JPWO2003018793A1 - A novel gene with a guanine nucleotide exchange factor-like sequence - Google Patents

Abstract

The present invention relates to a novel polypeptide having a human and mouse guanine nucleotide exchange factor-like (SecL-1) sequence, a gene encoding the same, a recombinant polypeptide using a DNA sequence encoding the polypeptide, and production thereof. Method, antibody specifically recognizing the same, method for producing the same, cell growth inhibitor, apoptosis inducer and therapeutic agent for liver disease containing the same, and screening of compounds that specifically regulate the activity of the protein using the same Provide methods and so on.

Description

アンカープライマーとしては５’ＲＡＣＥ Ｓｙｓｔｅｍに付属の試薬、５’ＲＡＣＥ Ａｂｒｉｄｇｅｄ Ａｎｃｈｏｒ Ｐｒｉｍｅｒ（ＡＡＰ）およびＡｂｒｉｄｇｅｄ Ｕｎｉｖｅｒｓａｌ Ａｍｐｌｉｆｉｃａｔｉｏｎ Ｐｒｉｍｅｒ（ＡＵＡＰ）を用いた。
Ｂａｌｂ／ｃマウス肝臓から自体公知の方法で取得した全ＲＮＡ１μｇに蒸留水を１４．０μｌ、２．５μＭのＳＦ２８プライマー（配列番号１９）を１μｌ添加し、７０℃で１０分間インキュベートし、５０℃に保温した溶液Ａを調製した。また、１０×ＰＣＲ Ｂｕｆｆｅｒ（２００ｍＭ Ｔｒｉｓ−ＨＣｌ、ｐＨ８．４；５００ｍＭ ＫＣｌを含む）を２．５μｌ、２５ｍＭ ＭｇＣｌ_２を２．５μｌ、１０ｍＭ ｄＮＴＰ ｍｉｘを１μｌ、０．１Ｍ ＤＴＴを２．５μｌそれぞれ混合し、５０℃で保温した溶液Ｂを調節した。溶液Ａと溶液ＢとＳｕｐｅｒ Ｓｃｒｉｐｔ^ＴＭＩＩ Ｒｅｖｅｒｓｅ Ｔｒａｎｓｃｒｉｐｔａｓｅ（２００ｕｎｉｔｓ／μｌ）を１μｌ混合し、４２℃で５０分間、７０℃で１５分間インキュベートした後、氷上に５分間静地した。ＲＮａｓｅ ｍｉｘ（ＲＮａｓｅ Ｈ＋ＲＮＡｓｅ Ｔ１）を１μｌ添加し、３７℃で３０分間インキュベートした。
上記で得られたｃＤＮＡはキット付属のＤＮＡ Ｐｕｒｉｆｉｃａｔｉｏｎ Ｓｙｓｔｅｍ（インビトロジェン社製）で精製を行った。上記反応液にｂｉｎｄｉｎｇ ｓｏｌｕｔｉｏｎ（６Ｍ ＮａＩ）を２２５μｌ添加し、ＧｌａｓｓＭＡＸ ＤＮＡ ｉｓｏｌａｔｉｏｎ ｓｐｉｎ ｃａｒｔｒｉｄｇｅｓにかけ、５０μｌの蒸留水で溶出し、精製ｃＤＮＡを得た。１０μｌの精製ｃＤＮＡに蒸留水を４μｌ、５×ｔａｉｌｉｎｇ ｂｕｆｆｅｒ（５０ｍＭ Ｔｒｉｓ−ＨＣｌ、ｐＨ８．４；１２５ｍＭ ＫＣｌ、７．５ｍＭ ＭｇＣｌ_２を含む）を５μｌ、１ｍＭ ｄＣＴＰを５μｌそれぞれ添加し、９４℃で２分間処理し、氷上に１分間放置した。Ｔｅｒｍｉｎａｌ ｄｅｏｘｙｎｕｃｌｅｏｔｉｄｙｌ ｔｒａｎｓｆｅｒａｓｅ（ｒＴｄＴ）を１μｌ添加し、３７℃で１０分間、６５℃で１０分間インキュベートした後、氷上に５分間放置し、５’ＲＡＣＥ用のｃＤＮＡを得た。
ＰＣＲ（Ｐｏｌｙｍｅｒａｓｅ Ｃｈａｉｎ Ｒｅａｃｔｉｏｎ）法はＴａＫａＲａ ＬＡ Ｔａｑ^ＴＭ（宝酒造社製）を用いて行った。上記で得られた５’ＲＡＣＥ用ｃＤＮＡ２．５μｌに、１０×ＰＣＲ Ｂｕｆｆｅｒを２．５μｌ、２．５ｍＭ ｄＮＴＰ ｍｉｘを４μｌ、１０μＭ ＡＡＰプライマーを１μｌ、１０μＭ プライマーＳＦ２９（配列番号２０）を１μｌ、ＴａＫａＲａ ＬＡ Ｔａｑ^ＴＭ（５Ｕ／μｌ）を０．２５μｌ、蒸留水を１３．７５μｌずつ混合した溶液を、９４℃で２分間を１回、９４℃で１分間、５５℃で１分間、７２℃で２分間のサイクルを３０回、７２℃で２分間を１回で１回目のＰＣＲを行った。１回目のＰＣＲ反応液を５０倍希釈した溶液２．５μｌに、１０×ＰＣＲ Ｂｕｆｆｅｒを２．５μｌ、２．５ｍＭ ｄＮＴＰ ｍｉｘを４μｌ、１０μＭ ＡＵＡＰプライマーを１μｌ、１０μＭ ＳＦ３０プライマーを１μｌ、ＴａＫａＲａ ＬＡ Ｔａｑ^ＴＭ（５Ｕ／μｌ）を０．２５μｌ、蒸留水を１３．７５μｌ混合した溶液を、９４℃で２分間を１回、９４℃で１分間、５５℃で１分間、７２℃で２分間のサイクルを２０回、７２℃で２分間を１回で２回目のＰＣＲを行った。２回目のＰＣＲ反応液を１．２％アガロースゲルで電気泳動し、約９００ｂｐのＤＮＡ断片を回収し、ＱＩＡＥＸ−ＩＩ Ｇｅｌ Ｅｘｔｒａｃｔｉｏｎ Ｓｙｓｔｅｍで精製した。精製したＤＮＡをプラスミドベクターｐＣＲ２（インビトロジェン社製）に挿入し、前述の方法で塩基配列の決定を行った。その結果、配列番号１１で表される８９１塩基からなる塩基配列（ＳＦ２１Ｅ）を得た。
得られた各種ＳＦ２１塩基配列の配置図を第１図に示しす。それを基にマウスＳＦ２１の全長ｃＤＮＡの塩基配列を決定した結果、マウスＳＦ２１は配列番号３で表される全長５１２４塩基および配列番号４で表される１７０８アミノ酸残基からなるタンパク質であることが明らかになった。
実施例２
ヒトｈＳＦ２１ｃＤＮＡの単離
マウスＳＦ２１とホモロジーを示すヒト遺伝子のクローニングを試みた。マウスＳＦ２１がコードするアミノ酸残基Ｐｒｏ−Ａｌａ−Ｖａｌ−Ａｒｇ−Ｌｙｓ−Ｓｅｒ−Ａｌａ−Ｇｌｙ−Ｇｌｎ−Ｔｈｒ（配列番号４：第１０３５番〜第１０４４番）およびＡｓｐ−Ｔｈｒ−Ａｌａ−Ｇｌｕ−Ｌｙｓ−Ｇｌｎ−Ｔｒｐ−Ａｌａ−Ｇｌｕ−Ｔｈｒ（配列番号４：第１１０２番〜第１１１１番）を基にＰＣＲ用のプライマーとして縮重オリゴヌクレオチド２種類を合成した。

ヒト肝臓ｃＤＮＡライブラリーＨｕｍａｎ Ｌｉｖｅｒ ５’−ＳＴＲＥＣＨ ＰＬＵＳ ｃＤＮＡ（クロンテック社製）を鋳型に、２種類のプライマーＳＦ−Ｄ３およびＳＦ−Ｄ４を用いてＰＣＲを行った。
まず、上記ヒト肝臓ｃＤＮＡライブラリー（２×１０^７ＰＦＵ／μｌ）０．５μｌにＴａＫａＲａ Ｅｘ Ｔａｑ^ＴＭ（宝酒造社製）１．２５ユニットにＳＦ−Ｄ３およびＳＦ−Ｄ４のプライマーを各５０ｐｍｏｌ、１０×Ｅｘ Ｔａｑ Ｂｕｆｆｅｒ（Ｍｇ^２＋ｐｌｕｓ）を５μｌおよびｄＮＴＰ Ｍｉｘｔｕｒｅ（各２．５ｍ）を最終濃度２００μＭに成るよう添加し、蒸留水を全量が５０μｌに成るよう添加しＰＣＲ用試料を調製した。９４℃で４分間を１回、９４℃で３０秒間、５５℃で３０秒間、７２℃で３０秒間のサイクルを３５回、７２℃で３分間を１回で、ＤＮＡサーマルサイクラーとして、Ｏｍｎｉ Ｇｅｎｅ（サーモハイベイド社）を用いてＰＣＲを行った。増幅された約２５０塩基のＤＮＡ断片をｐＣＲ２ベクター（インビトロジェン社製）にクローニングし、前述の方法と同様にして塩基配列の決定を行った。その結果、配列番号１２で表される２３０塩基から成る塩基配列（ｈＳＦ２１Ａ）を有するプラスミドｐｈＳＦ２１Ａを単離した。得られたｃＤＮＡはマウスＳＦ２１の相当領域と塩基配列で９５％、アミノ酸配列で１００％の相同性を有していた。このことから単離したｃＤＮＡはマウスＳＦ２１のヒトに相当する遺伝子の一部と考えられた。
ヒトｈＳＦ２１全長ｃＤＮＡを得るために、ｈＳＦ２１ＡをプローブとしてＨｕｍａｎ Ｌｉｖｅｒ ５’−ＳＴＲＥＣＨ ＰＬＵＳ ｃＤＮＡをプラークハイブリダイゼーションによりスクリーニングした。プラークハイブリダイゼーションはマウスＳＦ２１全長ｃＤＮＡの取得した時の方法に準じて行った。この方法により配列番号１３で表される１１６６塩基からなる塩基配列（ｈＳＦ２１Ｃ）を有するクローンｐｈＳＦ２１Ｃ、配列番号１４で表される２８２５塩基からなる塩基配列（ｈＳＦ２１Ｅ）を有するクローンｐｈＳＦ２１Ｅおよび配列番号１５で表される３１３９塩基からなる塩基配列（ｈＳＦ２１Ｆ）を有するクローンｐｈＳＦ２１Ｆの３種のクローンを得た。得られたヒトｈＳＦ２１ＡからＦの塩基配列を基に解析を行った結果、５５１７塩基（配列番号１：第２５０番目〜第５７６７番目）からなるｃＤＮＡを得た。
マウス同様にヒトにおいても翻訳領域の５’末端周辺の塩基配列を決定するためにＲＡＣＥ法で５’末端配列を単離した。操作は前述の方法に従って行った。
５’ＲＡＣＥにはＨｕｍａｎ Ｌｉｖｅｒ ５’−ＳＴＲＥＣＨ ＰＬＵＳ ｃＤＮＡを鋳型とし、プライマーとして上記で得られた５５１７塩基からなるヒトｃＤＮＡ配列を基に１種類を合成した。

