JP4219808B2 - A novel gene with a guanine nucleotide exchange factor-like sequence - Google Patents

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 includes a polypeptide having a human guanine nucleotide exchange factor-like sequence described in SEQ ID NO: 1; a polynucleotide encoding a polypeptide of the present invention; an expression vector comprising the polynucleotide of the present invention; A transformant having the expression vector of the invention; a production method using the transformant of the invention; an antibody specifically recognizing the polypeptide of the invention; a therapeutic agent for a disease containing the polypeptide of the invention; The present invention relates to a compound screening method 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 term for proteins that promote the activation reaction of a GTP binding protein by exchanging GDP (Guanosine 5'-diphosphate) bound to the GTP binding protein with GTP (Guanosine 5'-triphosphate). . GTP-binding protein, which is a substrate of guanine nucleotide exchange factor, is a generic term for proteins that specifically bind to GTP and GDP and hydrolyze the bound GTP to GDP. (1) Translation factor of mRNA into protein , (2) G protein, (3) low molecular weight GTP binding protein, and (4) microtubule constituent tubulin. For example, G proteins are coupled to G protein-coupled receptors, and many of these receptors perform intracellular signal transduction through activation of GTP-binding proteins. G protein-coupled receptors exist in various cells and organs in the body and play a physiologically important role as receptors for hormones and physiologically active substances that regulate their functions. Signal is transmitted into the cell via this signal, and the function of the cell is regulated by receiving this signal. G protein-coupled receptors are present on the cell membrane and are involved when hormones, neurotransmitters, etc. bind to transmit information into the cell. Low molecular weight GTP protein is an oncogene product such as Ras and Rho proteins, and is involved in cell proliferation / differentiation and intracellular transport / secretion of proteins. It functions as a molecular switch in the various signal transduction pathways involved, and plays an important role as a signal transduction factor for cell function control such as cytoskeleton formation, vesicle transport, and cell proliferation. Since guanine nucleotide exchange factor is involved in the regulation of GTP-binding protein activity, it plays an important role in regulating the functions of various living cells and organs. It plays a very important role in the regulation of signal transduction into cells, cell growth / differentiation, intracellular vesicle transport, and cell function regulation by protein transport to the nucleus. Can be the target of
As guanine nucleotide exchange factors using low molecular weight GTP proteins Ras and Rho as substrates, Sos (Son of Sevenless) and Trio have been reported, respectively. In addition, Sec7 was identified from a mutant strain exhibiting abnormalities in yeast vesicle transport, 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 have been identified in mammals including humans, such as BIG1, BIG2, ARNO, cytohesin 1 and GRP1, and these are also reported to be involved in vesicular 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 been reported yet.
Recently, various genome analyzes have progressed rapidly, and the complete base sequence decoding of humans, nematodes (Caenorhabditis elegans), Drosophila (Drosophila melanogaster), etc. has been completed, and many proteins with unknown functions have been reported in the database. . Among them, there are clearly different Sec7 family proteins that show homology to the Sec7 family but are known so far. For example, F11A10.4 (accession number: CAA92830) is registered as gene sequence information in GeneBank, and CG8863 (accession number: AE003620) is registered as sequence information of Drosophila in NCBI. There are no reports on the function of 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 an important role in vivo, but can also serve as 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 new substances and cloning genes can be a useful tool in drug development. New physiological activity modulators can be used in the development of therapeutic / preventive drugs and diagnostic drugs for the diseases involved. In addition, if a new family is found, new target diseases and functions will be elucidated, and it may be applied to search for the development of unprecedented therapeutic / preventive drugs and diagnostic drugs.
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 a partial peptide, a recombinant vector containing the polynucleotide, and the recombinant vector. Transformant to be retained, method for producing the polypeptide or partial peptide, method for screening a compound that specifically regulates the activity of the polypeptide, compound obtained by the screening, medicament containing the compound, and the polypeptide Alternatively, an antibody against the partial peptide is provided.
As a result of intensive research, the present inventors have found that yeast guanine nucleotides are based on a cDNA library prepared by subtraction cloning of a gene that is highly expressed in the liver cells of FLS mice, which are naturally occurring liver models of fatty liver. A novel gene encoding a novel mouse polypeptide having a Sec7-like sequence as an exchange factor was found. Moreover, 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 research, it was confirmed that the polypeptide of the present invention is involved in cell growth suppression and apoptosis induction. As a result of further studies based on these findings, the present inventors 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, it comprises an amino acid sequence having at least one mutation selected from deletion, substitution or addition of one or several amino acids, and inhibits cell growth or induces apoptosis A polypeptide having activity or a salt thereof;
(4) The partial peptide of the polypeptide according to any one of (1) to (3) above or a salt thereof;
(5) The partial peptide or salt thereof according to (4), comprising the amino acid sequence shown in SEQ ID NO: 5;
(6) a polynucleotide having a base sequence encoding the polypeptide or partial peptide of any one of (1) to (5);
(7) The polynucleotide according to (6) above, which has the 117th to 5267th positions of the base sequence shown in SEQ ID NO: 1;
(8) The polynucleotide according to (6) above, which has the 66th to 5189th nucleotide sequence of SEQ ID NO: 3;
(9) The polynucleotide according to (6), which has the base sequence represented by SEQ ID NO: 6;
(10) hybridizing under stringent conditions with a polynucleotide comprising the 117th to 5267th base sequence of SEQ ID NO: 1 or the 66th to 5189th base sequence of the base sequence shown in SEQ ID NO: 3; 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) above into a host;
(13) The transformant according to the above (12), wherein the host is an animal cell strain and an E. coli strain;
(14) The method according to any one of (1) to (5), comprising the step of culturing the transformant according to (12) or (13) and the step of recovering the produced recombinant polypeptide from the culture medium. A method for producing the polypeptide or partial peptide according to any one of the above;
(15) an antibody that specifically recognizes the polypeptide or partial peptide of any one of (1) to (5);
(16) A cell growth inhibitor containing the polypeptide or partial peptide of any one of (1) to (5) above;
(17) An apoptosis-inducing agent containing the polypeptide or partial peptide according to any one of (1) to (5) above;
(18) A therapeutic agent for liver disease comprising the polypeptide or partial peptide according to any one of (1) to (5) above;
(19) A compound that specifically modulates the activity of the polypeptide according to any one of (1) to (5) above using the polypeptide or partial peptide according to any one of (1) to (5) above 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. The salt includes a physiologically acceptable salt with an acid or a base, and is preferably a physiologically acceptable acid addition salt. Examples of such salts include inorganic acids such as hydrochloric acid, phosphoric acid, and sulfuric acid, and salts with organic acids such as acetic acid, formic acid, citric acid, and succinic acid.
