JP2004081196A - New protein and dna of the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new protein having activity of transporting organic anions, to provide a DNA which encodes the protein, to provide a method for screening a compound which promotes or inhibits the activity of the protein, and to provide the compound obtained by the method for screening the compound. <P>SOLUTION: This protein is useful as a diagnostic marker or the like of, for example, renal diseases, hepatic diseases, pancreatic diseases, immunological diseases associated with thymic disorders, genital diseases, gastrointestinal diseases, splenic diseases, cancers, respiratory diseases, myelitis, diabetes, hypertension, postischemic reperfusion injury, retinitis, central nervous diseases, dermal diseases, and diseases related to thyroid hormone. The compound which promotes or inhibits the activity of the protein is obtained by the method for screening the compound in which the protein is used. Further, the compound is used, for example, as a prophylactic or remedy for the diseases. <P>COPYRIGHT: (C)2004,JPO

Description

【００８２】
実施例５
マウスＴＣＨ２２９タンパク質をコードするｃＤＮＡの５’上流端のクローニング
ＰＣＲクローニングおよび５’ＲＡＣＥ　ＰＣＲ　クローニングによりマウスＴＣＨ２２９タンパク質をコードするｃＤＮＡの５’上流塩基配列を明らかにした。
２種のプライマーＤＮＡ〔プライマーｈ２４３Ｆ（配列番号：２８）およびプライマーｍＲ１（配列番号：２９）〕を用いて、マウス腎臓Ｍａｒａｔｈｏｎ−Ｒｅａｄｙ　ｃＤＮＡ（クロンテック社製）に対して、Ａｄｖａｎｔａｇｅ　２　ＤＮＡ　Ｐｏｌｙｍｅｒａｓｅ（クロンテック社製）により、以下の条件でＰＣＲを行った。
（１）９４℃３０秒間
（２）９４℃１０秒間−６３℃１０秒間−６８℃２分間を３５サイクル
（３）６８℃３分間
上記ＰＣＲ反応液５μｌにＰＣＲ　Ｐｒｏｄｕｃｔ　Ｐｒｅ−Ｓｅｑｕｅｎｃｉｎｇ　Ｋｉｔ（ユーエスビ社製）中のＥｘｏｎｕｃｌｅａｓｅＩとＳｈｒｉｍｐ　Ａｌｋａｌｉｎｅ　Ｐｏｓｐｈａｔａｓｅをそれぞれ１μｌ加え、３７℃・１５分、８５℃・１５分の反応を行った。これをプライマーｈ２４３Ｆ（配列番号：２８）、プライマーｍＲ１（配列番号：２９）およびＢｉｇＤｙｅ　Ｔｅｒｍｉｎａｔｏｒ　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　Ｋｉｔ（アプライドバイオシステムズ社製）を用いて反応を行い、増幅したＤＮＡ断片の塩基配列をＤＮＡシークエンサーＡＢＩ　ＰＲＩＳＭ　３１００　ＤＮＡアナライザ（アプライドバイオシステムズ社製）を用いて決定した。その結果、配列番号：４８に示す塩基配列を得た。
配列番号：４８を基にプライマーｍｒｒ５（配列番号：３０）およびプライマーｍｒｒ６（配列番号：３１）を設計した。２種のプライマーＤＮＡ〔プライマーＡＰ１（配列番号：３）およびプライマーｍｒｒ６（配列番号：３１）〕を用いて、マウス腎臓Ｍａｒａｔｈｏｎ−Ｒｅａｄｙ　ｃＤＮＡ（クロンテック社製）に対して、Ａｄｖａｎｔａｇｅ　２　ＤＮＡ　Ｐｏｌｙｍｅｒａｓｅ（クロンテック社製）により、以下の条件で一次ＰＣＲを行った。
（１）９４℃３０秒間
（２）９４℃１０秒間−６４℃４分間を４０サイクル
さらに、この一次ＰＣＲの産物を鋳型として、プライマーＡＰ２（配列番号：５）とプライマーｍｒｒ５（配列番号：３０）を用いて、Ａｄｖａｎｔａｇｅ　２　ＤＮＡ　Ｐｏｌｙｍｅｒａｓｅ（クロンテック社製）により以下の条件でｎｅｓｔｅｄ　ＰＣＲを行った。
（３）９４℃３０秒間
（４）９４℃１０秒間−６４℃３分間を３５サイクル
上記ｎｅｓｔｅｄ　ＰＣＲ反応液５μｌにＰＣＲ　Ｐｒｏｄｕｃｔ　Ｐｒｅ−Ｓｅｑｕｅｎｃｉｎｇ　Ｋｉｔ（ユーエスビ社製）中のＥｘｏｎｕｃｌｅａｓｅＩとＳｈｒｉｍｐ　Ａｌｋａｌｉｎｅ　Ｐｏｓｐｈａｔａｓｅをそれぞれ１μｌ加え、３７℃・１５分、８５℃・１５分の反応を行った。これをプライマーｍｒｒ５（配列番号：３０）およびＢｉｇＤｙｅ　Ｔｅｒｍｉｎａｔｏｒ　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　Ｋｉｔ（アプライドバイオシステムズ社製）を用いて反応を行い、増幅したＤＮＡ断片の塩基配列をＤＮＡシークエンサーＡＢＩ　ＰＲＩＳＭ　３１００　ＤＮＡアナライザ（アプライドバイオシステムズ社製）を用いて決定した。その結果、配列番号：４９に示す塩基配列を得た。
【００８３】
実施例６
マウスＴＣＨ２２９タンパク質をコードするｃＤＮＡの３’下流端のクローニング
３’ＲＡＣＥ　ＰＣＲ　クローニングによりマウスＴＣＨ２２９タンパク質をコードするｃＤＮＡの３’上流塩基配列を明らかにした。
２種のプライマーＤＮＡ〔プライマーＡＰ１（配列番号：３）およびプライマーｍＦ１（配列番号：３２）〕を用いて、マウス腎臓Ｍａｒａｔｈｏｎ−Ｒｅａｄｙ　ｃＤＮＡ（クロンテック社製）に対して、Ａｄｖａｎｔａｇｅ　２　ＤＮＡ　Ｐｏｌｙｍｅｒａｓｅ（クロンテック社製）により、以下の条件で一次ＰＣＲを行った。
（１）９４℃３０秒間
（２）９４℃１０秒間−６１℃４分間を４０サイクル
さらに、この一次ＰＣＲの産物を鋳型として、プライマーＡＰ２（配列番号：５）とプライマーｍｆｆ２（配列番号：３３）を用いて、Ａｄｖａｎｔａｇｅ　２　ＤＮＡ　Ｐｏｌｙｍｅｒａｓｅ（クロンテック社製）により以下の条件でｎｅｓｔｅｄ　ＰＣＲを行った。
（３）９４℃３０秒間
（４）９４℃１０秒間−６１℃３分間を３５サイクル
上記ｎｅｓｔｅｄ　ＰＣＲ反応液５μｌにＰＣＲ　Ｐｒｏｄｕｃｔ　Ｐｒｅ−Ｓｅｑｕｅｎｃｉｎｇ　Ｋｉｔ（ユーエスビ社製）中のＥｘｏｎｕｃｌｅａｓｅＩとＳｈｒｉｍｐ　Ａｌｋａｌｉｎｅ　Ｐｏｓｐｈａｔａｓｅをそれぞれ１μｌ加え、３７℃・１５分、８５℃・１５分の反応を行った。これをプライマーｍｆｆ２（配列番号：３３）およびＢｉｇＤｙｅ　Ｔｅｒｍｉｎａｔｏｒ　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　Ｋｉｔ（アプライドバイオシステムズ社製）を用いて反応を行い、増幅したＤＮＡ断片の塩基配列をＤＮＡシークエンサーＡＢＩ　ＰＲＩＳＭ　３１００　ＤＮＡアナライザ（アプライドバイオシステムズ社製）を用いて決定した。その結果、配列番号：５０に示す塩基配列を得た。
【００８４】
実施例７
マウスＴＣＨ２２９タンパク質をコードするｃＤＮＡのクローニング
２種のプライマーＤＮＡ〔プライマーｍＯＲＦＦ（４）（配列番号：３４）およびプライマーｍＯＲＦＲ（２１９５）（配列番号：３５）〕を用いて、マウス腎臓Ｍａｒａｔｈｏｎ−Ｒｅａｄｙ　ｃＤＮＡ（クロンテック社製）に対して、ｐｆｕ　ｔｕｒｂｏ　ＤＮＡ　Ｐｏｌｙｍｅｒａｓｅ（ストラタジーン社製）により、以下の条件（１）〜（３）で一次ＰＣＲを行った。
（１）９４℃３０秒間
（２）９４℃１０秒間−５６℃１０秒間−７２℃２分間を３５サイクル
（３）７２℃５分間
さらに、この一次ＰＣＲの産物を鋳型として、プライマーｍＯＲＦＦ（ａｔｇ）（配列番号：３６）とプライマーｍＯＲＦＲ（ｔｇａ）（配列番号：３７）を用いて、ｐｆｕ　ｔｕｒｂｏ　ＤＮＡ　Ｐｏｌｙｍｅｒａｓｅ（ストラタジーン社製）により以下の条件（４）〜（６）でｎｅｓｔｅｄ　ＰＣＲを行った。
（４）９４℃３０秒間
（５）９４℃１０秒間−５６℃１０秒間−７２℃２分間を３０サイクル
（６）７２℃５分間
上記ｎｅｓｔｅｄ　ＰＣＲ反応液１０μｌにＡｄｖａｎｔａｇｅ　２　ＤＮＡ　Ｐｏｌｙｍｅｒａｓｅ（クロンテック社製）を１．５μｌ加え、７２℃、１０分間の反応の後、Ｍｉｎ　Ｅｌｕｔｅ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　Ｋｉｔ（キアゲン社製）を用いて精製した。このＤＮＡを、ＴＯＰＯ　ＴＡ　Ｃｌｏｎｉｎｇ　Ｋｉｔ　（インビトロジェン社製）のプロトコールに従ってｐＣＲ２．１−ＴＯＰＯベクターへクローニングした。これをエシェリヒア　コリ（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）ＴＯＰ１０　ｃｏｍｐｅｔｅｎｔ　ｃｅｌｌ（インビトロジェン社製）に導入して形質転換した後、ｃＤＮＡ挿入断片を持つクローンをカナマイシンを含むＬＢ寒天培地で選択し、形質転換体を得た。個々のクローンをカナマイシンを含むＬＢ培地で一晩培養し、ＱＩＡｗｅｌｌ　８　Ｐｌａｓｍｉｄ　Ｋｉｔ　（キアゲン社製）を用いてプラスミドＤＮＡを調製し、プラスミドクローンｐＣＲ２．１−ｍＴＣＨ２２９の２クローン＃１、＃２および＃３を得た。これをプライマーＤＮＡ〔プライマーＭ１３Ｆ（配列番号：１５）、プライマーＭ１３Ｒ（配列番号：１６）、プライマーｈ２４３Ｆ（配列番号：２８）　、プライマーｍＲ１（配列番号：２９）　、プライマーｈ６５０Ｆ（配列番号：３８）、プライマーｈ９１０Ｆ（配列番号：３９）、プライマーｍｒｒ１（配列番号：４０）、プライマーｍｒｒ２（配列番号：４１）、プライマーｍｒｒ３（配列番号：４２）、プライマーＦ（４５）（配列番号：４３）　、プライマーｍ５２１Ｒ（配列番号：４４）、プライマーｍＦ１（配列番号：３２）、プライマーｍｆｆ２（配列番号：３３）、プライマーｍｒｒ６（配列番号：３１）〕およびＢｉｇＤｙｅ　Ｔｅｒｍｉｎａｔｏｒ　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　Ｋｉｔ（アプライドバイオシステムズ社製）を用いて反応を行い、挿入されているｃＤＮＡ断片の塩基配列をＤＮＡシークエンサーＡＢＩ　ＰＲＩＳＭ　３１００　ＤＮＡアナライザ（アプライドバイオシステムズ社製）を用いて決定した。その結果、取得した３クローンは同一のＤＮＡ断片を含んでおり２１６９個の塩基配列を有していた（配列番号：５１）。断片には７２２個のアミノ酸配列（配列番号：２６）がコードされており（配列番号：２７）、配列番号：２６で表されるアミノ酸配列を含有するタンパク質を、マウスＴＣＨ２２９タンパク質と命名した。
該ｃＤＮＡ断片を含むプラスミドを有する形質転換体を、エシェリヒア・コリ（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）ＴＯＰ１０／ｐＣＲ２．１−ｍＴＣＨ２２９と命名した。
Ｂｌａｓｔ　Ｐ（Ｎｕｃｌｅｉｃ　Ａｃｉｄｓ　Ｒｅｓ．　第２５巻、３３８９頁、１９９７年）を用いて公知データベースに対してホモロジー検索を行ったところ、該ｃＤＮＡはマウスで報告されている有機アニオントランスポータであるマウスＳＬＣ２１Ａ１１（Ｇｅｎｂａｎｋ：ＮＰ＿０７６３９７　）と塩基レベルで４３．２％、アミノ酸レベルで３７％の相同性を示した。またマウスＴＣＨ２２９は有機アニオントランスポータファミリーに属する新規遺伝子であるヒトＴＣＨ２２９と塩基レベルで８３％、アミノ酸レベルで８１％の相同性を示したことより、ヒトＴＣＨ２２９のマウスホモログであることが判明した（図５）。図中、膜貫通領域をＴＭ１〜１２で、またファミリー間で高く保存されているアミノ酸箇所で＊で示した。
【００８５】
実施例８
マウスＴＣＨ２２９遺伝子産物の組織分布の解析
マウスＴＣＨ２２９の配列から設計した２種のプライマーＤＮＡ、プライマーｍＴＭＦ（配列番号：４５）およびプライマーｍＴＭＲ（配列番号：４６）と、ＴａｑＭａｎプローブｍＰ１（配列番号：４７）を用いて、マウスの各組織のｃＤＮＡにおけるマウスＴＣＨ２２９の発現量をＴａｑＭａｎ　ＰＣＲにより測定した。
反応はＴａｑＭａｎ　Ｕｎｉｖｅｒｓａｌ　ＰＣＲ　Ｍａｓｔｅｒ　Ｍｉｘ（アプライドバイオシステムズ社製）を用いて、ＡＢＩ　ＰＲＩＳＭ　７９００　ｓｅｑｕｅｎｃｅ　ｄｅｔｅｃｔｉｏｎ　ｓｙｓｔｅｍ（アプライドバイオシステムズ社製）にて最初５０℃２分間、さらに９５℃１０分間おいた後で、９５℃で１５秒、６０℃で１分を１反応サイクルとして４０サイクル繰り返し、同時に検出を行った。
結果を図６に示す。
マウスＴＣＨ２２９遺伝子産物（ｍＲＮＡ）は、ｍａｕｓｅ　ＭＴＣ　ｐａｎｅｌ　ＩおよびＭＴＣ　ｐａｎｅｌ　ＩＩにおいては、前立腺で強い発現が見られ、骨髄、目、肺、腎臓においても比較的高い発現が見られた。
【００８６】
実施例９
ラットＴＣＨ２２９タンパク質をコードするｃＤＮＡの５’上流クローニングＰＣＲクローニングおよび５’ＲＡＣＥ　ＰＣＲ　クローニングによりラットＴＣＨ２２９タンパク質をコードするｃＤＮＡの５’上流塩基配列を明らかにした。
マウスＴＣＨ２２９の配列（配列番号：５１）を基に設計した２種のプライマーＤＮＡ、プライマーｍ１６３Ｆ（配列番号：５６）およびプライマーｍ２０８７Ｒ（配列番号：５７）を用いて、ラット腎臓Ｍａｒａｔｈｏｎ−Ｒｅａｄｙ　ｃＤＮＡ（クロンテック社製）に対して、Ａｄｖａｎｔａｇｅ　２　ＤＮＡ　Ｐｏｌｙｍｅｒａｓｅ（クロンテック社製）により、以下の条件でＰＣＲを行った。
（１）９４℃３０秒間
（２）９４℃１０秒間−６０℃１０秒間−６８℃２．５分間を３５サイクル
（３）６８℃５分間
上記ＰＣＲ反応液　５μｌにＰＣＲ　Ｐｒｏｄｕｃｔ　Ｐｒｅ−Ｓｅｑｕｅｎｃｉｎｇ　Ｋｉｔ（ユーエスビ社製）中のＥｘｏｎｕｃｌｅａｓｅＩとＳｈｒｉｍｐ　Ａｌｋａｌｉｎｅ　Ｐｏｓｐｈａｔａｓｅをそれぞれ１μｌ加え、３７℃・１５分、８５℃・１５分の反応を行った。これをプライマーｍ１６３Ｆ（配列番号：５６）、プライマーｍ２０８７Ｒ（配列番号：５７）およびＢｉｇＤｙｅ　Ｔｅｒｍｉｎａｔｏｒ　Ｃｙｃｌｅ　ＳｅｑｕｅｎｃｉｎｇＫｉｔ（アプライドバイオシステムズ社製）を用いて反応を行い、増幅したＤＮＡ断片の塩基配列をＤＮＡシークエンサーＡＢＩ　ＰＲＩＳＭ　３１００　ＤＮＡアナライザ（アプライドバイオシステムズ社製）を用いて決定した。その結果、配列番号：７７に示す塩基配列を得た。
配列番号７７を基にプライマーｒ２２９−ｒｒ１（配列番号：５８）およびプライマーｒ２２９−ｒｒ２（配列番号：５９）を設計した。２種のプライマーＤＮＡ、プライマーＡＰ１（配列番号：３）およびプライマーｒ２２９−ｒｒ１（配列番号：５８）を用いて、ラット腎臓Ｍａｒａｔｈｏｎ−Ｒｅａｄｙ　ｃＤＮＡ（クロンテック社製）に対して、Ａｄｖａｎｔａｇｅ　２　ＤＮＡ　Ｐｏｌｙｍｅｒａｓｅ（クロンテック社製）により、以下の条件で一次ＰＣＲを行った。
（１）９４℃３０秒間
（２）９４℃１０秒間−６４℃２分間を３５サイクル
さらに、この一次ＰＣＲの産物を鋳型として、プライマーＡＰ２（配列番号：５）とプライマーｒ２２９−ｒｒ２（配列番号：５９）を用いて、Ａｄｖａｎｔａｇｅ　２　ＤＮＡ　Ｐｏｌｙｍｅｒａｓｅ（クロンテック社製）により以下の条件でｎｅｓｔｅｄ　ＰＣＲを行った。
（３）９４℃３０秒間
（４）９４℃１０秒間−６４℃２分間を３５サイクル
上記ｎｅｓｔｅｄ　ＰＣＲ反応液　５μｌにＰＣＲ　Ｐｒｏｄｕｃｔ　Ｐｒｅ−Ｓｅｑｕｅｎｃｉｎｇ　Ｋｉｔ（ユーエスビ社製）中のＥｘｏｎｕｃｌｅａｓｅＩとＳｈｒｉｍｐ　Ａｌｋａｌｉｎｅ　Ｐｏｓｐｈａｔａｓｅをそれぞれ１μｌ加え、３７℃・１５分、８５℃・１５分の反応を行った。これをプライマーｒ２２９−ｒｒ２（配列番号：５９）とＢｉｇＤｙｅ　Ｔｅｒｍｉｎａｔｏｒ　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　Ｋｉｔ（アプライドバイオシステムズ社製）を用いて反応を行い、増幅したＤＮＡ断片の塩基配列をＤＮＡシークエンサーＡＢＩ　ＰＲＩＳＭ　３１００　ＤＮＡアナライザ（アプライドバイオシステムズ社製）を用いて決定した。その結果、配列番号：７８に示す塩基配列を得た。
【００８７】
実施例１０
ラットＴＣＨ２２９タンパク質をコードするｃＤＮＡの３’下流端のクローニング
３’ＲＡＣＥ　ＰＣＲ　クローニングによりラットＴＣＨ２２９タンパク質をコードするｃＤＮＡの３’下流塩基配列を明らかにした。
２種のプライマーＤＮＡ、プライマーＡＰ１（配列番号：３）およびプライマーｒｆｆ１（配列番号：６０）を用いて、ラット腎臓Ｍａｒａｔｈｏｎ−Ｒｅａｄｙ　ｃＤＮＡ（クロンテック社製）に対して、Ａｄｖａｎｔａｇｅ　２　ＤＮＡ　Ｐｏｌｙｍｅｒａｓｅ（クロンテック社製）により、以下の条件で一次ＰＣＲを行った。
（１）９４℃３０秒間
（２）９４℃１０秒間−６４℃２分間を３５サイクル
さらに、この一次ＰＣＲの産物を鋳型として、プライマーＡＰ２（配列番号：５）とプライマーｒｆｆ３（配列番号：６１）を用いて、Ａｄｖａｎｔａｇｅ　２　ＤＮＡ　Ｐｏｌｙｍｅｒａｓｅ（クロンテック社製）により以下の条件でｎｅｓｔｅｄ　ＰＣＲを行った。
（３）９４℃３０秒間
（４）９４℃１０秒間−６４℃２分間を３０サイクル
上記ｎｅｓｔｅｄ　ＰＣＲ反応液　５μｌにＰＣＲ　Ｐｒｏｄｕｃｔ　Ｐｒｅ−Ｓｅｑｕｅｎｃｉｎｇ　Ｋｉｔ（ユーエスビ社製）中のＥｘｏｎｕｃｌｅａｓｅＩとＳｈｒｉｍｐ　Ａｌｋａｌｉｎｅ　Ｐｏｓｐｈａｔａｓｅをそれぞれ１μｌ加え、３７℃・１５分、８５℃・１５分の反応を行った。これをプライマーｒｆｆ３（配列番号：６１）およびＢｉｇＤｙｅ　Ｔｅｒｍｉｎａｔｏｒ　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　Ｋｉｔ（アプライドバイオシステムズ社製）を用いて反応を行い、増幅したＤＮＡ断片の塩基配列をＤＮＡシークエンサーＡＢＩ　ＰＲＩＳＭ　３１００　ＤＮＡアナライザ（アプライドバイオシステムズ社製）を用いて決定した。その結果、配列番号：７９に示す塩基配列を得た。
【００８８】
実施例１１
ラットＴＣＨ２２９タンパク質をコードするｃＤＮＡのクローニング
２種のプライマーＤＮＡ、プライマーｒＯＲＦＦ１（配列番号：６２）およびプライマーｒＯＲＦＲ１２（配列番号：６３）を用いて、ラット腎臓Ｍａｒａｔｈｏｎ−Ｒｅａｄｙ　ｃＤＮＡ（クロンテック社製）に対して、ｐｆｕ　ｔｕｒｂｏ　ＤＮＡ　Ｐｏｌｙｍｅｒａｓｅ（ストラタジーン社製）により、以下の条件（１）〜（３）で一次ＰＣＲを行った。
（１）９４℃３０秒間
（２）９４℃１０秒間−５６℃１０秒間−７２℃２．５分間を３５サイクル
（３）７２℃５分間
さらに、この一次ＰＣＲの産物を鋳型として、プライマーｒＯＲＦＦ（ａｔｇ）（配列番号：６４）とプライマーｒＯＲＦＲ（ｔｇａ２）（配列番号：６５）を用いて、ｐｆｕ　ｔｕｒｂｏ　ＤＮＡＰｏｌｙｍｅｒａｓｅ（ストラタジーン社製）により以下の条件（４）〜（６）でｎｅｓｔｅｄ　ＰＣＲを行った。
（４）９４℃３０秒間
（５）９４℃１０秒間−５６℃１０秒間−７２℃２．５分間を３０サイクル
（６）７２℃５分間
上記ｎｅｓｔｅｄ　ＰＣＲ反応液１０μｌにＡｄｖａｎｔａｇｅ　２　ＤＮＡ　Ｐｏｌｙｍｅｒａｓｅ（クロンテック社製）を１．５μｌ加え、７２℃、１０分間の反応の後、Ｍｉｎ　Ｅｌｕｔｅ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　Ｋｉｔ（キアゲン社製）を用いて精製した。このＤＮＡを、ＴＯＰＯ　ＴＡ　Ｃｌｏｎｉｎｇ　Ｋｉｔ　（インビトロジェン社製）のプロトコールに従ってｐＣＲＩＩ−ＴＯＰＯベクターへクローニングした。これをエシェリヒア　コリ（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）ＴＯＰ１０　ｃｏｍｐｅｔｅｎｔ　ｃｅｌｌ（インビトロジェン社製）に導入して形質転換した後、ｃＤＮＡ挿入断片を持つクローンをカナマイシンを含むＬＢ寒天培地で選択し、形質転換体を得た。個々のクローンをカナマイシンを含むＬＢ培地で一晩培養し、ＱＩＡｗｅｌｌ　８　Ｐｌａｓｍｉｄ　Ｋｉｔ　（キアゲン社製）を用いてプラスミドＤＮＡを調製し、プラスミドクローンｐＣＲＩＩ−ｒＴＣＨ２２９の４クローン＃１、＃２、＃３および＃４を得た。これをプライマーＤＮＡ〔プライマーＴ７（配列番号：６６）、プライマーＳＰ６（配列番号：６７）、プライマーｒＯＲＦＦ（ａｔｇ）（配列番号：６４）、プライマーｒＯＲＦＲ（ｔｇａ２）（配列番号：６５）、プライマーｒＦ１（配列番号：６８）、プライマーｒＴＭＲ（配列番号：６９）、プライマーｒＴＭＦ（配列番号：７０）、プライマーｒｆｆ３（配列番号：６１）、プライマーｒｆｆ２（配列番号：７１）、プライマーｒＲ１（配列番号：７２）　、プライマーｒ９７２Ｆ（配列番号：７３）、プライマーｒ１１２３Ｆ（配列番号：７４）、プライマーｒ１７４６Ｒ（配列番号：７５）〕およびＢｉｇＤｙｅ　Ｔｅｒｍｉｎａｔｏｒ　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　Ｋｉｔ（アプライドバイオシステムズ社製）を用いて反応を行い、挿入されているｃＤＮＡ断片の塩基配列をＤＮＡシークエンサーＡＢＩ　ＰＲＩＳＭ　３１００　ＤＮＡアナライザ（アプライドバイオシステムズ社製）を用いて決定した。その結果、取得した４クローンは２種類のＤＮＡ断片（Ｎｏ．１およびＮｏ．２）を含んでいた。Ｎｏ．１は２１７５個の塩基配列を有しており（配列番号：８０）、断片には７２４個のアミノ酸配列（配列番号：５２）がコードされており（配列番号：５３）、配列番号：５２で表されるアミノ酸配列を含有していた。Ｎｏ．２は２１７５個の塩基配列を有しており（配列番号：８１）、断片には７２４個のアミノ酸配列（配列番号：５４）がコードされており（配列番号：５５）、配列番号：５５で表されるアミノ酸配列を含有していた。それぞれのタンパク質を、ラットＴＣＨ２２９タンパク質Ｎｏ．１およびラットＴＣＨ２２９タンパク質Ｎｏ．２と命名した。該ｃＤＮＡ断片を含むプラスミドを有する形質転換体をそれぞれ、エシェリヒア・コリ（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）ＴＯＰ１０／ｐＣＲＩＩ−ｒＴＣＨ２２９Ｎｏ．１およびエシェリヒア・コリ（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）ＴＯＰ１０／ｐＣＲＩＩ−ｒＴＣＨ２２９Ｎｏ．２と命名した。
ラットＴＣＨ２２９タンパク質Ｎｏ．１およびラットＴＣＨ２２９タンパク質Ｎｏ．２には、４箇所（配列番号：５３で表される塩基配列の１４４番目、２３１番目、１２５２番目および１５３９番目）の塩基置換が認められた。４箇所のうちＡ１４４Ｔ（Ｇｌｕ→Ａｓｐ）、Ｃ２３１Ａ（Ｓｅｒ→Ａｒｇ）、Ａ１２５２Ｔ（ＩＩｅ→Ｐｈｅ）の３箇所は、それぞれ括弧内に示したアミノ酸置換を伴うものであったが、Ａ１５３９Ｇはアミノ酸置換は無かった。これらの塩基置換は遺伝子多型（ＳＮＰｓ）に由来する可能性があると考えられる。
Ｂｌａｓｔ　Ｐ（Ｎｕｃｌｅｉｃ　Ａｃｉｄｓ　Ｒｅｓ．　第２５巻、３３８９頁、１９９７年）を用いて公知データベースに対してホモロジー検索を行ったところ、ラットＴＣＨ２２９Ｎｏ．１およびＮｏ．２は、ラットで報告されている有機アニオントランスポータであるラットｏａｔｐ−Ｅ（Ｅｎｄｏｃｒｉｎｏｌｏｇｙ　第１４２巻（５）、２００５頁、２００１年）と塩基レベルで約５０％、アミノ酸レベルで約４１％の相同性を示した。またラットＴＣＨ２２９Ｎｏ．１およびＮｏ．２は有機アニオントランスポータファミリーに属する新規遺伝子であるヒトＴＣＨ２２９と塩基レベルで約８１．４％、アミノ酸レベルで約８１．６％の相同性を示したことより、ヒトＴＣＨ２２９のラットホモログであることが判明した（図７および図８）。図中、膜貫通領域をＴＭ１〜１２で示す。
【００８９】
実施例１２
ラットＴＣＨ２２９遺伝子産物の組織分布の解析
ラットＴＣＨ２２９の配列から設計した２種のプライマーＤＮＡ、プライマーｒＴＭＦ（配列番号：７０）およびプライマーｒＴＭＲ（配列番号：６９）と、ＴａｑＭａｎプローブｒＰ１（配列番号：７６）を用いて、ラットの各組織（脾臓、胸腺、精巣、小腸、胃、皮膚、心臓、脳、肺、筋肉、腎臓）のｃＤＮＡにおけるラットＴＣＨ２２９の発現量をＴａｑＭａｎ　ＰＣＲにより測定した。
反応はＴａｑＭａｎ　Ｕｎｉｖｅｒｓａｌ　ＰＣＲ　Ｍａｓｔｅｒ　Ｍｉｘ（アプライドバイオシステムズ社製）を用いて、ＡＢＩ　ＰＲＩＳＭ　７９００　ｓｅｑｕｅｎｃｅ　ｄｅｔｅｃｔｉｏｎ　ｓｙｓｔｅｍ（アプライドバイオシステムズ社製）にて最初５０℃２分間、さらに９５℃１０分間おいた後で、９５℃で１５秒、６０℃で１分を１反応サイクルとして４０サイクル繰り返し、同時に検出を行った。結果を図９に示す。
ラットＴＣＨ２２９遺伝子産物（ｍＲＮＡ）は、Ｍｕｌｔｉｐｌｅ　Ｃｈｏｉｃｅ　ｃＤＮＡｓ　；　ＲａｔＫｉｔＩおよびＲａｔＫｉｔＩＩ（オリジーン社製）においては、肺と腎臓で強い発現が見られ、皮膚、脳においても発現が見られた。
【００９０】
実施例１３
マウスＴＣＨ２２９遺伝子産物の７週齢ＢＡＬＢ／ｃマウスにおける組織分布の解析
（１）正常マウス各組織のｃＤＮＡの調製
７週齢ＢＡＬＢ／ｃマウスの各組織〔大脳、小脳、海馬、延髄、脊髄、坐骨神経、皮膚、骨格筋、眼球、心臓、肺、気管、膵臓、腎臓、肝臓、前胃、後胃、十二指腸、空回腸、盲腸、結腸、直腸、脾臓、胸腺、骨髄、卵巣、子宮、前立腺、精巣　（卵巣および子宮は雌から、それ以外は雄から、各１〜１０匹分を採取）〕より、ＩＳＯＧＥＮ（ニッポンジーン社製）、またはＲＮｅａｓｙ　Ｍｉｎｉ　Ｋｉｔ（キアゲン社製）を用いてｔｏｔａｌ　ＲＮＡを調製した。調製したｔｏｔａｌ　ＲＮＡ　に対してＴａｑＭａｎ　Ｒｅｖｅｒｓｅ　Ｔｒａｎｓｃｒｉｐｔｉｏｎ　Ｒｅａｇｅｎｔｓ（アプライドバイオシステムズ社製）を用いて逆転写反応を行いｃＤＮＡを調製した。
（２）マウスＴＣＨ２２９遺伝子産物の組織分布の解析
実施例８で用いた２種のプライマーＤＮＡ、プライマーｍＴＭＦ（配列番号：４５）およびプライマーｍＴＭＲ（配列番号：４６）と、ＴａｑＭａｎプローブｍＰ１（配列番号：４７）を用いて、上記のマウス各組織のｃＤＮＡにおけるマウスＴＣＨ２２９の発現量（コピー数）をＴａｑＭａｎ　ＰＣＲにより測定した。同じｃＤＮＡについてＴａｑＭａｎ　ｒｏｄｅｎｔ　ＧＡＰＤＨ　ｃｏｎｔｒｏｌ　ｒｅａｇｅｎｔｓ（アプライドバイオシステムズ社製）を用いてｒｏｄｅｎｔ　ｇｌｙｃｅｒａｌｄｅｈｉｄｅ−３−ｐｈｏｓｐｈａｔｅ　ｄｅｈｙｄｒｏｇｅｎａｓｅ　（ＧＡＰＤＨ）の発現量（コピー数）も測定した。反応はＴａｑＭａｎ　Ｕｎｉｖｅｒｓａｌ　ＰＣＲ　Ｍａｓｔｅｒ　Ｍｉｘ（アプライドバイオシステムズ社製）を用いて、ＡＢＩ　ＰＲＩＳＭ　７９００　ｓｅｑｕｅｎｃｅ　ｄｅｔｅｃｔｉｏｎ　ｓｙｓｔｅｍ（アプライドバイオシステムズ社製）にて最初５０℃２分間、さらに９５℃１０分間おいた後で、９５℃で１５秒、６０℃で１分を１反応サイクルとして４０サイクル繰り返し、同時に検出を行った。
結果を図１０に示す。
マウスＴＣＨ２２９遺伝子産物（ｍＲＮＡ）は７週齢ＢＡＬＢ／ｃマウスの各組織においては、前立腺、気管、腎臓、骨髄で若干の発現が見られ、肺で高い発現が見られた。
【００９１】
実施例１４
（１）正常ラット各組織のｃＤＮＡの調製
１２週齢Ｗｉｓｔａｒラット雄の各組織（大脳、小脳、肝臓、腎臓、前立腺、心臓、肺、十二指腸、空回腸、結腸、皮膚、眼球）より、ＲＮｅａｓｙ　Ｍｉｎｉ　Ｋｉｔ（キアゲン社製）を用いてｔｏｔａｌ　ＲＮＡを調製した。調製したｔｏｔａｌ　ＲＮＡ　に対してＴａｑＭａｎ　Ｒｅｖｅｒｓｅ　Ｔｒａｎｓｃｒｉｐｔｉｏｎ　Ｒｅａｇｅｎｔｓ（アプライドバイオシステムズ社製）を用いて逆転写反応を行いｃＤＮＡを調製した。
（２）ラットＴＣＨ２２９遺伝子産物の組織分布の解析
実施例１２で用いた２種のプライマーＤＮＡ、プライマーｒＴＭＦ（配列番号：７０）およびプライマーｒＴＭＲ（配列番号：６９）と、ＴａｑＭａｎプローブｒＰ１（配列番号：７６）を用いて、上記のラット各組織のｃＤＮＡにおけるラットＴＣＨ２２９の発現量（コピー数）をＴａｑＭａｎ　ＰＣＲにより測定した。同じｃＤＮＡについてＴａｑＭａｎ　ｒｏｄｅｎｔ　ＧＡＰＤＨ　ｃｏｎｔｒｏｌ　ｒｅａｇｅｎｔｓ（アプライドバイオシステムズ社製）を用いてｒｏｄｅｎｔ　ｇｌｙｃｅｒａｌｄｅｈｉｄｅ−３−ｐｈｏｓｐｈａｔｅ　ｄｅｈｙｄｒｏｇｅｎａｓｅ　（ＧＡＰＤＨ）の発現量（コピー数）も測定した。反応はＴａｑＭａｎ　Ｕｎｉｖｅｒｓａｌ　ＰＣＲ　Ｍａｓｔｅｒ　Ｍｉｘ（アプライドバイオシステムズ社製）を用いて、ＡＢＩ　ＰＲＩＳＭ　７９００　ｓｅｑｕｅｎｃｅ　ｄｅｔｅｃｔｉｏｎ　ｓｙｓｔｅｍ（アプライドバイオシステムズ社製）にて最初５０℃２分間、さらに９５℃１０分間おいた後で、９５℃で１５秒、６０℃で１分を１反応サイクルとして４０サイクル繰り返し、同時に検出を行った。
