JP2004089179A - New transcription factor, dna and use of the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new transcription factor useful as a preventing, treating and diagnostic agent of nervous system diseases and a cholinergic neural differentiation-accelerating agent, etc. <P>SOLUTION: This protein is a diagnostic marker of the neural system diseases and has a cholinergic neural differentiation-suppressing activity. The compound adjusting (preferably inhibiting) the activity of the protein or the expression of gene of the protein, or its salt, its antisense polynucleotide, its antibody, etc., can be used as the preventing/treating agent of the nervous system diseases as the excellent cholinergic neural differentiation-acceleration agent. <P>COPYRIGHT: (C)2004,JPO

Description

【００７３】
本願明細書の配列表の配列番号は、以下の配列を示す。
〔配列番号：１〕
実施例１で得られたマウス由来のタンパク質ｍＮＨＥＳのアミノ酸配列を示す。
〔配列番号：２〕
配列番号：１で表されるアミノ酸配列を有するタンパク質ｍＮＨＥＳをコードするＤＮＡの塩基配列を示す。
〔配列番号：３〕
実施例１で得られたタンパク質をコードする全長遺伝子を含むＤＮＡの塩基配列を示す。
〔配列番号：４〕
実施例１で得られた塩基配列を示す。
〔配列番号：５〕
実施例３で得られたヒト由来のタンパク質ｈＮＨＥＳのアミノ酸配列を示す。〔配列番号：６〕
配列番号：５で表されるアミノ酸配列を有するタンパク質ｈＮＨＥＳをコードするＤＮＡの塩基配列を示す。
〔配列番号：７〕
実施例３で得られた塩基配列を示す。
〔配列番号：８〕
実施例１〜５で用いられたプライマー１の塩基配列を示す。
〔配列番号：９〕
実施例１、実施例３および実施例４で用いられたプライマー２の塩基配列を示す。
〔配列番号：１０〕
実施例１および実施例５で用いられたプライマー３の塩基配列を示す。
〔配列番号：１１〕
実施例１および実施例２で用いられたプライマー４の塩基配列を示す。
〔配列番号：１２〕
実施例３で用いられたプライマー５の塩基配列を示す。
〔配列番号：１３〕
実施例３で用いられたプライマー６の塩基配列を示す。
〔配列番号：１４〕
実施例４で用いられたプライマー７の塩基配列を示す。
〔配列番号：１５〕
実施例４で用いられたプライマー８の塩基配列を示す。
〔配列番号：１６〕
実施例４で得られたＤＮＡの塩基配列を示す。
〔配列番号：１７〕
配列番号：２６で表されるアミノ酸配列を有するタンパク質ｒＮＨＥＳをコードするＤＮＡの塩基配列を示す。
〔配列番号：１８〕
実施例６で用いられたプライマーの塩基配列を示す。