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 cytostatic activity or apoptosis-inducing activity. One or several mutations to be deleted, substituted or added are mutations of amino acids to the extent possible by site-directed mutagenesis and the like, and are 50 or less, preferably 30 or less, more preferably 10 or less. Means amino acids. Furthermore, the polypeptide of the present invention may be a polypeptide having cytostatic activity or apoptosis-inducing activity even by deletion, substitution or addition.
The partial peptide of the polypeptide of the present invention includes a polypeptide composed of a part of the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 or a salt thereof. Such a polypeptide may not have a cytostatic activity or an apoptosis-inducing activity. Moreover, those partial polypeptides should just be a polypeptide which can be used in order to produce the antibody with respect to the polypeptide of this invention. In addition, even if the polypeptide itself is not antigenic, any peptide can be used as long as it can produce an antibody by binding to other proteins such as bovine serum albumin. Good. Such a peptide is a polypeptide comprising 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 DNA encoding the polypeptide of the present invention. Examples of the polynucleotide of the present invention include polynucleotides containing the base sequences represented by SEQ ID NOs: 1, 3, and 6. In addition, the polynucleotide of the present invention can include a polynucleotide that hybridizes with the polynucleotide of the present invention under stringent conditions. Preferably, the nucleotide may encode a polypeptide having cytostatic activity or apoptosis inducing activity.
In the present specification, “a polynucleotide that hybridizes under stringent conditions” refers to a Southern hybridization method, a colony hybridization method, or a plaque using a polynucleotide selected from the polynucleotide of the present invention or a part thereof as a probe. It is a polynucleotide that can be obtained by using various hybridization assay methods such as a hybridization method. Specifically, 6 × SSC (saline sodium citrate: 150 mM NaCl, 15 mM C) using the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3 or a part thereof as a probe. ₆ H ₅ Na ₃ O ₇ ) This is a condition in which a hybridization reaction is carried out in a solution at 42 ° C. and then washed in a 0.1 × SSC solution at 68 ° C. Hybridization: Molecular Cloning: A Laboratory Manual Second Edition (Cold Spring Harbor Laboratory press, New York (1989).) And DNA Cloning1: Core Technique, Ara d. It can be performed according to the method described in the experiment document.
The “expression vector” of the present invention is, for example, a polynucleotide encoding the protein of the present invention, ATG as a translation initiation codon on the 5 ′ end side, and translation stop codons TAA, TGA or 3 ′ end side. It may have a TAG and can be constructed by ligating to the 5 ′ end of the promoter in the expression vector. Vectors include plasmids such as pBR322, pUC19, pSV. SPORT1, pBK-CMV, pGEX-6P-1, etc. can be used. Examples of promoters that control transcription include trp promoter, lac promoter, T7 promoter, SV40 promoter, and the like. In addition, an expression vector containing an enhancer, a splicing signal, a selection marker, a replication origin, a DNA sequence encoding an additional protein, or the like can also be used.
The “transformant” of the present invention is prepared by selecting an expression vector and a host such as a cell suitable for the expression vector, and transforming the host with the expression vector. Examples of the host to be transformed include prokaryotic organisms such as Escherichia coli, unicellular eukaryotic organisms such as yeast, animal cells such as hamsters, plant cells such as tobacco, and insect cells such as mushrooms. Etc. Preferably, Escherichia coli such as Escherichia coli BL21 (DE3) strain, animal cells such as human embryonic kidney-derived cells 293 cells, monkey cells COS-7, Chinese hamster cells CHO and the like 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, yeast, animal cell, plant cell, insect cell using an expression vector incorporating a polynucleotide encoding the polypeptide of the present invention. The transformant obtained by transforming a host such as the above is cultured by a culture method suitable for the host, the polypeptide of the present invention is produced, and recovered from the medium or the transformant. Or the method of manufacturing 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 includes fragments, derivatives, conjugates, modifications and the like thereof. An antibody that recognizes the polypeptide of the present invention is preferred, and an antibody that specifically recognizes the polypeptide of the present invention is more preferred. Such an antibody may be either a monoclonal antibody or a polyclonal antibody.
The “cell growth inhibitor” of the present invention is used for prevention or treatment by suppressing cell growth of a disease associated with cell growth because the polypeptide of the present invention has an action of suppressing cell growth. It can be done. The “apoptosis inducing agent” of the present invention is used for diseases that can be prevented or treated by inducing apoptosis because the polypeptide of the present invention has an effect of inducing apoptosis. Such a prophylactic or therapeutic agent preferably contains the polypeptide of the present invention and can be used for the prophylaxis or treatment of specific diseases such as cancer.
Furthermore, since the polypeptide of the present invention is highly expressed in the liver, the “liver disease therapeutic agent” of the present invention can be used for the prevention or treatment of diseases related to liver function. Such a prophylactic or therapeutic agent preferably contains the polypeptide of the present invention, and can be used for the prophylaxis or treatment of specific diseases such as liver cancer.
The “compound that specifically modulates 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 vivo, for example, a low molecular compound, a polynucleotide, a polypeptide Etc. 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 NO: 1, 3, 6 can be used. Such compounds may also be novel compounds or known compounds.
The “screening method” of the present invention is a method for selecting a compound that specifically modulates 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 screening method for the specific activity regulator are described below.
In the present invention, gene recombination techniques, E. coli, recombinant protein production methods including separation and purification, and antibody production methods known in the art are employed unless otherwise specified.
A cDNA library is prepared from the liver tissue by a conventional method well known and commonly used in the art.
As a method for preparing a cDNA library, a method described in Molecular Cloning: A Laboratory Manual Second Edition (Cold Spring Harbor Laboratory Press, New York (1989)), or a commercially available kit, for example, CLS Examples include a method using 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 clone containing the DNA fragment of the present invention is selected by purifying the plasmid of each clone from the cDNA library prepared above and analyzing the base sequence. As a base sequence analysis method, Sanger et al.'S dideoxy method (Proceeding of the National Academy of Sciences USA, 74, 5463 (1977).) Or a commercially available device such as DNA sequencer 373S (manufactured by Applied Biosystems), etc. The method to use etc. are mentioned.
Screening the cDNA library by plaque hybridization using the DNA fragment obtained above as a probe, and the whole mRNA by the Rapid Amplification of cDNA Ends (RACE) method in order to obtain the nucleotide of the translation region at the 5 ′ end Thus, the polynucleotide of the present invention can be obtained.