結果を図１１に示す。
ラットＴＣＨ２２９遺伝子産物（ｍＲＮＡ）は１２週齢Ｗｉｓｔａｒラットの組織においては、腎臓、肺で高い発現が見られた。
【００９２】
実施例１５
ヒトＴＣＨ２２９発現ベクターの構築
ヒトＴＣＨ２２９（配列番号：１）発現ベクターを、以下の方法により作成した。
実施例１により得られたプラスミド１０ｎｇを鋳型として、プライマー２２９ＯＦ２（配列番号：８２）およびプライマー２２９ＯＲ２（配列番号：８３）を用いて、ｐｆｕ　ｔｕｒｂｏ　ＤＮＡ　Ｐｏｌｙｍｅｒａｓｅ（ストラタジーン社製）により以下の条件（１）〜（３）でＰＣＲを行った。５’末端側プライマー２２９ＯＦ２および３’末端側プライマー２２９ＯＲ２は、ベクターへのクローニングのために５’末端側にそれぞれＥｃｏＲＶサイトおよびＸｈｏＩサイトを付加するように設計した。
（１）９４℃３０秒間
（２）９４℃１０秒間−５５℃１０秒間−７０℃２．５分間を３５サイクル
（３）７０℃５分間
上記ＰＣＲ反応液をゲル電気泳動後、主要バンドを精製した。これにより得られたＰＣＲ断片を、制限酵素ＥｃｏＲＶおよびＸｈｏＩを用いて、３７℃で１時間保温することにより消化し、この反応液をゲル電気泳動後、精製した。これを、動物細胞発現ベクターであるｐｃＤＮＡ３．１（＋）（インビトロジェン社製）のＥｃｏＲＶサイトおよびＸｈｏＩサイトに、Ｔａｋａｒａ　ｌｉｇａｔｉｏｎ　ｋｉｔ　ｖｅｒ．２（タカラバイオ社製）を用いてライゲーションした。このライゲーション反応液をエタノール沈殿処理後、コンピーテント細胞である大腸菌（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）ＴＯＰ１０（インビトロジェン社製）に形質転換した。これにより得られた複数のコロニーからプラスミドを調製し、この塩基配列をプライマーＤＮＡ〔プライマーＢＧＨ　ＲＶ（配列番号：８４）、プライマーＴ７（配列番号：６６）、プライマー２２９ＯＦ２（配列番号：８２）、プライマー２２９ＯＲ２（配列番号：８３）、プライマーｒｒ２（配列番号：６）、プライマーＡ１（配列番号：１７）、プライマーＢ２（配列番号：１８）、プライマーｆｆ２（配列番号：１０）、プライマーｒｒ１（配列番号：４）、プライマーｆｆ１（配列番号：９）〕〕、およびＢｉｇＤｙｅ　Ｔｅｒｍｉｎａｔｏｒ　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　Ｋｉｔ（アプライドバイオシステムズ社製）を用いて反応を行い、塩基配列をＤＮＡシークエンサーＡＢＩ　ＰＲＩＳＭ　３１００　ＤＮＡアナライザ（アプライドバイオシステムズ社製）を用いて確認した。このプラスミドを有する形質転換体を、Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ　ＴＯＰ１０／ｐＣＤＮＡ３．１（＋）−ＴＣＨ２２９と命名した。
【００９３】
実施例１６
ヒトＴＣＨ２２９発現ＣＨＯ細胞株の作製および導入遺伝子発現量の測定
Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ　ＴＯＰ１０／ｐＣＤＮＡ３．１（＋）−ＴＣＨ２２９を培養し、この大腸菌体からＥｎｄｏＦｒｅｅ　Ｐｌａｓｍｉｄ　Ｍａｘｉ　Ｋｉｔ（キアゲン社製）を用いてプラスミドＤＮＡを調製した。このプラスミドＤＮＡをＦｕＧＥＮＥ　６　Ｔｒａｎｓｆｅｃｔｉｏｎ　Ｒｅａｇｅｎｔ（ロシュ社製）を用いて添付のプロトコールに従ってＣＨＯ　ｄｈｆｒ⁻細胞に導入した。２μｇのプラスミドＤＮＡとトランスフェクション試薬との混合液を、２４時間前に２×１０^５個のＣＨＯ　ｄｈｆｒ⁻細胞を播種した直径６　ｃｍシャーレに添加した。１０％ウシ胎児血清（ＪＲＨバイオサイエンス社製）を含むＭＥＭα培地（インビトロジェン社製）で１日間培養した後培地に１．０ｍｇ／ｍｌのジェネティシン（インビトロジェン社製）を添加し、その後さらにＴＣＨ２２９発現細胞を培養した。途中何回か培地交換を行い、約１０日間後トリプシン処理により細胞をはがし、回収した細胞を１ｗｅｌｌあたり０．５〜１個で９６ｗｅｌｌ　ｐｌａｔｅに播種し、その後約１０日間１．０ｍｇ／ｍｌのジェネティシンを含んだ培地中でＴＣＨ２２９発現細胞を選択した。１ｗｅｌｌあたり１コロニーの増殖が見られたｗｅｌｌについて、増殖した細胞からＲＮｅａｓｙ　Ｍｉｎｉ　ＫｉｔまたはＲＮｅａｓｙ　９６　Ｋｉｔ（ともにキアゲン社製）を用いてｔｏｔａｌ　ＲＮＡを調製した。調製したｔｏｔａｌ　ＲＮＡ　に対してＴａｑＭａｎ　Ｒｅｖｅｒｓｅ　Ｔｒａｎｓｃｒｉｐｔｉｏｎ　Ｒｅａｇｅｎｔｓ（アプライドバイオシステムズ社製）を用いて逆転写反応を行いｃＤＮＡを調製した。これについて、実施例４で用いた２種のプライマーＤＮＡ、プライマーＴＭＦ（配列番号：１９）およびプライマーＴＭＲ（配列番号：２０）と、ＴａｑＭａｎプローブＰ１（配列番号：２１）を用いて、ヒトＴＣＨ２２９の発現量をＴａｑＭａｎ　ＰＣＲにより測定した。反応はＴａｑＭａｎ　Ｕｎｉｖｅｒｓａｌ　ＰＣＲ　Ｍａｓｔｅｒ　Ｍｉｘ（アプライドバイオシステムズ社製）を用いて、ＡＢＩ　ＰＲＩＳＭ　７９００　ｓｅｑｕｅｎｃｅ　ｄｅｔｅｃｔｉｏｎ　ｓｙｓｔｅｍ（アプライドバイオシステムズ社製）にて、最初５０℃２分間、さらに９５℃１０分間おいた後で、９５℃で１５秒、６０℃で１分を１反応サイクルとして４０サイクル繰り返し、同時に検出を行った。ヒトＴＣＨ２２９遺伝子高発現細胞株（モノクローナル）として、クローンＮｏ．７１を選択した。
【００９４】
実施例１７
ヒトＴＣＨ２２９遺伝子の市販正常ヒト細胞における発現解析
（１）正常ヒト細胞ｃＤＮＡの調製
正常ヒト細胞はＣａｍｂｒｅｘ　ＢｉｏＳｃｉｅｎｃｅ　Ｗａｌｋｅｒｓｖｉｌｌ社製品を購入し、製品添付の使用説明書記載の方法に従って培養した。実験に使用した細胞を表２に示す。
【表２】

各細胞を７５ｃｍ^２培養フラスコにサブコンフルエントになるよう培養した。表中の＊印のついた細胞については、ＴＮＦ−α、ＩＬ−１βおよびＩＬ−６が各々最終濃度が１０ｎｇ／ｍｌになるように加えた。＊印が付されていない細胞については培養開始から１６時間後に、＊印が付された細胞についてはＴＮＦ−α、ＩＬ−１βおよびＩＬ−６の混合物を添加してから８時間後に、各細胞を、トリプシン−ＥＤＴＡ処理により回収した。回収した細胞から、ＩＳＯＧＥＮ（ニッポンジーン社製）、またはＲＮｅａｓｙ　Ｍｉｎｉ　Ｋｉｔ（キアゲン社製）を用いてｔｏｔａｌ　ＲＮＡを調製した（いずれの場合もＤＮａｓｅ処理により混入ＤＮＡを除去した）。調製したｔｏｔａｌ　ＲＮＡ　に対してＴａｑＭａｎ　Ｒｅｖｅｒｓｅ　Ｔｒａｎｓｃｒｉｐｔｉｏｎ　Ｒｅａｇｅｎｔｓ（アプライドバイオシステムズ社製）を用いて逆転写反応を行いｃＤＮＡを調製した。
（２）種々の刺激を加えたヒト正常細胞ｃＤＮＡの調製
ａ）腎臓近位尿細管上皮細胞（ＲＰＴＥＣ）および腎臓皮質上皮細胞（ＨＲＣＥ）（いずれもＣａｍｂｒｅｘ　ＢｉｏＳｃｉｅｎｃｅ　Ｗａｌｋｅｒｓｖｉｌｌ社製）をコラーゲンＩコートした２４ｗｅｌｌプレートに１×１０^５個ずつ播種し、一晩培養後、トリヨードチロニン（Ｔ３）フリーのブレッドキットＲＥＧＭ（ＢＩＯ　ＷＨＩＴＴＡＫＥＲ社製）に置換し、更に３０分培養した。
ｂ）刺激剤としては、ＴＧＦ−β１（和光純薬社製）、ＴＮＦ−α（ジェンザイム社製）、ＩＬ−１β（ジェンザイム社製）、ＩＬ−６（ジェンザイム社製）およびＰＭＡ（和光純薬社製）を用いた。これら５種の刺激剤をそれぞれ、Ｔ３フリーのブレッドキットＲＥＧＭ（ＢＩＯ　ＷＨＩＴＴＡＫＥＲ社製）で最終濃度が１０ｎｇ／ｍｌになるよう希釈した溶液を準備した。
ｃ）上記ａ）のＲＰＴＥＣおよびＨＲＣＥの培養上清を除去後、直ちに上記ｂ）で調製した溶液を、１ｗｅｌｌあたり５００μｌずつ添加し、培養を行なった。コントロールとして、上記５種の刺激剤が溶けている溶媒の混合物を、Ｔ３フリーのブレッドキットＲＥＧＭ（ＢＩＯ　ＷＨＩＴＴＡＫＥＲ社製）で刺激剤と同じ割合で希釈した溶液を準備し、ＲＰＴＥＣおよびＨＲＣＥに１ｗｅｌｌあたり５００μｌずつ添加し、培養を行なった。
ｄ）刺激剤添加後、０．５、１、２、４、６および８時間後に、それぞれ細胞を回収し、ＲＮｅａｓｙ　Ｍｉｎｉ　Ｋｉｔ（キアゲン社製）を用いてｔｏｔａｌ　ＲＮＡを調製した。調製したｔｏｔａｌＲＮＡ　に対してＴａｑＭａｎ　Ｒｅｖｅｒｓｅ　Ｔｒａｎｓｃｒｉｐｔｉｏｎ　Ｒｅａｇｅｎｔｓ（アプライドバイオシステムズ社製）を用いて逆転写反応を行いｃＤＮＡを調製した。
（３）ヒトＴＣＨ２２９遺伝子の市販正常ヒト細胞における発現解析
実施例４で用いた２種のプライマーＤＮＡ、プライマーＴＭＦ（配列番号：１９）およびプライマーＴＭＲ（配列番号：２０）と、ＴａｑＭａｎプローブＰ１（配列番号：２１）を用いて、上記の正常ヒト細胞および刺激剤で刺激したＰＲＴＥＣおよびＨＲＣＥからそれぞれ調製したｃＤＮＡにおいて、ヒトＴＣＨ２２９の発現量（コピー数）をＴａｑＭａｎ　ＰＣＲにより測定した。上記の正常ヒト細胞および刺激剤で刺激したＲＰＴＥＣおよびＨＲＣＥからそれぞれ調製したｃＤＮＡについて、Ｅｕｋａｒｙｏｔｉｃ　１８Ｓ　ｒＲＮＡ　Ｐｒｅ−Ｄｅｖｅｌｏｐｅｄ　ＴａｑＭａｎ　Ａｓｓａｙ　Ｒｅａｇｅｎｔｓ（アプライドバイオシステムズ社製）を用いてｒｉｂｏｓｏｍａｌ　ＲＮＡ（１８Ｓ）の発現量（コピー数）、またはＨｕｍａｎ　ＧＡＰＤ（ＧＡＰＤＨ）（アプライドバイオシステムズ社製）を用いてｈｕｍａｎ　ｇｌｙｃｅｒａｌｄｅｈｉｄｅ−３−ｐｈｏｓｐｈａｔｅ　ｄｅｈｙｄｒｏｇｅｎａｓｅ　（ＧＡＰＤＨ）の発現量（コピー数）も測定した。反応は、ＴａｑＭａｎ　Ｕｎｉｖｅｒｓａｌ　ＰＣＲ　Ｍａｓｔｅｒ　Ｍｉｘ（アプライドバイオシステムズ社製）を用いて、ＡＢＩ　ＰＲＩＳＭ　７９００　ｓｅｑｕｅｎｃｅ　ｄｅｔｅｃｔｉｏｎ　ｓｙｓｔｅｍ（アプライドバイオシステムズ社製）にて最初５０℃２分間、さらに９５℃１０分間おいた後で、９５℃で１５秒、６０℃で１分を１反応サイクルとして４０サイクル繰り返し、同時に検出を行った。
ヒト正常細胞におけるヒトＴＣＨ２２９の発現変動を図１２に示す。表２において＊印が付された細胞についてはＴＮＦ−α、ＩＬ−１βおよびＩＬ−６（各々１０ｎｇ／ｍｌ）で刺激したが、無刺激の場合と比較して発現変動ははほとんど認められず、かつ発現もほとんど認められなかった。
刺激剤で刺激したＲＰＴＥＣにおけるヒトＴＣＨ２２９の発現変動を図１３に、刺激剤で刺激したＨＲＣＥにおけるヒトＴＣＨ２２９の発現変動を図１４に示す。
ヒトＴＣＨ２２９遺伝子産物（ｍＲＮＡ）は腎臓近位尿細管上皮細胞で特異的な発現が見られ、腎臓皮質上皮細胞で若干の発現が見られた。また、ＲＰＴＥＣよびＨＲＣＥを、ＴＧＦ−β１、ＴＮＦ−α、ＩＬ−１βまたはＰＭＡで各々で刺激したときにコントロールと比較して、ヒトＴＣＨ２２９の発現が培養時間依存的に減少することが確認できた。
【００９５】
実施例１８
腎疾患モデル（Ｗｉｓｔａｒ　Ｆａｔｔｙ，　ＳＨＣ，　Ｚｕｃｋｅｒ　Ｆａｔｔｙ）ラット腎臓におけるラットＴＣＨ２２９遺伝子産物の発現解析
疾患モデル動物として、Ｗｉｓｔａｒ　Ｆａｔｔｙ　ラット（ＷＦラット）、Ｚｕｃｋｅｒ　Ｆａｔｔｙ　ラット（ＺＦラット）、自然発症高コレステロール血症ラット（ＳＨＣラット）の３種類を用い、それぞれに対応する対照として、ＷＦラットに対してはＷｉｓｔａｒ　Ｌｅａｎ　Ｒａｔ（ＷＬラット）、ＺＦラットに対してはＺｕｃｋｅｒ　Ｌｅａｎ　Ｒａｔ（ＺＬラット）、ＳＨＣラットに対してはＳＤ　Ｒａｔ（ＳＤラット）を使用した。
Ｗｉｓｔａｒ　Ｆａｔｔｙラット（ＷＦラット）は、腎機能低下症状に加え糖尿病に特徴的は症状を示すことから、糖尿病性腎症モデルとして（Ｆｒｏｎｔｉｅｒｓ　ｉｎ　ｄｉａｂｅｔｅｓ　ｒｅｓｅａｒｃｈ，　ｌｅｓｓｏｎｓ　ｆｒｏｍ　ａｎｉｍａｌ　ｄｉａｂｅｔｅｓ　ＩＩ，　５３５−５４１，　１９８８年）、Ｚｕｃｋｅｒ　Ｆａｔｔｙラット（ＺＦラット）は、ＷＦラットと同様に腎機能低下と糖尿病の症状を合わせて呈することから、糖尿病性腎症モデルとして（Ｋｉｄｎｅｙ　ｉｎｔｅｒｎａｔｉｏｎａｌ，　５２巻，　Ｓ２１８−Ｓ２２０頁，　１９９７年）、自然発症高コレステロール血症ラット（ＳＨＣラット）は、ヒト巣状糸球体硬化症と類似の症状を示すことから、巣状糸球体硬化症モデルとして（日本腎臓学界誌，　３７巻，　９１−９９頁，　１９９５年）報告されている。
（１）疾患モデル動物と対照動物を同じ条件で飼育し、ＷＦラットおよびＷＬラットでは生後１３週（発症前）、２０週（発症後）、４２週（進展期）および６８週（腎機能低下期）に、ＳＨＣラットおよびＳＤラットでは生後６週（発症前）、１２週（発症後）、２０週（進展期）および２６〜３０週（腎機能低下期）に、ＺＦラットおよびＺＬラットでは生後８週（発症前）および２７週（発症後）に、以下のようにｔｏｔａｌ　ＲＮＡを抽出した。
ＷＦラット群（各週齢ｎ＝５）、ＷＬラット群（各週齢ｎ＝５）、ＳＨＣラット群（各週齢ｎ＝５）、ＳＤラット群（各週齢ｎ＝５）、ＺＦラット群（各週齢ｎ＝９）、ＺＬラット群（各週齢ｎ＝９）の腎臓を、乳頭部を含む割断面で約１００ｍｇになるようにスライスし、ＩＳＯＧＥＮを用いて、添付のマニュアルに従い、ｔｏｔａｌ　ＲＮＡを抽出した。さらにＱＩＡＧＥＮ　ＲＮｅａｓｙ　Ｍｉｎｉ　ｋｉｔ（キアゲン社製）およびＲＮａｓｅ−Ｆｒｅｅ　ＤＮａｓｅ　ｓｅｔ（キアゲン社製）を用いて、混入ＤＮＡを除去した。調製したｔｏｔａｌ　ＲＮＡ　に対してＴａｑＭａｎ　Ｒｅｖｅｒｓｅ　Ｔｒａｎｓｃｒｉｐｔｉｏｎ　Ｒｅａｇｅｎｔｓ（アプライドバイオシステムズ社製）を用いて逆転写反応を行いｃＤＮＡを調製した。
【００９６】
（２）　腎疾患モデルラット腎臓におけるラットＴＣＨ２２９遺伝子産物の発現解析
実施例１２で用いた２種のプライマーＤＮＡ、プライマーｒＴＭＦ（配列番号：７０）およびプライマーｒＴＭＲ（配列番号：６９）と、ＴａｑＭａｎプローブｒＰ１（配列番号：７６）を用いて、上記（１）で調製した腎疾患モデルラット腎臓ｃＤＮＡにおけるラットＴＣＨ２２９の発現量（コピー数）をＴａｑＭａｎ　ＰＣＲにより測定した。同じｃＤＮＡについてＴａｑＭａｎ　ｒｏｄｅｎｔ　ＧＡＰＤＨ　ｃｏｎｔｒｏｌ　ｒｅａｇｅｎｔｓ（アプライドバイオシステムズ社製）を用いてｒｏｄｅｎｔ　ｇｌｙｃｅｒａｌｄｅｈｉｄｅ−３−ｐｈｏｓｐｈａｔｅ　ｄｅｈｙｄｒｏｇｅｎａｓｅ　（ＧＡＰＤＨ）の発現量（コピー数）も測定した。反応はＴａｑＭａｎ　Ｕｎｉｖｅｒｓａｌ　ＰＣＲ　Ｍａｓｔｅｒ　Ｍｉｘ（アプライドバイオシステムズ社製）を用いて、ＡＢＩ　ＰＲＩＳＭ　７９００　ｓｅｑｕｅｎｃｅ　ｄｅｔｅｃｔｉｏｎ　ｓｙｓｔｅｍ（アプライドバイオシステムズ社製）にて最初５０℃２分間、さらに９５℃１０分間おいた後で、９５℃で１５秒、６０℃で１分を１反応サイクルとして４０サイクル繰り返し、同時に検出を行った。ＷＦラット群の結果を図１５、ＳＨＣラット群の結果を図１６に、ＺＦラット群の結果を図１７にそれぞれ示す。
ラットＴＣＨ２２９は３種類すべての腎疾患モデルラット腎臓で、腎障害が発症した後にその発現が各対照群と比べて減少した。このことから、ＴＣＨ２２９は腎症などの腎疾患に関与する可能性が考えられる。
【００９７】
【発明の効果】
本発明のタンパク質、ポリヌクレオチドおよび抗体などは、例えば腎疾患（例、腎不全、糸球体腎炎、糖尿病性腎症、巣状糸球体硬化症、ネフローゼ症候群、腎性浮腫、間質性腎炎、腎硬化症、尿毒症など）、肝臓疾患（例、肝硬変、肝炎、アルコール性肝臓疾患など）、膵臓疾患（例、膵炎など）、胸腺異常に伴う免疫疾患、生殖器疾患（例、前立腺肥大症、前立腺炎、精巣神経症、卵巣嚢腫など）、消化器性疾患（例、過敏性腸症候群、潰瘍性大腸炎、虚血性大腸炎、胃炎など）、脾臓疾患（例、脾機能亢進症、脾腫性症候群など）、癌（例、肺癌、腎臓癌、肝臓癌、非小細胞肺癌、卵巣癌、前立腺癌、胃癌、膀胱癌、乳癌、子宮頸部癌、結腸癌、直腸癌、膵臓癌、甲状腺癌、骨髄性白血病など）、呼吸器疾患（例、胸膜炎、肺炎、慢性閉塞性肺疾患、喘息など）、骨髄炎、糖尿病、高血圧、虚血後再灌流障害、網膜炎、中枢神経疾患（例、てんかん、アルツハイマー病、パーキンソン症候群、精神分裂病など）、皮膚疾患（例、アトピー性皮膚炎、脂漏性皮膚炎など、甲状腺ホルモン関連疾患（例、甲状腺ホルモン不応症、バセドウ病、クレチン病、甲状腺機能亢進症など））などの診断マーカー等として有用である。該タンパク質、ポリヌクレオチドまたは抗体などを用いるスクリーニング法により得られる該タンパク質の活性を促進または阻害する化合物は、該タンパク質遺伝子の発現を促進または阻害する化合物、該タンパク質の発現を促進または阻害する化合物などは、例えば、腎疾患（例、腎不全、糸球体腎炎、糖尿病性腎症、巣状糸球体硬化症、ネフローゼ症候群、腎性浮腫、間質性腎炎、腎硬化症、尿毒症など）、肝臓疾患（例、肝硬変、肝炎、アルコール性肝臓疾患など）、膵臓疾患（例、膵炎など）、胸腺異常に伴う免疫疾患、生殖器疾患（例、前立腺肥大症、前立腺炎、精巣神経症、卵巣嚢腫など）、消化器性疾患（例、過敏性腸症候群、潰瘍性大腸炎、虚血性大腸炎、胃炎など）、脾臓疾患（例、脾機能亢進症、脾腫性症候群など）、癌（例、肺癌、腎臓癌、肝臓癌、非小細胞肺癌、卵巣癌、前立腺癌、胃癌、膀胱癌、乳癌、子宮頸部癌、結腸癌、直腸癌、膵臓癌、甲状腺癌、骨髄性白血病など）、呼吸器疾患（例、胸膜炎、肺炎、慢性閉塞性肺疾患、喘息など）、骨髄炎、糖尿病、高血圧、虚血後再灌流障害、網膜炎、中枢神経疾患（例、てんかん、アルツハイマー病、パーキンソン症候群、精神分裂病など）、皮膚疾患（例、アトピー性皮膚炎、脂漏性皮膚炎など）、甲状腺ホルモン関連疾患（例、甲状腺ホルモン不応症、バセドウ病、クレチン病、甲状腺機能亢進症など）などの予防・治療剤などとして使用することができる。好ましくは腎疾患、甲状腺ホルモン関連疾患などの予防・治療剤として、さらに好ましくは糖尿病性腎症などの予防・治療剤として使用する。
【００９８】
【配列表】

【図面の簡単な説明】
【図１】ヒトＴＣＨ２２９、ヒトＳＬＣ２１Ａ１２およびヒトＯＡＴＰＲＰ４のアミノ酸配列の比較を表す図である。図中、ＴＣＨ２２９はヒトＴＣＨ２２９のアミノ酸配列を、ＳＬＣ２１Ａ１２はヒトＳＬＣ２１Ａ１２のアミノ酸配列を、ＯＡＴＰＲＰ４はヒトＯＡＴＰＲＰ４のアミノ酸配列を、ＴＭ１〜ＴＭ１２は膜貫通領域を示す。□は、ヒトＴＣＨ２２９に一致するアミノ酸を示す。（図２へつづく）
【図２】ヒトＴＣＨ２２９、ヒトＳＬＣ２１Ａ１２およびヒトＯＡＴＰＲＰ４のアミノ酸配列の比較を表す図である。図中、ＴＣＨ２２９はヒトＴＣＨ２２９のアミノ酸配列を、ＳＬＣ２１Ａ１２はヒトＳＬＣ２１Ａ１２のアミノ酸配列を、ＯＡＴＰＲＰ４はヒトＯＡＴＰＲＰ４のアミノ酸配列を、ＴＭ１〜ＴＭ１２は膜貫通領域を示す。□は、ヒトＴＣＨ２２９に一致するアミノ酸を示す。（図１のつづき）
【図３】ヒトＴＣＨ２２９遺伝子産物の各組織における発現量を表す図である。発現量はｃＤＮＡ溶液１μｌ当たりのコピー数で表した。
【図４】ヒトＴＣＨ２２９遺伝子産物の各組織における発現量を表す図である。発現量はｃＤＮＡ溶液１μｌ当たりのコピー数で表した。
【図５】ヒトＴＣＨ２２９およびマウスＴＣＨ２２９のアミノ酸配列の比較を表す図である。図中、ＴＣＨ２２９はヒトＴＣＨ２２９のアミノ酸配列を、ｍＴＣＨ２２９はマウスＴＣＨ２２９のアミノ酸配列を、膜貫通領域はＴＭ１〜１２、ファミリー間で高く保存されているアミノ酸箇所は＊で示した。□は、両者に一致するアミノ酸を示す。
【図６】マウスＴＣＨ２２９遺伝子産物の各組織における発現量を表す図である。発現量はｃＤＮＡ溶液１μｌ当たりのコピー数で表した。
【図７】ヒトＴＣＨ２２９とラットＴＣＨ２２９Ｎｏ．１およびＮｏ．２のアミノ酸配列の比較を表す図である。図中、ＴＣＨ２２９はヒトＴＣＨ２２９のアミノ酸配列を、ｒＴＣＨ２２９Ｎｏ．１はラットＴＣＨ２２９Ｎｏ．１のアミノ酸配列を、ｒＴＣＨ２２９Ｎｏ．２はラットＴＣＨ２２９Ｎｏ．２のアミノ酸配列を示す。ＴＭ１〜ＴＭ１２は膜貫通領域を示す。□は、３者に一致するアミノ酸を示す。（図８へつづく）
【図８】ヒトＴＣＨ２２９とラットＴＣＨ２２９Ｎｏ．１およびＮｏ．２のアミノ酸配列の比較を表す図である。図中、ＴＣＨ２２９はヒトＴＣＨ２２９のアミノ酸配列を、ｒＴＣＨ２２９Ｎｏ．１はラットＴＣＨ２２９Ｎｏ．１のアミノ酸配列を、ｒＴＣＨ２２９Ｎｏ．２はラットＴＣＨ２２９Ｎｏ．２のアミノ酸配列を示す。ＴＭ１〜ＴＭ１２は膜貫通領域を示す。□は、３者に一致するアミノ酸を示す。（図７のつづき）
【図９】ラットＴＣＨ２２９遺伝子産物の各組織における発現量を表す図である。発現量はｃＤＮＡ溶液１μｌ当たりのコピー数で表した。
【図１０】マウスＴＣＨ２２９遺伝子産物の各組織における発現量を表す図である。発現量は、ｃＤＮＡ溶液１μｌ当たりのマウスＴＣＨ２２９のコピー数を、等量の各組織ｃＤＮＡにおけるｒｏｄｅｎｔ　ＧＡＰＤＨのコピー数で割った値で表した。
【図１１】ラットＴＣＨ２２９遺伝子産物の各組織における発現量を表す図である。発現量は、相対的発現量を１００倍した値で表した。
【図１２】ヒト正常細胞におけるヒトＴＣＨ２２９の発現変動を表す図である。図中、縦軸は、ヒトＴＣＨ２２９遺伝子発現値のヒト１８Ｓ発現値に対する相対的発現値を１０００００倍した値を示す。横軸は細胞名を示す。■はＴＮＦ−α、ＩＬ−１βおよびＩＬ−６（各々１０ｎｇ／ｍｌ）刺激無し、□はＴＮＦ−α、ＩＬ−１βおよびＩＬ−６（各々１０ｎｇ／ｍｌ）刺激有りを示す。
【図１３】刺激剤で刺激したＲＰＴＥＣにおけるヒトＴＣＨ２２９の発現変動を表す図である。図中、縦軸は、ヒトＴＣＨ２２９遺伝子発現値のヒトＧＡＰＤＨ発現値に対する相対的発現値を１００倍した値を示す。横軸は刺激剤での反応時間を示す。−●−はＴＧＦ−β１、−■−はＰＭＡ、−▲−はＴＮＦ−α、−＊−はＩＬ−１β、−○−はＩＬ−６、−△−はコントロールを示す。
【図１４】刺激剤で刺激したＨＲＣＥにおけるヒトＴＣＨ２２９の発現変動を表す図である。図中、縦軸は、ヒトＴＣＨ２２９遺伝子発現値のヒトＧＡＰＤＨ発現値に対する相対的発現値を１００倍した値を示す。横軸は刺激剤での反応時間を示す。−●−はＴＧＦ−β１、−■−はＰＭＡ、−▲−はＴＮＦ−α、−＊−はＩＬ−１β、−○−はＩＬ−６、−△−はコントロールを示す。
【図１５】ラットＴＣＨ２２９遺伝子発現量を表す図である。図中、縦軸はラットＴＣＨ２２９遺伝子発現値のｒｏｄｅｎｔ　ＧＡＰＤＨ発現値に対する相対的発現値を１０００倍した値を、横軸は腎臓を採取した週齢を示す。□は実験群（ＷＦラット）、■は対照群（ＷＬラット）の結果である。
【図１６】ラットＴＣＨ２２９遺伝子発現量を表す図である。図中、縦軸はラットＴＣＨ２２９遺伝子発現値のｒｏｄｅｎｔ　ＧＡＰＤＨ発現値に対する相対的発現値を１０００倍した値を、横軸は腎臓を採取した週齢を示す。□は実験群（ＳＨＣラット）、■は対照群（ＳＤラット）の結果である。
【図１７】ラットＴＣＨ２２９遺伝子発現量を表す図である。図中、縦軸はラットＴＣＨ２２９遺伝子発現値のｒｏｄｅｎｔ　ＧＡＰＤＨ発現値に対する相対的発現値を１０００倍した値を、横軸は腎臓を採取した週齢を示す。□は実験群（ＺＦラット）、■は対照群（ＺＬラット）の結果である。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention provides a novel organic anion transporter (oatp / LST) protein, a DNA encoding the protein, a method for screening a compound that promotes or inhibits the activity of the protein, a compound obtained by the screening method, and the like.
[0002]
[Prior art]
The oatp / LST gene group (SLC21 family), which is an organic anion transporter, is a sodium-independent transporter, and takes up thyroid hormones into the central nervous system via the blood-brain barrier in vivo and bile from blood. It is known to play an essential role in maintaining homeostasis such as transfer of acids and drugs to the liver, removal of inflammatory mediators such as prostaglandins and leukotrienes, and excretion of foreign substances into bile and urine. ing. To date, 13 types of oatp / LST groups have been used in humans (eg, SLC21A3, SLC21A6, SLC21A11, SLC21A12 (Non-Patent Document 1 Biochemical and Biophysical Research Communications, 273, 251 pages, 2000)), and 10 in rats. For example, Slc21a1 and Slc21a5) have been reported, and the expression distribution is roughly classified into three types: those that are brain-specific, those that are liver-specific, and those that are widely expressed.
[Non-patent document 1]
Biochemical and Biophysical Research Communications, 273, 251 (2000)
[0003]
[Problems to be solved by the invention]
Organic anion transporters transport not only bile acids as substrates but also physiologically active substances such as thyroid hormones and conjugated steroids. Since many of these substrates are ligands for nuclear receptors, it is thought that the organic anion transporter plays an important role in first taking up ligands for nuclear receptors into cells. However, the detailed mechanism is not well understood. Therefore, elucidation of the role of these transporters will lead to the development of therapeutic agents for various diseases.
[0004]
[Means for Solving the Problems]
The present inventors have conducted intensive studies and, as a result, have found a novel organic anion transporter protein. As a method for suppressing the protein, for example, it is conceivable to inhibit the transport of an organic anion or to suppress the transcription of the protein to lower the expression level. Examples of a method for activating the protein include enhancing the expression level by promoting transport of organic anions, activating a promoter of the protein, or stabilizing mRNA.
The present inventors have further studied based on these findings, and have completed the present invention.
