JP2004041003A - New protein, its dna and use thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new protein useful for prophylaxis/treatment of heart diseases, central nerve diseases or genital diseases, a polynucleotide encoding the protein, a method for screening a compound regulating the activity of the protein, the compound, etc., obtained by the method for screening. <P>SOLUTION: The protein and polynucleotide are useful as a diagnostic marker, etc., for the heart diseases, central nerve diseases or genital diseases, etc. The compound is obtained by screening using the protein, polynucleotide or an antibody. A neutralizing antibody inhibiting the activity of the protein can be used as a prophylactic agent/therapeutic agent for, e.g. the heart diseases, central nerve diseases or genital diseases. <P>COPYRIGHT: (C)2004,JPO

Description

【００７６】
本願明細書の配列表の配列番号は、以下の配列を示す。
〔配列番号：１〕
実施例１で得られた塩基配列を示す。
〔配列番号：２〕
実施例１で用いられたプライマーの塩基配列を示す。
〔配列番号：３〕
実施例１で用いられたプライマーの塩基配列を示す。
〔配列番号：４〕
実施例１で得られた塩基配列を示す。
〔配列番号：５〕
実施例１で得られた塩基配列を示す。
〔配列番号：６〕
実施例１で用いられたプライマーの塩基配列を示す。
〔配列番号：７〕
実施例１で用いられたプライマーの塩基配列を示す。
〔配列番号：８〕
実施例１で得られた塩基配列を示す。
〔配列番号：９〕
実施例１〜実施例５、実施例８および実施例９で用いられたプライマーの塩基配列を示す。
〔配列番号：１０〕
実施例１で用いられたプライマーの塩基配列を示す。
〔配列番号：１１〕
実施例１で得られた塩基配列を示す。
〔配列番号：１２〕
実施例１で得られたｒ１６４−１ｈ全長遺伝子を含むＤＮＡの塩基配列を示す。
〔配列番号：１３〕
ラット由来新規タンパク質ｒ１６４−１ｈのアミノ酸配列を示す。
〔配列番号：１４〕
実施例１で用いられたプライマーの塩基配列を示す。
〔配列番号：１５〕
実施例１で用いられたプライマーの塩基配列を示す。
〔配列番号：１６〕
実施例１で得られたｒ１６４−１ｈ全長遺伝子を含むＤＮＡの塩基配列を示す。
〔配列番号：１７〕
実施例２〜実施例５で用いられたプライマーの塩基配列を示す。
〔配列番号：１８〕
実施例２で得られた塩基配列を示す。
〔配列番号：１９〕
実施例２で用いられたプライマーの塩基配列を示す。
〔配列番号：２０〕
実施例２で用いられたプライマーの塩基配列を示す。
〔配列番号：２１〕
実施例２で得られた塩基配列を示す。
〔配列番号：２２〕
実施例２で用いられたプライマーの塩基配列を示す。
〔配列番号：２３〕
実施例２および実施例６で用いられたプライマーの塩基配列を示す。
〔配列番号：２４〕
実施例２で得られた塩基配列を示す。
〔配列番号：２５〕
ヒト由来新規タンパク質ｈ１６４−１ｈのアミノ酸配列を示す。
〔配列番号：２６〕
ヒト由来新規タンパク質ｈ１６４−１ｈをコードするＤＮＡの塩基配列を示す。
〔配列番号：２７〕
ラット由来新規タンパク質ｒ１６４−１ｈをコードするＤＮＡの塩基配列を示す。
〔配列番号：２８〕
ヒト由来新規タンパク質ｈ１６４−１ｂのアミノ酸配列を示す。
〔配列番号：２９〕
ヒト由来新規タンパク質ｈ１６４−１ｂをコードするＤＮＡの塩基配列を示す。
〔配列番号：３０〕
ラット由来新規タンパク質ｒ１６４−１ｂのアミノ酸配列を示す。
〔配列番号：３１〕
ラット由来新規タンパク質ｒ１６４−１ｂをコードするＤＮＡの塩基配列を示す。
〔配列番号：３２〕
実施例６で用いられたプライマーの塩基配列を示す。
〔配列番号：３３〕
実施例６で得られた塩基配列を示す。
〔配列番号：３４〕
実施例７で用いられたプライマーの塩基配列を示す。
〔配列番号：３５〕
実施例７で用いられたプライマーの塩基配列を示す。
〔配列番号：３６〕
実施例７で得られた塩基配列を示す。
〔配列番号：３７〕
実施例７で用いられたプライマーの塩基配列を示す。
〔配列番号：３８〕
実施例７で用いられたプライマーの塩基配列を示す。
〔配列番号：３９〕
実施例７で得られた塩基配列を示す。
〔配列番号：４０〕
実施例７で用いられたプライマーの塩基配列を示す。
〔配列番号：４１〕
実施例７で用いられたプライマーの塩基配列を示す。
〔配列番号：４２〕
実施例７で得られた塩基配列を示す。
〔配列番号：４３〕
実施例７で得られた塩基配列を示す。
〔配列番号：４４〕
実施例８および実施例９で用いられたプライマーの塩基配列を示す。
【００７７】
後述の実施例１で得られた形質転換体　Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ　ＪＭ１０９／ｐＴＢ２２６８　は、２００２年６月１２日から日本国茨城県つくば市東１丁目１番地１　中央第６（郵便番号３０５−８５６６）の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受託番号　ＦＥＲＭ　ＢＰ−８０７１として寄託されている。
後述の実施例２で得られた形質転換体　Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ　ＪＭ１０９／ｐＴＢ２２６９　は、２００２年６月１２日から日本国茨城県つくば市東１丁目１番地１　中央第６（郵便番号３０５−８５６６）の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受託番号　ＦＥＲＭ　ＢＰ−８０７２として寄託されている。
後述の実施例６で得られた形質転換体　Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ　ＪＭ１０９／ｐＴＢ２２７０　は、２００２年６月１２日から日本国茨城県つくば市東１丁目１番地１　中央第６（郵便番号３０５−８５６６）の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受託番号　ＦＥＲＭ　ＢＰ−８０７３として寄託されている。
【実施例】
以下に、実施例を挙げて本発明をさらに具体的に説明するが、本発明はこれに限定されるものではない。
実施例１
ｒ１６４−１ｈ遺伝子のクローニング
（１）心筋梗塞モデルラットの作製
渡邉らの報告（サーキュレーションリサーチ、第６９巻、３７０−３７７頁、１９９１年）に従い雄性ウイスターラット（１１週齢：体重３００−４００ｇ）をペントバルビタール（５０　ｍｇ／ｋｇ，ｉ．ｐ．）で麻酔し、人工呼吸下で正中にて開胸した。心嚢膜を切開後、心臓を露出させた。冠動脈の左前下行枝起始部で、糸付き縫合針（エルプ社、５−０シルク）にて心筋ごと冠動脈を絹糸で縛った後閉胸した。偽手術群は糸を縛らずに閉胸した。麻酔から回復後、通常飼育した。
（２）Ｔｏｔａｌ　ＲＮＡの抽出
術後１週経過、８週経過、２０週経過および３０週経過したラットをペントバルビタール麻酔下で開胸し、心臓を摘出した後、生理食塩水で大動脈より逆行性に冠動脈を潅流して血液を洗い流した。摘出した心臓からハサミで左心室以外の組織を取り除いた後、梗塞形成を確認した後に梗塞領域（スカー形成部位）を取り除き、非梗塞領域のみとした。これをハサミで細かく細断した後、ＩＳＯＧＥＮ（和光純薬社製）を用いてＴｏｔａｌ　ＲＮＡを抽出した。
【００７８】
（３）ｒ１６４−１ｈ遺伝子のクローニングと発現ベクターの構築
Ｔｏｔａｌ　ＲＮＡからゲノムＤＮＡを除去する目的でｅｎｚｙｍｅ　ｓｅｔ　ｆｏｒ　ＤＤ（宝酒造社製）を用いてＤＮＡ分解操作を加えた後、Ｆｌｕｏｒｅｓｃｅｎｃｅ　Ｄｉｆｆｅｒｅｎｔｉａｌ　Ｄｉｓｐｌａｙ　ｋｉｔ　Ｆｌｕｏｒｅｓｃｅｉｎ　ｖｅｒｓｉｏｎ（宝酒造社製）を用いてディファレンシャルディスプレー（ＤＤ）を行った。対象組織として偽手術した後８週経過した左心室由来のＴｏｔａｌ　ＲＮＡを用いた。その結果、対象組織と比較して術後１週、２０週、３０週経過した組織で増加を示すバンドを見出した。このバンドをアクリルアミドゲルからカッターで切り出し、滅菌蒸留水に懸濁し、９５℃、１０分間加熱することによりゲルから遺伝子断片を抽出した。次にＰＣＲで再増幅した後、ＤＮＡ塩基配列を解読した。そこで明らかとなった塩基配列（配列番号：１）を公のデータベースであるＥＳＴデータベースを用いてＢｌａｓｔＮによるホモロジー検索を行ったところ、ＡＷ４３５３２９　ＵＩ−Ｒ−ＢＪ０ｐ−ａｆｃ−ｃ−０５−０−ＵＩ．ｓ１　ＵＩ−Ｒ−ＢＪ０ｐ　Ｒａｔｔｕｓ　ｎｏｒｖｅｇｉｃｕｓ　ｃＤＮＡ　ｃｌｏｎｅ　ＵＩ−Ｒ−ＢＪ０ｐ−ａｆｃ−ｃ−０５−０−ＵＩ　３’，　ｍＲＮＡ　ｓｅｑｕｅｎｃｅとほぼ一致することがわかった。そこでこの配列を基に２種のプライマー（配列番号：２および配列番号：３）を設計し、これらのプライマーを用いｒａｔ　ｈｅａｒｔ　Ｍａｒａｔｈｏｎ−Ｒｅａｄｙ　ｃＤＮＡ（ＣＬＯＮＴＥＣＨ社）に対し、プロトコールに従って５’　ＲＡＣＥ　ＰＣＲを行った。その結果、３’側の配列が一致した２種の塩基配列（配列番号：４および配列番号：５）が得られた。また、得られた配列番号：４で表される塩基配列を公のデータベースであるｎｔデータベースを用いてＢｌａｓｔＮによるホモロジー検索を行ったところ、Ｈｏｍｏ　ｓａｐｉｅｎｓ　ｈｙｐｏｔｈｅｔｉｃａｌ　ｐｒｏｔｅｉｎ　ＭＧＣ１４１６１　（ＭＧＣ１４１６１）　ｍＲＮＡ（Ｇｅｎｂａｎｋ　ＩＤ：ＸＭ＿０３２３７７）と相同性が高いことがわかった。そこで配列番号：４で表される塩基配列を基にフォワードプライマー（配列番号：６）を、配列番号：５で表される塩基配列を基にリバースプライマー（配列番号：７）を作成し、これらのプライマーを用いｒａｔ　ｈｅａｒｔ　Ｍａｒａｔｈｏｎ−Ｒｅａｄｙ　ｃＤＮＡ（ＣＬＯＮＴＥＣＨ社）に対し、ＰＣＲを行い、配列番号：８で表される塩基配列を得た。また配列番号：４で表される塩基配列を基に２種のプライマー（配列番号：９および配列番号：１０）を設計し、これらのプライマーを用いｒａｔ　ｈｅａｒｔ　Ｍａｒａｔｈｏｎ−Ｒｅａｄｙ　ｃＤＮＡ（ＣＬＯＮＴＥＣＨ社）に対し、プロトコールに従って再度５’　ＲＡＣＥ　ＰＣＲを行った。その結果、配列番号：１１の塩基配列が得られた。
配列番号：４、配列番号：８および配列番号：１１の３種の塩基配列を合わせることにより、配列番号：１２で表される塩基配列を得、この塩基配列には２５６アミノ酸残基からなるタンパク質（配列番号：１３）をコードする配列番号：２７で表される塩基配列が含まれていた。配列番号：１３で表されるアミノ酸配列を含有するタンパク質を、ｒ１６４−１ｈと命名した。
配列番号：１２で表される塩基配列を基に２種のプライマー（配列番号：１４および配列番号：１５）を設計し、これらのプライマーを用いｒａｔ　ｈｅａｒｔ　Ｍａｒａｔｈｏｎ−Ｒｅａｄｙ　ｃＤＮＡ（ＣＬＯＮＴＥＣＨ社）に対し、ＰＣＲを行い、得られた配列番号：１６で表される塩基配列を有する１０７２ｂｐの断片をｐＴＡＲＧＥＴ　Ｍａｍｍａｌｉａｎ　Ｅｘｐｒｅｓｓｉｏｎ　Ｖｅｃｔｏｒ　ｓｙｓｔｅｍ（Ｐｒｏｍｅｇａ社）の処方に従いプラスミドベクターｐＴＡＲＧＥＴベクター（Ｐｒｏｍｅｇａ社）へサブクローニングした。得られた発現ベクターをｒ１６４１ｈｏｎＴＡＲＧＥと命名した。
ｒ１６４−１ｈ（配列番号：１３）は、Ｈｏｍｏ　ｓａｐｉｅｎｓ　ｈｙｐｏｔｈｅｔｉｃａｌ　ｐｒｏｔｅｉｎ　ＭＧＣ１４１６１とアミノ酸レベルで７７％の相同性があった。
配列番号：１６で表される塩基配列を含むプラスミドｐＴＢ２２６８を有する形質転換体を、エシェリヒア・コリ（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）ＪＭ１０９／ｐＴＢ２２６８と命名した。
【００７９】
実施例２
ｈ１６４−１ｈ遺伝子のクローニングと発現ベクターの構築
ｒ１６４−１ｈをコードする塩基配列を基に設計したプライマー（配列番号：１７および配列番号：９）を用い、ｈｕｍａｎ　ｈｅａｒｔ　Ｍａｒａｔｈｏｎ−Ｒｅａｄｙ　ｃＤＮＡ（ＣＬＯＮＴＥＣＨ社）に対し、ＰＣＲを行い、配列番号：１８で表される塩基配列を得た。また配列番号：１８で表される塩基配列を基に２種のプライマー（配列番号：１９および配列番号：２０）を設計し、これらのプライマーを用いｈｕｍａｎ　ｈｅａｒｔ　Ｍａｒａｔｈｏｎ−Ｒｅａｄｙ　ｃＤＮＡ（ＣＬＯＮＴＥＣＨ社）に対し、プロトコールに従って５’　ＲＡＣＥ　ＰＣＲを行った。その結果、配列番号：２１で表される塩基配列が得られた。配列番号：２１の塩基配列を基に設計したプライマー（配列番号：２２）およびＭＧＣ１４１６１の塩基配列（実施例１参照）を基に設計したプライマー（配列番号：２３）を用い、ｈｕｍａｎ　ｈｅａｒｔ　Ｍａｒａｔｈｏｎ−Ｒｅａｄｙ　ｃＤＮＡ（ＣＬＯＮＴＥＣＨ社）に対し、ＰＣＲを行い、配列番号：２４で表される塩基配列を得た。この塩基配列には２４９アミノ酸残基からなるタンパク質（配列番号：２５）をコードする配列番号：２６で表される塩基配列が含まれていた。配列番号：２５で表されるアミノ酸配列を含有するタンパク質を、ｈ１６４−１ｈと命名した。
得られた断片（配列番号：２４）を再度、２種のプライマー（配列番号：２２および配列番号：２３）を用いてＰＣＲで増幅させ、得られた断片をｐＴＡＲＧＥＴ　Ｍａｍｍａｌｉａｎ　Ｅｘｐｒｅｓｓｉｏｎ　Ｖｅｃｔｏｒ　ｓｙｓｔｅｍ（Ｐｒｏｍｅｇａ社）の処方に従いプラスミドベクターｐＴＡＲＧＥＴベクター（Ｐｒｏｍｅｇａ社）へサブクローニングした。得られた発現ベクターをｈ１６４１ｈｏｎＴＡＲＧＥと命名した。
ｈ１６４−１ｈ（配列番号：２５）は、Ｈｏｍｏ　ｓａｐｉｅｎｓ　ｈｙｐｏｔｈｅｔｉｃａｌ　ｐｒｏｔｅｉｎ　ＭＧＣ１４１６１とアミノ酸レベルで８２％の相同性があった。また、ｈ１６４−１ｈ（配列番号：２５）と実施例１で得られたｒ１６４−１ｈ（配列番号：１３）とは、アミノ酸レベルで９０％の相同性があった。
配列番号：２４で表される塩基配列を含むプラスミドｐＴＢ２２６９を有する形質転換体を、エシェリヒア・コリ（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）ＪＭ１０９／ｐＴＢ２２６９と命名した。
【００８０】
実施例３
心筋梗塞モデルラットでのｒ１６４−１ｈ遺伝子の経時変化の解析
実施例１（２）に記載した心筋梗塞形成術後１週、８週、２０週および３０週経過ラットの左心室の非梗塞領域由来のＴｏｔａｌ　ＲＮＡと、その対象として偽手術した後１週、８週、２０週および３０週経過ラットの左心室由来のＴｏｔａｌ　ＲＮＡからＴａｑＭａｎ　Ｒｅｖｅｒｓｅ　Ｔｒａｎｓｃｒｉｐｔｉｏｎ　Ｒｅａｇｅｎｔｓ（ＰＥアプライドバイオシステムズ社製）を用いてｃＤＮＡを合成した。次にｒ１６４−１ｈを特異的に増幅できるプライマーである配列番号：１７および配列番号：９でそれぞれ表される塩基配列を有するプライマーを用い、ＰＣＲによるｒ１６４−１ｈ遺伝子のコピー数の定量をＡＢＩ　Ｐｒｉｓｍ　７９００　ｓｅｑｕｅｎｃｅ　Ｄｅｔｅｃｔｉｏｎ　Ｓｙｓｔｅｍによって行った。なお、反応は、ＳＹＢＲ　Ｇｒｅｅｎ　ＰＣＲ　Ｍａｓｔｅｒ　Ｍｉｘ（ＰＥアプライドバイオシステムズ社製）を使用し、その全ての方法は添付されている説明書に従って行った。定量化用スタンダードの調製法を以下に記す。心筋梗塞形成術後１週経過ラット左心室の非梗塞領域由来のＴｏｔａｌ　ＲＮＡをＴａｑＭａｎ　Ｒｅｖｅｒｓｅ　Ｔｒａｎｓｃｒｉｐｔｉｏｎ　Ｒｅａｇｅｎｔｓ（ＰＥアプライドバイオシステムズ社製）を用いてｃＤＮＡを合成した。次にｒ１６４−１ｈを特異的に増幅できるプライマーである配列番号：１７および配列番号：９でそれぞれ表されるプライマーを用いてＰＣＲを行い、得られたｒ１６４−１ｈ遺伝子の部分配列を有する遺伝子断片をそのスタンダードとした。更に算出されたコピー数を内部コントロールとしてｒ１６４−１ｈ遺伝子のコピー数と同様にＴａｑＭａｎ　Ｒｏｄｅｎｔ　ＧＡＰＤＨ　Ｃｏｎｔｒｏｌ　ｒｅｇｅｎｔｓ　ＶＩＣ（ＰＥアプライドバイオシステムズ社製）を用いて算出したグリセロール３りん酸脱水酵素のコピー数で補正した後、その値を発現量として対象組織と比較し、変動率をデータ化した。
その結果、ｒ１６４−１ｈ遺伝子は術後１週、２０週および３０週でそれぞれ、１．３倍、１．３倍および１．４倍それぞれ発現が増加することが明らかとなった。
【００８１】
実施例４
正常ラットにおけるｒ１６４−１ｈ遺伝子の組織分布の解析
ｒ１６４−１ｈを特異的に増幅できるプライマーとして配列番号：１７および配列番号：９でそれぞれ表される塩基配列を有するプライマーを用い、脳、心臓、腎臓、脾臓、胸腺、肝臓、胃、小腸、筋肉、肺、精巣および皮膚の各臓器由来のＭａｒａｔｈｏｎ−Ｒｅａｄｙ　ｃＤＮＡ　ｌｉｂｒａｒｙ（ＣＬＯＮＴＥＣＨ社）を鋳型にＰＣＲを行い、ｒ１６４−１ｈ遺伝子のコピー数の定量をＡＢＩ　Ｐｒｉｓｍ　７９００　ｓｅｑｕｅｎｃｅ　Ｄｅｔｅｃｔｉｏｎ　Ｓｙｓｔｅｍによって行った。なお、反応はＳＹＢＲ　Ｇｒｅｅｎ　ＰＣＲ　Ｍａｓｔｅｒ　Ｍｉｘ（ＰＥアプライドバイオシステムズ社製）を使用し、その全ての方法は添付されている説明書に従って行った。定量化用スタンダードの調製法を以下に記す。心筋梗塞形成術後１週経過ラット左心室の非梗塞領域由来のＴｏｔａｌ　ＲＮＡをＴａｑＭａｎ　Ｒｅｖｅｒｓｅ　Ｔｒａｎｓｃｒｉｐｔｉｏｎ　Ｒｅａｇｅｎｔｓ（ＰＥアプライドバイオシステムズ社製）を用いてｃＤＮＡを合成した。次にｒ１６４−１ｈを特異的に増幅できるプライマーである配列番号：１７および配列番号：９でそれぞれ表されるプライマーを用いてＰＣＲを行い、得られたｒ１６４−１ｈ遺伝子の部分配列を有する遺伝子断片をそのスタンダードとした。更に算出されたコピー数を内部コントロールとしてｒ１６４−１ｈ遺伝子のコピー数と同様にＴａｑＭａｎ　Ｒｏｄｅｎｔ　ＧＡＰＤＨ　Ｃｏｎｔｒｏｌ　ｒｅｇｅｎｔｓ　ＶＩＣ（ＰＥアプライドバイオシステムズ社製）を用いて算出したグリセロール３りん酸脱水酵素のコピー数で補正した後、その値を発現量としてデータ化した。
その結果、ｒ１６４−１ｈ遺伝子の心臓での発現が０．００４１と最大で、精巣では０．０００５の発現が見られた。他の臓器では、ｒ１６４−１ｈ遺伝子の発現は０．０００２以下であり、心臓がｒ１６４−１ｈ遺伝子の主要発現部位であることがわかった。
【００８２】
実施例５
ヒトにおけるｈ１６４−１ｈ遺伝子の組織分布の解析
配列番号：１７および配列番号：９で表される塩基配列をそれぞれ有するプライマーを用いて、脳、心臓、腎臓　、脾臓、胸腺　、肝臓、膵臓、小腸、骨格筋、肺、精巣および大動脈の各臓器由来のＭａｒａｔｈｏｎ−Ｒｅａｄｙ　ｃＤＮＡ　ｌｉｂｒａｒｙ（ＣＬＯＮＴＥＣＨ社）を鋳型にＰＣＲを行い、ＰＣＲによるｈ１６４−１ｈ遺伝子のコピー数の定量を、ＡＢＩ　Ｐｒｉｓｍ　７９００　ｓｅｑｕｅｎｃｅ　Ｄｅｔｅｃｔｉｏｎ　Ｓｙｓｔｅｍによって行った。なお、反応は、ＳＹＢＲ　Ｇｒｅｅｎ　ＰＣＲ　Ｍａｓｔｅｒ　Ｍｉｘ（ＰＥアプライドバイオシステムズ社製）を使用し、その全ての方法は添付されている説明書に従って行った。定量化用スタンダードの調製法を以下に記す。ｈｕｍａｎ　ｈｅａｒｔ　Ｍａｒａｔｈｏｎ−Ｒｅａｄｙ　ｃＤＮＡ（ＣＬＯＮＴＥＣＨ社）を用いて上記プライマー（配列番号：１７および配列番号：９）でＰＣＲを行い、得られたｈ１６４−１ｈ遺伝子の部分配列を有する遺伝子断片をそのスタンダードとした。更に算出されたコピー数を内部コントロールとしてｈ１６４−１ｈ遺伝子のコピー数と同様にＴａｑＭａｎ　ＧＡＰＤＨ　Ｃｏｎｔｒｏｌ　ｒｅｇｅｎｔｓ（ＰＥアプライドバイオシステムズ社製）を用いて算出したグリセロール３りん酸脱水酵素のコピー数で補正した後、その値を発現量としてデータ化した。
その結果、心臓でのｈ１６４−１ｈ遺伝子の発現が０．００２１と最大で、精巣では０．０００３の発現が見られた。他の臓器ではｈ１６４−１ｈ遺伝子の発現は０．０００１以下であり、心臓がｈ１６４−１ｈ遺伝子の主要発現部位であることがわかった。
【００８３】
実施例６
ｈ１６４−１ｂ遺伝子のクローニングと発現ベクターの構築
ＭＧＣ１４１６１をコードする塩基配列を基に設計したプライマー　（配列番号：３２および配列番号：２３）を用い、ｈｕｍａｎ　ｈｅａｒｔ　Ｍａｒａｔｈｏｎ−Ｒｅａｄｙ　ｃＤＮＡ（ＣＬＯＮＴＥＣＨ社）に対し、ＰＣＲを行い、配列番号：３３で表される塩基配列を得た。この塩基配列には４８１アミノ酸残基からなるタンパク質（配列番号：２８）をコードする配列番号：２９で表される塩基配列が含まれていた。配列番号：２８で表されるアミノ酸配列を含有するタンパク質を、ｈ１６４−１ｂと命名した。ｈ１６４−１ｂ（配列番号：２８）は、Ｈｏｍｏ　ｓａｐｉｅｎｓ　ｈｙｐｏｔｈｅｔｉｃａｌ　ｐｒｏｔｅｉｎ　ＭＧＣ１４１６１とアミノ酸レベルで７４％の相同性があった。
配列番号：３３で表される塩基配列を再度増幅後、得られた断片をｐＴＡＲＧＥＴ　Ｍａｍｍａｌｉａｎ　Ｅｘｐｒｅｓｓｉｏｎ　Ｖｅｃｔｏｒ　ｓｙｓｔｅｍ（Ｐｒｏｍｅｇａ社）の処方に従いプラスミドベクターｐＴＡＲＧＥＴベクター（Ｐｒｏｍｅｇａ社）へサブクローニングした。得られた発現ベクターをｈ１６４１ｂｏｎＴＡＲＧＥと命名した。
配列番号：３３で表される塩基配列を含むプラスミドｐＴＢ２２７０を有する形質転換体を、エシェリヒア・コリ（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）ＪＭ１０９／ｐＴＢ２２７０と命名した。
【００８４】
実施例７
ｒ１６４−１ｂ遺伝子の塩基配列の決定
ＭＧＣ１４１６１をコードする塩基配列を基に設計したプライマー（配列番号：３４）および配列番号：１２で表される塩基配列を基に設計したプライマー（配列番号：３５）を用い、ｒａｔ　ｂｒａｉｎ　Ｍａｒａｔｈｏｎ−Ｒｅａｄｙ　ｃＤＮＡ（ＣＬＯＮＴＥＣＨ社）に対し、ＰＣＲを行い、配列番号：３６で表される塩基配列を得た。ＭＧＣ１４１６１をコードする塩基配列を基に設計したプライマー（配列番号：３７）および配列番号：１２で表される塩基配列を基に設計したプライマー（配列番号：３８）を用い、ｒａｔ　ｂｒａｉｎ　Ｍａｒａｔｈｏｎ−Ｒｅａｄｙ　ｃＤＮＡ（ＣＬＯＮＴＥＣＨ社）に対し、ＰＣＲを行い、配列番号：３９で表される塩基配列を得た。また配列番号：３９で表される塩基配列を基に２種のプライマー（配列番号：４０および配列番号：４１）を設計し、これらのプライマーを用いｒａｔ　ｂｒａｉｎ　Ｍａｒａｔｈｏｎ−Ｒｅａｄｙ　ｃＤＮＡ（ＣＬＯＮＴＥＣＨ社）、ｒａｔ　ｈｅａｒｔ　Ｍａｒａｔｈｏｎ−Ｒｅａｄｙ　ｃＤＮＡ（ＣＬＯＮＴＥＣＨ社）、ｒａｔ　ｔｅｓｔｉｓ　Ｍａｒａｔｈｏｎ−Ｒｅａｄｙ　ｃＤＮＡ（ＣＬＯＮＴＥＣＨ社）、に対し、プロトコールに従って５’　ＲＡＣＥ　ＰＣＲを行った。その結果、配列番号：４２で表される塩基配列が得られた。配列番号１２、配列番号：３６、配列番号：３９、および配列番号：４２の塩基配列を合わせることにより、配列番号：４３の塩基配列を得た。この塩基配列には４８１アミノ酸残基からなるタンパク質（配列番号：３０）をコードする配列番号：３１の塩基配列が含まれていた。配列番号：３０で表されるアミノ酸配列を含有するタンパク質を、ｒ１６４−１ｂと命名した。
ｒ１６４−１ｂ（配列番号：３０）は、Ｈｏｍｏ　ｓａｐｉｅｎｓ　ｈｙｐｏｔｈｅｔｉｃａｌ　ｐｒｏｔｅｉｎ　ＭＧＣ１４１６１とアミノ酸レベルで４８％の相同性があった。また、ｒ１６４−１ｂ（配列番号：３０）と実施例１で得られたｒ１６４−１ｈ（配列番号：１３）とは、アミノ酸レベルで７８％の相同性があった。また、ｒ１６４−１ｂ（配列番号：３０）と実施例６で得られたｈ１６４−１ｂ（配列番号：２８）とは、アミノ酸レベルで９８％の相同性があった。
【００８５】
実施例８
心筋梗塞モデルラットでのｒ１６４−１ｂ遺伝子の経時変化の解析
実施例１（２）に記載した心筋梗塞形成術後１週、８週、２０週および３０週経過ラットの左心室の非梗塞領域由来のＴｏｔａｌ　ＲＮＡと、その対象として偽手術した後１週、８週、２０週および３０週経過ラットの左心室由来のＴｏｔａｌ　ＲＮＡからＴａｑＭａｎ　Ｒｅｖｅｒｓｅ　Ｔｒａｎｓｃｒｉｐｔｉｏｎ　Ｒｅａｇｅｎｔｓ（ＰＥアプライドバイオシステムズ社製）を用いてｃＤＮＡを合成した。次にｒ１６４−１ｂを特異的に増幅させるプライマーとして、配列番号：４４および配列番号：９でそれぞれ表される塩基配列を有するプライマーを用い、ＰＣＲによるｒ１６４−１ｂ遺伝子のコピー数の定量をＡＢＩ　Ｐｒｉｓｍ　７９００　ｓｅｑｕｅｎｃｅ　Ｄｅｔｅｃｔｉｏｎ　Ｓｙｓｔｅｍによって行った。なお、反応は、ＳＹＢＲ　Ｇｒｅｅｎ　ＰＣＲ　Ｍａｓｔｅｒ　Ｍｉｘ（ＰＥアプライドバイオシステムズ社製）を使用し、その全ての方法は添付されている説明書に従って行った。定量化用スタンダードの調製法を以下に記す。Ｒａｔ　Ｂｒａｉｎ　Ｍａｒａｔｈｏｎ−Ｒｅａｄｙ　ｃＤＮＡ（ＣＬＯＮＴＥＣＨ社）と、配列番号：４４および配列番号：９でそれぞれ表される塩基配列を有するプライマーとを用いてＰＣＲを行い、得られたｒ１６４−１ｂ遺伝子の部分配列を有する遺伝子断片をそのスタンダードとした。更に算出されたコピー数を内部コントロールとしてｒ１６４−１ｂ遺伝子のコピー数と同様にＴａｑＭａｎ　Ｒｏｄｅｎｔ　ＧＡＰＤＨ　Ｃｏｎｔｒｏｌ　ｒｅｇｅｎｔｓ　ＶＩＣ（ＰＥアプライドバイオシステムズ社製）を用いて算出したグリセロール３りん酸脱水酵素のコピー数で補正した後、その値を発現量として対象組織と比較し、変動率をデータ化した。
その結果、ｒ１６４−１ｂ遺伝子は術後１週、８週、２０週および３０週でそれぞれ、２．２倍、２．２倍、２．７倍および３．３倍それぞれ発現が増加することが明らかとなった。
【００８６】
実施例９
正常ラットにおけるｒ１６４−１ｂ遺伝子の組織分布の解析
ｒ１６４−１ｂを特異的に増幅させるプライマーとして配列番号：４４および配列番号：９でそれぞれ表される塩基配列を有するプライマーを用い、脳、心臓、腎臓、脾臓、胸腺、肝臓、胃、小腸、筋肉、肺、精巣および皮膚の各臓器由来のＭａｒａｔｈｏｎ−Ｒｅａｄｙ　ｃＤＮＡ　ｌｉｂｒａｒｙ（ＣＬＯＮＴＥＣＨ社）を鋳型にＰＣＲを行い、ｒ１６４−１ｂ遺伝子のコピー数の定量をＡＢＩ　Ｐｒｉｓｍ　７９００　ｓｅｑｕｅｎｃｅ　Ｄｅｔｅｃｔｉｏｎ　Ｓｙｓｔｅｍによって行った。なお、反応はＳＹＢＲ　Ｇｒｅｅｎ　ＰＣＲ　Ｍａｓｔｅｒ　Ｍｉｘ（ＰＥアプライドバイオシステムズ社製）を使用し、その全ての方法は添付されている説明書に従って行った。定量化用スタンダードの調製法を以下に記す。Ｒａｔ　Ｂｒａｉｎ　Ｍａｒａｔｈｏｎ−Ｒｅａｄｙ　ｃＤＮＡ（ＣＬＯＮＴＥＣＨ社）と配列番号：４４および配列番号：９でそれぞれ表される塩基配列を有するプライマーとを用いてＰＣＲを行い、得られたｒ１６４−１ｂ遺伝子の部分配列を有する遺伝子断片をそのスタンダードとした。更に算出されたコピー数を内部コントロールとしてｒ１６４−１ｂ遺伝子のコピー数と同様にＴａｑＭａｎ　Ｒｏｄｅｎｔ　ＧＡＰＤＨ　Ｃｏｎｔｒｏｌ　ｒｅｇｅｎｔｓ　ＶＩＣ（ＰＥアプライドバイオシステムズ社製）を用いて算出したグリセロール３りん酸脱水酵素のコピー数で補正した後、その値を発現量としてデータ化した。
その結果、ｒ１６４−１ｂ遺伝子発現は精巣での発現が０．００３９と最大で、脳では０．０００５の発現が見られた。心臓での発現は０．００００２であり、他の臓器では、ｒ１６４−１ｂ遺伝子の発現は０．０００１以下であった。精巣と脳がｒ１６４−１ｂ遺伝子の主要発現部位であることがわかった。
【００８７】
【発明の効果】
本発明のタンパク質およびポリヌクレオチドは、心疾患（例、心筋症、心筋梗塞、心不全、狭心症など）、中枢神経疾患（例、アルツハイマー病、パーキンソン症候群、精神分裂病など）、生殖器疾患（例、精巣過敏症、卵巣機能不全、不妊症など）などの診断マーカー等として有用であり、該タンパク質、ポリヌクレオチドまたは該タンパク質に対する抗体を用いるスクリーニングにより得られる化合物またはその塩、該タンパク質の活性を阻害する中和抗体などは、例えば、心疾患（例、心筋症、心筋梗塞、心不全、狭心症など）、中枢神経疾患（例、アルツハイマー病、パーキンソン症候群、精神分裂病など）、生殖器疾患（例、精巣過敏症、卵巣機能不全、不妊症など）などの予防・治療剤、または避妊薬として使用することができる。
また、本発明のアンチセンスヌクレオチドは、本発明のタンパク質の発現を抑制することができ、例えば、心疾患（例、心筋症、心筋梗塞、心不全、狭心症など）、中枢神経疾患（例、アルツハイマー病、パーキンソン症候群、精神分裂病など）、生殖器疾患（例、精巣過敏症、卵巣機能不全、不妊症など）などの予防・治療剤、または避妊薬などとして使用することができる。さらに、本発明のポリヌクレオチドは、例えば心疾患（例、心筋症、心筋梗塞、心不全、狭心症など）中枢神経疾患（例、アルツハイマー病、パーキンソン症候群、精神分裂病など）、生殖器疾患（例、精巣過敏症、卵巣機能不全、不妊症など）などの診断、予防または治療に有用である。
【００８８】
【配列表】

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a novel protein, a DNA encoding the protein, a method for screening a compound that regulates the activity of the protein, a compound obtained by the screening method, and the like. More specifically, the present invention relates to a novel protein or the like useful as a prophylactic / therapeutic agent or diagnostic agent for heart disease.
[0002]
[Prior art]