Using the DNA fragment obtained above as a probe, it is 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 such as Mouse Liver 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 plaque hybridization using Clontech (manufactured by Clontech), Human Liver 5′-STRETCH PLUS cDNA (Clontech) or the like. Moreover, total mRNA is prepared by a conventional method well known and commonly used in the art. Examples of the method for preparing total mRNA include the method described in Molecular Cloning: A Laboratory Manual Second Edition (Cold Spring Harbor Laboratory Press, New York (1989)), or a commercially available kit such as quat, QuT. And the like. The RACE method is described in Flohman, M .; A. (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 Corporation). . Commercially available total mRNA, for example, PolyA + RNA Mouse River (manufactured by Amersham Pharmacia Biotech) can also be used.
The DNA fragment obtained by the above method is treated with a restriction enzyme, if necessary, and then 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 incorporating and analyzing the base sequence by the above 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, those represented by SEQ ID NOs: 1 and 3 And a polynucleotide having a base sequence.
The polynucleotide obtained as described above is incorporated 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 in which the obtained expression vector is introduced into an appropriate host cell and cultured. Further, it may be separated and purified from human or mammalian cells or tissues by a conventional method well known in the art. Furthermore, it may be obtained according to a chemical peptide synthesis method. Whether the protein of the present invention thus obtained has cell growth inhibitory activity or apoptosis-inducing activity can be examined 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. The DNA encoding the polypeptide of the present invention is placed at the 5 ′ end side. ATG as a translation initiation codon may have a translation termination codon TAA, TAG or TGA on the 3 ′ end side, and can be constructed by ligating to the 5 ′ end side of the promoter in the expression vector. Such vectors include plasmids such as pBR322, pUC19, pSV. SPORT1, pBK-CMV, pGEX-6P-1, etc. can be used. Examples of promoters that control transcription include trp promoter, lac promoter, T7 promoter, SV40 promoter, and the like. In addition, an expression vector containing an enhancer, a splicing signal, a selection marker, a replication origin, a DNA sequence encoding an additional protein, or the like can also be used. For example, glutathione S transferase (GST) or thioredoxin is used as the additional protein, and the DNA sequence encoding the target protein is linked to the 3 ′ end of the DNA sequence encoding the additional protein to express the fusion protein. A vector can be produced. The fusion protein expression vector may contain a DNA sequence encoding an amino acid sequence that can be cleaved by a protease between the additional protein and the target protein. Examples of the protease include thrombin protease, factor Xa and BLase. Is used. Further, it may contain a cloning DNA sequence for efficiently inserting the DNA sequence encoding the target protein into the fusion protein expression vector. The cloning DNA sequence can be cleaved with a restriction enzyme, for example. A DNA sequence, preferably a DNA sequence that is 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 prokaryotic organisms such as Escherichia coli, unicellular eukaryotic organisms such as yeast, insect cells such as sugar beet, and multicellular eukaryotic organisms such as animal cells such as hamsters. Preferably, Escherichia coli, for example, Escherichia coli BL21 (DE3) strain, and animal cells such as human embryonic kidney-derived cells 293 cells, monkey cells COS-7, Chinese hamster cells CHO, etc. Preferably, 293 cells can be used.
Methods for transforming host cells can be implemented based on many textbooks and literature such as, for example, the above-mentioned Molecular Cloning: A Laboratory Manual Second Edition (Cold Spring Harbor Laboratory Press, New York (1989).). . When transforming E. coli, for example, Proceeding of the National Academy of the Sciences of the USA, 69, 2110 (1972). Etc. according to the method described in the above. When transforming animal cells, for example, electroporation method (Cytotechnology, 3, 133 (1990).), Calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), lipofection method (Proceeding of the Science of Science USA, 84, 7413 (1987)., Virology, Vol. 52, 456 (1973).).
The transformant of the present invention is cultured by a conventional method well-known in the art. A medium suitable for the transformed host cell can be used, and a liquid medium is suitable as the medium used for the culture. Specifically, MEM medium (Science, 122, 501 (1952).), D-MEM medium (Virology, 8, 396 (1959).), RPMI 1640 medium (The Journal of the American Medical Association, 199, 519) 1967).), YT medium, BEM medium and the like are used. 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 host is an animal cell, for example, a MEM medium, D-MEM medium, RPMI medium, or the like to which fetal bovine serum (FCS) is added in an appropriate amount is used. In order to enhance the transcription activity of the promoter of the expression vector as necessary, it may contain a substance that promotes the transcription activity, for example, using isopropyl-1-thio-β-D-galactopyranosin (IPTG) or the like. it can.
The medium contains nutrients necessary for the transformant to grow, such as glucose, amino acids, peptone, vitamins, hormones, serum, preferably fetal bovine serum, calcium chloride, magnesium chloride, etc. 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. Culturing is performed under conditions such as pH 6.0 to 8.0, 25 to 40 ° C., and 5% CO 2.
The recombinant protein produced from the transformant of the present invention is recovered from the culture medium by a conventional method well known in the art.
The culture solution obtained by culturing the transformant as described above is separated into cells or cells and a medium, for example, by a method such as centrifugation, and when the recombinant protein is present in the cells, the cells or cells are After collecting and suspending in an appropriate buffer such as STE solution, cells or cells can be crushed with lysozyme, ultrasonic waves, etc., and collected by centrifugation or filtration. The buffer used for separation / purification may contain an appropriate amount of a surfactant, for example, sodium lauryl sulfate (SDS), sodium N-lauroyl sarcosine (sarcosyl) and the like. The method for purifying and separating the target protein contained in the obtained crude product can be performed by combining various separation and purification methods known per se. As these known methods, for example, salting out, dialysis, ultrafiltration, gel filtration, various chromatographic methods such as ion exchange chromatography, various electrophoresis such as SDS-polyacrylamide gel electrophoresis, etc. are used. It is done. When the target protein is expressed as a fusion protein, the target protein can be purified by separating the fusion protein using an antibody specific for 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 by, for example, site-directed mutagenesis described in Molecular Cloning: A Laboratory Manual Second Edition (Cold Spring Harbor Laboratory Press, New York (1989)). And a method such as PCR is used to introduce a mutation into the DNA sequence of the present invention, and an appropriate vector and host are used. For example, Molecular Cloning: A Laboratory Manual Second Edition (Cold Spring Harbor Laboratory Press, New York) (198) .)) By expressing genetically engineered DNA with mutations. It is possible.