〔配列番号：１９〕
実施例６で用いられたプライマーの塩基配列を示す。
〔配列番号：２０〕
実施例６で用いられたＴａｑＭａｎプローブの塩基配列を示す。
〔配列番号：２１〕
実施例８で用いられたプライマーの塩基配列を示す。
〔配列番号：２２〕
実施例８で用いられたプライマーの塩基配列を示す。
〔配列番号：２３〕
実施例８で用いられたＴａｑＭａｎプローブの塩基配列を示す。
〔配列番号：２４〕
実施例５で用いられたプライマー９の塩基配列を示す。
〔配列番号：２５〕
実施例５で得られたＤＮＡの塩基配列を示す。
〔配列番号：２６〕
実施例５で得られたラット由来のタンパク質ｒＮＨＥＳのアミノ酸配列を示す。
〔配列番号：２７〕
実施例７で用いられたプライマーの塩基配列を示す。
〔配列番号：２８〕
実施例７で用いられたプライマーの塩基配列を示す。
〔配列番号：２９〕
実施例７で用いられたプローブの塩基配列を示す。５’末端にリポーター色素としてＦＡＭ（６−ｃａｒｂｏｘｙ−ｆｌｕｏｒｅｓｃｅｉｎ）を、３’末端にはクエンチャーとしてＴＡＭＲＡ（６−ｃａｒｂｏｘｙ−ｔｅｔｒａｍｅｔｈｙｌ−ｒｈｏｄａｍｉｎｅ）を標識した。
後述の実施例１で取得された大腸菌（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）ＪＭ１０９／ｐＴＢ２２６４は、２００２年４月１日から、日本国茨城県つくば市東１−１−１　中央第６（郵便番号３０５−８５６６）の独立行政法人産業技術総合研究所　特許生物寄託センターに、寄託番号ＦＥＲＭ　ＢＰ−７９９２として、２００２年３月１９日から、大阪府大阪市淀川区十三本町２−１７−８５（郵便番号５３２−８６８６）の財団法人　発酵研究所（ＩＦＯ）に受託番号ＩＦＯ　１６７７９として寄託されている。
後述の実施例４で取得された大腸菌（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）ＪＭ１０９／ｐＴＢ２２７４は、２００２年７月４日から、日本国茨城県つくば市東１−１−１　中央第６（郵便番号３０５−８５６６）の独立行政法人産業技術総合研究所　特許生物寄託センターに、寄託番号ＦＥＲＭ　ＢＰ−８１１１として寄託されている。
後述の実施例５で取得された大腸菌（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）ＪＭ１０９／ｒＮＨＥＳＴＯＰＯは、２００２年２月２１日から、日本国茨城県つくば市東１−１−１　中央第６（郵便番号３０５−８５６６）の独立行政法人産業技術総合研究所　特許生物寄託センターに、寄託番号ＦＥＲＭ　ＢＰ−８２９９として寄託されている。
【００７４】
以下において、実施例により本発明をより具体的にするが、この発明はこれらに限定されるものではない。
実施例１
マウス脳由来のＭａｒａｔｈｏｎ−Ｒｅａｄｙ　ｃＤＮＡ（Ｃｌｏｎｔｅｃｈ社）を鋳型とし、２個のプライマー、プライマー１（配列番号：８）およびプライマー２（配列番号：９）を用いてＰＣＲ反応を行った。該反応における反応液の組成は上記ｃＤＮＡ　１μｌを鋳型として使用し、ＱｕａｎｔｉＴｅｃｔ　ＳＹＢＲ　Ｇｒｅｅｎ　ＰＣＲ　ｋｉｔ　（ＱＩＡＧＥＮ社）の　２Ｘ　ＳＹＢＥＲ　Ｇｒｅｅｎ　Ｍａｓｔｅｒ　Ｍｉｘ　１０μｌ、プライマー１およびプライマー２を各０．４μＭ、滅菌蒸留水７μｌを加え、２０μｌの液量とした。ＰＣＲ反応は、９５℃・１５分の後、９５℃・２０秒、６０℃・２０秒、７２℃・１分のサイクルを４０回繰り返し、最後に７２℃・４分の伸長反応を行った。該ＰＣＲ反応産物を再度プライマー３（配列番号：１０）およびプライマー２を用いてＰＣＲ反応を行った。