Heart failure is considered to be contractile dysfunction of the myocardium, and the mechanism of its onset is as follows. Myocardial damage, mechanical abnormalities of the heart pump, functional abnormalities of the heart pump, pressure overload due to hypertension or pulmonary hypertension, and volume overload such as anemia or acute nephritis. Due to these factors, the heart operates a sympathetic nervous system, a nerve-fluid-endocrine system, etc., in response to a state in which the heart cannot pump blood volume according to the demand of the living body, thereby causing Try to maintain homeostasis. As a result, cardiac hypertrophy occurs due to the enlargement of cardiomyocytes. However, if the above-mentioned disorder or load is chronically sustained, the compensatory mechanism will fail. In other words, not enough blood is supplied to the enlarged cardiomyocytes, resulting in ischemia, resulting in myocardial damage such as myocardial contractile dysfunction, decreased cardiac output, impaired organ circulation, venous congestion, and fluid retention. It will lead to heart failure syndrome with such. Treatment for this requires improvement of myocardial cell damage, enhancement of cardioprotective effects, recovery of cardiac dysfunction due to myocardial contractile insufficiency, and suppression of destructive failure of the living body or improvement of excessive compensation mechanisms. . At present, in treating the heart failure syndrome, (1) cardiac glycosides such as digoxin, (2) sympathomimetics such as dobutamine, and (3) phosphodiesterase inhibitors such as amrinone are clinically used as inotropic drugs. As vasodilators, hydralazine, calcium antagonists, angiotensin exchange enzyme inhibitors, angiotensin receptor antagonists and the like have been used. For treating dilated cardiomyopathy, β-blockers and the like are used. On the other hand, there is no therapeutic agent from the viewpoint of suppressing excessive compensatory mechanisms or suppressing compensatory failure (including suppression of apoptosis).
[0003]
[Problems to be solved by the invention]
There is a strong need for the development of an excellent therapeutic agent for heart failure with few side effects from the viewpoint of suppressing excessive compensation mechanism or compensation failure.
[0004]
[Means for Solving the Problems]
The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, have found a gene whose expression is increased at the onset of heart failure in a myocardial infarction model rat due to coronary artery ligation. As a result of further studies based on this finding, the present invention has been completed.
That is, the present invention
(1) a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 13 or SEQ ID NO: 30, or a salt thereof,
(2) the protein according to (1) or a salt thereof, which comprises the amino acid sequence represented by SEQ ID NO: 25;
(3) the protein of the above-mentioned (1), which comprises the amino acid sequence represented by SEQ ID NO: 28, or a salt thereof,
(4) the protein or a salt thereof according to the above (1), which comprises the amino acid sequence represented by SEQ ID NO: 13;
(5) The protein or a salt thereof according to the above (1), comprising the amino acid sequence represented by SEQ ID NO: 30;
(6) a partial peptide of the protein according to (1) or a salt thereof,
(7) a polynucleotide containing a polynucleotide encoding the protein of (1) or the partial peptide of (6),
(8) the polynucleotide according to the above (7), which is a DNA;
(9) The polynucleotide according to the above (8), comprising the nucleotide sequence represented by SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 27 or SEQ ID NO: 31.
(10) a recombinant vector containing the polynucleotide according to (7),
(11) a transformant transformed with the recombinant vector according to (10),
(12) The transformant according to (1), wherein the transformant according to (11) is cultured to produce and accumulate the protein according to (1) or the partial peptide according to (6), and collect the protein. )) The method for producing the protein or the partial peptide according to (6) or a salt thereof,
(13) a medicament comprising the protein according to (1) or a partial peptide thereof or a salt thereof;
(14) a medicine comprising the polynucleotide according to (7),
(15) a diagnostic agent comprising the polynucleotide according to (7),
(16) an antibody against the protein according to (1) or the partial peptide according to (6) or a salt thereof,
(17) a diagnostic agent comprising the antibody according to the above (16),
(18) a medicine comprising the antibody according to the above (16),
(19) an antisense nucleotide comprising a nucleotide sequence complementary or substantially complementary to the nucleotide sequence of the polynucleotide according to (7) or a part thereof,
(20) a medicine comprising the antisense nucleotide according to (19), (21) the protein according to (1), the partial peptide according to (6), or a salt thereof, A method for screening a compound or a salt thereof that regulates the activity of the protein of (1) or the partial peptide of (6) or a salt thereof;
(22) The protein according to (1), the partial peptide according to (6), or a salt thereof, comprising the protein according to (1), the partial peptide according to (6), or a salt thereof. A kit for screening a compound or a salt thereof that regulates the activity of
(23) The activity of the protein of (1), the partial peptide of (6), or a salt thereof, which can be obtained by using the screening method of (21) or the screening kit of (22). A modulating compound or a salt thereof,
(24) a medicament comprising the compound according to (23) or a salt thereof,
(25) A method for screening a compound or a salt thereof that regulates the expression of the protein gene according to (1), which comprises using the polynucleotide according to (7).
(26) A kit for screening a compound or a salt thereof that regulates the expression of the protein gene according to (1), which comprises the polynucleotide according to (7).
(27) A compound or a salt thereof that regulates the expression of the protein gene according to (1), which can be obtained by using the screening method according to (25) or the screening kit according to (26).
(28) a medicament comprising the compound of (27) or a salt thereof,
(29) The method for quantifying a protein according to (1), comprising using the antibody according to (16);
(30) a method for diagnosing a disease associated with the function of the protein according to (1), which comprises using the quantification method according to (29);
(31) a method for screening a compound or a salt thereof that regulates the expression of the protein according to (1), which comprises using the antibody according to (16);
(32) A kit for screening a compound or a salt thereof that regulates the expression of the protein according to (1), comprising the antibody according to (16);
(33) A compound or a salt thereof that regulates the expression of the protein according to (1), obtained by using the screening method according to (31) or the screening kit according to (32).
(34) a medicament comprising the compound according to (33) or a salt thereof,
(35) The medicament according to the above (13), (14), (18), (20), (24), (28) or (34), which is a prophylactic / therapeutic agent for heart disease.
(36) The medicament according to the above (24), (28) or (34), wherein the compound is a compound that suppresses excessive compensatory mechanism or compensatory failure.
(37) The medicament according to the above (35) or (36), which is an agent for preventing or treating cardiomyopathy, myocardial infarction, heart failure or angina.
(38) The diagnostic agent according to the above (15) or (17), which is a diagnostic agent for heart disease, is provided.
Moreover,
(39) the protein is (i) the amino acid sequence represented by SEQ ID NO: 25 or SEQ ID NO: 13, or (ii) one or more of the amino acid sequences represented by SEQ ID NO: 25 or SEQ ID NO: 13 (For example, about 1 to 30, preferably about 1 to 10, and more preferably number (1 to 5)) of amino acid sequences deleted, (iii) represented by SEQ ID NO: 25 or SEQ ID NO: 13. An amino acid sequence obtained by adding one or more amino acids (for example, about 1 to 30, preferably about 1 to 10, more preferably number (1 to 5)) to the amino acid sequence to be prepared, (iv) SEQ ID NO: : 25 or one or more (eg, about 1 to 30, preferably about 1 to 10, more preferably number (1 to 5)) amino acids are inserted into the amino acid sequence represented by SEQ ID NO: 13. (V) one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 25 or SEQ ID NO: 13 (for example, about 1 to 30, preferably about 1 to 10, more preferably The protein according to the above (1), which is a protein containing an amino acid sequence in which (1 to 5) amino acids are substituted with another amino acid, or (vi) an amino acid sequence obtained by combining the above (ii) to (v). ,
(40) Also provided is a polynucleotide that hybridizes with the polynucleotide of the above (5) under conditions of high stringency.
[0005]
BEST MODE FOR CARRYING OUT THE INVENTION
A protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 13 or SEQ ID NO: 30 (hereinafter also referred to as the protein of the present invention) Are) human or non-human warm-blooded animals (eg, guinea pigs, rats, mice, chickens, rabbits, pigs, sheep, cattle, monkeys, etc.) (eg, hepatocytes, splenocytes, nerve cells, glial cells, Pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, goblet cells, endothelial cells, smooth muscle cells, fibroblasts, fiber cells, muscle cells, adipocytes, immune cells (eg, macrophages, T cells) Cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, osteocytes, osteoblasts, ruptures Cells, mammary cells, hepatocytes or stromal cells, or precursors of these cells, stem cells or cancer cells) or any tissue in which these cells are present, for example, the brain, various parts of the brain (eg, olfactory bulb, tonsil nucleus) , Basal sphere, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla, cerebellum), spinal cord, pituitary, stomach, pancreas, kidney, liver, gonads, thyroid, gallbladder, bone marrow, adrenal gland, skin, muscle, lung, digestion It may be a protein derived from a duct (eg, large intestine, small intestine), blood vessel, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testicle, ovary, placenta, uterus, bone, joint, skeletal muscle, etc. Or a synthetic protein.
[0006]
As the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 25, about 85% or more, preferably about 90% or more, and more preferably about 95% or more, of the amino acid sequence represented by SEQ ID NO: 25 And more preferably an amino acid sequence having about 97% or more homology.
Examples of the protein containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 25 include, for example, a protein containing the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 25; A protein having substantially the same activity as the protein having the amino acid sequence represented by No. 25 is preferred.
The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 28 is 75% or more, preferably about 80% or more, about 85% or more, and preferably about 75% or more of the amino acid sequence represented by SEQ ID NO: 28. Amino acid sequences having 90% or more, more preferably about 95% or more, more preferably about 97% or more homology, and the like.
Examples of the protein containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 28 include, for example, a protein containing the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 28; A protein having substantially the same activity as the protein having the amino acid sequence represented by No. 28 is preferable.
The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 13 is about 80% or more, preferably about 85% or more, preferably about 90% or more as the amino acid sequence represented by SEQ ID NO: 13. More preferably, the amino acid sequence has about 95% or more, more preferably about 97% or more homology.
Examples of the protein containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 13 include, for example, a protein containing the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 13; A protein having substantially the same activity as the protein having the amino acid sequence represented by No. 13 is preferred.
The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 29 includes about 50% or more, preferably about 60% or more, preferably about 70% or more, of the amino acid sequence represented by SEQ ID NO: 29. Amino acid sequences having about 80% or more, preferably about 85% or more, preferably about 90% or more, more preferably about 95% or more, and more preferably about 97% or more homology.
Examples of the protein containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 29 include, for example, a protein containing the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 29; A protein having substantially the same activity as the protein having the amino acid sequence represented by No. 29 is preferred.
Examples of substantially equivalent activities include, for example, cardiac function promoting or suppressing activity, cardiomyocyte functional activity or inactivating effect, and the like. Specific examples include cardiac function-promoting activity as one of the compensatory mechanisms of cardiac function, and cardiac function-suppressing activity due to compensatory failure caused by excessive activation of the compensatory mechanism. The cardiomyocyte function activating action also includes a cytoprotective action.
