NO: 45: PCR antisense primer for amplifying DNA encoding a part of GST-CC
SEQ ID NO: 46: PCR antisense primer for amplifying DNA encoding a part of GST-CCdel
SEQ ID NO: 47: PCR antisense primer for amplifying DNA encoding a part of GST-C1
SEQ ID NO: 48: oligonucleotide for inserting sequence encoding histidine tag
SEQ ID NO: 49: oligonucleotide for inserting sequence encoding histidine tag
SEQ ID NO: 50: sense / antisense primer for amplifying a partial fragment of plasmid pME18S / KhAP5 / His
SEQ ID NO: 51: PCR sense primer for amplifying DNA encoding a part of GST-ARP4N
SEQ ID NO: 52: PCR antisense primer for amplifying DNA encoding a part of GST-ARP4N
SEQ ID NO: 55: PCR sense primer for amplifying DNA encoding a part of GST-ARP4Full
SEQ ID NO: 56: PCR antisense primer for amplifying DNA encoding a part of GST-ARP4Full
SEQ ID NO: 57: {Sequencing primer} -21M13 forward
SEQ ID NO: 58: << sequencing primer >> M13 reverse
SEQ ID NO: 59: PCR primer 1
SEQ ID NO: 60: PCR primer 2
SEQ ID NO: 61: oligonucleotide for preparing insert of plasmid pGL13-1
SEQ ID NO: 62: {Oligonucleotide for preparing insert of plasmid pGL13-1
SEQ ID NO: 63: {circle around (2)} PCR sense primer to which a sequence specific to restriction enzyme KpnI has been added.
SEQ ID NO: 64: PCR antisense primer to which a sequence specific to restriction enzyme HindIII has been added
SEQ ID NO: 65: {circle around (2)} PCR sense primer to which a sequence specific to restriction enzyme KpnI has been added.
SEQ ID NO: 66: PCR antisense primer to which a sequence specific to restriction enzyme HindIII has been added
[0583]
[Sequence list]

[Brief description of the drawings]
FIG. 1 is a diagram showing the results of Northern blot analysis on a sample derived from a KK mouse organ.
FIG. 2 is a diagram showing the results of Western blot analysis using a transgenic COS-1 cell culture supernatant as a sample.
FIG. 3 is a diagram showing the results of Western blot analysis using a sample of a culture supernatant of a transfected HeLa cell.
FIG. 4. KK / infected with recombinant adenovirusSnkThe figure showing the measurement result of the neutral fat concentration in the peripheral blood of the mouse.
FIG. 5. KK mice and KK / infected with recombinant adenovirus.SnkThe figure showing the result of the Northern blot analysis about the sample derived from the liver of a mouse.
FIG. 6 shows a calibration curve of purified “angiopoietin-related protein 3” by ELISA.
FIG. 7 shows the structure of “angiopoietin-related proteins 1 to 5”. The left side is the amino terminal and the right side is the carboxyl terminal. Angiopoietin-related protein consists of a signal peptide, a helical region, and a fibrinogen-like region from the amino terminus.
FIG. 8: KK /SnkThe figure which showed the effect on the blood neutral fat concentration in a mouse.
FIG. 9 is a diagram showing the results of Western blot analysis using the plasma of a mouse infected with a recombinant adenovirus as a sample.
FIG. 10 shows the results of measuring the concentration of neutral fat in peripheral blood of mice infected with recombinant adenovirus.
FIG. 11 is a graph showing the effect of angiopoietin-related protein 3 in which a mutation has been introduced into a heparin binding site on plasma triglycerides.