[0005]
That is, the present invention
(1) a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 26 or SEQ ID NO: 52, or a salt thereof;
(2) a protein comprising an amino acid sequence represented by SEQ ID NO: 1 or a salt thereof;
(3) a protein consisting of the amino acid sequence represented by SEQ ID NO: 26 or a salt thereof;
(4) a protein consisting of the amino acid sequence represented by SEQ ID NO: 52 or a salt thereof;
(5) the protein or salt thereof according to the above (1), wherein the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 52 is the amino acid sequence represented by SEQ ID NO: 54;
(6) a protein consisting of the amino acid sequence represented by SEQ ID NO: 54 or a salt thereof;
(7) a partial peptide of the protein according to (1) or a salt thereof;
(8) a polynucleotide containing a polynucleotide encoding the protein of (1) or the partial peptide of (7),
(9) the polynucleotide according to the above (8), which is a DNA;
(10) a polynucleotide having a base sequence represented by SEQ ID NO: 2, SEQ ID NO: 27, SEQ ID NO: 53, or SEQ ID NO: 55,
(11) a recombinant vector containing the polynucleotide according to (8),
(12) a transformant transformed with the recombinant vector according to (11),
(13) The transformant according to (1), wherein the transformant according to (12) is cultured to produce and accumulate the protein according to (1) or the partial peptide according to (7), and collect the protein. ) A method for producing the protein or the partial peptide according to (7) or a salt thereof,
(14) a medicine comprising the protein of (1) or the partial peptide of (7) or a salt thereof,
(15) a medicine comprising the polynucleotide according to the above (8),
(16) a diagnostic agent comprising the polynucleotide according to the above (8),
(17) an antibody against the protein according to (1) or the partial peptide according to (7) or a salt thereof,
(18) a diagnostic agent comprising the antibody according to (17),
(19) a medicine comprising the antibody according to (17),
(20) a polynucleotide comprising a nucleotide sequence complementary to or substantially complementary to the polynucleotide of (8) or a part thereof;
(21) a diagnostic agent comprising the polynucleotide according to (20),
(22) a medicine comprising the polynucleotide according to the above (20),
(23) The protein according to (1), the partial peptide according to (7), or a salt thereof, wherein the protein according to (1), the partial peptide according to (7), or a salt thereof is used. A method for screening a compound or a salt thereof that promotes or inhibits the activity,
(24) The activity of the protein according to (1) or the partial peptide according to (7) or a salt thereof, comprising the protein according to (1) or the partial peptide according to (7) or a salt thereof. A kit for screening for a compound that promotes or inhibits or a salt thereof,
(25) Promotes the activity of the protein of (1), the partial peptide of (7) or a salt thereof obtained by using the screening method of (23) or the screening kit of (24). Or a compound that inhibits or a salt thereof,
(25a) a compound or salt thereof which promotes the activity of the protein of (1) or the partial peptide of (7) or a salt thereof,
(26) a medicament comprising the compound according to (25) or a salt thereof,
(26a) a medicament comprising the compound of (25a) or a salt thereof,
(27) A method for screening a compound or a salt thereof that promotes or inhibits the expression of the protein gene according to (1), which comprises using the polynucleotide according to (8).
(28) A kit for screening a compound or a salt thereof that promotes or inhibits the expression of the protein gene according to (1), comprising the polynucleotide according to (8),
(29) A compound or a salt thereof that promotes or inhibits the expression of the protein gene according to (1), which is obtained by using the screening method according to (27) or the screening kit according to (28),
(29a) a compound that promotes expression of the protein gene according to (1) or a salt thereof,
(30) a medicament comprising the compound according to (29) or a salt thereof,
(30a) a medicine comprising the compound of the above (29a) or a salt thereof,
(31) The method for quantifying a protein according to (1), comprising using the antibody according to (17);
(32) A method for diagnosing a disease associated with the function of the protein according to (1), characterized by using the quantification method according to (31);
(33) a method for screening a compound or a salt thereof that promotes or inhibits the expression of the protein according to (1), which comprises using the antibody according to (17);
(34) A kit for screening a compound or a salt thereof that promotes or inhibits the expression of the protein according to (1), comprising the antibody according to (17);
(35) A compound or a salt thereof that promotes or inhibits the expression of the protein of (1), obtained by using the screening method of (33) or the screening kit of (34),
(35a) a compound or a salt thereof that promotes expression of the protein according to (1),
(36) a medicament comprising the compound according to (35) or a salt thereof,
(36a) a medicament comprising the compound of (35a) or a salt thereof,
(37) The medicament according to the above (14), (15), (19), (22), (26), (30) or (36), which is a prophylactic / therapeutic agent for kidney disease.
(37a) The medicament according to the above (26a), (30a) or (36a), which is a prophylactic / therapeutic agent for kidney disease.
(37b) the medicament according to the above (37) or (37a), wherein the renal disease is diabetic nephropathy;
(37c) The medicament according to the above (14), (15), (19), (22), (26), (30) or (36), which is a prophylactic / therapeutic agent for a thyroid hormone-related disease.
(37d) The medicament according to the above (26a), (30a) or (36a), which is a preventive / therapeutic agent for a thyroid hormone-related disease.
(37e) the medicament according to the above (37c) or (37d), wherein the thyroid hormone-related disease is thyroid hormone refractory;
(38) a method for preventing or treating renal disease, which comprises administering to a mammal an effective amount of the compound or a salt thereof according to (25), (29) or (35);
(39) Use of the compound according to (25), (29) or (35) or a salt thereof for producing a prophylactic / therapeutic agent for kidney disease is provided.
[0006]
BEST MODE FOR CARRYING OUT THE INVENTION
A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 26 or SEQ ID NO: 52 (hereinafter sometimes referred to as the protein of the present invention) is a human And cells of warm-blooded animals (eg, guinea pig, rat, mouse, chicken, rabbit, pig, sheep, cow, monkey, etc.) (eg, hepatocytes, spleen cells, nerve cells, glial cells, pancreatic β cells, bone marrow cells, Mesangial cells, Langerhans cells, epidermal cells, epithelial cells, goblet cells, endothelial cells, smooth muscle cells, fibroblasts, fiber cells, muscle cells, adipocytes, immune cells (eg, macrophages, T cells, B cells, natural killers) Cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts, mammary cells, liver cells Or stromal cells, or precursors of these cells, stem cells, or cancer cells) or any tissue in which those cells are present, such as the brain, various parts of the brain (eg, olfactory bulb, amygdala, basal sphere, hippocampus, Thalamus, hypothalamus, cerebral cortex, medulla, cerebellum), spinal cord, pituitary, stomach, pancreas, kidney, liver, gonad, thyroid, gall bladder, bone marrow, adrenal gland, skin, muscle, lung, digestive tract (eg, large intestine, small intestine) ), Proteins derived from blood vessels, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testis, ovary, placenta, uterus, bones, joints, skeletal muscle, etc., or synthetic proteins Good.
[0007]
The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 is about 50% or more, preferably about 60% or more, preferably about 70% or more as the amino acid sequence represented by SEQ ID NO: 1. Preferably, the amino acid sequence has a homology of about 80% or more, preferably about 90% or more, more preferably about 95% or more, and particularly preferably about 99% or more.
Examples of the protein containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 include, for example, a protein containing the same amino acid sequence as the above-mentioned amino acid sequence represented by SEQ ID NO: 1. And a protein having substantially the same activity as the protein having the amino acid sequence represented by SEQ ID NO: 1.
As the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 26, about 50% or more, preferably about 60% or more, preferably about 70% or more of the amino acid sequence represented by SEQ ID NO: 26 Preferably, the amino acid sequence has a homology of about 80% or more, preferably about 90% or more, more preferably about 95% or more, and particularly preferably about 99% or more.
Examples of the protein having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 26 include, for example, a protein having the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 26 described above. And a protein having substantially the same activity as the protein having the amino acid sequence represented by SEQ ID NO: 26.
As the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 52, about 50% or more, preferably about 60% or more, preferably about 70% or more as the amino acid sequence represented by SEQ ID NO: 52 Preferably, the amino acid sequence has a homology of about 80% or more, preferably about 90% or more, more preferably about 95% or more, and particularly preferably about 99% or more.
Examples of the protein having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 52 include, for example, a protein having the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 52 described above. And a protein having substantially the same activity as the protein having the amino acid sequence represented by SEQ ID NO: 52. Examples of the protein containing the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 52 include a protein containing the amino acid sequence represented by SEQ ID NO: 54.
Examples of the substantially equivalent activity include, for example, organic anion transport activity. Substantially homogenous indicates that the properties are homogenous (eg, physiologically or pharmacologically). Accordingly, it is preferable that the organic anions have the same transport activity (eg, about 0.01 to 100 times, preferably about 0.1 to 10 times, more preferably 0.5 to 2 times). Quantitative factors, such as the degree of molecular weight and protein molecular weight, may be different.
Examples of the organic anion include bile acids such as glucuronic acid, glutathione, and cholic acid, and thyroid hormone.
The activity such as the transport activity of the organic anion can be measured according to a known method. For example, the activity is measured according to the method described in Biochemical and Biophysical Research Communications, Vol. can do.
[0008]
Examples of the protein of the present invention include (1) (i) one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 (eg, about 1 to 200, preferably about 1 to 150, Preferably about 1 to 100, preferably about 1 to 50, preferably about 1 to 30, preferably about 1 to 10, and more preferably several (1 to 5) amino acids Sequence, (ii) one or two or more (for example, about 1 to 200, preferably about 1 to 150, preferably about 1 to 100, preferably 1 to 50) amino acid sequences represented by SEQ ID NO: 1. Amino acid sequence to which about 1, preferably about 1 to 30, preferably about 1 to 10, and more preferably number (1 to 5) amino acids have been added, (iii) the amino acid represented by SEQ ID NO: 1 Into an array Or two or more (eg, about 1 to 200, preferably about 1 to 150, preferably about 1 to 100, preferably about 1 to 50, preferably about 1 to 30, preferably 1 to 10 Degree, more preferably an amino acid sequence in which a number (1 to 5) of amino acids are inserted, and (iv) one or more (eg, about 1 to 200) in the amino acid sequence represented by SEQ ID NO: 1. Preferably about 1 to 150 pieces, preferably about 1 to 100 pieces, preferably about 1 to 50 pieces, preferably about 1 to 30 pieces, preferably about 1 to 10 pieces, and more preferably number (1 to 5) pieces. )) An amino acid sequence in which the amino acid is replaced with another amino acid, or (v) a so-called mutein such as a protein containing an amino acid sequence obtained by combining them.
(2) (i) one or more amino acids in the amino acid sequence represented by SEQ ID NO: 26 (for example, about 1 to 200, preferably about 1 to 150, preferably about 1 to 100, preferably 1 An amino acid sequence in which about 50 to about 50 amino acids are deleted, preferably about 1 to 30 amino acids, preferably about 1 to 10 amino acids, and more preferably several (1 to 5) amino acids. 1 or 2 or more (for example, about 1 to 200, preferably about 1 to 150, preferably about 1 to 100, preferably about 1 to 50, preferably about 1 to 30, Preferably, about 1 to 10, more preferably number (1 to 5) amino acids are added, and (iii) one or more (for example, 1 to 2) amino acid sequences represented by SEQ ID NO: 26 200 pieces Degree, preferably about 1 to 150 pieces, preferably about 1 to 100 pieces, preferably about 1 to 50 pieces, preferably about 1 to 30 pieces, preferably about 1 to 10 pieces, and more preferably a number (1 to 5 pieces). ) Amino acid sequence, and (iv) one or more (eg, about 1 to 200, preferably about 1 to 150, preferably about 1 to 200 in the amino acid sequence represented by SEQ ID NO: 26) About 1 to 100, preferably about 1 to 50, preferably about 1 to 30, preferably about 1 to 10, and more preferably several (1 to 5) amino acids are substituted with another amino acid. A so-called mutein such as a protein containing an amino acid sequence or (v) an amino acid sequence combining them;
(3) (i) 1 or 2 or more (eg, about 1 to 200, preferably about 1 to 150, preferably about 1 to 100, preferably 1) in the amino acid sequence represented by SEQ ID NO: 52 An amino acid sequence in which about 50, preferably about 1 to 30, preferably about 1 to 10, and more preferably a number (1 to 5) of amino acids have been deleted; (ii) an amino acid sequence represented by SEQ ID NO: 52 1 or 2 or more (for example, about 1 to 200, preferably about 1 to 150, preferably about 1 to 100, preferably about 1 to 50, preferably about 1 to 30, Preferably about 1 to 10, more preferably number (1 to 5) amino acids are added, and (iii) one or more (for example, 1 to 2) to the amino acid sequence represented by SEQ ID NO: 52 200 pieces Degree, preferably about 1 to 150 pieces, preferably about 1 to 100 pieces, preferably about 1 to 50 pieces, preferably about 1 to 30 pieces, preferably about 1 to 10 pieces, and more preferably a number (1 to 5 pieces). ) Amino acid sequence, and (iv) one or more amino acids in the amino acid sequence represented by SEQ ID NO: 52 (for example, about 1 to 200, preferably about 1 to 150, and preferably About 1 to 100, preferably about 1 to 50, preferably about 1 to 30, preferably about 1 to 10, and more preferably several (1 to 5) amino acids are substituted with another amino acid. So-called muteins, such as proteins containing the amino acid sequence or (v) an amino acid sequence combining them, are also included.
When the amino acid sequence is inserted, deleted or substituted as described above, the position of the insertion, deletion or substitution is not particularly limited.
[0009]
In the protein in the present specification, the left end is the N-terminus (amino terminus) and the right end is the C-terminus (carboxyl terminus) according to the convention of peptide labeling. The protein of the present invention including the protein containing the amino acid sequence represented by SEQ ID NO: 1 has a carboxyl group (—COOH), a carboxylate (—COO) ⁻ ), Amide (-CONH ₂ ) Or an ester (—COOR).
Here, as R in the ester, for example, C, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, etc. _1-6 Alkyl groups such as C, cyclopentyl, cyclohexyl, etc. _3-8 Cycloalkyl groups such as phenyl, α-naphthyl and the like; _6-12 Aryl groups, for example phenyl-C such as benzyl, phenethyl, etc. _1-2 An alkyl group or α-naphthyl-C such as α-naphthylmethyl _1-2 C such as an alkyl group _7-14 An aralkyl group, a pivaloyloxymethyl group and the like are used. When the protein of the present invention has a carboxyl group (or carboxylate) other than the C-terminus, the protein of the present invention includes a carboxyl group amidated or esterified. As the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.
Furthermore, in the protein of the present invention, the amino group of the N-terminal amino acid residue (eg, methionine residue) has a protecting group (eg, formyl group, acetyl group, etc.). _1-6 C such as alkanoyl _1-6 Acyl group), N-terminal glutamine residue generated by cleavage in vivo, pyroglutamine oxidation, substituent on the side chain of amino acid in the molecule (eg, -OH, -SH , An amino group, an imidazole group, an indole group, a guanidino group, etc.) are suitable protecting groups (for example, formyl group, acetyl group, etc.). _1-6 C such as alkanoyl group _1-6 (Eg, an acyl group), or a complex protein such as a so-called glycoprotein to which a sugar chain is bound.
Specific examples of the protein of the present invention include, for example, a protein containing the amino acid sequence represented by SEQ ID NO: 1, a protein containing the amino acid sequence represented by SEQ ID NO: 26, and a protein containing SEQ ID NO: 52 A protein containing the amino acid sequence; a protein containing the amino acid sequence represented by SEQ ID NO: 54;
[0010]
The partial peptide of the protein of the present invention is the partial peptide of the protein of the present invention described above, and preferably any peptide having the same properties as the protein of the present invention described above.
For example, of the constituent amino acid sequences of the protein of the present invention, for example, 10 or more, preferably 20 or more, preferably 50 or more, preferably 70 or more, preferably 100 or more, preferably 200 or more And the like.
The partial peptide used in the present invention has one or two or more (preferably about 1 to 20, more preferably about 1 to 10, and still more preferably about 1 to 5) amino acid sequences in the amino acid sequence. Or 1 or 2 or more (preferably about 1 to 20, more preferably about 1 to 10, and still more preferably, about 1 to 5) amino acids in the amino acid sequence. 1 or 2 or more (preferably about 1 to 20, more preferably about 1 to 10, and still more preferably, about 1 to 5) amino acids are added to the amino acid sequence, or In the amino acid sequence, one or more (preferably about 1 to 20, more preferably about 1 to 10, and still more preferably, about 1 to 5) amino acids are substituted with another amino acid. hand Good.
As the partial peptide of the present invention, for example, in the amino acid sequence represented by SEQ ID NO: 1, for example, the amino acid sequence at positions 340 to 370 and 490 to 520, the amino acid sequence represented by SEQ ID NO: 26 In the amino acid sequence represented by, for example, the 335th to 365th, the 490th to 520th, the amino acid sequence represented by SEQ ID NO: 52, for example, the 335th to 365th, the 490th to 520th amino acid sequences; In the amino acid sequence represented by SEQ ID NO: 54, for example, a peptide containing the 335th to 365th amino acids and the 490th to 520th amino acids is preferable.
[0011]
The partial peptide used in the present invention has a carboxyl group (—COOH) and a carboxylate (—COO) at the C-terminus. ⁻ ) Amide (-CONH ₂ )) Or an ester (—COOR).
Further, the partial peptides used in the present invention include those having a carboxyl group (or carboxylate) other than the C-terminus and N-terminal amino acid residues (eg, methionine), as in the above-mentioned protein of the present invention. Residue), the amino group of which is protected by a protecting group, the N-terminal side is cleaved in vivo, and the generated glutamine residue is pyroglutamine-oxidized, and the substituent on the side chain of the amino acid in the molecule is appropriate. Also included are those protected with a protecting group or complex peptides such as so-called glycopeptides to which sugar chains are bonded.
The partial peptide used in the present invention can also be used as an antigen for preparing an antibody. For example, for the purpose of preparing the antibody of the present invention described below, for example, in the amino acid sequence represented by SEQ ID NO: 1, for example, the amino acid sequence at positions 340 to 370 and 490 to 520, SEQ ID NO: 26 In the amino acid sequence represented by, for example, the 335th to 365th and 490th to 520th amino acid sequences, and in the amino acid sequence represented by SEQ ID NO: 52, for example, the 335th to 365th and 490th to In the 520th amino acid sequence and the amino acid sequence represented by SEQ ID NO: 54, for example, a peptide containing the 335th to 365th amino acids, the 490th to 520th amino acids, and the like is preferable.
[0012]
As the salt of the protein or partial peptide of the present invention, a salt with a physiologically acceptable acid (eg, an inorganic acid or an organic acid) or a base (eg, an alkali metal or the like) is used. Preferred acid addition salts are: Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid) Acids, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like are used.
The protein of the present invention, a partial peptide thereof, or a salt thereof can be produced from the above-mentioned method for purifying a protein from human or warm-blooded animal cells or tissues, or a transformant containing a DNA encoding the protein. Can also be produced by culturing. Further, it can be produced according to the peptide synthesis method described later.
When producing from human or mammalian tissues or cells, the homogenized human or mammalian tissues or cells are extracted with an acid or the like, and the extract is subjected to chromatography such as reverse phase chromatography or ion exchange chromatography. Can be purified and isolated.
[0013]
For the synthesis of the protein or partial peptide of the present invention, or a salt thereof, or an amide thereof, a commercially available resin for protein synthesis can be generally used. Examples of such a resin include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, and 4-hydroxymethylmethyl. Phenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ′, 4′-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2 ′, 4′-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin, and the like. it can. Using such a resin, an amino acid having an α-amino group and a side chain functional group appropriately protected is condensed on the resin in accordance with the sequence of the target protein according to various known condensation methods. At the end of the reaction, the protein or partial peptide is cleaved from the resin, and at the same time, various protecting groups are removed.In addition, an intramolecular disulfide bond formation reaction is performed in a highly diluted solution to obtain the target protein or partial peptide or an amide thereof. I do.
For the condensation of the above protected amino acids, various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable. As the carbodiimides, DCC, N, N′-diisopropylcarbodiimide, N-ethyl-N ′-(3-dimethylaminoprolyl) carbodiimide and the like are used. For activation by these, the protected amino acid was directly added to the resin together with a racemization inhibitor additive (for example, HOBt, HOOBt), or the protected amino acid was previously activated as a symmetric acid anhydride or HOBt ester or HOOBt ester. Later it can be added to the resin.
[0014]
The solvent used for activating the protected amino acid or condensing with the resin can be appropriately selected from solvents known to be usable for the protein condensation reaction. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol, and dimethyl sulfoxide. Sulfoxides, ethers such as pyridine, dioxane, and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, and appropriate mixtures thereof are used. The reaction temperature is appropriately selected from a range known to be usable for a protein bond formation reaction, and is usually appropriately selected from a range of about -20 ° C to 50 ° C. The activated amino acid derivative is usually used in a 1.5 to 4-fold excess. As a result of the test using the ninhydrin reaction, when the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. When sufficient condensation cannot be obtained even by repeating the reaction, unreacted amino acids can be acetylated using acetic anhydride or acetylimidazole to prevent the subsequent reaction from being affected.
[0015]
Examples of the protecting group for the amino group of the starting material include Z, Boc, t-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, trifluoroacetyl , Phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like.
The carboxyl group may be, for example, a linear, branched or cyclic alkyl ester such as an alkyl esterified ester (eg, methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl). ), Aralkyl esterification (eg, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarbonylhydrazide, t-butoxy It can be protected by carbonyl hydrazide, trityl hydrazide and the like.
The hydroxyl group of serine can be protected, for example, by esterification or etherification. Suitable groups for this esterification include, for example, lower (C _1-6 A) Groups derived from carbonic acid such as an aroyl group such as an alkanoyl group and a benzoyl group, and a benzyloxycarbonyl group and an ethoxycarbonyl group. Examples of the group suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group.
Examples of the protecting group for the phenolic hydroxyl group of tyrosine include Bzl, Cl ₂ -Bzl, 2-nitrobenzyl, Br-Z, t-butyl and the like are used.
As the imidazole protecting group for histidine, for example, Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like are used.
[0016]
Examples of the activated carboxyl group of the raw material include, for example, a corresponding acid anhydride, azide, active ester [alcohol (eg, pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, Cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, and an ester with HOBt). As the activated amino group of the raw material, for example, a corresponding phosphoric amide is used.
As a method for removing (eliminating) the protecting group, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, or hydrogen fluoride anhydride, methanesulfonic acid, trifluoromethane, etc. Acid treatment with dichloromethane, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., reduction with sodium in liquid ammonia, and the like are also used. The elimination reaction by the above acid treatment is generally performed at a temperature of about -20 ° C to 40 ° C. In the acid treatment, for example, anisole, phenol, thioanisole, metacresol, paracresol, dimethylsulfide, 1,4 -Addition of a ligand-agonizing cation scavenger such as butanedithiol, 1-2-ethanedithiol and the like is effective. Further, the 2,4-dinitrophenyl group used as an imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as an indole protecting group of tryptophan is the above-mentioned 1-2-ethanedithiol, 1,4-butane. In addition to deprotection by acid treatment in the presence of dithiol or the like, it is also removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia or the like.
[0017]
The protection of the functional group that should not be involved in the reaction of the raw materials, the protective group, the elimination of the protective group, the activation of the functional group involved in the reaction, and the like can be appropriately selected from known groups or known means.
As another method for obtaining an amide form of a protein or a partial peptide, for example, after amidating and protecting the α-carboxyl group of the carboxy terminal amino acid, a peptide (protein) chain is added to the amino group side to a desired chain length. After the elongation, a protein or partial peptide from which only the protecting group for the N-terminal α-amino group of the peptide chain has been removed and a protein or partial peptide from which only the protecting group for the C-terminal carboxyl group has been removed, and these are produced. The protein or peptide is condensed in a mixed solvent as described above. Details of the condensation reaction are the same as described above. After purifying the protected protein or peptide obtained by the condensation, all the protecting groups are removed by the above-mentioned method to obtain a desired crude protein or peptide. This crude protein or peptide is purified by various known purification means, and the main fraction is lyophilized to obtain an amide of the desired protein or peptide.
In order to obtain an ester of a protein or peptide, for example, after condensing the α-carboxyl group of the carboxy terminal amino acid with a desired alcohol to form an amino acid ester, the desired protein or An ester of the peptide can be obtained.
[0018]
The partial peptide or a salt thereof used in the present invention can be produced according to a known peptide synthesis method or by cleaving the protein of the present invention with an appropriate peptidase. As a method for synthesizing a peptide, for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, the partial peptide or amino acid that can constitute the partial peptide used in the present invention is condensed with the remaining portion, and when the product has a protecting group, the protecting group is eliminated to produce the target peptide. it can. Examples of the known condensation method and elimination of the protecting group include the methods described in the following (a) to (e).
(A) M.I. Bodanszky and M.A. A. Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966)
(B) Schroeder and Luebke, The Peptide, Academic Press, New York (1965)
(C) Nobuo Izumiya et al., Basics and experiments of peptide synthesis, Maruzen Co., Ltd. (1975)
(D) Haruaki Yajima and Shunpei Sakakibara, Biochemistry Laboratory Course 1, Protein Chemistry IV, 205, (1977)
(E) Supervised by Haruaki Yajima, Development of Continuing Drugs, Volume 14, Peptide Synthesis, Hirokawa Shoten
After the reaction, the partial peptide used in the present invention can be purified and isolated by a combination of ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization. When the partial peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method or a method analogous thereto, and conversely, when the partial peptide is obtained as a salt, a known method or analogous method It can be converted into a free form or another salt by a method.
[0019]
The polynucleotide encoding the protein of the present invention may be any polynucleotide containing the above-described nucleotide sequence encoding the protein of the present invention. Preferably it is DNA. The DNA may be any of genomic DNA, genomic DNA library, cDNA derived from the above-described cells / tissues, cDNA library derived from the aforementioned cells / tissues, and synthetic DNA.
The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Alternatively, amplification can be performed directly by Reverse Transcriptase Polymerase Chain Reaction (hereinafter abbreviated as RT-PCR method) using a total RNA or mRNA fraction prepared from the above-described cells / tissues.
The DNA encoding the protein of the present invention includes, for example, (i) a DNA containing the base sequence represented by SEQ ID NO: 2 or a base sequence represented by SEQ ID NO: 2 hybridized under high stringent conditions. DNA encoding a protein having a soybean base sequence and having substantially the same properties as the protein containing the amino acid sequence represented by SEQ ID NO: 1, (ii) a base sequence represented by SEQ ID NO: 25 Or a protein having a nucleotide sequence that hybridizes under high stringent conditions with the nucleotide sequence represented by SEQ ID NO: 25 and substantially containing the amino acid sequence represented by SEQ ID NO: 1 DNA encoding a protein having the same properties as described above, (iii) DNA containing the nucleotide sequence represented by SEQ ID NO: 27, or SEQ ID NO: : A protein having a nucleotide sequence that hybridizes with the nucleotide sequence represented by SEQ ID NO: 27 under highly stringent conditions, and having substantially the same properties as the protein containing the amino acid sequence represented by SEQ ID NO: 26. Encoding DNA, (iv) a DNA containing the nucleotide sequence represented by SEQ ID NO: 51, or a nucleotide sequence that hybridizes under high stringent conditions to the nucleotide sequence represented by SEQ ID NO: 51; DNA encoding a protein having substantially the same properties as the protein containing the amino acid sequence represented by SEQ ID NO: 26, (v) DNA containing the base sequence represented by SEQ ID NO: 53, or SEQ ID NO: : 53, which has a base sequence hybridizable with the base sequence represented by SEQ ID NO: 53 under high stringent conditions, and is represented by SEQ ID NO: 52. DNA encoding a protein having substantially the same properties as the protein containing the amino acid sequence, (vi) DNA containing the base sequence represented by SEQ ID NO: 80, or base represented by SEQ ID NO: 80 DNA encoding a protein having a nucleotide sequence that hybridizes under high stringent conditions to a sequence and having substantially the same properties as a protein containing the amino acid sequence represented by SEQ ID NO: 52, (vii) A DNA containing the nucleotide sequence represented by SEQ ID NO: 55, or a nucleotide sequence that hybridizes with the nucleotide sequence represented by SEQ ID NO: 55 under high stringency conditions, and represented by SEQ ID NO: 54 DNA encoding a protein having substantially the same properties as the protein containing the amino acid sequence, (viii) SEQ ID NO: 8 DNA having the nucleotide sequence represented by SEQ ID NO: 1 or an amino acid sequence represented by SEQ ID NO: 54 which has a nucleotide sequence hybridizable under high stringent conditions to the nucleotide sequence represented by SEQ ID NO: 81 Any DNA may be used as long as it encodes a protein having substantially the same properties as a protein containing.
[0020]
Examples of the DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 2 or SEQ ID NO: 25 under high stringency conditions include, for example, a nucleotide sequence represented by SEQ ID NO: 2 or SEQ ID NO: 25 and about 50 % Or more, preferably about 60% or more, preferably about 70% or more, preferably about 80% or more, preferably about 90% or more, more preferably about 95% or more, and particularly preferably about 99% or more. DNA having a base sequence having the same is used.
Examples of the DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 27 or SEQ ID NO: 51 under high stringency conditions include, for example, a nucleotide sequence represented by SEQ ID NO: 27 or SEQ ID NO: 51 and about 50 % Or more, preferably about 60% or more, preferably about 70% or more, preferably about 80% or more, preferably about 90% or more, more preferably about 95% or more, and particularly preferably about 99% or more. DNA having a base sequence having the same is used.
Examples of the DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 53 or SEQ ID NO: 80 under high stringency conditions include, for example, a nucleotide sequence represented by SEQ ID NO: 53 or about 50 % Or more, preferably about 60% or more, preferably about 70% or more, preferably about 80% or more, preferably about 90% or more, more preferably about 95% or more, and particularly preferably about 99% or more. DNA having a base sequence having the same is used.
Examples of the DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 55 or SEQ ID NO: 81 under high stringency conditions include, for example, a nucleotide sequence represented by SEQ ID NO: 55 or about 50 % Or more, preferably about 60% or more, preferably about 70% or more, preferably about 80% or more, preferably about 90% or more, more preferably about 95% or more, and particularly preferably about 99% or more. DNA having a base sequence having the same is used.
Hybridization can be performed according to a known method or a method analogous thereto, for example, a method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. More preferably, it can be performed under high stringent conditions.
The high stringent conditions are, for example, conditions in which the sodium concentration is about 19 to 40 mM, preferably about 19 to 20 mM, and the temperature is about 50 to 70 ° C, preferably about 60 to 65 ° C. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C. is most preferable.
More specifically, the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 1 is a DNA containing the base sequence represented by SEQ ID NO: 2, and represented by SEQ ID NO: 25 Examples of the DNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 26, such as DNA having the nucleotide sequence represented by SEQ ID NO: 27, include DNA having the nucleotide sequence represented by SEQ ID NO: 27, and SEQ ID NO: 51. Examples of the DNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 52, such as the DNA containing the nucleotide sequence represented by SEQ ID NO: 52, include the DNA containing the nucleotide sequence represented by SEQ ID NO: 53, No.:80 as a DNA encoding a protein containing an amino acid sequence represented by SEQ ID NO: 54, and the like. Column Number: DNA containing the base sequence represented by 55, SEQ ID NO: DNA containing the base sequence represented by 81 is used.
[0021]
The polynucleotide (eg, DNA) encoding the partial peptide used in the present invention may be any polynucleotide containing the above-described nucleotide sequence encoding the partial peptide used in the present invention. Further, any of genomic DNA, genomic DNA library, cDNA derived from the above-mentioned cells / tissues, cDNA library derived from the aforementioned cells / tissues, and synthetic DNA may be used.
Examples of the DNA encoding the partial peptide used in the present invention include (1) DNA having a part of the DNA having the base sequence represented by SEQ ID NO: 2 or SEQ ID NO: 25, or SEQ ID NO: 2 or the sequence DNA containing a base sequence hybridizable with the base sequence represented by No. 25 under high stringent conditions, and containing a part of a DNA encoding a protein having substantially the same activity as the protein of the present invention (2) a DNA having a portion of the DNA having the nucleotide sequence of SEQ ID NO: 27, or a nucleotide sequence that hybridizes with the nucleotide sequence of SEQ ID NO: 27 under high stringency conditions; A DNA containing a portion of a DNA encoding a protein having substantially the same activity as the protein of the present invention; 3) a DNA comprising a portion of the DNA having the nucleotide sequence of SEQ ID NO: 53 or a nucleotide sequence which hybridizes under high stringency conditions with the nucleotide sequence of SEQ ID NO: 53; DNA containing a part of a DNA encoding a protein having substantially the same activity as the protein of the present invention, (4) DNA having a part of a DNA having a base sequence represented by SEQ ID NO: 55, or SEQ ID NO: DNA containing a part of DNA encoding a protein having a nucleotide sequence that hybridizes under high stringent conditions with the nucleotide sequence represented by 55 and encoding a protein having substantially the same activity as the protein of the present invention; Used.
DNAs capable of hybridizing with the base sequence represented by SEQ ID NO: 2, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 53 or SEQ ID NO: 55 have the same significance as described above.
The same hybridization method and high stringency conditions as described above are used.
[0022]
As a means for cloning a DNA that completely encodes the protein or partial peptide of the present invention (hereinafter, in the description of the cloning and expression of DNAs encoding them, these may be simply abbreviated to the protein of the present invention), Amplifying by a PCR method using a synthetic DNA primer having a part of the nucleotide sequence encoding the protein of the present invention, or incorporating a DNA incorporated in an appropriate vector into a DNA encoding a part or the whole region of the protein of the present invention. Selection can be performed by hybridization with fragments or those labeled with synthetic DNA. Hybridization can be performed, for example, according to the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual.
Conversion of the base sequence of DNA can be performed by PCR or a known kit, for example, Mutan. ^TM -Super Express Km (Takara Shuzo Co., Ltd.), Mutan ^TM Using -K (Takara Shuzo Co., Ltd.) or the like, it can be carried out according to a known method such as the ODA-LA PCR method, the Gapped Duplex method or the Kunkel method, or a method analogous thereto.
The DNA encoding the cloned protein can be used as it is or as desired, after digestion with a restriction enzyme or addition of a linker, if desired. The DNA may have ATG as a translation initiation codon at the 5 'end and TAA, TGA or TAG as a translation termination codon at the 3' end. These translation initiation codon and translation termination codon can also be added using an appropriate synthetic DNA adapter.
The expression vector for the protein of the present invention includes, for example, (a) cutting out a DNA fragment of interest from DNA encoding the protein of the present invention, and (b) ligating the DNA fragment downstream of a promoter in an appropriate expression vector. It can be manufactured by the following.
[0023]
Examples of the vector include plasmids derived from Escherichia coli (eg, pBR322, pBR325, pUC12, pUC13), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, pC194), plasmids derived from yeast (eg, pSH19, pSH15), λ phage and the like. In addition to animal viruses such as bacteriophage, retrovirus, vaccinia virus, and baculovirus, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo, and the like are used.
The promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression. For example, when an animal cell is used as a host, SRα promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and the like can be mentioned.
Of these, it is preferable to use a CMV (cytomegalovirus) promoter, SRα promoter, or the like. When the host is a bacterium belonging to the genus Escherichia, trp promoter, lac promoter, recA promoter, λPL promoter, lpp promoter, T7 promoter, etc., and when the host is a bacterium belonging to the genus Bacillus, SPO1 promoter, SPO2 promoter, penP promoter, etc. When the host is yeast, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable. When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable.
[0024]
In addition to the above, an expression vector containing an enhancer, a splicing signal, a polyA addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori), and the like may be used, if desired. it can. Examples of the selectable marker include a dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [methotrexate (MTX) resistance] and an ampicillin resistance gene (hereinafter Amp). ^r Neomycin resistance gene (hereinafter Neo). ^r G418 resistance). In particular, when the dhfr gene is used as a selection marker using Chinese hamster cells deficient in dhfr gene, the target gene can be selected using a thymidine-free medium.