Using the protein of the present invention, a partial peptide thereof or a polypeptide containing a partial peptide as an antigen, it can be prepared according to a method for producing an antibody or antiserum known per se. For example, an antibody can be produced by administering the protein of the present invention, a partial peptide thereof or a polypeptide containing a partial peptide as an antigen to a mammal. The antigen itself may be used, or an antigen bound to a carrier such as bovine serum albumin (BSA) may be used. In addition, in order to enhance the immune response due to the antigen, Freund's complete adjuvant (CFA) and incomplete adjuvant (IFA) may be administered, for example. As mammals used for immunization, mice, rats, rabbits, goats and the like can be used.
Polyclonal antibodies 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))), etc. After the first administration of an antigen to a mammal, It can be produced by obtaining immunized mammals by administering 3 to 10 times every 2 weeks, and collecting and purifying serum from those mammals, which can be purified by various chromatography, centrifugation, and salting out. It can be performed by combining dialysis and the like.
After obtaining a sufficient antibody titer as described above, the monoclonal antibody is obtained by collecting spleen or lymph nodes from those mammals and fusing the antibody-producing cells obtained from them with myeloma cells. A monoclonal antibody-producing hybridoma can be obtained. As myeloma cells, cell lines established from mice or rats are used. The cell fusion method 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 liver disease therapeutic agent containing the polypeptide of the present invention, it is possible to administer the polypeptide of the present invention alone, but usually it is pharmacologically acceptable. It is desirable to provide a pharmaceutical preparation mixed with one or more carriers and manufactured in a manner well known in the art of pharmaceutics.
As the administration route, it is desirable to adopt the most effective method for the disease, and examples thereof include oral administration, parenteral administration such as intravenous, subcutaneous and intraperitoneal administration. The dosage form is preferably a form capable of efficiently and effectively transporting the active ingredient against a disease, and examples thereof include capsules, granules, tablets, powders, injections, transdermal agents, suppositories, and emulsions.
As a screening method for a compound that specifically changes the activity of the polypeptide of the present invention, the 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 for substances that alter the activity of the protein of the present invention, such as proteins, peptides, nucleic acids, non-peptide compounds, and the like, and include agonists and antagonists. Measuring and comparing 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 or salt thereof. A screening method for the substance of the present invention is provided. Further, the activity of the polypeptide of the present invention and the cells of the transformant when a mammalian substance or transformant expressing the polypeptide of the present invention is cultured and the test substance is contacted with the expressed polypeptide of the present invention. It is possible to screen for a substance that changes the activity of the polypeptide of the present invention or a salt thereof, characterized by measuring and comparing the number, mRNA expression level, and the like.
The compound obtained by the screening method of the present invention or a salt thereof is a compound having an action of changing the activity of the polypeptide of the present invention or a salt thereof. For example, it binds to the polypeptide of the present invention or a salt thereof and inhibits the activity. Or a salt thereof, or a compound or a salt thereof having an activity of binding to a DNA sequence encoding the polypeptide of the present invention and suppressing the expression of the protein. Examples of the compound include proteins, peptides, non-peptides, nucleic acids and the like. For example, an antibody against the polypeptide of the present invention and a DNA sequence described in SEQ ID NO: 1 or / and SEQ ID NO: 3 or a partial sequence thereof A base sequence having a complementary sequence can be used. These compounds may be novel compounds or known compounds.
Example
The invention is illustrated 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 increased expression from the FLS mouse liver by the cDNA subtraction method
Total RNA was prepared from the livers of 15-week-old FLS mice and DS mice by the AGPC (Acid Guanidinium Phenol Chloroform Method) method (Analytical Biochemistry, 162, 156-159 (1987)), and mRNA Purification Kit Poly (A) + RNA was prepared using CLONTECH PCR-Select using poly (A) + RNA derived from FLS mouse as a tester and Poly (A) + RNA derived from DS mouse as a driver ^TM DNA subtraction was performed using a cDNA Subtraction Kit (Clontech) according to the attached manual. The obtained cDNA was purified by QIAEX-II Gel Extraction System (Qiagen), and the pCR2 vector (Invitrogen) was used for DNA Ligation Kit Ver. 2 (Takara Shuzo Co., Ltd.) was used and reacted at 16 ° C. for 4 hours for insertion. The constructed vector was introduced into Escherichia coli JM109 strain and transformed, and then ampicillin (50 μg / ml), 5-Bromo-4-Chloro-3-indolyl-β-D-galactoside (40 μg / ml) and Isopropyl- The solution was applied to an LB agar medium containing β-D-Thio-Galactopyranoside (100 μM) and cultured at 37 ° C. overnight. After culturing, colonies that appeared on the plate were inoculated into 2 ml of LB liquid medium containing 50 μg / ml ampicillin, cultured at 37 ° C. overnight, and then collected by centrifugation. Recombinant DNA was extracted from the collected cells using QIAprep Spin Plasmid Miniprep Kit (manufactured by Qiagen) and purified. The obtained cDNA library was determined by a dye terminator method using a DNA sequencer 373S (Applied Biosystems). As a result of analyzing the base sequence of the obtained cDNA library using the analysis software BLAST, the amino acid sequence 1026-1126 and 66% of the amino acid sequence encoded by Drosophila melanogaster function unknown gene CG8863 and the nematode (Caenorhabditis elegans) function unknown gene F11A10 A clone pSF21A having a base sequence consisting of 329 bases represented by SEQ ID NO: 7 (SF21A) having 43% homology with the remaining 1023 to 1118 region encoded by .4 was obtained.
(2) Isolation of mouse SF21 full-length cDNA
Since SF21A does 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 (Takara Shuzo), fractionated by 0.8% agarose gel electrophoresis, DNA fragments were extracted, and QIAEX-II Gel Extraction System (Qiagen) Purification was performed using The obtained DNA was 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.
E. coli strain Y1090 in which mouse liver cDNA library Mouse Liver 5′-STRETCH PLUS cDNA (Clontech) was cultured overnight. ⁻ Added to the culture and incubated for 15 minutes at 37 ° C. To this, LB-top agarose (1 liter: 10 g tryptone, 5 g yeast extract, 5 g NaCl, 10 mM MgSO) previously warmed to 50 ° C. ₄ 7.2 g agarose), seeded uniformly on an LB-aggar plate, and cultured at 37 ° C. for 5 hours. After the plaque-formed plate was cooled at 4 ° C. for 1 hour, the plaque was transferred to a nylon membrane Hybond-N + (manufactured by Amersham Pharmacia Biotech). The membrane was alkali-denatured with a 0.5 M NaOH (containing 1.5 M NaCl) solution, DNA was immobilized, and then neutralized with 0.5 M Tris-HCl, pH 7.2 (containing 1.5 M NaCl). And washed with 2 × SSC. Hybridization was performed overnight at 55 ° C. using the probe prepared above for this membrane. The membrane was washed with 2 × SSC (containing 0.1% SDS) solution at room temperature twice for 10 minutes and with 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.