Claims (87)

A method for testing the effect of treating or preventing a test substance for one or more diseases selected from hyperlipidemia, arteriosclerosis, and hyperglycemia, which comprises the following steps:
1) culturing cultured cells derived from a mammal in the presence or absence of a test substance;
2) detecting the expression level of mRNA having the nucleotide sequence described in any one of the following a) to e) (where t in the sequence is replaced by u) in the cultured cells obtained in 1) above: Do:
a) a nucleotide sequence represented by nucleotide numbers 47 to 1411 of SEQ ID NO: 1 in the sequence listing;
b) a nucleotide sequence represented by nucleotide numbers 78 to 1457 of SEQ ID NO: 3 in the sequence listing;
c) Transformed E. coli. a nucleotide sequence of a DNA inserted into a phagemid carried by E. coli pBK / m55-1 SANK 72199 (FERM BP-6940);
d) Transformed E. coli. nucleotide sequence of a DNA inserted into a phagemid carried by E. coli pTrip / h55-1 SANK 72299 (FERM BP-6941);
e) a polynucleotide having an activity of hybridizing under stringent conditions with a polynucleotide comprising an antisense sequence of the nucleotide sequence according to any one of the above a) to d) to increase the concentration of neutral fat in blood. A nucleotide sequence encoding a peptide; and 3) comparing the expression level of the detected mRNA between cells cultured in the absence of the test substance and cells cultured in the presence of the test substance, as a result of the above step 2) I do. The method according to claim 1, wherein the cultured cells derived from a mammal are derived from a liver. The method according to claim 1 or 2, wherein the cultured cells derived from mammals are derived from primates or rodents. The method according to any one of claims 1 to 3, wherein the cultured cells derived from a mammal are derived from a human or a mouse. The method according to any one of claims 1 to 4, wherein the means for detecting the expression level of mRNA is a Northern blot, a dot blot or a slot blot. The method according to any one of claims 1 to 4, wherein the means for detecting the expression level of mRNA is RT-PCR. The method according to any one of claims 1 to 4, wherein the means for detecting the expression level of mRNA is a ribonuclease protection assay. The method according to any one of claims 1 to 4, wherein the means for detecting the expression level of mRNA is a run-on assay. The method according to any one of claims 1 to 4, wherein the means for detecting the expression level of mRNA uses a complementary DNA group derived from animal tissue or cells or a gene chip prepared from a partial sequence of the DNA. A method characterized in that: A method for testing the effect of treating or preventing a test substance for one or more diseases selected from hyperlipidemia, arteriosclerosis, and hyperglycemia, which comprises the following steps:
1) culturing the cultured cells in the presence or absence of a test substance;
2) The production amount of the amino acid sequence encoded by the nucleotide sequence described in any one of the following a) to e) or a polypeptide comprising a part thereof in the cultured cell supernatant obtained in 1) above: Detect using an antibody that specifically binds to the polypeptide:
a) a nucleotide sequence represented by nucleotide numbers 47 to 1411 of SEQ ID NO: 1 in the sequence listing;
b) a nucleotide sequence represented by nucleotide numbers 78 to 1457 of SEQ ID NO: 3 in the sequence listing;
c) Transformed E. coli. a nucleotide sequence of a DNA inserted into a phagemid carried by E. coli pBK / m55-1 SANK 72199 (FERM BP-6940);
d) Transformed E. coli. nucleotide sequence of a DNA inserted into a phagemid carried by E. coli pTrip / h55-1 SANK 72299 (FERM BP-6941);
e) a polynucleotide having an activity of hybridizing under stringent conditions with a polynucleotide comprising an antisense sequence of the nucleotide sequence according to any one of the above a) to d) to increase the concentration of neutral fat in blood. Nucleotide sequence encoding a peptide; and 3) comparing the amount of polypeptide detected between cells cultured in the absence of the test substance and cells cultured in the presence of the test substance as a result of step 2) above I do. 11. The method according to claim 10, wherein the antibody that specifically binds to the polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence according to any one of a) to e) or a part thereof is selected from the sequence list. A polypeptide consisting of the amino acid sequence represented by amino acid Nos. 17-455 of SEQ ID NO: 2 or a part thereof; a polypeptide consisting of the amino acid sequence represented by amino acid Nos. 19-455 or a part thereof; 460. A method specifically binding to a polypeptide comprising the amino acid sequence shown in 460 or a part thereof. 