If necessary, a signal sequence suitable for the host is added to the N-terminal side of the protein of the present invention. When the host is a genus Escherichia, a PhoA signal sequence, an OmpA signal sequence, or the like is used. When the host is a Bacillus genus, an α-amylase signal sequence, a subtilisin signal sequence, or the like is used. In some cases, MFα signal sequence, SUC2 signal sequence, etc., and when the host is an animal cell, insulin signal sequence, α-interferon signal sequence, antibody molecule, signal sequence, etc. can be used.
A transformant can be produced using the vector containing the DNA encoding the protein of the present invention thus constructed.
[0025]
As the host, for example, Escherichia, Bacillus, yeast, insect cells, insects, animal cells and the like are used.
Specific examples of the genus Escherichia include, for example, Escherichia coli K12.DH1 [Proc. Natl. Acad. Sci. USA, 60, 160 (1968)], JM103 [Nucleic Acids Research, 9, 309 (1981)], JA221 [Journal of Molecular Biology, 120, 517 (1978)], HB101 [Journalolgo olegolando]. Vol., 459 (1969)], C600 [Genetics, 39, 440 (1954)] and the like.
As the Bacillus bacterium, for example, Bacillus subtilis MI114 [Gene, 24, 255 (1983)], 207-21 [Journal of Biochemistry, 95, 87 (1984)] and the like are used.
Examples of yeast include, for example, Saccharomyces cerevisiae AH22, AH22R ⁻ , NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036, Pichia pastoris KM71 and the like.
[0026]
When the virus is AcNPV, for example, when the virus is AcNPV, a cell line derived from a larva of night roth moth (Spodoptera frugiperda cell; Sf cell), an MG1 cell derived from the midgut of Trichoplusia ni, and a Figh cell derived from Trichoplusia ni egg. And cells derived from Mamestra brassicae or cells derived from Estigmena acrea. When the virus is BmNPV, a silkworm-derived cell line (Bombyx mori N cell; BmN cell) or the like is used. As the Sf cells, for example, Sf9 cells (ATCC CRL 1711), Sf21 cells (Vaughn, JL, et al., In Vivo, 13, 213-217, (1977)) and the like are used. .
As insects, for example, silkworm larvae are used [Maeda et al., Nature, 315, 592 (1985)].
Examples of animal cells include monkey cells COS-7, Vero, Chinese hamster cells CHO (hereinafter abbreviated as CHO cells), dhfr gene-deficient Chinese hamster cells CHO (hereinafter CHO (dhfr). ⁻ ) Cells), mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells, and the like.
For transformation of Escherichia, for example, Proc. Natl. Acad. Sci. USA, 69, 2110 (1972), and Gene, 17, 107 (1982).
[0027]
Transformation of Bacillus spp. Can be performed, for example, according to the method described in Molecular & General Genetics, Vol. 168, 111 (1979).
To transform yeast, see, for example, Methods in Enzymology, Vol. 194, 182-187 (1991), Proc. Natl. Acad. Sci. USA, Vol. 75, 1929 (1978).
Insect cells or insects can be transformed, for example, according to the method described in Bio / Technology, 6, 47-55 (1988).
To transform animal cells, for example, see Cell Engineering Separate Volume 8, New Cell Engineering Experiment Protocol. 263-267 (1995) (published by Shujunsha) and Virology, Vol. 52, 456 (1973).
Thus, a transformant transformed with the expression vector containing the DNA encoding the protein can be obtained.
When culturing a transformant whose host is a genus Escherichia or Bacillus, a liquid medium is suitable as a medium used for the culturing, and a carbon source necessary for growth of the transformant is used therein. Nitrogen sources, inorganic substances, etc. are included. As a carbon source, for example, glucose, dextrin, soluble starch, sucrose, etc., as a nitrogen source, for example, ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal, potato extract and the like Examples of the inorganic or organic substance and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride. In addition, yeast extract, vitamins, growth promoting factors and the like may be added. The pH of the medium is preferably about 5 to 8.
[0028]
As a medium for culturing the genus Escherichia, for example, an M9 medium containing glucose and casamino acid [Journal of Experiments in Molecular Genetics, 431-433, Cold Spring Harbor Laboratory, New York] is preferable. If necessary, an agent such as 3β-indolylacrylic acid can be added in order to make the promoter work efficiently.
When the host is a bacterium belonging to the genus Escherichia, the cultivation is usually performed at about 15 to 43 ° C. for about 3 to 24 hours, and if necessary, aeration and stirring can be added.
When the host is a bacterium belonging to the genus Bacillus, the cultivation is usually carried out at about 30 to 40 ° C. for about 6 to 24 hours, and if necessary, aeration and stirring may be added.
When culturing a transformant in which the host is yeast, for example, Burkholder's minimum medium [Bostian, K. et al. L. Proc. Natl. Acad. Sci. USA, Vol. 77, 4505 (1980)] or an SD medium containing 0.5% casamino acid [Bitter, G. et al. A. Proc. Natl. Acad. Sci. USA, 81, 5330 (1984)]. Preferably, the pH of the medium is adjusted to about 5-8. The cultivation is usually performed at about 20 to 35 ° C. for about 24 to 72 hours, and aeration and / or agitation are added as necessary.
When culturing a transformant in which the host is an insect cell or an insect, as a medium, an additive such as 10% bovine serum immobilized in Grace's Insect Medium (Nature, 195, 788 (1962)) is appropriately used. The added one is used. The pH of the medium is preferably adjusted to about 6.2 to 6.4. The cultivation is usually performed at about 27 ° C. for about 3 to 5 days, and if necessary, aeration or stirring is added.
When culturing a transformant in which the host is an animal cell, examples of the medium include a MEM medium containing about 5 to 20% fetal bovine serum [Science, Vol. 122, 501 (1952)], a DMEM medium [Virology, 8, 396 (1959)], RPMI 1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], 199 medium [Proceeding of the Society for Biotechnology, etc., 1973, etc.]. Used. Preferably, the pH is between about 6 and 8. The cultivation is usually performed at about 30 to 40 ° C. for about 15 to 60 hours, and if necessary, aeration and stirring are added.
As described above, the protein of the present invention can be produced in cells, cell membranes or extracellular cells of the transformant.
[0029]
The protein of the present invention can be separated and purified from the culture by, for example, the following method.
When extracting the protein of the present invention from cultured cells or cells, the cells or cells are collected by a known method after culturing, suspended in an appropriate buffer, and subjected to sonication, lysozyme and / or freeze-thawing. After the cells or cells are destroyed by the method, a method of obtaining a crude extract of the protein by centrifugation or filtration is used as appropriate. In a buffer, a protein denaturant such as urea or guanidine hydrochloride, or Triton X-100 is used. ^TM And the like. When the protein is secreted into the culture solution, after the culture, the supernatant is separated from the cells or cells by a known method, and the supernatant is collected.
The protein contained in the thus obtained culture supernatant or extract can be purified by appropriately combining known separation and purification methods. These known separation and purification methods include methods utilizing solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, mainly molecular weight. Using differences in charge, methods using charge differences such as ion exchange chromatography, methods using specific affinity such as affinity chromatography, and using differences in hydrophobicity such as reversed-phase high-performance liquid chromatography A method utilizing a difference between isoelectric points, such as an isoelectric focusing method, is used.
[0030]
When the thus obtained protein is obtained in a free form, it can be converted to a salt by a known method or a method analogous thereto, and conversely, when the protein is obtained as a salt, by a known method or a method analogous thereto, It can be converted to a free form or other salts.
The protein produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by applying an appropriate protein modifying enzyme before or after purification. As the protein modifying enzyme, for example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used.
The presence of the protein of the present invention thus produced can be measured by an enzyme immunoassay using a specific antibody, Western blotting, or the like.
[0031]
The antibody against the protein or partial peptide of the present invention or a salt thereof may be any of a polyclonal antibody and a monoclonal antibody as long as it can recognize the protein or partial peptide of the present invention or a salt thereof.
An antibody against the protein or partial peptide of the present invention or a salt thereof (hereinafter, these may be simply abbreviated as the protein of the present invention) using the protein of the present invention as an antigen, is a known antibody or It can be produced according to the method for producing antiserum.
[Preparation of monoclonal antibody]
(A) Preparation of monoclonal antibody-producing cells
The protein of the present invention is administered to a warm-blooded animal itself or together with a carrier and a diluent at a site capable of producing an antibody upon administration. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. The administration is usually performed once every 2 to 6 weeks, for a total of about 2 to 10 times. Examples of the warm-blooded animal used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, and chickens, and mice and rats are preferably used.
When preparing monoclonal antibody-producing cells, a warm-blooded animal immunized with an antigen, for example, an individual having an antibody titer is selected from a mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization and contained in them. By fusing the antibody-producing cells thus obtained with myeloma cells of the same or different species, a monoclonal antibody-producing hybridoma can be prepared. The antibody titer in the antiserum can be measured, for example, by reacting a labeled protein described below with the antiserum, and then measuring the activity of a labeling agent bound to the antibody. The fusion operation can be performed according to a known method, for example, the method of Kohler and Milstein [Nature, 256, 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used.
[0032]
Examples of myeloma cells include myeloma cells of warm-blooded animals such as NS-1, P3U1, SP2 / 0, and AP-1, but P3U1 is preferably used. The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG1000 to PEG6000) is added at a concentration of about 10 to 80%. By incubating at 20 to 40 ° C, preferably 30 to 37 ° C for 1 to 10 minutes, cell fusion can be carried out efficiently. Various methods can be used to screen for monoclonal antibody-producing hybridomas. For example, a hybridoma culture supernatant is added to a solid phase (eg, a microplate) on which a protein antigen is directly or adsorbed together with a carrier, and then radioactive substances or A method for detecting a monoclonal antibody bound to a solid phase by adding an anti-immunoglobulin antibody labeled with an enzyme or the like (an anti-mouse immunoglobulin antibody is used when cells used for cell fusion are mice) or protein A; A method in which a hybridoma culture supernatant is added to a solid phase to which globulin antibodies or protein A is adsorbed, a protein labeled with a radioactive substance, an enzyme, or the like is added, and a monoclonal antibody bound to the solid phase is detected.
The selection of the monoclonal antibody can be performed according to a known method or a method analogous thereto. Usually, it can be performed in a medium for animal cells supplemented with HAT (hypoxanthine, aminopterin, thymidine). As a selection and breeding medium, any medium can be used as long as a hybridoma can grow. For example, RPMI 1640 medium containing 1 to 20%, preferably 10 to 20% fetal bovine serum, GIT medium containing 1 to 10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.) or serum-free for hybridoma culture A medium (SFM-101, Nissui Pharmaceutical Co., Ltd.) or the like can be used. The culturing temperature is usually 20 to 40 ° C, preferably about 37 ° C. The culture time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks. The culture can be usually performed under 5% carbon dioxide gas. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.
[0033]
(B) Purification of monoclonal antibody
Monoclonal antibodies can be separated and purified by known methods, for example, immunoglobulin separation and purification methods [eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, absorption by ion exchanger (eg, DEAE)]. Desorption method, ultracentrifugation method, gel filtration method, antigen-binding solid phase or specific purification method of collecting only the antibody with an active adsorbent such as protein A or protein G and dissociating the bond to obtain the antibody] it can.
[0034]
(Preparation of polyclonal antibody)
The polyclonal antibody of the present invention can be produced according to a known method or a method analogous thereto. For example, a immunizing antigen (protein antigen) itself or a complex thereof with a carrier protein is formed, and immunization is performed on a warm-blooded animal in the same manner as in the above-described method for producing a monoclonal antibody. It can be produced by collecting a substance and separating and purifying the antibody. Regarding a complex of an immunizing antigen and a carrier protein used to immunize a warm-blooded animal, the type of the carrier protein and the mixing ratio of the carrier protein and the hapten are determined by the antibody against the hapten immunized by crosslinking the carrier protein. Any material may be crosslinked at any ratio as long as it can be efficiently produced.For example, bovine serum albumin, bovine thyroglobulin, hemocyanin, etc. are used in a weight ratio of about 0.1 to 20, preferably 1 to hapten 1 with respect to hapten. A method of coupling at a rate of about 1 to 5 is used.
In addition, various condensing agents can be used for coupling the hapten and the carrier protein, and glutaraldehyde, carbodiimide, maleimide active ester, an active ester reagent containing a thiol group or a dithioviridyl group, or the like is used.
The condensation product is administered to a warm-blooded animal itself or together with a carrier or diluent at a site where antibody production is possible. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. The administration is usually performed once every about 2 to 6 weeks, about 3 to 10 times in total.
The polyclonal antibody can be collected from the blood, ascites, or the like, preferably from the blood of a warm-blooded animal immunized by the above method.
The measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the antiserum described above. The separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as the above-mentioned separation and purification of the monoclonal antibody.
[0035]
Complementary or substantially complementary to the base sequence of DNA encoding the protein or partial peptide of the present invention (hereinafter, these DNAs may be abbreviated as the DNA of the present invention in the description of the antisense polynucleotide). The antisense polynucleotide having a complementary base sequence or a part thereof has a base sequence complementary to or substantially complementary to the base sequence of the DNA of the present invention or a part thereof, and Any antisense polynucleotide may be used as long as it has an action of suppressing expression, but antisense DNA is preferable.
The nucleotide sequence substantially complementary to the DNA of the present invention is, for example, about 70% of the total nucleotide sequence or a partial nucleotide sequence of the nucleotide sequence complementary to the DNA of the present invention (that is, the complementary strand of the DNA of the present invention). % Or more, preferably about 80% or more, more preferably about 90% or more, most preferably about 95% or more. In particular, of the entire base sequence of the complementary strand of the DNA of the present invention, the base sequence of the portion encoding the N-terminal portion of the protein of the present invention (for example, the base sequence near the start codon, etc.) is at least about 70% or more of the complementary strand. Antisense polynucleotides having preferably at least about 80%, more preferably at least about 90%, most preferably at least about 95% homology are suitable.
Specifically, a nucleotide sequence complementary or substantially complementary to the nucleotide sequence of DNA having the nucleotide sequence represented by SEQ ID NO: 2, SEQ ID NO: 27, SEQ ID NO: 53 or SEQ ID NO: 55, Or a base sequence complementary to a base sequence of a DNA having a base sequence represented by SEQ ID NO: 2, SEQ ID NO: 27, SEQ ID NO: 53 or SEQ ID NO: 55, Or an antisense polynucleotide having a part thereof.
The antisense polynucleotide is usually composed of about 10 to 40, preferably about 15 to 30 bases.
In order to prevent degradation by a hydrolase such as nuclease, a phosphate residue (phosphate) of each nucleotide constituting the antisense DNA may be, for example, a chemically modified phosphate residue such as phosphorothioate, methylphosphonate, or phosphorodithionate. It may be substituted. These antisense polynucleotides can be produced using a known DNA synthesizer or the like.
[0036]
According to the present invention, the antisense polynucleotide (nucleic acid) corresponding to the protein gene of the present invention, which can inhibit the replication or expression of the gene, is cloned or the base of DNA encoding the determined protein is determined. It can be designed and synthesized based on sequence information. Such antisense polynucleotides can hybridize to the RNA of the protein gene of the present invention and inhibit the synthesis or function of the RNA, or through interaction with the protein-related RNA of the present invention. The expression of the protein gene of the present invention can be regulated and controlled. Polynucleotides that are complementary to a selected sequence of the protein-associated RNA of the present invention, and that can specifically hybridize with the protein-associated RNA of the present invention, can be used in vivo and in vitro to produce the protein gene of the present invention. It is useful for regulating and controlling the expression of, and also useful for treating or diagnosing diseases and the like. The term “corresponding” means having homology or being complementary to a specific sequence of nucleotides, base sequences or nucleic acids including genes. The "correspondence" between a nucleotide, base sequence or nucleic acid and a protein usually refers to the amino acid of the protein (as indicated) derived from the nucleotide (nucleic acid) sequence or its complement. 5 'end hairpin loop of protein gene, 5' end 6-base pair repeat, 5 'end untranslated region, polypeptide translation start codon, protein coding region, ORF translation stop codon, 3' end untranslated region, 3 ' Although a terminal palindrome region or a 3′-end hairpin loop can be selected as a preferable target region, any region within a protein gene can be selected as a target. Regarding the relationship between the target nucleic acid and a polynucleotide complementary to at least a part of the target region, if the target nucleic acid can hybridize with the target region, the target nucleic acid is Can be said to be "antisense." Antisense polynucleotides are polydeoxyribonucleotides containing 2-deoxy-D-ribose, polyribonucleotides containing D-ribose, N-glycosides of purine or pyrimidine bases and other types of polynucleotides. , Other polymers with non-nucleotide backbones (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing specialized linkages, such as those found in DNA or RNA. Base pairing and nucleotides having a configuration that allows base attachment). They may be double stranded DNA, single stranded DNA, double stranded RNA, single stranded RNA, DNA: RNA hybrids, and may further comprise unmodified polynucleotides (or unmodified oligonucleotides), known modifications. Additions, e.g., those with labels known in the art, capped, methylated, substituted for one or more natural nucleotides with analogs, modified with intramolecular nucleotides Having an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.), having a charged or sulfur-containing bond (eg, phosphorothioate, phosphorodithioate, etc.) Such as proteins (eg, nucleases, nuclease inhibitors, toxins, antibodies, Nal peptides, poly-L-lysine, etc.) or sugars (eg, monosaccharides, etc.) having side chain groups, interactive compounds (eg, acridine, psoralen, etc.), chelating compounds (eg, , Metals, radioactive metals, boron, oxidizable metals, etc.), those containing alkylating agents, those having modified bonds (eg, α-anomeric nucleic acids, etc.) Is also good. Here, "nucleoside", "nucleotide", and "nucleic acid" may include not only those containing purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, e.g., where one or more hydroxyl groups have been replaced with halogens, aliphatic groups, or the like, or with functional groups such as ethers, amines, and the like. It may have been converted.
The antisense polynucleotide of the present invention is RNA, DNA or a modified nucleic acid (RNA, DNA). Specific examples of the modified nucleic acid include a sulfur derivative of a nucleic acid, a thiophosphate derivative, a nucleic acid which is resistant to degradation of polynucleoside amide and oligonucleoside amide and the like. The antisense polynucleotide of the present invention can be designed, for example, as follows. That is, the antisense polynucleotide in a cell is made more stable, the cell permeability of the antisense polynucleotide is increased, the affinity for the target sense strand is increased, and if the toxicity is low. In some cases, the toxicity of the antisense polynucleotide is reduced. Many such modifications are reported in, for example, Pharm Tech Japan, 8, 247 or 395, 1992, Antisense Research and Applications, CRC Press, 1993.
The antisense polynucleotides of the present invention may contain altered or modified sugars, bases, bonds, and may be provided in special forms such as liposomes, microspheres, or applied by gene therapy. , Can be provided in an added form. Thus, in the form of addition, polycations such as polylysine which acts to neutralize the charge of the phosphate skeleton, lipids which enhance the interaction with the cell membrane or increase the uptake of nucleic acids ( Hydrophobic substances such as, for example, phospholipids, cholesterol, etc.). Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chloroformate, cholic acid, etc.). These can be attached to the 3 'or 5' end of the nucleic acid and can be attached via a base, sugar, intramolecular nucleoside linkage. Other groups include cap groups specifically arranged at the 3 ′ end or 5 ′ end of a nucleic acid for preventing degradation by nucleases such as exonuclease and RNase. Such capping groups include, but are not limited to, hydroxyl-protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.
The inhibitory activity of the antisense polynucleotide can be examined using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention, or the in vivo or in vitro translation system of the protein of the present invention. .
[0037]
Hereinafter, the protein or partial peptide of the present invention or a salt thereof (hereinafter, may be abbreviated as the protein of the present invention), a polynucleotide encoding the protein or partial peptide of the present invention, for example, DNA (hereinafter, DNA of the present invention) ), An antibody against the protein or partial peptide of the present invention or a salt thereof (hereinafter, may be abbreviated as the antibody of the present invention), and an antisense polynucleotide of the DNA of the present invention (hereinafter, the present invention). May be abbreviated as “antisense polynucleotide”).
Pharmaceuticals containing a compound or a salt thereof that inhibits the activity of the protein of the present invention can be used, for example, by suppressing the transport activity of organic anions to obtain, for example, renal diseases (eg, renal failure, glomerulonephritis, diabetic nephropathy) , Focal glomerulosclerosis, nephrotic syndrome, renal edema, interstitial nephritis, renal sclerosis, uremic disease, etc., liver disease (eg, cirrhosis, hepatitis, alcoholic liver disease, etc.), pancreatic disease (eg, Pancreatitis), immune disorders associated with thymic abnormalities, genital disorders (eg, benign prostatic hyperplasia, prostatitis, testicular neurosis, ovarian cysts), gastrointestinal disorders (eg, irritable bowel syndrome, ulcerative colitis, illness) Blood colitis, gastritis, etc.), spleen disease (eg, hypersplenia, splenomegaly syndrome, etc.), cancer (eg, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder) Cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, pancreas , Thyroid cancer, myeloid leukemia, etc.), respiratory diseases (eg, pleurisy, pneumonia, chronic obstructive pulmonary disease, asthma, etc.), osteomyelitis, diabetes, hypertension, post-ischemic reperfusion injury, retinitis, central nervous disease (E.g., epilepsy, Alzheimer's disease, Parkinson's syndrome, schizophrenia), skin disorders (e.g., atopic dermatitis, seborrheic dermatitis, etc.), thyroid hormone-related disorders (e.g., thyroid hormone refractory disease, Basedow's disease, Cretinism, hyperthyroidism, etc.). It is preferably used as a prophylactic / therapeutic agent for renal diseases and thyroid hormone-related diseases, and more preferably as a prophylactic / therapeutic agent for diabetic nephropathy. On the other hand, a drug containing a compound or a salt thereof that promotes the activity of the protein of the present invention can be used, for example, by promoting the transport activity of an organic anion to obtain, for example, a renal disease (eg, renal failure, glomerulonephritis, diabetic Nephropathy, focal glomerulosclerosis, nephrotic syndrome, renal edema, interstitial nephritis, nephrosclerosis, uremic disease, etc., liver disease (eg, cirrhosis, hepatitis, alcoholic liver disease, etc.), pancreatic disease ( E.g., pancreatitis), immune disorders associated with thymic abnormalities, genital disorders (e.g., benign prostatic hyperplasia, prostatitis, testicular neurosis, ovarian cysts), gastrointestinal disorders (e.g., irritable bowel syndrome, ulcerative colitis) , Ischemic colitis, gastritis, etc.), spleen disease (eg, hypersplenism, splenomegaly syndrome, etc.), cancer (eg, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer) , Bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer Pancreatic cancer, thyroid cancer, myeloid leukemia, etc.), respiratory diseases (eg, pleurisy, pneumonia, chronic obstructive pulmonary disease, asthma, etc.), osteomyelitis, diabetes, hypertension, post-ischemic reperfusion injury, retinitis, central Neurological diseases (eg, epilepsy, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), skin diseases (eg, atopic dermatitis, seborrheic dermatitis, etc.), thyroid hormone-related diseases (eg, thyroid hormone refractory disease, Basedow) Disease, cretinism, hyperthyroidism, etc.). It is preferably used as a prophylactic / therapeutic agent for renal diseases and thyroid hormone-related diseases, and more preferably as a prophylactic / therapeutic agent for diabetic nephropathy.
[0038]
[1] A preventive / therapeutic agent for various diseases related to the protein of the present invention
The protein of the present invention has an organic anion transport activity and the like, takes up thyroid hormones into the central nervous system through the blood brain barrier in vivo, transfers bile acids or drugs from the blood to the liver, It plays an important role in maintaining homeostasis, such as removing inflammatory mediators such as gin or leukotriene, and excreting foreign substances into bile or urine.
Therefore, when the DNA encoding the protein of the present invention is abnormal or defective, or when the expression level of the protein of the present invention is reduced, for example, renal diseases (eg, renal failure, glomeruli) Nephritis, diabetic nephropathy, focal glomerulosclerosis, nephrotic syndrome, renal edema, interstitial nephritis, nephrosclerosis, uremia, etc., liver disease (eg, cirrhosis, hepatitis, alcoholic liver disease, etc.) , Pancreatic disorders (eg, pancreatitis), immune disorders associated with thymic abnormalities, genital disorders (eg, benign prostatic hyperplasia, prostatitis, testicular neurosis, ovarian cysts), gastrointestinal disorders (eg, irritable bowel syndrome, Ulcerative colitis, ischemic colitis, gastritis, etc.), spleen disease (eg, hypersplenism, splenomegaly syndrome, etc.), cancer (eg, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer, Prostate, gastric, bladder, breast, cervical, Intestinal cancer, rectal cancer, pancreatic cancer, thyroid cancer, myeloid leukemia, etc.), respiratory disease (eg, pleurisy, pneumonia, chronic obstructive pulmonary disease, asthma, etc.), osteomyelitis, diabetes, hypertension, reperfusion after ischemia Disorders, retinitis, central nervous system disorders (eg, epilepsy, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), skin disorders (eg, atopic dermatitis, seborrheic dermatitis, etc.), thyroid hormone related disorders (eg, Various diseases such as thyroid hormone refractory disease, Basedow's disease, cretinism, and hyperthyroidism develop.
Therefore, the protein of the present invention and the DNA of the present invention can be used, for example, in renal diseases (eg, renal failure, glomerulonephritis, diabetic nephropathy, focal glomerulosclerosis, nephrotic syndrome, renal edema, interstitial nephritis) , Renal sclerosis, uremic disease, etc.), liver disease (eg, cirrhosis, hepatitis, alcoholic liver disease, etc.), pancreatic disease (eg, pancreatitis, etc.), immune disease associated with thymic abnormalities, genital disease (eg, prostatic hypertrophy) , Prostatitis, testicular neurosis, ovarian cysts, etc.), digestive disorders (eg, irritable bowel syndrome, ulcerative colitis, ischemic colitis, gastritis, etc.), spleen disorders (eg, hypersplenism, splenomegaly) Sexual syndrome, etc.), cancer (eg, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, pancreatic cancer, thyroid gland Cancer, myeloid leukemia, etc.), respiratory diseases (eg, pleurisy, pneumonia, Obstructive pulmonary disease, asthma, etc.), osteomyelitis, diabetes, hypertension, post-ischemic reperfusion injury, retinitis, central nervous system diseases (eg, epilepsy, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), skin diseases (eg, , Atopic dermatitis, seborrheic dermatitis, etc.) and thyroid hormone-related diseases (eg, thyroid hormone refractory disease, Basedow's disease, cretin disease, hyperthyroidism, etc.) be able to. It is preferably used as a prophylactic / therapeutic agent for renal diseases and thyroid hormone-related diseases, and more preferably as a prophylactic / therapeutic agent for diabetic nephropathy.
For example, when the protein of the present invention is reduced or deficient in a living body, and there is a patient who cannot sufficiently or normally exhibit the organic anion transport activity, (a) the DNA of the present invention is added to the patient. By administering and expressing the protein of the present invention in vivo, (ii) by inserting the DNA of the present invention into cells and expressing the protein of the present invention, and then transplanting the cells into a patient; or (C) The role of the protein of the present invention in the patient can be sufficiently or normally exerted by administering the protein of the present invention to the patient.
When the DNA of the present invention is used as the above-mentioned prophylactic / therapeutic agent, after inserting the DNA alone or into an appropriate vector such as a retrovirus vector, adenovirus vector, adenovirus associated virus vector, etc. It can be administered to humans or warm-blooded animals. The DNA of the present invention can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as an auxiliary agent for promoting uptake, using a gene gun or a catheter such as a hydrogel catheter.
When the protein of the present invention is used as the above-mentioned prophylactic / therapeutic agent, it is preferable to use a protein purified to at least 90%, preferably 95% or more, more preferably 98% or more, and still more preferably 99% or more. preferable.
[0039]
The protein of the present invention can be used, for example, as a sugar-coated tablet, capsule, elixir, microcapsule, orally, or aseptic with water or other pharmaceutically acceptable liquids. It can be used parenterally in the form of injections, such as solutions or suspensions. For example, the protein or the like of the present invention is mixed with physiologically acceptable carriers, flavors, excipients, vehicles, preservatives, stabilizers, binders, and the like in a unit dosage form generally required for the practice of preparations. Can be manufactured. The amount of the active ingredient in these preparations is such that a proper dosage in the specified range can be obtained.
Examples of additives that can be incorporated into tablets, capsules, and the like include binders such as gelatin, corn starch, tragacanth, and acacia, excipients such as crystalline cellulose, corn starch, gelatin, and alginic acid. Useful bulking agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, reddish oil or cherry and the like are used. When the preparation unit form is a capsule, a liquid carrier such as oil and fat can be further contained in the above-mentioned type of material. Sterile compositions for injection can be formulated according to normal pharmaceutical practice such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil, coconut oil and the like.
Aqueous liquids for injection include, for example, physiological saline, isotonic solutions containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.), and suitable solubilizing agents. For example, alcohols (eg, ethanol, etc.), polyalcohols (eg, propylene glycol, polyethylene glycol, etc.), nonionic surfactants (eg, polysorbate 80) ^TM , HCO-50, etc.). Examples of the oily liquid include sesame oil and soybean oil, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. Also, buffers (eg, phosphate buffer, sodium acetate buffer, etc.), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.), You may mix | blend with a preservative (for example, benzyl alcohol, phenol etc.), an antioxidant, etc. The prepared injection is usually filled in an appropriate ampoule.
The vector into which the DNA of the present invention has been inserted is also formulated in the same manner as described above, and is usually used parenterally.
[0040]
The preparation obtained in this way is safe and low toxic, and thus, for example, is useful for warm-blooded animals (eg, human, rat, mouse, guinea pig, rabbit, bird, sheep, pig, cow, horse, cat, dog, monkey). , Chimpanzees, etc.).
The dose of the protein of the present invention varies depending on the target disease, the subject of administration, the administration route, and the like. For example, when the protein or the like of the present invention is orally administered for the purpose of treating renal insufficiency, generally the adult (body weight 60 kg) is used. )), The protein is administered in an amount of about 0.1-100 mg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg per day. When administered parenterally, a single dose of the protein may vary depending on the administration subject, target disease, and the like. For example, the protein of the present invention may be administered in the form of an injection to an adult (body weight 60 kg) for the treatment of renal failure. ) Is conveniently administered by injecting about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg of the protein per day into the affected area. It is. In the case of other animals, the amount can be administered in terms of the weight per 60 kg.
[0041]
[2] Screening of drug candidate compounds for disease
The protein of the present invention is useful as a reagent for screening a compound or a salt thereof that promotes or inhibits the activity of the protein of the present invention.
The present invention relates to (1) a compound or a salt thereof which promotes or inhibits the activity of the protein of the present invention (for example, an organic anion transporting activity and the like) characterized by using the protein of the present invention (hereinafter referred to as a promoter, Abbreviated as inhibitor). More specifically, for example,
(2) (i) the organic anion transport activity of a cell capable of producing the protein of the present invention and (ii) the organic anion transport activity of a mixture of a test compound and a cell capable of producing the protein of the present invention. There is provided a method of screening for an accelerator or an inhibitor characterized by performing a comparison.
Specifically, in the above screening method, for example, in the cases of (i) and (ii), the transport activity of an organic anion is measured with a fluorescent dye, and compared as an index of the transport activity of the organic anion. Is what you do.
[0042]
Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like. Or a known compound.
In order to carry out the above-mentioned screening method, cells having the ability to produce the protein of the present invention are prepared by suspending them in a buffer suitable for screening. The buffer may be any buffer such as a phosphate buffer or a borate buffer having a pH of about 4 to 10 (preferably, a pH of about 6 to 8) as long as it does not inhibit the organic anion transport activity of the protein of the present invention.
As a cell having the ability to produce the protein of the present invention, for example, a host (transformant) transformed with a vector containing the DNA encoding the protein of the present invention described above is used. As a host, for example, animal cells such as CHO cells are preferably used. For the screening, for example, a transformant in which the protein of the present invention is expressed on a cell membrane by culturing by the method described above is preferably used.
[0043]
The transport activity of the protein of the present invention for organic anions can be measured according to a known method, for example, the method described in Biochemical and Biophysical Research Communications, vol. 273, p. 251, 2000 or a method analogous thereto.
For example, a test compound which promotes the organic anion transport activity in the case (ii) above by about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the case (i) is used. It can be selected as a compound or a salt thereof that promotes the activity of the protein of the invention.
In addition, for example, the transport activity of the organic anion in the case (ii) is inhibited (or suppressed) by about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the case (i). ) Can be selected as a compound that inhibits the activity of the protein of the present invention or a salt thereof.
In addition, a gene such as secretory alkaline phosphatase or luciferase is inserted downstream of the promoter of the protein gene of the present invention, expressed in the above-described various cells, and activated to activate the enzyme activity when the test compound is brought into contact with the cells. By searching for an inhibitory compound or a salt thereof, a compound or a salt thereof that promotes or suppresses the expression of the protein of the present invention (that is, promotes or inhibits the activity of the protein of the present invention) can be screened.
[0044]
The polynucleotide encoding the protein of the present invention is useful as a reagent for screening a compound or a salt thereof that promotes or inhibits the expression of the protein gene of the present invention.
The present invention relates to (3) a compound or a salt thereof which promotes or inhibits the expression of the protein gene of the present invention, which is characterized by using a polynucleotide encoding the protein of the present invention (hereinafter referred to as a promoter and an inhibitor, respectively). May be provided), more specifically, for example,
(4) A comparison is made between (iii) culturing cells capable of producing the protein of the present invention and (iv) culturing a mixture of cells capable of producing the protein of the present invention and a test compound. A method for screening for an accelerator or an inhibitor is provided.
In the above-mentioned screening method, for example, in the cases (iii) and (iv), the expression level of the protein gene of the present invention (specifically, the amount of the protein of the present invention or the amount of mRNA encoding the protein) is measured. And compare.
Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like. Or a known compound.
In order to carry out the above-mentioned screening method, cells having the ability to produce the protein of the present invention are prepared by suspending them in a buffer suitable for screening. The buffer may be any buffer such as a phosphate buffer or a borate buffer having a pH of about 4 to 10 (preferably, a pH of about 6 to 8) as long as it does not inhibit the organic anion transport activity of the protein of the present invention.
As a cell having the ability to produce the protein of the present invention, for example, a host (transformant) transformed with a vector containing the DNA encoding the protein of the present invention described above is used. As a host, for example, animal cells such as CHO cells are preferably used. For the screening, for example, a transformant in which the protein of the present invention is expressed on a cell membrane by culturing by the method described above is preferably used.
The amount of the protein of the present invention can be measured by a known method, for example, using an antibody recognizing the protein of the present invention, and analyzing the protein present in a cell extract or the like by a method such as Western analysis, ELISA, or the like. It can be measured according to the method.
The expression level of the protein gene of the present invention can be determined by a known method, for example, a method such as Northern blotting, Reverse transcription-polymerase chain reaction (RT-PCR), a real-time PCR analysis system (manufactured by ABI, TaqMan polymerase chain reaction), or the like. It can be measured according to a corresponding method.
For example, a test compound that promotes the expression of the protein gene of the present invention in the case of the above (iv) by about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the case of the above (iii). Can be selected as a compound or its salt that promotes the expression of the protein gene of the present invention.