Plaque groups containing plaques corresponding to positive signals detected for radioactivity were isolated from the plate and 1 ml SM buffer (Tris-HCl, pH 7.5; 10 mM MgSO ₄ , 0.1 M NaCl, 0.01% gelatin, 50 mM) and eluted the phages at 4 ° C. for 16 hours, and then the phages were recovered in the supernatant by centrifugation at 13,000 rpm for 10 minutes. LB-MgSO so that the phage solution appears to be 10, 100, 1000 plaques ₄ It applied to the plate and positive plaques were screened by the same method as described above. As a result, the positive plaque was successfully isolated as a completely isolated plaque, the single clone was grown, DNA was extracted from the phage particle 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 (Stratagene), and DNA Ligation Kit Ver. 2 (Takara Shuzo Co., Ltd.) was used for ligation reaction at 16 ° C. for 4 hours. Escherichia coli strain JM109 was transformed with these plasmids, and after culturing by a method known per se, the cells were collected and DNA was extracted and purified. The base sequence was determined in the same manner as described above. As a result, clone pSF21B having the base sequence (SF21B) consisting of 2402 bases represented by SEQ ID NO: 8 was isolated.
Next, by preparing a radioisotope labeled probe from clone pSF21B by the same method as described above and screening a mouse cDNA library, a clone containing the base sequence (SF21C) consisting of 2016 base represented by SEQ ID NO: 9 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 each base sequence of the obtained SF21A to D, cDNA consisting of 4661 bases (SEQ ID NO: 3: 798th to 5459th) was obtained.
Furthermore, 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 mice as a template, and 5' RACE System for Rapid Amplification of cDNA Ends, version 2.0, in vitro manufactured by Gen. ) And operated according to the attached manual.
Three kinds of primers were synthesized based on the cDNA sequence consisting of 4661 bases obtained above.

As the anchor primer, the reagent attached to the 5 ′ RACE System, 5 ′ RACE Abbreviated Anchor Primer (AAP) and Abbreviated Universal Amplification Primer (AUAP) were used.
1 μg of distilled water and 1 μl of 2.5 μM SF28 primer (SEQ ID NO: 19) were added to 1 μg of total RNA obtained from a Balb / c mouse liver by a method known per se, and incubated at 70 ° C. for 10 minutes. A warmed solution A was prepared. In addition, 10 μl PCR Buffer (200 mM Tris-HCl, pH 8.4; containing 500 mM KCl) 2.5 μl, 25 mM MgCl ₂ Was mixed with 2.5 μl, 1 μl of 10 mM dNTP mix and 2.5 μl of 0.1 M DTT, respectively, and solution B kept at 50 ° C. was adjusted. Solution A, Solution B, and Super Script ^TM II Reverse Transcriptase (200 units / μl) was mixed at 1 μl, incubated at 42 ° C. for 50 minutes and 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 with the DNA Purification System (Invitrogen) attached to the kit. 225 μl of binding solution (6M NaI) was added to the reaction solution, subjected to GlassMAX DNA isolation spin cartridges and eluted with 50 μl of distilled water to obtain purified cDNA. 10 μl of purified cDNA with 4 μl of distilled water, 5 × tailing buffer (50 mM Tris-HCl, pH 8.4; 125 mM KCl, 7.5 mM MgCl ₂ 5 μl and 1 mM dCTP (5 μl) were added, treated at 94 ° C. for 2 minutes, and left on ice for 1 minute. 1 μl of terminal deoxynucleotidyl transferase (rTdT) was added and incubated at 37 ° C. for 10 minutes and at 65 ° C. for 10 minutes, and then left on ice for 5 minutes to obtain 5 ′ RACE cDNA.
The PCR (Polymerase Chain Reaction) method is TaKaRa LA Taq. ^TM (Takara Shuzo Co., Ltd.) was used. To 2.5 μl of the 5 ′ RACE cDNA 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), TaKaRa LA Taq ^TM (5 U / μl) mixed with 0.25 μl and distilled water (13.75 μl) once at 94 ° C for 2 minutes, 94 ° C for 1 minute, 55 ° C for 1 minute, 72 ° C for 2 minutes The first PCR was performed 30 times at 72 ° C. for 2 minutes once. 2.5 μl of 50 × diluted 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, TaKaRa LA Taq ^TM (5 U / μl) mixed with 0.25 μl and distilled water (13.75 μl) in a cycle of 94 ° C. for 2 minutes, 94 ° C. for 1 minute, 55 ° C. for 1 minute, 72 ° C. for 2 minutes. The second PCR was performed 20 times at 72 ° C. for 2 minutes once. The second PCR reaction solution was electrophoresed on a 1.2% agarose gel, and a DNA fragment of about 900 bp was collected and purified by QIAEX-II Gel Extraction System. The purified DNA was inserted into plasmid vector pCR2 (manufactured by Invitrogen), and the base 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 layout of the various SF21 base sequences obtained. As a result of determining the base sequence of the full-length cDNA of mouse SF21 based on this, it is clear that mouse SF21 is a protein consisting of 5124 bases in total length represented by SEQ ID NO: 3 and 1708 amino acid residues represented in SEQ ID NO: 4 Became.
Example 2
Isolation of human hSF21 cDNA
An attempt was made to clone a human gene showing homology with mouse SF21. Amino acid residues encoded by mouse SF21 Pro-Ala-Val-Arg-Lys-Ser-Ala-Gly-Gln-Thr (SEQ ID NO: 1035 to 1044) and Asp-Thr-Ala-Glu-Lys Two types of degenerate oligonucleotides were synthesized as primers for PCR based on -Gln-Trp-Ala-Glu-Thr (SEQ ID NO: 4: Nos. 1102 to 1111).

PCR was carried out using two types of primers SF-D3 and SF-D4 using human liver cDNA library Human River 5′-STRECH PLUS cDNA (Clontech) as a template.