12. The method according to claim 10 or 11, wherein the antibody that specifically binds to a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence according to any one of a) to e) or a part thereof is selected from the group consisting of: A method for specifically recognizing an amino acid sequence represented by amino acid numbers 1 to 13 of SEQ ID NO: 9 or an amino acid sequence represented by amino acid numbers 1 to 14 of SEQ ID NO: 10 in the sequence listing. The method according to any one of claims 10 to 12, wherein the production amount of the polypeptide comprising the amino acid sequence encoded by the nucleotide sequence according to any one of (a) to (e) or a part thereof is determined. A method, wherein the operation of detecting using an antibody that specifically binds to the polypeptide is a Western blot, a dot blot, or a slot blot. The method according to any one of claims 10 to 12, wherein the production amount of the polypeptide comprising the amino acid sequence encoded by the nucleotide sequence according to any one of (a) to (e) or a part thereof is determined. A method characterized in that the operation of detecting using an antibody that specifically binds to the polypeptide is enzyme-linked immunosorbent assay (ELISA) or radioisotope immunoassay (RIA). The method according to claim 14, wherein the monoclonal antibody Mab45B1 produced by the mouse hybridoma 45B1 is used as the antibody immobilized on the plate in the ELISA method according to claim 14. The hybridoma 45B1 has been deposited with the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary on March 13, 2002 under the deposit number FERM @ BP-7963. Testing a therapeutic or prophylactic agent for one or more diseases selected from hyperlipidemia, arteriosclerosis and hyperglycemia, which comprises the antibody described in i) and / or ii) below. Kit for:
i) an antibody that specifically binds to a polypeptide consisting of an amino acid sequence encoded by the nucleotide sequence according to any one of the following a) to e) or a part thereof:
a) a nucleotide sequence represented by nucleotide numbers 47 to 1411 of SEQ ID NO: 1 in the sequence listing;
b) a nucleotide sequence represented by nucleotide numbers 78 to 1457 of SEQ ID NO: 3 in the sequence listing;
c) Transformed E. coli. a nucleotide sequence of a DNA inserted into a phagemid carried by E. coli pBK / m55-1 SANK 72199 (FERM BP-6940);
d) Transformed E. coli. nucleotide sequence of a DNA inserted into a phagemid carried by E. coli pTrip / h55-1 SANK 72299 (FERM BP-6941);
e) a polynucleotide having an activity of hybridizing under stringent conditions with a polynucleotide comprising an antisense sequence of the nucleotide sequence according to any one of the above a) to d) to increase the concentration of neutral fat in blood. A nucleotide sequence encoding a peptide;
ii) An antibody which specifically binds to an amino acid sequence represented by amino acid numbers 1 to 13 of SEQ ID NO: 9 or an amino acid sequence represented by amino acid numbers 1 to 14 of SEQ ID NO: 10 in the sequence listing. A high fat containing, as an active ingredient, DNA or RNA having a nucleotide sequence containing an antisense sequence of a nucleotide sequence consisting of 15 to 30 consecutive nucleotides in the nucleotide sequence of nucleotide numbers 78 to 1457 of SEQ ID NO: 3 in the sequence listing A composition for gene therapy of one or more diseases selected from bloodemia, arteriosclerosis and hyperglycemia. One continuous amino acid sequence contained in the amino acid sequence shown in SEQ ID NO: 12 in the sequence listing, wherein the amino acid at amino acid No. 17 is an amino terminal, and any one of amino acids 147 to 207 is a carboxyl terminal. A polypeptide consisting of an amino acid sequence. 19. The polypeptide according to claim 18, comprising an amino acid sequence represented by amino acid numbers 17 to 207 of SEQ ID NO: 12 in the sequence listing. 19. The polypeptide according to claim 18, comprising an amino acid sequence represented by amino acids 17 to 195 of SEQ ID NO: 12 in the sequence listing. 