The activity of promoting or suppressing cardiac function and the activity of activating or inactivating cardiomyocyte function of the protein of the present invention are measured by measuring cardiac function.
The measurement of cardiac function can be measured according to a known method or a method analogous thereto. For example, the measurement is performed by an echocardiograph (Cell, Vol. 97, pp. 189-198, 1999) or cardiac function measurement using a cardiac catheter (circulation research, Vol. 69, pp. 370-377, 1991). Furthermore, for example, the enhancement of renin-angiotensin system (RAS) such as angiotensin I converting enzyme (ACE) is measured using a commercially available measurement kit (eg, manufactured by Peninsula, Phoenix, etc.), and the activity of increasing catecholamine in blood is measured. (Tosoh Corp., fully automatic catecholamine analyzer) is used to measure cardiac function.
When the activation or inactivation of cardiomyocyte function is measured using cells having the ability to produce the protein of the present invention, for example, the respiratory activity of the cells is measured, or a heart failure marker gene [eg, ANP (atrial sodium A change in expression of a diuretic peptide) or BNP (such as brain natriuretic peptide) is measured or a production amount of a heart failure marker gene product is measured.The respiratory activity of cells can be measured according to a known method or a method analogous thereto. For example, the MTT (3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium) method, the trypan blue staining method, and the TUNNEL staining method (Terminal deoxytransferase-measured dUTP-X nickend) abeling,
Cell, Vol. 97, pp. 189-198, 1999).
Substantially equivalent indicates that the activity is qualitatively (eg, physiologically or pharmacologically) equivalent. Therefore, the activity of promoting or suppressing cardiac function and the activity of activating or inactivating cardiomyocyte function are equivalent (eg, about 0.01 to 100 times, preferably about 0.1 to 10 times, more preferably 0.5 to 10 times). However, quantitative factors such as the degree of the activity and the molecular weight of the protein may be different.
[0007]
Examples of the protein of the present invention include, for example, (1) (i) one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 25 (for example, about 1 to 30, preferably about 1 to 10, More preferably, an amino acid sequence in which a number (1 to 5) of amino acids are deleted, and (ii) one or more (for example, about 1 to 30, preferably 1 to 30) amino acids in the amino acid sequence represented by SEQ ID NO: 25 An amino acid sequence to which about 10 to 10, more preferably a number (1 to 5) amino acids have been added; (iii) one or more (for example, about 1 to 30) amino acids to the amino acid sequence represented by SEQ ID NO: 25 (Preferably about 1 to 10, more preferably number (1 to 5) amino acids), and (iv) one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 25 ( For example, 1 to 30 (Preferably about 1 to 10, more preferably number (1 to 5) amino acids) in which an amino acid sequence is substituted with another amino acid, or (v) a protein containing an amino acid sequence obtained by combining them. So-called mutein,
(2) (i) 1 or 2 or more in the amino acid sequence represented by SEQ ID NO: 28 (for example, about 1 to 30, preferably about 1 to 10, more preferably number (1 to 5)) (Ii) one or two or more amino acids (e.g., about 1 to 30, preferably about 1 to 10, more preferably about 1 to 10 amino acids) in the amino acid sequence represented by SEQ ID NO: 28; (Iii) 1 or 2 or more amino acids (e.g., about 1 to 30, preferably about 1 to 10, more preferably about 1 to 10) in the amino acid sequence represented by SEQ ID NO: 28. Is an amino acid sequence in which several (1 to 5) amino acids are inserted, and (iv) one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 28 (for example, about 1 to 30, preferably 1 to 30) About 10 or more Properly a few (1-5)) amino acids are substituted by other amino acids, or (v) the so-called muteins such as proteins containing the amino acid sequence comprising a combination thereof,
(3) (i) 1 or 2 or more in the amino acid sequence represented by SEQ ID NO: 13 (eg, about 1 to 30, preferably about 1 to 10, more preferably number (1 to 5)) (Ii) one or two or more amino acids (for example, about 1 to 30, preferably about 1 to 10, more preferably about 1 to 10 amino acids in the amino acid sequence represented by SEQ ID NO: 13) (Iii) 1 or 2 or more amino acids (e.g., about 1 to 30, preferably about 1 to 10, more preferably about 1 to 10 amino acids) in the amino acid sequence represented by SEQ ID NO: 13. Is an amino acid sequence in which several (1 to 5) amino acids have been inserted, and (iv) one or more (for example, about 1 to 30, preferably 1 to 3) in the amino acid sequence represented by SEQ ID NO: 13. About 10 or more Properly a few (1-5)) amino acids are substituted by other amino acids, or (v) the so-called muteins such as proteins containing the amino acid sequence comprising a combination thereof,
(4) (i) 1 or 2 or more in the amino acid sequence represented by SEQ ID NO: 30 (for example, about 1 to 30, preferably about 1 to 10, more preferably number (1 to 5)) (Ii) one or more amino acids (for example, about 1 to 30, preferably about 1 to 10, more preferably about 1 to 10 amino acids) in the amino acid sequence represented by SEQ ID NO: 30; (Iii) 1 or 2 or more amino acids (e.g., about 1 to 30, preferably about 1 to 10, more preferably about 1 to 10) to the amino acid sequence represented by SEQ ID NO: 30. Is an amino acid sequence in which several (1 to 5) amino acids are inserted, and (iv) one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 30 (for example, about 1 to 30, preferably 1 to 3) About 10 or more Properly it is also included so-called muteins such as proteins containing the amino acid sequence amino acid is a combination amino acid sequence are substituted with other amino acids, or (v) their number (1-5) pieces).
[0008]
In the protein in the present specification, the left end is the N-terminus (amino terminus) and the right end is the C-terminus (carboxyl terminus) according to the convention of peptide labeling. The protein of the present invention has a carboxyl group (—COOH) and a carboxylate (—COO) ⁻ ), Amide (-CONH ₂ )) Or an ester (—COOR).
Here, as R in the ester, for example, C, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, etc. _1-6 Alkyl groups such as C, cyclopentyl, cyclohexyl, etc. _3-8 Cycloalkyl groups such as phenyl, α-naphthyl and the like; _6-12 Aryl groups, for example phenyl-C such as benzyl, phenethyl, etc. _1-2 An alkyl group or α-naphthyl-C such as α-naphthylmethyl _1-2 C such as an alkyl group _7-14 An aralkyl group, a pivaloyloxymethyl group and the like are used.
When the protein of the present invention has a carboxyl group (or carboxylate) other than the C-terminus, the protein of the present invention includes a carboxyl group amidated or esterified. As the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.
Furthermore, in the protein of the present invention, the amino group of the N-terminal amino acid residue (eg, methionine residue) has a protecting group (eg, formyl group, acetyl group, etc.). _1-6 C such as alkanoyl _1-6 Acyl group), N-terminal glutamine residue generated by cleavage in vivo, pyroglutamine oxidation, substituent on the side chain of amino acid in the molecule (eg, -OH, -SH , An amino group, an imidazole group, an indole group, a guanidino group, etc.) are suitable protecting groups (for example, formyl group, acetyl group, etc.). _1-6 C such as alkanoyl group _1-6 (Eg, an acyl group), or a complex protein such as a so-called glycoprotein to which a sugar chain is bound.
Specific examples of the protein of the present invention include, for example, a protein containing the amino acid sequence represented by SEQ ID NO: 25, a protein containing the amino acid sequence represented by SEQ ID NO: 28, and a protein containing SEQ ID NO: 13 A protein containing the amino acid sequence; a protein containing the amino acid sequence represented by SEQ ID NO: 30;
[0009]
The partial peptide of the protein of the present invention is a partial peptide of the protein of the present invention, and preferably any peptide having the same properties as the protein of the present invention described above. For example, peptides having at least 20 or more, preferably 50 or more, preferably 70 or more, preferably 100 or more, preferably 200 or more amino acid sequences among the constituent amino acid sequences of the protein of the present invention are used. .
[0010]
In the partial peptide of the present invention, one or more (preferably about 1 to 10, and more preferably, about 1 to 5) amino acids in the amino acid sequence are deleted, or One or two or more (preferably about 1 to 20, more preferably about 1 to 10, and still more preferably, about 1 to 5) amino acids are added to the amino acid sequence, or the amino acid sequence is added to the amino acid sequence. One or more (preferably about 1 to 20, more preferably about 1 to 10, and still more preferably, about 1 to 5) amino acids are inserted, or one or more amino acids in the amino acid sequence are inserted. Two or more (preferably about 1 to 10, more preferably several, and still more preferably about 1 to 5) amino acids may be substituted with another amino acid.
More specifically, as the partial peptide of the present invention, for example, (1) the second to 57th or 58th to 249th amino acid residues from the N-terminal in the amino acid sequence represented by SEQ ID NO: 25 A partial peptide having a partial amino acid sequence having a group,
(2) a partial peptide having a partial amino acid sequence having the second to 239th or 240th to 481th amino acid residues from the N-terminus in the amino acid sequence represented by SEQ ID NO: 28,
(3) a partial peptide having a partial amino acid sequence having the 2nd to 64th or 65th to 256th amino acid residues from the N-terminus in the amino acid sequence represented by SEQ ID NO: 13,
(4) In the amino acid sequence represented by SEQ ID NO: 30, a partial peptide having a partial amino acid sequence having the 2nd to 239th or 240th to 481th amino acid residues from the N-terminus and the like can be mentioned.
The partial peptide of the present invention has a carboxyl group (—COOH) and a carboxylate (—COO) ⁻ ), Amide (-CONH ₂ )) Or an ester (—COOR).
Furthermore, the partial peptide of the present invention has a carboxyl group (or carboxylate) other than the C-terminus, an N-terminal amino acid residue (eg, a methionine residue), similarly to the above-described protein of the present invention. ) Wherein the amino group is protected with a protecting group, glutamine residues generated by cleavage of the N-terminal side in vivo are pyroglutamine-oxidized, and the substituent on the side chain of the amino acid in the molecule is an appropriate protecting group. Or a complex peptide such as a so-called glycopeptide to which a sugar chain is bound.
The partial peptide of the present invention can also be used as an antigen for producing an antibody. As the salt of the protein or partial peptide of the present invention, salts with physiologically acceptable acids (eg, inorganic acids, organic acids) and bases (eg, alkali metal salts) are used. Preferred acid addition salts are: Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid) Acids, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like are used.
[0011]
The protein of the present invention, its partial peptide, or a salt thereof can be produced from the above-mentioned human or non-human warm-blooded animal cells or tissues by a known method for purifying a protein, or a protein containing a DNA encoding the protein. It can also be produced by culturing the transformant. Further, it can be produced according to the peptide synthesis method described later.
When producing from human or non-human mammal tissues or cells, after homogenizing human or non-human mammal tissues or cells, extraction is performed with an acid or the like, and the extract is subjected to reverse phase chromatography or ion exchange chromatography. Purification and isolation can be performed by combining chromatography such as described above.
[0012]
For the synthesis of the protein or partial peptide of the present invention, or a salt thereof, or an amide thereof, a commercially available resin for protein synthesis can be generally used. Examples of such a resin include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, and 4-hydroxymethylmethyl. Phenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ′, 4′-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2 ′, 4′-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin, and the like. it can. Using such a resin, an amino acid having an α-amino group and a side chain functional group appropriately protected is condensed on the resin in accordance with the sequence of the target protein according to various known condensation methods. At the end of the reaction, the protein or partial peptide is cleaved from the resin, and at the same time, various protecting groups are removed.In addition, an intramolecular disulfide bond formation reaction is performed in a highly diluted solution to obtain the target protein or partial peptide or an amide thereof. I do.
For the condensation of the above protected amino acids, various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable. As the carbodiimides, DCC, N, N′-diisopropylcarbodiimide, N-ethyl-N ′-(3-dimethylaminoprolyl) carbodiimide and the like are used. For activation by these, the protected amino acid is directly added to the resin together with a racemization inhibitor (eg, HOBt, HOOBt), or the protected amino acid is previously activated as a corresponding acid anhydride or HOBt ester or HOOBt ester. After that, it can be added to the resin.
[0013]
The solvent used for activating the protected amino acid or condensing with the resin can be appropriately selected from solvents known to be usable for the protein condensation reaction. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol, and dimethyl sulfoxide. Sulfoxides, ethers such as pyridine, dioxane, and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, and appropriate mixtures thereof are used. The reaction temperature is appropriately selected from a range known to be usable for a protein bond formation reaction, and is usually appropriately selected from a range of about -20 ° C to 50 ° C. The activated amino acid derivative is usually used in a 1.5 to 4-fold excess. As a result of the test using the ninhydrin reaction, when the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. When sufficient condensation cannot be obtained even by repeating the reaction, unreacted amino acids can be acetylated using acetic anhydride or acetylimidazole to prevent the subsequent reaction from being affected.
[0014]
Examples of the protecting group for the amino group of the starting material include Z, Boc, t-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, trifluoroacetyl , Phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like.
The carboxyl group may be, for example, a linear, branched or cyclic alkyl ester such as an alkyl esterified ester (eg, methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl). ), Aralkyl esterification (eg, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarbonylhydrazide, t-butoxy It can be protected by carbonyl hydrazide, trityl hydrazide and the like.
The hydroxyl group of serine can be protected, for example, by esterification or etherification. Suitable groups for this esterification include, for example, lower (C _1-6 A) Groups derived from carbonic acid such as an aroyl group such as an alkanoyl group and a benzoyl group, and a benzyloxycarbonyl group and an ethoxycarbonyl group. Examples of the group suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group.
Examples of the protecting group for the phenolic hydroxyl group of tyrosine include Bzl, Cl ₂ -Bzl, 2-nitrobenzyl, Br-Z, t-butyl and the like are used.
As the imidazole protecting group for histidine, for example, Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like are used.
[0015]
Examples of the activated carboxyl group of the raw material include, for example, a corresponding acid anhydride, azide, active ester [alcohol (eg, pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, Cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, and an ester with HOBt). As the activated amino group of the raw material, for example, a corresponding phosphoric amide is used.
As a method for removing (eliminating) the protecting group, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, or hydrogen fluoride anhydride, methanesulfonic acid, trifluoromethane, etc. Acid treatment with dichloromethane, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., reduction with sodium in liquid ammonia, and the like are also used. The elimination reaction by the above acid treatment is generally performed at a temperature of about -20 ° C to 40 ° C. In the acid treatment, for example, anisole, phenol, thioanisole, metacresol, paracresol, dimethylsulfide, 1,4 -Addition of a cation scavenger such as butanedithiol, 1,2-ethanedithiol and the like is effective. Further, the 2,4-dinitrophenyl group used as an imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as an indole protecting group of tryptophan is the above 1,2-ethanedithiol, 1,4-butane. In addition to deprotection by acid treatment in the presence of dithiol or the like, it is also removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia or the like.
[0016]
The protection of the functional group that should not be involved in the reaction of the raw materials, the protective group, the elimination of the protective group, the activation of the functional group involved in the reaction, and the like can be appropriately selected from known groups or known means.
As another method for obtaining an amide form of a protein or a partial peptide, for example, after amidating and protecting the α-carboxyl group of the carboxy terminal amino acid, a peptide (protein) chain is added to the amino group side to a desired chain length. After the elongation, a protein or partial peptide from which only the protecting group for the N-terminal α-amino group of the peptide chain has been removed and a protein or partial peptide from which only the protecting group for the C-terminal carboxyl group has been removed, and these are produced. The protein or peptide is condensed in a mixed solvent as described above. Details of the condensation reaction are the same as described above. After purifying the protected protein or peptide obtained by the condensation, all the protecting groups are removed by the above-mentioned method to obtain a desired crude protein or peptide. This crude protein or peptide is purified by various known purification means, and the main fraction is lyophilized to obtain an amide of the desired protein or peptide.
In order to obtain an ester of a protein or peptide, for example, after condensing the α-carboxyl group of the carboxy terminal amino acid with a desired alcohol to form an amino acid ester, the desired protein or An ester of the peptide can be obtained.
[0017]
The partial peptide of the present invention or a salt thereof can be produced according to a known peptide synthesis method, or by cleaving the protein of the present invention with an appropriate peptidase. As a method for synthesizing a peptide, for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, the target peptide can be produced by condensing a partial peptide or amino acid that can constitute the partial peptide of the present invention with the remaining portion, and when the product has a protecting group, removing the protecting group. Known condensation methods and elimination of protecting groups include, for example, the methods described in the following (i) to (v).
(I) M.I. Bodanszky and M.A. A. Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966)
(Ii) Schroeder and Luebke, The Peptide, Academic Press, New York (1965)
(Iii) Nobuo Izumiya et al., Basics and Experiments of Peptide Synthesis, Maruzen Co., Ltd. (1975)
(Iv) Haruaki Yajima and Shunpei Sakakibara, Laboratory of Biochemistry 1, Protein Chemistry IV, 205, (1977)
(V) Supervisor of Haruaki Yajima, Development of Continuing Drugs, Volume 14, Peptide Synthesis, Hirokawa Shoten
After the reaction, the partial peptide of the present invention can be purified and isolated by a combination of ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization. When the partial peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method or a method analogous thereto, and conversely, when the partial peptide is obtained as a salt, a known method or analogous method It can be converted into a free form or another salt by a method.
[0018]
The polynucleotide encoding the protein of the present invention may be any polynucleotide as long as it contains a base sequence (DNA or RNA, preferably DNA) encoding the protein of the present invention. The polynucleotide is RNA such as DNA or mRNA encoding the protein of the present invention, and may be double-stranded or single-stranded. In the case of double-stranded, it may be double-stranded DNA, double-stranded RNA or DNA: RNA hybrid. In the case of a single strand, it may be a sense strand (ie, a coding strand) or an antisense strand (ie, a non-coding strand).
Using the polynucleotide encoding the protein of the present invention, for example, the mRNA of the protein of the present invention can be obtained by the method described in the well-known experimental medicine special edition “New PCR and its Applications” 15 (7), 1997 or a method analogous thereto. It can be quantified.
The DNA encoding the protein of the present invention may be any DNA as long as it contains the above-described nucleotide sequence encoding the protein of the present invention. Further, any of genomic DNA, genomic DNA library, cDNA derived from the above-mentioned cells / tissues, cDNA library derived from the aforementioned cells / tissues, and synthetic DNA may be used.
The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Alternatively, it can be directly amplified by Reverse Transcriptase Polymerase Chain Reaction (hereinafter abbreviated as RT-PCR method) using a preparation of a total RNA or mRNA fraction from the above-described cells / tissues.
The DNA encoding the protein of the present invention includes, for example, (1) a DNA containing the base sequence represented by SEQ ID NO: 26, or a base sequence represented by SEQ ID NO: 26 under high stringent conditions. A DNA containing a hybridizing base sequence and encoding a protein having substantially the same properties as a protein containing the amino acid sequence represented by SEQ ID NO: 25;
(2) a DNA containing the nucleotide sequence represented by SEQ ID NO: 29, or a nucleotide sequence that hybridizes with the nucleotide sequence represented by SEQ ID NO: 29 under high stringency conditions, and SEQ ID NO: 28 A DNA encoding a protein having substantially the same properties as a protein containing an amino acid sequence represented by
(3) a DNA containing the nucleotide sequence represented by SEQ ID NO: 27, or a nucleotide sequence that hybridizes under high stringent conditions to the nucleotide sequence represented by SEQ ID NO: 27; A DNA encoding a protein having substantially the same properties as a protein containing the amino acid sequence represented by
(4) a DNA containing the base sequence represented by SEQ ID NO: 31 or a base sequence that hybridizes under high stringent conditions to the base sequence represented by SEQ ID NO: 31; Any DNA may be used as long as it encodes a protein having substantially the same properties as the protein containing the amino acid sequence represented by
Examples of the DNA that can hybridize with the base sequence represented by SEQ ID NO: 26 under high stringent conditions include, for example, about 85% or more, preferably about 90% or more with the base sequence represented by SEQ ID NO: 26. More preferably, a DNA containing a base sequence having a homology of about 95% or more, more preferably about 97% or more is used.
Examples of the DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 29 under high stringency conditions include, for example, 75% or more, preferably about 80% or more, DNA containing a base sequence having a homology of 85% or more, preferably about 90% or more, more preferably about 95% or more, and more preferably about 97% or more is used.
Examples of the DNA that can hybridize with the base sequence represented by SEQ ID NO: 27 under high stringency conditions include, for example, about 80% or more, preferably about 85% or more with the base sequence represented by SEQ ID NO: 27. Preferably, a DNA containing a base sequence having a homology of about 90% or more, more preferably about 95% or more, more preferably about 97% or more is used.
Examples of the DNA that can hybridize with the base sequence represented by SEQ ID NO: 31 under high stringent conditions include, for example, about 50% or more, preferably about 60% or more with the base sequence represented by SEQ ID NO: 31. Preferably containing a base sequence having a homology of about 70% or more, about 80% or more, preferably about 85% or more, preferably about 90% or more, more preferably about 95% or more, and more preferably about 97% or more. DNA or the like is used.
[0019]
Hybridization can be performed according to a known method or a method analogous thereto, for example, the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. More preferably, it can be performed under high stringent conditions.
The high stringent conditions are, for example, conditions in which the sodium concentration is about 19 to 40 mM, preferably about 19 to 20 mM, and the temperature is about 50 to 70 ° C, preferably about 60 to 65 ° C. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C. is most preferable.
[0020]
More specifically, (1) the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 25 includes a DNA containing the base sequence represented by SEQ ID NO: 26; The DNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 28 includes the DNA having the nucleotide sequence represented by SEQ ID NO: 29, and (3) the DNA having the amino acid sequence represented by SEQ ID NO: 13. As the DNA encoding the protein, a DNA containing the base sequence represented by SEQ ID NO: 27, and (4) as a DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 30, SEQ ID NO: For example, a DNA containing the base sequence represented by 31 is used.
The DNA encoding the partial peptide of the present invention may be any DNA containing the above-described nucleotide sequence encoding the partial peptide of the present invention. Further, any of genomic DNA, genomic DNA library, cDNA derived from the above-mentioned cells / tissues, cDNA library derived from the aforementioned cells / tissues, and synthetic DNA may be used.
Examples of the DNA encoding the partial peptide of the present invention include a DNA having a part of the DNA having the base sequence represented by SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 27 or SEQ ID NO: 31, or It contains a nucleotide sequence that hybridizes under high stringent conditions to the nucleotide sequence represented by SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 27 or SEQ ID NO: 31, and comprises SEQ ID NO: 25, SEQ ID NO: 28 A DNA containing a part of a DNA encoding a protein having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 13 or SEQ ID NO: 30 is used.
DNAs capable of hybridizing with the nucleotide sequence represented by SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 27 or SEQ ID NO: 31 have the same significance as described above.
The same hybridization method and high stringency conditions as described above are used.
As a means for cloning a DNA that completely encodes the protein or partial peptide of the present invention (hereinafter, in the description of the cloning and expression of DNAs encoding them, these may be simply abbreviated to the protein of the present invention), Amplifying by a PCR method using a synthetic DNA primer having a part of the nucleotide sequence encoding the protein of the present invention, or incorporating a DNA incorporated in an appropriate vector into a DNA encoding a part or the whole region of the protein of the present invention. Selection can be performed by hybridization with fragments or those labeled with synthetic DNA. Hybridization can be performed, for example, according to the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual.
[0021]
Conversion of the base sequence of DNA can be performed by PCR or a known kit, for example, Mutan. ^TM -Super Express Km (Takara Shuzo Co., Ltd.), Mutan ^TM Using -K (Takara Shuzo Co., Ltd.) or the like, it can be carried out according to a known method such as the ODA-LA PCR method, the Gapped Duplex method, the Kunkel method, or a method analogous thereto.