For example, a test compound that inhibits the expression of the protein gene of the present invention in the case of the above (iv) by about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the case of the above (iii). Can be selected as a compound that inhibits the expression of the protein gene of the present invention or a salt thereof.
Furthermore, the antibody of the present invention is useful as a reagent for screening a compound or a salt thereof that promotes or inhibits the expression of the protein of the present invention.
The present invention relates to (5) a compound or a salt thereof which promotes or inhibits the expression (production) of the protein of the present invention, which is characterized by using the antibody of the present invention (hereinafter referred to as a promoter or an inhibitor, respectively). Is provided), more specifically, for example,
(6) A comparison is made between (v) culturing cells capable of producing the protein of the present invention and (vi) culturing a mixture of cells capable of producing the protein of the present invention and a test compound. A method for screening for an accelerator or an inhibitor is provided.
In the above screening method, for example, the expression level of the protein of the present invention (specifically, the protein level of the present invention) in the cases (v) and (vi) is measured using the antibody of the present invention (for example, The expression of the protein of the present invention is detected, the expression level of the protein of the present invention is quantified, and the like, and compared.
Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like. Or a known compound.
In order to carry out the above-mentioned screening method, cells having the ability to produce the protein of the present invention are prepared by suspending them in a buffer suitable for screening. The buffer may be any buffer such as a phosphate buffer or a borate buffer having a pH of about 4 to 10 (preferably, a pH of about 6 to 8) as long as it does not inhibit the organic anion transport activity of the protein of the present invention.
As a cell having the ability to produce the protein of the present invention, for example, a host (transformant) transformed with a vector containing the DNA encoding the protein of the present invention described above is used. As a host, for example, animal cells such as CHO cells are preferably used. For the screening, for example, a transformant in which the protein of the present invention is expressed on a cell membrane by culturing by the method described above is preferably used.
The amount of the protein of the present invention can be measured by a known method, for example, using an antibody recognizing the protein of the present invention, and analyzing the protein present in a cell extract or the like by a method such as Western analysis, ELISA, or the like. It can be measured according to the method.
For example, a test compound that promotes the expression of the protein of the present invention in the case (vi) above by about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the case (v). It can be selected as a compound or a salt thereof that promotes the expression of the protein of the present invention.
For example, a test compound that inhibits the expression of the protein of the present invention in the case of the above (vi) by about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the case of the above (v) It can be selected as a compound that inhibits the expression of the protein of the present invention or a salt thereof.
[0045]
The screening kit of the present invention contains the protein or partial peptide of the present invention or a salt thereof, or a cell capable of producing the protein or partial peptide of the present invention.
Compounds or salts thereof obtained by using the screening method or the screening kit of the present invention are test compounds described above, for example, peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts. A compound selected from animal tissue extract, plasma, and the like, or a salt thereof that promotes or inhibits the activity of the protein of the present invention (eg, organic anion transport activity).
As the salt of the compound, those similar to the aforementioned salts of the protein of the present invention are used.
Compounds or salts thereof that promote the activity of the protein of the present invention include, for example, renal diseases (eg, renal failure, glomerulonephritis, diabetic nephropathy, focal glomerulosclerosis, nephrotic syndrome, renal edema, interstitium) Hepatic disease (eg, cirrhosis, hepatitis, alcoholic liver disease, etc.), pancreatic disease (eg, pancreatitis, etc.), immune disease associated with thymic abnormalities, genital disease (eg, prostate gland) Hypertrophy, prostatitis, testicular neurosis, ovarian cysts, etc.), digestive disorders (eg, irritable bowel syndrome, ulcerative colitis, ischemic colitis, gastritis, etc.), spleen disorders (eg, hypersplenism) , Splenomatous syndrome, etc.), cancer (eg, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectum cancer, pancreatic cancer , Thyroid cancer, myeloid leukemia, etc.), respiratory diseases (eg, pleurisy, pneumonia, Obstructive pulmonary disease, asthma, etc.), osteomyelitis, diabetes, hypertension, post-ischemic reperfusion injury, retinitis, central nervous system diseases (eg, epilepsy, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), skin diseases ( For example, useful as prophylactic / therapeutic agents such as atopic dermatitis, seborrheic dermatitis, etc., thyroid hormone-related diseases (eg, thyroid hormone refractory disease, Basedow's disease, cretinism, hyperthyroidism, etc.) It is. It is preferably used as a prophylactic / therapeutic agent for renal diseases and thyroid hormone-related diseases, and more preferably as a prophylactic / therapeutic agent for diabetic nephropathy.
Further, the compound or a salt thereof that inhibits the activity of the protein of the present invention includes, for example, renal diseases (eg, renal failure, glomerulonephritis, diabetic nephropathy, focal glomerulosclerosis, nephrotic syndrome, renal edema, Interstitial nephritis, renal sclerosis, uremic disease, etc.), liver disease (eg, cirrhosis, hepatitis, alcoholic liver disease, etc.), pancreatic disease (eg, pancreatitis, etc.), immune disease associated with thymic abnormalities, genital disease (eg, , Prostatic hyperplasia, prostatitis, testicular neurosis, ovarian cysts), gastrointestinal disorders (eg, irritable bowel syndrome, ulcerative colitis, ischemic colitis, gastritis, etc.), spleen disorders (eg, spleen function) Hypertension, splenomegaly syndrome, etc.), cancer (eg, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, Pancreatic cancer, thyroid cancer, myeloid leukemia, etc.), respiratory diseases (eg, pleurisy, Inflammation, chronic obstructive pulmonary disease, asthma, etc.), osteomyelitis, diabetes, hypertension, post-ischemic reperfusion injury, retinitis, central nervous system diseases (eg, epilepsy, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), skin Preventive / therapeutic agents for diseases (eg, atopic dermatitis, seborrheic dermatitis, etc.), thyroid hormone-related diseases (eg, thyroid hormone refractory disease, Basedow's disease, cretinism, hyperthyroidism, etc.) Useful as a medicine. It is preferably used as a prophylactic / therapeutic agent for renal diseases and thyroid hormone-related diseases, and more preferably as a prophylactic / therapeutic agent for diabetic nephropathy.
Compounds or salts thereof that promote the expression of the protein gene of the present invention include, for example, renal diseases (eg, renal failure, glomerulonephritis, diabetic nephropathy, focal glomerulosclerosis, nephrotic syndrome, renal edema, Hepatic disease (eg, cirrhosis, hepatitis, alcoholic liver disease, etc.), pancreatic disease (eg, pancreatitis, etc.), immune diseases associated with thymic abnormalities, genital diseases (eg, Prostatic hyperplasia, prostatitis, testicular neurosis, ovarian cysts, etc., gastrointestinal disorders (eg, irritable bowel syndrome, ulcerative colitis, ischemic colitis, gastritis, etc.), spleen disorders (eg, hypersplenism) Disease, splenomegaly syndrome, etc.), cancer (eg, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, pancreas) Cancer, thyroid cancer, myeloid leukemia, etc.), respiratory diseases (eg, pleurisy, Inflammation, chronic obstructive pulmonary disease, asthma, etc.), osteomyelitis, diabetes, hypertension, post-ischemic reperfusion injury, retinitis, central nervous system diseases (eg, epilepsy, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), skin Preventive / therapeutic agents for diseases (eg, atopic dermatitis, seborrheic dermatitis, etc.), thyroid hormone-related diseases (eg, thyroid hormone refractory disease, Basedow's disease, cretinism, hyperthyroidism, etc.) Useful as a medicine. It is preferably used as a prophylactic / therapeutic agent for renal diseases and thyroid hormone-related diseases, and more preferably as a prophylactic / therapeutic agent for diabetic nephropathy.
Further, the compound of the present invention that inhibits the expression of the protein gene or a salt thereof includes, for example, renal diseases (eg, renal failure, glomerulonephritis, diabetic nephropathy, focal glomerulosclerosis, nephrotic syndrome, renal edema) , Interstitial nephritis, renal sclerosis, uremic disease, etc.), liver disease (eg, cirrhosis, hepatitis, alcoholic liver disease, etc.), pancreatic disease (eg, pancreatitis, etc.), immune diseases associated with thymic abnormalities, genital diseases ( Eg, benign prostatic hyperplasia, prostatitis, testicular neurosis, ovarian cysts), gastrointestinal disorders (eg, irritable bowel syndrome, ulcerative colitis, ischemic colitis, gastritis, etc.), spleen disorders (eg, spleen) Hyperfunction, splenomegaly syndrome, etc.), cancer (eg, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer) , Pancreatic cancer, thyroid cancer, myeloid leukemia, etc.), respiratory diseases (eg, breast Inflammation, pneumonia, chronic obstructive pulmonary disease, asthma), osteomyelitis, diabetes, hypertension, post-ischemic reperfusion injury, retinitis, central nervous system diseases (eg, epilepsy, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.) , Skin diseases (eg, atopic dermatitis, seborrheic dermatitis, etc.), thyroid hormone-related diseases (eg, thyroid hormone refractory disease, Basedow's disease, cretinism, hyperthyroidism, etc.), etc. It is useful as a medicament. It is preferably used as a prophylactic / therapeutic agent for renal diseases and thyroid hormone-related diseases, and more preferably as a prophylactic / therapeutic agent for diabetic nephropathy.
Compounds or salts thereof that promote the expression of the protein of the present invention include, for example, renal diseases (eg, renal failure, glomerulonephritis, diabetic nephropathy, focal glomerulosclerosis, nephrotic syndrome, renal edema, interstitial) Hepatic disease (eg, cirrhosis, hepatitis, alcoholic liver disease, etc.), pancreatic disease (eg, pancreatitis, etc.), immune disease associated with thymic abnormalities, genital disease (eg, prostate gland) Hypertrophy, prostatitis, testicular neurosis, ovarian cysts, etc.), digestive disorders (eg, irritable bowel syndrome, ulcerative colitis, ischemic colitis, gastritis, etc.), spleen disorders (eg, hypersplenism) , Splenomatous syndrome, etc.), cancer (eg, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectum cancer, pancreatic cancer , Thyroid cancer, myeloid leukemia, etc.), respiratory diseases (eg, pleurisy, pneumonia, Obstructive pulmonary disease, asthma, etc.), osteomyelitis, diabetes, hypertension, post-ischemic reperfusion injury, retinitis, central nervous system diseases (eg, epilepsy, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), skin diseases ( For example, drugs for the prevention and treatment of thyroid hormone-related diseases (eg, thyroid hormone refractory disease, Basedow's disease, cretin disease, hyperthyroidism, etc.), atopic dermatitis, seborrheic dermatitis, etc. Useful. It is preferably used as a prophylactic / therapeutic agent for renal diseases and thyroid hormone-related diseases, and more preferably as a prophylactic / therapeutic agent for diabetic nephropathy.
Further, the compound or a salt thereof that inhibits the expression of the protein of the present invention includes, for example, renal diseases (eg, renal failure, glomerulonephritis, diabetic nephropathy, focal glomerulosclerosis, nephrotic syndrome, renal edema, Interstitial nephritis, renal sclerosis, uremic disease, etc.), liver disease (eg, cirrhosis, hepatitis, alcoholic liver disease, etc.), pancreatic disease (eg, pancreatitis, etc.), immune disease associated with thymic abnormalities, genital disease (eg, , Prostatic hyperplasia, prostatitis, testicular neurosis, ovarian cysts), gastrointestinal disorders (eg, irritable bowel syndrome, ulcerative colitis, ischemic colitis, gastritis, etc.), spleen disorders (eg, spleen function) Hypertension, splenomegaly syndrome, etc.), cancer (eg, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, Pancreatic cancer, thyroid cancer, myeloid leukemia, etc.), respiratory diseases (eg, pleurisy, Inflammation, chronic obstructive pulmonary disease, asthma, etc.), osteomyelitis, diabetes, hypertension, post-ischemic reperfusion injury, retinitis, central nervous system diseases (eg, epilepsy, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), skin Medications for the prevention and treatment of diseases (eg, atopic dermatitis, seborrheic dermatitis, etc.), thyroid hormone-related diseases (eg, thyroid hormone refractory disease, Basedow's disease, cretinism, hyperthyroidism, etc.) Useful as It is preferably used as a prophylactic / therapeutic agent for renal diseases and thyroid hormone-related diseases, and more preferably as a prophylactic / therapeutic agent for diabetic nephropathy.
Compounds or salts thereof obtained using the screening method or screening kit of the present invention include compounds or salts thereof that promote the activity of the protein of the present invention, compounds or salts thereof that promote the expression of the protein gene of the present invention, Compounds or salts thereof that promote the expression of the protein of the present invention are preferred.
[0046]
When the compound or a salt thereof obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated according to a conventional method. For example, tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions and the like can be used.
The preparations obtained in this way are safe and low toxic, for example, human or warm-blooded animals (eg, mouse, rat, rabbit, sheep, pig, cow, horse, bird, cat, dog, monkey, chimpanzee, etc.) ) Can be administered orally or parenterally.
The dose of the compound or a salt thereof varies depending on its action, target disease, administration subject, administration route and the like.For example, a compound or a salt thereof that promotes the activity of the protein of the present invention for the purpose of treating renal failure is used. When administered orally, in general, for an adult (with a body weight of 60 kg), the compound or a salt thereof is used in an amount of about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 50 mg per day. Administer 20 mg. When administered parenterally, the single dose of the compound or a salt thereof varies depending on the administration subject, target disease, and the like. For example, a compound or a compound that promotes the activity of the protein of the present invention for the purpose of treating renal failure When the salt is administered to an adult (with a body weight of 60 kg) usually in the form of an injection, the compound or a salt thereof is used in an amount of about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 20 mg per day. Advantageously, 1 to 10 mg is administered by intravenous injection. In the case of other animals, the amount can be administered in terms of the weight per 60 kg.
[0047]
[3] Quantification of the protein of the present invention, its partial peptide or its salt
Since the antibody of the present invention can specifically recognize the protein of the present invention, it can be used for quantification of the protein of the present invention in a test solution, particularly for quantification by sandwich immunoassay.
That is, the present invention
(I) reacting the antibody of the present invention with a test solution and a labeled protein of the present invention competitively, and measuring the ratio of the labeled protein of the present invention bound to the antibody; A method for quantifying the protein of the present invention in a test solution, and
(Ii) After reacting a test solution with the antibody of the present invention immobilized on a carrier and another labeled antibody of the present invention simultaneously or continuously, the activity of the labeling agent on the insolubilized carrier is measured. A method for quantifying the protein of the present invention in a test solution is provided.
In the quantitative method (ii), it is desirable that one antibody is an antibody that recognizes the N-terminal of the protein of the present invention and the other antibody is an antibody that reacts with the C-terminal of the protein of the present invention.
[0048]
In addition, the protein of the present invention can be quantified using a monoclonal antibody against the protein of the present invention (hereinafter sometimes referred to as the monoclonal antibody of the present invention), and can also be detected by tissue staining or the like. For these purposes, the antibody molecule itself may be used, and the F (ab ') ₂ , Fab ′ or Fab fraction may be used.
The method for quantifying the protein of the present invention using the antibody of the present invention is not particularly limited, and the antibody, antigen, or antibody-antigen complex corresponding to the amount of the antigen (eg, the amount of the protein) in the test solution is measured. Any measurement method may be used as long as the amount is detected by chemical or physical means, and the amount is calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephelometry, competition method, immunometric method and sandwich method are suitably used, but it is particularly preferable to use the sandwich method described later in terms of sensitivity and specificity.
As a labeling agent used in a measurement method using a labeling substance, for example, a radioisotope (eg, [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ¹⁴ C], [ ³² P], [ ³³ P], [ ³⁵ S], etc.), fluorescent substances [eg, cyanine fluorescent dyes (eg, Cy2, Cy3, Cy5, Cy5.5, Cy7 (manufactured by Amersham Bioscience)), fluorescamine, fluorescein isothiocyanate, etc.], enzymes ( Examples include β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase, etc., luminescent substances (eg, luminol, luminol derivatives, luciferin, lucigenin, etc.), biotin, lanthanide elements and the like. Further, a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.
[0049]
For the insolubilization of an antigen or an antibody, physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing and immobilizing a protein or an enzyme may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide, and silicon; and glass.
In the sandwich method, the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further reacted with another labeled monoclonal antibody of the present invention (secondary reaction). By measuring the activity of the labeling agent, the amount of the protein of the present invention in the test solution can be determined. The primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at staggered times. The labeling agent and the method of insolubilization can be in accordance with those described above. In addition, in the immunoassay by the sandwich method, the antibody used for the solid phase antibody or the labeling antibody is not necessarily one kind, and a mixture of two or more kinds of antibodies is used for the purpose of improving measurement sensitivity and the like. You may.
In the method for measuring the protein of the present invention by the sandwich method of the present invention, as the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction, an antibody having a different site to which the protein of the present invention binds is preferably used. That is, the antibody used in the primary reaction and the secondary reaction is preferably, for example, when the antibody used in the secondary reaction recognizes the C-terminal of the protein of the present invention, the antibody used in the primary reaction is preferably An antibody that recognizes, for example, the N-terminal other than the C-terminal is used.
[0050]
The monoclonal antibody of the present invention can be used in a measurement system other than the sandwich method, for example, a competition method, an immunometric method, a nephrometry, or the like.
In the competition method, after the antigen in the test solution and the labeled antigen are allowed to react competitively with the antibody, the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated. (B / F separation), the amount of any of B and F is measured, and the amount of antigen in the test solution is quantified. In this reaction method, a soluble antibody is used as the antibody, B / F separation is performed using polyethylene glycol, a liquid phase method using a second antibody against the antibody, or a solid phase antibody is used as the first antibody. A solid-phase method using a soluble antibody as the first antibody and using a solid-phased antibody as the second antibody is used.
In the immunometric method, the antigen in the test solution and the immobilized antigen are subjected to a competitive reaction with a fixed amount of the labeled antibody, and then the solid phase and the liquid phase are separated. And an excess amount of the labeled antibody, and then immobilized antigen is added to bind unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Next, the amount of label in any phase is measured to determine the amount of antigen in the test solution.
In nephelometry, the amount of insoluble sediment produced as a result of an antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test solution is small and only a small amount of sediment is obtained, laser nephrometry utilizing laser scattering is preferably used.
[0051]
In applying these individual immunological measurement methods to the quantification method of the present invention, no special conditions, operations, and the like need to be set. The protein measurement system of the present invention may be constructed by adding ordinary technical considerations of those skilled in the art to ordinary conditions and operation methods in each method. For details of these general technical means, reference can be made to reviews, books, and the like.
For example, Hiroe Irie, "Radio Immunoassay" (Kodansha, published in 1974), Hiroshi Irie, "Sequence Radioimmunoassay" (Kodansha, published in 1974), Eiji Ishikawa et al., "Enzyme Immunoassay" (Medical Shoin, Showa Ed. Ishikawa et al., "Enzyme Immunoassay" (2nd edition) (Issue Shoin, published in 1982), Eiji Ishikawa et al., "Enzyme Immunoassay" (3rd edition) (Medical Shoin, Showa) 62)), "Methods in ENZYMOLOGY" Vol. 70 (Immunochemical Techniques (Part A)), ibid., Vol. 73 (Immunochemical Technologies (Part B)), ibid., Vol. 74 (Immunochemical Technologies (Part C)), ibid., Vol. 84 (Immunochemical Technologies (Part D: Selected Immunoassays)), ibid., Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)), ibid., Vol. 121 (Immunochemical Technologies (Part I: Hybridoma Technology and Monoclonal Antibodies)) (all published by Academic Press) can be referred to.
As described above, the protein of the present invention can be quantified with high sensitivity by using the antibody of the present invention.
Furthermore, when a decrease in the concentration of the protein of the present invention is detected by quantifying the concentration of the protein of the present invention using the antibody of the present invention, for example, a renal disease (eg, renal failure, glomerulonephritis, Diabetic nephropathy, focal glomerulosclerosis, nephrotic syndrome, renal edema, interstitial nephritis, nephrosclerosis, uremia, etc., liver disease (eg, cirrhosis, hepatitis, alcoholic liver disease, etc.), pancreas Diseases (eg, pancreatitis), immune disorders associated with thymic abnormalities, genital diseases (eg, prostatic hypertrophy, prostatitis, testicular neurosis, ovarian cysts), gastrointestinal disorders (eg, irritable bowel syndrome, ulcerative) Colitis, ischemic colitis, gastritis, etc., spleen disease (eg, hypersplenism, splenomegaly syndrome, etc.), cancer (eg, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer) , Gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer Pancreatic cancer, thyroid cancer, myeloid leukemia, etc.), respiratory diseases (eg, pleurisy, pneumonia, chronic obstructive pulmonary disease, asthma, etc.), osteomyelitis, diabetes, hypertension, post-ischemic reperfusion injury, retinitis, central Neurological diseases (eg, epilepsy, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), skin diseases (eg, atopic dermatitis, seborrheic dermatitis, etc.), thyroid hormone-related diseases (eg, thyroid hormone refractory disease, Basedow) Disease, cretinism, hyperthyroidism, etc.) can be diagnosed as having a high possibility. Conversely, for example, when an increase in the concentration of the protein of the present invention is detected, for example, renal diseases (eg, renal failure, glomerulonephritis, diabetic nephropathy, focal glomerulosclerosis, nephrotic syndrome, nephrotic syndrome) Edema, interstitial nephritis, renal sclerosis, uremia, etc.), liver disease (eg, cirrhosis, hepatitis, alcoholic liver disease, etc.), pancreatic disease (eg, pancreatitis, etc.), immune disease associated with thymic abnormalities, genital disease (Eg, benign prostatic hyperplasia, prostatitis, testicular neurosis, ovarian cysts), gastrointestinal disorders (eg, irritable bowel syndrome, ulcerative colitis, ischemic colitis, gastritis, etc.), spleen disorders (eg, Hypersplenism, splenomegaly syndrome, etc.), cancer (eg, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectum Cancer, pancreatic cancer, thyroid cancer, myeloid leukemia, etc., respiratory diseases (eg, pleurisy) Pneumonia, chronic obstructive pulmonary disease, asthma), osteomyelitis, diabetes, hypertension, post-ischemic reperfusion injury, retinitis, central nervous system diseases (eg, epilepsy, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), skin Diseases (eg, atopic dermatitis, seborrheic dermatitis, etc.) and thyroid hormone-related diseases (eg, thyroid hormone refractory disease, Graves' disease, cretinism, hyperthyroidism, etc.) It can be diagnosed as high.
Further, the antibody of the present invention can be used for detecting the protein of the present invention present in a subject such as a body fluid or a tissue. Further, for preparation of an antibody column used for purifying the protein of the present invention, detection of the protein of the present invention in each fraction at the time of purification, analysis of the behavior of the protein of the present invention in test cells, etc. Can be used.
[0052]
[4] Gene diagnostics
The DNA of the present invention can be used, for example, as a probe to produce a human or warm-blooded animal (eg, rat, mouse, guinea pig, rabbit, bird, sheep, pig, cow, horse, cat, dog, monkey, chimpanzee, etc.). Can detect an abnormality (genetic abnormality) in DNA or mRNA encoding the protein of the present invention or a partial peptide thereof in, for example, damage, mutation or decrease in expression of the DNA or mRNA, or decrease in the DNA or mRNA. It is useful as a diagnostic agent for a gene such as increase or overexpression.
The above-described genetic diagnosis using the DNA of the present invention can be performed, for example, by the known Northern hybridization or PCR-SSCP method (Genomics, Vol. 5, pp. 874-879 (1989), Proceedings of the National Academy of Sciences of the United States). of America, Vol. 86, pp. 2766-2770 (1989)) and the like.
For example, when increased expression is detected by Northern hybridization, for example, renal diseases (eg, renal failure, glomerulonephritis, diabetic nephropathy, focal glomerulosclerosis, nephrotic syndrome, renal edema, interstitial nephritis) , Renal sclerosis, uremic disease, etc.), liver disease (eg, cirrhosis, hepatitis, alcoholic liver disease, etc.), pancreatic disease (eg, pancreatitis, etc.), immune disease associated with thymic abnormalities, genital disease (eg, prostatic hypertrophy) , Prostatitis, testicular neurosis, ovarian cysts, etc.), digestive disorders (eg, irritable bowel syndrome, ulcerative colitis, ischemic colitis, gastritis, etc.), spleen disorders (eg, hypersplenism, splenomegaly) Sexual syndrome, etc.), cancer (eg, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, pancreatic cancer, thyroid gland Cancer, myeloid leukemia, etc.), respiratory diseases (eg, breast Inflammation, pneumonia, chronic obstructive pulmonary disease, asthma), osteomyelitis, diabetes, hypertension, post-ischemic reperfusion injury, retinitis, central nervous system diseases (eg, epilepsy, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.) , Skin diseases (eg, atopic dermatitis, seborrheic dermatitis, etc.), thyroid hormone-related diseases (eg, thyroid hormone refractory disease, Basedow's disease, cretinism, hyperthyroidism, etc.) Can be diagnosed. Conversely, when a decrease in expression is detected or a DNA mutation is detected by the PCR-SSCP method, for example, renal diseases (eg, renal failure, glomerulonephritis, diabetic nephropathy, focal glomeruli) Sclerosis, nephrotic syndrome, renal edema, interstitial nephritis, renal sclerosis, uremic disease, etc., liver disease (eg, cirrhosis, hepatitis, alcoholic liver disease, etc.), pancreatic disease (eg, pancreatitis, etc.), thymus Immune diseases associated with abnormalities, genital diseases (eg, prostatic hypertrophy, prostatitis, testicular neurosis, ovarian cysts, etc.), gastrointestinal diseases (eg, irritable bowel syndrome, ulcerative colitis, ischemic colitis, gastritis) Etc.), spleen disease (eg, hypersplenia, splenomegaly syndrome, etc.), cancer (eg, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, uterus Cervical, colon, rectal, pancreatic, thyroid, myeloid leukemia Etc.), respiratory diseases (eg, pleurisy, pneumonia, chronic obstructive pulmonary disease, asthma, etc.), osteomyelitis, diabetes, hypertension, post-ischemic reperfusion injury, retinitis, central nervous system diseases (eg, epilepsy, Alzheimer's disease) , Parkinson's syndrome, schizophrenia, etc.), skin diseases (eg, atopic dermatitis, seborrheic dermatitis, etc.), thyroid hormone-related diseases (eg, thyroid hormone refractory disease, Graves' disease, cretin disease, hyperthyroidism) Etc.) can be diagnosed as having a high possibility.
[0053]
[5] Pharmaceutical and diagnostic agents containing antisense polynucleotide
The antisense polynucleotide of the present invention capable of binding complementarily to the DNA of the present invention and suppressing the expression of the DNA has low toxicity, and the function of the protein of the present invention or the function of the DNA of the present invention in vivo (eg, For example, renal disease (eg, renal failure, glomerulonephritis, diabetic nephropathy, focal glomerulosclerosis, nephrotic syndrome, renal edema, interstitial) Hepatic disease (eg, cirrhosis, hepatitis, alcoholic liver disease, etc.), pancreatic disease (eg, pancreatitis, etc.), immune disease associated with thymic abnormalities, genital disease (eg, prostate gland) Hypertrophy, prostatitis, testicular neurosis, ovarian cysts, etc.), digestive disorders (eg, irritable bowel syndrome, ulcerative colitis, ischemic colitis, gastritis, etc.), spleen disorders (eg, hypersplenism) , Splenomegaly syndrome) Cancer (eg, lung, kidney, liver, non-small cell lung, ovarian, prostate, gastric, bladder, breast, cervical, colon, rectal, pancreatic, thyroid, myeloid leukemia ), Respiratory diseases (eg, pleurisy, pneumonia, chronic obstructive pulmonary disease, asthma, etc.), osteomyelitis, diabetes, hypertension, reperfusion injury after ischemia, retinitis, central nervous system diseases (eg, epilepsy, Alzheimer's disease) , Parkinson's syndrome, schizophrenia, etc.), skin diseases (eg, atopic dermatitis, seborrheic dermatitis, etc.), thyroid hormone-related diseases (eg, thyroid hormone refractory disease, Graves' disease, cretin disease, hyperthyroidism) ) Can be used as a prophylactic / therapeutic agent. It is preferably used as a prophylactic / therapeutic agent for renal diseases and thyroid hormone-related diseases, and more preferably as a prophylactic / therapeutic agent for diabetic nephropathy. Further, the antisense polynucleotide of the present invention is also used for diagnosis of the above-mentioned diseases.
When the above-mentioned antisense polynucleotide is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated and administered according to a known method.
For example, when the antisense polynucleotide is used, the antisense polynucleotide is inserted alone or into a suitable vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, or the like, and then inserted into a human or mammal according to a conventional method. It can be administered orally or parenterally to animals (eg, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). The antisense polynucleotide can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as an auxiliary agent for promoting uptake, and can be administered using a gene gun or a catheter such as a hydrogel catheter.
The dose of the antisense polynucleotide varies depending on the target disease, the administration subject, the administration route, and the like.For example, when the antisense polynucleotide of the present invention is locally administered to the kidney for the purpose of treating renal failure, it is generally used. For an adult (body weight 60 kg), about 0.1 to 100 mg of the antisense polynucleotide is administered per day. Furthermore, the antisense polynucleotide can also be used as a diagnostic oligonucleotide probe for examining the presence or expression of the DNA of the present invention in tissues or cells.
[0054]
Further, the present invention provides
(I) a double-stranded RNA containing a part of RNA encoding the protein of the present invention and RNA complementary thereto,
(Ii) a medicine comprising the double-stranded RNA,
(Iii) a ribozyme containing a part of RNA encoding the protein of the present invention,
(Iv) a medicine comprising the ribozyme,
(V) Also provided is an expression vector containing the gene (DNA) encoding the ribozyme.
Like the above-mentioned antisense polynucleotide, double-stranded RNA, ribozyme and the like can also destroy RNA transcribed from the DNA of the present invention or suppress the function thereof, and can be used in vivo for the protein of the present invention or the present invention. Since the function of DNA used can be suppressed, for example, renal diseases (eg, renal failure, glomerulonephritis, diabetic nephropathy, focal glomerulosclerosis, nephrotic syndrome, renal edema, interstitial nephritis) , Renal sclerosis, uremic disease, etc.), liver disease (eg, cirrhosis, hepatitis, alcoholic liver disease, etc.), pancreatic disease (eg, pancreatitis, etc.), immune disease associated with thymic abnormalities, genital disease (eg, prostatic hypertrophy) , Prostatitis, testicular neurosis, ovarian cysts, etc.), digestive disorders (eg, irritable bowel syndrome, ulcerative colitis, ischemic colitis, gastritis, etc.), spleen disorders (eg, hypersplenism, splenomegaly) Sexual disorder Group, etc.), cancer (eg, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, pancreatic cancer, thyroid cancer , Myeloid leukemia), respiratory disease (eg, pleurisy, pneumonia, chronic obstructive pulmonary disease, asthma, etc.), osteomyelitis, diabetes, hypertension, post-ischemic reperfusion injury, retinitis, central nervous system disease (eg, Epilepsy, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), skin diseases (eg, atopic dermatitis, seborrheic dermatitis), thyroid hormone-related diseases (eg, thyroid hormone refractory disease, Graves' disease, Cretin disease, For example, hyperthyroidism, etc.). It is preferably used as a prophylactic / therapeutic agent for renal diseases and thyroid hormone-related diseases, and more preferably as a prophylactic / therapeutic agent for diabetic nephropathy.
The double-stranded RNA can be produced by designing based on the sequence of the polynucleotide of the present invention according to a known method (eg, Nature, 411, 494, 2001).
A ribozyme can be designed and produced based on the sequence of the polynucleotide of the present invention according to a known method (eg, TRENDS in Molecular Medicine, Vol. 7, pp. 221, 2001). For example, it can be produced by substituting a part of a known ribozyme sequence with a part of RNA encoding the protein of the present invention. As a part of the RNA encoding the protein of the present invention, a sequence in the vicinity of a consensus sequence NUX (where N represents all bases and X represents a base other than G) which can be cleaved by a known ribozyme, and the like are included. No.
When the above-mentioned double-stranded RNA or ribozyme is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated and administered in the same manner as an antisense polynucleotide. The expression vector (v) is used in the same manner as known gene therapy methods and used as the above-mentioned prophylactic / therapeutic agent.
[0055]
[6] A drug containing the antibody of the present invention
The antibody of the present invention, which has the activity of neutralizing the activity of the protein of the present invention, can be used, for example, in renal diseases (eg, renal failure, glomerulonephritis, diabetic nephropathy, focal glomerulosclerosis, nephrotic syndrome, renal Edema, interstitial nephritis, renal sclerosis, uremia, etc.), liver disease (eg, cirrhosis, hepatitis, alcoholic liver disease, etc.), pancreatic disease (eg, pancreatitis, etc.), immune disease associated with thymic abnormalities, genital disease (Eg, benign prostatic hyperplasia, prostatitis, testicular neurosis, ovarian cysts), gastrointestinal disorders (eg, irritable bowel syndrome, ulcerative colitis, ischemic colitis, gastritis, etc.), spleen disorders (eg, Hypersplenism, splenomegaly syndrome, etc.), cancer (eg, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectum Cancer, pancreatic cancer, thyroid cancer, myeloid leukemia, etc.), respiratory diseases (eg, pleurisy, Inflammation, chronic obstructive pulmonary disease, asthma, etc.), osteomyelitis, diabetes, hypertension, post-ischemic reperfusion injury, retinitis, central nervous system diseases (eg, epilepsy, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), skin Used as a preventive / therapeutic agent for diseases (eg, atopic dermatitis, seborrheic dermatitis, etc.) and thyroid hormone-related diseases (eg, thyroid hormone refractory disease, Basedow's disease, cretinism, hyperthyroidism, etc.) can do. It is preferably used as a prophylactic / therapeutic agent for renal diseases and thyroid hormone-related diseases, and more preferably as a prophylactic / therapeutic agent for diabetic nephropathy.
The prophylactic / therapeutic agent for the above-mentioned diseases containing the antibody of the present invention has low toxicity and is used as it is as a liquid or as a pharmaceutical composition of an appropriate dosage form, in humans or mammals (eg, rat, rabbit, sheep, pig, It can be administered orally or parenterally (eg, intra-articular administration) to cattle, cats, dogs, monkeys, and the like. The dose varies depending on the administration subject, target disease, symptoms, administration route, and the like.For example, when used for the treatment or prevention of diabetic nephropathy, the antibody of the present invention is usually used as a single dose. About 0.01 to 20 mg / kg body weight, preferably about 0.1 to 10 mg / kg body weight, more preferably about 0.1 to 5 mg / kg body weight, about 1 to 5 times a day, preferably 1 to 5 times a day It is convenient to administer intravenously about three times. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If the symptoms are particularly severe, the dose may be increased according to the symptoms.
The antibodies of the present invention can be administered as such or as a suitable pharmaceutical composition. The pharmaceutical composition used for the administration contains the antibody or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient. Such compositions are provided in dosage forms suitable for oral or parenteral administration (eg, intravenous, intra-articular administration). It is preferably provided as an inhalant.
Each of the above-mentioned compositions may contain another active ingredient as long as the composition does not cause an undesirable interaction with the antibody.
[0056]
[7] Creation of an animal having the DNA of the present invention
The present invention relates to a non-protein having a DNA encoding an exogenous protein of the present invention (hereinafter abbreviated as the exogenous DNA of the present invention) or a mutant DNA thereof (sometimes abbreviated as the exogenous mutant DNA of the present invention). A human mammal is provided.