19. The polypeptide according to claim 18, comprising an amino acid sequence represented by amino acid numbers 17 to 147 of SEQ ID NO: 12 in the sequence listing. A polypeptide comprising a continuous one amino acid sequence contained in the amino acid sequence represented by SEQ ID NO: 16 in the sequence listing, the amino acid sequence having amino acid number 26 as an amino terminal and amino acid number 143 as a carboxyl terminal. A continuous amino acid sequence contained in the amino acid sequence represented by SEQ ID NO: 16 in the sequence listing, wherein the amino acid sequence has amino acid number 26 as an amino terminal and amino acid number 144 to 183 as a carboxyl terminal. Polypeptide. A polyamino acid sequence comprising one continuous amino acid sequence contained in the amino acid sequence of SEQ ID NO: 18 in the sequence listing, the amino acid sequence having amino acid number 22 as an amino terminal and any one of amino acid numbers 202 to 276 as a carboxyl terminal. peptide. A continuous amino acid sequence contained in the amino acid sequence of SEQ ID NO: 20 in the sequence listing, wherein the amino acid sequence is amino acid sequence 23, and any one of amino acid sequences 206 to 275 is a carboxyl sequence. peptide. A polyamino acid sequence comprising one continuous amino acid sequence contained in the amino acid sequence of SEQ ID NO: 22 in the sequence listing, wherein the amino acid sequence has amino acid number 21 as an amino terminal and any one of amino acid numbers 185 to 258 as a carboxyl terminal. peptide. A continuous amino acid sequence contained in the amino acid sequence represented by SEQ ID NO: 24 in the sequence listing, wherein the amino acid sequence has amino acid number 27 as an amino terminal and amino acid number 122 to 129 as a carboxyl terminal. Polypeptide. A polypeptide comprising an amino acid sequence having amino acid number 26 in SEQ ID NO: 16 as the amino terminal and 406 as the carboxyl terminal in the sequence listing. A polypeptide comprising an amino acid sequence having amino acid number 22 of SEQ ID NO: 18 in the sequence listing as an amino terminal and 491 as a carboxyl terminal. A polypeptide comprising an amino acid sequence having amino acid number 23 of SEQ ID NO: 20 in the sequence listing as an amino terminal and 493 as a carboxyl terminal. A polypeptide consisting of an amino acid sequence having amino acid number 21 in amino acid number 21 and amino acid number 470 in carboxyl terminal of SEQ ID NO: 22 in the sequence listing. A polypeptide comprising an amino acid sequence having amino acid number 27 in SEQ ID NO: 24 as the amino terminal and 346 as the carboxyl terminal in the sequence listing. A polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 26 in the sequence listing. 34. Neutral fat in the blood of a mammal, wherein one or several amino acid residues are deleted, inserted, added or substituted at one or several sites of the amino acid sequence according to claims 18 to 33. An equivalent of a polypeptide having an activity of increasing the concentration. A DNA encoding the polypeptide according to any one of claims 18 to 34. One continuous sequence contained in the nucleotide sequence shown in SEQ ID NO: 11 in the sequence listing, wherein the nucleotide of nucleotide No. 18 is the 5 'end, and any one of nucleotides 458 to 638 is the 3' end. DNA consisting of a nucleotide sequence that: One continuous sequence contained in the nucleotide sequence shown in SEQ ID NO: 11 in the sequence listing, wherein the nucleotide at nucleotide number 18 is the 5 'end, and any one of nucleotide numbers 458 to 602 is the 3' end. DNA consisting of a nucleotide sequence that: A DNA comprising a continuous sequence contained in the nucleotide sequence shown in SEQ ID NO: 15 in the sequence listing, the nucleotide sequence having the nucleotide of nucleotide number 173 as the 5 'end and the nucleotide number 601 as the 3' end. A DNA consisting of a continuous sequence contained in the nucleotide sequence shown in SEQ ID NO: 15 in the sequence listing and having a nucleotide at nucleotide number 173 as a 5'-terminal and nucleotide number 1393 as a 3'-terminal. One continuous sequence contained in the nucleotide sequence shown in SEQ ID NO: 17 in the sequence listing, wherein the nucleotide at nucleotide number 434 is the 5 'end, and any one nucleotide from nucleotide numbers 1039 to 1261 is the 3' end. DNA consisting of a nucleotide sequence that: A DNA comprising a continuous sequence contained in the nucleotide sequence shown in SEQ ID NO: 17 in the sequence listing, the nucleotide sequence having nucleotide 434 as a 5 'end and nucleotide 1909 as a 3' end. One continuous sequence contained in the nucleotide sequence shown in SEQ ID NO: 19 in the