また、クローニングベクターλｇｔ１１配列に特異的なプライマーとしてＧＴ１１−ＬＤＦを合成した。

ヒト肝臓ｃＤＮＡライブラリーＨｕｍａｎ Ｌｉｖｅｒ ５’−ＳＴＲＥＣＨ ＰＬＵＳ ｃＤＮＡを蒸留水で１００倍に希釈した溶液０．５μｌに、ＫｌｅｎＴａｑ ＬＡ Ｐｏｌｙｍｅｒａｓｅ Ｍｉｘ（クロンテック社製）を１．２５ユニット、上記各プライマーを５０ｐｍｏｌ、１０×ＰＣＲ Ｂｕｆｆｅｒ（２００ｍＭ Ｔｒｉｓ−ＨＣｌ、ｐＨ８．３；２０ｍＭ ＭｇＳＯ_４、１００ｍＭ （ＮＨ_４）_２ＳＯ_４、１００ｍＭ ＫＣｌ、１％ ＴｒｉｔｏＸ−１００、１ｍｇ／ｍｌ ＢＳＡを含む）を５μｌおよびｄＮＴＰｓを最終濃度が２００μＭになるように添加し、全量が５０μｌになるよう蒸留水を添加しＰＣＲ用の試料を調製した。試料は９４℃で４分間を１回、９４℃で３０秒間、６８℃で３分間の反応サイクルを２５回、６８℃で３分間を１回でＤＮＡサーマルサイクラーＯｍｎｉ Ｇｅｎｅを用いてＰＣＲを行った。ＰＣＲ反応液を、１．２％アガロースゲルで電気泳動し、約５５０ｂｐのＤＮＡ断片を回収し、精製した。精製したＤＮＡ断片をｐＣＲ２ベクターに挿入し、ＤＮＡシークエンサー３７３Ｓを用い、ダイターミネーター法で塩基配列を決定した。その結果、配列番号１６で表される４３３塩基からなる塩基配列（ｈＳＦ２１Ｇ）を得た。
得られた各種ｈＳＦ２１塩基配列の位置を第１図に示す。それを基にヒトｈＳＦ２１の全長ｃＤＮＡの塩基配列を決定した結果、ヒトｈＳＦ２１はそれぞれ配列番号１で表される全長５８８３塩基からなる塩基配列および配列番号２で表される１７１７アミノ酸残基からなるタンパク質であることが明らかになった。単離したヒトの塩基配列はマウスの塩基配列と８２％の相同性を示すことから、得られたヒト遺伝子はマウス遺伝子に相当するものであると考えられた。
ヒト全長ｈＳＦ２１ｃＤＮＡの推定アミノ酸配列について公開データベースＧｅｎＢａｎｋをＢＬＡＳＴ検索したところ、ショウジョウバエ（Ｄｒｏｓｏｐｈｉｌａ ｍｅｌａｎｏｇａｓｔｅｒ）機能未知遺伝子ＣＧ８６８３（１６６９残基）と４３％、センチュウ（Ｃａｅｎｏｒｈａｂｄｉｔｉｓ ｅｌｅｇａｎｓ）機能未知遺伝子Ｆ１１Ａ１０．４（１６４６残基）と２９％、分裂酵母（Ｓｃｈｚｏｓａｃｃｈａｒｏｍｙｃｅｓ ｐｏｍｂｅ）機能未知遺伝子Ｃ２３Ｄ３．１３Ｃ（１６１６残基）と１７％のホモロジーをそれぞれ示した。また、ヒトｈＳＦ２１のアミノ酸残基第２１３目〜第３９７目および第６３９目〜第９３１目の領域がそれぞれＳｅｃ７／ＢＩＧ１ファミリーに属するヒトｂｒｅｆｅｌｄｉｎ Ａ阻害グアニンヌクレオチド交換因子１タンパク質（ＢＩＧ１）のアミノ酸残基第４２１目〜第５９１目および第９８９目〜第１３０６目と２３％のホモロジーを示した。また、ヒトｈＳＦ２１のアミノ酸残基第１７８目〜第３９２目および第７４６目〜第９１９目の領域がそれぞれ酵母（Ｓｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ）のＳｅｃ７ｐのアミノ酸残基第４７０目〜第６７１目および第１２７０目〜第１４４３目と２２％および１９％のホモロジーを示した。しかし、ヒトｈＳＦ２１はＢＩＧ１およびＳｅｃ７ｐの基質であるＡＲＦに対する触媒ドメインであるＳｅｃ７ドメインに相当する領域のホモロジーは低く、ＡＲＦに対するＧＥＦ活性に必要とされているＳｅｃ７ドメインに保存されたアミノ酸配列が認められないことから、Ｓｅｃ７／ＢＩＧ１ファミリーと異なる新規タンパク質をコードする遺伝子と考えられた。よって、ヒトｈＳＦ２１タンパク質をコードする遺伝子は上記のような特徴を持つことから、Ｓｅｃ７ Ｌｉｋｅ−１（ＳｅｃＬ−１）遺伝子と命名した。
実施例３
ヒトの各種組織におけるＳｅｃＬ−１の発現
ヒトの各種組織（脳、心臓、骨格筋、大腸、胸腺、脾臓、腎臓、肝臓、小腸、胎盤、肺、白血球）におけるＳｅｃＬ−１遺伝子発現の有無を調べた。
各種ヒト組織由来のｍＲＮＡとしてＨｕｍａｎ Ｍｕｌｔｉｐｌｅ Ｔｉｓｓｕｅ Ｎｏｒｔｈｅｒｎ（ＭＴＮ^ＴＭ） Ｂｌｏｔ（クロンテック社製）を、また、プローブとしてヒトＳｅｃＬ−１ｃＤＮＡを制限酵素ＳｐｈＩおよびＳｐｅＩで消化することで得られる、配列番号１記載の第４５０３番目〜第５２０７番目で示される７０５ｂｐの塩基配列を前述と同様の方法で放射性同位体［α−^３２Ｐ］ｄＣＴＰ（アマシャムファルマシアバイオテク社製）標識したものを用いてノーザンブロット解析を行った。
ＥｘｐｒｅｓｓＨｙｂ^ＴＭ Ｈｙｂｒｉｄｉｚａｔｉｏｎ Ｓｏｌｕｔｉｏｎ（クロンテック社製）１０ｍｌに上記で作製したプローブ５μｌ添加し、ハイブリダイゼーション溶液を調製した。その様にして調製したハイブリダイゼーション溶液中でＭＴＮ Ｂｌｏｔを、６８℃で１６時間インキュベーションし、ハイブリダイゼーションを行った。そのメンブランを２×ＳＳＣ（０．１％ ＳＤＳを含む）溶液で、室温にて１５分間を４回、０．１×ＳＳＣ（０．１％ ＳＤＳを含む）溶液で、５０℃にて２０分間を２回行って洗浄した後、−７０℃で５日間オートラジオグラフィーを行った。その結果、全ての組織でＳｅｃＬ−１の発現が見とめられ、特に脳、心臓、腎臓、肝臓、胎盤では強いシグナルが認められた。ｍＲＮＡのバンドのサイズが、単離されたｃＤＮＡより長いことから、ＳｅｃＬ−１のｍＲＮＡは長い非翻訳部分を持つと考えられた。
実施例４
ヒトＳｅｃＬ−１発現ベクターの構築
ヒトＳｅｃＬ−１遺伝子の機能解析を行うために、哺乳動物細胞での発現ベクターを構築した。ヒトＳｅｃＬ−１全長ＤＮＡ配列を基に６種類のプライマーを合成した。なお、プライマーの５’末端側にＨＳＦ５３、ＨＳＦ６２およびＨＳＦ６６はＸｈｏＩが認識する配列を、ＨＳＦ６３はＢｓｔＸＩが認識する配列を、ＨＳＦ６７はＡｐａＩが認識する配列を、ＨＳＦ６９はＫｐｎＩが認識する配列を付加した。

ＨＥＫ２９３細胞より調製したｍＲＮＡを鋳型にし、６種類のプライマーをＨＳＦ５３およびＨＳＦ６３、ＨＳＦ６２およびＨＳＦ６７、ＨＳＦ６６およびＨＳＦ６９で組合わせ、ヒトＳｅｃＬ−１が３分割されるようＰＣＲ法を行った。
まず、ＨＥＫ２９３細胞からの全ＲＮＡ調製にはＲＮｅａｓｙ Ｍｉｎｉ Ｋｉｔ（キアゲン社製）を用い、添付のマニュアルにしたがって全ＲＮＡを調製した。調製した全ＲＮＡからｃＤＮＡへの逆転写反応にはＳｕｐｅｒＳｃｒｉｐｔＩＩ ＲｎａｓｅＨ−Ｒｅｖｅｒｓｅ Ｔｒａｎｓｃｒｉｐｔａｓｅ（インビトロジェン社製）を用い、添付のマニュアルにしたがってｃＤＮＡを調製した。調製したｃＤＮＡ０．５μｌにＰｆｕ Ｔｕｒｂｏ ＤＮＡ Ｐｏｌｙｍｅｒａｓｅ（ストラタジーン社製）１．２５ユニットにプライマーを上記の組合わせで各５０ｐｍｏｌ、１０×ＰＣＲ Ｂｕｆｆｅｒ（２００ｍＭ Ｔｒｉｓ−ＨＣｌ、ｐＨ８．７５；２０ｍＭ ＭｇＳＯ_４、１００ｍＭ （ＮＨ_４）_２ＳＯ_４、１００ｍＭ ＫＣｌ、１％ ＴｒｉｔｏｎＸ−１００、１ｍｇ／ｍｌ ＢＳＡを含む）を５μｌおよびｄＮＴＰｓを最終濃度２００μＭに成るよう添加し、全量が５０μｌに成るよう蒸留水を添加し試料を調製した。試料は９９℃で２分間を１回、９４℃で３０秒間、６５℃で３０秒間、７５℃で３分間のサイクルを３５回、７２℃で７分間を１回で、ＤＮＡサーマルサイクラーＯｍｎｉ Ｇｅｎｅを用いてＰＣＲを行った。
ＰＣＲ反応液を１．２％アガロースゲルで電気泳動し、目的のＤＮＡ断片を回収し、ＧＦＸ ＰＣＲ ＤＮＡ ａｎｄ Ｇｅｌ Ｂａｎｄ Ｐｕｒｉｆｉｃａｔｉｏｎ Ｋｉｔ（アマシャムファルマシアバイオテク社製）を用いて精製した。これらの各種ＤＮＡ断片をプラスミドベクターｐＣＲ−Ｂｌｕｎｔ（インビトロジェン社製）に挿入し、プラスミドｐＣＲ−Ｂｌｕｎｔ／ＨＳＦ６６−６９、ｐＣＲ−Ｂｌｕｎｔ／ＨＳＦ６２−６７およびｐＣＲ−Ｂｌｕｎｔ／ＨＳＦ５３−６３を構築し、各ＤＮＡ断片の塩基配列の決定をおこなった。
次に、上記で得られたプラスミド３種類を制限酵素で消化し、各ＤＮＡ断片を哺乳類細胞発現用ベクターに組込み、全長ヒトＳｅｃＬ−１ｃＤＮＡを再構築し、発現ベクターを構築した。
プラスミドｐＣＲ−Ｂｌｕｎｔ／ＨＳＦ６６−６９を制限酵素ＸｈｏＩおよびＫｐｎＩで消化後、１．２％アガロースゲルによる電気泳動で分画し、目的の断片を回収した。回収したＤＮＡ断片はＧＦＸ ＰＣＲ ＤＮＡ ａｎｄ Ｇｅｌ Ｂａｎｄ Ｐｕｒｉｆｉｃａｔｉｏｎ Ｋｉｔ（アマシャムファルマシアバイオテク社製）で添付のマニュアルに従って操作して精製した。ｐＢＫ−ＣＭＶベクター（ストラタジーン社製）も同様の制限酵素で消化し、ＨＳＦ６６−ＨＳＦ６９断片を組込み、プラスミドｐＫＢ−ＣＭＶ／ＨＳＦ６６−６９を得た。同様にして、ｐＣＲ−Ｂｌｕｎｔ／ＨＳＦ６２−６７を制限酵素ＸｈｏＩおよびＡｐａＩで消化後、１．２％アガロースゲルによる電気泳動で分画し、目的の断片を回収・精製した。ｐＫＢ−ＣＭＶ／ＨＳＦ６６−６９を同様の制限酵素で消化し、ＨＳＦ６２−６７断片を組込み、プラスミドｐＫＢ−ＣＭＶ／ＨＳＦ６６−６９・ＨＳＦ６２−６７を得た。次いで、ｐＣＲ−Ｂｌｕｎｔ／ＨＳＦ５３−６３を制限酵素ＸｈｏＩおよびＢｓｔＸＩで消化後、１．２％アガロースゲルによる電気泳動で分画し、目的の断片を回収・精製した。ｐＫＢ−ＣＭＶ／ＨＳＦ６６−６９・ＨＳＦ６２−６７を同様の制限酵素で消化し、ＨＳＦ５３−６３断片を組込み、発現プラスミドｐＫＢ−ＣＭＶ／ＨＳＦ６６−６９・ＨＳＦ６２−６７・ＨＳＦ５３−６３（ｐＫＢ−ＣＭＶ／ｈＳｅｃＬ−１）を得た。
ヒト胎児腎臓由来細胞株２９３細胞（ＡＴＣＣ：ＣＲＬ−１５７３）をバイオコート ポリＤリシン（Ｐｏｌｙ−Ｄ−Ｌｙｓｉｎｅ：ｓｙｎｔｈｅｔｉｃ）６ウェルマイクロテストプレート（ベクトンディッキンソン社製）に５×１０^５個／ウェルで分注し、Ｄ−ＭＥＭ（ｈｉｇｈ ｇｌｕｃｏｓｅ）培地（インビトロジェン社製）（１０％ ＦＢＳ（インビトロジェン社製）を含む）を適量添加し、５％ ＣＯ_２中、３７℃で一晩培養を行った。２μｇのｈＳｅｃＬ−１発現ベクターｐＢＫ−ＣＭＶ／ｈＳｅｃＬ−１をＯｐｔｉ−ＭＥＭ Ｒｅｄｕｃｅｄ−Ｓｅｒｕｍ Ｍｅｄｉｕｍ（１×）溶液（インビトロジェン社製）で最終容量１００μｌに成るよう調製し、ＳｕｐｅｒＦｅｃｔ Ｔｒａｎｓｆｅｃｔｉｏｎ Ｒｅａｇｅｎｔ（キアゲン社製）を１０μｌ添加し良く混和した後、室温で５〜１０分間インキュベーションしＳｕｐｅｒＦｅｃｔ−ＤＮＡ複合体を形成した。同様の操作で発現ベクターｐＢＫ−ＣＭＶを細胞に導入したものを対照として用いた。これらの各ベクターにＤ−ＭＥＭ（１０％ ＦＢＳを含む）を６００μｌ添加し、ピペッティングで良く混和した後、上記で培養した２９３細胞培養液から培養液を吸引除去し、ＰＢＳ溶液で１回洗浄した細胞に添加した。この細胞培養液は５％ ＣＯ_２中、３７℃で３時間培養した後、培養液を吸引除去し、ＰＢＳ溶液で１回洗浄した。このようにして形質転換した細胞にＤ−ＭＥＭ培地（１０％ ＦＢＳを含む）を２ｍｌ添加し、５％ ＣＯ_２中、３７℃で４８時間培養することでｈＳｅｃＬ−１発現組換え細胞を得た。
実施例５
ｈＳｅｃＬ−１抗体の作製
抗原として配列番号５で表されるヒトＳｅｃＬ−１のアミノ末端１４６残基からなるポリペプチドを用いた。抗原ペプチドはＧＳＴ Ｇｅｎｅ Ｆｕｓｉｏｎ Ｓｙｓｔｅｍ（アマシャムファルマシアバイオテク社製）を用い、添付のマニュアルにしたがって操作し、ＧＳＴ融合タンパク質を作製し、次いでＧＳＴ除去組換えタンパク質を作製した。
まず、上記で得られた発現プラスミドｐＢＫ−ＣＭＶ／ｈＳｅｃＬ−１を鋳型とし、配列番号６記載の塩基配列を基に２種類のＰＣＲ用プライマーを合成した。ＨＳＦＳ１は配列番号６記載の第１番目から第２６番目の塩基配列に５’末端側にＥｃｏＲＩが認識する配列を、ＨＳＦＳ２は第４１３番目から第４３８番目の塩基配列に３’末端側にストップコドンおよびＸｈｏＩが認識する配列を付加した。