That is, the present invention
(1) a non-human mammal having the exogenous DNA of the present invention or a mutant DNA thereof,
(2) The animal according to (1) above, wherein the non-human mammal is a rodent.
(3) The animal according to (2), wherein the rodent is a mouse or a rat, and
(4) A recombinant vector or the like containing the exogenous DNA of the present invention or its mutant DNA and capable of being expressed in mammals is provided.
Non-human mammals having the exogenous DNA of the present invention or the mutant DNA thereof (hereinafter abbreviated as the DNA-transferred animal of the present invention) can be used for non-fertilized eggs, fertilized eggs, germ cells including spermatozoa and their progenitor cells, and the like. And preferably at the stage of embryonic development in non-human mammal development (more preferably at the stage of single cells or fertilized eggs and generally before the 8-cell stage), the calcium phosphate method, the electric pulse method, the lipofection method, the agglutination method, It can be produced by introducing a target DNA by a microinjection method, a particle gun method, a DEAE-dextran method, or the like. Further, by the DNA introduction method, the exogenous DNA of the present invention can be introduced into somatic cells, organs of living organisms, tissue cells, and the like, and used for cell culture, tissue culture, and the like. The DNA-transfected animal of the present invention can also be produced by fusing the above-mentioned germ cells with a known cell fusion method.
As the non-human mammal, for example, cow, pig, sheep, goat, rabbit, dog, cat, guinea pig, hamster, mouse, rat and the like are used. Among them, rodents, which are relatively short in ontogeny and biological cycle in terms of preparation of disease animal model systems, and are easy to breed, especially mice (for example, hybrids such as C57BL / 6 strain and DBA2 strain as pure strains) B6C3F as a system ₁ System, BDF ₁ System, B6D2F ₁ Strain, BALB / c strain, ICR strain, etc.) or rat (eg, Wistar, SD etc.) is preferred.
"Mammals" in a recombinant vector that can be expressed in mammals include humans in addition to the above-mentioned non-human mammals.
[0057]
The exogenous DNA of the present invention refers to the DNA of the present invention once isolated and extracted from a mammal, not the DNA of the present invention originally possessed by a non-human mammal.
As the mutant DNA of the present invention, those having a mutation (for example, mutation) in the base sequence of the original DNA of the present invention, specifically, base addition, deletion, substitution with another base, etc. The resulting DNA or the like is used, and also includes abnormal DNA.
The abnormal DNA means a DNA that expresses an abnormal protein of the present invention, and for example, a DNA that expresses a protein that suppresses the function of the normal protein of the present invention is used.
The exogenous DNA of the present invention may be derived from a mammal that is the same or different from the animal of interest. When introducing the DNA of the present invention into a target animal, it is generally advantageous to use the DNA as a DNA construct linked downstream of a promoter capable of being expressed in animal cells. For example, when the human DNA of the present invention is introduced, DNAs derived from various mammals (eg, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.) having the DNA of the present invention having high homology thereto are expressed. Highly expressing the DNA of the present invention by microinjecting a DNA construct (eg, vector, etc.) in which the human DNA of the present invention is bound downstream of various promoters that can be made into a fertilized egg of a target mammal, for example, a mouse fertilized egg. DNA-introduced mammals can be created.
[0058]
Examples of the expression vector of the protein of the present invention include plasmids derived from Escherichia coli, plasmids derived from Bacillus subtilis, plasmids derived from yeast, bacteriophages such as λ phage, retroviruses such as Moloney leukemia virus, animal viruses such as vaccinia virus or baculovirus. Are used. Among them, a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis or a plasmid derived from yeast is preferably used.
Examples of the promoter for controlling the DNA expression include, for example, (i) a promoter of a DNA derived from a virus (eg, simian virus, cytomegalovirus, Moloney leukemia virus, JC virus, breast cancer virus, poliovirus, etc.); ) Promoters derived from various mammals (human, rabbit, dog, cat, guinea pig, hamster, rat, mouse, etc.), for example, albumin, insulin II, uroplakin II, elastase, erythropoietin, endothelin, muscle creatine kinase, glial fibrillary acid Protein, glutathione S-transferase, platelet-derived growth factor β, keratins K1, K10 and K14, collagen types I and II, cyclic AMP-dependent protein kinase βI subunit, dyst Lophine, tartrate-resistant alkaline phosphatase, atrial natriuretic factor, endothelial receptor tyrosine kinase (commonly abbreviated as Tie2), sodium potassium adenosine 3 kinase (Na, K-ATPase), neurofilament light chain, metallothionein I and IIA, metalloproteinase 1 tissue inhibitor, MHC class I antigen (H-2L), H-ras, renin, dopamine β-hydroxylase, thyroid peroxidase (TPO), polypeptide chain elongation factor 1α (EF-1α), β Actin, α and β myosin heavy chains, myosin light chains 1 and 2, myelin basic protein, thyroglobulin, Thy-1, immunoglobulin, heavy chain variable region (VNP), serum amyloid P component, myoglobin, troponin C, blunt α-actin, preproenkephalin A, such as a promoter, such as vasopressin are used. Among them, a cytomegalovirus promoter capable of high expression throughout the body, a promoter of human polypeptide chain elongation factor 1α (EF-1α), human and chicken β-actin promoters, and the like are preferable.
The above vector preferably has a sequence that terminates transcription of the target mRNA in a DNA-transfected mammal (generally called a terminator). For example, a sequence of each DNA derived from a virus and from various mammals is used. Preferably, a simian virus SV40 terminator or the like is used.
[0059]
In addition, a splicing signal of each DNA, an enhancer region, a part of an intron of a eukaryotic DNA, and the like are placed 5 ′ upstream of the promoter region, between the promoter region and the translation region, or between the translation region for the purpose of further expressing the desired foreign DNA. It is also possible to connect 3 ′ downstream of the above depending on the purpose.
The normal translation region of the protein of the present invention can be obtained from liver, kidney, thyroid cells, fibroblast-derived DNA derived from humans or various mammals (eg, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.) and commercially available DNAs. From a variety of genomic DNA libraries, or as a starting material, a complementary DNA prepared by known methods from RNA derived from liver, kidney, thyroid cells, and fibroblasts. In addition, a translation region obtained by mutating a translation region of a normal polypeptide obtained from the above-mentioned cells or tissues by a point mutagenesis method can be used for the exogenous abnormal DNA.
The translation region can be prepared as a DNA construct that can be expressed in an introduced animal by a usual DNA engineering technique in which the translation region is ligated downstream of the promoter and, if desired, upstream of the transcription termination site.
Introduction of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal. The presence of the exogenous DNA of the present invention in the germinal cells of the produced animal after the introduction of the DNA means that all progeny of the produced animal will retain the exogenous DNA of the present invention in all of the germ cells and somatic cells. means. The offspring of such animals that have inherited the exogenous DNA of the present invention have the exogenous DNA of the present invention in all of their germ cells and somatic cells.
The non-human mammal into which the exogenous normal DNA of the present invention has been introduced can be subcultured in a normal breeding environment as an animal having the DNA after confirming that the exogenous DNA is stably maintained by mating.
Introduction of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in excess in all germ cells and somatic cells of the target mammal. Excessive presence of the exogenous DNA of the present invention in the germinal cells of the produced animal after DNA introduction means that all the offspring of the produced animal have the exogenous DNA of the present invention in all of their germinal and somatic cells. Means Progeny of such animals that have inherited the exogenous DNA of the present invention have an excess of the exogenous DNA of the present invention in all of their germinal and somatic cells.
By obtaining a homozygous animal having the introduced DNA on both homologous chromosomes and mating the male and female animals, all offspring can be bred to have the DNA in excess.
[0060]
The non-human mammal having the normal DNA of the present invention expresses the normal DNA of the present invention at a high level and promotes the function of the endogenous normal DNA to eventually cause hyperactivity of the protein of the present invention. Occasionally, it can be used as a disease model animal. For example, using the normal DNA-transfected animal of the present invention, it is possible to elucidate the pathological mechanism of the hyperactivity of the protein of the present invention and diseases associated with the protein of the present invention, and to examine a method for treating these diseases. It is possible.
Further, since the mammal into which the exogenous normal DNA of the present invention has been introduced has an increased symptom of the released protein of the present invention, it can be used for a screening test for a therapeutic drug for a disease associated with the protein of the present invention. .
On the other hand, a non-human mammal having the exogenous abnormal DNA of the present invention can be subcultured in an ordinary breeding environment as an animal having the DNA after confirming that the exogenous DNA is stably maintained by mating. Furthermore, the desired foreign DNA can be used as a raw material by incorporating it into the aforementioned plasmid. The DNA construct with the promoter can be prepared by a usual DNA engineering technique. Introduction of the abnormal DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal. The presence of the abnormal DNA of the present invention in the germinal cells of the produced animal after DNA transfer means that all the offspring of the produced animal have the abnormal DNA of the present invention in all of its germ cells and somatic cells. The progeny of such animals that have inherited the exogenous DNA of the present invention have the abnormal DNA of the present invention in all of their germinal and somatic cells. A homozygous animal having the introduced DNA on both homologous chromosomes is obtained, and by crossing these male and female animals, it is possible to breed the cells so that all offspring have the DNA.
[0061]
The non-human mammal having the abnormal DNA of the present invention, in which the abnormal DNA of the present invention is highly expressed, inhibits the function of endogenous normal DNA, and finally, the functionally inactive form of the protein of the present invention. It can be refractory and can be used as a model animal for the disease. For example, by using the abnormal DNA-introduced animal of the present invention, it is possible to elucidate the pathological mechanism of the function-inactive refractory of the protein of the present invention and to examine a method for treating this disease.
Further, as a specific possibility, the abnormal DNA-highly expressing animal of the present invention exhibits a function of inhibiting the normal protein function (dominant negative action) by the abnormal protein of the present invention in the inactive refractory disease of the protein of the present invention. A model to elucidate. In addition, since the mammal into which the foreign abnormal DNA of the present invention has been introduced has an increased symptom of the released protein of the present invention, it can be used for a therapeutic drug screening test for the protein of the present invention or a functionally inactive refractory disease thereof. It is.
In addition, other possible uses of the two kinds of the DNA-introduced animals of the present invention include, for example,
(I) use as a cell source for tissue culture;
(Ii) a protein specifically expressed or activated by the protein of the present invention, by directly analyzing DNA or RNA in the tissue of the DNA-transfected animal of the present invention, or analyzing a polypeptide tissue expressed by the DNA; Analysis of the relationship with
(Iii) culturing cells of DNA-bearing tissue by standard tissue culture techniques and using them to study the function of cells from tissues that are generally difficult to culture;
(Iv) screening for an agent that enhances the function of the cell by using the cell described in (iii) above, and
(V) Isolation and purification of the mutant protein of the present invention and production of its antibody can be considered.
Furthermore, using the DNA-introduced animal of the present invention, it is possible to examine the clinical symptoms of diseases related to the protein of the present invention, including the inactive refractory disease of the protein of the present invention, etc. More detailed pathological findings in each organ of a disease model related to the disease can be obtained, which can contribute to the development of a new treatment method, and further to the research and treatment of a secondary disease caused by the disease.
In addition, it is possible to take out each organ from the DNA-introduced animal of the present invention, cut it into small pieces, and then use a proteolytic enzyme such as trypsin to obtain the released DNA-introduced cells, culture them, or systematize the cultured cells. . Furthermore, it is possible to examine the specificity of the protein-producing cell of the present invention, its relationship with apoptosis, differentiation or proliferation, or its signal transduction mechanism, and its abnormalities. It is an effective research material for
Further, in order to develop a therapeutic agent for a disease associated with the protein of the present invention, including a functionally inactive refractory type of the protein of the present invention, using the DNA-transfected animal of the present invention, It is possible to provide an effective and rapid screening method for a therapeutic agent for the disease by using a method or the like. In addition, using the DNA-introduced animal of the present invention or the exogenous DNA expression vector of the present invention, it is possible to examine and develop a DNA treatment method for a disease associated with the protein of the present invention.
[0062]
[8] Knockout animal
The present invention provides a non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated, and a non-human mammal deficient in expression of the DNA of the present invention.
That is, the present invention
(1) a non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated,
(2) The embryonic stem cell according to (1), wherein the DNA is inactivated by introducing a reporter gene (eg, a β-galactosidase gene derived from Escherichia coli).
(3) The embryonic stem cell according to (1), which is neomycin-resistant,
(4) the embryonic stem cell according to (1), wherein the non-human mammal is a rodent;
(5) The embryonic stem cell according to (4), wherein the rodent is a mouse,
(6) a non-human mammal deficient in expression of the DNA of the present invention, wherein the DNA is inactivated;
(7) The above (6), wherein the DNA is inactivated by introducing a reporter gene (eg, a β-galactosidase gene derived from Escherichia coli), and the reporter gene can be expressed under the control of a promoter for the DNA of the present invention. The non-human mammal of the description,
(8) the non-human mammal according to the above (6), wherein the non-human mammal is a rodent;
(9) The non-human mammal according to (8), wherein the rodent is a mouse, and
(10) A method for screening a compound or a salt thereof that promotes or inhibits promoter activity on DNA of the present invention, which comprises administering a test compound to the animal according to (7) and detecting the expression of a reporter gene. provide.
A non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated is an artificially mutated DNA of the present invention possessed by the non-human mammal, which suppresses the expression ability of the DNA, or By substantially losing the activity of the protein of the present invention encoded by the DNA, the DNA does not substantially have the ability to express the protein of the present invention (hereinafter, may be referred to as the knockout DNA of the present invention). ) Non-human mammalian embryonic stem cells (hereinafter abbreviated as ES cells).
As the non-human mammal, the same one as described above is used.
[0063]
The method of artificially mutating the DNA of the present invention can be performed, for example, by deleting a part or all of the DNA sequence and inserting or substituting another DNA by a genetic engineering technique. With these mutations, the knockout DNA of the present invention may be prepared, for example, by shifting the codon reading frame or disrupting the function of the promoter or exon.
Specific examples of the non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated (hereinafter abbreviated as the DNA-inactivated ES cells of the present invention or the knockout ES cells of the present invention) include, for example, The DNA of the present invention possessed by a non-human mammal is isolated, and its exon portion is a drug resistance gene represented by a neomycin resistance gene or a hygromycin resistance gene, or lacZ (β-galactosidase gene), cat (chloramphenicol acetyl). A reporter gene or the like typified by a transferase gene) to disrupt the exon function, or insert a DNA sequence that terminates gene transcription into an intron between exons (for example, a polyA addition signal); By making it impossible to synthesize complete mRNA, A DNA strand having a DNA sequence constructed so as to eventually disrupt the gene (hereinafter abbreviated as a targeting vector) is introduced into the chromosome of the animal by, for example, homologous recombination, and the resulting ES cells are used in the present invention. Analysis by Southern hybridization analysis using a DNA sequence on or in the vicinity of the DNA as a probe, or PCR using a DNA sequence on a targeting vector and a DNA sequence of a neighboring region other than the DNA of the present invention used for the preparation of the targeting vector as primers However, it can be obtained by selecting the knockout ES cells of the present invention.
[0064]
As the original ES cells for inactivating the DNA of the present invention by the homologous recombination method or the like, for example, those already established as described above may be used, or according to the known method of Evans and Kaufman. May be newly established. For example, in the case of mouse ES cells, currently, 129-line ES cells are generally used. For example, for the purpose of obtaining ES cells in which BDF is clearly known, for example, BDF in which the number of eggs collected by C57BL / 6 mice or C57BL / 6 has been improved by hybridization with DBA / 2 ₁ Mouse (F of C57BL / 6 and DBA / 2) ₁ ) Can also be used favorably. BDF ₁ In addition to the advantage that the number of eggs collected and the eggs are robust, the mice have C57BL / 6 mice as a background, and thus, the ES cells obtained using the C57BL / 6 mice were used to produce C57BL / 6 mice. / 6 mice can be advantageously used in that the genetic background can be replaced by C57BL / 6 mice by backcrossing.
In addition, when ES cells are established, blastocysts 3.5 days after fertilization are generally used. In addition, a large number of blastocysts can be efficiently obtained by collecting embryos at the 8-cell stage and culturing them until blastocysts are used. Early embryos can be obtained.
Either male or female ES cells may be used, but generally male ES cells are more convenient for producing a germline chimera. It is also desirable to discriminate between males and females as soon as possible in order to reduce cumbersome culture labor.
As a method for determining the sex of ES cells, for example, a method of amplifying and detecting a gene in a sex-determining region on the Y chromosome by a PCR method can be mentioned. If this method is used, it is conventionally necessary to perform about 10 karyotype analyses. ⁶ Since the number of ES cells is required, the number of ES cells of about 1 colony (approximately 50) is sufficient, so that primary selection of ES cells in the early stage of culture can be performed by discriminating between male and female. In addition, by enabling selection of male cells at an early stage, labor in the initial stage of culture can be significantly reduced.
[0065]
The secondary selection can be performed, for example, by confirming the number of chromosomes by the G-banding method. The number of chromosomes in the obtained ES cells is desirably 100% of the normal number. However, when it is difficult due to physical operations at the time of establishment, after knocking out the gene of the ES cells, the normal cells (for example, chromosomes in mice) are knocked out. It is desirable to clone again into cells (number 2n = 40).
The embryonic stem cell line obtained in this way usually has very good growth properties, but it must be carefully subcultured because it tends to lose its ontogenic ability. For example, on a suitable feeder cell such as STO fibroblast in the presence of LIF (1 to 10,000 U / ml) in a carbon dioxide incubator (preferably 5% carbon dioxide, 95% air or 5% oxygen, 5% The cells are cultured by a method such as culturing at about 37 ° C. in carbon dioxide gas and 90% air. At the time of subculture, for example, trypsin / EDTA solution (usually 0.001 to 0.5% trypsin / 0.1 to 5 mM EDTA, Preferably, a method is used in which cells are made into single cells by treatment with about 0.1% trypsin / 1 mM EDTA and seeded on freshly prepared feeder cells. Such passage is usually carried out every 1 to 3 days. At this time, it is desirable to observe the cells and, if any morphologically abnormal cells are found, discard the cultured cells.
ES cells are differentiated into various types of cells, such as parietal muscle, visceral muscle, and cardiac muscle, by monolayer culture up to high density or suspension culture until cell clumps are formed under appropriate conditions. [M. J. Evans and M.E. H. Kaufman, Nature, 292, 154, 1981; R. Martin Proc. Of National Academy of Sciences USA (Proc. Natl. Acad. Sci. USA) 78, 7634, 1981; C. Doetschman et al., Journal of Embryology and Experimental Morphology, Vol. 87, p. 27, 1985], that the DNA-deficient cell of the present invention obtained by differentiating the ES cell of the present invention is characterized by in vitro Is useful in cell biological studies of the protein of the present invention.
The non-human mammal deficient in DNA expression of the present invention can be distinguished from a normal animal by measuring the mRNA level of the animal using a known method and indirectly comparing the expression level.
As the non-human mammal, those similar to the aforementioned can be used.
[0066]
The non-human mammal deficient in expression of the DNA of the present invention is, for example, a DNA in which the targeting vector prepared as described above is introduced into mouse embryonic stem cells or mouse egg cells, and the DNA of the targeting vector is inactivated by the introduction. The DNA of the present invention can be knocked out by homologous recombination in which the sequence replaces the DNA of the present invention on the chromosome of mouse embryonic stem cells or mouse egg cells by homologous recombination.
Cells in which the DNA of the present invention has been knocked out can be used as a DNA sequence on a Southern hybridization analysis or targeting vector using the DNA sequence on or near the DNA of the present invention as a probe, and the DNA of the present invention derived from the mouse used for the targeting vector. It can be determined by analysis by PCR using a DNA sequence of a nearby region other than DNA as a primer. When non-human mammalian embryonic stem cells are used, a cell line in which the DNA of the present invention has been inactivated by gene homologous recombination is cloned, and the cells are cultured at an appropriate time, for example, at the 8-cell stage of non-human mammalian cells. The chimeric embryo is injected into an animal embryo or blastocyst, and the resulting chimeric embryo is transplanted into the uterus of the pseudopregnant non-human mammal. The produced animal is a chimeric animal comprising both cells having the normal DNA locus of the present invention and cells having the artificially mutated DNA locus of the present invention.
When a part of the germ cells of the chimeric animal has a mutated DNA locus of the present invention, all tissues are artificially mutated from a population obtained by crossing such a chimeric individual with a normal individual. It can be obtained by selecting individuals composed of cells having the added DNA locus of the present invention, for example, by judging coat color or the like. The individual thus obtained is usually an individual with a heterozygous expression of the protein of the present invention, which is crossed with an individual with a heterozygous expression of the protein of the present invention. Defective individuals can be obtained.
In the case of using an egg cell, for example, a transgenic non-human mammal having a targeting vector introduced into a chromosome can be obtained by injecting a DNA solution into a nucleus of an egg by a microinjection method, and these transgenic non-human mammals can be obtained. Compared to mammals, they can be obtained by selecting those having a mutation in the DNA locus of the present invention by homologous recombination.
In this manner, the individual in which the DNA of the present invention is knocked out can be bred in an ordinary breeding environment after confirming that the DNA of the animal obtained by the breeding is also knocked out.
Further, the acquisition and maintenance of the germ line may be performed according to a conventional method. That is, by crossing male and female animals having the inactivated DNA, a homozygous animal having the inactivated DNA on both homologous chromosomes can be obtained. The obtained homozygous animal can be efficiently obtained by rearing the mother animal in such a manner that there are one normal animal and one homozygote. By mating male and female heterozygous animals, homozygous and heterozygous animals having the inactivated DNA are bred and subcultured.
The non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated are extremely useful for producing a non-human mammal deficient in expressing the DNA of the present invention.
In addition, since the non-human mammal deficient in expression of the DNA of the present invention lacks various biological activities that can be induced by the protein of the present invention, a model of a disease caused by inactivation of the biological activity of the protein of the present invention. Therefore, it is useful for investigating the causes of these diseases and examining treatment methods.
[0067]
[8a] A method for screening a compound having a therapeutic / preventive effect on diseases caused by DNA deficiency or damage of the present invention
The non-human mammal deficient in expression of the DNA of the present invention can be used for screening for a compound having a therapeutic / preventive effect against diseases caused by deficiency or damage of the DNA of the present invention.
That is, the present invention is characterized by administering a test compound to a non-human mammal deficient in expressing the DNA of the present invention, and observing and measuring a change in the animal. Provided is a method for screening a compound or a salt thereof having a therapeutic / prophylactic effect against a disease.
Examples of the non-human mammal deficient in expressing DNA of the present invention used in the screening method include the same as described above.
Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma, and the like. These compounds are novel compounds. Or a known compound.
Specifically, a non-human mammal deficient in expression of the DNA of the present invention is treated with a test compound, compared with an untreated control animal, and tested using changes in each organ, tissue, disease symptoms, etc. of the animal as an index. The therapeutic / preventive effects of the compounds can be tested.
As a method of treating a test animal with a test compound, for example, oral administration, intravenous injection and the like are used, and it can be appropriately selected according to the symptoms of the test animal, the properties of the test compound, and the like. The dose of the test compound can be appropriately selected according to the administration method, the properties of the test compound, and the like.
For example, when screening for a compound having a prophylactic or therapeutic effect on renal failure, a test compound is administered to a non-human mammal deficient in expressing DNA of the present invention, and the amount of creatinine in blood and the amount of urinary protein in the animal are determined. Measure over time.
The compound obtained by using the screening method is a compound selected from the test compounds described above, and has a prophylactic / therapeutic effect against a disease caused by deficiency or damage of the protein of the present invention. It can be used as a drug for safe and low toxic prophylactic / therapeutic agents. Furthermore, compounds derived from the compounds obtained by the above screening can be used in the same manner.
[0068]
The compound obtained by the screening method may form a salt. Examples of the salt of the compound include physiologically acceptable acids (eg, inorganic acids, organic acids, etc.) and bases (eg, alkali metals, etc.). ) Is used, and a physiologically acceptable acid addition salt is particularly preferable. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, For example, salts with succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and the like are used.
A drug containing the compound or a salt thereof obtained by the screening method can be produced in the same manner as the above-mentioned drug containing the protein of the present invention.
The preparations obtained in this way are safe and low toxic, and thus, for example, human or other mammals (eg, rat, mouse, guinea pig, rabbit, sheep, pig, cow, horse, cat, dog, monkey, etc.) ) Can be administered.
The dose of the compound or a salt thereof varies depending on the target disease, the administration subject, the administration route, and the like. For example, when the compound is orally administered, it is generally used in an adult patient (with a body weight of 60 kg) having renal failure. Administers about 0.1-100 mg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg of the compound per day. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, and the like. For example, the compound is usually in the form of an injection in an adult patient (with a body weight of 60 kg) having renal failure. It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg of the compound by intravenous injection per day. In the case of other animals, the amount can be administered in terms of the weight per 60 kg.
[0069]
[8b] A method for screening a compound that promotes or inhibits the activity of a promoter for the DNA of the present invention
The present invention provides a compound which promotes or inhibits the activity of a promoter for the DNA of the present invention, which comprises administering a test compound to a non-human mammal deficient in expressing the DNA of the present invention, and detecting the expression of a reporter gene, or a compound thereof. A method for screening a salt is provided.
In the above-mentioned screening method, the non-human mammal deficient in expression of DNA of the present invention may be a non-human mammal deficient in expression of DNA of the present invention in which the DNA of the present invention is inactivated by introducing a reporter gene, A reporter gene that can be expressed under the control of a promoter for the DNA of the present invention is used.
Examples of the test compound include the same compounds as described above.
As the reporter gene, the same one as described above is used, and a β-galactosidase gene (lacZ), a soluble alkaline phosphatase gene, a luciferase gene and the like are preferable.
[0070]
In a non-human mammal deficient in expression of the DNA of the present invention in which the DNA of the present invention is replaced with a reporter gene, since the reporter gene is under the control of the promoter for the DNA of the present invention, the expression of the substance encoded by the reporter gene is traced. By doing so, the activity of the promoter can be detected.
For example, when a part of the DNA region encoding the protein of the present invention is replaced with a β-galactosidase gene (lacZ) derived from Escherichia coli, the tissue that expresses the protein of the present invention originally replaces the protein of the present invention. Β-galactosidase is expressed. Therefore, for example, the present invention can be easily performed by staining with a reagent serving as a substrate for β-galactosidase such as 5-bromo-4-chloro-3-indolyl-β-galactopyranoside (X-gal). Can be observed in the animal body. Specifically, the protein-deficient mouse of the present invention or a tissue section thereof is fixed with glutaraldehyde or the like, washed with phosphate buffered saline (PBS), and then stained with X-gal at room temperature or around 37 ° C. After reacting for about 30 minutes to 1 hour, the β-galactosidase reaction may be stopped by washing the tissue specimen with a 1 mM EDTA / PBS solution, and coloration may be observed. Further, mRNA encoding lacZ may be detected according to a conventional method.
The compound or a salt thereof obtained by using the above-mentioned screening method is a compound selected from the above-mentioned test compounds, and is a compound that promotes or inhibits the promoter activity for the DNA of the present invention.
[0071]
The compound obtained by the screening method may form a salt, and the salt of the compound may include a physiologically acceptable acid (eg, an inorganic acid) and a base (eg, an alkali metal). Are used, and especially preferred are physiologically acceptable acid addition salts. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, For example, salts with succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and the like are used.
Since the compound or its salt that promotes the promoter activity of the DNA of the present invention can promote the expression of the protein of the present invention and promote the function of the protein, it can be used for, for example, renal diseases (eg, renal failure, glomerulus) Nephritis, diabetic nephropathy, focal glomerulosclerosis, nephrotic syndrome, renal edema, interstitial nephritis, nephrosclerosis, uremia, etc., liver disease (eg, cirrhosis, hepatitis, alcoholic liver disease, etc.) , Pancreatic disorders (eg, pancreatitis), immune disorders associated with thymic abnormalities, genital disorders (eg, benign prostatic hyperplasia, prostatitis, testicular neurosis, ovarian cysts), gastrointestinal disorders (eg, irritable bowel syndrome, Ulcerative colitis, ischemic colitis, gastritis, etc.), spleen disease (eg, hypersplenism, splenomegaly syndrome, etc.), cancer (eg, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer, Prostate cancer, stomach cancer, bladder cancer, breast cancer, child Cervical, colon, rectal, pancreatic, thyroid, myeloid leukemia, etc., respiratory diseases (eg, pleurisy, pneumonia, chronic obstructive pulmonary disease, asthma, etc.), osteomyelitis, diabetes, hypertension, illness Post-blood reperfusion injury, retinitis, central nervous system disorders (eg, epilepsy, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), skin disorders (eg, atopic dermatitis, seborrheic dermatitis, etc.), thyroid hormone-related It is useful as a medicament such as a prophylactic or therapeutic agent for diseases (eg, thyroid hormone refractory disease, Basedow's disease, cretin disease, hyperthyroidism, etc.). It is preferably used as a prophylactic / therapeutic agent for renal diseases and thyroid hormone-related diseases, and more preferably as a prophylactic / therapeutic agent for diabetic nephropathy.
[0072]
In addition, the compound of the present invention or a salt thereof that inhibits the promoter activity for the DNA can inhibit the expression of the protein of the present invention and inhibit the function of the protein. Glomerulonephritis, diabetic nephropathy, focal glomerulosclerosis, nephrotic syndrome, renal edema, interstitial nephritis, nephrosclerosis, uremia, etc., liver disease (eg, cirrhosis, hepatitis, alcoholic liver disease, etc.) ), Pancreatic disease (eg, pancreatitis), immune disease associated with thymic abnormalities, genital disease (eg, prostatic hypertrophy, prostatitis, testicular neurosis, ovarian cysts, etc.), gastrointestinal disease (eg, irritable bowel syndrome) , Ulcerative colitis, ischemic colitis, gastritis, etc.), spleen disease (eg, hypersplenism, splenomegaly syndrome, etc.), cancer (eg, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer) , Prostate cancer, stomach cancer, bladder cancer, breast cancer Cervical, colon, rectal, pancreatic, thyroid, myeloid leukemia, etc., respiratory diseases (eg, pleurisy, pneumonia, chronic obstructive pulmonary disease, asthma, etc.), osteomyelitis, diabetes, hypertension, Post-ischemic reperfusion injury, retinitis, central nervous system disorders (eg, epilepsy, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), skin disorders (eg, atopic dermatitis, seborrheic dermatitis, etc.), thyroid hormone It is useful as a medicament such as an agent for preventing or treating related diseases (eg, thyroid hormone refractory disease, Basedow's disease, cretin disease, hyperthyroidism, etc.). It is preferably used as a prophylactic / therapeutic agent for renal diseases and thyroid hormone-related diseases, and more preferably as a prophylactic / therapeutic agent for diabetic nephropathy.
Among the above compounds, a compound or a salt thereof that promotes the promoter activity of the DNA of the present invention is preferable.
Furthermore, compounds derived from the compounds obtained by the above screening can be used in the same manner.
A drug containing the compound or a salt thereof obtained by the screening method can be produced in the same manner as the above-mentioned drug containing the protein of the present invention or a salt thereof.
The preparations obtained in this way are safe and low toxic, and thus, for example, human or other mammals (eg, rat, mouse, guinea pig, rabbit, sheep, pig, cow, horse, cat, dog, monkey, etc.) ) Can be administered.
The dose of the compound or a salt thereof varies depending on the disease to be treated, the subject of administration, the administration route, and the like. For example, when the compound of the present invention that promotes the promoter activity for DNA is orally administered, generally the adult (body weight) In patients with renal failure (as 60 kg), the compound is administered at about 0.1-100 mg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg per day. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, and the like. For example, the compound of the present invention that promotes the promoter activity for DNA is usually administered in the form of an injection to an adult ( When administered to a patient with renal failure (with a body weight of 60 kg), about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg of the compound is administered by intravenous injection per day. It is convenient. In the case of other animals, the amount can be administered in terms of the weight per 60 kg.
[0073]
On the other hand, for example, when a compound of the present invention that inhibits the promoter activity for DNA is orally administered, generally, in an adult (assuming a body weight of 60 kg) renal failure patient, about 0.1 to 100 mg of the compound per day is used. Preferably about 1.0-50 mg, more preferably about 1.0-20 mg is administered. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, and the like. For example, the compound of the present invention that inhibits the promoter activity for DNA is usually administered in the form of an injection to an adult ( When administered to a patient with renal failure (with a body weight of 60 kg), about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg of the compound is administered by intravenous injection per day. It is convenient. In the case of other animals, the amount can be administered in terms of the weight per 60 kg.
As described above, the non-human mammal deficient in expression of the DNA of the present invention is extremely useful for screening for a compound or a salt thereof that promotes or inhibits the activity of the promoter for the DNA of the present invention, It can greatly contribute to investigating the cause of various diseases caused by it or to develop preventive and therapeutic drugs.
In addition, using a DNA containing the promoter region of the protein of the present invention, genes encoding various proteins are ligated downstream thereof and injected into egg cells of an animal to produce a so-called transgenic animal (transgenic animal). Once created, it will also be possible to specifically synthesize the polypeptide and study its effects in living organisms. Furthermore, if a suitable reporter gene is bound to the above promoter portion, and a cell line that expresses the reporter gene is established, a low-molecular compound having an action of specifically promoting or suppressing the production ability of the protein itself of the present invention in the body. Can be used as a search system.
[0074]
In the present specification and drawings, bases, amino acids, and the like are indicated by abbreviations based on the abbreviations by IUPAC-IUB Commission on Biochemical Nomenclature or by abbreviations commonly used in the art, and examples thereof are described below. When an amino acid can have an optical isomer, the L-form is indicated unless otherwise specified.
DNA: deoxyribonucleic acid
cDNA: complementary deoxyribonucleic acid
A: Adenine
T: Thymine
G: Guanine
C: Cytosine
RNA: ribonucleic acid
mRNA: messenger ribonucleic acid
dATP: deoxyadenosine triphosphate
dTTP: deoxythymidine triphosphate
dGTP: deoxyguanosine triphosphate
dCTP: deoxycytidine triphosphate
ATP: Adenosine triphosphate
EDTA: Ethylenediaminetetraacetic acid
SDS: Sodium dodecyl sulfate
Gly: glycine
Ala: Alanine
Val: Valine
Leu: Leucine
Ile: Isoleucine
Ser: Serine
Thr: Threonine
Cys: cysteine
Met: methionine
Glu: glutamic acid
Asp: Aspartic acid
Lys: lysine
Arg: Arginine
His: histidine
Phe: phenylalanine
Tyr: Tyrosine
Trp: Tryptophan
Pro: Proline
Asn: Asparagine
Gln: Glutamine
pGlu: pyroglutamic acid
[0075]
Further, substituents, protecting groups and reagents frequently used in the present specification are represented by the following symbols.