The DNA encoding the cloned protein can be used as it is or as desired, after digestion with a restriction enzyme or addition of a linker, if desired. The DNA may have ATG as a translation initiation codon at the 5 'end and TAA, TGA or TAG as a translation termination codon at the 3' end. These translation initiation codon and translation termination codon can also be added using an appropriate synthetic DNA adapter.
[0022]
The expression vector for the protein of the present invention can be obtained, for example, by (A) cutting out a DNA fragment of interest from a DNA (eg, cDNA) containing a DNA encoding the protein of the present invention, and (B) expressing the DNA fragment appropriately. It can be produced by ligating downstream of a promoter in a vector.
Examples of the vector include plasmids derived from Escherichia coli (eg, pBR322, pBR325, pUC12, pUC13), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, pC194), plasmids derived from yeast (eg, pSH19, pSH15), λ phage and the like. In addition to animal viruses such as bacteriophage, retrovirus, vaccinia virus, and baculovirus, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo, and the like are used.
[0023]
The promoter of the present invention may be any promoter as long as it is appropriate for the host used for gene expression. For example, when an animal cell is used as a host, SRα promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and the like can be mentioned.
Of these, it is preferable to use a CMV (cytomegalovirus) promoter, SRα promoter, or the like. When the host is Escherichia, trp promoter, lac promoter, recA promoter, λP _L Promoter, lpp promoter, T7 promoter, etc., when the host is Bacillus, SPO1 promoter, SPO2 promoter, penP promoter, etc., and when the host is yeast, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, etc. Is preferred. When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable. In addition to the above, an expression vector containing an enhancer, a splicing signal, a polyA addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori), and the like may be used, if desired. it can. Examples of the selectable marker include a dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [methotrexate (MTX) resistance] and an ampicillin resistance gene (hereinafter Amp). ^r Neomycin resistance gene (hereinafter Neo). ^r G418 resistance). In particular, when the dhfr gene is used as a selection marker using dhfr gene-deficient Chinese hamster cells, the target gene can be selected using a thymidine-free medium.
If necessary, a signal sequence suitable for the host is added to the N-terminal side of the protein of the present invention. When the host is a genus Escherichia, a PhoA signal sequence, an OmpA signal sequence, or the like is used. When the host is a Bacillus genus, an α-amylase signal sequence, a subtilisin signal sequence, or the like is used. In some cases, MFα signal sequence, SUC2 signal sequence, etc., and when the host is an animal cell, insulin signal sequence, α-interferon signal sequence, antibody molecule, signal sequence, etc. can be used.
[0024]
A transformant can be produced using the vector containing the DNA encoding the protein of the present invention thus constructed.
As the host, for example, Escherichia, Bacillus, yeast, insect cells, insects, animal cells and the like are used.
Specific examples of the genus Escherichia include, for example, Escherichia coli K12.DH1 [Procedures of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 60, 160 (1968)], JM103 [Nucleic Acids Research, 9, 309 (1981)], JA221 [Journal of Molecular biology (Journal of Molecular biology)]. Biology), 120, 517 (1978)], HB101 [Journal of Molecular Biology, 41, 459 (1969)], C600 [Genetic Genetics, Vol. 39, 440 (1954)].
Examples of Bacillus spp. Include, for example, Bacillus subtilis MI114 [Gene, 24, 255 (1983)], 207-21 [Journal of Biochemistry, 95, 87 (1984)]. )] Is used.
Examples of yeast include, for example, Saccharomyces cerevisiae AH22, AH22R ⁻ , NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036, Pichia pastoris KM71 and the like.
When the virus is AcNPV, for example, when the virus is AcNPV, a cell line derived from a larva of Spodoptera litura (Spodoptera frugiperda cell; Sf cell), an MG1 cell derived from the middle intestine of Trichoplusia ni, and a Hig derived from egg of Trichoplusia ni ni. ^TM Cells, cells derived from Maestrabrassicae, cells derived from Estigmena acrea, and the like are used. When the virus is BmNPV, a silkworm-derived cell line (Bombyx mori N cell; BmN cell) or the like is used. As the Sf cells, for example, Sf9 cells (ATCC CRL 1711), Sf21 cells (Vaughn, JL, et al., In Vivo, 13, 213-217, (1977)) and the like are used. .
As insects, for example, silkworm larvae are used [Maeda et al., Nature, 315, 592 (1985)].
Examples of animal cells include monkey cells COS-7, Vero, Chinese hamster cells CHO (hereinafter abbreviated as CHO cells), dhfr gene-deficient Chinese hamster cells CHO (hereinafter CHO (dhfr). ⁻ ) Cells), mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells, H9c2 cells, and the like.
[0025]
In order to transform Escherichia, for example, Procesings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 69, 2110 ( 1972) and Gene, Vol. 17, 107 (1982).
Bacillus spp. Can be transformed, for example, according to the method described in Molecular & General Genetics, Vol. 168, 111 (1979).
To transform yeast, for example, Methods in Enzymology, Vol. 194, 182-187 (1991), Processings of the National Academy of Sciences of Science -The USA (Proc. Natl. Acad. Sci. USA), Vol. 75, 1929 (1978).
Insect cells or insects can be transformed, for example, according to the method described in Bio / Technology, 6, 47-55 (1988). To transform animal cells, for example, see Cell Engineering Separate Volume 8, New Cell Engineering Experiment Protocol. 263-267 (1995) (published by Shujunsha) and Virology, Vol. 52, 456 (1973).
Thus, a transformant transformed with the expression vector containing the DNA encoding the protein can be obtained.
[0026]
When culturing a transformant whose host is a genus Escherichia or Bacillus, a liquid medium is suitable as a medium used for the culturing, and a carbon source necessary for growth of the transformant is used therein. Nitrogen sources, inorganic substances, etc. are included. As a carbon source, for example, glucose, dextrin, soluble starch, sucrose, etc., as a nitrogen source, for example, ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal, potato extract and the like Examples of the inorganic or organic substance and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride. In addition, yeast extract, vitamins, growth promoting factors and the like may be added. The pH of the medium is preferably about 5 to 8.
Examples of a medium for culturing Escherichia include, for example, an M9 medium containing glucose and casamino acid [Miller, Journal of Experiments in Molecular Genetics]. , 431-433, Cold Spring Harbor Laboratory, New York 1972]. If necessary, an agent such as 3β-indolylacrylic acid can be added in order to make the promoter work efficiently. When the host is a bacterium belonging to the genus Escherichia, the cultivation is usually performed at about 15 to 43 ° C. for about 3 to 24 hours, and if necessary, aeration and stirring can be added.
When the host is a bacterium belonging to the genus Bacillus, the cultivation is usually carried out at about 30 to 40 ° C. for about 6 to 24 hours, and if necessary, aeration and stirring may be added.
When culturing a transformant in which the host is yeast, for example, Burkholder's minimum medium [Bostian, K. et al. L. Procurings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 77, 4505 (1980)] and 0.5% casamino acid. SD medium containing [Bitter, G .; A. Processing of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 81, 5330 (1984)]. Preferably, the pH of the medium is adjusted to about 5-8. The cultivation is usually performed at about 20 ° C. to 35 ° C. for about 24 to 72 hours, and if necessary, aeration and stirring are added.
When culturing a transformant whose host is an insect cell or an insect, the medium is inactivated by Grace's Insect Medium (Grace, TCC, Nature, 195, 788 (1962)). For example, those to which an additive such as 10% bovine serum or the like is appropriately added are used. The pH of the medium is preferably adjusted to about 6.2 to 6.4. The cultivation is usually performed at about 27 ° C. for about 3 to 5 days, and if necessary, aeration or stirring is added.
When culturing a transformant in which the host is an animal cell, examples of the medium include a MEM medium containing about 5 to 20% fetal bovine serum [Science, 122, 501 (1952)], a DMEM medium. [Virology, 8, 396 (1959)], RPMI 1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], 199 medium. [Proceding of the Society for the Biological Medicine, Vol. 73, 1 (1950) [Proceding of the Society for the Biological Medicine] ], Such as is used. Preferably, the pH is between about 6 and 8. The cultivation is usually performed at about 30 ° C. to 40 ° C. for about 15 to 60 hours, and if necessary, aeration and stirring are added.
As described above, the protein of the present invention can be produced in cells, cell membranes or extracellular cells of the transformant.
[0027]
The protein of the present invention can be separated and purified from the culture by, for example, the following method.
When extracting the protein of the present invention from cultured cells or cells, the cells or cells are collected by a known method after culturing, suspended in an appropriate buffer, and subjected to sonication, lysozyme and / or freeze-thawing. After the cells or cells are destroyed by the method, a method of obtaining a crude extract of the protein by centrifugation or filtration is used as appropriate. In a buffer, a protein denaturant such as urea or guanidine hydrochloride, or Triton X-100 is used. ^TM And the like. When the protein is secreted into the culture solution, after the culture, the supernatant is separated from the cells or cells by a known method, and the supernatant is collected.
The protein contained in the thus obtained culture supernatant or extract can be purified by appropriately combining known separation and purification methods. These known separation and purification methods include methods utilizing solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, mainly molecular weight. Using differences in charge, methods using charge differences such as ion exchange chromatography, methods using specific affinity such as affinity chromatography, and using differences in hydrophobicity such as reversed-phase high-performance liquid chromatography A method utilizing a difference between isoelectric points, such as an isoelectric focusing method, is used.
When the protein thus obtained is obtained in a free form, it can be converted to a salt by a known method or a method analogous thereto, and conversely, when the protein is obtained in the form of a salt, the known method or analogous method Depending on the method, it can be converted into a free form or another salt.
The protein produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by allowing a suitable protein modifying enzyme to act on the protein before or after purification. As the protein modifying enzyme, for example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used.
[0028]
The presence of the protein of the present invention thus produced can be measured by an enzyme immunoassay using a specific antibody, Western blotting, or the like.
The antibody against the protein or partial peptide of the present invention or a salt thereof may be any of a polyclonal antibody and a monoclonal antibody as long as it can recognize the protein or partial peptide of the present invention or a salt thereof.
An antibody against the protein or partial peptide of the present invention or a salt thereof (hereinafter, these may be simply abbreviated as the protein of the present invention) using the protein of the present invention as an antigen, is a known antibody or It can be produced according to the method for producing antiserum.
[0029]
[Preparation of monoclonal antibody]
(A) Preparation of monoclonal antibody-producing cells
The protein of the present invention is administered to a warm-blooded animal itself or together with a carrier and a diluent at a site capable of producing an antibody upon administration. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. The administration is usually performed once every 2 to 6 weeks, for a total of about 2 to 10 times. Examples of the warm-blooded animal used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, and chickens, and mice and rats are preferably used.
When preparing monoclonal antibody-producing cells, a warm-blooded animal immunized with an antigen, for example, an individual having an antibody titer is selected from a mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization and contained in them. By fusing the antibody-producing cells thus obtained with myeloma cells of the same or different species, a monoclonal antibody-producing hybridoma can be prepared. The antibody titer in the antiserum can be measured, for example, by reacting a labeled protein described below with the antiserum, and then measuring the activity of a labeling agent bound to the antibody. The fusion operation can be performed according to a known method, for example, the method of Koehler and Milstein [Nature, 256, 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used.
Examples of myeloma cells include myeloma cells of warm-blooded animals such as NS-1, P3U1, SP2 / 0, and AP-1, but P3U1 is preferably used. The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG1000 to PEG6000) is added at a concentration of about 10 to 80%. By incubating at 20 to 40 ° C, preferably 30 to 37 ° C for 1 to 10 minutes, cell fusion can be carried out efficiently. Various methods can be used to screen for monoclonal antibody-producing hybridomas. For example, a hybridoma culture supernatant is added to a solid phase (eg, a microplate) on which a protein antigen is directly or adsorbed together with a carrier, and then radioactive substances or A method for detecting a monoclonal antibody bound to a solid phase by adding an anti-immunoglobulin antibody labeled with an enzyme or the like (an anti-mouse immunoglobulin antibody is used when cells used for cell fusion are mice) or protein A; A method in which a hybridoma culture supernatant is added to a solid phase to which globulin antibodies or protein A is adsorbed, a protein labeled with a radioactive substance, an enzyme, or the like is added, and a monoclonal antibody bound to the solid phase is detected.
The selection of monoclonal antibodies can be performed according to a known method or a method analogous thereto. Usually, it can be performed in a medium for animal cells to which HAT (hypoxanthine, aminopterin, thymidine) is added. As a selection and breeding medium, any medium can be used as long as a hybridoma can grow. For example, RPMI 1640 medium containing 1 to 20%, preferably 10 to 20% fetal bovine serum, GIT medium containing 1 to 10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.) or serum-free for hybridoma culture A medium (SFM-101, Nissui Pharmaceutical Co., Ltd.) or the like can be used. The culturing temperature is usually 20 to 40 ° C, preferably about 37 ° C. The culture time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks. The culture can be usually performed under 5% carbon dioxide gas. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.
[0030]
(B) Purification of monoclonal antibody
Monoclonal antibodies can be separated and purified by known methods, for example, immunoglobulin separation and purification methods [eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, absorption by ion exchanger (eg, DEAE)]. Desorption method, ultracentrifugation method, gel filtration method, antigen-binding solid phase or specific purification method in which only antibody is collected using an active adsorbent such as protein A or protein G and the bond is dissociated to obtain the antibody]. it can.
[0031]
(Preparation of polyclonal antibody)
The polyclonal antibody of the present invention can be produced according to a known method or a method analogous thereto. For example, a immunizing antigen (protein antigen) itself or a complex thereof with a carrier protein is formed, and a warm-blooded animal is immunized in the same manner as in the above-described method for producing a monoclonal antibody. It can be produced by collecting a substance and separating and purifying the antibody.
Regarding a complex of an immunizing antigen and a carrier protein used to immunize a warm-blooded animal, the type of carrier protein and the mixing ratio of the carrier and the hapten are such that the antibody is efficiently cross-linked to the hapten immunized by cross-linking with the carrier. If possible, any material may be cross-linked at any ratio. For example, bovine serum albumin, bovine thyroglobulin, hemocyanin and the like are used in a weight ratio of about 0.1 to 20, preferably about 1 to 20, relative to hapten 1. A method of coupling at a rate of ~ 5 is used. Various coupling agents can be used for coupling the hapten and the carrier, and glutaraldehyde, carbodiimide, a maleimide active ester, an active ester reagent containing a thiol group or a dithioviridyl group, or the like is used.
The condensation product is administered to a warm-blooded animal itself or together with a carrier or diluent at a site where antibody production is possible. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. The administration is usually performed once every about 2 to 6 weeks, about 3 to 10 times in total.
The polyclonal antibody can be collected from the blood, ascites, or the like, preferably from the blood of a warm-blooded animal immunized by the above method.
The measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the antiserum described above. The polyclonal antibody can be separated and purified according to the same method for separating and purifying immunoglobulins as in the above-described method for separating and purifying a monoclonal antibody.
[0032]
Complementary or substantially complementary to the base sequence of DNA encoding the protein or partial peptide of the present invention (hereinafter, these DNAs may be abbreviated as the DNA of the present invention in the description of antisense nucleotides) The antisense nucleotide having a specific base sequence or a part thereof has a base sequence complementary to or substantially complementary to the base sequence of the DNA of the present invention or a part thereof, and is capable of expressing the DNA. Any antisense nucleotide may be used as long as it has an inhibitory effect, but antisense DNA is preferred.
The nucleotide sequence substantially complementary to the DNA of the present invention is, for example, about 70% of the total nucleotide sequence or a partial nucleotide sequence of the nucleotide sequence complementary to the DNA of the present invention (that is, the complementary strand of the DNA of the present invention). % Or more, preferably about 80% or more, more preferably about 90% or more, most preferably about 95% or more. In particular, of the entire base sequence of the complementary strand of the DNA of the present invention, the base sequence of the portion encoding the N-terminal portion of the protein of the present invention (for example, the base sequence near the start codon, etc.) is at least about 70% or more of the complementary strand. Antisense nucleotides having a homology of preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more are suitable.
Specifically, a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of DNA having the nucleotide sequence represented by SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 27 or SEQ ID NO: 31, Or a base sequence complementary to a base sequence of a DNA having a base sequence represented by SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 27 or SEQ ID NO: 31, for example, Or an antisense nucleotide having a part thereof (more preferably, a nucleotide sequence complementary to the nucleotide sequence of DNA having the nucleotide sequence represented by SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 27 or SEQ ID NO: 31, or (An antisense nucleotide having a part thereof).
[0033]
The antisense nucleotide is usually composed of about 10 to 40, preferably about 15 to 30 bases.
In order to prevent degradation by a hydrolase such as nuclease, the phosphate residue (phosphate) of each nucleotide constituting the antisense DNA is replaced with, for example, a chemically modified phosphate residue such as phosphorothioate, methylphosphonate or phosphorodithionate. It may be substituted. These antisense nucleotides can be produced using a known DNA synthesizer or the like.
According to the present invention, an antisense nucleotide (nucleic acid) capable of inhibiting the replication or expression of the protein gene of the present invention is designed based on the cloned or determined nucleotide sequence information of the DNA encoding the protein. , Can be synthesized. Such a polynucleotide (nucleic acid) can hybridize with the RNA of the protein gene of the present invention and inhibit the synthesis or function of the RNA, or through the interaction with the protein-related RNA of the present invention. Thus, the expression of the protein gene of the present invention can be regulated and controlled. Polynucleotides that are complementary to a selected sequence of the protein-associated RNA of the present invention, and that can specifically hybridize with the protein-associated RNA of the present invention, can be used in vivo and in vitro to produce the protein gene of the present invention. It is useful for regulating and controlling the expression of, and also useful for treating or diagnosing diseases and the like. The term “corresponding” means having homology or being complementary to a specific sequence of nucleotides, base sequences or nucleic acids including genes. "Corresponding" between a nucleotide, base sequence or nucleic acid and a peptide (protein) usually refers to the amino acid of the peptide (protein) as directed by the nucleotide (nucleic acid) sequence or its complement. . 5 'end hairpin loop of protein gene, 5' end 6-base pair repeat, 5 'end untranslated region, polypeptide translation start codon, protein coding region, ORF translation stop codon, 3' end untranslated region, 3 ' The terminal palindrome region and the 3'-end hairpin loop may be selected as preferred regions of interest, but any region within the protein gene may be selected as a target.
[0034]
The relationship between the target nucleic acid and a polynucleotide complementary to at least a part of the target region or the relationship between the target nucleic acid and a polynucleotide that can hybridize with the target can be said to be “antisense”. Antisense nucleotides include polynucleotides containing 2-deoxy-D-ribose, polynucleotides containing D-ribose, other types of polynucleotides that are N-glycosides of purine or pyrimidine bases, or non-nucleotides. Other polymers having a nucleotide backbone (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special bonds, provided that the polymer is composed of bases such as those found in DNA or RNA. Pairing and nucleotides having a configuration that allows base attachment)). They can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and also DNA: RNA hybrids, and can be unmodified polynucleotides (or unmodified oligonucleotides), or even known. Such as labeled, capped, methylated, one or more naturally-occurring nucleotides replaced by analogs, intramolecular nucleotides Modified, eg, those having an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.), charged or sulfur-containing bonds (eg, phosphorothioate, phosphorodithioate, etc.) ), Such as proteins (nucleases, nuclease inhibitors, One having a side chain group such as syn, antibody, signal peptide, poly-L-lysine or sugar (for example, monosaccharide), one having an interactive compound (for example, acridine, psoralen, etc.), Those containing chelating compounds (eg, metals, radioactive metals, boron, oxidizing metals, etc.), those containing alkylating agents, those with modified bonds (eg, α-anomeric nucleic acids, etc.) ). Here, "nucleoside", "nucleotide", and "nucleic acid" may include not only those containing purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, e.g., where one or more hydroxyl groups have been replaced by halogens, aliphatic groups, etc., or by functional groups such as ethers, amines, etc. It may have been converted.
[0035]
The antisense nucleotide (nucleic acid) of the present invention is RNA, DNA, or a modified nucleic acid (RNA, DNA). Specific examples of the modified nucleic acid include, but are not limited to, sulfur derivatives and thiophosphate derivatives of nucleic acids, and those that are resistant to degradation of polynucleoside amides and oligonucleoside amides. The antisense nucleic acid of the present invention can be preferably designed according to the following policy. That is, to make the antisense nucleic acid more stable in the cell, to make the antisense nucleic acid more cell permeable, to have a greater affinity for the target sense strand, and to be more toxic if it is toxic. Minimize the toxicity of sense nucleic acids.
Thus, many modifications are known in the art, for example, Kawakami et al. , Pharm Tech Japan, Vol. 8, pp. 247, 1992; Vol. 8, pp. 395, 1992; T. Crooke et al. ed. , Antisense Research and Applications, CRC Press, 1993.
The antisense nucleic acid of the present invention may contain altered or modified sugars, bases or bonds, may be provided in a special form such as liposomes or microspheres, may be applied by gene therapy, It could be provided in an added form. Thus, in the form of addition, polycations such as polylysine which acts to neutralize the charge of the phosphate skeleton, lipids which enhance the interaction with the cell membrane or increase the uptake of nucleic acids ( For example, hydrophobic substances such as phospholipid and cholesterol can be mentioned. Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chloroformate, cholic acid, etc.). These can be attached to the 3 'end or 5' end of the nucleic acid, and can be attached via a base, sugar, or intramolecular nucleoside bond. Other groups include cap groups specifically arranged at the 3 ′ end or 5 ′ end of a nucleic acid for preventing degradation by nucleases such as exonuclease and RNase. Such capping groups include, but are not limited to, hydroxyl-protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.
The inhibitory activity of the antisense nucleic acid can be examined using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention, or the in vivo or in vitro translation system of the protein of the present invention. The nucleic acid can be applied to cells by various known methods.
[0036]
Hereinafter, the protein or partial peptide of the present invention or a salt thereof (hereinafter sometimes abbreviated as the protein of the present invention), the DNA encoding the protein or partial peptide of the present invention (hereinafter referred to as the DNA of the present invention) Antibody) against the protein or partial peptide of the present invention or a salt thereof (hereinafter, may be abbreviated as the antibody of the present invention), and the antisense nucleotide of the DNA of the present invention (hereinafter referred to as the antisense nucleotide of the present invention). (May be abbreviated).
The protein of the present invention can be used as a disease marker because its expression is increased in the heart at the stage of heart failure transition (heart failure decompensation / heart failure decompensation) after myocardial infarction. That is, for example, heart disease (eg, cardiomyopathy, myocardial infarction, heart failure, angina pectoris, etc.), central nervous system disease (eg, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), genital disease (eg, testicular hypersensitivity, ovary) It is useful as a marker for early diagnosis of dysfunction, infertility, etc.), determination of symptom severity, and prediction of disease progression.
Antisense nucleotide of a gene encoding the protein of the present invention (protein gene of the present invention), a compound or its salt that regulates the activity of the protein of the present invention, a compound or a salt thereof that regulates the expression of the protein gene of the present invention, or Pharmaceuticals containing an antibody against the protein of the present invention include, for example, diseases characterized by reduced cardiac function, such as heart diseases (eg, cardiomyopathy, myocardial infarction, heart failure, angina pectoris, etc.), central nervous system diseases (eg, Alzheimer's disease) Disease, Parkinson's syndrome, schizophrenia, etc.), reproductive diseases (eg, testicular hypersensitivity, ovarian insufficiency, infertility, etc.) and the like, or can be used as a contraceptive.