０．５μｌのｐＢＫ−ＣＭＶ／ｈＳｅｃＬ−１にＰｆｕＴｕｒｂｏ ＤＮＡ Ｐｏｌｙｍｅｒａｓｅ（ストラタジーン社製）１．２５ユニット、プライマーＨＳＦＳ１およびＨＳＦＳ２を各５０ｐｍｏｌ、１０×ＰＣＲ Ｂｕｆｆｅｒ（２００ｍＭ Ｔｒｉｓ−ＨＣｌ、ｐＨ８．３；２０ｍＭ ＭｇＳＯ_４、１００ｍＭ （ＮＨ_４）_２ＳＯ_４、１００ｍＭ ＫＣｌ、１％ ＴｒｉｔｏｎＸ−１００、１ｍｇ／ｍｌ ＢＳＡを含む）を５μｌおよびｄＮＴＰｓを最終濃度２００μＭに成るよう添加し、全量が５０μｌに成るよう蒸留水を添加しＰＣＲ用試料を調製した。試料は９９℃で２分間を１回、９４℃で３０秒間、６０℃で３０秒間、７２℃で９０秒間のサイクルを３５回、７２℃で７分間を１回でサーマルサイクラーＯｍｎｉ Ｇｅｎｅを用いてＰＣＲを行った。ＰＣＲ反応溶液を１．２％アガロースによる電気泳動にて分画後、４５２ｂｐのＤＮＡ断片を回収した。回収したＤＮＡ断片はＧＦＸ ＰＣＲ ＤＮＡ ａｎｄ Ｇｅｌ Ｂａｎｄ Ｐｕｒｉｆｉｃａｔｉｏｎ Ｋｉｔを用いて精製後、制限酵素ＥｃｏＲＩとＸｈｏＩで消化した。
キット付属のＧＳＴ融合タンパク質発現ベクターｐＧＥＸ−６Ｐ−１を制限酵素ＥｃｏＲＩおよびＸｈｏＩで消化し、上記で得られたＤＮＡ断片を挿入し、融合タンパク質発現プラスミドｐＧＥＸ−ＨＳＦＳを得た。得られた発現プラスミドｐＧＥＸ−ＨＳＦＳを大腸菌ＢＬ２１（ＤＥ３）株に導入し、形質転換体を得た。この形質転換体を１００ｍｌの２×ＹＴ培地に添加し、３７℃で１８時間の前培養を行った。前培養液２５ｍｌを２．５Ｌの２×ＹＴ培地に加え、ＯＤ値が０．８になるまで培養を行い、その後、培養液にイソプロピル−１−チオ−β−Ｄ−ガラクトピラノシド（シグマアルドリッチ社製）を最終濃度が０．１５ｍＭに成るよう添加し、３０分間培養することでタンパク質の発現誘導を行った。培養液を８，０００回転で遠心分離して採集した菌体にＳＴＥ溶液（１０ｍＭ Ｔｒｉｓ−ＨＣｌ、ｐＨ８．０；１５０ｍＭ ＮａＣｌ、１ｍＭ ＥＤＴＡ・２Ｎａを含む）を１００ｍｌ添加して懸濁し、Ｌｙｓｏｚｙｍｅ（シグマアルドリッチ社製）の１０ｍｇ／ｍｌ溶液を１ｍｌ加え、氷中に３０分間放置した。これに１０％サルコシル（シグマアルドリッチ社製）を含むＳＴＥ溶液１５ｍｌを添加した後、菌体を超音波処理したものを９，０００回転で遠心分離することで融合タンパク質を含む上清を得た。この上清から融合タンパク質をアフィニティー精製するためにＧｌｕｔａｔｈｉｏｎ Ｓｅｐｈａｒｏｓｅ ４Ｂゲル（アマシャムファルマシアバイオテク社製）を１０ｍｌ加えて３０分間ゆっくりと混和し、融合タンパク質を担体に吸着させた。この担体をカラムに充填し、ＳＴＥ溶液２０ｍｌで３回洗浄した後、溶出バッファー（７５ｍＭ ＨＥＰＥＳ、ｐＨ７．４；１５０ｍＭ ＮａＣｌ、１０ｍＭ 還元型ｇｌｕｔａｔｈｉｏｎｅ、５ｍＭ ｄｉｔｈｉｏｔｈｒｅｉｔｏｌ、２％ Ｎ−ｏｃｔｙｌ ｇｌｕｃｏｓｉｄｅを含む）でＧＳＴ−ｈＳｅｃＬ−１融合タンパク質を溶出した。溶出した融合タンパク質はＰＢＳ溶液中で一晩透析し、ＰＢＳ置換を行った後、試料にＰｒｅＳｃｉｓｓｉｏｎ Ｐｒｏｔｅａｓｅ溶液（アマシャムファルマシアバイオテク社製）を２５０μｌ加え４℃で１時間処理した。この試料をＧｌｕｔａｔｈｉｏｎｅ Ｓｅｐｈａｒｏｓｅ ４Ｂカラム（アマシャムファルマシアバイオテク社製）に添加し、ＧＳＴを担体に吸着させることでＧＳＴを除去した。このようにして得られたｈＳｅｃＬ−１部分ペプチドはＰＢＳ溶液中で一晩透析し、ＰＢＳ置換を行った。培養液１５Ｌから約４ｍｇのｈＳｅｃＬ−１部分ペプチドが得られた。
ｈＳｅｃＬ−１部分ペプチド１００μｇをフロインドの完全アジュバント（インビトロジェン社製）１ｍｌと十分混和し、ニュージーランドホワイトウサギに注射した。この操作を数週間に数回行ったウサギより血液を採取し、ｈＳｅｃＬ−１部分ペプチドに対する抗体を含む血清を得た。得られた抗血清と西洋ワサビペルオキシダーゼ標識抗ウサギＩｇＧ抗体（アマシャムファルマシアバイオテク社製）を用いてＴｏｗｂｉｎら（Ｐｒｏｃｅｅｄｉｎｇ ｏｆ ｔｈｅ Ｎａｔｉｏｎａｌ Ａｃａｄｅｍｙ ｏｆ Ｓｃｉｅｎｃｅｓ、ＵＳＡ，７６，４３５０−４３５４（１９７９）．）の方法と同様にしてウェスタンブロット分析を行った。
まず、上記で得られたｈＳｅｃＬ−１発現組換え細胞を培養液から回収し、Ｅｘｒａｃｔｉｏｎ Ｂｕｆｆｅｒ（２０ｍＭ Ｔｒｉｓ−ＨＣｌ、ｐＨ７．５；０．２５Ｍ ｓｕｃｒｏｓｅ、１ｍＭ ＥＤＴＡ・２Ｎａ、３ｍＭ ＭｇＣｌ_２、１０ｍＭ ２−ｍｅｒｃａｐｔｏｅｔｈａｎｏｌ、ｐｒｏｔｅａｓｅ ｉｎｈｉｂｉｔｏｒ ｍｉｘｔｕｒｅ（カルビオケム社製）を含む）を０．１ｍｌ添加し、細胞抽出液を調製した。その様にして調製した細胞抽出液をＳＤＳ−ポリアクリルアミドゲル（３−８％）で電気泳動により分画後、ゲルからＰＶＤＦメンブラン（ミリポア社製）に蛋白質を電気泳動法で転写し、そのメンブランをＰＢＳ（５％ スキムミルク、０．０５％ Ｔｗｅｅｎ２０を含む）でブロッキングした。次に、上記で得られた抗血清をＰＢＳ（５％ スキムミルク、０．０５％ Ｔｗｅｅｎ２０を含む）で４０００倍希釈した抗体溶液中にブロッキング後のメンブランを浸し、室温で１時間インキュベートした。その後、ＰＢＳ（０．０５％ Ｔｗｅｅｎ２０を含む）で１５分間、２回洗浄し、西洋ワサビペルオキシダーゼ標識抗ウサギＩｇＧ抗体（アマシャムファルマシアバイオテク社製）をＰＢＳで４０００倍希釈した標識抗体溶液に洗浄後のメンブランを浸し、室温で１時間インキュベートした。その後、ＰＢＳ（０．０５％ Ｔｗｅｅｎ２０を含む）で１５分間、２回洗浄し、洗浄後のメンブランはＥＣＬ（Ｅｎｈａｎｃｅｄ ｃｈｅｍｉｌｕｍｉｎｅｓｃｅｎｓｅ）法で発光させたシグナルをエックス線フィルムに感光させ、抗体と結合する蛋白質を検出した。その結果、ｈＳｅｃＬ−１発現ベクターを導入した細胞から調製した試料はヒトＳｅｃＬ−１から予測される１９０ｋＤａのバンドが検出され、得られた抗血清が組換えヒトＳｅｃＬ−１タンパク質と特異的に反応を示したことが判明した。
実施例６
ヒトＳｅｃＬ−１の細胞増殖およびアポトーシスに及ぼす影響
上記同様にヒト胎児腎臓由来細胞株２９３細胞（ＡＴＣＣ：ＣＲＬ−１５７３）をｐＢＫ−ＣＭＶ／ｈＳｅｃＬ−１で形質転換したヒトＳｅｃＬ−１発現組換え細胞および対照としてｐＢＫ−ＣＭＶを導入した細胞に各ウェル２ｍｌのＤ−ＭＥＭ（１０％ ＦＢＳを含む）培地を添加し、５％ ＣＯ_２中、３７℃で培養を行った。培養後１日目および５日目の細胞数をＣｅｌｌＴｉｔｅｒ ９６ Ａ Ｑｕｅｏｕｓ Ｏｎｅ Ｓｏｌｕｔｉｏｎ Ｃｅｌｌ Ｐｒｏｌｉｆｅｒａｔｉｏｎ Ａｓｓａｙ（プロメガ社製）で添付のマニュアルにしたがって実施した。この結果、ヒトＳｅｃＬ−１高発現細胞株は対照細胞株に比べて細胞増殖の低下が認められた（第２図）。
この細胞増殖抑制へのアポトーシスの関与を検討した。Ｃａｓｐａｓｅ−３がアポトーシス誘導時に特異的に活性化されることが知られている（Ｃｅｌｌ，８８，３５５−３６５（１９９７）．）。そこで、ｐＢＫ−ＣＭＶ／ｈＳｅｃＬ−１導入細胞およびｐＢＫ−ＣＭＶ導入細胞のＣａｓｐａｓｅ−３活性を測定した。Ｃａｓｐａｓｅ−３の活性測定はＣａｓｐＡＣＥ^ＴＭ Ａｓｓａｙ Ｓｙｓｔｅｍ Ｃｏｌｏｒｉｍｅｔｒｉｃ（プロメガ社製）を用い、添付のマニュアルにしたがって実施した。その結果、ヒトＳｅｃＬ−１高発現細胞では対照細胞に比べ約３倍のＣａｓｐａｓｅ−３活性上昇が認められた（第３図）。
次に、ヒトＳｅｃＬ−１高発現細胞でアポトーシスを誘導した際に内在性のＳｅｃＬ−１遺伝子の発現上昇が認められるが検討した。過酸化水素水はアポトーシス誘導を刺激することが知られている（ＦＥＢＳ Ｌｅｔｔｅｒ，４８８，１２３−１３２（２００１）．）。そこで、過酸化水素水を細胞培養液中に添加し、ｈＳｅｃＬ−１遺伝子発現量としてそのｍＲＮＡをＲＴ−ＰＣＲ法で経時的に定量することにより検討した。ＰＣＲ用のプライマーとしてＨＳＦ２８およびＨＳＦ２９の２種類を合成した。

まず、ヒトＳｅｃＬ−１発現細胞培養液に０．１μＭ、０．３μＭ、０．６μＭおよび０．９μＭの４段階の濃度で調製した過酸化水素水を添加し、５％ ＣＯ_２中、３７℃で２４時間培養した後、生細胞数およびヒトＳｅｃＬ−１のｍＲＮＡ量を測定した。対照として過酸化水素水を添加していない細胞を同様の条件で培養した。各細胞からの全ＲＮＡ調製にはＲＮｅａｓｙ Ｍｉｎｉ Ｋｉｔ（キアゲン社製）を用い、添付のマニュアルにしたがって調製した。調製した全ＲＮＡからｃＤＮＡへの逆転写反応にはＳｕｐｅｒＳｃｒｉｐｔＩＩ ＲｎａｓｅＨ− Ｒｅｖｅｒｓｅ Ｔｒａｎｓｃｒｉｐｔａｓｅ（インビトロジェン社製）を用い、添付のマニュアルにしたがってｃＤＮＡを調製した。調製したｃＤＮＡ０．５μｌにＰｆｕＴｕｒｂｏ ＤＮＡ Ｐｏｌｙｍｅｒａｓｅ（ストラタジーン社製）１．２５ユニット、１０μＭの各プライマーを２μｌ、１０×ＰＣＲ Ｂｕｆｆｅｒを５μｌ、２．５ｍＭのｄＮＴＰｓを４μｌ、蒸留水５０μｌを混合し、９９℃で２分間を１回、９４℃で３０秒間、６０℃で３０秒間、７２℃で３０秒間のサイクルを３５回、７２℃で５分間を１回で、ＤＮＡサーマルサイクラーＯｍｎｉ Ｇｅｎｅを用いてＰＣＲを行った。ＰＣＲ反応液を１．２％アガロースゲルで電気泳動し、約４８０ｂｐのバンドとして確認した。その結果、対照区と比較して、過酸化水素添加した細胞は濃度に反比例して生細胞数の減少が認められた（第４図）。また、ヒトＳｅｃＬ−１のｍＲＮＡ量は過酸化水素を処理することで著しい増加が認められた。
以上の結果から、ヒトＳｅｃＬ−１はアポトーシス誘導を伴う細胞増殖抑制に関与していると考えられた。
産業上の利用可能性
本発明のタンパク質、その部分ペプチドまたはそれらの塩、およびそれらをコードする塩基配列は新規生理機能の探索、抗体の作製、組換え体発現系の構築、同発現系を用いた生理活性調節物質アッセイ系および医薬品候補化合物のスクリーニング系の開発、診断に有用な試薬作成のための試薬、予防および治療薬などの医薬品の開発等に用いることができる。
【配列表】