Me: methyl group
Et: ethyl group
Bu: butyl group
Ph: phenyl group
TC: thiazolidine-4 (R) -carboxamide group
Tos: p-toluenesulfonyl
CHO: Formyl
Bzl: benzyl
Cl ₂ -Bzl: 2,6-dichlorobenzyl
Bom: benzyloxymethyl
Z: benzyloxycarbonyl
Cl-Z: 2-chlorobenzyloxycarbonyl
Br-Z: 2-bromobenzyloxycarbonyl
Boc: t-butoxycarbonyl
DNP: dinitrophenyl
Trt: Trityl
Bum: t-butoxymethyl
Fmoc: N-9-fluorenylmethoxycarbonyl
HOBt: 1-hydroxybenztriazole
HOOBt: 3,4-dihydro-3-hydroxy-4-oxo-1-2,3-benzotriazine
HONB: 1-hydroxy-5-norbornene-2,3-dicarboximide
DCC: N, N'-dicyclohexylcarbodiimide
[0076]
The sequence numbers in the sequence listing in the present specification indicate the following sequences.
[SEQ ID NO: 1]
3 shows the amino acid sequence of human TCH229 protein obtained in Example 3.
[SEQ ID NO: 2]
1 shows the nucleotide sequence of DNA encoding human TCH229 protein having the amino acid sequence represented by SEQ ID NO: 1.
[SEQ ID NO: 3]
The base sequence of the primer AP1 used in Example 1, Example 2, Example 5, Example 6, Example 9, and Example 10 is shown.
[SEQ ID NO: 4]
15 shows the base sequence of primer rr1 used in Examples 1, 3 and 15.
[SEQ ID NO: 5]
The base sequence of the primer AP2 used in Example 1, Example 2, Example 5, Example 6, Example 9, and Example 10 is shown.
[SEQ ID NO: 6]
15 shows the base sequence of primer rr2 used in Examples 1, 3 and 15.
[SEQ ID NO: 7]
2 shows the base sequence of primer rr3 used in Example 1.
[SEQ ID NO: 8]
2 shows the base sequence of primer rr4 used in Example 1.
[SEQ ID NO: 9]
Example 2 This shows the base sequence of primer ff1 used in Examples 3 and 15.
[SEQ ID NO: 10]
15 shows the base sequence of primer ff2 used in Examples 2, 3, and 15.
[SEQ ID NO: 11]
4 shows the nucleotide sequence of primer ORFF1 used in Example 3.
[SEQ ID NO: 12]
3 shows the nucleotide sequence of primer ORFR1 used in Example 3.
[SEQ ID NO: 13]
4 shows the nucleotide sequence of primer ORFF2 used in Example 3.
[SEQ ID NO: 14]
3 shows the nucleotide sequence of primer ORFR2 used in Example 3.
[SEQ ID NO: 15]
13 shows the nucleotide sequence of primer M13F used in Examples 3 and 7.
[SEQ ID NO: 16]
13 shows the base sequence of primer M13R used in Examples 3 and 7.
[SEQ ID NO: 17]
14 shows the base sequence of primer A1 used in Examples 3 and 15.
[SEQ ID NO: 18]
14 shows the base sequence of primer B2 used in Examples 3 and 15.
[SEQ ID NO: 19]
17 shows the nucleotide sequence of primer TMF used in Examples 4, 16 and 17.
[SEQ ID NO: 20]
17 shows the nucleotide sequence of primer TMR used in Examples 4, 16 and 17.
[SEQ ID NO: 21]
17 shows the base sequence of TaqMan probe P1 used in Examples 4, 16 and 17.
[SEQ ID NO: 22]
1 shows the base sequence obtained in Example 1.
[SEQ ID NO: 23]
1 shows the base sequence obtained in Example 1.
[SEQ ID NO: 24]
3 shows the base sequence obtained in Example 2.
[SEQ ID NO: 25]
3 shows the base sequence obtained in Example 3.
[SEQ ID NO: 26]
7 shows the amino acid sequence of mouse TCH229 protein obtained in Example 7.
[SEQ ID NO: 27]
This shows the base sequence of DNA encoding the mouse TCH229 protein having the amino acid sequence represented by SEQ ID NO: 26.
[SEQ ID NO: 28]
13 shows the base sequences of primers used in Examples 5 and 7.
[SEQ ID NO: 29]
13 shows the base sequences of primers used in Examples 5 and 7.
[SEQ ID NO: 30]
14 shows the base sequence of a primer used in Example 5.
[SEQ ID NO: 31]
13 shows the base sequences of primers used in Examples 5 and 7.
[SEQ ID NO: 32]
13 shows the base sequences of primers used in Examples 6 and 7.
[SEQ ID NO: 33]
13 shows the base sequences of primers used in Examples 6 and 7.
[SEQ ID NO: 34]
14 shows the base sequence of a primer used in Example 7.
[SEQ ID NO: 35]
14 shows the base sequence of a primer used in Example 7.
[SEQ ID NO: 36]
14 shows the base sequence of a primer used in Example 7.
[SEQ ID NO: 37]
14 shows the base sequence of a primer used in Example 7.
[SEQ ID NO: 38]
14 shows the base sequence of a primer used in Example 7.
[SEQ ID NO: 39]
14 shows the base sequence of a primer used in Example 7.
[SEQ ID NO: 40]
14 shows the base sequence of a primer used in Example 7.
[SEQ ID NO: 41]
14 shows the base sequence of a primer used in Example 7.
[SEQ ID NO: 42]
14 shows the base sequence of a primer used in Example 7.
[SEQ ID NO: 43]
14 shows the base sequence of a primer used in Example 7.
[SEQ ID NO: 44]
14 shows the base sequence of a primer used in Example 7.
[SEQ ID NO: 45]
14 shows the base sequences of primers used in Examples 8 and 13.
[SEQ ID NO: 46]
14 shows the base sequences of primers used in Examples 8 and 13.
[SEQ ID NO: 47]
14 shows the nucleotide sequences of probes used in Examples 8 and 13.
[SEQ ID NO: 48]
7 shows the base sequence obtained in Example 5.
[SEQ ID NO: 49]
7 shows the base sequence obtained in Example 5.
[SEQ ID NO: 50]
7 shows the base sequence obtained in Example 6.
[SEQ ID NO: 51]
7 shows the base sequence obtained in Example 7.
[SEQ ID NO: 52]
Rat TCH229 protein No. obtained in Example 11 1 shows the amino acid sequence of 1.
[SEQ ID NO: 53]
Rat TCH229 protein No. having the amino acid sequence represented by SEQ ID NO: 52. 1 shows the nucleotide sequence of DNA encoding 1.
[SEQ ID NO: 54]
Rat TCH229 protein No. obtained in Example 11 2 shows the amino acid sequence of 2.
[SEQ ID NO: 55]
Rat TCH229 protein No. having the amino acid sequence represented by SEQ ID NO: 54 2 shows the nucleotide sequence of DNA encoding No. 2.
[SEQ ID NO: 56]
14 shows the base sequence of a primer used in Example 9.
[SEQ ID NO: 57]
14 shows the base sequence of a primer used in Example 9.
[SEQ ID NO: 58]
14 shows the base sequence of a primer used in Example 9.
[SEQ ID NO: 59]
14 shows the base sequence of a primer used in Example 9.
[SEQ ID NO: 60]
14 shows the base sequence of a primer used in Example 10.
[SEQ ID NO: 61]
13 shows the base sequences of primers used in Examples 10 and 11.
[SEQ ID NO: 62]
14 shows the base sequence of a primer used in Example 11.
[SEQ ID NO: 63]
14 shows the base sequence of a primer used in Example 11.
[SEQ ID NO: 64]
14 shows the base sequence of a primer used in Example 11.
[SEQ ID NO: 65]
14 shows the base sequence of a primer used in Example 11.
[SEQ ID NO: 66]
15 shows the base sequences of primers used in Examples 11 and 15.
[SEQ ID NO: 67]
14 shows the base sequence of a primer used in Example 11.
[SEQ ID NO: 68]
14 shows the base sequence of a primer used in Example 11.
[SEQ ID NO: 69]
The base sequences of the primers used in Examples 11, 12, 14, and 18 are shown.
[SEQ ID NO: 70]
The base sequences of the primers used in Examples 11, 12, 14, and 18 are shown.
[SEQ ID NO: 71]
14 shows the base sequence of a primer used in Example 11.
[SEQ ID NO: 72]
14 shows the base sequence of a primer used in Example 11.
[SEQ ID NO: 73]
14 shows the base sequence of a primer used in Example 11.
[SEQ ID NO: 74]
14 shows the base sequence of a primer used in Example 11.
[SEQ ID NO: 75]
14 shows the base sequence of a primer used in Example 11.
[SEQ ID NO: 76]
19 shows the nucleotide sequences of probes used in Examples 12, 14, and 18.
[SEQ ID NO: 77]
13 shows the base sequence obtained in Example 9.
[SEQ ID NO: 78]
13 shows the base sequence obtained in Example 9.
[SEQ ID NO: 79]
13 shows the base sequence obtained in Example 10.
[SEQ ID NO: 80]
13 shows the base sequence obtained in Example 11.
[SEQ ID NO: 81]
13 shows the base sequence obtained in Example 11.
[SEQ ID NO: 82]
14 shows the nucleotide sequence of a primer used in Example 15.
[SEQ ID NO: 83]
14 shows the nucleotide sequence of a primer used in Example 15.
[SEQ ID NO: 84]
14 shows the nucleotide sequence of a primer used in Example 15.
[0077]
Escherichia coli TOP10 / pCR-BluntII-TCH229 obtained in Example 3 to be described later has been used since March 27, 2002, 1-1-1 Higashi 1-1-1 Tsukuba City, Ibaraki Prefecture, Japan, postal code 305-8566) at the National Institute of Advanced Industrial Science and Technology (AIST) under the deposit number FERM BP-7983 from March 12, 2002, at 2-17-85, Jusanhoncho, Yodogawa-ku, Osaka-shi, Osaka, Japan. No. 532-8686) and deposited at the Fermentation Research Institute (IFO) under the accession number IFO 16767.
Escherichia coli TOP10 / pCR2.1-mTCH229 obtained in Example 7 described below has been used since June 13, 2002, 1-1-1 Higashi, 1-1, Tsukuba, Ibaraki, Japan Central Japan No. 6 (postal code) 305-8566) at the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary, under the deposit number FERM BP-8076 from June 4, 2002, 2-17-85, Jusanhoncho, Yodogawa-ku, Osaka-shi, Osaka No. 532-8686) and deposited at the Fermentation Research Institute (IFO) under the accession number IFO16800.
【Example】
Hereinafter, the present invention will be described in more detail with reference to Examples, but these do not limit the scope of the present invention. In addition, the gene manipulation method using Escherichia coli followed the method described in Molecular cloning (Molecular cloning).
[0078]
Example 1
Cloning of 5 'upstream end of cDNA encoding human TCH229 protein
5 ′ RACE PCR cloning revealed the 5 ′ upstream nucleotide sequence of cDNA encoding human TCH229 protein.
Using two types of primer DNAs, primer AP1 (SEQ ID NO: 3) and primer rr1 (SEQ ID NO: 4), an Advantage 2 DNA Polymerase (Clontech) against human kidney Marathon-Ready cDNA (Clontech) ), Primary PCR was performed under the following conditions.
(1) 94 ° C for 30 seconds
(2) 35 cycles of 94 ° C for 10 seconds and -63 ° C for 3 minutes
Furthermore, using the product of this primary PCR as a template, nested PCR was performed under the following conditions using Advantage 2 DNA Polymerase (manufactured by Clontech) using primer AP2 (SEQ ID NO: 5) and primer rr2 (SEQ ID NO: 6). Was.
(3) 94 ° C for 30 seconds
(4) 30 cycles of 94 ° C for 10 seconds and -63 ° C for 3 minutes
To 5 μl of the above nested PCR reaction solution, 1 μl each of Exonuclease I and Shrimp Alkaline Posphorase in PCR Product Pre-Sequencing Kit (manufactured by Usb) were added, and the reaction was carried out at 37 ° C. for 15 minutes and at 85 ° C. for 15 minutes. This was reacted using primer AP2 (SEQ ID NO: 5), primer rr2 (SEQ ID NO: 6) and BigDye Terminator Cycle Sequencing Kit (manufactured by Applied Biosystems), and the base sequence of the amplified DNA fragment was subjected to DNA sequencer ABI. It was determined using a PRISM 3100 DNA analyzer (manufactured by Applied Biosystems). As a result, the nucleotide sequence represented by SEQ ID NO: 22 was obtained.
Based on the nucleotide sequence shown in SEQ ID NO: 22, primer rr3 (SEQ ID NO: 7) and primer rr4 (SEQ ID NO: 8) were designed. Using the primer AP1 (SEQ ID NO: 3) and the primer rr3 (SEQ ID NO: 7), the following conditions were applied to the human kidney Marathon-Ready cDNA (Clontech) by Advantage 2 DNA Polymerase (Clontech). The primary PCR was performed.
(5) 94 ° C for 30 seconds
(6) 35 cycles of 94 ° C for 10 seconds and -63 ° C for 2 minutes
Further, nested PCR was performed under the following conditions using Advantage 2 DNA Polymerase (manufactured by Clontech) using the product of this primary PCR as a template, primer AP2 (SEQ ID NO: 5) and primer rr4 (SEQ ID NO: 8), under the following conditions. Was.
(7) 94 ° C for 30 seconds
(8) 30 cycles of 94 ° C for 10 seconds and -63 ° C for 2 minutes
To 5 μl of the above nested PCR reaction solution, 1 μl each of Exonuclease I and Shrimp Alkaline Posphorase in PCR Product Pre-Sequencing Kit (manufactured by Usb) were added, and the reaction was carried out at 37 ° C. for 15 minutes and at 85 ° C. for 15 minutes. This was reacted using primer AP2 (SEQ ID NO: 5), primer rr4 (SEQ ID NO: 8), and BigDye Terminator Cycle Sequencing Kit (manufactured by Applied Biosystems), and the base sequence of the amplified DNA fragment was subjected to DNA sequencer ABI. It was determined using a PRISM 3100 DNA analyzer (manufactured by Applied Biosystems). As a result, the nucleotide sequence represented by SEQ ID NO: 23 was obtained.
[0079]
Example 2
Cloning of 3 'downstream end of cDNA encoding human TCH229 protein
3'RACE PCR cloning revealed the base sequence 3 'under the cDNA encoding human TCH229 protein.
Using two types of primer DNAs, primer AP1 (SEQ ID NO: 3) and primer ff1 (SEQ ID NO: 9), an Advantage 2 DNA Polymerase (Clontech) against human kidney Marathon-Ready cDNA (Clontech) ), Primary PCR was performed under the following conditions.
(1) 94 ° C for 30 seconds
(2) 35 cycles of 94 ° C for 10 seconds and -63 ° C for 3 minutes
Further, nested PCR was performed under the following conditions using Advantage 2 DNA Polymerase (manufactured by Clontech) using the product of this primary PCR as a template and primer AP2 (SEQ ID NO: 5) and primer ff2 (SEQ ID NO: 10) under the following conditions. Was.
(3) 94 ° C for 30 seconds
(4) 30 cycles of 94 ° C for 10 seconds and -63 ° C for 3 minutes
To 5 μl of the above nested PCR reaction solution, 1 μl each of Exonuclease I and Shrimp Alkaline Posphorase in PCR Product Pre-Sequencing Kit (manufactured by Usb) were added, and the reaction was carried out at 37 ° C. for 15 minutes and at 85 ° C. for 15 minutes. This was reacted using primer AP2 (SEQ ID NO: 5), primer ff2 (SEQ ID NO: 10), and BigDye Terminator Cycle Sequencing Kit (manufactured by Applied Biosystems), and the base sequence of the amplified DNA fragment was subjected to DNA sequencer ABI. It was determined using a PRISM 3100 DNA analyzer (manufactured by Applied Biosystems). As a result, the nucleotide sequence represented by SEQ ID NO: 24 was obtained.
[0080]
Example 3
Cloning of cDNA encoding human TCH229 protein
Using two kinds of primer DNAs, primer ORFF1 (SEQ ID NO: 11) and primer ORFR1 (SEQ ID NO: 12), pfu turbo DNA Polymerase (Stratagene) against human kidney Marathon-Ready cDNA (Clontech) Primary PCR was performed under the following conditions (1) to (3).
(1) 94 ° C for 30 seconds
(2) 35 cycles of 94 ° C for 10 seconds -57 ° C for 10 seconds -72 ° C for 2.5 minutes
(3) 72 ° C for 5 minutes
Further, using the product of this primary PCR as a template and the primer ORFF2 (SEQ ID NO: 13) and the primer ORFR2 (SEQ ID NO: 14), the following conditions (4) to pfu turbo DNA Polymerase (Stratagene) were used. Nested PCR was performed in (6).
(4) 94 ° C for 30 seconds
(5) 30 cycles of 94 ° C. for 10 seconds, −56 ° C. for 10 seconds, and −72 ° C. for 2.5 minutes
(6) 72 ° C for 5 minutes
The nested PCR reaction solution was purified using Min Elute Gel Extraction Kit (Qiagen). This DNA was cloned into the pCR-Blunt II-TOPO vector according to the protocol of Zero Blunt TOPO PCR Cloning Kit (manufactured by Invitrogen). This was introduced into Escherichia coli TOP10 competent cell (manufactured by Invitrogen) for transformation, and a clone having a cDNA insert was selected on an LB agar medium containing kanamycin to obtain a transformant. Each clone was cultured overnight in an LB medium containing kanamycin, and a plasmid DNA was prepared using QIAwell 8 Plasmid Kit (manufactured by Qiagen). Two clones # 1, # 2, and # 2 of the plasmid clone pCR-BluntII-TCH229 were prepared. 3 was obtained. This was combined with primer DNA [primer M13F (SEQ ID NO: 15), primer M13R (SEQ ID NO: 16), primer ORFF2 (SEQ ID NO: 13), primer ORFR2 (SEQ ID NO: 14), primer rr2 (SEQ ID NO: 6), Primer A1 (SEQ ID NO: 17), Primer B2 (SEQ ID NO: 18), Primer ff2 (SEQ ID NO: 10), Primer rr1 (SEQ ID NO: 4), Primer ff1 (SEQ ID NO: 9)], and BigDye Terminator Cycle Sequencing The reaction was carried out using Kit (manufactured by Applied Biosystems), and the base sequence of the inserted cDNA fragment was determined using a DNA sequencer ABI PRISM 3100 DNA Analyzer (manufactured by Applied Biosystems). . As a result, the obtained three clones contained the same DNA fragment and had 2251 base sequences (SEQ ID NO: 25). The fragment encodes 724 amino acid sequences (SEQ ID NO: 1) (SEQ ID NO: 2), and the protein containing the amino acid sequence represented by SEQ ID NO: 1 was named human TCH229 protein.
A transformant having a plasmid containing the cDNA fragment was named Escherichia coli TOP10 / pCR-BluntII-TCH229.
A homology search was performed on a known database using Blast P (Nucleic Acids Res., Vol. 25, p. 3389, 1997), and the cDNA was found to be a novel gene belonging to the organic anion transporter. (FIGS. 1 and 2). In the figure, the transmembrane regions are indicated by TM1 to TM12. It has 41% homology at the amino acid level with SLC21A12 (Biochemical and Biophysical Research Communications, vol. 273, p. 251, 2000), which is an organic anion transporter reported in humans, and OATPRP4 (GenBank Accession No. 030958) showed 36% homology at the amino acid level, suggesting that the protein has a 12-transmembrane structure.
[0081]
Example 4
Analysis of tissue distribution of human TCH229 gene product
Using two kinds of primer DNAs designed from the sequence of human TCH229, primer TMF (SEQ ID NO: 19) and primer TMR (SEQ ID NO: 20), and TaqMan probe P1 (SEQ ID NO: 21), The expression level of human TCH229 in the cDNA was measured by TaqMan PCR.
The reaction was first performed at 50 ° C. for 2 minutes and then at 95 ° C. for 10 minutes using ABI PRISM 7900 sequence detection system (Applied Biosystems) using TaqMan Universal PCR Master Mix (Applied Biosystems), and then 95%. Forty cycles were repeated at 15 ° C. for 15 seconds and 60 ° C. for one minute as one reaction cycle, and detection was performed simultaneously.
Table 1 shows the cDNA of each human tissue used for the measurement.
The results are shown in FIGS.
Human TCH229 gene product (mRNA) showed strong expression in human MTC panel I and MTC panel II in kidney and weak expression in lung, liver and pancreas.
In the human digestive system MTC panel, relatively strong expression was observed in the liver, but only slight expression was observed in all sites from the stomach to the rectum.
[Table 1]

[0082]
Example 5
Cloning of 5 'upstream end of cDNA encoding mouse TCH229 protein
PCR cloning and 5′RACE PCR cloning revealed the 5 ′ upstream nucleotide sequence of the cDNA encoding the mouse TCH229 protein.
Using two types of primer DNAs [primer h243F (SEQ ID NO: 28) and primer mR1 (SEQ ID NO: 29)], mouse 2 Marathon-Ready cDNA (Clontech) and Advantage 2 DNA Polymerase (Clontech) Was performed under the following conditions.
(1) 94 ° C for 30 seconds
(2) 35 cycles of 94 ° C for 10 seconds -63 ° C for 10 seconds -68 ° C for 2 minutes
(3) 68 ° C for 3 minutes
To 5 μl of the above PCR reaction solution, 1 μl of Exonuclease I and Shrimp Alkaline Posthase in a PCR Product Pre-Sequencing Kit (manufactured by Usb) were added, and the reaction was carried out at 37 ° C. for 15 minutes and 85 ° C. for 15 minutes. This was reacted using primer h243F (SEQ ID NO: 28), primer mR1 (SEQ ID NO: 29), and BigDye Terminator Cycle Sequencing Kit (manufactured by Applied Biosystems), and the base sequence of the amplified DNA fragment was subjected to DNA sequencer ABI. It was determined using a PRISM 3100 DNA analyzer (manufactured by Applied Biosystems). As a result, the nucleotide sequence represented by SEQ ID NO: 48 was obtained.
Based on SEQ ID NO: 48, primer mrr5 (SEQ ID NO: 30) and primer mrr6 (SEQ ID NO: 31) were designed. Using two types of primer DNAs [Primer AP1 (SEQ ID NO: 3) and primer mrr6 (SEQ ID NO: 31)], an Advantage 2 DNA Polymerase (Clontech) against mouse kidney Marathon-Ready cDNA (Clontech). Primary PCR was performed under the following conditions.
(1) 94 ° C for 30 seconds
(2) 40 cycles of 94 ° C for 10 seconds and -64 ° C for 4 minutes
Further, nested PCR was performed under the following conditions using Advantage 2 DNA Polymerase (manufactured by Clontech) using the product of this primary PCR as a template and primer AP2 (SEQ ID NO: 5) and primer mrr5 (SEQ ID NO: 30) under the following conditions. Was.
(3) 94 ° C for 30 seconds
(4) 35 cycles of 94 ° C for 10 seconds and -64 ° C for 3 minutes
To 5 μl of the nested PCR reaction solution, 1 μl of each of Exonuclease I and Shrimp Alkaline Posphorase in PCR Product Pre-Sequencing Kit (manufactured by Usb) were added, and the reaction was carried out at 37 ° C. for 15 minutes and at 85 ° C. for 15 minutes. This was reacted using primer mrr5 (SEQ ID NO: 30) and BigDye Terminator Cycle Sequencing Kit (manufactured by Applied Biosystems), and the base sequence of the amplified DNA fragment was analyzed using the DNA sequencer ABI PRISM 3100 DNA Analyzer (Applied Biosystems). Was used for the determination. As a result, the nucleotide sequence represented by SEQ ID NO: 49 was obtained.
[0083]
Example 6
Cloning of 3 'downstream end of cDNA encoding mouse TCH229 protein
3 ′ RACE PCR cloning revealed the 3 ′ upstream nucleotide sequence of cDNA encoding mouse TCH229 protein.
Using two types of primer DNAs [Primer AP1 (SEQ ID NO: 3) and primer mF1 (SEQ ID NO: 32)], an Advantage 2 DNA Polymerase (Clontech) against mouse kidney Marathon-Ready cDNA (Clontech). Primary PCR was performed under the following conditions.
(1) 94 ° C for 30 seconds
(2) 40 cycles of 94 ° C for 10 seconds and -61 ° C for 4 minutes
Further, nested PCR was performed under the following conditions using Advantage 2 DNA Polymerase (manufactured by Clontech) using the product of the primary PCR as a template and primer AP2 (SEQ ID NO: 5) and primer mff2 (SEQ ID NO: 33) under the following conditions. Was.
(3) 94 ° C for 30 seconds
(4) 35 cycles of 94 ° C for 10 seconds and -61 ° C for 3 minutes
To 5 μl of the nested PCR reaction solution, 1 μl of each of Exonuclease I and Shrimp Alkaline Posphorase in PCR Product Pre-Sequencing Kit (manufactured by Usb) were added, and the reaction was carried out at 37 ° C. for 15 minutes and at 85 ° C. for 15 minutes. This was reacted using primer mff2 (SEQ ID NO: 33) and BigDye Terminator Cycle Sequencing Kit (manufactured by Applied Biosystems), and the base sequence of the amplified DNA fragment was analyzed using the DNA sequencer ABI PRISM 3100 DNA Analyzer (Applied Biosystems). Was used for the determination. As a result, the nucleotide sequence represented by SEQ ID NO: 50 was obtained.
[0084]
Example 7
Cloning of cDNA encoding mouse TCH229 protein
Using two types of primer DNAs (primer mORFF (4) (SEQ ID NO: 34) and primer mORFR (2195) (SEQ ID NO: 35)), pfu was obtained for mouse kidney Marathon-Ready cDNA (Clontech). Primary PCR was performed using turbo DNA Polymerase (manufactured by Stratagene) under the following conditions (1) to (3).
(1) 94 ° C for 30 seconds
(2) 35 cycles of 94 ° C for 10 seconds -56 ° C for 10 seconds -72 ° C for 2 minutes
(3) 72 ° C for 5 minutes
Further, using the product of this primary PCR as a template and primer mORFF (atg) (SEQ ID NO: 36) and primer mORFR (tga) (SEQ ID NO: 37), pfu turbo DNA Polymerase (manufactured by Stratagene) Nested PCR was performed under the conditions (4) to (6).
(4) 94 ° C for 30 seconds
(5) 30 cycles of 94 ° C for 10 seconds -56 ° C for 10 seconds -72 ° C for 2 minutes
(6) 72 ° C for 5 minutes
1.5 μl of Advantage 2 DNA Polymerase (Clontech) was added to 10 μl of the nested PCR reaction solution, and the mixture was reacted at 72 ° C. for 10 minutes and purified using Min Elute Gel Extraction Kit (Qiagen). This DNA was cloned into the pCR2.1-TOPO vector according to the protocol of TOPO TA Cloning Kit (Invitrogen). This was introduced into Escherichia coli TOP10 competent cell (manufactured by Invitrogen) for transformation, and a clone having a cDNA insert was selected on an LB agar medium containing kanamycin to obtain a transformant. Each clone was cultured overnight in an LB medium containing kanamycin, and a plasmid DNA was prepared using QIAwell 8 Plasmid Kit (manufactured by Qiagen). Two clones # 1, # 2 and # of plasmid clone pCR2.1-mTCH229 were prepared. 3 was obtained. This was combined with primer DNA [primer M13F (SEQ ID NO: 15), primer M13R (SEQ ID NO: 16), primer h243F (SEQ ID NO: 28), primer mR1 (SEQ ID NO: 29), primer h650F (SEQ ID NO: 38), Primer h910F (SEQ ID NO: 39), primer mrr1 (SEQ ID NO: 40), primer mrr2 (SEQ ID NO: 41), primer mrr3 (SEQ ID NO: 42), primer F (45) (SEQ ID NO: 43), primer m521R (SEQ ID NO: 44), primer mF1 (SEQ ID NO: 32), primer mff2 (SEQ ID NO: 33), primer mrr6 (SEQ ID NO: 31)] and BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) And the base sequence of the inserted cDNA fragment was determined using a DNA sequencer ABI PRISM 3100 DNA Analyzer (manufactured by Applied Biosystems). As a result, the obtained three clones contained the same DNA fragment and had 2169 base sequences (SEQ ID NO: 51). The fragment encodes 722 amino acid sequences (SEQ ID NO: 26) (SEQ ID NO: 27), and a protein containing the amino acid sequence represented by SEQ ID NO: 26 was designated as mouse TCH229 protein.
A transformant having a plasmid containing the cDNA fragment was designated as Escherichia coli TOP10 / pCR2.1-mTCH229.
When a homology search was performed on a known database using Blast P (Nucleic Acids Res., Vol. 25, p. 3389, 1997), the cDNA was found to be mouse SLC21A11 (an organic anion transporter reported in mice). Genbank: NP — 076397) and 43.2% homology at the base level and 37% at the amino acid level. Further, mouse TCH229 showed a homology of 83% at the base level and 81% at the amino acid level with human TCH229, a novel gene belonging to the organic anion transporter family, indicating that it is a mouse homolog of human TCH229 ( (Fig. 5). In the figure, transmembrane regions are indicated by TM1 to 12 and amino acids highly conserved among families are indicated by *.
[0085]
Example 8
Analysis of tissue distribution of mouse TCH229 gene product
Using two kinds of primer DNAs designed from the sequence of mouse TCH229, primer mTMF (SEQ ID NO: 45) and primer mTMR (SEQ ID NO: 46), and TaqMan probe mP1 (SEQ ID NO: 47), The expression level of mouse TCH229 in the cDNA was measured by TaqMan PCR.
The reaction was first performed at 50 ° C. for 2 minutes and then at 95 ° C. for 10 minutes using ABI PRISM 7900 sequence detection system (Applied Biosystems) using TaqMan Universal PCR Master Mix (Applied Biosystems), and then 95%. Forty cycles were repeated at 15 ° C. for 15 seconds and 60 ° C. for one minute as one reaction cycle, and detection was performed simultaneously.
FIG. 6 shows the results.
In the mouse MTC panel I and MTC panel II, the mouse TCH229 gene product (mRNA) showed strong expression in the prostate and relatively high expression in the bone marrow, eyes, lungs, and kidneys.
[0086]
Example 9
5 ′ upstream cloning PCR cloning of cDNA encoding rat TCH229 protein and 5 ′ RACE PCR cloning revealed the 5 ′ upstream base sequence of cDNA encoding rat TCH229 protein.
Rat kidney Marathon-Ready cDNA (Clontech) was used using two kinds of primer DNAs designed based on the sequence of mouse TCH229 (SEQ ID NO: 51), primer m163F (SEQ ID NO: 56) and primer m2087R (SEQ ID NO: 57). PCR) was performed using Advantage 2 DNA Polymerase (manufactured by Clontech) under the following conditions.
(1) 94 ° C for 30 seconds
(2) 35 cycles of 94 ° C. for 10 seconds, −60 ° C. for 10 seconds, and −68 ° C. for 2.5 minutes
(3) 68 ° C for 5 minutes
To 5 μl of the PCR reaction solution, 1 μl each of Exonuclease I and Shrimp Alkaline Posphorase in the PCR Product Pre-Sequencing Kit (manufactured by Usb) were added, and the reaction was carried out at 37 ° C. for 15 minutes and 85 ° C. for 15 minutes. This was reacted using primer m163F (SEQ ID NO: 56), primer m2087R (SEQ ID NO: 57) and BigDye Terminator Cycle Sequencing Kit (manufactured by Applied Biosystems), and the base sequence of the amplified DNA fragment was analyzed using a DNA sequencer ABI PRISM. It was determined using a 3100 DNA analyzer (manufactured by Applied Biosystems). As a result, the nucleotide sequence represented by SEQ ID NO: 77 was obtained.
Based on SEQ ID NO: 77, primers r229-rr1 (SEQ ID NO: 58) and primer r229-rr2 (SEQ ID NO: 59) were designed. Using two types of primer DNAs, a primer AP1 (SEQ ID NO: 3) and a primer r229-rr1 (SEQ ID NO: 58), an Advantage 2 DNA Polymerase (Clontech) was applied to rat kidney Marathon-Ready cDNA (Clontech). Primary PCR was performed under the following conditions.
(1) 94 ° C for 30 seconds
(2) 35 cycles of 94 ° C for 10 seconds and -64 ° C for 2 minutes
Furthermore, using the product of this primary PCR as a template, primer AP2 (SEQ ID NO: 5) and primer r229-rr2 (SEQ ID NO: 59), Advantage 2 DNA Polymerase (manufactured by Clontech) and nested PCR under the following conditions: Was done.
(3) 94 ° C for 30 seconds
(4) 35 cycles of 94 ° C for 10 seconds and -64 ° C for 2 minutes
To 5 μl of the above nested PCR reaction solution, 1 μl each of Exonuclease I and Shrimp Alkaline Posphorase in PCR Product Pre-Sequencing Kit (manufactured by Usb) were added, and the reaction was carried out at 37 ° C. for 15 minutes and at 85 ° C. for 15 minutes. This was reacted with primers r229-rr2 (SEQ ID NO: 59) and BigDye Terminator Cycle Sequencing Kit (manufactured by Applied Biosystems), and the base sequence of the amplified DNA fragment was analyzed using a DNA sequencer ABI PRISM 3100 DNA Analyzer (Applied Biosystems). (Manufactured by Systems Inc.). As a result, the nucleotide sequence represented by SEQ ID NO: 78 was obtained.
[0087]
Example 10
Cloning of 3 'downstream end of cDNA encoding rat TCH229 protein
The 3 ′ downstream nucleotide sequence of the cDNA encoding the rat TCH229 protein was revealed by 3 ′ RACE PCR cloning.
Using two types of primer DNAs, primer AP1 (SEQ ID NO: 3) and primer rff1 (SEQ ID NO: 60), an Advantage 2 DNA Polymerase (Clontech) against rat kidney Marathon-Ready cDNA (Clontech). ), Primary PCR was performed under the following conditions.
(1) 94 ° C for 30 seconds
(2) 35 cycles of 94 ° C for 10 seconds and -64 ° C for 2 minutes
Further, using the product of this primary PCR as a template, nested PCR was performed under the following conditions by Advantage 2 DNA Polymerase (Clontech) using primer AP2 (SEQ ID NO: 5) and primer rff3 (SEQ ID NO: 61). Was.
(3) 94 ° C for 30 seconds
(4) 30 cycles of 94 ° C for 10 seconds and -64 ° C for 2 minutes
To 5 μl of the above nested PCR reaction solution, 1 μl each of Exonuclease I and Shrimp Alkaline Posphorase in PCR Product Pre-Sequencing Kit (manufactured by Usb) were added, and the reaction was carried out at 37 ° C. for 15 minutes and at 85 ° C. for 15 minutes. This was reacted using primer rff3 (SEQ ID NO: 61) and BigDye Terminator Cycle Sequencing Kit (manufactured by Applied Biosystems), and the base sequence of the amplified DNA fragment was analyzed using the DNA sequencer ABI PRISM 3100 DNA Analyzer (Applied Biosystems). Was used for the determination. As a result, the nucleotide sequence represented by SEQ ID NO: 79 was obtained.
[0088]
Example 11
Cloning of cDNA encoding rat TCH229 protein
Using two types of primer DNAs, primer rORFF1 (SEQ ID NO: 62) and primer rORFR12 (SEQ ID NO: 63), pfu turbo DNA Polymerase (Stratagene) against rat kidney Marathon-Ready cDNA (Clontech) Primary PCR was performed under the following conditions (1) to (3).