sequence listing, wherein the nucleotide at nucleotide number 22 is the 5 'end, and any one of nucleotide numbers 639 to 846 is the 3' end. DNA consisting of a nucleotide sequence that: A DNA consisting of a continuous sequence contained in the nucleotide sequence shown in SEQ ID NO: 19 in the sequence listing, having a nucleotide at nucleotide 5 at the 5 'end and nucleotide at 1503 at the 3' end. One continuous sequence contained in the nucleotide sequence shown in SEQ ID NO: 21 in the sequence listing, wherein the nucleotide at nucleotide number 242 is the 5 'end, and any one nucleotide from nucleotide numbers 796 to 1015 is the 3' end. DNA consisting of a nucleotide sequence that: A DNA comprising a continuous sequence contained in the nucleotide sequence shown in SEQ ID NO: 21 in the sequence listing, the nucleotide sequence having the nucleotide of nucleotide number 242 as the 5 'end and nucleotide number 1654 as the 3' end. A single continuous sequence contained in the nucleotide sequence shown in SEQ ID NO: 23 of the sequence listing, wherein the nucleotide at nucleotide number 86 is the 5 'end, and any one of nucleotide numbers 373 to 394 is the 3' end. DNA consisting of a nucleotide sequence that: A DNA consisting of a continuous sequence contained in the nucleotide sequence shown in SEQ ID NO: 23 in the sequence listing and having a nucleotide at nucleotide number 86 as a 5'-terminal and nucleotide number 1048 as a 3'-terminal. A DNA comprising a nucleotide sequence represented by nucleotide numbers 18 to 662 of SEQ ID NO: 25 in the sequence listing. A recombinant vector comprising the DNA according to any one of claims 35 to 48. 50. The recombinant vector according to claim 49, which is an adenovirus vector. A host cell transformed with the recombinant vector according to claim 49 or 50. A culture of the host cell according to claim 51, wherein a polypeptide having an activity of increasing the neutral fat concentration in the blood of the mammal is recovered from the culture. A method for producing a polypeptide having an activity of increasing neutral fat concentration. A method for testing an agent for treating or preventing one or more diseases selected from hyperlipidemia, arteriosclerosis and hyperglycemia, comprising the following steps:
1) administering to a non-human mammal one or more polypeptides selected from the polypeptides according to claims 18 to 34, and a test substance; and 2) blood in the animal of 1) above. Measure the triglyceride concentration. A method for testing an agent for treating or preventing one or more diseases selected from hyperlipidemia, arteriosclerosis and hyperglycemia, comprising the following steps:
1) infecting a non-human mammal with an adenovirus carrying the recombinant adenovirus vector of claim 50;
2) The test substance is administered to the animal in the above step 1); and 3) The neutral fat concentration in the blood of the animal in the above step 2) is measured. A method for testing an agent for treating or preventing one or more diseases selected from hyperlipidemia, arteriosclerosis, and hyperglycemia, which comprises lipoprotein lipase (hereinafter, referred to as “LPL”) and / or hepatic bird. In a system for reacting acyl glycerol lipase (hereinafter referred to as “HTGL”) with its substrate, a polypeptide comprising an amino acid sequence represented by amino acid numbers 17 to 460 of SEQ ID NO: 4 in the sequence listing together with a test substance; A polypeptide lacking residues 1 to 253, or the polypeptide according to claims 18 to 34, is allowed to coexist, and then the inhibitory effect of the test substance on the inhibitory effect of the polypeptide on LPL and / or HTGL activity is examined. Features method. Test method for substance regulating LPL activity, including the following steps: 1) Prepare a mixture in which the materials described in the following (1) -a to (3) and the test substance coexist:
(1) -a material containing LPL;
{Circle around (2)}-a Material containing LPL substrate;
(3) a polypeptide comprising an amino acid sequence represented by amino acids 17 to 460 of SEQ ID NO: 4 in the sequence listing, a polypeptide lacking 1 to 253 residues at the carboxyl terminus thereof, or a polypeptide according to claims 18 to 34;
2) A mixture consisting only of the above-mentioned materials 1) -a to 3) is prepared separately from 1);