[0037]
[1] Screening of drug candidate compounds for disease
Since the expression of the protein of the present invention and the protein gene of the present invention (heart failure decompensation / heart failure decompensation) increases with decreased cardiac function, a compound or a salt thereof that regulates the activity of the protein of the present invention, and the protein of the present invention Compounds or salts thereof that regulate gene expression include, for example, heart diseases (eg, cardiomyopathy, myocardial infarction, heart failure, angina pectoris, etc.), central nervous system diseases (eg, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), It can be used as a prophylactic / therapeutic agent for genital diseases (eg, testicular hypersensitivity, ovarian dysfunction, infertility, etc.), or as a medicament such as a contraceptive.
Therefore, the protein of the present invention is useful as a reagent for screening a compound or its salt that regulates the activity of the protein of the present invention, and the polynucleotide encoding the protein regulates the expression of the gene of the protein of the present invention. Useful as a reagent for screening a compound or a salt thereof.
That is, the present invention provides (1) regulating the activity of the protein of the present invention (for example, promoting or suppressing cardiac function, activating or inactivating cardiomyocyte function) characterized by using the protein of the present invention. (2) a compound for regulating (promoting or inhibiting) the expression of the protein gene of the present invention, which comprises using a polynucleotide encoding the protein of the present invention; A method for screening a salt thereof, and (3) a method for screening a compound or a salt thereof that regulates (promotes or inhibits) the expression of the protein of the present invention, which comprises using the antibody of the present invention, and the like.
[0038]
(1) A method for screening a compound or a salt thereof that regulates the activity of the protein of the present invention using the protein of the present invention
Specific examples of the screening method include (i) culturing a cell capable of producing the protein of the present invention and (ii) culturing a mixture of a cell capable of producing the protein of the present invention and a test compound. A screening method for a modulator characterized by performing a comparison with the case.
In the above screening method, for example, in the cases of (i) and (ii), the activity of the protein of the present invention for promoting or suppressing cardiac function, activating or inactivating cardiomyocyte function, and the like are measured and compared.
The activity of promoting or suppressing cardiac function and the activity of activating or inactivating cardiomyocyte function of the protein of the present invention are measured by measuring cardiac function.
The measurement of cardiac function can be measured according to a known method or a method analogous thereto. For example, the measurement is performed by an echocardiograph (Cell, Vol. 97, pp. 189-198, 1999) or cardiac function measurement using a cardiac catheter (circulation research, Vol. 69, pp. 370-377, 1991). Furthermore, for example, the enhancement of renin-angiotensin system (RAS) such as angiotensin I converting enzyme (ACE) is measured using a commercially available measurement kit (eg, manufactured by Peninsula, Phoenix, etc.), and the activity of increasing catecholamine in blood is measured. (Tosoh Corp., fully automatic catecholamine analyzer) is used to measure cardiac function.
When a cell having the ability to produce the protein of the present invention is used, for example, the respiratory activity of the cell is measured, or the expression of a heart failure marker gene [eg, ANP (atrial natriuretic peptide), BNP (brain natriuretic peptide, etc.)] The respiratory activity of the cells can be measured according to a known method or a method analogous thereto, for example, MTT (3- (4,5-Dimethyl-2). -Thiazolyl) -2,5-diphenyl-2H-tetrazolium method, trypan blue staining method, TUNNEL staining method (Terminal deoxytransferase-meditedd UTP-X nickel end labeling, Cell, Vol. 97, 189-19) Page, 1999) the like.
Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like. Or a known compound.
In order to carry out the above-mentioned screening method, cells having the ability to produce the protein of the present invention are prepared by suspending them in a buffer suitable for screening. The buffer may be any of a phosphate buffer and a borate buffer having a pH of about 4 to 10 (preferably, a pH of about 6 to 8).
As a cell having the ability to produce the protein of the present invention, for example, a host (transformant) transformed with a vector containing the DNA encoding the protein of the present invention described above is used. As a host, for example, animal cells such as H9c2 cells are preferably used. For the screening, for example, a transformant in which the protein of the present invention is expressed in cells by culturing by the above-described method is preferably used.
For example, a test compound which promotes the cardiomyocyte function activating effect in the case (ii) above by about 20% or more, preferably 30% or more, and more preferably about 50% or more as compared with the case (i). It can be selected as a compound or a salt thereof that promotes the activity of the protein of the present invention. A test compound which inhibits the cardiomyocyte function inactivating effect in the case (ii) above by about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the case (i). It can be selected as a compound that inhibits the activity of the protein of the invention or a salt thereof.
[0039]
(2) A method for screening a compound or a salt thereof that regulates the expression of the protein gene of the present invention using a polynucleotide encoding the protein of the present invention
Specific examples of the screening method include (iii) culturing a cell capable of producing the protein of the present invention and (iv) culturing a mixture of a cell capable of producing the protein of the present invention and a test compound. , The expression level of the protein gene of the present invention (specifically, the amount of the protein of the present invention or the amount of mRNA encoding the protein) is measured and compared.
For example, (iii ') cells having the ability to produce the protein of the present invention when cultured under hypoxic conditions with application of stretching stimulation, and (iv') cells having the ability to produce the protein of the present invention and a test compound. Of the protein gene of the present invention (specifically, the amount of the protein of the present invention or the amount of mRNA encoding the protein) in a case where the mixture of the above was cultured under hypoxic conditions with extension stimulation applied thereto. ,Compare.
The hypoxic condition refers to, for example, 20% O 2 ₂ The following oxygen concentration means a condition of, for example, 2% (Nature, 394, 485-490, 1998).
The stretching stimulus is, for example, a stimulus in which a target cell is cultured on a stretchable silicon film and a mechanical load is applied by pulling the silicon film (JBC, Vol. 271, 33592-33597). , 1996, Circulation, Vol. 89, pp. 2204-221, 1994, JBC, Vol. 271, 3221-2228, 1996).
For example, (iii '') a cell capable of producing the protein of the present invention is cultured under lethal conditions, and (iv '') a mixture of the cell capable of producing the protein of the present invention and a test compound. Are cultured under lethal conditions, and the expression level of the protein gene of the present invention (specifically, the amount of the protein of the present invention or the amount of mRNA encoding the protein) is measured and compared.
The culture under the above lethal condition includes, for example, culturing in the absence of serum or culturing with addition of an anticancer agent (eg, adriamycin which is relatively toxic to cardiomyocytes).
The amount of the protein of the present invention can be measured by a known method, for example, using an antibody recognizing the protein of the present invention, and analyzing the protein present in a cell extract or the like by a method such as Western analysis, ELISA, or the like. It can be measured according to the method.
The expression level of the protein gene of the present invention can be determined by a known method, for example, Northern blotting, reverse transcription-polymerase chain reaction (RT-PCR), a real-time PCR analysis system (manufactured by ABI, TaqMan polymerase chain reaction) or the like. It can be measured according to the method.
Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like. Or a known compound.
In order to carry out the above-mentioned screening method, cells having the ability to produce the protein of the present invention are prepared by suspending them in a buffer suitable for screening. The buffer may be any buffer that does not inhibit the activity of the protein of the present invention, such as a phosphate buffer or a borate buffer having a pH of about 4 to 10 (preferably, a pH of about 6 to 8).
As a cell having the ability to produce the protein of the present invention, for example, a host (transformant) transformed with a vector containing the DNA encoding the protein of the present invention described above is used. As a host, for example, animal cells such as H9c2 cells are preferably used. For the screening, for example, a transformant in which the protein of the present invention is expressed in cells by culturing by the above-described method is preferably used.
For example, a test in which the expression level of the protein gene of the present invention in the case of the above (iv) is promoted by about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the case of the above (iii). The compound can be selected as a compound that promotes the expression of the protein gene of the present invention or a salt thereof. A test compound that inhibits the expression level of the protein gene of the present invention in the case of the above (iv) by about 20% or more, preferably 30% or more, more preferably about 50% or more compared to the case of the above (iii) It can be selected as a compound that inhibits the expression of the protein gene of the present invention or a salt thereof.
[0040]
(3) A method for screening a compound or a salt thereof that regulates the expression of the protein of the present invention using the antibody of the present invention
Specific examples of the screening method include (v) culturing a cell capable of producing the protein of the present invention and (vi) culturing a mixture of a cell capable of producing the protein of the present invention and a test compound. Compare with. In the above screening method, the expression level of the protein of the present invention (specifically, the protein level of the present invention) in the cases (v) and (vi) is measured using the antibody of the present invention (eg, the expression level of the protein). Detection, quantification of expression level, etc.) and compare.
The amount of the protein of the present invention can be measured by a known method, for example, using an antibody recognizing the protein of the present invention, and analyzing the protein present in a cell extract or the like by a method such as Western analysis, ELISA, or the like. It can be measured according to the method.
Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like. Or a known compound.
As a cell having the ability to produce the protein of the present invention, for example, a host (transformant) transformed with a vector containing the DNA encoding the protein of the present invention described above is used. As a host, for example, animal cells such as H9c2 cells are preferably used. For the screening, for example, a transformant in which the protein of the present invention is expressed in cells by culturing by the above-described method is preferably used.
For example, a test compound that promotes the expression level of the protein of the present invention in the case of the above (vi) by about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the case of the above (v). Can be selected as a compound or its salt that promotes the expression of the protein of the present invention. A test compound that inhibits the expression level of the protein of the present invention in the case of the above (vi) by about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the case of the above (v). It can be selected as a compound that inhibits the expression of the protein of the invention or a salt thereof.
[0041]
The screening kit of the present invention has the ability to produce the protein or partial peptide of the present invention or a salt thereof, the polypeptide encoding the protein or partial peptide of the present invention, the antibody of the present invention, or the protein or partial peptide of the present invention. It contains cells.
Compounds or salts thereof obtained by using the screening method or the screening kit of the present invention are test compounds described above, for example, peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts. , A compound selected from animal tissue extract, plasma, and the like, or a salt thereof, and the activity of the protein of the present invention (eg, cardiac function promoting or inhibiting activity, cardiomyocyte function activating or inactivating effect, etc.), the present invention Or a salt thereof that regulates (promotes or inhibits) the expression of the protein gene of the present invention or the expression of the protein of the present invention.
As the salt of the compound, those similar to the aforementioned salts of the protein of the present invention are used.
[0042]
It is thought that excessively enhanced expression of the gene encoding the protein of the present invention causes excessive activation of cells and accelerates cell fatigue. On the other hand, moderate expression is thought to enhance the compensation mechanism. Therefore, it is considered important to appropriately control the expression level of the gene encoding the protein of the present invention. When it is considered that cell damage is caused by promotion of the expression of the gene encoding the protein of the present invention, for example, in the acute phase of heart failure and the decompensated phase of heart failure, a compound that inhibits the activity of the protein of the present invention, A compound that inhibits the expression level of the protein gene of the present invention or a compound that inhibits the expression level of the protein of the present invention or a salt thereof (inhibitor) is administered, and conversely, the expression of the gene encoding the protein of the present invention decreases. When it is considered that cell damage has occurred, a compound that promotes the activity of the protein of the present invention, a compound that promotes the expression level of the protein gene of the present invention or a compound that promotes the expression level of the protein of the present invention, or a compound thereof Administer salt (promoter).
Since the DNA (gene) encoding the protein of the present invention has a function of regulating cardiac function, an extremely low expression of the gene encoding the protein of the present invention impairs cell function, and conversely, excessive expression is unnecessary. It is considered that the activation of the cell causes the cell damage.
Therefore, when an excessive decrease in the expression of the protein of the present invention or the DNA of the present invention is found, administration of the protein of the present invention or the DNA of the present invention, or the promoter obtained by the above-described screening method is performed. In the case where an overexpression of the protein of the present invention or the DNA of the present invention is found, the inhibitor obtained by the above-mentioned screening method is administered. By doing so, the disorder of the cell function can be prevented and treated.
A compound or a salt thereof that inhibits the activity of the protein of the present invention, the expression of the protein gene of the present invention, or the expression of the protein of the present invention, obtained by the above-described screening method, is used for the expression of DNA (gene) encoding the protein of the present invention. Administration in the acute phase or decompensated phase of heart failure, where enhancement is observed, can be expected to restore cardiac function. Further, a compound or a salt thereof that promotes the activity of the protein of the present invention, the expression of the gene of the protein of the present invention, or the expression of the protein of the present invention, when the expression of DNA (gene) encoding the protein of the present invention is reduced. Is administered, the compensation mechanism is strengthened, and a heart protective effect by protecting cardiomyocytes can be expected.
The compound or a salt thereof obtained by using the screening method or the screening kit of the present invention has excellent cardiac function promoting activity and cardiomyocyte function activating activity (such as cytoprotective activity) and the like. Cardiomyopathy, myocardial infarction, heart failure, angina, etc.), central nervous system disorders (eg, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), genital disorders (eg, testicular hypersensitivity, ovarian insufficiency, infertility, etc.) It is useful as a prophylactic / therapeutic agent or a medicament such as a contraceptive.
The compound or a salt thereof obtained by using the screening method or the screening kit of the present invention can be safely administered as it is or as an appropriate drug. The medicament used for the administration contains the compound or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient, and is provided as a pharmaceutical composition suitable for oral or parenteral administration. .
For example, compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, and capsules (including soft capsules). Syrup, emulsion, suspension and the like. Such a composition is produced by a known method and contains a carrier, diluent or excipient commonly used in the field of pharmaceuticals. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.
As the composition for parenteral administration, for example, injections, suppositories, etc. are used, and the injections are in the form of intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, intravenous injections and the like. Is included. Such injections are prepared according to a known method, for example, by dissolving, suspending or emulsifying the antibody or a salt thereof in a sterile aqueous or oily liquid commonly used for injections. As an aqueous liquid for injection, for example, physiological saline, isotonic solution containing glucose and other auxiliary agents and the like are used, and suitable solubilizing agents, for example, alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) add-on of hydro- genated castor oil)) and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil, and the like are used, and benzyl benzoate, benzyl alcohol, and the like may be used in combination as a solubilizing agent. The prepared injection is usually filled in an appropriate ampoule. Suppositories to be used for rectal administration are prepared by mixing the above antibody or a salt thereof with a conventional suppository base.
[0043]
The above-mentioned oral or parenteral pharmaceutical composition is conveniently prepared in a unit dosage form so as to be compatible with the dosage of the active ingredient. Examples of such dosage unit dosage forms include tablets, pills, capsules, injections (ampoules), suppositories, etc., and usually 5 to 500 mg for each dosage unit dosage, 5 to 100 mg for injections, and others. The above-mentioned dosage form preferably contains 10 to 250 mg of the above compound.
[0044]
The preparations obtained in this way are safe and low toxic, for example, human or warm-blooded animals (eg, mouse, rat, rabbit, sheep, pig, cow, horse, bird, cat, dog, monkey, chimpanzee, etc.) ) Can be administered orally or parenterally.
The dose of the compound or its salt that regulates the activity of the protein of the present invention varies depending on its action, target disease, subject to be administered, administration route, etc., for example, the activity of the protein of the present invention for the purpose of treating heart failure. When orally administering the modulating compound or salt thereof, generally 0.1 to 100 mg, preferably about 1.0 to 50 mg, of the compound or salt thereof per day for adults (with a body weight of 60 kg). Preferably, about 1.0 to 20 mg is administered. When administered parenterally, the single dose of the compound or a salt thereof varies depending on the administration subject, the target disease, and the like. For example, a compound or a compound thereof that regulates the activity of the protein of the present invention for the purpose of treating heart failure. When a salt is usually administered to an adult (as 60 kg) in the form of an injection, about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 20 mg of the compound or its salt per day is administered. It is convenient to administer about 1 to 10 mg by intravenous injection. In the case of other animals, the amount can be administered in terms of 60 kg.
[0045]
[2] Quantification of the protein of the present invention, its partial peptide or its salt
An antibody against the protein of the present invention (hereinafter sometimes abbreviated as the antibody of the present invention) can specifically recognize the protein of the present invention. It can be used for quantification by sandwich immunoassay and the like.
That is, the present invention
(I) reacting the antibody of the present invention with a test solution and a labeled protein of the present invention competitively, and measuring the ratio of the labeled protein of the present invention bound to the antibody; A method for quantifying the protein of the present invention in a test solution, and
(Ii) After reacting a test solution with the antibody of the present invention immobilized on a carrier and another labeled antibody of the present invention simultaneously or continuously, the activity of the labeling agent on the insolubilized carrier is measured. A method for quantifying the protein of the present invention in a test solution is provided.
In the quantitative method (ii), it is desirable that one antibody is an antibody that recognizes the N-terminal of the protein of the present invention and the other antibody is an antibody that reacts with the C-terminal of the protein of the present invention.
In addition, the protein of the present invention can be quantified using a monoclonal antibody against the protein of the present invention (hereinafter sometimes referred to as the monoclonal antibody of the present invention), and can also be detected by tissue staining or the like. For these purposes, the antibody molecule itself may be used, and the F (ab ') ₂ , Fab ′ or Fab fraction may be used.
[0046]
The method for quantifying the protein of the present invention using the antibody of the present invention is not particularly limited, and the antibody, antigen, or antibody-antigen complex corresponding to the amount of the antigen (eg, the amount of the protein) in the test solution is measured. Any measurement method may be used as long as the amount is detected by chemical or physical means, and the amount is calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephelometry, competition method, immunometric method and sandwich method are suitably used, but it is particularly preferable to use the sandwich method described later in terms of sensitivity and specificity.
As a labeling agent used in a measuring method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like are used. As the radioisotope, for example, [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ¹⁴ C] is used. As the above-mentioned enzyme, a stable enzyme having a large specific activity is preferable. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, a luminol derivative, luciferin, lucigenin and the like are used. Further, a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.
For the insolubilization of an antigen or an antibody, physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing and immobilizing a protein or an enzyme may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide, and silicon; and glass.
[0047]
In the sandwich method, the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further reacted with another labeled monoclonal antibody of the present invention (secondary reaction). By measuring the activity of the labeling agent, the amount of the protein of the present invention in the test solution can be determined. The primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at staggered times. The labeling agent and the method of insolubilization can be in accordance with those described above. In addition, in the immunoassay by the sandwich method, the antibody used for the solid phase antibody or the labeling antibody is not necessarily one kind, and a mixture of two or more kinds of antibodies is used for the purpose of improving measurement sensitivity and the like. You may.
In the method for measuring the protein of the present invention by the sandwich method of the present invention, as the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction, an antibody having a different site to which the protein of the present invention binds is preferably used. That is, the antibody used in the primary reaction and the secondary reaction is preferably, for example, when the antibody used in the secondary reaction recognizes the C-terminal of the protein of the present invention, the antibody used in the primary reaction is preferably An antibody that recognizes, for example, the N-terminal other than the C-terminal is used.
The monoclonal antibody of the present invention can be used in a measurement system other than the sandwich method, for example, a competition method, an immunometric method, a nephrometry, or the like.
In the competition method, after the antigen in the test solution and the labeled antigen are allowed to react competitively with the antibody, the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated. (B / F separation), the amount of any of B and F is measured, and the amount of antigen in the test solution is quantified. In this reaction method, a soluble antibody is used as the antibody, B / F separation is performed using polyethylene glycol, a liquid phase method using a second antibody against the antibody, or a solid phase antibody is used as the first antibody. A solid-phase method using a soluble antibody as the first antibody and using a solid-phased antibody as the second antibody is used.
[0048]
In the immunometric method, the antigen in the test solution and the immobilized antigen are subjected to a competitive reaction with a fixed amount of the labeled antibody, and then the solid phase and the liquid phase are separated. And an excess amount of the labeled antibody, and then immobilized antigen is added to bind unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Next, the amount of label in any phase is measured to determine the amount of antigen in the test solution.
In nephelometry, the amount of insoluble sediment produced as a result of an antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test solution is small and only a small amount of sediment is obtained, laser nephrometry utilizing laser scattering is preferably used.
In applying these individual immunological measurement methods to the quantification method of the present invention, no special conditions, operations, and the like need to be set. The protein measurement system of the present invention may be constructed by adding ordinary technical considerations of those skilled in the art to ordinary conditions and operation methods in each method. For details of these general technical means, reference can be made to reviews, books, and the like.
For example, Hiroe Irie, "Radio Immunoassay" (Kodansha, published in 1974), Hiroshi Irie, "Radio Immunoassay" (Kodansha, published in 1974), Eiji Ishikawa et al., "Enzyme Immunoassay" (Medical Shoin, Showa Ed. Ishikawa et al., “Enzyme Immunoassay” (2nd edition) (Issue Shoin, 1982), Eiji Ishikawa et al. “Enzyme Immunoassay” (Third Edition), 3rd edition (Medical Shoin, Showa) 62)), "Methods in ENZYMOLOGY" Vol. 70 (Immunochemical Techniques (Part A)), ibid., Vol. 73 (Immunochemical Technologies (Part B)), ibid., Vol. 74 (Immunochemical Technologies (Part C)), ibid., Vol. 84 (Immunochemical Technologies (Part D: Selected Immunoassays)), ibid., Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)), ibid., Vol. 121 (Immunochemical Technologies (Part I: Hybridoma Technology and Monoclonal Antibodies)) (all published by Academic Press) can be referred to.
As described above, the protein of the present invention can be quantified with high sensitivity by using the antibody of the present invention.
Furthermore, when an increase in the concentration of the protein of the present invention is detected by quantifying the concentration of the protein of the present invention using the antibody of the present invention, for example, heart disease (eg, cardiomyopathy, myocardial infarction, heart failure) , Angina, etc.), diseases such as central nervous system disorders (eg, Alzheimer's disease, Parkinson's syndrome, schizophrenia), genital disorders (eg, testicular hypersensitivity, ovarian dysfunction, infertility, etc.), or in the future It can be diagnosed as having a high likelihood of being affected.
Further, the antibody of the present invention can be used for detecting the protein of the present invention present in a subject such as a body fluid or a tissue. Further, for preparation of an antibody column used for purifying the protein of the present invention, detection of the protein of the present invention in each fraction at the time of purification, analysis of the behavior of the protein of the present invention in test cells, etc. Can be used.
[0049]
[3] Gene diagnostic agent
The DNA of the present invention can be used, for example, as a probe to produce a human or warm-blooded animal (eg, rat, mouse, guinea pig, rabbit, bird, sheep, pig, cow, horse, cat, dog, monkey, chimpanzee, etc.). Can detect an abnormality (genetic abnormality) in DNA or mRNA encoding the protein of the present invention or a partial peptide thereof in, for example, damage, mutation or decrease in expression of the DNA or mRNA, or decrease in the DNA or mRNA. It is useful as a diagnostic agent for genes such as increase or overexpression.
The above-described genetic diagnosis using the DNA of the present invention can be performed, for example, by the known Northern hybridization or PCR-SSCP method (Genomics, Vol. 5, pp. 874-879 (1989), Processings of the * Proceedings of the National Academy of Sciences of the United States of America, Vol. 86, pp. 2766-2770 (1989), and the like. For example, when overexpression is detected by Northern hybridization or DNA mutation is detected by PCR-SSCP method, for example, heart disease (eg, cardiomyopathy, myocardial infarction, heart failure, angina pectoris, etc.) Can be diagnosed as likely to be a disease, such as a central nervous system disorder (eg, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.) or a genital disease (eg, testicular hypersensitivity, ovarian insufficiency, infertility, etc.) it can.
[0050]
[4] a drug containing an antisense nucleotide
The antisense nucleotide of the present invention, which complementarily binds to the DNA of the present invention and can suppress the expression of the DNA, has low toxicity, and functions of the protein of the present invention or the DNA of the present invention in vivo (eg, Since it can suppress cardiac function inhibitory activity, cardiomyocyte function inactivating effect, etc., for example, heart disease (eg, cardiomyopathy, myocardial infarction, heart failure, angina pectoris, etc.), central nervous system disease (eg, Alzheimer's disease) Disease, Parkinson's syndrome, schizophrenia, etc.), reproductive diseases (eg, testicular hypersensitivity, ovarian insufficiency, infertility, etc.) and the like, or can be used as a contraceptive.