【図面の簡単な説明】
図１ ヒトおよびマウスからクローニングされた各種ＳｅｃＬ−１ｃＤＮＡ断片の配置を示している。
図２ ヒト胎児腎臓由来細胞株２９３細胞にヒトＳｅｃＬ−１ｃＤＮＡを挿入した発現ベクターｐＢＫ−ＣＭＶ／ｈＳｅｃＬ−１を導入し形質転換したヒトＳｅｃＬ−１発現細胞株（□）およびベクターｐＢＫ−ＣＭＶを導入した対照細胞株（●）における細胞増殖率の培養時間（日）における経時的変化を示している。
図３ ヒト胎児腎臓由来細胞株２９３細胞にヒトＳｅｃＬ−１ｃＤＮＡを挿入した発現ベクターｐＢＫ−ＣＭＶ／ｈＳｅｃＬ−１を導入し形質転換したヒトＳｅｃＬ−１発現細胞株（■）およびベクターｐＢＫ−ＣＭＶを導入した対照細胞株（□）における各細胞株のＣａｓｐａｓｅ−３活性を示している。
図４ ヒト胎児腎臓由来細胞株２９３細胞にヒトＳｅｃＬ−１ｃＤＮＡを挿入した発現ベクターｐＢＫ−ＣＭＶ／ｈＳｅｃＬ−１を導入し形質転換したヒトＳｅｃＬ−１発現細胞株における細胞増殖への各種濃度での過酸化水素の影響を示している。Technical field
The present invention relates to a polypeptide having the human or mouse guanine nucleotide exchange factor-like sequence of SEQ ID NO: 1; a polynucleotide encoding the polypeptide of the present invention; an expression vector containing the polynucleotide of the present invention; A transformant having the expression vector of the present invention; a production method using the transformant of the present invention; an antibody that specifically recognizes the polypeptide of the present invention; a therapeutic agent for a disease containing the polypeptide of the present invention; A method for screening a compound using a polypeptide; a compound obtained by the screening method of the present invention and the like.
Background art
A guanine nucleotide exchange factor is a generic name of proteins that exchange GDP (Guanosine 5′-diphosphate) bound to a GTP-binding protein with GTP (Guanosine 5′-triphosphate) to promote an activation reaction of the GTP-binding protein. . A GTP-binding protein that is a substrate of a guanine nucleotide exchange factor specifically binds to GTP and GDP, and is a generic term for a protein having an enzymatic activity of hydrolyzing bound GTP to GDP. , (2) G protein, (3) low molecular weight GTP binding protein, and (4) tubulin, a component of microtubules. For example, G proteins are coupled to G protein-coupled receptors, and many of these receptors transduce intracellular signals through activation of GTP-binding proteins. G protein-coupled receptors are present in various cells and organs in living organisms and play a physiologically important role as receptors for hormones and bioactive substances that regulate their functions. Signals are transmitted into cells via the cells, and the signals are transmitted to regulate cell functions. G protein-coupled receptors are present on cell membranes and are involved when hormones, neurotransmitters and the like bind to transmit their information into cells. Low molecular weight GTP proteins are oncogene products such as Ras and Rho proteins, and are involved in cell growth / differentiation and intracellular transport / secretion of proteins. In various related signal transduction pathways, it functions as a molecular switch and plays an important role as a signal transduction factor for controlling cell functions such as cytoskeleton formation, vesicle transport, and cell proliferation. Guanine nucleotide exchange factors are involved in regulating the activity of GTP-binding proteins, and therefore play an important role in regulating the functions of cells and organs of various living organisms. For example, guanine nucleotide exchange factors include hormones, neurotransmitters, and physiologically active substances. It plays a very important role in regulating signal transduction into cells, regulating cell function by cell proliferation / differentiation, intracellular vesicle transport, and protein transport to the nucleus. Target.
Sos (Son of Sevenless) and Trio have been reported as guanine nucleotide exchange factors using Ras and Rho as low molecular weight GTP proteins as substrates. In addition, Sec7 was identified from a mutant showing an abnormality in vesicle transport in yeast, and was subsequently shown to be a guanine nucleotide exchange factor using ARF (ADP-ribosylation factor) belonging to a low molecular weight GTP protein as a substrate. Various guanine nucleotide exchange factors having homology to Sec7, such as BIG1, BIG2, ARNO, cytohesin 1 and GRP1, have been identified in mammals including humans, and these have also been reported to be involved in vesicle transport. ing. Recently, mammalian Sec7-like guanine nucleotide exchange factors have been classified into subgroups according to their BFA (Brefeldin A) sensitivity, molecular size, and domain organization (Trends Cell Biology, 10 (2), 60-67 (2000)). .). However, many guanine nucleotide exchange factors have not yet been reported.
Recently, various genomic analyzes have rapidly progressed, and the decoding of all nucleotide sequences of humans, nematodes (Caenorhabditis elegans), and Drosophila (Drosophila melanogaster) has been completed, and many predicted proteins of unknown functions have been reported in the database. . Among them, there are those which show homology to the Sec7 family and are obviously different from the known Sec7 family proteins. For example, in GeneBank, F11A10.4 (accession number: CAA92830) is registered as nematode sequence information, and in NCBI, CG8863 (accession number: AE003620) is registered as drosophila sequence information. No function has been reported for proteins.
It has been identified from various organisms as a guanine nucleotide exchange factor, and has been reported to be involved in signal transduction into cells. In addition, since oncogene products and the like are a family of these exchange factors, they not only play important roles in vivo, but can also be target molecules for disease treatment. However, not all substances that regulate such physiological activities have been found, and a new family may exist. Finding such a novel substance and cloning the gene can be a useful tool in drug development. New physiological activity modulators can be used in developing therapeutic / prophylactic and diagnostic agents for diseases associated with them. In addition, if a new family is found, new target diseases and functions will be elucidated, and it may be applicable to search for the development of unprecedented therapeutic / preventive drugs and diagnostics.
Disclosure of the invention
The present invention relates to a novel polypeptide having a guanine nucleotide exchange factor-like sequence, a partial peptide of the polypeptide, a polynucleotide encoding the polypeptide or the partial peptide, a recombinant vector containing the polynucleotide, and a recombinant vector. Retained transformant, method for producing the polypeptide or partial peptide, method for screening for a compound that specifically regulates the activity of the polypeptide, compound obtained by the screening, drug containing the compound, and polypeptide Alternatively, an antibody against a partial peptide thereof is provided.
The present inventors have conducted intensive studies and found that yeast guanine nucleotides based on a cDNA library created by subtraction cloning of a gene highly expressed in the liver cells of FLS mice, which are spontaneous fatty liver model mice. A novel gene encoding a novel mouse polypeptide having a Sec7-like sequence that is an exchange factor was found. Further, based on the polynucleotide sequence, a novel gene encoding a novel human polypeptide corresponding to a novel mouse gene was found from a human liver cDNA library. As a result of further study, it was confirmed that the polypeptide of the present invention is involved in cell growth suppression and apoptosis induction. The present inventors have further studied based on these findings, and have completed the present invention.
That is, the present invention
(1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 or a salt thereof;
(2) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4 or a salt thereof;
(3) In the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, one or several amino acids have an amino acid sequence having at least one mutation selected from deletion, substitution or addition, and have cell growth inhibitory activity or apoptosis induction An active polypeptide or a salt thereof;
(4) a partial peptide of the polypeptide according to any one of (1) to (3) or a salt thereof;
(5) the partial peptide or the salt thereof according to the above (4), which comprises the amino acid sequence shown in SEQ ID NO: 5;
(6) a polynucleotide having a nucleotide sequence encoding the polypeptide or partial peptide according to any of (1) to (5);
(7) the polynucleotide according to (6), which has the 117th to 5267th positions of the nucleotide sequence shown in SEQ ID NO: 1;
(8) the polynucleotide according to (6), which has the 66th position to the 5189th position of the nucleotide sequence shown in SEQ ID NO: 3;
(9) the polynucleotide according to (6), which has the nucleotide sequence shown in SEQ ID NO: 6;
(10) a hub hybridizing with a polynucleotide consisting of the 117th to 5267th nucleotide sequence of the nucleotide sequence shown in SEQ ID NO: 1 or the 66th to 5189th nucleotide sequence of the nucleotide sequence shown in SEQ ID NO: 3 under stringent conditions; And a polynucleotide encoding a polypeptide having cytostatic activity or apoptosis-inducing activity;
(11) an expression vector having the polynucleotide according to any one of (6) to (10);
(12) a transformant obtained by introducing the expression vector according to (11) into a host;
(13) the transformant according to (12), wherein the host is an animal cell strain or an E. coli strain;
(14) Any of the above (1) to (5), including a step of culturing the transformant according to (12) or (13) and a step of recovering the produced recombinant polypeptide from a culture medium. A method for producing a polypeptide or partial peptide according to
(15) an antibody that specifically recognizes the polypeptide or partial peptide according to any of (1) to (5);
(16) a cell growth inhibitor comprising the polypeptide or partial peptide according to any of (1) to (5);
(17) an apoptosis-inducing agent containing the polypeptide or partial peptide according to any of (1) to (5);
(18) A therapeutic agent for liver disease containing the polypeptide or partial peptide according to any of (1) to (5);
(19) A compound that specifically regulates the activity of the polypeptide according to any of (1) to (5), using the polypeptide or partial peptide according to any of (1) to (5). Screening method;
(20) a compound obtained by the screening method according to (19),
About.
The polypeptide of the present invention includes a polypeptide containing the amino acid sequence represented by SEQ ID NO: 2 or a salt thereof, and a polypeptide containing the amino acid sequence represented by SEQ ID NO: 4 or a salt thereof. Salts include physiologically acceptable salts with acids or bases, preferably physiologically acceptable acid addition salts. As such a salt, for example, a salt with an inorganic acid such as hydrochloric acid, phosphoric acid, and sulfuric acid, or a salt with an organic acid such as acetic acid, formic acid, citric acid, and oxalic acid is used.
The polypeptide of the present invention includes a polypeptide having a mutation selected from one or several deletions, substitutions or additions in the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4, or a salt thereof. . Such a polypeptide has a cytostatic activity or an apoptosis-inducing activity. The one or several mutations to be deleted, substituted or added are mutations of the number of amino acids as much as possible by site-directed mutagenesis or the like, and are 50 or less, preferably 30 or less, more preferably 10 or less. Means the amino acid. Further, the polypeptide of the present invention may be a polypeptide having a cell growth inhibitory activity or an apoptosis-inducing activity even by deletion, substitution or addition.
The partial peptide of the polypeptide of the present invention includes, among the amino acid sequences represented by SEQ ID NO: 2 or SEQ ID NO: 4, a polypeptide composed of a part of those sequences or a salt thereof. Such a polypeptide may not have cytostatic activity or apoptosis-inducing activity. Further, those partial polypeptides may be polypeptides that can be used for preparing an antibody against the polypeptide of the present invention. In addition, even if the polypeptide itself does not have antigenicity, any peptide can be used to produce an antibody by binding to other proteins, for example, bovine serum albumin, etc. Good. Such a peptide is a polypeptide consisting of 6 or more amino acids, preferably 20 or more, more preferably 50 or more amino acids, for example, a polypeptide having the amino acid sequence represented by SEQ ID NO: 5.
The polynucleotide of the present invention includes a DNA encoding the polypeptide of the present invention. Examples of the polynucleotide of the present invention include polynucleotides containing the nucleotide sequences represented by SEQ ID NOS: 1, 3, and 6. In addition, the polynucleotide of the present invention may include a polynucleotide that hybridizes with the polynucleotide of the present invention under stringent conditions. Preferably, the nucleotide may encode a polypeptide having cytostatic or apoptosis-inducing activity.
As used herein, the term "polynucleotide that hybridizes under stringent conditions" refers to a polynucleotide selected from the polynucleotides of the present invention or a part thereof as a probe using a Southern hybridization method, a colony hybridization method, or a plaque. It is a polynucleotide that can be obtained by using various hybridization assay methods such as a hybridization method. Specifically, using the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3 or a part thereof as a probe, 6 × SSC (Saline sodium citrate: 150 mM NaCl, 15 mM C ₆ H ₅ Na ₃ O ₇ ) The conditions are such that a hybridization reaction is carried out at 42 ° C. in a solution and then washed at 68 ° C. in a 0.1 × SSC solution. Hybridization, Molecular Cloning: A Lboratory Manual Second Edition (Cold Spring Harber Laboratory press, New York (1989).) And DNA Cloning1: Core Techniques, A Practical Approach, Second Edition (. Oxford University Press (1995)), such as It can be performed according to the method described in the experiment book.
The "expression vector" of the present invention includes, for example, a polynucleotide encoding the protein of the present invention, ATG as a translation initiation codon at the 5 'end, and a translation stop codon TAA, TGA or 3' at the 3 'end. It may have a TAG and can be constructed by ligating to the 5 'end of a promoter in an expression vector. Vectors include plasmids such as pBR322, pUC19, pSV. SPORT1, pBK-CMV, pGEX-6P-1 and the like can be used. As a promoter for controlling transcription, for example, a trp promoter, a lac promoter, a T7 promoter, an SV40 promoter and the like can be used. In addition, an expression vector containing an enhancer, a splicing signal, a selectable marker, a replication origin, a DNA sequence encoding an additional protein, and the like can also be used.
The “transformant” of the present invention is prepared by selecting a host such as an expression vector and a cell suitable for the expression vector, and transforming the host with the expression vector. Examples of the host to be transformed include multicellular eukaryotes including prokaryotes such as Escherichia coli, unicellular eukaryotes such as yeast, animal cells such as hamsters, plant cells such as tobacco, and insect cells such as armyworm. And the like. Preferably, Escherichia coli, for example, Escherichia coli BL21 (DE3) strain, or animal cells, for example, human embryonic kidney-derived cells 293 cells, monkey cells COS-7, and Chinese hamster cells CHO are used.
The "method for producing a polypeptide, a partial peptide thereof or a salt thereof" of the present invention refers to a prokaryotic cell, a yeast, an animal cell, a plant cell, an insect cell using an expression vector incorporating a polynucleotide encoding the polypeptide of the present invention. A transformant obtained by transforming a host such as the above is cultured by a culture method suitable for the host to produce the polypeptide of the present invention, and the polypeptide of the present invention is recovered from the medium or the transformant. Or a method for producing a partial peptide.
The “antibody” of the present invention is an immunoglobulin capable of reacting with the polypeptide or partial peptide of the present invention, and also includes fragments, derivatives, conjugates, and modified forms thereof. Preferably, it is an antibody that recognizes the polypeptide of the present invention, and more preferably, it is an antibody that specifically recognizes the polypeptide of the present invention. Such antibodies can be either monoclonal or polyclonal.
The "cell growth inhibitor" of the present invention can be used for prevention or treatment by suppressing cell growth of a disease involving cell growth, since the polypeptide of the present invention has an effect of suppressing cell growth. You can do it. The “apoptosis-inducing agent” of the present invention is used for a disease which can be prevented or treated by inducing apoptosis, since the polypeptide of the present invention has an apoptosis-inducing action. Such a prophylactic or therapeutic agent preferably contains the polypeptide of the present invention and can be used for the prevention or treatment of a specific disease, for example, cancer.
Further, the "agent for treating liver disease" of the present invention can be used for the prevention or treatment of a disease associated with liver function since the polypeptide of the present invention is highly expressed in the liver. Such a prophylactic or therapeutic agent preferably contains the polypeptide of the present invention and can be used for the prevention or treatment of a specific disease, for example, liver cancer.
The “compound that specifically regulates the activity” of the present invention is a compound having an action of activating or inhibiting the function of the polypeptide of the present invention in a living body, and includes, for example, a low-molecular compound, a polynucleotide, and a polypeptide. And the like. As such a compound, an antibody against the polypeptide of the present invention, a polynucleotide having a sequence complementary to the polynucleotide sequence represented by SEQ ID NOS: 1, 3, or 6 can be used. Also, such compounds may be novel or known compounds.
The “screening method” of the present invention is a method for selecting a compound that specifically regulates the activity of the polynucleotide of the present invention, and examples thereof include a bioassay, a binding assay, and an immunoassay.
BEST MODE FOR CARRYING OUT THE INVENTION
The preparation of the polynucleotide of the present invention, the preparation of the polypeptide of the present invention and its partial peptide, the expression vector, the transformant, the production method, the antibody, and the method of screening for a specific activity modulator will be described below.
In the present invention, unless otherwise specified, a gene recombination technique known in the art, Escherichia coli, a method for producing a recombinant protein including separation and purification, and a method for producing an antibody are employed.
From the liver tissue, a cDNA library is prepared by a conventional method well known and used in the art.
Examples of a method for preparing a cDNA library include a method described in Molecular Cloning: A Laboratory Manual Second Edition (Cold Spring Harbor Laboratory Press, New York (1989)), and a commercially available kit, such as, for example, a cDNA kit such as, for example, a cDNA kit, a kit such as, for example, a cDNA kit, a kit such as, for example, a cDNA kit, a kit such as ct-cion, and so on. (Manufactured by Clontech).
The DNA fragment of the present invention is inserted into a vector. Examples of the vector include pCR2, pCR-Blunt (manufactured by Invitrogen), pBK-CMV (manufactured by Stratagene) and the like. Using the vector thus obtained, Escherichia coli JM109 strain is transformed to prepare a cDNA library. A clone containing the target DNA is selected from the prepared cDNA library by the following method.
A plasmid containing each DNA fragment of the present invention is selected by purifying the plasmid of each clone from the cDNA library prepared above and analyzing the nucleotide sequence. Examples of the nucleotide sequence analysis method include the dideoxy method of Sanger et al. (Proceeding of the National Academy of Sciences USA, 74, 5463 (1977)) or a commercially available device such as a DNA sequencer 373S (manufactured by Applied Biosystems). And the method used.
Using the DNA fragment obtained above as a probe, a cDNA library is screened by plaque hybridization, and all mRNAs are screened by a Rapid Amplification of cDNA Ends (RACE) method in order to obtain nucleotides in the 5'-terminal translated region. To obtain the polynucleotide of the present invention.
The DNA fragment obtained above is used as a probe, labeled with a radioisotope or the like, and a cDNA library is screened. A cDNA library prepared as described above, or a commercially available cDNA library, for example, Mouse River 5′-STRETCH PLUS cDNA, Human River 5′-STRETCH PLUS cDNA (manufactured by Clontech), Human River 5′-STRETCH PLUS cDNA A DNA fragment containing the nucleotide of the present invention can be obtained by performing plaque hybridization using (Clontech), Human River 5'-STRETCH PLUS cDNA (Clontech) and the like. In addition, total mRNA is prepared by a conventional method which is well known and used in the art. As a method for preparing total mRNA, for example, a method described in Molecular Cloning: A Laboratory Manual Second Edition (Cold Spring Harbor Laboratory Press, New York (1989)) or the like, or a commercially available kit, such as, for example, a tik kit, a kipEk tik, or a commercially available kit. And the like. The RACE method is described in Frohman, M .; A. And the like (Proceeding of the National Academy of Sciences, USA, 85, 8998 (1998)) or a commercially available kit, for example, a method using 5'RACE System, version 2.0 (manufactured by Invitrogen). . Alternatively, commercially available total mRNA, for example, PolyA + RNA Mouse Liver (manufactured by Amersham Pharmacia Biotech) or the like can also be used.
The DNA fragment obtained by the above method is treated with a restriction enzyme or the like, if necessary, and then the method is described in Molecular Cloning: A Laboratory Manual Second Edition (Cold Spring Harbor Laboratory Press, New York (1989)). The polynucleotide of the present invention can be obtained by incorporation and analyzing the base sequence by the above-described base sequence analysis method.
Examples of the polynucleotide obtained by the method of the present invention include a polynucleotide encoding the polypeptide represented by SEQ ID NO: 2 or SEQ ID NO: 4, and specifically, represented by SEQ ID NOs: 1 and 3. And a polynucleotide having a base sequence of
The polynucleotide obtained as described above is integrated into an expression vector to construct an expression plasmid. The protein of the present invention or a salt thereof can be obtained by a genetic engineering technique of culturing a product obtained by introducing the obtained expression vector into a suitable host cell and recovering the resultant from the culture solution. Further, those which are separated and purified from human or mammalian cells or tissues by a commonly used and well-known method in the art may be used. Further, it may be obtained according to a chemical peptide synthesis method. It is possible to examine whether the thus-obtained protein of the present invention has a cell growth inhibitory activity and an apoptosis-inducing activity by using the cell concentration or caspase activity as an index.
As the expression vector of the present invention, any vector can be used as long as it contains the DNA of the present invention and can be expressed in animal cells. A DNA encoding the polypeptide of the present invention may have a 5′-terminal ATG may be provided as a translation initiation codon and a translation termination codon TAA, TAG or TGA may be provided at the 3 'end, and can be constructed by ligating to the 5' end of a promoter in an expression vector. Such vectors include plasmids such as pBR322, pUC19, pSV. SPORT1, pBK-CMV, pGEX-6P-1 and the like can be used. As a promoter for controlling transcription, for example, a trp promoter, a lac promoter, a T7 promoter, an SV40 promoter and the like can be used. In addition, an expression vector containing an enhancer, a splicing signal, a selectable marker, a replication origin, a DNA sequence encoding an additional protein, and the like can also be used. As the additional protein, for example, glutathione S-transferase (GST), thioredoxin, or the like is used. The DNA sequence encoding the target protein is linked to the 3 ′ end of the DNA sequence encoding the additional protein to express a fusion protein. A vector can be prepared. The fusion protein expression vector may contain a DNA sequence encoding an amino acid sequence cleavable by a protease between the additional protein and the target protein. Examples of the protease include thrombin protease, factor Xa, and BLase. Is used. It may also contain a cloning DNA sequence for efficiently inserting the DNA sequence encoding the target protein into the fusion protein expression vector. Examples of the cloning DNA sequence include those which can be cleaved with a restriction enzyme. It is a DNA sequence, preferably a DNA sequence cleaved with restriction enzymes BamHI, EcoRI, SamI, SalI, XhoI, NotI, and the like.
A transformant can be prepared by selecting an expression vector of the present invention and a host suitable for the expression vector. Examples of the host to be transformed include multicellular eukaryotes such as prokaryotes such as Escherichia coli, unicellular eukaryotes such as yeast, insect cells such as armyworm, and animal cells such as hamster. Preferably, Escherichia coli, for example, Escherichia coli BL21 (DE3) strain, and animal cells, for example, human embryonic kidney-derived cells 293 cells, monkey cells COS-7, Chinese hamster cells CHO, and the like can be mentioned. Preferably, 293 cells can be used.
Methods for transforming host cells can be found in many textbooks and literature, including, for example, the aforementioned Molecular Cloning: A Laboratory Manual Second Edition (Cold Spring Harbor Laboratory Press, New York (1989)). . When transforming Escherichia coli, for example, Proceeding of the National Academy of the Sciences of the USA, 69, 2110 (1972). And the like. When animal cells are transformed, for example, electroporation (Cytotechnology, 3,133 (1990).), Calcium phosphate method (Japanese Unexamined Patent Publication No. 2-227075), lipofection method (Proceeding of the National Academy of Sciences USA, 84, 7413 (1987), Virology, Vol. 52, 456 (1973)).
The transformant of the present invention is cultured by a conventional method commonly used in the art. The culture can be performed using a medium suitable for the transformed host cell, and a liquid medium is suitable as the medium used for the culture. Specifically, a MEM medium (Science, 122, 501 (1952).), A D-MEM medium (Virology, 8, 396 (1959).), And an RPMI1640 medium (The Journal of the American Medical Association, 199, 519). 1967).), YT medium, BEM medium and the like. As a medium for culturing a transformant whose host is Escherichia coli, for example, a YT medium containing bactotryptone, yeast extract and sodium chloride is preferable. As a medium for culturing a transformant whose animal host is an animal cell, for example, a medium obtained by adding an appropriate amount of fetal calf serum (FCS) to a MEM medium, a D-MEM medium, an RPMI medium, or the like is used. If necessary, in order to increase the transcription activity of the promoter of the expression vector, a substance which promotes the transcription activity may be contained. For example, isopropyl-1-thio-β-D-galactopyranosine (IPTG) may be used. it can.
The medium contains nutrients necessary for the transformant to grow, for example, glucose, amino acids, peptone, vitamins, hormones, serum, preferably fetal calf serum, calcium chloride, magnesium chloride, and the like. A medium having any composition can be used as long as it is such a medium, and a commercially available medium can also be used. Cultivation is performed under conditions such as pH 6.0 to 8.0, 25 to 40 ° C., and the presence of 5% CO 2.
The recombinant protein produced from the transformant of the present invention is recovered from the culture medium by a conventional method commonly used in the art.
The culture solution obtained by culturing the transformant as described above is, for example, separated into cells or cells and a medium by a method such as centrifugation, and when the recombinant protein is present in the cells, the cells or cells are separated. After being collected and suspended in an appropriate buffer such as an STE solution, the cells or cells can be crushed with lysozyme, ultrasonic waves or the like, and recovered by centrifugation or filtration. The buffer used for the separation / purification may contain an appropriate amount of a surfactant, for example, sodium lauryl sulfate (SDS) or N-lauroyl sarcosine sodium (sarkosyl). The method of purifying / separating the target protein contained in the obtained crudely purified product can be performed by combining various known / separated methods. As these known methods, for example, various chromatographic methods such as salting out, dialysis, ultrafiltration, gel filtration, and ion exchange chromatography, and various electrophoresis such as SDS-polyacrylamide gel electrophoresis are used. Can be If the target protein is expressed as a fusion protein, the target protein can be purified by separating the fusion protein using an antibody specific to the additional protein and cleaving the additional protein with a protease or the like.
Deletion, substitution or addition of the amino acid sequence of the polypeptide of the present invention can be performed, for example, by site-specific mutation described in Molecular Cloning: A Laboratory Manual Second Edition (Cold Spring Harbor Laboratory Press, New York (1989)). Mutation is introduced into the DNA sequence of the present invention using a method such as PCR or PCR, and using an appropriate vector and host, for example, Molecular Cloning: A Laboratory Manual Second Edition (Cold Spring Harbor Laboratory, New York, 1989) )) By genetically engineering the DNA into which the mutation has been introduced. It is possible.
Using the protein of the present invention, a partial peptide thereof or a polypeptide containing the partial peptide, etc. as an antigen, it can be prepared according to a known method for producing an antibody or antiserum. Antibodies can be prepared by, for example, administering the protein of the present invention, its partial peptide or a polypeptide containing the partial peptide to a mammal using the antigen as an antigen. The antigen may be the antigen itself, or may be one bound to a carrier such as bovine serum albumin (BSA) or the like. Further, in order to enhance an immune response by the antigen, for example, Freund's complete adjuvant (CFA) and incomplete adjuvant (IFA) may be administered. As mammals used for immunization, mice, rats, rabbits, goats and the like can be used.
The polyclonal antibody can be produced, for example, according to the method of Lane et al. (Antibodies; A Laboratory Manual (Cold Spring Harbor Laboratory Press, New York (1989)).) After the first administration of the antigen to the mammal, 1 The immunized mammals can be obtained by administering 3 to 10 times every 2 weeks to obtain immunized mammals, collecting serum from those mammals, and purifying the immunized mammals by various kinds of chromatography, centrifugation, and salting out. , Dialysis and the like.
Monoclonal antibodies are obtained by obtaining spleen or lymph nodes from those mammals after sufficient antibody titers are obtained as described above, and fusing antibody-producing cells obtained therefrom with myeloma (myeloma) cells. A hybridoma producing a monoclonal antibody can be obtained. Cell lines established from mice or rats are used as myeloid cells. The cell fusion can be performed by a known method, for example, according to the method of Kohler and Milstein (Nature, 256, 495-497 (1975)).
As the cell growth inhibitor, apoptosis inducer and therapeutic agent for liver disease containing the polypeptide of the present invention, it is possible to administer the polypeptide of the present invention alone, but it is usually one that is pharmacologically acceptable. Desirably, it is mixed with one or more carriers and provided as a pharmaceutical preparation manufactured by methods well known in the art of pharmacy.
As the administration route, it is desirable to employ the most effective method for the disease, and examples thereof include oral administration, parenteral administration such as intravenous, subcutaneous, and intraperitoneal administration. The administration form is desirably a form capable of efficiently and effectively delivering the active ingredient to a disease, and examples thereof include capsules, granules, tablets, powders, injections, transdermals, suppositories, and emulsions.
As a method for screening a compound that specifically changes the activity of the polypeptide of the present invention, a polypeptide of the present invention or a partial peptide thereof or a salt thereof, or a recombinant protein expression system is constructed, and an assay using the expression system is performed. This can be done by using a system. The screening method of the present invention can screen substances that alter the activity of the protein of the present invention, for example, proteins, peptides, nucleic acids, non-peptide compounds and the like, and include agonists and antagonists. The activity of the polypeptide of the present invention when a substance (test compound) that changes the activity of the polypeptide of the present invention is brought into contact with the polypeptide of the present invention or a partial peptide thereof or a salt thereof is measured and compared. A method for screening a substance of the present invention is provided. Further, when a mammalian cell or a transformant expressing the polypeptide of the present invention is cultured, and the test substance is brought into contact with the expressed polypeptide of the present invention, the activity of the polypeptide of the present invention, the cells of the transformant It is possible to screen for a substance that alters the activity of the polypeptide of the present invention or a salt thereof, which is characterized in that the number, mRNA expression level, etc. are measured and compared.
The compound or a salt thereof obtained by the screening method of the present invention is a compound having an action of changing the activity of the polypeptide of the present invention or a salt thereof. For example, the compound binds to the polypeptide of the present invention or a salt thereof and has an inhibitory activity. Or a salt thereof, or a compound having the activity of binding to a DNA sequence encoding the polypeptide of the present invention and suppressing the expression of the protein, or a salt thereof. Examples of the compound include proteins, peptides, non-peptides, and nucleic acids. For example, antibodies to the polypeptide of the present invention and the DNA sequence described in SEQ ID NO: 1 or / and / or SEQ ID NO: 3 or a partial sequence thereof A base sequence having a complementary sequence can be used. Further, these compounds may be novel compounds or known compounds.
Example
The invention is described in more detail in the following examples, which do not limit the scope of the invention.
Example 1
Isolation of mouse SF21 cDNA
(1) Isolation of a gene showing an increased expression from the FLS mouse liver by cDNA subtraction (Subtraction) method
Total RNA was prepared from livers of 15-week-old FLS mice and DS mice by AGPC (Acid Guanidinium Phenol Chloroform Method) method (Analytical Biochemistry, 162, 156-159 (1987).), And mRNA Purification Biotechnology was performed. (Poly (A) + RNA). Using a poly (A) + RNA derived from an FLS mouse as a tester and a Poly (A) + RNA derived from a DS mouse as a driver, CLONTECH PCR-Select ^TM DNA subtraction was performed using cDNA Subtraction Kit (Clontech) according to the attached manual. The obtained cDNA was purified by QIAEX-II Gel Extraction System (manufactured by Qiagen), and was added to pCR2 vector (manufactured by Invitrogen) using DNA Ligation Kit Ver. Using 2 (manufactured by Takara Shuzo), the reaction was carried out at 16 ° C. for 4 hours and inserted. After introducing the constructed vector into Escherichia coli JM109 strain and performing transformation, ampicillin (50 μg / ml), 5-Bromo-4-Chloro-3-indolyl-β-D-galactoside (40 μg / ml) and Isopropyl- The cells were spread on an LB agar medium containing β-D-Thio-Galactopyranoside (100 μM) and cultured at 37 ° C. overnight. After the culture, the colonies that appeared on the plate were inoculated into 2 ml of an LB liquid medium containing 50 μg / ml of ampicillin, cultured at 37 ° C. overnight, and then the cells were collected by centrifugation. The recombinant DNA was extracted and purified from the collected cells using QIAprep Spin Plasmid Miniprep Kit (manufactured by Qiagen). The nucleotide sequence of the obtained cDNA library was determined by a DNA terminator method using a DNA sequencer 373S (manufactured by Applied Biosystems). As a result of analyzing the nucleotide sequence of the obtained cDNA library with the analysis software BLAST, the Drosophila melanogaster function unknown gene CG8863 encodes 1026 to 1126 regions and 66% of the amino acid sequence encoded by the gene CG8863, and the nematode (Caenorhabditis elegans) function unknown gene F11A10 A clone pSF21A having a nucleotide sequence consisting of 329 bases (SF21A) represented by SEQ ID NO: 7 and having 43% homology with the remaining 1023 to 1118 regions of the amino acid residues encoded by 0.4 was obtained.
(2) Isolation of mouse SF21 full-length cDNA
Since SF21A did not contain the entire translation region, isolation of full-length cDNA was attempted. First, a probe used for full-length cDNA isolation was prepared from plasmid pSF21A. 5 μg of plasmid pSF21A was digested with 20 units of restriction enzyme EcoRI (manufactured by Takara Shuzo), fractionated by 0.8% agarose gel electrophoresis, DNA fragment was extracted, and QIAEX-II Gel Extraction System (manufactured by Qiagen) Was used for purification. The obtained DNA was purified using RTG DNA Labeling Beads (manufactured by Amersham Pharmacia Biotech) according to the attached manual. ³² [P] dCTP (Amersham Pharmacia Biotech) was used as a probe.
An Escherichia coli strain Y1090 in which a mouse liver cDNA library, Mouse Liver 5'-STRETCH PLUS cDNA (Clontech) was cultured overnight. ⁻ It was added to the culture solution and incubated at 37 ° C. for 15 minutes. To this was added LB-top agarose (10 g tryptone, 5 g yeast extract, 5 g NaCl, 10 mM MgSO 4) pre-warmed to 50 ° C. ₄ , 7.2 g agarose), inoculated uniformly on an LB-agagar plate, and cultured at 37 ° C for 5 hours. After cooling the plate on which the plaque was formed at 4 ° C. for 1 hour, the plaque was transferred to a nylon membrane Hybond-N + (manufactured by Amersham Pharmacia Biotech). This membrane is alkali-denatured with a 0.5 M NaOH (containing 1.5 M NaCl) solution, and after immobilizing the DNA, neutralized with 0.5 M Tris-HCl, pH 7.2 (containing 1.5 M NaCl). And washed with 2 × SSC. Hybridization was carried out at 55 ° C. overnight using the probe prepared above for this membrane. The membrane is washed twice with a 2 × SSC (containing 0.1% SDS) solution at room temperature for 10 minutes and with a 0.1 × SSC (containing 0.1% SDS) solution at 55 ° C. for 15 minutes. Was washed twice, and the target plaque was detected by autoradiography.
A plaque group containing plaques corresponding to the detected positive signal of radioactivity was isolated from the plate and 1 ml of SM buffer (Tris-HCl, pH 7.5; 10 mM MgSO 4) ₄ , 0.1 M NaCl, 0.01% gelatin, 50 mM), eluted the phage at 4 ° C. for 16 hours, and collected the phage in the supernatant by centrifugation at 13,000 rpm for 10 minutes. This phage solution was diluted with LB-MgSO4 so that the number of plaques that appeared was 10, 100, or 1000. ₄ It was applied to a plate and positive plaques were screened in the same manner as described above. As a result, a positive plaque was successfully isolated as a completely isolated plaque, the single clone was expanded, DNA was extracted from phage particles, and purified. All of these operations followed a method known per se. The purified DNA was digested with a restriction enzyme, EcoRI, and similarly digested with EcoRI, pBluescript II (manufactured by Stratagene) and DNA Ligation Kit Ver. Ligation reaction was performed at 16 ° C. for 4 hours using No. 2 (Takara Shuzo). Escherichia coli strain JM109 was transformed with these plasmids, cultured by a method known per se, and the cells were collected, followed by DNA extraction and purification. The nucleotide sequence was determined in the same manner as described above. As a result, a clone pSF21B having a base sequence (SF21B) consisting of 2402 bases represented by SEQ ID NO: 8 was isolated.
Next, a radioisotope-labeled probe was prepared from clone pSF21B in the same manner as described above, and a mouse cDNA library was screened. A clone pSF21D containing pSF21C and a base sequence (SF21D) consisting of 2414 bases represented by SEQ ID NO: 10 was obtained. As a result of analysis based on the obtained nucleotide sequences of SF21A to SF21D, cDNA consisting of 4661 bases (SEQ ID NO: 798th to 5459th) was obtained.
Further, in order to determine the base sequence around the 5 'end of the translation region, the 5' end sequence was isolated by the RACE (Rapid Amplification of cDNA Ends) method. The 5 'end RACE (Rapid Amplification of cDNA Ends) method uses poly (A) + RNA prepared from the liver of Balb / c mouse as a template, and 5' RACE System for Rapid Amplification of cDNA Ends, version 2.0, in vitro. ) Was operated according to the attached manual.
As a primer, three types were synthesized based on the cDNA sequence consisting of 4661 bases obtained above.