該反応における反応液の組成は、上記ＰＣＲ反応産物１μｌを鋳型として使用し、ＱｕａｎｔｉＴｅｃｔ　ＳＹＢＲ　Ｇｒｅｅｎ　ＰＣＲ　ｋｉｔ　（ＱＩＡＧＥＮ社）の　２Ｘ　ＳＹＢＥＲ　Ｇｒｅｅｎ　Ｍａｓｔｅｒ　Ｍｉｘ　１０μｌ、プライマー２およびプライマー３を各０．４μＭ、滅菌蒸留水７μｌを加え、２０μｌの液量とした。ＰＣＲ反応は、９５℃・１５分の後、９５℃・２０秒、６０℃・２０秒、７２℃・１分のサイクルを１０回繰り返し、最後に７２℃・４分の伸長反応を行った。ＴＯＰＯ　ＴＡクローニングキット（Ｉｎｖｉｔｒｏｇｅｎ社）の処方に従いプラスミドベクターｐＣＲＩＩ−ＴＯＰＯベクター（Ｉｎｖｉｔｒｏｇｅｎ社）へサブクローニングした。これを大腸菌（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）ＴＯＰ１０Ｆ’に導入し、ｃＤＮＡを持つクローンをアンピシリンを含むＬＢ寒天培地中で選択した。個々のクローンの配列を解析した結果、塩基性へリックス・ループ・へリックス型転写因子タンパク質をコードするｃＤＮＡ配列（配列番号：３）を得た。また、マウス脳由来のＭａｒａｔｈｏｎ−Ｒｅａｄｙ　ｃＤＮＡ（Ｃｌｏｎｔｅｃｈ社）を鋳型とし、２個のプライマー、プライマー１およびプライマー４（配列番号：１１）を用いてＰＣＲ反応を行った。該反応における反応液の組成は上記ｃＤＮＡ　１μｌを鋳型として使用し、ＱｕａｎｔｉＴｅｃｔ　ＳＹＢＲ　Ｇｒｅｅｎ　ＰＣＲ　ｋｉｔ　（ＱＩＡＧＥＮ社）の　２Ｘ　ＳＹＢＥＲ　Ｇｒｅｅｎ　Ｍａｓｔｅｒ　Ｍｉｘ　１０μｌ、プライマー１およびプライマー４を各０．４μＭ、滅菌蒸留水７μｌを加え、２０μｌの液量とした。ＰＣＲ反応は、９５℃・１５分の後、９５℃・２０秒、６０℃・２０秒、７２℃・１分のサイクルを４０回繰り返し、最後に７２℃・４分の伸長反応を行った。該ＰＣＲ反応産物を再度プライマー１およびプライマー４を用いてＰＣＲ反応を行った。該反応における反応液の組成は上記ＰＣＲ反応産物１μｌを鋳型として使用し、ＱｕａｎｔｉＴｅｃｔ　ＳＹＢＲ　Ｇｒｅｅｎ　ＰＣＲ　ｋｉｔ　（ＱＩＡＧＥＮ社）の　２Ｘ　ＳＹＢＥＲ　Ｇｒｅｅｎ　Ｍａｓｔｅｒ　Ｍｉｘ　１０μｌ、プライマー１およびプライマー４を各０．４μＭ、滅菌蒸留水７μｌを加え、２０μｌの液量とした。ＰＣＲ反応は、９５℃・１５分の後、９５℃・２０秒、６０℃・２０秒、７２℃・１分のサイクルを１０回繰り返し、最後に７２℃・４分の伸長反応を行った。得られたＤＮＡ断片の塩基配列を解析した結果、得られたＤＮＡ断片は配列番号：３で表される塩基配列と同一の部分を含む、４３７ｂｐの断片であった（配列番号：４）。配列番号：３で表されるｃＤＮＡ断片には２４０個のアミノ酸配列（配列番号：１）がコードされていた（配列番号：２）。配列番号：１で表されるアミノ酸配列を含有するタンパク質をｍＮＨＥＳと命名した。ｍＮＨＥＳ（配列番号：１）と、塩基性へリックス・ループ・へリックス型転写因子タンパク質Ｈｅｓｒ−１（ＳＷＩＳＳ−ＰＲＯＴ　ＩＤ：　Ｑ９ＷＶ９３；Ｂｉｏｃｈｅｍ．　Ｂｉｏｐｈｙｓ．　Ｒｅｓ．　Ｃｏｍｍｕｎ．　２６０　巻　４５９−４６５頁　１９９９年）とは、アミノ酸レベルで、４７％の相同性を示した。配列番号：３で表される塩基配列を有するプラスミドをｐＴＢ２２６４と命名し、得られた形質転換体を大腸菌（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）ＪＭ１０９／ｐＴＢ２２６４と命名した。
【００７５】
実施例２