When the antisense nucleotide is used as the prophylactic / therapeutic agent, it can be formulated and administered according to a known method.
For example, when the antisense nucleotide is used, after inserting the antisense nucleotide alone or into an appropriate vector such as a retrovirus vector, adenovirus vector, adenovirus associated virus vector, etc., human or non-human mammalian It can be administered orally or parenterally to animals (eg, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). The antisense nucleotide can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as an auxiliary agent for promoting uptake, and then administered using a gene gun or a catheter such as a hydrogel catheter. Alternatively, they can be aerosolized and administered locally into the trachea as an inhalant.
The dose of the antisense nucleotide varies depending on the target disease, the administration subject, the administration route, and the like. For example, when the antisense nucleotide of the present invention is orally administered for the purpose of treating heart failure, it is generally adult (60 kg body weight). )), About 0.1 to 100 mg of the antisense nucleotide is administered per day.
Furthermore, the antisense nucleotide can also be used as a diagnostic oligonucleotide probe for examining the presence of the DNA of the present invention in tissues or cells and the state of expression thereof.
[0051]
The invention further provides
(I) a double-stranded RNA containing a part of RNA encoding the protein of the present invention, (ii) a medicine containing the double-stranded RNA,
(Iii) a ribozyme containing a part of RNA encoding the protein of the present invention; and (iv) a medicine containing the ribozyme.
These double-stranded RNAs (RNAi; RNA interference method), ribozymes, and the like can suppress the expression of the polynucleotide (eg, DNA) of the present invention in the same manner as the antisense nucleotide, and Can inhibit the activity or function (eg, cardiac function inhibitory activity, cardiomyocyte function inactivating action, etc.) of the peptide of the present invention or the polynucleotide (eg, DNA) of the present invention. Disorders, myocardial infarction, heart failure, angina, etc.), central nervous system disorders (eg, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), genital disorders (eg, testicular hypersensitivity, ovarian dysfunction, infertility, etc.) It can be used as a prophylactic / therapeutic agent or as a contraceptive.
The double-stranded RNA can be produced by designing based on the sequence of the polynucleotide of the present invention according to a known method (eg, Nature, 411, 494, 2001).
A ribozyme can be designed and produced based on the sequence of the polynucleotide of the present invention according to a known method (eg, TRENDS in Molecular Medicine, Vol. 7, pp. 221, 2001). For example, it can be produced by linking a known ribozyme to a part of RNA encoding the peptide of the present invention. Examples of a part of the RNA encoding the peptide of the present invention include a portion (RNA fragment) close to a cleavage site on the RNA of the present invention that can be cleaved by a known ribozyme.
When the above-mentioned double-stranded RNA or ribozyme is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated and administered in the same manner as the antisense nucleotide.
[0052]
[5] A drug containing the antibody of the present invention
The antibody of the present invention having an activity of neutralizing the activity of the protein of the present invention may be used for heart disease (eg, cardiomyopathy, myocardial infarction, heart failure, angina pectoris, etc.), central nervous disease (eg, Alzheimer's disease, Parkinson's syndrome, It can be used as a medicament for diseases such as schizophrenia) and genital diseases (eg, testicular hypersensitivity, ovarian dysfunction, infertility, etc.).
The prophylactic / therapeutic agent for the above-mentioned diseases containing the antibody of the present invention has low toxicity, and is used as it is as a liquid or as a pharmaceutical composition of an appropriate dosage form, as a human or non-human mammal (eg, rat, rabbit, sheep, Pigs, cattle, cats, dogs, monkeys, etc.) orally or parenterally. The dose varies depending on the administration subject, target disease, symptoms, administration route, and the like. For example, when used for the treatment of heart failure in adults, the antibody of the present invention is usually administered in a single dose of 0.01%. About 20 to about 20 mg / kg body weight, preferably about 0.1 to 10 mg / kg body weight, more preferably about 0.1 to 5 mg / kg body weight, about 1 to 5 times a day, preferably about 1 to 3 times a day. It is conveniently administered by intravenous injection. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If the symptoms are particularly severe, the dose may be increased according to the symptoms.
The antibodies of the present invention can be administered as such or as a suitable pharmaceutical composition. The pharmaceutical composition used for the administration contains the antibody or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient. Such compositions are provided in dosage forms suitable for oral or parenteral administration.
That is, for example, compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (soft capsules and the like). Syrup, emulsion, suspension and the like. Such a composition is produced by a known method and contains a carrier, diluent or excipient commonly used in the field of pharmaceuticals. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.
As the composition for parenteral administration, for example, injections, suppositories, etc. are used, and the injections are in the form of intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, intravenous injections and the like. Is included. Such injections are prepared according to a known method, for example, by dissolving, suspending or emulsifying the antibody or a salt thereof in a sterile aqueous or oily liquid commonly used for injections. As an aqueous liquid for injection, for example, physiological saline, isotonic solution containing glucose and other auxiliary agents and the like are used, and suitable solubilizing agents, for example, alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) add-on of hydro- genated castor oil)) and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil, and the like are used, and benzyl benzoate, benzyl alcohol, and the like may be used in combination as a solubilizing agent. The prepared injection is usually filled in an appropriate ampoule. Suppositories to be used for rectal administration are prepared by mixing the above antibody or a salt thereof with a conventional suppository base.
The above-mentioned oral or parenteral pharmaceutical composition is conveniently prepared in a unit dosage form so as to be compatible with the dosage of the active ingredient. Examples of such dosage unit dosage forms include tablets, pills, capsules, injections (ampoules), suppositories, and the like. Usually, each dosage unit dosage form has a dosage of 5 to 500 mg, especially 5 to 100 mg for an injection. Other dosage forms preferably contain 10 to 250 mg of the above antibody.
Each of the above-mentioned compositions may contain another active ingredient as long as the composition does not cause an undesirable interaction with the antibody.
[0053]
[6] A drug containing the protein or DNA of the present invention
The protein of the present invention or DNA encoding the protein of the present invention (hereinafter sometimes abbreviated as the DNA of the present invention) may be used for heart disease (eg, cardiomyopathy, myocardial infarction, heart failure, angina pectoris, etc.), central nervous system, etc. It is also useful as a prophylactic / therapeutic agent for diseases (eg, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), genital diseases (eg, testicular hypersensitivity, ovarian insufficiency, infertility, etc.), or pharmaceuticals such as contraceptives. is there. When the protein of the present invention is used as the above medicine, it can be formulated according to conventional means. When the DNA of the present invention is used as the above-mentioned prophylactic / therapeutic agent, the DNA of the present invention is used alone or after insertion into a suitable vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, and the like, followed by a conventional method. Can be implemented. The DNA of the present invention can be administered as it is or together with an auxiliary agent for promoting uptake, using a gene gun or a catheter such as a hydrogel catheter.
For example, the protein of the present invention or the DNA of the present invention is orally acceptable as a sugar-coated tablet, capsule, elixir, microcapsule or the like, or water or other pharmaceutically acceptable, if necessary. It can be used parenterally in the form of a sterile solution with liquid or injection, such as a suspension. For example, the protein of the present invention or the DNA of the present invention is required for the generally accepted formulation practice with known carriers, flavors, excipients, vehicles, preservatives, stabilizers, binders, etc., which are physiologically recognized. It can be manufactured by mixing in unit dosage form. The amount of the active ingredient in these preparations is such that a proper dosage in the specified range can be obtained.
The dosage of the protein of the present invention varies depending on the administration subject, target organ, condition, administration method, and the like. However, in the case of oral administration, for example, in a heart failure patient (as 60 kg), about 0% per day is generally used. 0.1 mg to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptoms, administration method, and the like. For example, usually in the form of an injection, for example, in a heart failure patient (as 60 kg), It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day by intravenous injection. In the case of other animals, the amount can be administered in terms of 60 kg.
The dose of the DNA of the present invention varies depending on the administration subject, target organ, condition, administration method, and the like. However, in the case of oral administration, for example, in a heart failure patient (as 60 kg), it is generally about 0% per day. 0.1 mg to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptoms, administration method, and the like. For example, usually in the form of an injection, for example, in a heart failure patient (as 60 kg), It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day by intravenous injection. In the case of other animals, the amount can be administered in terms of 60 kg.
[0054]
[7] Creation of an animal having the DNA of the present invention
The present invention relates to a non-protein having a DNA encoding an exogenous protein of the present invention (hereinafter abbreviated as the exogenous DNA of the present invention) or a mutant DNA thereof (sometimes abbreviated as the exogenous mutant DNA of the present invention). A human mammal is provided.
That is, the present invention
(1) a non-human mammal having the exogenous DNA of the present invention or a mutant DNA thereof,
(2) The animal according to (1) above, wherein the non-human mammal is a rodent.
(3) The animal according to (2), wherein the rodent is a mouse or a rat, and
(4) A recombinant vector or the like containing the exogenous DNA of the present invention or its mutant DNA and capable of being expressed in mammals is provided.
Non-human mammals having the exogenous DNA of the present invention or the mutant DNA thereof (hereinafter abbreviated as the DNA-transferred animal of the present invention) can be used for non-fertilized eggs, fertilized eggs, germ cells including spermatozoa and their progenitor cells, and the like. And preferably at the stage of embryonic development in non-human mammal development (more preferably at the stage of single cells or fertilized eggs and generally before the 8-cell stage), the calcium phosphate method, the electric pulse method, the lipofection method, the agglutination method, It can be produced by introducing a target DNA by a microinjection method, a particle gun method, a DEAE-dextran method, or the like. Further, by the DNA introduction method, the exogenous DNA of the present invention can be introduced into somatic cells, organs of living organisms, tissue cells, and the like, and used for cell culture, tissue culture, and the like. The DNA-transfected animal of the present invention can also be produced by fusing the above-mentioned germ cells with a known cell fusion method.
As the non-human mammal, for example, cow, pig, sheep, goat, rabbit, dog, cat, guinea pig, hamster, mouse, rat and the like are used. Among them, rodents, which are relatively short in ontogeny and biological cycle in terms of preparation of disease animal model systems, and are easy to breed, especially mice (for example, hybrids such as C57BL / 6 strain and DBA2 strain as pure strains) B6C3F as a system ₁ System, BDF ₁ System, B6D2F ₁ Strain, BALB / c strain, ICR strain, etc.) or rat (eg, Wistar, SD etc.) is preferred.
"Mammals" in a recombinant vector that can be expressed in mammals include humans in addition to the above-mentioned non-human mammals.
[0055]
The exogenous DNA of the present invention refers to the DNA of the present invention once isolated and extracted from a mammal, not the DNA of the present invention originally possessed by a non-human mammal.
As the mutant DNA of the present invention, those having a mutation (for example, mutation) in the base sequence of the original DNA of the present invention, specifically, base addition, deletion, substitution with another base, etc. The resulting DNA or the like is used, and also includes abnormal DNA.
The abnormal DNA means a DNA that expresses an abnormal protein of the present invention, and for example, a DNA that expresses a protein that suppresses the function of the normal protein of the present invention is used.
The exogenous DNA of the present invention may be derived from a mammal that is the same or different from the animal of interest. When introducing the DNA of the present invention into a target animal, it is generally advantageous to use the DNA as a DNA construct linked downstream of a promoter capable of being expressed in animal cells. For example, when the human DNA of the present invention is introduced, DNAs derived from various mammals (eg, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.) having the DNA of the present invention having high homology thereto are expressed. Highly expressing the DNA of the present invention by microinjecting a DNA construct (eg, vector, etc.) in which the human DNA of the present invention is bound downstream of various promoters that can be made into a fertilized egg of a target mammal, for example, a mouse fertilized egg. DNA-introduced mammals can be created.
[0056]
Examples of the expression vector of the protein of the present invention include plasmids derived from Escherichia coli, plasmids derived from Bacillus subtilis, plasmids derived from yeast, bacteriophages such as λ phage, retroviruses such as Moloney leukemia virus, animal viruses such as vaccinia virus or baculovirus. Are used. Among them, a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis or a plasmid derived from yeast is preferably used.
Examples of the promoter for controlling the DNA expression include, but are not limited to, promoters of DNA derived from (1) viruses (eg, simian virus, cytomegalovirus, Moloney leukemia virus, JC virus, breast cancer virus, poliovirus, etc.), and various types of mammals. Promoters derived from animals (human, rabbit, dog, cat, guinea pig, hamster, rat, mouse, etc.), for example, albumin, insulin II, uroplakin II, elastase, erythropoietin, endothelin, muscle creatine kinase, glial fibrillary acidic protein, glutathione S-transferase, platelet-derived growth factor β, keratins K1, K10 and K14, collagen types I and II, cyclic AMP-dependent protein kinase βI subunit, dystrophin , Tartrate-resistant alkaline phosphatase, atrial natriuretic factor, endothelial receptor tyrosine kinase (commonly abbreviated as Tie2), sodium potassium adenosine 3 kinase (Na, K-ATPase), neurofilament light chain, metallothionein I and IIA , Metalloproteinase 1 tissue inhibitor, MHC class I antigen (H-2L), H-ras, renin, dopamine β-hydroxylase, thyroid peroxidase (TPO), polypeptide chain elongation factor 1α (EF-1α), β actin , Α and β myosin heavy chain, myosin light chain 1 and 2, myelin basic protein, thyroglobulin, Thy-1, immunoglobulin, heavy chain variable region (VNP), serum amyloid P component, myoglobin, troponin C, smooth muscle α Aku Down, pre-pro-enkephalin A, such as a promoter, such as vasopressin is used. Among them, a cytomegalovirus promoter capable of high expression throughout the body, a promoter of human polypeptide chain elongation factor 1α (EF-1α), human and chicken β-actin promoters, and the like are preferable.
The vector preferably has a sequence that terminates transcription of a target messenger RNA in a DNA-transfected mammal (generally called a terminator). For example, a sequence of each DNA derived from a virus and various mammals can be used. A simian virus SV40 terminator or the like is preferably used.
[0057]
In addition, a splicing signal of each DNA, an enhancer region, a part of an intron of a eukaryotic DNA, and the like are placed 5 ′ upstream of the promoter region, between the promoter region and the translation region, or between the translation region for the purpose of further expressing the desired foreign DNA. It is also possible to connect 3 ′ downstream of the above depending on the purpose.
The normal translation region of the protein of the present invention can be obtained from liver, kidney, thyroid cells, fibroblast-derived DNA derived from humans or various mammals (eg, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.) and commercially available DNAs. From a variety of genomic DNA libraries, or as a starting material, a complementary DNA prepared by known methods from RNA derived from liver, kidney, thyroid cells, and fibroblasts. In addition, a translation region obtained by mutating a translation region of a normal polypeptide obtained from the above-mentioned cells or tissues by a point mutagenesis method can be used for the exogenous abnormal DNA.
The translation region can be prepared as a DNA construct that can be expressed in a DNA-transduced animal by a conventional DNA engineering technique in which the translation region is ligated downstream of the promoter and, if desired, upstream of the transcription termination site.
Introduction of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal. The presence of the exogenous DNA of the present invention in the germinal cells of the produced animal after the introduction of the DNA means that all progeny of the produced animal will retain the exogenous DNA of the present invention in all of the germ cells and somatic cells. means. The offspring of such animals that have inherited the exogenous DNA of the present invention have the exogenous DNA of the present invention in all of their germ cells and somatic cells.
The non-human mammal to which the exogenous normal DNA of the present invention has been transferred can be subcultured in a normal breeding environment as an animal having the DNA after confirming that the exogenous DNA is stably retained by mating. .
Introduction of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in excess in all germ cells and somatic cells of the target mammal. Excessive presence of the exogenous DNA of the present invention in the germinal cells of the produced animal after DNA introduction means that all the offspring of the produced animal have the exogenous DNA of the present invention in all of their germinal and somatic cells. Means Progeny of such animals that have inherited the exogenous DNA of the present invention have an excess of the exogenous DNA of the present invention in all of their germinal and somatic cells.
By obtaining a homozygous animal having the introduced DNA on both homologous chromosomes and mating the male and female animals, all offspring can be bred to have the DNA in excess.
[0058]
The non-human mammal having the normal DNA of the present invention expresses the normal DNA of the present invention at a high level and promotes the function of the endogenous normal DNA to eventually cause hyperactivity of the protein of the present invention. Occasionally, it can be used as a disease model animal. For example, using the normal DNA-transfected animal of the present invention, it is possible to elucidate the pathological mechanism of the hyperactivity of the protein of the present invention and diseases associated with the protein of the present invention, and to examine a method for treating these diseases. It is possible.
Further, since the mammal into which the exogenous normal DNA of the present invention has been introduced has an increased symptom of the released protein of the present invention, it can be used for screening tests for therapeutic drugs for diseases related to the protein of the present invention. is there.
On the other hand, a non-human mammal having the exogenous abnormal DNA of the present invention can be subcultured in an ordinary breeding environment as an animal having the DNA after confirming that the exogenous DNA is stably maintained by mating. Furthermore, the desired foreign DNA can be incorporated into the above-described plasmid and used as a source material. The DNA construct with the promoter can be prepared by a usual DNA engineering technique. Introduction of the abnormal DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal. The presence of the abnormal DNA of the present invention in the germinal cells of the produced animal after DNA introduction means that all the offspring of the produced animal have the abnormal DNA of the present invention in all of its germinal and somatic cells. The progeny of such animals that have inherited the exogenous DNA of the present invention have the abnormal DNA of the present invention in all of their germinal and somatic cells. A homozygous animal having the introduced DNA on both homologous chromosomes is obtained, and by crossing these male and female animals, it is possible to breed the cells so that all offspring have the DNA.
[0059]
The non-human mammal having the abnormal DNA of the present invention, in which the abnormal DNA of the present invention is highly expressed, inhibits the function of endogenous normal DNA, and finally, the functionally inactive form of the protein of the present invention. It can be refractory and can be used as a model animal for the disease. For example, by using the abnormal DNA-introduced animal of the present invention, it is possible to elucidate the pathological mechanism of the function-inactive refractory of the protein of the present invention and to examine a method for treating this disease.
Further, as a specific possibility, the abnormal DNA-highly expressing animal of the present invention exhibits a function of inhibiting the normal protein function (dominant negative action) by the abnormal protein of the present invention in the inactive refractory disease of the protein of the present invention. A model to elucidate. Further, since the mammal into which the foreign abnormal DNA of the present invention has been introduced has an increased symptom of the released protein of the present invention, it is also used for a therapeutic drug screening test for the protein of the present invention or a functionally inactive refractory disease thereof. It is possible.
In addition, other possible uses of the two kinds of the DNA-introduced animals of the present invention include, for example,
(I) use as a cell source for tissue culture;
(Ii) a protein specifically expressed or activated by the protein of the present invention, by directly analyzing DNA or RNA in the tissue of the DNA-transfected animal of the present invention, or analyzing a polypeptide tissue expressed by the DNA; Analysis of the relationship with
(Iii) culturing cells of DNA-bearing tissue by standard tissue culture techniques and using them to study the function of cells from tissues that are generally difficult to culture;
(Iv) screening for an agent that enhances the function of the cell by using the cell described in (iii) above, and
(V) Isolation and purification of the mutant protein of the present invention and production of its antibody can be considered.
Furthermore, using the DNA-introduced animal of the present invention, it is possible to examine the clinical symptoms of diseases related to the protein of the present invention, including the inactive refractory disease of the protein of the present invention, etc. More detailed pathological findings in each organ of a disease model related to the disease can be obtained, which can contribute to the development of a new treatment method, and further to the research and treatment of a secondary disease caused by the disease.
In addition, it is possible to take out each organ from the DNA-introduced animal of the present invention, cut it into small pieces, and then use a proteolytic enzyme such as trypsin to obtain the released DNA-introduced cells, culture them, or systematize the cultured cells. . Furthermore, it is possible to examine the specificity of the protein-producing cell of the present invention, its relationship with apoptosis, differentiation or proliferation, or its signal transduction mechanism, and its abnormalities. It is an effective research material for
[0060]
As a specific example, a test compound is administered to the DNA-introduced animal of the present invention, and the heart function, electrocardiogram, heart weight, and the like of the animal are measured. Heart weight is a parameter of cardiac hypertrophy. Specifically, the heart structure can be examined by calculating the heart weight per body weight, the left ventricular weight per body weight, and the left ventricular weight per right ventricle weight. Since the above parameters increase when cardiac hypertrophy occurs, the test compound can be evaluated using the suppression of this increase as an index. After administering the test compound to the DNA-introduced animal of the present invention, a myocardial infarction is performed, and the heart function, electrocardiogram, heart weight and the like of the animal are measured. After the myocardial infarction operation, the infarct progress-inhibitory activity of the test compound can be examined by weighing the infarct layer. Administration of the test compound may be after infarction surgery. In addition, a new heart failure model can be prepared by breeding the animal with a genetic hypertension model rat such as an SHR rat. The compound is administered to the heart failure model prepared as described above, and the heart function, electrocardiogram, heart weight, infarction progress inhibitory activity and the like of the animal are examined.
[0061]
[8] Knockout animal
The present invention provides a non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated, and a non-human mammal deficient in expression of the DNA of the present invention.
That is, the present invention
(1) a non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated,
(2) The embryonic stem cell according to (1), wherein the DNA is inactivated by introducing a reporter gene (eg, a β-galactosidase gene derived from Escherichia coli).
(3) The embryonic stem cell according to (1), which is neomycin-resistant,
(4) the embryonic stem cell according to (1), wherein the non-human mammal is a rodent;
(5) The embryonic stem cell according to (4), wherein the rodent is a mouse,
(6) a non-human mammal deficient in expression of the DNA of the present invention, wherein the DNA is inactivated;
(7) The above (6), wherein the DNA is inactivated by introducing a reporter gene (eg, a β-galactosidase gene derived from Escherichia coli), and the reporter gene can be expressed under the control of a promoter for the DNA of the present invention. The non-human mammal of the description,
(8) the non-human mammal according to the above (6), wherein the non-human mammal is a rodent;
(9) The non-human mammal according to (8), wherein the rodent is a mouse, and
(10) A compound that promotes or inhibits (preferably inhibits) the promoter activity of the DNA of the present invention, which comprises administering a test compound to the animal according to (7) and detecting expression of a reporter gene, or a compound thereof. A method for screening a salt is provided.
A non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated is an artificially mutated DNA of the present invention possessed by the non-human mammal, which suppresses the expression ability of the DNA, or By substantially losing the activity of the protein of the present invention encoded by the DNA, the DNA does not substantially have the ability to express the protein of the present invention (hereinafter, may be referred to as the knockout DNA of the present invention). ) Non-human mammalian embryonic stem cells (hereinafter abbreviated as ES cells).
As the non-human mammal, the same one as described above is used.
[0062]
The method of artificially mutating the DNA of the present invention can be performed, for example, by deleting a part or all of the DNA sequence and inserting or substituting another DNA by a genetic engineering technique. With these mutations, the knockout DNA of the present invention may be prepared, for example, by shifting the codon reading frame or disrupting the function of the promoter or exon.
Specific examples of the non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated (hereinafter abbreviated as the DNA-inactivated ES cells of the present invention or the knockout ES cells of the present invention) include, for example, The DNA of the present invention possessed by a non-human mammal is isolated and its exon portion is a drug resistance gene typified by a neomycin resistance gene or a hygromycin resistance gene, or lacZ (β-galactosidase gene), cat (chloramphenicol acetyl). A reporter gene or the like typified by a transferase gene) to disrupt the exon function, or insert a DNA sequence that terminates gene transcription into an intron between exons (for example, a polyA addition signal); Inability to synthesize complete messenger RNA Thus, a DNA strand having a DNA sequence constructed so as to eventually destroy the gene (hereinafter abbreviated as a targeting vector) is introduced into the chromosome of the animal by, for example, homologous recombination, and the resulting ES cells PCR using the DNA sequence on or near the DNA of the present invention as a probe, and using the DNA sequence on the targeting vector and the DNA sequence in the neighboring region other than the DNA of the present invention used for the preparation of the targeting vector as primers And selecting the knockout ES cells of the present invention.