As an anchor primer, a reagent attached to 5'RACE System, 5'RACE Abridged Anchor Primer (AAP) and an Abbreviated Universal Amplification Primer (AUAP) were used.
To 1 μg of total RNA obtained from a Balb / c mouse liver by a method known per se, 14.0 μl of distilled water and 1 μl of 2.5 μM SF28 primer (SEQ ID NO: 19) were added, and the mixture was incubated at 70 ° C. for 10 minutes, and heated to 50 ° C. A solution A kept warm was prepared. 2.5 μl of 10 × PCR Buffer (200 mM Tris-HCl, pH 8.4; containing 500 mM KCl), 25 mM MgCl 2 ₂ Was mixed with 1 μl of 10 mM dNTP mix and 2.5 μl of 0.1 M DTT to prepare a solution B kept at 50 ° C. Solution A, Solution B, and Super Script ^TM After mixing 1 μl of II Reverse Transcriptase (200 units / μl), the mixture was incubated at 42 ° C. for 50 minutes and at 70 ° C. for 15 minutes, and then allowed to stand on ice for 5 minutes. 1 μl of RNase mix (RNase H + RNAse T1) was added and incubated at 37 ° C. for 30 minutes.
The cDNA obtained above was purified using the DNA Purification System (Invitrogen) included in the kit. 225 μl of binding solution (6 M NaI) was added to the above reaction solution, applied to GlassMAX DNA isolation spin cartridges, and eluted with 50 μl of distilled water to obtain a purified cDNA. 4 μl of distilled water was added to 10 μl of the purified cDNA, 5 × tailing buffer (50 mM Tris-HCl, pH 8.4; 125 mM KCl, 7.5 mM MgCl 2) ₂ 5 μl and 5 μl of 1 mM dCTP, respectively, treated at 94 ° C. for 2 minutes, and left on ice for 1 minute. 1 μl of Terminal deoxynucleotidyl transferase (rTdT) was added, and the mixture was incubated at 37 ° C. for 10 minutes and at 65 ° C. for 10 minutes, and then left on ice for 5 minutes to obtain cDNA for 5′RACE.
The PCR (Polymerase Chain Reaction) method is TaKaRa LA Taq. ^TM (Manufactured by Takara Shuzo). To 2.5 μl of the cDNA for 5′RACE obtained above, 2.5 μl of 10 × PCR Buffer, 4 μl of 2.5 mM dNTP mix, 1 μl of 10 μM AAP primer, 1 μl of 10 μM primer SF29 (SEQ ID NO: 20), 1 μl of TaKaRa LA Taq ^TM A solution obtained by mixing 0.25 μl of (5 U / μl) and 13.75 μl of distilled water was cycled once at 94 ° C. for 2 minutes, at 94 ° C. for 1 minute, at 55 ° C. for 1 minute, and at 72 ° C. for 2 minutes. The first PCR was performed 30 times, once at 72 ° C. for 2 minutes. 2.5 μl of a 50 × dilution of the first PCR reaction solution, 2.5 μl of 10 × PCR Buffer, 4 μl of 2.5 mM dNTP mix, 1 μl of 10 μM AUAP primer, 1 μl of 10 μM SF30 primer, 1 μl of TaKaRa LA Taq ^TM A solution obtained by mixing 0.25 μl of (5 U / μl) and 13.75 μl of distilled water was subjected to a cycle of 94 ° C. for 2 minutes, 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 2 minutes. The second PCR was performed 20 times, once at 72 ° C. for 2 minutes. The second PCR reaction solution was electrophoresed on a 1.2% agarose gel, and a DNA fragment of about 900 bp was recovered and purified by QIAEX-II Gel Extraction System. The purified DNA was inserted into a plasmid vector pCR2 (manufactured by Invitrogen), and the nucleotide sequence was determined by the method described above. As a result, a base sequence (SF21E) consisting of 891 bases represented by SEQ ID NO: 11 was obtained.
FIG. 1 shows the arrangement of the obtained SF21 base sequences. As a result of determining the nucleotide sequence of the full-length cDNA of mouse SF21 based on this, it was revealed that mouse SF21 was a protein consisting of 5124 nucleotides in full length represented by SEQ ID NO: 3 and 1708 amino acid residues represented by SEQ ID NO: 4. Became.
Example 2
Isolation of human hSF21 cDNA
Cloning of a human gene showing homology with mouse SF21 was attempted. Amino acid residues Pro-Ala-Val-Arg-Lys-Ser-Ala-Gly-Gln-Thr (SEQ ID NO: 4: Nos. 1035 to 1044) and Asp-Thr-Ala-Glu-Lys encoded by mouse SF21 Based on -Gln-Trp-Ala-Glu-Thr (SEQ ID NO: 4: Nos. 1102 to 1111), two types of degenerate oligonucleotides were synthesized as primers for PCR.