First, the human liver cDNA library (2 × 10 ⁷ PFU / μl) TaKaRa Ex Taq to 0.5 μl ^TM (Manufactured by Takara Shuzo Co., Ltd.) 1.25 units of SF-D3 and SF-D4 primers each 50 pmol, 10 × Ex Taq Buffer (Mg ²⁺ plus) 5 μl and dNTP Mixture (2.5 m each) were added to a final concentration of 200 μM, and distilled water was added to a total volume of 50 μl to prepare a sample for PCR. Omni Gene (4 minutes at 94 ° C., 30 seconds at 94 ° C., 30 seconds at 55 ° C., 35 cycles at 72 ° C. for 30 seconds, and 3 minutes at 72 ° C. as a DNA thermal cycler. PCR was carried out using Thermo High Bayed). The amplified DNA fragment of about 250 bases was cloned into pCR2 vector (manufactured by Invitrogen), and the base 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 with the corresponding region of mouse SF21 in nucleotide sequence and 100% in amino acid sequence. From this, the isolated cDNA was considered to be a part of the gene corresponding to human of mouse SF21.
In order to obtain a full-length human hSF21 cDNA, Human Liver 5′-STRECH PLUS cDNA was screened by plaque hybridization using hSF21A as a probe. Plaque hybridization was performed according to the method used when mouse full-length cDNA of SF21 was obtained. By this method, a clone phSF21C having a base sequence consisting of 1166 bases (hSF21C) represented by SEQ ID NO: 13, a clone phSF21E having a base sequence consisting of 2825 bases (hSF21E) represented by SEQ ID NO: 14 and a table represented by SEQ ID NO: 15 Three clones of clone phSF21F having a base sequence (hSF21F) consisting of 3139 bases were obtained. As a result of analysis based on the base sequence of the obtained human hSF21A to F, cDNA comprising 5517 bases (SEQ ID NO: 1st to 250th to 5767th) was obtained.
In order to determine the base sequence around the 5 ′ end of the translation region in human as well as mouse, the 5 ′ end sequence was isolated by RACE method. 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 based on the human cDNA sequence consisting of 5517 bases obtained above as a primer.

In addition, GT11-LDF was synthesized as a primer specific for the cloning vector λgt11 sequence.

Human liver cDNA library Human Liver 5′-STRECH PLUS cDNA was diluted 100-fold with distilled water to 0.5 μl, 1.25 units of KlenTaq LA Polymerase Mix (Clontech), 50 pmol of each of the above primers, 10 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, containing 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 for PCR did. The sample was subjected to PCR using the DNA thermal cycler Omni Gene at 94 ° C. for 4 minutes once, 94 ° C. for 30 seconds, 68 ° C. for 3 minutes 25 times, and 68 ° C. for 3 minutes once. . 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 the pCR2 vector, and the nucleotide sequence was determined by the dye terminator method using the 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 various hSF21 nucleotide sequences are shown in FIG. As a result of determining the base sequence of the full-length cDNA of human hSF21 based on that, human hSF21 is a protein consisting of a base sequence consisting of 5883 bases represented by SEQ ID NO: 1 and a 1717 amino acid residue represented by SEQ ID NO: 2, respectively. It became clear that. Since the isolated human base sequence showed 82% homology with the mouse base sequence, the obtained human gene was considered to correspond to the mouse gene.
A BLAST search of the public database GenBank for the deduced amino acid sequence of human full-length hSF21 cDNA revealed that Drosophila melanogaster function unknown gene CG8863 (1669 residues) and 43%, nematode (Caenorhabditis elegans) function unknown gene F11A10.4 (1646 residues) ) And 29%, and homology of Schzosaccharomyces pombe function unknown gene C23D3.13C (1616 residues) and 17%, respectively. In addition, amino acid residues of human brefeldin A-inhibiting guanine nucleotide exchange factor 1 protein (BIG1) in which the regions of amino acid residues 213 to 397 and 639 to 931 of human hSF21 belong to the Sec7 / BIG1 family, respectively. It showed 23% homology with 421st to 591st and 989th to 1306th. In addition, the regions of amino acid residues 178 to 392 and 746 to 919 of human hSF21 are the Sec7p amino acid residues 470 to 671 and 1270 to 270 of yeast (Sccharomyces cerevisiae), respectively. It showed homology of 1443 with 22% and 19%. However, human hSF21 has a low homology in the region corresponding to the Sec7 domain, which is a catalytic domain for ARF, which is a substrate for BIG1 and Sec7p, and an amino acid sequence conserved in the Sec7 domain required for GEF activity against ARF is observed. Therefore, it was considered to be a gene encoding a novel protein different from the Sec7 / BIG1 family. Therefore, since the gene encoding human hSF21 protein has the characteristics as described above, 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 ) Base sequence of 705 bp represented by the 4503rd to 5207th positions described in SEQ ID NO: 1, obtained by digesting Blot (manufactured by Clontech) with human SecL-1 cDNA using restriction enzymes SphI and SpeI as probes Isotope [α- ³² Northern blot analysis was performed using the one labeled with P] dCTP (Amersham Pharmacia Biotech).
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. Hybridization was carried out by incubating MTN Blot in the hybridization solution thus prepared at 68 ° C. for 16 hours. The membrane was washed with 2 × SSC (containing 0.1% SDS) solution at room temperature four times for 15 minutes, and with 0.1 × SSC (containing 0.1% SDS) solution at 50 ° C. for 20 minutes. After washing twice, autoradiography was performed at −70 ° C. for 5 days. As a result, SecL-1 expression was observed in all tissues, and strong signals were observed particularly in the brain, heart, kidney, liver, and placenta. Since the size of the mRNA band was longer than that of the isolated cDNA, it was considered that the SecL-1 mRNA had 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 were added with a sequence recognized by XhoI, HSF63 was added with a sequence recognized by BstXI, HSF67 was added with a sequence recognized by ApaI, and HSF69 was added with a sequence recognized by KpnI at the 5 ′ end of the primer. .

Using the mRNA prepared from HEK293 cells as a template, PCR was performed so that 6 types of primers were combined with HSF53 and HSF63, HSF62 and HSF67, HSF66 and HSF69, and human SecL-1 was divided into three.