[0063]
As the ES cells from which the DNA of the present invention is inactivated by the homologous recombination method or the like, for example, those already established as described above may be used, or according to the known method of Evans and Kaufma. May be newly established. For example, in the case of mouse ES cells, currently, 129-line ES cells are generally used. For example, for the purpose of obtaining ES cells in which BDF is clearly known, for example, BDF in which the number of eggs collected by C57BL / 6 mice or C57BL / 6 has been improved by hybridization with DBA / 2 ₁ Mouse (F of C57BL / 6 and DBA / 2) ₁ ) Can also be used favorably. The BDF1 mouse has the advantage that the number of eggs collected is large and the eggs are robust. In addition, since the C57BL / 6 mouse is used as a background, the ES cells obtained using the mouse have It can be advantageously used in that the genetic background can be replaced by C57BL / 6 mice by backcrossing with C57BL / 6 mice.
In addition, when ES cells are established, blastocysts 3.5 days after fertilization are generally used. In addition, a large number of blastocysts can be efficiently obtained by collecting embryos at the 8-cell stage and culturing them until blastocysts are used. Early embryos can be obtained.
Either male or female ES cells may be used, but generally male ES cells are more convenient for producing a germline chimera. It is also desirable to discriminate between males and females as soon as possible in order to reduce cumbersome culture labor.
As a method for determining the sex of ES cells, for example, a method of amplifying and detecting a gene in a sex-determining region on the Y chromosome by a PCR method can be mentioned. If this method is used, it is conventionally necessary to perform about 10 karyotype analyses. ⁶ Since the number of ES cells is required, the number of ES cells of about 1 colony (approximately 50) is sufficient, so that primary selection of ES cells in the early stage of culture can be performed by discriminating between male and female. In addition, by enabling selection of male cells at an early stage, labor in the initial stage of culture can be significantly reduced.
[0064]
The secondary selection can be performed, for example, by confirming the number of chromosomes by the G-banding method. The number of chromosomes in the obtained ES cells is desirably 100% of the normal number. However, when it is difficult due to physical operations at the time of establishment, after knocking out the gene of the ES cells, the normal cells (for example, chromosomes in mice) are knocked out. It is desirable to clone again into cells (number 2n = 40).
The embryonic stem cell line obtained in this way usually has very good growth properties, but it must be carefully subcultured because it tends to lose its ontogenic ability. For example, on a suitable feeder cell such as STO fibroblast, in the presence of LIF (1-10000 U / ml) in a carbon dioxide incubator (preferably, 5% carbon dioxide, 95% air or 5% oxygen, 5% The cells are cultured by a method such as culturing at about 37 ° C. in carbon dioxide gas and 90% air. At the time of subculture, for example, a trypsin / EDTA solution (usually 0.001-0.5% trypsin / 0.1-5 mM EDTA, Preferably, a method is used in which cells are converted into single cells by treatment with about 0.1% trypsin / 1 mM EDTA and seeded on newly prepared feeder cells. Such subculture is usually performed every 1 to 3 days. At this time, it is desirable to observe the cells and, if any morphologically abnormal cells are found, discard the cultured cells.
ES cells are differentiated into various types of cells, such as parietal muscle, visceral muscle, and cardiac muscle, by monolayer culture up to high density or suspension culture until cell clumps are formed under appropriate conditions. [M. J. Evans and M.E. H. Kaufman, Nature, 292, 154, 1981; R. Martin Proc. Of National Academy of Sciences USA (Proc. Natl. Acad. Sci. USA) 78, 7634, 1981; C. Doetschman et al., Journal of Embryology and Experimental Morphology, Vol. 87, p. 27, 1985], that the DNA-deficient cell of the present invention obtained by differentiating the ES cell of the present invention is characterized by in vitro Is useful in cell biological studies of the protein of the present invention.
The non-human mammal deficient in DNA expression of the present invention can be distinguished from a normal animal by measuring the mRNA level of the animal using a known method and indirectly comparing the expression level.
As the non-human mammal, those similar to the aforementioned can be used.
[0065]
The non-human mammal deficient in expression of the DNA of the present invention is, for example, a DNA in which the targeting vector prepared as described above is introduced into mouse embryonic stem cells or mouse egg cells, and the DNA of the targeting vector is inactivated by the introduction. The DNA of the present invention can be knocked out by homologous recombination in which the sequence replaces the DNA of the present invention on the chromosome of mouse embryonic stem cells or mouse egg cells by homologous recombination.
Cells in which the DNA of the present invention has been knocked out can be used as a DNA sequence on a Southern hybridization analysis or targeting vector using the DNA sequence on or near the DNA of the present invention as a probe, and the DNA of the present invention derived from the mouse used for the targeting vector. It can be determined by analysis by PCR using a DNA sequence of a nearby region other than DNA as a primer. When non-human mammalian embryonic stem cells are used, a cell line in which the DNA of the present invention has been inactivated by gene homologous recombination is cloned, and the cells are cultured at an appropriate time, for example, at the 8-cell stage of non-human mammalian cells. The chimeric embryo is injected into an animal embryo or blastocyst, and the resulting chimeric embryo is transplanted into the uterus of the pseudopregnant non-human mammal. The produced animal is a chimeric animal comprising both cells having the normal DNA locus of the present invention and cells having the artificially mutated DNA locus of the present invention. When a part of the germ cells of the chimeric animal has a mutated DNA locus of the present invention, all tissues are artificially mutated from a population obtained by crossing such a chimeric individual with a normal individual. It can be obtained by selecting individuals composed of cells having the added DNA locus of the present invention, for example, by judging coat color or the like. The individual thus obtained is usually an individual with a heterozygous expression of the protein of the present invention, which is crossed with an individual with a heterozygous expression of the protein of the present invention. Defective individuals can be obtained.
In the case of using an egg cell, for example, a transgenic non-human mammal having a targeting vector introduced into a chromosome can be obtained by injecting a DNA solution into a nucleus of an egg by a microinjection method, and these transgenic non-human mammals can be obtained. Compared to mammals, they can be obtained by selecting those having a mutation in the DNA locus of the present invention by homologous recombination.
In this manner, the individual in which the DNA of the present invention is knocked out can be bred in an ordinary breeding environment after confirming that the DNA of the animal obtained by the breeding is also knocked out.
Further, the acquisition and maintenance of the germ line may be performed according to a conventional method. That is, by crossing male and female animals having the inactivated DNA, a homozygous animal having the inactivated DNA on both homologous chromosomes can be obtained. The obtained homozygous animal can be efficiently obtained by rearing the mother animal in such a manner that there are one normal animal and one homozygote. By mating male and female heterozygous animals, homozygous and heterozygous animals having the inactivated DNA are bred and subcultured.
The non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated are extremely useful for producing a non-human mammal deficient in expressing the DNA of the present invention.
In addition, since the non-human mammal deficient in expression of the DNA of the present invention lacks various biological activities that can be induced by the protein of the present invention, a model of a disease caused by inactivation of the biological activity of the protein of the present invention. Therefore, it is useful for investigating the causes of these diseases and examining treatment methods.
[0066]
[8a] A method for screening a compound having a therapeutic / preventive effect on diseases caused by DNA deficiency or damage of the present invention
The non-human mammal deficient in expression of the DNA of the present invention can be used for screening for a compound having a therapeutic / preventive effect against diseases caused by deficiency or damage of the DNA of the present invention.
That is, the present invention is characterized by administering a test compound to a non-human mammal deficient in expressing the DNA of the present invention, and observing and measuring a change in the animal. Provided is a method for screening a compound or a salt thereof having a therapeutic / prophylactic effect against a disease.
Examples of the non-human mammal deficient in expressing DNA of the present invention used in the screening method include the same as described above.
Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma and the like. These compounds are novel compounds. Or a known compound.
Specifically, a non-human mammal deficient in expression of the DNA of the present invention is treated with a test compound, compared with an untreated control animal, and tested using changes in each organ, tissue, disease symptoms, etc. of the animal as an index. The therapeutic / preventive effects of the compounds can be tested.
As a method of treating a test animal with a test compound, for example, oral administration, intravenous injection and the like are used, and it can be appropriately selected according to the symptoms of the test animal, the properties of the test compound, and the like. The dose of the test compound can be appropriately selected according to the administration method, the properties of the test compound, and the like.
For example, diseases characterized by reduced cardiac function (eg, heart diseases (eg, cardiomyopathy, myocardial infarction, heart failure, angina pectoris, etc.), central nervous system diseases (eg, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.) When screening for a compound having a preventive / therapeutic effect on genital diseases (eg, testicular hypersensitivity, ovarian dysfunction, infertility, etc.), a test compound is administered to a non-human mammal deficient in expressing the DNA of the present invention. Then, the heart function, electrocardiogram, heart weight and the like of the animal are measured. Heart weight is a parameter of cardiac hypertrophy. Specifically, the heart structure can be examined by calculating the heart weight per body weight, the left ventricular weight per body weight, and the left ventricular weight per right ventricle weight. Since the above parameters increase when cardiac hypertrophy occurs, the test compound can be evaluated using the suppression of this increase as an index. After administering the test compound to the non-human mammal deficient in expression of the DNA of the present invention, a myocardial infarction is performed, and the heart function, electrocardiogram, heart weight, and the like of the animal are measured. After the myocardial infarction operation, the infarct progress-inhibitory activity of the test compound can be examined by weighing the infarct layer. Administration of the test compound may be after infarction surgery. In addition, a new heart failure model can be prepared by breeding the animal with a genetic hypertension model rat such as an SHR rat. The compound is administered to the heart failure model prepared as described above, and the heart function, electrocardiogram, heart weight, infarction progress inhibitory activity and the like of the animal are examined.
[0067]
The compound obtained by the screening method may form a salt. Examples of the salt of the compound include physiologically acceptable acids (eg, inorganic acids, organic acids, etc.) and bases (eg, alkali metals, etc.). ) Is used, and a physiologically acceptable acid addition salt is particularly preferable. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, For example, salts with succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and the like are used.
A drug containing the compound or a salt thereof obtained by the screening method can be produced in the same manner as the above-mentioned drug containing the protein of the present invention.
The preparations obtained in this way are safe and have low toxicity, for example, human or non-human mammals (eg, rat, mouse, guinea pig, rabbit, sheep, pig, cow, horse, cat, dog, monkey, etc.) ) Can be administered.
The dose of the compound or a salt thereof varies depending on the target disease, the subject of administration, the administration route, and the like. For example, when the compound is orally administered, generally in an adult (with a body weight of 60 kg) heart failure patient, About 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg of the compound is administered per day. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, and the like. For example, the compound is usually administered as an injection to an adult (60 kg) heart failure patient. In this case, it is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg of the compound per day by intravenous injection. In the case of other animals, the amount can be administered in terms of 60 kg.
[0068]
[8b] A method for screening a compound that promotes or inhibits the activity of a promoter for the DNA of the present invention
The present invention provides a compound which promotes or inhibits the activity of a promoter for the DNA of the present invention, which comprises administering a test compound to a non-human mammal deficient in expressing the DNA of the present invention, and detecting the expression of a reporter gene, or a compound thereof. A method for screening a salt is provided.
In the above-mentioned screening method, the non-human mammal deficient in expression of DNA of the present invention may be a non-human mammal deficient in expression of DNA of the present invention in which the DNA of the present invention is inactivated by introducing a reporter gene, A reporter gene that can be expressed under the control of a promoter for the DNA of the present invention is used.
Examples of the test compound include the same compounds as described above.
As the reporter gene, the same one as described above is used, and a β-galactosidase gene (lacZ), a soluble alkaline phosphatase gene, a luciferase gene and the like are preferable.
[0069]
In a non-human mammal deficient in expression of the DNA of the present invention in which the DNA of the present invention is replaced with a reporter gene, since the reporter gene is under the control of the promoter for the DNA of the present invention, the expression of the substance encoded by the reporter gene is traced. By doing so, the activity of the promoter can be detected.
For example, when a part of the DNA region encoding the protein of the present invention is replaced with a β-galactosidase gene (lacZ) derived from Escherichia coli, the tissue that expresses the protein of the present invention originally replaces the protein of the present invention. Β-galactosidase is expressed. Therefore, for example, the present invention can be easily performed by staining with a reagent serving as a substrate for β-galactosidase such as 5-bromo-4-chloro-3-indolyl-β-galactopyranoside (X-gal). Can be observed in the animal body. Specifically, the protein-deficient mouse of the present invention or a tissue section thereof is fixed with glutaraldehyde or the like, washed with phosphate buffered saline (PBS), and then stained with X-gal at room temperature or around 37 ° C. After reacting for about 30 minutes to 1 hour, the β-galactosidase reaction may be stopped by washing the tissue specimen with a 1 mM EDTA / PBS solution, and coloration may be observed. Further, mRNA encoding lacZ may be detected according to a conventional method.
The compound or a salt thereof obtained by using the above-mentioned screening method is a compound selected from the above-mentioned test compounds, and is a compound that promotes or inhibits the promoter activity for the DNA of the present invention.
[0070]
The compound obtained by the screening method may form a salt, and the salt of the compound may include a physiologically acceptable acid (eg, an inorganic acid) and a base (eg, an organic acid). And particularly preferably a physiologically acceptable acid addition salt. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, For example, salts with succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and the like are used.
[0071]
In addition, the compound of the present invention or a salt thereof that inhibits the promoter activity of the DNA can inhibit the expression of the protein of the present invention and inhibit the function of the protein. Agents for the prevention and treatment of central nervous system disorders (eg, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), genital disorders (eg, testicular hypersensitivity, ovarian insufficiency, infertility, etc.) Or as a medicament such as a contraceptive.
Furthermore, compounds derived from the compounds obtained by the above screening can be used in the same manner.
A drug containing the compound or a salt thereof obtained by the screening method can be produced in the same manner as the above-mentioned drug containing the protein of the present invention or a salt thereof.
The preparations obtained in this way are safe and have low toxicity, for example, human or non-human mammals (eg, rat, mouse, guinea pig, rabbit, sheep, pig, cow, horse, cat, dog, monkey, etc.) ) Can be administered.
The dose of the compound or a salt thereof varies depending on the disease to be treated, the subject of administration, the administration route, and the like. For example, when the compound of the present invention that promotes the promoter activity for DNA is orally administered, generally the adult (body weight) is used. For patients with heart failure (as 60 kg), about 0.1-100 mg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg of the compound is administered per day. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, and the like. For example, the compound of the present invention that promotes the promoter activity for DNA is usually administered in the form of an injection to an adult ( When administered to a patient with heart failure (as 60 kg), about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg of the compound is administered by intravenous injection per day. It is convenient to do so. In the case of other animals, the amount can be administered in terms of 60 kg.
[0072]
On the other hand, for example, when the compound of the present invention that inhibits the promoter activity for DNA is orally administered, generally in an adult (assuming a body weight of 60 kg) heart failure patient, the compound is preferably about 0.1 to 100 mg per day, Is administered in an amount of about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, and the like. For example, the compound of the present invention that inhibits the promoter activity for DNA is usually administered in the form of an injection to an adult ( When administered to a patient with heart failure (as 60 kg), about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg of the compound is administered by intravenous injection per day. It is convenient to do so. In the case of other animals, the amount can be administered in terms of 60 kg.
As described above, the non-human mammal deficient in expression of the DNA of the present invention is extremely useful for screening for a compound or a salt thereof that promotes or inhibits the activity of the promoter for the DNA of the present invention, It can greatly contribute to investigating the cause of various diseases caused by it or to develop preventive and therapeutic drugs.
Further, using a DNA containing the promoter region of the protein of the present invention, genes encoding various proteins are ligated downstream thereof and injected into egg cells of an animal to produce a so-called transgenic animal (transgenic animal). Once created, it will also be possible to specifically synthesize the polypeptide and study its effects in living organisms. Furthermore, if a suitable reporter gene is bound to the above promoter portion, and a cell line that expresses the reporter gene is established, a low-molecular compound having an action of specifically promoting or suppressing the production ability of the protein itself of the present invention in the body. Can be used as a search system.
[0073]
In the present specification, bases, amino acids, and the like are represented by abbreviations based on the abbreviations by IUPAC-IUB Commission on Biochemical Nomenclature or abbreviations commonly used in the art, and examples thereof are described below. When an amino acid can have an optical isomer, the L-form is indicated unless otherwise specified.
DNA: deoxyribonucleic acid
cDNA: Complementary deoxyribonucleic acid
A: Adenine
T: Thymine
G: Guanine
C: Cytosine
R: adenine (A) or guanine (G)
Y: cytosine (C) or thymine (T)
K: guanine (G) or thymine (T)
M: adenine (A) or cytosine (C)
S: guanine (G) or cytosine (C)
W: adenine (A) or thymine (T)
N: adenine (A), cytosine (C), guanine (G) or thymine (T)
RNA: ribonucleic acid
mRNA: messenger ribonucleic acid
dATP: deoxyadenosine triphosphate
dTTP: deoxythymidine triphosphate
dGTP: deoxyguanosine triphosphate
dCTP: deoxycytidine triphosphate
ATP: Adenosine triphosphate
EDTA: Ethylenediaminetetraacetic acid
SDS: Sodium dodecyl sulfate
[0074]
Gly: glycine
Ala: Alanine
Val: Valine
Leu: Leucine
Ile: Isoleucine
Ser: Serine
Thr: Threonine
Cys: cysteine
Met: methionine
Glu: glutamic acid
Asp: Aspartic acid
Lys: lysine
Arg: Arginine
His: histidine
Phe: phenylalanine
Tyr: Tyrosine
Trp: Tryptophan
Pro: Proline
Asn: Asparagine
Gln: Glutamine
pGlu: pyroglutamic acid
[0075]
Further, substituents, protecting groups and reagents frequently used in the present specification are represented by the following symbols.

[0076]
The sequence numbers in the sequence listing in the present specification indicate the following sequences.
[SEQ ID NO: 1]
1 shows the base sequence obtained in Example 1.
[SEQ ID NO: 2]
1 shows the nucleotide sequence of a primer used in Example 1.
[SEQ ID NO: 3]
1 shows the nucleotide sequence of a primer used in Example 1.
[SEQ ID NO: 4]
1 shows the base sequence obtained in Example 1.
[SEQ ID NO: 5]
1 shows the base sequence obtained in Example 1.
[SEQ ID NO: 6]
1 shows the nucleotide sequence of a primer used in Example 1.
[SEQ ID NO: 7]
1 shows the nucleotide sequence of a primer used in Example 1.
[SEQ ID NO: 8]
1 shows the base sequence obtained in Example 1.
[SEQ ID NO: 9]
The base sequences of the primers used in Examples 1 to 5, Example 8, and Example 9 are shown.
[SEQ ID NO: 10]
1 shows the nucleotide sequence of a primer used in Example 1.
[SEQ ID NO: 11]
1 shows the base sequence obtained in Example 1.
[SEQ ID NO: 12]
1 shows the nucleotide sequence of the DNA containing the r164-1h full-length gene obtained in Example 1.
[SEQ ID NO: 13]
1 shows the amino acid sequence of a novel rat-derived protein r164-1h.
[SEQ ID NO: 14]
1 shows the nucleotide sequence of a primer used in Example 1.
[SEQ ID NO: 15]
1 shows the nucleotide sequence of a primer used in Example 1.
[SEQ ID NO: 16]
1 shows the nucleotide sequence of the DNA containing the r164-1h full-length gene obtained in Example 1.
[SEQ ID NO: 17]
6 shows the base sequences of primers used in Examples 2 to 5.
[SEQ ID NO: 18]
3 shows the base sequence obtained in Example 2.
[SEQ ID NO: 19]
3 shows the base sequence of a primer used in Example 2.
[SEQ ID NO: 20]
3 shows the base sequence of a primer used in Example 2.
[SEQ ID NO: 21]
3 shows the base sequence obtained in Example 2.
[SEQ ID NO: 22]
3 shows the nucleotide sequence of a primer used in Example 2.
[SEQ ID NO: 23]
7 shows the base sequences of primers used in Examples 2 and 6.
[SEQ ID NO: 24]
3 shows the base sequence obtained in Example 2.
[SEQ ID NO: 25]
1 shows the amino acid sequence of human novel protein h164-1h.
[SEQ ID NO: 26]
1 shows the nucleotide sequence of DNA encoding a novel human-derived protein h164-1h.
[SEQ ID NO: 27]
1 shows the nucleotide sequence of DNA encoding a novel rat-derived protein r164-1h.
[SEQ ID NO: 28]
1 shows the amino acid sequence of a novel human-derived protein h164-1b.
[SEQ ID NO: 29]
1 shows the nucleotide sequence of a DNA encoding a novel human-derived protein h164-1b.
[SEQ ID NO: 30]
1 shows the amino acid sequence of a rat-derived novel protein r164-1b.
[SEQ ID NO: 31]
1 shows the nucleotide sequence of DNA encoding a novel rat-derived protein r164-1b.
[SEQ ID NO: 32]
14 shows the base sequence of a primer used in Example 6.
[SEQ ID NO: 33]
7 shows the base sequence obtained in Example 6.
[SEQ ID NO: 34]
14 shows the base sequence of a primer used in Example 7.
[SEQ ID NO: 35]
14 shows the base sequence of a primer used in Example 7.
[SEQ ID NO: 36]
7 shows the base sequence obtained in Example 7.
[SEQ ID NO: 37]
14 shows the base sequence of a primer used in Example 7.
[SEQ ID NO: 38]
14 shows the base sequence of a primer used in Example 7.
[SEQ ID NO: 39]
7 shows the base sequence obtained in Example 7.
[SEQ ID NO: 40]
14 shows the base sequence of a primer used in Example 7.
[SEQ ID NO: 41]
14 shows the base sequence of a primer used in Example 7.
[SEQ ID NO: 42]
7 shows the base sequence obtained in Example 7.
[SEQ ID NO: 43]
7 shows the base sequence obtained in Example 7.
[SEQ ID NO: 44]
10 shows the base sequences of primers used in Examples 8 and 9.
[0077]
The transformant Escherichia coli JM109 / pTB2268 obtained in Example 1 described below has been administered from June 12, 2002, at 1-1, Higashi 1-1, Tsukuba, Ibaraki, Japan, Chuo No. 6 (zip code 305-8566). Deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology under the accession number FERM BP-8071.
The transformant Escherichia coli JM109 / pTB2269 obtained in Example 2 described below has been administered from June 12, 2002 at 1-1, Higashi 1-1, Tsukuba, Ibaraki, Japan, Chuo No. 6 (Zip code 305-8566). Deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology under the accession number FERM BP-8072.
The transformant Escherichia coli JM109 / pTB2270 obtained in Example 6, which will be described later, was obtained from June 12, 2002, at 1-1, Higashi 1-1, Tsukuba City, Ibaraki Prefecture, Japan, Chuo No. 6 (zip code 305-8566). Deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology under the accession number FERM BP-8073.
【Example】
Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited thereto.
Example 1
Cloning of r164-1h gene
(1) Preparation of myocardial infarction model rat
According to the report of Watanabe et al. (Circulation Research, Vol. 69, pp. 370-377, 1991), male Wistar rats (11 weeks old, weighing 300-400 g) were treated with pentobarbital (50 mg / kg, ip). Anesthesia was performed and the chest was opened at the midline under artificial respiration. After incision of the pericardium, the heart was exposed. At the origin of the left anterior descending branch of the coronary artery, the coronary artery and the myocardium were tied with a silk thread using a suture needle with a thread (ELP, 5-0 silk), and the chest was closed. The sham-operated group closed the chest without tying the thread. After recovery from anesthesia, the animals were usually raised.