配列番号：１で表されるアミノ酸配列を含有するタンパク質遺伝子の脳組織での発現プロファイルを明らかにするため、マウスブレインｃＤＮＡパネル（Ｃｅｍｉｎｅｓ社）を材料とし、ＡＢＩ　Ｐｒｉｓｍ　７９００　Ｓｅｑｕｅｎｃｅ　Ｄｅｔｅｃｔｉｏｎ　Ｓｙｓｔｅｍ（Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍｓ社）を用いて、リアルタイムＰＣＲ反応を行った。
該反応における反応液の組成は上記ｃＤＮＡパネル中の各ｃＤＮＡ　１μｌを鋳型として使用し、プライマー１およびプライマー４を各０．３μＭ、２Ｘ　ＳＹＢＥＲ　Ｇｒｅｅｎ　Ｍａｓｔｅｒ　Ｍｉｘ　１０μｌ、滅菌蒸留水７μｌを加え、２０μｌの液量とした。ＰＣＲ反応は、５０℃・２分−９５℃・１０分の後、９５℃・１５秒−６０℃・１分のサイクルを４０回繰り返して行った。算出されたコピー数は、内部コントロールとして、配列番号：１で表されるアミノ酸配列を含有するタンパク質遺伝子のコピー数と同様に算出したグリセロール３リン酸脱水酵素のコピー数で補正した。
その結果、実施例１で得られたタンパク質の遺伝子の発現は、胎児期の脳で生後に比べ高い発現を示した（図１）。
これより実施例１で得られたｍＮＨＥＳタンパク質の遺伝子は胎児期の脳で多く発現していることが明らかとなった。
【００７６】
実施例３
ヒト腎臓由来のＭａｒａｔｈｏｎ−Ｒｅａｄｙ　ｃＤＮＡ（Ｃｌｏｎｔｅｃｈ社）を鋳型とし、２個のプライマー、プライマー５（配列番号：１２）およびプライマー６（配列番号：１３）を用いてＰＣＲ反応を行った。該反応における反応液の組成は上記ｃＤＮＡ　１μｌを鋳型として使用し、ＱｕａｎｔｉＴｅｃｔ　ＳＹＢＲ　Ｇｒｅｅｎ　ＰＣＲ　ｋｉｔ　（ＱＩＡＧＥＮ社）の　２Ｘ　ＳＹＢＥＲ　Ｇｒｅｅｎ　Ｍａｓｔｅｒ　Ｍｉｘ　１０μｌ、プライマー１およびプライマー２を各０．４μＭ、滅菌蒸留水７μｌを加え、２０μｌの液量とした。ＰＣＲ反応は、９５℃・１５分の後、９５℃・２０秒−５０℃・２０秒−７２℃・１分のサイクルを５０回繰り返し、最後に７２℃・４分の伸長反応を行った。得られた約９００ｂｐのＤＮＡ断片を回収し、塩基配列を解析した結果、９１９塩基からなる配列を得た（配列番号：７）。配列番号：７で表されるｃＤＮＡ断片には２４１個のアミノ酸配列（配列番号：５）がコードされていた（配列番号：６）。
配列番号：５で表されるアミノ酸配列を含有するタンパク質をｈＮＨＥＳと命名した。
実施例１で得られた配列番号：２で表される塩基配列と、配列番号：６で表される塩基配列には８５％の相同性が、配列番号：１で表されるアミノ酸配列と、配列番号：５で表されるアミノ酸配列には９１％の相同性があった。
ｈＮＨＥＳ（配列番号：５）は、予測配列として報告されている塩基性へリックス・ループ・へリックス型転写因子タンパク質（Ｇｅｎｂａｎｋ　ＩＤ：ＸＰ　０７０２８３）と、アミノ酸レベルで７３％の相同性を示した。また、ｈＮＨＥＳ（配列番号：５）は、塩基性へリックス・ループ・へリックス型転写因子タンパク質Ｈｅｙ１（Ｇｅｎｂａｎｋ　ＩＤ：　ＮＰ　０３６３９０　；Ｇｅｎｏｍｉｃｓ、６６巻、１９５−２０３頁、２０００年）と、アミノ酸レベルで２６％の相同性を示した。
【００７７】
実施例４