PCR was performed using two types of primers, SF-D3 and SF-D4, with a human liver cDNA library Human River 5'-STRECH PLUS cDNA (manufactured by Clontech) as a template.
First, the human liver cDNA library (2 × 10 ⁷ (PFU / μl) 0.5 μl to TaKaRa Ex Taq ^TM (Takara Shuzo Co., Ltd.) 1.25 units of SF-D3 and SF-D4 primers were added at 50 pmol each and 10 × Ex Taq Buffer (Mg). ²⁺ plus 5 μl and dNTP Mixture (2.5 m each) to a final concentration of 200 μM, and distilled water to a total volume of 50 μl to prepare a sample for PCR. A cycle of 94 ° C. for 4 minutes, 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, 72 ° C. for 30 seconds 35 times, 72 ° C. for 3 minutes once, as a DNA thermal cycler, Omni Gene ( PCR was carried out using Thermo-Hybide Co., Ltd.). The amplified DNA fragment of about 250 bases was cloned into a pCR2 vector (manufactured by Invitrogen), and the nucleotide sequence was determined in the same manner as described above. As a result, a plasmid phSF21A having a base sequence (hSF21A) consisting of 230 bases represented by SEQ ID NO: 12 was isolated. The obtained cDNA had 95% homology in base sequence and 100% amino acid sequence with the corresponding region of mouse SF21. From this, the isolated cDNA was considered to be part of the human SF21 mouse gene.
In order to obtain human full-length hSF21 cDNA, Human River 5'-STRECH PLUS cDNA was screened by plaque hybridization using hSF21A as a probe. Plaque hybridization was performed according to the method used when obtaining mouse full-length SF21 cDNA. According to this method, clone phSF21C having a base sequence of 1166 bases (hSF21C) represented by SEQ ID NO: 13, clone phSF21E having a base sequence of 2825 bases (hSF21E) represented by SEQ ID NO: 14 and a clone represented by SEQ ID NO: 15 Thus, three clones of clone phSF21F having a base sequence (hSF21F) consisting of 3139 bases were obtained. As a result of performing analysis based on the nucleotide sequence of the obtained human hSF21A to F, a cDNA consisting of 5517 bases (SEQ ID NO: 250th to 5767th) was obtained.
The human 5'-terminal sequence was isolated by the RACE method in order to determine the nucleotide sequence around the 5'-terminal of the translation region in the same manner as in the mouse. The operation was performed according to the method described above.
For 5′RACE, Human River 5′-STRECH PLUS cDNA was used as a template, and one kind was synthesized as a primer based on the human cDNA sequence consisting of 5517 bases obtained above.