(2) Extraction of total RNA
Rats 1 week, 8 weeks, 20 weeks and 30 weeks after the operation were opened for chest under pentobarbital anesthesia, the heart was excised, and the coronary artery was perfused retrograde from the aorta with physiological saline to obtain blood. Was washed away. After removing the tissue other than the left ventricle from the extracted heart with scissors, after confirming infarction formation, the infarct region (scar formation site) was removed, leaving only the non-infarction region. This was finely cut with scissors, and then the total RNA was extracted using ISOGEN (manufactured by Wako Pure Chemical Industries, Ltd.).
[0078]
(3) Cloning of r164-1h gene and construction of expression vector
In order to remove genomic DNA from the total RNA, a DNA degradation operation is performed using an enzyme set for DD (manufactured by Takara Shuzo), and then a Fluorescence Differential Display kit Fluorescein version (manufactured by Dharansha) is sprayed using a sprayer (manufactured by Takara Shuzo). went. Total RNA derived from the left ventricle 8 weeks after sham operation was used as a target tissue. As a result, a band showing an increase was found in the tissues 1 week, 20 weeks, and 30 weeks after the operation compared to the target tissues. This band was cut out of the acrylamide gel with a cutter, suspended in sterilized distilled water, and heated at 95 ° C. for 10 minutes to extract a gene fragment from the gel. Next, after re-amplification by PCR, the DNA base sequence was decoded. The base sequence (SEQ ID NO: 1) revealed therefrom was subjected to homology search by BlastN using an EST database which is a public database, and AW435329 UI-R-BJ0p-afc-c-05-0-UI. s1 UI-R-BJ0p Rattus norvegicus cDNA clone UI-R-BJ0p-afc-c-05-0-UI 3 ′, mRNA sequence was found to be almost identical. Therefore, two types of primers (SEQ ID NO: 2 and SEQ ID NO: 3) were designed based on this sequence, and 5 ′ RACE PCR was performed on rat heart Marathon-Ready cDNA (CLONTECH) using these primers according to the protocol. went. As a result, two types of base sequences (SEQ ID NO: 4 and SEQ ID NO: 5) in which the sequences on the 3 ′ side were identical were obtained. Further, when the obtained nucleotide sequence represented by SEQ ID NO: 4 was subjected to homology search by BlastN using an nt database which is a public database, Homo sapiens hypothetical protein MGC14161 (MGC14161) mRNA (Genbank ID: XM_03377) And high homology. Therefore, a forward primer (SEQ ID NO: 6) was prepared based on the base sequence represented by SEQ ID NO: 4, and a reverse primer (SEQ ID NO: 7) was prepared based on the base sequence represented by SEQ ID NO: 5. PCR was performed on rat heart Marathon-Ready cDNA (CLONTECH) using the above primers to obtain a base sequence represented by SEQ ID NO: 8. Also, two kinds of primers (SEQ ID NO: 9 and SEQ ID NO: 10) were designed based on the base sequence represented by SEQ ID NO: 4, and using these primers, rat heart Marathon-Ready cDNA (CLONTECH) was used. 5 'RACE PCR was performed again according to the protocol. As a result, the nucleotide sequence of SEQ ID NO: 11 was obtained.
By combining the three nucleotide sequences of SEQ ID NO: 4, SEQ ID NO: 8 and SEQ ID NO: 11, a nucleotide sequence represented by SEQ ID NO: 12 is obtained, and this nucleotide sequence has a protein consisting of 256 amino acid residues. The nucleotide sequence represented by SEQ ID NO: 27 encoding (SEQ ID NO: 13) was included. The protein containing the amino acid sequence represented by SEQ ID NO: 13 was named r164-1h.
Two kinds of primers (SEQ ID NO: 14 and SEQ ID NO: 15) were designed based on the nucleotide sequence represented by SEQ ID NO: 12, and rat heart Marathon-Ready cDNA (CLONTECH) was designed using these primers. PCR was performed, and the obtained 1072 bp fragment having the base sequence represented by SEQ ID NO: 16 was subcloned into a plasmid vector pTARGET vector (Promega) according to the prescription of pTARGET Mammarian Expression Vector system (Promega). The obtained expression vector was named r1641honTARGE.
r164-1h (SEQ ID NO: 13) had 77% homology at the amino acid level with Homo sapiens hypothetical protein MGC14161.
A transformant having the plasmid pTB2268 containing the nucleotide sequence represented by SEQ ID NO: 16 was designated as Escherichia coli JM109 / pTB2268.
[0079]
Example 2
Cloning of h164-1h gene and construction of expression vector
Using primers designed based on the base sequence encoding r164-1h (SEQ ID NO: 17 and SEQ ID NO: 9), PCR was performed on human heart Marathon-Ready cDNA (CLONTECH), and SEQ ID NO: 18 The nucleotide sequence represented was obtained. Also, two types of primers (SEQ ID NO: 19 and SEQ ID NO: 20) were designed based on the base sequence represented by SEQ ID NO: 18, and human primers were used for human heart Marathon-Ready cDNA (CLONTECH). 5 ′ RACE PCR was performed according to the protocol. As a result, the base sequence represented by SEQ ID NO: 21 was obtained. Using a primer designed based on the base sequence of SEQ ID NO: 21 (SEQ ID NO: 22) and a primer designed based on the base sequence of MGC14161 (see Example 1) (SEQ ID NO: 23), human heart Marathon-Ready PCR was performed on the cDNA (CLONTECH) to obtain a base sequence represented by SEQ ID NO: 24. This nucleotide sequence contained the nucleotide sequence represented by SEQ ID NO: 26, which codes for a protein consisting of 249 amino acid residues (SEQ ID NO: 25). The protein containing the amino acid sequence represented by SEQ ID NO: 25 was designated as h164-1h.
The obtained fragment (SEQ ID NO: 24) is again amplified by PCR using two types of primers (SEQ ID NO: 22 and SEQ ID NO: 23), and the obtained fragment is pTARGET Mammalian Expression Vector system (Promega) Was subcloned into a plasmid vector pTARGET vector (Promega) according to the prescription. The obtained expression vector was designated as h1641honTARGE.
h164-1h (SEQ ID NO: 25) has 82% homology at the amino acid level with Homo sapiens hypothetical protein MGC14161. Further, h164-1h (SEQ ID NO: 25) and r164-1h (SEQ ID NO: 13) obtained in Example 1 had 90% homology at the amino acid level.
A transformant having the plasmid pTB2269 containing the nucleotide sequence represented by SEQ ID NO: 24 was designated as Escherichia coli JM109 / pTB2269.
[0080]
Example 3
Analysis of time course of r164-1h gene in myocardial infarction model rat
1 week, 8 weeks, 20 weeks, and 30 weeks after myocardial infarction described in Example 1 (2) Total RNA derived from the non-infarcted region of the left ventricle of the rat, and 1 week after sham operation as a subject, CDNA was synthesized from total RNA derived from the left ventricle of a rat after 8 weeks, 20 weeks, and 30 weeks using TaqMan Reverse Transcription Reagents (manufactured by PE Applied Biosystems). Next, using a primer having a nucleotide sequence represented by SEQ ID NO: 17 or SEQ ID NO: 9, which is a primer capable of specifically amplifying r164-1h, the copy number of the r164-1h gene was quantified by PCR using ABI Prism. Performed by 7900 sequence Detection System. The reaction was performed using SYBR Green PCR Master Mix (manufactured by PE Applied Biosystems), and all the methods were performed according to the attached instructions. The method for preparing the standard for quantification is described below. One week after myocardial infarction surgery Total RNA derived from the non-infarcted region of the left ventricle of the rat was synthesized with cDNA using TaqMan Reverse Transcription Reagents (manufactured by PE Applied Biosystems). Next, PCR was performed using primers represented by SEQ ID NO: 17 and SEQ ID NO: 9, which are primers capable of specifically amplifying r164-1h, and a gene fragment having a partial sequence of the obtained r164-1h gene was obtained. Was the standard. Further, the calculated copy number was used as an internal control and corrected by the copy number of glycerol triphosphate dehydratase calculated using TaqMan Rodent GAPDH Control reagents VIC (manufactured by PE Applied Biosystems) in the same manner as the copy number of the r164-1h gene. After that, the value was compared with the target tissue as the expression level, and the fluctuation rate was converted into data.
As a result, it was revealed that the expression of the r164-1h gene increased 1.3-fold, 1.3-fold, and 1.4-fold at 1, 20, and 30 weeks after the operation, respectively.
[0081]
Example 4
Analysis of tissue distribution of r164-1h gene in normal rats
Using a primer having a nucleotide sequence represented by SEQ ID NO: 17 or SEQ ID NO: 9 as a primer capable of specifically amplifying r164-1h, brain, heart, kidney, spleen, thymus, liver, stomach, small intestine, muscle PCR was performed using Marathon-Ready cDNA library (CLONTECH) derived from organs such as lung, testis and skin, and the copy number of the r164-1h gene was quantified by ABI Prism 7900 Sequence Detection System. The reaction was performed using SYBR Green PCR Master Mix (manufactured by PE Applied Biosystems), and all the methods were performed according to the attached instructions. The method for preparing the standard for quantification is described below. One week after myocardial infarction surgery Total RNA derived from the non-infarcted region of the left ventricle of the rat was synthesized with cDNA using TaqMan Reverse Transcription Reagents (manufactured by PE Applied Biosystems). Next, PCR was performed using primers represented by SEQ ID NO: 17 and SEQ ID NO: 9, which are primers capable of specifically amplifying r164-1h, and a gene fragment having a partial sequence of the obtained r164-1h gene was obtained. Was the standard. Further, the calculated copy number was used as an internal control and corrected by the copy number of glycerol triphosphate dehydratase calculated using TaqMan Rodent GAPDH Control reagents VIC (manufactured by PE Applied Biosystems) in the same manner as the copy number of the r164-1h gene. After that, the value was converted into data as the expression level.
As a result, the expression of the r164-1h gene in the heart was as high as 0.0041, and the expression in the testis was 0.0005. In other organs, the expression of the r164-1h gene was 0.0002 or less, indicating that the heart was the main expression site of the r164-1h gene.
[0082]
Example 5
Analysis of tissue distribution of h164-1h gene in human
Using primers having the nucleotide sequences represented by SEQ ID NO: 17 and SEQ ID NO: 9, organs such as brain, heart, kidney, spleen, thymus, liver, pancreas, small intestine, skeletal muscle, lung, testis and aorta PCR was performed using the derived Marathon-Ready cDNA library (CLONTECH) as a template, and the copy number of the h164-1h gene was quantified by ABI Prism 7900 Sequence Detection System by PCR. The reaction was performed using SYBR Green PCR Master Mix (manufactured by PE Applied Biosystems), and all the methods were performed according to the attached instructions. The method for preparing the standard for quantification is described below. Using human heart Marathon-Ready cDNA (CLONTECH), PCR was performed with the above primers (SEQ ID NO: 17 and SEQ ID NO: 9), and the obtained gene fragment having a partial sequence of the h164-1h gene was used as its standard. . Furthermore, after correcting the calculated copy number as an internal control with the copy number of glycerol triphosphate dehydratase calculated using TaqMan GAPDH Control reagents (manufactured by PE Applied Biosystems) in the same manner as the copy number of the h164-1h gene, , And the value was used as the expression level as data.
As a result, the expression of the h164-1h gene in the heart was as high as 0.0021, and the expression in the testis was 0.0003. In other organs, the expression of the h164-1h gene was 0.0001 or less, indicating that the heart was the main expression site of the h164-1h gene.
[0083]
Example 6
Cloning of h164-1b gene and construction of expression vector
Using primers designed based on the nucleotide sequence encoding MGC14161 (SEQ ID NO: 32 and SEQ ID NO: 23), PCR was performed on human heart Marathon-Ready cDNA (CLONTECH), and represented by SEQ ID NO: 33. Base sequence was obtained. This nucleotide sequence contained the nucleotide sequence represented by SEQ ID NO: 29, which codes for a protein consisting of 481 amino acid residues (SEQ ID NO: 28). The protein containing the amino acid sequence represented by SEQ ID NO: 28 was designated as h164-1b. h164-1b (SEQ ID NO: 28) had 74% homology at the amino acid level with Homo sapiens hypothetical protein MGC14161.
After re-amplifying the base sequence represented by SEQ ID NO: 33, the obtained fragment was subcloned into a plasmid vector pTARGET vector (Promega) according to the prescription of pTARGET Mammarian Expression Vector system (Promega). The obtained expression vector was designated as h1641bonTARGE.
A transformant having the plasmid pTB2270 containing the nucleotide sequence represented by SEQ ID NO: 33 was designated as Escherichia coli JM109 / pTB2270.
[0084]
Example 7
Determination of base sequence of r164-1b gene
Using a primer designed based on the nucleotide sequence encoding MGC14161 (SEQ ID NO: 34) and a primer designed based on the nucleotide sequence represented by SEQ ID NO: 12 (SEQ ID NO: 35), rat brain Marathon-Ready cDNA (CLONTECH) was subjected to PCR to obtain a base sequence represented by SEQ ID NO: 36. Rat brain Marathon-Ready cDNA was prepared using a primer designed based on the nucleotide sequence encoding MGC14161 (SEQ ID NO: 37) and a primer designed based on the nucleotide sequence represented by SEQ ID NO: 12 (SEQ ID NO: 38). (CLONTECH) was subjected to PCR to obtain a nucleotide sequence represented by SEQ ID NO: 39. Also, two types of primers (SEQ ID NO: 40 and SEQ ID NO: 41) were designed based on the base sequence represented by SEQ ID NO: 39, and rat brain Marathon-Ready cDNA (CLONTECH), rat Heart Marathon-Ready cDNA (CLONTECH) and rat testis Marathon-Ready cDNA (CLONTECH) were subjected to 5 ′ RACE PCR according to the protocol. As a result, the nucleotide sequence represented by SEQ ID NO: 42 was obtained. The nucleotide sequence of SEQ ID NO: 43 was obtained by combining the nucleotide sequences of SEQ ID NO: 12, SEQ ID NO: 36, SEQ ID NO: 39, and SEQ ID NO: 42. This nucleotide sequence contained the nucleotide sequence of SEQ ID NO: 31 encoding a protein consisting of 481 amino acid residues (SEQ ID NO: 30). The protein containing the amino acid sequence represented by SEQ ID NO: 30 was named r164-1b.
r164-1b (SEQ ID NO: 30) had 48% homology at the amino acid level with Homo sapiens hypothetical protein MGC14161. Further, r164-1b (SEQ ID NO: 30) and r164-1h (SEQ ID NO: 13) obtained in Example 1 had 78% homology at the amino acid level. Further, r164-1b (SEQ ID NO: 30) and h164-1b (SEQ ID NO: 28) obtained in Example 6 had 98% homology at the amino acid level.
[0085]
Example 8
Analysis of time course of r164-1b gene in myocardial infarction model rat
1 week, 8 weeks, 20 weeks, and 30 weeks after myocardial infarction described in Example 1 (2) Total RNA derived from the non-infarcted region of the left ventricle of the rat, and 1 week after sham operation as a subject, CDNA was synthesized from total RNA derived from the left ventricle of a rat after 8 weeks, 20 weeks, and 30 weeks using TaqMan Reverse Transcription Reagents (manufactured by PE Applied Biosystems). Next, as a primer for specifically amplifying r164-1b, a primer having a nucleotide sequence represented by SEQ ID NO: 44 or SEQ ID NO: 9 was used, and the copy number of the r164-1b gene was quantified by ABI Prism by PCR. Performed by 7900 sequence Detection System. The reaction was performed using SYBR Green PCR Master Mix (manufactured by PE Applied Biosystems), and all the methods were performed according to the attached instructions. The method for preparing the standard for quantification is described below. PCR was performed using Rat Brain Marathon-Ready cDNA (CLONTECH) and primers having the nucleotide sequences represented by SEQ ID NO: 44 and SEQ ID NO: 9, and the obtained r164-1b gene partial sequence was subjected to PCR. The gene fragment contained was used as the standard. Further, the calculated copy number was used as an internal control and corrected by the copy number of glycerol triphosphate dehydratase calculated using TaqMan Rodent GAPDH Control reagents VIC (manufactured by PE Applied Biosystems) in the same manner as the copy number of the r164-1b gene. After that, the value was compared with the target tissue as the expression level, and the fluctuation rate was converted into data.
As a result, the expression of the r164-1b gene may be increased 2.2, 2.2, 2.7 and 3.3 times at 1, 8, 20, and 30 weeks after the operation, respectively. It became clear.
[0086]
Example 9
Analysis of tissue distribution of r164-1b gene in normal rats
Using a primer having a nucleotide sequence represented by SEQ ID NO: 44 or SEQ ID NO: 9 as a primer for specifically amplifying r164-1b, brain, heart, kidney, spleen, thymus, liver, stomach, small intestine, muscle PCR was performed using Marathon-Ready cDNA library (CLONTECH) derived from organs such as lung, testis and skin, and the copy number of r164-1b gene was quantified by ABI Prism 7900 Sequence Detection System. The reaction was performed using SYBR Green PCR Master Mix (manufactured by PE Applied Biosystems), and all the methods were performed according to the attached instructions. The method for preparing the standard for quantification is described below. PCR is performed using Rat Brain Marathon-Ready cDNA (CLONTECH) and primers having the nucleotide sequences represented by SEQ ID NO: 44 and SEQ ID NO: 9, respectively, and has a partial sequence of the obtained r164-1b gene. The gene fragment was used as the standard. Further, the calculated copy number was used as an internal control, and similarly to the copy number of the r164-1b gene, similarly to the copy number of the r164-1b gene, the copy number of glycerol 3-phosphate dehydratase was corrected using TaqMan Rodent GAPDH Control reagents VIC (manufactured by PE Applied Biosystems). After that, the value was converted into data as the expression level.
As a result, the expression of the r164-1b gene was as high as 0.0039 in the testis, and 0.0005 in the brain. The expression in the heart was 0.00002, and in other organs, the expression of the r164-1b gene was 0.0001 or less. The testis and brain were found to be the major expression sites for the r164-1b gene.
[0087]
【The invention's effect】
The proteins and polynucleotides of the present invention may be used for heart diseases (eg, cardiomyopathy, myocardial infarction, heart failure, angina pectoris, etc.), central nervous system diseases (eg, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), genital diseases (eg, , Testicular hypersensitivity, ovarian insufficiency, infertility, etc.) and useful as diagnostic markers, etc., and compounds or salts thereof obtained by screening using the protein, polynucleotide or an antibody against the protein, or inhibiting the activity of the protein. Examples of neutralizing antibodies include heart diseases (eg, cardiomyopathy, myocardial infarction, heart failure, angina pectoris, etc.), central nervous system diseases (eg, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), genital diseases (eg, , Testicular hypersensitivity, ovarian dysfunction, infertility, etc.), or as a contraceptive .
In addition, the antisense nucleotide of the present invention can suppress the expression of the protein of the present invention. Examples thereof include heart diseases (eg, cardiomyopathy, myocardial infarction, heart failure, angina pectoris, etc.), central nervous system diseases (eg, It can be used as a prophylactic / therapeutic agent for Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc., genital diseases (eg, testicular hypersensitivity, ovarian dysfunction, infertility, etc.), or as a contraceptive. Furthermore, the polynucleotide of the present invention may be used for, for example, heart diseases (eg, cardiomyopathy, myocardial infarction, heart failure, angina pectoris, etc.), central nervous diseases (eg, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), genital diseases (eg, , Testicular hypersensitivity, ovarian insufficiency, infertility, etc.).
[0088]
[Sequence list]

Claims (38)

A protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 13 or SEQ ID NO: 30, or a salt thereof. The protein according to claim 1, which comprises the amino acid sequence represented by SEQ ID NO: 25, or a salt thereof. 2. The protein according to claim 1, which comprises the amino acid sequence represented by SEQ ID NO: 28, or a salt thereof. The protein according to claim 1, which comprises the amino acid sequence represented by SEQ ID NO: 13, or a salt thereof. The protein according to claim 1, which comprises the amino acid sequence represented by SEQ ID NO: 30, or a salt thereof. A partial peptide of the protein according to claim 1 or a salt thereof. A polynucleotide comprising a polynucleotide encoding the protein according to claim 1 or the partial peptide according to claim 6. The polynucleotide according to claim 7, which is DNA. The polynucleotide according to claim 8, comprising the nucleotide sequence represented by SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 27 or SEQ ID NO: 31. A recombinant vector containing the polynucleotide according to claim 7. A transformant transformed with the recombinant vector according to claim 10. The protein according to claim 1, wherein the transformant according to claim 11 is cultured to produce and accumulate the protein according to claim 1 or the partial peptide according to claim 6, and the collected protein is collected. 7. The method for producing the partial peptide or a salt thereof according to 6. A medicament comprising the protein according to claim 1 or a partial peptide thereof or a salt thereof. A medicament comprising the polynucleotide according to claim 7. A diagnostic agent comprising the polynucleotide according to claim 7. An antibody against the protein according to claim 1, the partial peptide according to claim 6, or a salt thereof. A diagnostic agent comprising the antibody according to claim 16. A medicament comprising the antibody according to claim 16. An antisense nucleotide comprising a nucleotide sequence complementary or substantially complementary to the nucleotide sequence of the polynucleotide according to claim 7, or a part thereof. A medicament comprising the antisense nucleotide according to claim 19. A compound that regulates the activity of the protein according to claim 1, the partial peptide according to claim 6, or a salt thereof, or a compound thereof, wherein the protein according to claim 1 or the partial peptide according to claim 6 or a salt thereof is used. Salt screening method. A compound which regulates the activity of the protein of claim 1, the partial peptide of claim 6, or a salt thereof, which comprises the protein of claim 1, the partial peptide of claim 6, or a salt thereof, or A kit for screening the salt. A compound or a salt thereof, which can be obtained using the screening method according to claim 21 or the screening kit according to claim 22, which regulates the activity of the protein according to claim 1, the partial peptide according to claim 6, or a salt thereof. A medicament comprising the compound according to claim 23 or a salt thereof. A method for screening a compound or a salt thereof that regulates the expression of the protein gene according to claim 1, wherein the polynucleotide according to claim 7 is used. A kit for screening a compound or a salt thereof that regulates the expression of the protein gene according to claim 1, comprising the polynucleotide according to claim 7. A compound or its salt that regulates the expression of the protein gene according to claim 1, which can be obtained by using the screening method according to claim 25 or the screening kit according to claim 26. A medicament comprising the compound according to claim 27 or a salt thereof. A method for quantifying a protein according to claim 1, wherein the antibody according to claim 16 is used. A method for diagnosing a disease associated with the function of the protein according to claim 1, wherein the method according to claim 29 is used. A method for screening a compound or a salt thereof, which regulates the expression of the protein according to claim 1, wherein the antibody according to claim 16 is used. A kit for screening a compound or a salt thereof that regulates expression of the protein according to claim 1, comprising the antibody according to claim 16. A compound or its salt that regulates the expression of the protein according to claim 1, which is obtained by using the screening method according to claim 31 or the screening kit according to claim 32. A medicament comprising the compound according to claim 33 or a salt thereof. The medicament according to claim 13, 14, 18, 20, 20, 24, 28 or 34, which is a prophylactic / therapeutic agent for heart disease. 35. The medicament according to claim 24, 28 or 34, wherein the compound is a compound that suppresses excessive compensatory mechanism or compensatory failure. 37. The medicament according to claim 35 or 36, which is an agent for preventing or treating cardiomyopathy, myocardial infarction, heart failure or angina. The diagnostic agent according to claim 15 or 17, which is a diagnostic agent for heart disease.