3) In the mixture of the above 1) and 2), the amount of the fatty acid released from the substrate is measured, respectively, and the measured values are compared. 57. The method according to claim 56, wherein the material containing LPL is a culture supernatant of a cultured adipocyte derived from a mammal. The method according to claim 56 or 57, wherein the material containing LPL is a culture supernatant of a cultured adipocyte derived from a rodent or a primate. 59. The method according to any one of claims 56 to 58, wherein the material comprising the substrate of LPL is glycerol trioleic acid. A method for testing a substance that modulates the activity of LPL and / or HTGL, comprising the steps of:
1) Prepare a mixture in which the following materials (1) -b to (3) are coexisted with the test substance:
(1) -b Material containing LPL and / or HTGL;
{Circle around (2)}-b A material containing a substrate of LPL and / or HTGL;
(3) a polypeptide comprising the amino acid sequence of amino acids 17 to 460 of SEQ ID NO: 4 in the sequence listing, a polypeptide lacking 1 to 253 residues at the carboxyl terminus thereof, or a polypeptide according to claims 18 to 34;
2) A mixture consisting only of the above-mentioned materials 1) -b to 3) is prepared separately from 1);
3) In the mixture of the above 1) and 2), the amount of the fatty acid released from the substrate is measured, and the measured values are compared. 61. The method of claim 60, wherein the material comprising LPL and / or HTGL is plasma from a mammal to which heparin has been administered intravenously. 62. The method according to claim 60 or 61, wherein the material comprising a substrate for LPL and / or HTGL is glycerol trioleate. The material according to claim 59, wherein the material containing LPL and / or HTGL is a culture supernatant of a cultured adipocyte derived from a mammal, or plasma derived from a mammal to which heparin has been intravenously administered. Method. 64. The method according to claim 59 or 63, wherein the material comprising LPL and / or HTGL is a culture supernatant of cultured adipocytes from rodents or primates. 65. The method according to any one of claims 59, 63 or 64, characterized in that the material comprising a substrate for LPL and / or HTGL is glycerol trioleate. DNA represented by any one of the following 1) to 3):
1) In the nucleotide sequence shown in SEQ ID NO: 27 in the sequence listing, a nucleotide sequence having the nucleotide shown by nucleotide No. 1 as the 5 ′ end and the nucleotide shown by any one of nucleotide numbers 510 to 526 as the 3 ′ end DNA consisting of:
2) DNA that hybridizes with the DNA of 1) above under stringent conditions and has promoter activity in mammalian liver-derived cells;
3) A DNA comprising a nucleotide sequence having 95% or more nucleotide sequence identity to the DNA described in 1) above and having promoter activity in mammalian liver-derived cells. A DNA comprising a nucleotide sequence represented by nucleotide numbers 1 to 510 of SEQ ID NO: 27 in the sequence listing. A DNA comprising a nucleotide sequence represented by nucleotide numbers 1 to 510 of SEQ ID NO: 27 in the sequence listing. A DNA comprising a nucleotide sequence represented by nucleotide numbers 1 to 526 of SEQ ID NO: 27 in the sequence listing. DNA represented by any one of the following 1) to 3):
1) a nucleotide sequence having a nucleotide represented by nucleotide No. 1 as a 5 ′ end and a nucleotide represented by any one of nucleotides 188 to 260 as a 3 ′ end in the nucleotide sequence represented by SEQ ID NO: 28 in the sequence listing DNA consisting of:
2) DNA that hybridizes with the DNA of 1) above under stringent conditions and has promoter activity in mammalian cells;
3) A DNA comprising a nucleotide sequence having 95% or more nucleotide sequence identity with the DNA described in 1) above and having promoter activity in mammalian cells. A DNA comprising a nucleotide sequence represented by nucleotide numbers 1 to 188 of SEQ ID NO: 28 in the sequence listing and having promoter activity in mammalian cells. A DNA comprising a nucleotide sequence represented by nucleotide numbers 1 to 188 of SEQ ID NO: 28 in the sequence listing. An expression vector comprising the DNA according to any one of claims 66 to 72. 73. An expression vector comprising the DNA of any one of claims 66 to 72 ligated upstream of the foreign gene in the same transcriptional direction as the sequence of the foreign gene, and capable of expressing the foreign gene in mammalian cells. 75. The expression vector according to claim 74, wherein the foreign gene sequence is a gene sequence encoding a protein selected from the group consisting of luciferase, horseradish peroxidase, alkaline phosphatase, β-galactosidase, and green fluorescent protein. 75. The expression vector according to claim 74, wherein the foreign gene sequence is a gene sequence encoding luciferase. Recombinant plasmid pGL8-3 (FERM @ BP-7627). Expression vector pGL11-4. A host cell transformed with the expression vector according to any one of claims 73 to 78. A method for testing the effect of treating or preventing a test substance for one or more diseases selected from hyperlipidemia, arteriosclerosis and hyperglycemia, comprising the following steps i) to iii):