First, RNeasy Mini Kit (manufactured by Qiagen) was used for preparation of total RNA from HEK293 cells, and total RNA was prepared according to the attached manual. For reverse transcription reaction from the prepared total RNA to cDNA, SuperScript II Rnase H-Reverse Transcriptase (manufactured by Invitrogen) was used, and cDNA was prepared according to the attached manual. 50 μmol each of the above-mentioned combinations of 1.25 units of Pfu Turbo DNA Polymerase (manufactured by Stratagene), 0.5 pL of the prepared cDNA, and 10 × PCR Buffer (200 mM Tris-HCl, pH 8.75; 20 mM MgSO) ₄ , 100 mM (NH ₄ ) ₂ SO ₄ And 100 mM KCl, 1% Triton X-100, containing 1 mg / ml BSA) and dNTPs were added to a final concentration of 200 μM, and distilled water was added to a total volume of 50 μl to prepare a sample. Samples were prepared at a temperature of 99 ° C for 2 minutes, 94 ° C for 30 seconds, 65 ° C for 30 seconds, 75 ° C for 3 minutes, 35 times, 72 ° C for 1 minute, and DNA thermal cycler Omni Gene. PCR was performed.
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 (manufactured by 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 base 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, full-length human SecL-1 cDNA was reconstructed, and an expression vector was constructed.
The plasmid pCR-Blunt / HSF66-69 was digested with restriction enzymes XhoI and KpnI, and fractionated by electrophoresis on 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 (Stratagene) was also digested with the same restriction enzymes, and the HSF66-HSF69 fragment was incorporated to obtain the plasmid pKB-CMV / HSF66-69. Similarly, pCR-Blunt / HSF62-67 was digested with restriction enzymes XhoI and ApaI, and 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 enzyme, and the HSF62-67 fragment was incorporated to obtain plasmid pKB-CMV / HSF66-69 / HSF62-67. Subsequently, pCR-Blunt / HSF53-63 was digested with restriction enzymes XhoI and BstXI, and fractionated by electrophoresis on a 1.2% agarose gel to recover and purify the target fragment. 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.
A human fetal kidney-derived cell line 293 cells (ATCC: CRL-1573) were applied to a biocoat poly-D-lysine (Poly-D-Lycine: synthetic) 6-well micro test plate (Becton Dickinson) at 5 × 10 ⁵ 1 / well, D-MEM (high glucose) medium (manufactured by Invitrogen) (including 10% FBS (manufactured by Invitrogen)) was added in an appropriate amount, and 5% CO 2 was added. ₂ Incubated overnight at 37 ° C. 2 μg of hSecL-1 expression vector pBK-CMV / hSecL-1 was prepared with Opti-MEM Reduced-Serum Medium (1 ×) solution (Invitrogen) to a final volume of 100 μl, and SuperFect Transfection Reagent (Qiagen) 10 μl was added and mixed well, followed by incubation at room temperature for 5 to 10 minutes to form a SuperFect-DNA complex. An expression vector pBK-CMV introduced into cells by the same operation was used as a control. After adding 600 μl of D-MEM (containing 10% FBS) to each of these vectors and mixing well by pipetting, the culture solution is aspirated and removed from the 293 cell culture solution cultured above and washed once with a PBS solution. Added to the cells. This cell culture is 5% CO ₂ After culturing at 37 ° C. for 3 hours, the culture medium was removed by suction and washed once with a PBS solution. 2 ml of D-MEM medium (containing 10% FBS) is added to the cells thus transformed, and 5% CO 2 is added. ₂ The medium was cultured at 37 ° C. for 48 hours to obtain hSecL-1-expressing recombinant cells.
Example 5
Production of hSecL-1 antibody
A polypeptide consisting of the amino terminal 146 residue of human SecL-1 represented by SEQ ID NO: 5 was used as an antigen. The antigen peptide was GST Gene Fusion System (manufactured by Amersham Pharmacia Biotech) and operated according to the attached manual to prepare a GST fusion protein, and then a GST-removed 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 base sequence described in SEQ ID NO: 6. HSFS1 is the first to 26th nucleotide sequence of SEQ ID NO: 6 and the sequence recognized by EcoRI on the 5 ′ end side, and HSFS2 is the 413th to 438th nucleotide sequence on the 3 ′ end side of the stop codon. And a sequence recognized by XhoI was added.

0.5 μl of pBK-CMV / hSecL-1 and 1.25 units of PfuTurbo DNA Polymerase (Stratagene), primers HSFS1 and HSFS2 each 50 pmol, 10 × PCR Buffer (200 mM Tris-HCl, pH 8.3; 20 mM MgSO ₄ , 100 mM (NH ₄ ) ₂ SO ₄ , 100 mM KCl, 1% Triton X-100, 1 mg / ml BSA) was added to 5 μl and dNTPs to a final concentration of 200 μM, and distilled water was added to a total volume of 50 μl to prepare a sample for PCR. Samples were cycled at 99 ° C. for 2 minutes, 94 ° C. for 30 seconds, 60 ° C. for 30 seconds, 72 ° C. for 90 seconds 35 times, 72 ° C. for 7 minutes once, using the thermal cycler Omni Gene. PCR was performed. After the PCR reaction solution was fractionated by electrophoresis with 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 BL21 (DE3) strain to obtain a transformant. This transformant was added to 100 ml of 2 × YT medium and pre-cultured at 37 ° C. for 18 hours. 25 ml of the preculture solution is added to 2.5 L of 2 × YT medium and cultured until the OD value becomes 0.8. Thereafter, isopropyl-1-thio-β-D-galactopyranoside (Sigma) is added to the culture solution. Aldrich) was added to a final concentration of 0.15 mM, and the protein expression was induced by culturing for 30 minutes. 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 collected by centrifugation at 8,000 rpm and suspended, and Lysozyme (Sigma) was added. 1 ml of a 10 mg / ml solution of Aldrich was added and left in ice for 30 minutes. To this was added 15 ml of an STE solution containing 10% sarkosyl (Sigma Aldrich), and then the sonicated cells were centrifuged at 9,000 rpm to obtain a supernatant containing the fusion protein. In order to affinity purify the fusion protein from this supernatant, 10 ml of Glutathion Sepharose 4B gel (manufactured by Amersham Pharmacia Biotech) was added and gently mixed for 30 minutes to adsorb the fusion protein to the carrier. This carrier is packed in a column, washed 3 times with 20 ml of STE solution, and then washed with GST in an elution buffer (containing 75 mM HEPES, pH 7.4; 150 mM NaCl, 10 mM reduced glutathione, 5 mM dithiothreitol, 2% N-octyl glucoside). -The hSecL-1 fusion protein was eluted. The eluted fusion protein was dialyzed overnight in a PBS solution and replaced with PBS. Then, 250 μl of PreScission Protease solution (manufactured by Amersham Pharmacia Biotech) was added to the sample and 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 replaced with PBS. About 4 mg of hSecL-1 partial peptide was obtained from 15 L of the culture solution.