ヒト腎臓由来のＭａｒａｔｈｏｎ−Ｒｅａｄｙ　ｃＤＮＡ（Ｃｌｏｎｔｅｃｈ社）を鋳型とし、２個のプライマー、プライマー７（配列番号：１４）およびプライマー８（配列番号：１５）を用いてＰＣＲ反応を行った。該反応における反応液の組成は上記ｃＤＮＡ　１μｌを鋳型として使用し、ＱｕａｎｔｉＴｅｃｔ　ＳＹＢＲ　Ｇｒｅｅｎ　ＰＣＲ　ｋｉｔ　（ＱＩＡＧＥＮ社）の　２Ｘ　ＳＹＢＥＲ　Ｇｒｅｅｎ　Ｍａｓｔｅｒ　Ｍｉｘ　１０μｌ、プライマー１およびプライマー２を各０．４μＭ、滅菌蒸留水７μｌを加え、２０μｌの液量とした。ＰＣＲ反応は、９５℃・１５分の後、９５℃・２０秒−５０℃・２０秒−７２℃・１分のサイクルを５０回繰り返し、最後に７２℃・４分の伸長反応を行った。その結果、得られた約８００ｂｐのＤＮＡ断片を回収した。回収後のＤＮＡ断片をＨｏｔＳｔａｒＴａｑ　ＤＮＡ　ｐｏｌｙｍｅｒａｓｅ（ＱＩＡＧＥＮ社）を用い説明書に従い、プライマー７およびプライマー８を用いて再度増幅させた。その結果得られた反応液に含まれるＤＮＡ断片を、ＱＩＡＧＥＮ　ＰＣＲ　ｃｌｏｎｉｎｇ　ｋｉｔ（ＱＩＡＧＥＮ社）を用い、説明書に従い、ｐＤｒｉｖｅ　ｃｌｏｎｉｎｇ　ｖｅｃｔｏｒにクローニングした。クローニングされたＤＮＡ断片の塩基配列は、解析の結果、８１９塩基からなるｈＮＨＥＳをコードする塩基配列（配列番号：１６）であった。配列番号：１６で表される塩基配列を有するプラスミドをｐＴＢ２２７４と命名し、得られた形質転換体を大腸菌（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）ＪＭ１０９／ｐＴＢ２２７４と命名した。
【００７８】
実施例５
ラット脳由来のＭａｒａｔｈｏｎ−Ｒｅａｄｙ　ｃＤＮＡ（Ｃｌｏｎｔｅｃｈ社）を鋳型とし、２個のプライマー、プライマー１（配列番号：８）およびプライマー９（配列番号：２４）を用いてＰＣＲ反応を行った。該反応における反応液の組成は上記ｃＤＮＡ　１μｌを鋳型として使用し、ＱｕａｎｔｉＴｅｃｔ　ＳＹＢＲ　Ｇｒｅｅｎ　ＰＣＲ　ｋｉｔ　（ＱＩＡＧＥＮ社）の　２Ｘ　ＳＹＢＥＲ　Ｇｒｅｅｎ　Ｍａｓｔｅｒ　Ｍｉｘ　１０μｌ、プライマー１（配列番号：８）およびプライマー９を各０．４μＭ、滅菌蒸留水７μｌを加え、２０μｌの液量とした。ＰＣＲ反応は、９５℃・１５分の後、９５℃・２０秒、５０℃・２０秒、７２℃・１分のサイクルを５０回繰り返し、最後に７２℃・４分の伸長反応を行った。該ＰＣＲ反応産物を、再度、プライマー１およびプライマー７を用いてＰＣＲ反応を行った。該反応における反応液の組成は上記ＰＣＲ反応産物１μｌを鋳型として使用し、ＨｏｔｓｔａｒＴａｑ　Ｍａｓｔｅｒ　Ｍｉｘ　ｋｉｔ　（ＱＩＡＧＥＮ社）の　ＨｏｔｓｔａｒＴａｑ　Ｍａｓｔｅｒ　Ｍｉｘ　１０μｌ、プライマー１およびプライマー７９を各０．４μＭ、滅菌蒸留水７μｌを加え、２０μｌの液量とした。ＰＣＲ反応は、９５℃・１５分の後、９５℃・２０秒、６０℃・２０秒、７２℃・１分のサイクルを１０回繰り返し、最後に７２℃・４分の伸長反応を行った。ＴＯＰＯ　ＴＡクローニングキット（Ｉｎｖｉｔｒｏｇｅｎ社）の処方に従い、プラスミドベクターｐＣＲＩＩ−ＴＯＰＯベクター（Ｉｎｖｉｔｒｏｇｅｎ社）へサブクローニングした。これを大腸菌（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）ＴＯＰ１０Ｆ’に導入し、ｃＤＮＡを持つクローンをアンピシリンを含むＬＢ寒天培地中で選択した。個々のクローンの配列を解析した結果、塩基性へリックス・ループ・へリックス型転写因子タンパク質をコードするｃＤＮＡ配列（配列番号：２５）を得た。配列番号：２５で表されるｃＤＮＡ断片には２４１個のアミノ酸配列（配列番号：２６）がコードされていた（配列番号：１７）。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ｒＮＨＥＳと命名した。ｒＮＨＥＳ（配列番号：２６）と、マウス由来の塩基性へリックス・ループ・へリックス型転写因子タンパク質Ｍｅｄａｎｅ（ＷＯ　０２／６０９２８号公報）とは、アミノ酸レベルで、９７％の相同性を示した。