i) a step of culturing a host cell transformed with the expression vector according to any one of claims 73 to 78 in the presence or absence of a test substance;
ii) a step of detecting or measuring a production amount of a protein encoded by a foreign gene in the expression vector according to any one of claims 73 to 78 in the host cell of i);
iii) a step of comparing the results of the above ii) between detection and measurement of cells cultured in the presence of the test substance and cells cultured in the absence of the test substance. A method for identifying a cDNA encoding a protein having a therapeutic or preventive effect on one or more diseases selected from hyperlipidemia, arteriosclerosis and hyperglycemia, comprising the following steps i) and ii):
i) (1) a host cell transformed with the expression vector according to any one of claims 73 to 78 (hereinafter referred to as "cell A"); Cells transiently or stably expressing the test cDNA (hereinafter referred to as "cell B") and (3) cell A by transformation with the recombinant plasmid incorporated in Individually culturing cells selected from the group consisting of cells (1) to (3) of cells that do not express the test cDNA (hereinafter referred to as “cell C”);
ii) The expression level of the protein encoded by the foreign gene in the expression vector according to any one of claims 73 to 78 in the cell A, the cell B and the cell C of the above i) is measured and compared. Process. A method for detecting a protein that regulates the "angiopoietin-related protein 3" gene promoter activity, comprising contacting a sample containing the protein with the DNA according to any one of claims 66 to 69, and then contacting the DNA with the DNA. A method comprising detecting the bound protein. A method for testing a substance having an activity of regulating the "angiopoietin-related protein 3" gene promoter activity, comprising the following steps i) and ii):
i) a DNA obtained by radiolabeling the DNA according to any one of claims 66 to 69 to which a nuclear extract of a mouse cell and a test substance have been added, and a product obtained by adding only a nuclear extract of a mouse cell. Prepare each;
ii) Electrophoresis of each mixture sample of the above i) and comparing the mobility between the band found in the sample to which only the nuclear extract of mouse cells is added and the band found in the sample to which the test substance is added. A DNA consisting of at least 10 contiguous nucleotides in the nucleotide sequence represented by nucleotide numbers 1 to 526 of SEQ ID NO: 27 in the sequence listing, a nucleic acid consisting of a nucleotide sequence including its antisense sequence, or a derivative thereof. A method for detecting a protein that regulates the "angiopoietin-related protein 4" gene promoter activity, comprising contacting a sample containing the protein with the DNA according to any one of claims 70 to 72, and then contacting the DNA with the DNA. A method comprising detecting the bound protein. A method for testing a substance having an activity of modulating the “angiopoietin-related protein 4” gene promoter activity, comprising the following steps i) and ii):
i) a DNA obtained by radiolabeling the DNA according to any one of claims 70 to 72 to which a mouse cell nuclear extract and a test substance have been added, and a mouse cell nuclear extract only. Prepare each;
ii) Electrophoresis of each mixture sample of the above i) and comparing the mobility between the band found in the sample to which only the nuclear extract of mouse cells is added and the band found in the sample to which the test substance is added. 73. A nucleotide sequence comprising at least 10 contiguous nucleotides in the nucleotide sequence represented by nucleotide numbers 1 to 260 of SEQ ID NO: 28 in the sequence listing, and a nucleotide sequence containing an antisense sequence thereof, according to any one of claims 70 to 72. A nucleic acid or a derivative thereof that promotes or inhibits the promoter activity of the DNA according to (1).

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