GT11-LDF was synthesized as a primer specific to the cloning vector λgt11 sequence.

1.25 units of KlenTaq LA Polymerase Mix (manufactured by Clontech) and 50 pmol of each of the above primers were added to 0.5 μl of a solution obtained by diluting human liver cDNA library Human Liver 5′-STRECH PLUS cDNA 100 times with distilled water. X PCR Buffer (200 mM Tris-HCl, pH 8.3; 20 mM MgSO ₄ , 100 mM (NH ₄ ) ₂ SO ₄ , 100 mM KCl, 1% TritoX-100, 1 mg / ml BSA), 5 μl of dNTPs to a final concentration of 200 μM, and distilled water to a total volume of 50 μl to prepare a sample for PCR. did. The sample was subjected to PCR using a DNA thermal cycler Omni Gene for 25 times at 94 ° C. for 4 minutes, 25 times at 94 ° C. for 30 seconds and 68 ° C. for 3 minutes, and once at 68 ° C. for 3 minutes. . The PCR reaction solution was electrophoresed on a 1.2% agarose gel, and a DNA fragment of about 550 bp was recovered and purified. The purified DNA fragment was inserted into a pCR2 vector, and the nucleotide sequence was determined by a DNA terminator method using a DNA sequencer 373S. As a result, a base sequence (hSF21G) consisting of 433 bases represented by SEQ ID NO: 16 was obtained.
The positions of the obtained base sequences of various hSF21 are shown in FIG. As a result of determining the base sequence of the full-length cDNA of human hSF21, human hSF21 was found to have a base sequence consisting of 5883 bases in total length represented by SEQ ID NO: 1 and a protein consisting of 1717 amino acid residues represented by SEQ ID NO: 2. It became clear that it was. Since the isolated human base sequence showed 82% homology with the mouse base sequence, the obtained human gene was considered to be equivalent to the mouse gene.
A BLAST search was performed on the public database GenBank for the deduced amino acid sequence of the human full-length hSF21 cDNA. ) And 29%, and 17% homology with fission yeast (Schzosaccharomyces pombe) function unknown gene C23D3.13C (1616 residues), respectively. In addition, amino acid residues 213 to 397 and 639 to 931 of human hSF21 have amino acid residues of human brefeldin A-inhibiting guanine nucleotide exchange factor 1 protein (BIG1) belonging to the Sec7 / BIG1 family, respectively. It showed a homology of 23% with the 421st to 591st and the 989th to 1306th. In addition, regions of amino acid residues 178 to 392 and 746 to 919 of human hSF21 are amino acid residues 470 to 671 and 1270 to Sec7p of yeast (Scharomyces cerevisiae), respectively. The 1443th showed 22% and 19% homology. However, human hSF21 has a low homology to a region corresponding to the Sec7 domain which is a catalytic domain for ARF which is a substrate of BIG1 and Sec7p, and an amino acid sequence conserved in the Sec7 domain required for GEF activity on ARF was recognized. Therefore, it was considered to be a gene encoding a novel protein different from the Sec7 / BIG1 family. Therefore, since the gene encoding the human hSF21 protein has the above characteristics, it was named Sec7 Like-1 (SecL-1) gene.
Example 3
Expression of SecL-1 in various human tissues
The presence or absence of SecL-1 gene expression in various human tissues (brain, heart, skeletal muscle, large intestine, thymus, spleen, kidney, liver, small intestine, placenta, lung, leukocytes) was examined.
Human Multiple Tissue Northern (MTN) as mRNA derived from various human tissues ^TM ) A 705 bp nucleotide sequence represented by SEQ ID NO: 1 from position 4503 to position 5207, obtained by digesting Blot (manufactured by Clontech) with human SecL-1 cDNA as a probe with restriction enzymes SphI and SpeI. In the same manner as described above. ³² P] dCTP (manufactured by Amersham Pharmacia Biotech) was used for Northern blot analysis.
ExpressHyb ^TM 5 μl of the probe prepared above was added to 10 ml of Hybridization Solution (manufactured by Clontech) to prepare a hybridization solution. In the hybridization solution thus prepared, MTN Blot was incubated at 68 ° C. for 16 hours to perform hybridization. The membrane was washed four times with a 2 × SSC (containing 0.1% SDS) solution at room temperature for 15 minutes and with a 0.1 × SSC (containing 0.1% SDS) solution at 50 ° C. for 20 minutes. Was performed twice, followed by autoradiography at -70 ° C for 5 days. As a result, SecL-1 expression was observed in all tissues, and particularly strong signals were observed in the brain, heart, kidney, liver, and placenta. Since the size of the mRNA band was longer than that of the isolated cDNA, SecL-1 mRNA was considered to have a long untranslated portion.
Example 4
Construction of human SecL-1 expression vector
In order to analyze the function of the human SecL-1 gene, an expression vector in mammalian cells was constructed. Six types of primers were synthesized based on the human SecL-1 full length DNA sequence. HSF53, HSF62, and HSF66 added a sequence recognized by XhoI, HSF63 added a sequence recognized by BstXI, HSF67 added a sequence recognized by ApaI, and HSF69 added a sequence recognized by KpnI to the 5 ′ end of the primer. .

Using mRNA prepared from HEK293 cells as a template, 6 kinds of primers were combined with HSF53 and HSF63, HSF62 and HSF67, HSF66 and HSF69, and a PCR method was performed so that human SecL-1 was divided into three.
First, RNeasy Mini Kit (manufactured by Qiagen) was used to prepare total RNA from HEK293 cells, and total RNA was prepared according to the attached manual. For the reverse transcription reaction from the prepared total RNA to cDNA, SuperScript II Rnase H-Reverse Transcriptase (manufactured by Invitrogen) was used to prepare cDNA according to the attached manual. Primers were added to 0.5 μl of the prepared cDNA and 1.25 units of Pfu Turbo DNA Polymerase (manufactured by Stratagene) in the above combination at 50 pmol each and 10 × PCR Buffer (200 mM Tris-HCl, pH 8.75; 20 mM MgSO 4) ₄ , 100 mM (NH ₄ ) ₂ SO ₄ , 100 mM KCl, 1% Triton X-100, 1 mg / ml BSA) and dNTPs to a final concentration of 200 μM, and distilled water to a total volume of 50 μl to prepare a sample. The samples were cycled once at 99 ° C. for 2 minutes, 35 cycles of 94 ° C. for 30 seconds, 65 ° C. for 30 seconds, 75 ° C. for 3 minutes, and once at 72 ° C. for 7 minutes, using the DNA Thermal Cycler Omni Gene. Was used to perform PCR.
The PCR reaction solution was electrophoresed on a 1.2% agarose gel, and the target DNA fragment was recovered and purified using GFX PCR DNA and Gel Band Purification Kit (Amersham Pharmacia Biotech). These various DNA fragments were inserted into a plasmid vector pCR-Blunt (manufactured by Invitrogen) to construct plasmids pCR-Blunt / HSF66-69, pCR-Blunt / HSF62-67 and pCR-Blunt / HSF53-63. The nucleotide sequence of the fragment was determined.
Next, the three types of plasmids obtained above were digested with restriction enzymes, each DNA fragment was incorporated into a mammalian cell expression vector, and the full-length human SecL-1 cDNA was reconstructed to construct an expression vector.
The plasmid pCR-Blunt / HSF66-69 was digested with the restriction enzymes XhoI and KpnI, and then fractionated by electrophoresis on a 1.2% agarose gel to recover the desired fragment. The recovered DNA fragment was purified using GFX PCR DNA and Gel Band Purification Kit (manufactured by Amersham Pharmacia Biotech) according to the attached manual. The pBK-CMV vector (manufactured by Stratagene) was also digested with the same restriction enzymes, and the HSF66-HSF69 fragment was integrated to obtain a plasmid pKB-CMV / HSF66-69. Similarly, pCR-Blunt / HSF62-67 was digested with restriction enzymes XhoI and ApaI, fractionated by electrophoresis on a 1.2% agarose gel, and the target fragment was recovered and purified. pKB-CMV / HSF66-69 was digested with the same restriction enzymes, and the HSF62-67 fragment was integrated to obtain plasmid pKB-CMV / HSF66-69.HSF62-67. Next, pCR-Blunt / HSF53-63 was digested with restriction enzymes XhoI and BstXI, and fractionated by electrophoresis on a 1.2% agarose gel, and the target fragment was recovered and purified. pKB-CMV / HSF66-69.HSF62-67 is digested with the same restriction enzymes, the HSF53-63 fragment is incorporated, and the expression plasmid pKB-CMV / HSF66-69.HSF62-67.HSF53-63 (pKB-CMV / hSecL). -1) was obtained.
Human embryonic kidney-derived cell line 293 cells (ATCC: CRL-1573) were placed in a biocoated poly-D-lysine (Synthetic) 6-well micro test plate (manufactured by Becton Dickinson) at 5 × 10 5. ⁵ And a suitable amount of D-MEM (high glucose) medium (Invitrogen) (containing 10% FBS (Invitrogen)) was added thereto. ₂ The medium was cultured overnight at 37 ° C. 2 μg of the hSecL-1 expression vector pBK-CMV / hSecL-1 was prepared to a final volume of 100 μl with an Opti-MEM Reduced-Serum Medium (1 ×) solution (manufactured by Invitrogen), and was prepared by SuperFect Transfection Reagent (Qiagen). Was added and mixed well, followed by incubation at room temperature for 5 to 10 minutes to form a SuperFect-DNA complex. A cell obtained by introducing the expression vector pBK-CMV into cells by the same operation was used as a control. To each of these vectors, add 600 μl of D-MEM (containing 10% FBS), mix well by pipetting, aspirate the culture from the 293 cell culture cultured above, and wash once with PBS. Was added to the harvested cells. This cell culture is 5% CO2 ₂ After culturing at 37 ° C. for 3 hours, the culture solution was removed by suction and washed once with a PBS solution. 2 ml of D-MEM medium (containing 10% FBS) was added to the cells thus transformed, and 5% CO 2 was added. ₂ By culturing at 37 ° C. for 48 hours, hSecL-1 expressing recombinant cells were obtained.
Example 5
Production of hSecL-1 antibody
As an antigen, a polypeptide consisting of the amino terminal 146 residues of human SecL-1 represented by SEQ ID NO: 5 was used. The antigen peptide was prepared using a GST Gene Fusion System (manufactured by Amersham Pharmacia Biotech) according to the attached manual to prepare a GST fusion protein, and then a GST-free recombinant protein.
First, using the expression plasmid pBK-CMV / hSecL-1 obtained above as a template, two types of PCR primers were synthesized based on the nucleotide sequence of SEQ ID NO: 6. HSFS1 is a sequence recognized by EcoRI at the 5 'end in the first to 26th base sequences described in SEQ ID NO: 6, and HSFS2 is a stop codon at the 3' end in the 413th to 438th base sequences. And sequences recognized by XhoI were added.