100 μg of hSecL-1 partial peptide was thoroughly mixed with 1 ml of Freund's complete adjuvant (Invitrogen) and injected into a New Zealand white rabbit. 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), 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 extracted with Extraction Buffer (20 mM Tris-HCl, pH 7.5; 0.25 M sucrose, 1 mM EDTA · 2Na, 3 mM MgCl. ₂ 0.1 ml of 10 mM 2-mercaptoethanol and protein inhibitor mixture (manufactured by Calbiochem) was added to prepare a cell extract. The cell extract thus prepared was fractionated by electrophoresis on SDS-polyacrylamide gel (3-8%), and then the protein was transferred from the gel to a PVDF membrane (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, 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 labeled antibody solution obtained by diluting horseradish peroxidase-labeled anti-rabbit IgG antibody (Amersham Pharmacia Biotech) 4000 times with PBS. The membrane was soaked and incubated for 1 hour at room temperature. Thereafter, the plate was washed twice with PBS (containing 0.05% Tween 20) for 15 minutes, and the washed membrane sensitized the signal emitted by the ECL (Enhanced Chemiluminescence) method to the X-ray film, and the protein that binds to the antibody was detected. Detected. As a result, a 190 kDa band predicted from human SecL-1 was detected in the sample prepared from the cells into which the hSecL-1 expression vector was introduced, and the obtained antiserum reacted specifically with the recombinant human SecL-1 protein. It turned out that it showed.
Example 6
Effects of human SecL-1 on cell proliferation and apoptosis
As described above, human SecL-1-expressing recombinant cells obtained by transforming human fetal kidney-derived cell line 293 cells (ATCC: CRL-1573) with pBK-CMV / hSecL-1, and cells into which pBK-CMV was introduced as a control were added. Add 2 ml of D-MEM (containing 10% FBS) medium to the well and add 5% CO ₂ Medium was cultured at 37 ° C. The number of cells on the first day and the fifth day after the culture was measured with CellTiter 96 A Queuous 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 highly expressing cell line compared to the control cell line (FIG. 2).
The involvement of apoptosis in cell growth inhibition 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 pBK-CMV / hSecL-1 introduced cells and pBK-CMV introduced cells was measured. The activity of Caspase-3 is measured by CaspACE ^TM The assay was performed using an Assay System Colorimetric (Promega) according to the attached manual. As a result, human SecL-1 highly expressing cells were found to have about 3-fold increase in caspase-3 activity compared to control cells (FIG. 3).
Next, an increase in the expression of the endogenous SecL-1 gene was observed when apoptosis was induced in human SecL-1 highly expressing cells. It is known that hydrogen peroxide solution stimulates apoptosis induction (FEBS Letter, 488, 123-132 (2001)). Thus, hydrogen peroxide water was added to the cell culture medium, and the mRNA was quantified over time by the RT-PCR method as the hSecL-1 gene expression level. Two types of primers, HSF28 and HSF29, were synthesized as PCR primers.

First, hydrogen peroxide water prepared at four levels of 0.1 μM, 0.3 μM, 0.6 μM, and 0.9 μM was added to human SecL-1-expressing 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 not added with hydrogen peroxide were cultured under the same conditions. Total RNA from each cell was prepared using RNeasy Mini Kit (Qiagen) according to the attached manual. For the reverse transcription reaction from the prepared total RNA to cDNA, SuperScriptII RnaseH-Reverse Transcriptase (manufactured by Invitrogen) was used, and cDNA was prepared according to the attached manual. 0.5 μl of the prepared cDNA was mixed with 1.25 units of Pfu Turbo DNA Polymerase (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, 99 PCR with DNA thermal cycler Omni Gene once at 2 ° C for 2 minutes, 94 ° C for 30 seconds, 60 ° C for 30 seconds, 72 ° C for 30 seconds, once at 72 ° C for 5 minutes Went. The PCR reaction solution was electrophoresed on a 1.2% agarose gel and confirmed as a band of about 480 bp. As a result, the number of viable cells decreased in inverse proportion to the concentration of cells added with hydrogen peroxide as compared with the control group (FIG. 4). In addition, the amount of human SecL-1 mRNA was significantly increased by treatment with hydrogen peroxide.
From the above results, it was considered that human SecL-1 is involved in suppression of cell proliferation accompanied by apoptosis induction.
Industrial applicability
The protein of the present invention, a partial peptide thereof or a salt thereof, and a base sequence encoding them are used to search for a new physiological function, to produce an antibody, to construct a recombinant expression system, and to assay a biological activity regulator using the expression system It can be used for development of screening systems for pharmaceutical systems and drug candidate compounds, development of reagents useful for diagnosis, development of pharmaceuticals such as prophylactic and therapeutic drugs, and the like.
[Sequence Listing]

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

Claims (15)

A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 or a salt thereof. A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4 or a salt thereof. The amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 comprises an amino acid sequence having at least one mutation selected from deletion, substitution or addition of one or several amino acids, and has cytostatic activity or apoptosis-inducing activity A polypeptide or a salt thereof. Polynucleotide having a nucleotide sequence encoding a polypeptide de according to any of claim 1, wherein 3. The polynucleotide according to claim 4 having the 117th to 5267th positions of the base sequence shown in SEQ ID NO: 1. The polynucleotide according to claim 4, which has the 66th to 5189th nucleotide sequence of SEQ ID NO: 3. It hybridizes under stringent conditions with a polynucleotide comprising the 117th to 5267th nucleotide sequences of the nucleotide sequence shown in SEQ ID NO: 1 or the 66th to 5189th nucleotide sequences of the nucleotide sequence shown in SEQ ID NO: 3, and cell proliferation A polynucleotide encoding a polypeptide having inhibitory activity or apoptosis-inducing activity. An expression vector comprising the polynucleotide according to any one of claims 4 to 7 . A transformant obtained by introducing the expression vector according to claim 8 into a host. The transformant according to claim 9 , wherein the host is an animal cell strain or an E. coli strain. Step of culturing the transformant according to claim 9 or 1 0 of claims and recovering the produced recombinant polypeptide from the culture medium, according to any of claim 1, wherein the third polypeptide de Production method. Antibodies that specifically recognize polypeptides de according to any of claim 1, wherein 3. Cytostatic agents containing polypeptide de according to any of claim 1, wherein 3. Apoptosis-inducing agent containing a polypeptide de according to any of claim 1, wherein 3. Using polypeptide de according to any range 1 to 3 claims a method of screening for a compound that specifically modulate the activity of a polypeptide according to any one of claims 1 3.

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