(1) 94 ° C for 30 seconds
(2) 35 cycles of 94 ° C for 10 seconds -56 ° C for 10 seconds -72 ° C for 2.5 minutes
(3) 72 ° C for 5 minutes
Further, using the product of this primary PCR as a template and the primer rORFF (atg) (SEQ ID NO: 64) and the primer rORFR (tga2) (SEQ ID NO: 65), the following reaction was performed by pfu turbo DNA Polymerase (manufactured by Stratagene). Nested PCR was performed under conditions (4) to (6).
(4) 94 ° C for 30 seconds
(5) 30 cycles of 94 ° C. for 10 seconds, −56 ° C. for 10 seconds, and −72 ° C. for 2.5 minutes
(6) 72 ° C for 5 minutes
1.5 μl of Advantage 2 DNA Polymerase (Clontech) was added to 10 μl of the nested PCR reaction solution, and the mixture was reacted at 72 ° C. for 10 minutes and purified using Min Elute Gel Extraction Kit (Qiagen). This DNA was cloned into the pCRII-TOPO vector according to the protocol of TOPO TA Cloning Kit (Invitrogen). This was introduced into Escherichia coli TOP10 competent cell (manufactured by Invitrogen) for transformation, and a clone having a cDNA insert was selected on an LB agar medium containing kanamycin to obtain a transformant. The individual clones were cultured overnight in an LB medium containing kanamycin, and plasmid DNA was prepared using QIAwell 8 Plasmid Kit (manufactured by Qiagen), and 4 clones # 1, # 2, # 3 and 4 of plasmid clone pCRII-rTCH229 were prepared. # 4 was obtained. This was combined with primer DNA [primer T7 (SEQ ID NO: 66), primer SP6 (SEQ ID NO: 67), primer rORFF (atg) (SEQ ID NO: 64), primer rORFR (tga2) (SEQ ID NO: 65), primer rF1 ( SEQ ID NO: 68), primer rTMR (SEQ ID NO: 69), primer rTMF (SEQ ID NO: 70), primer rff3 (SEQ ID NO: 61), primer rff2 (SEQ ID NO: 71), primer rR1 (SEQ ID NO: 72) Primer r972F (SEQ ID NO: 73), primer r1123F (SEQ ID NO: 74), primer r1746R (SEQ ID NO: 75)] and BigDye Terminator Cycle Sequencing Kit (manufactured by Applied Biosystems). Was carried out, the base sequence of the cDNA fragment inserted was determined using DNA sequencer ABI PRISM 3100 DNA Analyzer (Applied Biosystems). As a result, the obtained four clones contained two types of DNA fragments (No. 1 and No. 2). No. 1 has a base sequence of 2175 (SEQ ID NO: 80), and the fragment encodes a 724 amino acid sequence (SEQ ID NO: 52) (SEQ ID NO: 53). It contained the amino acid sequence represented. No. 2 has a base sequence of 2175 (SEQ ID NO: 81), and the fragment encodes 724 amino acid sequences (SEQ ID NO: 54) (SEQ ID NO: 55). It contained the amino acid sequence represented. Each protein was designated as rat TCH229 protein No. 1 and rat TCH229 protein no. No. 2. The transformant having the plasmid containing the cDNA fragment was individually subjected to Escherichia coli TOP10 / pCRII-rTCH229No. 1 and Escherichia coli TOP10 / pCRII-rTCH229No. No. 2.
Rat TCH229 protein no. 1 and rat TCH229 protein no. In No. 2, four base substitutions (144th, 231, 1252, and 1539 in the base sequence represented by SEQ ID NO: 53) were observed. Of the four positions, three of A144T (Glu → Asp), C231A (Ser → Arg), and A1252T (IIe → Phe) each had the amino acid substitution shown in parentheses, but A1539G had no amino acid substitution. There was no. It is thought that these base substitutions may be derived from genetic polymorphisms (SNPs).
When a homology search was performed on a known database using Blast P (Nucleic Acids Res. 25, 3389, 1997), rat TCH229No. 1 and No. 2 has a homology of about 50% at the base level and about 41% at the amino acid level with rat oatp-E (Endocrinology Vol. 142 (5), p. 2005, 2001) which is an organic anion transporter reported in rats. Showed sex. In addition, rat TCH229No. 1 and No. 2 has a homology of about 81.4% at the base level and about 81.6% at the amino acid level with human TCH229, a novel gene belonging to the organic anion transporter family, indicating that it is a rat homolog of human TCH229. (FIGS. 7 and 8). In the drawing, the transmembrane regions are denoted by TM1 to TM12.
[0089]
Example 12
Analysis of tissue distribution of rat TCH229 gene product
Using two kinds of primer DNAs designed from the sequence of rat TCH229, primer rTMF (SEQ ID NO: 70) and primer rTMR (SEQ ID NO: 69), and TaqMan probe rP1 (SEQ ID NO: 76), each rat tissue ( The expression level of rat TCH229 in cDNA of spleen, thymus, testis, small intestine, stomach, skin, heart, brain, lung, muscle, kidney) was measured by TaqMan PCR.
The reaction was first performed at 50 ° C. for 2 minutes and then at 95 ° C. for 10 minutes using ABI PRISM 7900 sequence detection system (Applied Biosystems) using TaqMan Universal PCR Master Mix (Applied Biosystems), and then 95%. Forty cycles were repeated at 15 ° C. for 15 seconds and 60 ° C. for one minute as one reaction cycle, and detection was performed simultaneously. FIG. 9 shows the results.
Rat TCH229 gene product (mRNA) showed strong expression in lung and kidney in Multiple Choice cDNAs; RatKitI and RatKitII (manufactured by Origene), and also expression in skin and brain.
[0090]
Example 13
Analysis of tissue distribution of mouse TCH229 gene product in 7-week-old BALB / c mice
(1) Preparation of cDNA for each tissue of normal mouse
7-week-old BALB / c mouse tissues [cerebrum, cerebellum, hippocampus, medulla oblongata, spinal cord, sciatic nerve, skin, skeletal muscle, eyeball, heart, lung, trachea, pancreas, kidney, liver, forestomach, posterior stomach, duodenum , Jejunum, cecum, colon, rectum, spleen, thymus, bone marrow, ovary, uterus, prostate, testis (1 to 10 ovaries and uterus are collected from females, and the rest are collected from males). (Manufactured by Nippon Gene) or RNeasy Mini Kit (manufactured by Qiagen) was used to prepare total RNA. The prepared total RNA was subjected to a reverse transcription reaction using TaqMan Reverse Transcription Reagents (manufactured by Applied Biosystems) to prepare cDNA.
(2) Analysis of tissue distribution of mouse TCH229 gene product
Using the two types of primer DNA, primer mTMF (SEQ ID NO: 45) and primer mTMR (SEQ ID NO: 46) used in Example 8, and TaqMan probe mP1 (SEQ ID NO: 47), The expression level (copy number) of mouse TCH229 in the cDNA was measured by TaqMan PCR. For the same cDNA, the expression level (copy number) of the rodent glycoderaldehyde-3-phosphate dehydrogenase (GAPDH) was also measured using TaqMan rodent GAPDH control reagents (manufactured by Applied Biosystems). The reaction was first performed at 50 ° C. for 2 minutes and then at 95 ° C. for 10 minutes using ABI PRISM 7900 sequence detection system (Applied Biosystems) using TaqMan Universal PCR Master Mix (Applied Biosystems), and then 95%. Forty cycles were repeated at 15 ° C. for 15 seconds and 60 ° C. for one minute as one reaction cycle, and detection was performed simultaneously.
The results are shown in FIG.
The mouse TCH229 gene product (mRNA) was slightly expressed in prostate, trachea, kidney, and bone marrow, and highly expressed in lung in each tissue of BALB / c mice at 7 weeks of age.
[0091]
Example 14
(1) Preparation of cDNA for normal rat tissues
Total RNA from 12-week-old Wistar rat male tissues (cerebrum, cerebellum, liver, kidney, prostate, heart, lung, duodenum, jejunum, colon, skin, eyeball) using RNeasy Mini Kit (Qiagen) Was prepared. The prepared total RNA was subjected to a reverse transcription reaction using TaqMan Reverse Transcription Reagents (manufactured by Applied Biosystems) to prepare cDNA.
(2) Analysis of tissue distribution of rat TCH229 gene product
Using the two types of primer DNA, primer rTMF (SEQ ID NO: 70) and primer rTMR (SEQ ID NO: 69) used in Example 12, and TaqMan probe rP1 (SEQ ID NO: 76), The expression level (copy number) of rat TCH229 in the cDNA was measured by TaqMan PCR. For the same cDNA, the expression level (copy number) of the rodent glycoderaldehyde-3-phosphate dehydrogenase (GAPDH) was also measured using TaqMan rodent GAPDH control reagents (manufactured by Applied Biosystems). The reaction was first performed at 50 ° C. for 2 minutes and then at 95 ° C. for 10 minutes using ABI PRISM 7900 sequence detection system (Applied Biosystems) using TaqMan Universal PCR Master Mix (Applied Biosystems), and then 95%. Forty cycles were repeated at 15 ° C. for 15 seconds and 60 ° C. for one minute as one reaction cycle, and detection was performed simultaneously.
The results are shown in FIG.
In the tissues of 12-week-old Wistar rats, high expression of the rat TCH229 gene product (mRNA) was observed in kidney and lung.
[0092]
Example 15
Construction of human TCH229 expression vector
A human TCH229 (SEQ ID NO: 1) expression vector was created by the following method.
Using 10 ng of the plasmid obtained in Example 1 as a template and primers 229OF2 (SEQ ID NO: 82) and 229OR2 (SEQ ID NO: 83), pfu turbo DNA Polymerase (manufactured by Stratagene) under the following conditions (1): ) To (3), PCR was performed. The 5′-end primer 229OF2 and the 3′-end primer 229OR2 were designed to add an EcoRV site and an XhoI site to the 5 ′ end for cloning into a vector, respectively.
(1) 94 ° C for 30 seconds
(2) 35 cycles of 94 ° C for 10 seconds -55 ° C for 10 seconds -70 ° C for 2.5 minutes
(3) 70 ° C for 5 minutes
After gel electrophoresis of the PCR reaction solution, the main band was purified. The PCR fragment thus obtained was digested with the restriction enzymes EcoRV and XhoI by incubating at 37 ° C. for 1 hour, and the reaction solution was purified by gel electrophoresis. This was added to Takara ligation kit ver. At the EcoRV site and XhoI site of pcDNA3.1 (+) (Invitrogen), which is an animal cell expression vector. 2 (manufactured by Takara Bio Inc.). After the ligation reaction mixture was subjected to ethanol precipitation, the ligation reaction mixture was transformed into competent E. coli (Escherichia coli) TOP10 (manufactured by Invitrogen). A plasmid was prepared from a plurality of colonies thus obtained, and this base sequence was used as a primer DNA [primer BGH RV (SEQ ID NO: 84), primer T7 (SEQ ID NO: 66), primer 229OF2 (SEQ ID NO: 82), primer 229OR2 (SEQ ID NO: 83), primer rr2 (SEQ ID NO: 6), primer A1 (SEQ ID NO: 17), primer B2 (SEQ ID NO: 18), primer ff2 (SEQ ID NO: 10), primer rr1 (SEQ ID NO: 4), primer ff1 (SEQ ID NO: 9)]], and a reaction using BigDye Terminator Cycle Sequencing Kit (manufactured by Applied Biosystems), and the base sequence was determined using the DNA sequencer ABI PRISM 3100 DNA Analyzer (App). It was confirmed by using the id Biosystems). A transformant having this plasmid was named Escherichia coli TOP10 / pCDNA3.1 (+)-TCH229.
[0093]
Example 16
Preparation of CHO cell line expressing human TCH229 and measurement of transgene expression level
Escherichia coli TOP10 / pCDNA3.1 (+)-TCH229 was cultured, and a plasmid DNA was prepared from the Escherichia coli body using EndoFree Plasmid Maxi Kit (manufactured by Qiagen). This plasmid DNA was subjected to CHO dhfr using FuGENE 6 Transfection Reagent (manufactured by Roche) according to the attached protocol. ⁻ The cells were introduced. A mixture of 2 μg of plasmid DNA and transfection reagent was added 2 × 10 4 hours before ⁵ CHO dhfr ⁻ Cells were added to a seeded 6 cm diameter petri dish. After culturing in MEMα medium (Invitrogen) containing 10% fetal bovine serum (JRH Bioscience) for 1 day, 1.0 mg / ml Geneticin (Invitrogen) was added to the medium, and then TCH229 expressing cells were further added. Was cultured. The medium was exchanged several times during the process. After about 10 days, the cells were separated by trypsin treatment, and the collected cells were seeded on a 96-well plate at 0.5 to 1 cell per well, and thereafter, 1.0 mg / ml of Geneticin was added for about 10 days. TCH229 expressing cells were selected in a medium containing. For wells in which one colony was grown per well, total RNA was prepared from the grown cells using RNeasy Mini Kit or RNeasy 96 Kit (both manufactured by Qiagen). The prepared total RNA was subjected to a reverse transcription reaction using TaqMan Reverse Transcription Reagents (manufactured by Applied Biosystems) to prepare cDNA. In this regard, human TCH229 was purified using the two types of primer DNA, primer TMF (SEQ ID NO: 19) and primer TMR (SEQ ID NO: 20) used in Example 4, and TaqMan probe P1 (SEQ ID NO: 21). The expression level was measured by TaqMan PCR. The reaction was first performed at 50 ° C. for 2 minutes and then at 95 ° C. for 10 minutes using ABI PRISM 7900 sequence detection system (Applied Biosystems) using TaqMan Universal PCR Master Mix (Applied Biosystems). Forty cycles were repeated at 95 ° C. for 15 seconds and 60 ° C. for 1 minute as one reaction cycle, and detection was performed simultaneously. As a human TCH229 gene high expression cell line (monoclonal), clone No. 71 was selected.
[0094]
Example 17
Expression analysis of human TCH229 gene in commercially available normal human cells
(1) Preparation of normal human cell cDNA
Normal human cells were purchased from Cambrex BioScience Walkersville and cultured according to the instructions provided with the product. Table 2 shows the cells used in the experiment.
[Table 2]

75cm each cell ² The cells were cultured in a culture flask so as to be subconfluent. For the cells marked with * in the table, TNF-α, IL-1β and IL-6 were added to a final concentration of 10 ng / ml. For the cells not marked with *, 16 hours after the start of culture, and for the cells marked with *, 8 hours after adding the mixture of TNF-α, IL-1β and IL-6, Was recovered by trypsin-EDTA treatment. Total RNA was prepared from the collected cells using ISOGEN (manufactured by Nippon Gene) or RNeasy Mini Kit (manufactured by Qiagen) (contaminated DNA was removed by DNase treatment in each case). The prepared total RNA was subjected to a reverse transcription reaction using TaqMan Reverse Transcription Reagents (manufactured by Applied Biosystems) to prepare cDNA.
(2) Preparation of cDNA for human normal cells subjected to various stimuli
a) Renal proximal tubular epithelial cells (RPTEC) and renal cortical epithelial cells (HRCE) (both manufactured by Cambrex BioScience Walkersville) were 1 × 10 4 in a collagen I-coated 24-well plate. ⁵ The seeds were inoculated individually, cultured overnight, replaced with a triiodothyronine (T3) -free bread kit REGM (manufactured by BIO WHITTAKER), and further cultured for 30 minutes.
b) As stimulants, TGF-β1 (manufactured by Wako Pure Chemical Industries), TNF-α (manufactured by Genzyme), IL-1β (manufactured by Genzyme), IL-6 (manufactured by Genzyme) and PMA (manufactured by Wako Pure Chemical) (Manufactured by the company). A solution was prepared by diluting each of these five stimulants with a T3-free bread kit REGM (manufactured by BIO WHITTAKER) to a final concentration of 10 ng / ml.
c) Immediately after removing the culture supernatant of RPTEC and HRCE in a) above, 500 μl of the solution prepared in b) was added per well, followed by culturing. As a control, a solution prepared by diluting a mixture of the solvents in which the above five kinds of stimulants are dissolved with a T3-free bread kit REGM (manufactured by BIO WHITTAKE) at the same ratio as the stimulant was prepared, and RPTEC and HRCE were used per well. The culture was performed by adding 500 μl each.
d) 0.5, 1, 2, 4, 6 and 8 hours after the addition of the stimulant, the cells were collected respectively, and total RNA was prepared using RNeasy Mini Kit (manufactured by Qiagen). A reverse transcription reaction was performed on the prepared total RNA using TaqMan Reverse Transcription Reagents (manufactured by Applied Biosystems) to prepare cDNA.
(3) Expression analysis of human TCH229 gene in commercially available normal human cells
Using the two types of primer DNA, primer TMF (SEQ ID NO: 19) and primer TMR (SEQ ID NO: 20) used in Example 4, and the TaqMan probe P1 (SEQ ID NO: 21), The expression level (copy number) of human TCH229 was measured by TaqMan PCR in cDNA prepared from PRTEC and HRCE stimulated with a stimulant, respectively. Expression amounts of ribosomal RNA (18S) of cDNA prepared from normal human cells and RPTEC and HRCE stimulated with a stimulant, respectively, using Eukaryotic 18S rRNA Pre-Developed TaqMan Assay Reagents (manufactured by Applied Biosystems). ) Or Human GAPDH (GAPDH) (manufactured by Applied Biosystems) was also used to measure the expression level (copy number) of human glycodealdehyde-3-phosphate dehydrogenase (GAPDH). The reaction was first carried out at 50 ° C. for 2 minutes and further at 95 ° C. for 10 minutes using ABI PRISM 7900 sequence detection system (Applied Biosystems) using TaqMan Universal PCR Master Mix (Applied Biosystems). Forty cycles were repeated at 95 ° C. for 15 seconds and 60 ° C. for one minute as one reaction cycle, and detection was performed simultaneously.
FIG. 12 shows changes in the expression of human TCH229 in human normal cells. Cells marked with * in Table 2 were stimulated with TNF-α, IL-1β and IL-6 (each at 10 ng / ml), but little change in expression was observed compared to no stimulation. And almost no expression was observed.
FIG. 13 shows changes in the expression of human TCH229 in RPTEC stimulated with the stimulant, and FIG. 14 shows changes in the expression of human TCH229 in HRCE stimulated with the stimulant.
The human TCH229 gene product (mRNA) was specifically expressed in renal proximal tubular epithelial cells and slightly expressed in renal cortical epithelial cells. In addition, when RPTEC and HRCE were stimulated with TGF-β1, TNF-α, IL-1β or PMA, the expression of human TCH229 was confirmed to be decreased in a culture time-dependent manner as compared with the control. .
[0095]
Example 18
Expression analysis of rat TCH229 gene product in renal disease model (Wistar Fatty, SHC, Zucker Fatty) rat kidney
As disease model animals, three types of Wistar Fatty rats (WF rats), Zucker Fatty rats (ZF rats), and spontaneously hypercholesterolemic rats (SHC rats) were used. As corresponding controls, WF rats were used. Used Wistar Lean Rat (WL rat), Zucker Lean Rat (ZL rat) for ZF rat, and SD Rat (SD rat) for SHC rat.
Wistar Fatty rats (WF rats) show symptoms characteristic of diabetes in addition to renal dysfunction symptoms. Therefore, as a diabetic nephropathy model (Frontiers in diabetes research, lessons from animal diabetes II, 535-541, 1988). ) And Zucker Fatty rats (ZF rats) exhibit renal dysfunction and diabetic symptoms in the same manner as WF rats. Therefore, as a diabetic nephropathy model (Kidney international, 52, S218-S220, 1997). ), Spontaneously hypercholesterolemic rats (SHC rats) show symptoms similar to human focal glomerulosclerosis, and are used as focal glomerulosclerosis model (Japanese Society of Nephrology, Vol. 37, 91). 99 pages, has been 1995 years) reported.
(1) Disease model animals and control animals are bred under the same conditions. For WF rats and WL rats, 13 weeks (before onset), 20 weeks (after onset), 42 weeks (for advanced stage) and 68 weeks (for reduced renal function) Phase), at 6 weeks (pre-onset), 12 weeks (post-onset), 20 weeks (expansion phase) and 26-30 weeks (renal dysfunction phase) in SHC rats and SD rats, and in ZF rats and ZL rats. At 8 weeks (before onset) and 27 weeks (after onset), total RNA was extracted as follows.
WF rat group (each week n = 5), WL rat group (each week n = 5), SHC rat group (each week n = 5), SD rat group (each week n = 5), ZF rat group (each week) n = 9), kidneys of the ZL rat group (each weekly age n = 9) were sliced so as to be about 100 mg in a fractured surface including the nipple, and total RNA was extracted using ISOGEN according to the attached manual. . Further, contaminating DNA was removed using QIAGEN RNeasy Mini kit (manufactured by Qiagen) and RNase-Free DNase set (manufactured by Qiagen). The prepared total RNA was subjected to a reverse transcription reaction using TaqMan Reverse Transcription Reagents (manufactured by Applied Biosystems) to prepare cDNA.
[0096]
(2) Expression analysis of rat TCH229 gene product in renal disease model rat kidney
Prepared in (1) above using the two kinds of primer DNA, primer rTMF (SEQ ID NO: 70) and primer rTMR (SEQ ID NO: 69) used in Example 12, and TaqMan probe rP1 (SEQ ID NO: 76). The expression level (copy number) of rat TCH229 in the renal disease model rat kidney cDNA was measured by TaqMan PCR. For the same cDNA, the expression level (copy number) of the rodent glycoderaldehyde-3-phosphate dehydrogenase (GAPDH) was also measured using TaqMan rodent GAPDH control reagents (manufactured by Applied Biosystems). The reaction was first performed at 50 ° C. for 2 minutes and then at 95 ° C. for 10 minutes using ABI PRISM 7900 sequence detection system (Applied Biosystems) using TaqMan Universal PCR Master Mix (Applied Biosystems), and then 95%. Forty cycles were repeated at 15 ° C. for 15 seconds and 60 ° C. for one minute as one reaction cycle, and detection was performed simultaneously. FIG. 15 shows the results of the WF rat group, FIG. 16 shows the results of the SHC rat group, and FIG. 17 shows the results of the ZF rat group.
The expression of rat TCH229 was reduced in all three types of renal disease model rat kidneys after the onset of renal dysfunction as compared to each control group. From this, TCH229 may be involved in renal diseases such as nephropathy.
[0097]
【The invention's effect】
The protein, polynucleotide and antibody of the present invention include, for example, renal diseases (eg, renal failure, glomerulonephritis, diabetic nephropathy, focal glomerulosclerosis, nephrotic syndrome, renal edema, interstitial nephritis, renal disease) Sclerosis, uremia, etc.), liver disease (eg, cirrhosis, hepatitis, alcoholic liver disease, etc.), pancreatic disease (eg, pancreatitis, etc.), immune disease associated with thymic abnormalities, genital disease (eg, prostatic hypertrophy, prostate gland) Inflammation, testicular neurosis, ovarian cysts, etc.), gastrointestinal disorders (eg, irritable bowel syndrome, ulcerative colitis, ischemic colitis, gastritis, etc.), spleen disorders (eg, hypersplenism, splenomegaly syndrome) ), Cancer (eg, lung, kidney, liver, non-small cell lung, ovarian, prostate, stomach, bladder, breast, cervical, colon, rectal, pancreatic, thyroid, Myeloid leukemia, etc.), respiratory diseases (eg, pleurisy, pneumonia, chronic Obstructive lung disease, asthma, etc.), osteomyelitis, diabetes, hypertension, post-ischemic reperfusion injury, retinitis, central nervous system diseases (eg, epilepsy, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), skin diseases (eg, It is useful as a diagnostic marker for thyroid hormone-related diseases such as atopic dermatitis and seborrheic dermatitis (eg, thyroid hormone refractory disease, Basedow's disease, cretin disease, hyperthyroidism, etc.). Compounds that promote or inhibit the activity of the protein obtained by a screening method using the protein, polynucleotide, or antibody include compounds that promote or inhibit the expression of the protein gene, compounds that promote or inhibit the expression of the protein, and the like. For example, renal diseases (eg, renal failure, glomerulonephritis, diabetic nephropathy, focal glomerulosclerosis, nephrotic syndrome, renal edema, interstitial nephritis, renal sclerosis, uremic, etc.), liver Diseases (eg, cirrhosis, hepatitis, alcoholic liver disease, etc.), pancreatic diseases (eg, pancreatitis, etc.), immune diseases associated with thymic abnormalities, genital diseases (eg, prostatic hypertrophy, prostatitis, testicular neurosis, ovarian cysts, etc.) ), Gastrointestinal disorders (eg, irritable bowel syndrome, ulcerative colitis, ischemic colitis, gastritis, etc.), spleen disorders (eg, hypersplenism, splenomegaly syndrome, etc.), (Eg, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, pancreatic cancer, thyroid cancer, myeloid leukemia, etc. ), Respiratory diseases (eg, pleurisy, pneumonia, chronic obstructive pulmonary disease, asthma, etc.), osteomyelitis, diabetes, hypertension, post-ischemic reperfusion injury, retinitis, central nervous system diseases (eg, epilepsy, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc., skin diseases (eg, atopic dermatitis, seborrheic dermatitis, etc.), thyroid hormone-related diseases (eg, thyroid hormone refractory disease, Basedow's disease, Cretin's disease, hyperthyroidism, etc.) ) Can be used as a prophylactic / therapeutic agent. It is preferably used as a prophylactic / therapeutic agent for renal diseases and thyroid hormone-related diseases, and more preferably as a prophylactic / therapeutic agent for diabetic nephropathy.
[0098]
[Sequence list]

[Brief description of the drawings]
FIG. 1 is a diagram showing a comparison of amino acid sequences of human TCH229, human SLC21A12 and human OATPRP4. In the figure, TCH229 indicates the amino acid sequence of human TCH229, SLC21A12 indicates the amino acid sequence of human SLC21A12, OATPRP4 indicates the amino acid sequence of human OATPRP4, and TM1 to TM12 indicate the transmembrane region. □ indicates an amino acid corresponding to human TCH229. (Continued to Fig. 2)
FIG. 2 shows a comparison of amino acid sequences of human TCH229, human SLC21A12 and human OATPRP4. In the figure, TCH229 indicates the amino acid sequence of human TCH229, SLC21A12 indicates the amino acid sequence of human SLC21A12, OATPRP4 indicates the amino acid sequence of human OATPRP4, and TM1 to TM12 indicate the transmembrane region. □ indicates an amino acid corresponding to human TCH229. (Continued from Fig. 1)
FIG. 3 is a diagram showing the expression level of human TCH229 gene product in each tissue. The expression level was represented by the number of copies per μl of the cDNA solution.
FIG. 4 is a diagram showing the expression level of human TCH229 gene product in each tissue. The expression level was represented by the number of copies per μl of the cDNA solution.
FIG. 5 is a diagram showing a comparison of amino acid sequences of human TCH229 and mouse TCH229. In the figure, TCH229 indicates the amino acid sequence of human TCH229, mTCH229 indicates the amino acid sequence of mouse TCH229, the transmembrane region is TM1-12, and the amino acid positions highly conserved between families are indicated by *. □ indicates an amino acid corresponding to both.
FIG. 6 is a diagram showing the expression level of mouse TCH229 gene product in each tissue. The expression level was represented by the number of copies per μl of the cDNA solution.
FIG. 7 shows human TCH229 and rat TCH229No. 1 and No. FIG. 3 is a diagram showing a comparison of amino acid sequences of two amino acids. In the figure, TCH229 is the amino acid sequence of human TCH229, rTCH229No. 1 is rat TCH229No. The amino acid sequence of rTCH229No. 2 is rat TCH229No. 2 shows the amino acid sequence of 2. TM1 to TM12 indicate transmembrane regions. □ indicates an amino acid corresponding to the three. (Continued to Fig. 8)
FIG. 8. Human TCH229 and rat TCH229No. 1 and No. FIG. 3 is a diagram showing a comparison of amino acid sequences of two amino acids. In the figure, TCH229 is the amino acid sequence of human TCH229, rTCH229No. 1 is rat TCH229No. The amino acid sequence of rTCH229No. 2 is rat TCH229No. 2 shows the amino acid sequence of 2. TM1 to TM12 indicate transmembrane regions. □ indicates an amino acid corresponding to the three. (Continued from Fig. 7)
FIG. 9 is a view showing the expression level of rat TCH229 gene product in each tissue. The expression level was represented by the number of copies per μl of the cDNA solution.
FIG. 10 is a diagram showing the expression level of mouse TCH229 gene product in each tissue. The expression level was expressed as a value obtained by dividing the copy number of mouse TCH229 per 1 μl of the cDNA solution by the copy number of rodent GAPDH in an equal amount of each tissue cDNA.
FIG. 11 is a view showing the expression level of rat TCH229 gene product in each tissue. The expression level was expressed as a value obtained by multiplying the relative expression level by 100.
FIG. 12 is a graph showing changes in the expression of human TCH229 in human normal cells. In the figure, the vertical axis indicates a value obtained by multiplying the relative expression value of human TCH229 gene expression value to human 18S expression value by 100,000. The horizontal axis indicates the cell name. ■ indicates no TNF-α, IL-1β and IL-6 (10 ng / ml each) stimulation, and □ indicates TNF-α, IL-1β and IL-6 (10 ng / ml each) stimulation.
FIG. 13 is a graph showing changes in the expression of human TCH229 in RPTEC stimulated with a stimulant. In the figure, the vertical axis indicates a value obtained by multiplying the relative expression value of the human TCH229 gene expression value to the human GAPDH expression value by 100. The horizontal axis shows the reaction time with the stimulant. -●-indicates TGF-β1,-■-indicates PMA,-▲-indicates TNF-α,-*-indicates IL-1β,-○-indicates IL-6, and-△-indicates control.
FIG. 14 is a graph showing changes in the expression of human TCH229 in HRCE stimulated with a stimulant. In the figure, the vertical axis indicates a value obtained by multiplying the relative expression value of the human TCH229 gene expression value to the human GAPDH expression value by 100. The horizontal axis shows the reaction time with the stimulant. -●-indicates TGF-β1,-■-indicates PMA,-▲-indicates TNF-α,-*-indicates IL-1β,-○-indicates IL-6, and-△-indicates control.
FIG. 15 is a graph showing the expression level of rat TCH229 gene. In the figure, the vertical axis represents the value obtained by multiplying the relative expression value of the rat TCH229 gene expression value to that of the parent GAPDH expression value by 1000, and the horizontal axis represents the age at which the kidney was collected. □ indicates the results of the experimental group (WF rats), and ■ indicates the results of the control group (WL rats).
FIG. 16 is a graph showing the expression level of rat TCH229 gene. In the figure, the vertical axis represents the value obtained by multiplying the relative expression value of the rat TCH229 gene expression value to that of the parent GAPDH expression value by 1000, and the horizontal axis represents the age at which the kidney was collected. □ indicates the results of the experimental group (SHC rats), and ■ indicates the results of the control group (SD rats).
FIG. 17 is a diagram showing the expression level of rat TCH229 gene. In the figure, the vertical axis represents the value obtained by multiplying the relative expression value of the rat TCH229 gene expression value to that of the parent GAPDH expression value by 1000, and the horizontal axis represents the age at which the kidney was collected. □ indicates the results of the experimental group (ZF rats), and ■ indicates the results of the control group (ZL rats).

Claims (39)

A protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 26 or SEQ ID NO: 52, or a salt thereof. A protein comprising an amino acid sequence represented by SEQ ID NO: 1 or a salt thereof. A protein comprising an amino acid sequence represented by SEQ ID NO: 26 or a salt thereof. A protein comprising an amino acid sequence represented by SEQ ID NO: 52 or a salt thereof. The protein or salt thereof according to claim 1, wherein the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 52 is the amino acid sequence represented by SEQ ID NO: 54. A protein consisting of the amino acid sequence represented by SEQ ID NO: 54 or a salt thereof. A partial peptide of the protein according to claim 1 or a salt thereof. A polynucleotide comprising a polynucleotide encoding the protein according to claim 1 or the partial peptide according to claim 7. The polynucleotide according to claim 8, which is DNA. A polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 2, SEQ ID NO: 27, SEQ ID NO: 53 or SEQ ID NO: 55. A recombinant vector containing the polynucleotide according to claim 8. A transformant transformed with the recombinant vector according to claim 11. The protein according to claim 1, wherein the transformant according to claim 12 is cultured to produce and accumulate the protein according to claim 1 or the partial peptide according to claim 7, and collect the protein. 8. The method for producing the partial peptide or a salt thereof according to 7. A medicament comprising the protein according to claim 1, the partial peptide according to claim 7, or a salt thereof. A medicament comprising the polynucleotide according to claim 8. A diagnostic agent comprising the polynucleotide according to claim 8. An antibody against the protein according to claim 1, the partial peptide according to claim 7, or a salt thereof. A diagnostic agent comprising the antibody according to claim 17. A medicament comprising the antibody according to claim 17. A polynucleotide comprising a base sequence complementary to or substantially complementary to the polynucleotide according to claim 8, or a part thereof. A diagnostic agent comprising the polynucleotide according to claim 20. A medicament comprising the polynucleotide according to claim 20. A compound that promotes or inhibits the activity of the protein according to claim 1, the partial peptide according to claim 7, or a salt thereof, wherein the protein according to claim 1 or the partial peptide according to claim 7 or a salt thereof is used. Or a method of screening for a salt thereof. A compound that promotes or inhibits the activity of the protein according to claim 1, the partial peptide according to claim 7, or a salt thereof, or a compound thereof, comprising the protein according to claim 1, the partial peptide according to claim 7, or a salt thereof. Salt screening kit. A compound or salt thereof that promotes or inhibits the activity of the protein of claim 1, or the partial peptide of claim 7, or a salt thereof, obtained by using the screening method of claim 23 or the screening kit of claim 24. . A medicament comprising the compound according to claim 25 or a salt thereof. A method for screening a compound or a salt thereof that promotes or inhibits expression of the protein gene according to claim 1, characterized by using the polynucleotide according to claim 8. A kit for screening a compound or a salt thereof which promotes or inhibits the expression of the protein gene according to claim 1, comprising the polynucleotide according to claim 8. A compound or a salt thereof that promotes or inhibits expression of the protein gene according to claim 1, which is obtained by using the screening method according to claim 27 or the screening kit according to claim 28. A medicament comprising the compound according to claim 29 or a salt thereof. A method for quantifying a protein according to claim 1, wherein the antibody according to claim 17 is used. A method for diagnosing a disease associated with the function of a protein according to claim 1, wherein the method according to claim 31 is used. A method for screening a compound or a salt thereof that promotes or inhibits expression of the protein according to claim 1, characterized by using the antibody according to claim 17. A kit for screening a compound or a salt thereof that promotes or inhibits expression of the protein according to claim 1, comprising the antibody according to claim 17. 34. A compound or a salt thereof, which promotes or inhibits the expression of the protein according to claim 1, obtained by using the screening method according to claim 33 or the screening kit according to claim 34. A medicament comprising the compound according to claim 35 or a salt thereof. The medicament according to claim 14, claim 15, claim 19, claim 22, claim 26, claim 30, or claim 36, which is a prophylactic / therapeutic agent for kidney disease. A method for preventing or treating renal disease, comprising administering to a mammal an effective amount of the compound according to claim 25, claim 29 or claim 35 or a salt thereof. Use of the compound according to claim 25, claim 29 or claim 35 or a salt thereof for producing a prophylactic / therapeutic agent for kidney disease.

Images