To 0.5 μl of pBK-CMV / hSecL-1, 1.25 units of PfuTurbo DNA Polymerase (manufactured by Stratagene), 50 pmol of each of the primers HSFS1 and HSFS2, 10 × PCR Buffer (200 mM Tris-HCl, pH 8.3; 20 mM MgSO4) ₄ , 100 mM (NH ₄ ) ₂ SO ₄ , 100 mM KCl, 1% Triton X-100, and 1 mg / ml BSA) and 5 μl of dNTPs to a final concentration of 200 μM, and distilled water to a total volume of 50 μl to prepare a sample for PCR. The sample was cycled once at 99 ° C. for 2 minutes, 35 cycles of 94 ° C. for 30 seconds, 60 ° C. for 30 seconds, 72 ° C. for 90 seconds, and once at 72 ° C. for 7 minutes using a thermal cycler Omni Gene. PCR was performed. After fractionating the PCR reaction solution by electrophoresis using 1.2% agarose, a 452 bp DNA fragment was recovered. The recovered DNA fragment was purified using GFX PCR DNA and Gel Band Purification Kit, and then digested with restriction enzymes EcoRI and XhoI.
The GST fusion protein expression vector pGEX-6P-1 attached to the kit was digested with restriction enzymes EcoRI and XhoI, and the DNA fragment obtained above was inserted to obtain a fusion protein expression plasmid pGEX-HSFS. The obtained expression plasmid pGEX-HSFS was introduced into Escherichia coli strain BL21 (DE3) to obtain a transformant. This transformant was added to 100 ml of 2 × YT medium, and precultured at 37 ° C. for 18 hours. 25 ml of the preculture was added to 2.5 L of 2 × YT medium, and culture was performed until the OD value reached 0.8. Thereafter, isopropyl-1-thio-β-D-galactopyranoside (Sigma) was added to the culture. (Aldrich) was added to a final concentration of 0.15 mM, and the mixture was cultured for 30 minutes to induce protein expression. The culture was centrifuged at 8,000 rpm, and 100 ml of an STE solution (10 mM Tris-HCl, pH 8.0; containing 150 mM NaCl, 1 mM EDTA · 2Na) was added to the cells and suspended, and then suspended in Lysozyme (Sigma). 1 ml of a 10 mg / ml solution (manufactured by Aldrich) was added, and the mixture was left on ice for 30 minutes. After adding 15 ml of an STE solution containing 10% sarkosyl (manufactured by Sigma-Aldrich), the cells were sonicated and centrifuged at 9,000 rpm to obtain a supernatant containing the fusion protein. To affinity-purify the fusion protein from this supernatant, 10 ml of Glutathion Sepharose 4B gel (manufactured by Amersham Pharmacia Biotech) was added, and the mixture was slowly mixed for 30 minutes to adsorb the fusion protein to the carrier. This carrier was packed in a column, washed three times with 20 ml of an STE solution, and then washed with an elution buffer (containing 75 mM HEPES, pH 7.4; 150 mM NaCl, 10 mM reduced glutathione, 5 mM dithiothreitol, and 2% N-octyl glucoside). The -hSecL-1 fusion protein was eluted. The eluted fusion protein was dialyzed overnight in a PBS solution, and after substitution with PBS, 250 μl of PreScission Protease solution (Amersham Pharmacia Biotech) was added to the sample, and the sample was treated at 4 ° C. for 1 hour. This sample was added to a Glutathione Sepharose 4B column (manufactured by Amersham Pharmacia Biotech), and GST was adsorbed on a carrier to remove GST. The thus obtained hSecL-1 partial peptide was dialyzed overnight in a PBS solution, and then replaced with PBS. About 15 mg of the hSecL-1 partial peptide was obtained from 15 L of the culture solution.
100 μg of the hSecL-1 partial peptide was thoroughly mixed with 1 ml of Freund's complete adjuvant (manufactured by Invitrogen) and injected into New Zealand white rabbits. Blood was collected from rabbits that had been subjected to this operation several times in several weeks to obtain serum containing an antibody against the hSecL-1 partial peptide. Using the obtained antiserum and horseradish peroxidase-labeled anti-rabbit IgG antibody (manufactured by Amersham Pharmacia Biotech), the method of Towbin et al. (Proceeding of the National Academy of Sciences, USA, 76, 4350-4354 (1979)). Similarly, Western blot analysis was performed.
First, the hSecL-1 expressing recombinant cells obtained above were collected from the culture solution, and Extraction Buffer (20 mM Tris-HCl, pH 7.5; 0.25 M sucrose, 1 mM EDTA · 2Na, 3 mM MgCl 2 ₂ 0.1 ml of 10 mM 2-mercaptoethanol and protease inhibitor mixture (manufactured by Calbiochem) were added to prepare a cell extract. After fractionating the cell extract thus prepared by SDS-polyacrylamide gel (3-8%) by electrophoresis, proteins are transferred from the gel to PVDF membrane (manufactured by Millipore) by electrophoresis. Was blocked with PBS (containing 5% skim milk, 0.05% Tween 20). Next, the membrane after blocking was immersed in an antibody solution obtained by diluting the antiserum obtained above with PBS (containing 5% skim milk and 0.05% Tween 20) 4000 times, and incubated at room temperature for 1 hour. Thereafter, the plate was washed twice with PBS (containing 0.05% Tween 20) for 15 minutes, and washed with a horseradish peroxidase-labeled anti-rabbit IgG antibody (manufactured by Amersham Pharmacia Biotech) in a labeled antibody solution diluted 4000-fold with PBS. The membrane was soaked and incubated at room temperature for 1 hour. Thereafter, the membrane is washed twice with PBS (containing 0.05% Tween 20) for 15 minutes, and the washed membrane is exposed to a signal emitted by an ECL (Enhanced chemiluminescense) method on an X-ray film, and a protein binding to an antibody is removed. Detected. As a result, in the sample prepared from the cells into which the hSecL-1 expression vector was introduced, a 190 kDa band predicted from human SecL-1 was detected, and the obtained antiserum reacted specifically with the recombinant human SecL-1 protein. It turned out that it showed.
Example 6
Effect of human SecL-1 on cell proliferation and apoptosis
In the same manner as described above, a human fetal kidney-derived cell line 293 cell (ATCC: CRL-1573) was added to a human SecL-1 expressing recombinant cell transformed with pBK-CMV / hSecL-1 and a cell into which pBK-CMV was introduced as a control. 2 ml of D-MEM (containing 10% FBS) medium was added to each well, and 5% CO 2 was added. ₂ Culture was performed at 37 ° C. in the medium. The number of cells on the first day and the fifth day after the culture was measured using CellTiter 96 A Queue One Solution Cell Proliferation Assay (manufactured by Promega) according to the attached manual. As a result, a decrease in cell proliferation was observed in the human SecL-1 high-expressing cell line as compared to the control cell line (FIG. 2).
The involvement of apoptosis in suppressing cell growth was examined. It is known that Caspase-3 is specifically activated when apoptosis is induced (Cell, 88, 355-365 (1997)). Therefore, the Caspase-3 activity of the pBK-CMV / hSecL-1 introduced cells and the pBK-CMV introduced cells was measured. The activity of Caspase-3 was measured by CaspACE. ^TM The assay was performed using Assay System Colorimetric (promega) according to the attached manual. As a result, it was found that Caspase-3 activity was increased about three times in human SecL-1 high expressing cells as compared to control cells (FIG. 3).
Next, when apoptosis was induced in human SecL-1 high-expressing cells, the expression of endogenous SecL-1 gene was increased. It is known that aqueous hydrogen peroxide stimulates apoptosis induction (FEBS Letter, 488, 123-132 (2001).). Then, an aqueous hydrogen peroxide solution was added to the cell culture solution, and the mRNA was determined as the expression amount of the hSecL-1 gene over time by the RT-PCR method. HSF28 and HSF29 were synthesized as primers for PCR.

First, an aqueous solution of hydrogen peroxide prepared at four concentrations of 0.1 μM, 0.3 μM, 0.6 μM and 0.9 μM was added to a human SecL-1 expression cell culture solution, and 5% CO 2 was added. ₂ After culturing at 37 ° C. for 24 hours, the number of viable cells and the amount of human SecL-1 mRNA were measured. As a control, cells to which no aqueous hydrogen peroxide was added were cultured under the same conditions. Total RNA was prepared from each cell using RNeasy Mini Kit (Qiagen) according to the attached manual. For the reverse transcription reaction from the prepared total RNA to cDNA, cDNA was prepared according to the attached manual using SuperScript II Rnase H-Reverse Transcriptase (manufactured by Invitrogen). To 0.5 μl of the prepared cDNA, 1.25 units of PfuTurbo DNA Polymerase (manufactured by Stratagene), 2 μl of each 10 μM primer, 5 μl of 10 × PCR Buffer, 4 μl of 2.5 mM dNTPs, and 50 μl of distilled water were mixed. PCR for 1 cycle at 2 ° C. for 2 minutes, 35 cycles at 94 ° C. for 30 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 30 seconds, and 1 cycle at 72 ° C. for 5 minutes using the DNA thermal cycler Omni Gene. Was done. The PCR reaction solution was electrophoresed on a 1.2% agarose gel and confirmed as a band of about 480 bp. As a result, as compared with the control group, the number of viable cells decreased in the cells added with hydrogen peroxide in inverse proportion to the concentration (FIG. 4). In addition, the amount of mRNA of human SecL-1 was significantly increased by treating with hydrogen peroxide.
From the above results, it was considered that human SecL-1 is involved in cell growth suppression accompanied with apoptosis induction.
Industrial applicability
The protein of the present invention, its partial peptide or a salt thereof, and the nucleotide sequence encoding them can be used to search for novel physiological functions, produce antibodies, construct a recombinant expression system, and assay biologically active modulators using the expression system. It can be used for the development of a screening system for system and drug candidate compounds, reagents for preparing reagents useful for diagnosis, and development of drugs such as prophylactic and therapeutic agents.
[Sequence list]

[Brief description of the drawings]
FIG. 1 shows the arrangement of various SecL-1 cDNA fragments cloned from humans and mice.
Fig. 2 Human fetal kidney-derived cell line 293 cells were transfected with an expression vector pBK-CMV / hSecL-1 into which human SecL-1 cDNA was inserted, and a transformed human SecL-1 expression cell line (□) and vector pBK-CMV were introduced. The time-dependent change in the culture time (days) of the cell growth rate in the control cell line (●) is shown.
Fig. 3 Human SecL-1 expression cell line (■) transformed by introducing an expression vector pBK-CMV / hSecL-1 into which human SecL-1 cDNA was inserted into human fetal kidney-derived cell line 293 cells, and a vector pBK-CMV were introduced. The Caspase-3 activity of each cell line in the control cell line (□) is shown.
Fig. 4 Transfection of human SecL-1 expressing cell line at various concentrations in a human SecL-1 expressing cell line transformed by introducing an expression vector pBK-CMV / hSecL-1 into which human SecL-1 cDNA was inserted into human fetal kidney-derived cell line 293 cells. This shows the effect of hydrogen oxide.

Claims (20)

A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 or a salt thereof. A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4 or a salt thereof. In the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4, one or several amino acids comprise an amino acid sequence having at least one mutation selected from deletion, substitution or addition, and have a cell growth inhibitory activity or an apoptosis inducing activity A polypeptide or a salt thereof. A partial peptide of the polypeptide according to any one of claims 1 to 3, or a salt thereof. 5. The partial peptide according to claim 4, which comprises the amino acid sequence shown in SEQ ID NO: 5, or a salt thereof. A polynucleotide having a nucleotide sequence encoding the polypeptide or partial peptide according to any one of claims 1 to 5. 7. The polynucleotide according to claim 6, which has the 117th to 5267th positions of the nucleotide sequence shown in SEQ ID NO: 1. 7. The polynucleotide according to claim 6, which has the 66th position to the 5189th position of the nucleotide sequence shown in SEQ ID NO: 3. The polynucleotide according to claim 6, which has the nucleotide sequence shown in SEQ ID NO: 6. Hybridizes under stringent conditions with a polynucleotide consisting of nucleotides 117 to 5267 of the nucleotide sequence of SEQ ID NO: 1 or nucleotides 66 to 5189 of the nucleotide sequence of SEQ ID NO: 3, and proliferates the cells A polynucleotide encoding a polypeptide having an inhibitory activity or an apoptosis-inducing activity. An expression vector comprising the polynucleotide according to any one of claims 6 to 10. A transformant obtained by introducing the expression vector according to claim 11 into a host. 13. The transformant according to claim 12, wherein the host is an animal cell strain or an E. coli strain. The polypeptide or part according to any one of claims 1 to 5, comprising a step of culturing the transformant according to claim 12 or 13, and a step of recovering the produced recombinant polypeptide from the culture medium. A method for producing a peptide. An antibody that specifically recognizes the polypeptide or partial peptide according to any one of claims 1 to 5. A cell growth inhibitor comprising the polypeptide or partial peptide according to any one of claims 1 to 5. An apoptosis-inducing agent comprising the polypeptide or partial peptide according to any one of claims 1 to 5. A therapeutic agent for liver disease, comprising the polypeptide or partial peptide according to any one of claims 1 to 5. A method for screening a compound that specifically regulates the activity of the polypeptide according to any one of claims 1 to 5, using the polypeptide or the partial peptide according to any one of claims 1 to 5. A compound obtained by the screening method according to claim 19.

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