配列番号：２５で表される塩基配列を有するプラスミドをｒＮＨＥＳＴＯＰＯと命名し、得られた形質転換体を、大腸菌（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）ＪＭ１０９／ｒＮＨＥＳＴＯＰＯと命名した。
ｒＮＨＥＳとｍＮＨＥＳとは、アミノ酸レベルで９６％の相同性が、塩基レベルで９４％の相同性がある。
ｒＮＨＥＳとｈＮＨＥＳとは、アミノ酸レベルで９１％の相同性が、塩基レベルで８５％の相同性がある。
【００７９】
実施例６
ｍＮＨＥＳ遺伝子と神経分化との関連を明らかにするため、マウスＰ１９胚性腫瘍細胞を培養し、コリン作動性神経分化させ、その発現プロファイルについて検討を行った。
Ｐ１９細胞を、５ｘ１０^５ｃｅｌｌ／ｍｌの細胞濃度で、５ｘ１０^−７Ｍ　レチノイン酸（和光純薬工業）および１０％　ＦＢＳ（Ｉｎｖｉｔｒｏｇｅｎ社）を含むα−ＭＥＭ培地（Ｉｎｖｉｔｒｏｇｅｎ社）に縣濁した。その細胞縣濁液　３ｍｌを、３．５ｃｍ浮遊培養用プレート（住友ベークライト社）を用い、２日間培養した。ついでトリプシンで消化し、５ｘ１０^４ｃｅｌｌ／ｍｌの細胞濃度で、１％　Ｎ−２　ｓｕｐｐｌｉｍｅｎｔ（Ｉｎｖｉｔｒｏｇｅｎ社）および１μｇ／μｌ　ファイブロネクチン（Ｉｎｖｉｔｒｏｇｅｎ社）を含むＤＭＥＭ／Ｆ１２培地（Ｉｎｖｉｔｒｏｇｅｎ社）に縣濁した。その細胞縣濁液　０．５ｍｌを、４８ｗｅｌｌ　ｐｏｌｙ−Ｄ−Ｌｙｓｉｎｅコートプレート（ベクトンデッキソン社）を用いて培養し、以下の実験に供した。培養開始後３日目に、シトシン　β−Ｄ−アラビノフラノシド（ＡｒａＣ）（シグマ社）を５μｇ／ｍｌとなるよう、培地に添加した。
培養後３、４、５および６日目に、ＲＮｅａｓｙ　９６ｋｉｔ（ＱＩＡＧＥＮ社）を用いて、細胞からｔｏｔａｌ　ＲＮＡを抽出した。得られたｔｏｔａｌ　ＲＮＡから、ＴａｑＭａｎ　Ｒｅｖｅｒｓｅ　ＴｒａｎｓｃｒｉｐｔｉｏｎＲｅａｇｅｎｔｓ（ＰＥアプライドバイオシステムズ社製）を用いてｃＤＮＡを合成した。ｍＮＨＥＳ遺伝子を特異的に増幅、検出できる２種の合成プライマー（配列番号：１８および配列番号：１９）およびＴａｑＭａｎプローブ（配列番号：２０）を用い、ＰＣＲによるｍＮＨＥＳ遺伝子のコピー数の定量を、ＡＢＩ　Ｐｒｉｓｍ　７９００　ｓｅｑｕｅｎｃｅ　Ｄｅｔｅｃｔｉｏｎ　Ｓｙｓｔｅｍを用いて行った。ＰＣＲ反応は、ＴａｑＭａｎ　ｕｎｉｖｅｒｓａｌ　ＰＣＲ　Ｍａｓｔｅｒ　Ｍｉｘ（ＰＥアプライドバイオシステムズ社製）を使用し、条件は添付されている説明書に従って行った。算出されたコピー数を内部コントロールとして、ｍＮＨＥＳ遺伝子のコピー数と同様にＴａｑＭａｎ　Ｒｏｄｅｎｔ　ＧＡＰＤＨ　Ｃｏｎｔｒｏｌ　ｒｅｇｅｎｔｓ　ＶＩＣ（ＰＥアプライドバイオシステムズ社製）を用いて算出したグリセロール３リン酸脱水酵素のコピー数で補正した後、値を発現量として、３日目の発現量を１として変動率を算出した。
その結果、ｍＮＨＥＳ遺伝子の発現変動率は、培養開始後４、５および６日目で、それぞれ、３．０、１５．７および１６．６となり、培養日数経過と共に発現が増加することが明らかとなった。
これよりｍＮＨＥＳ遺伝子は、神経分化と共に発現誘導されることが明らかとなった。
【００８０】
実施例７
ｍＮＨＥＳ遺伝子とコリン作動性神経分化との関連を明らかにするため、Ｐ１９胚性腫瘍細胞のコリン作動性神経分化に対するｍＮＨＥＳ遺伝子強制発現の影響を検討した。
実施例１で構築したｐＴＢ２２７４を、制限酵素ＢａｍＨ　ＩおよびＸｈｏ　Ｉ（ともにタカラ酒造社）で消化し、約９００ｂｐの断片を回収後、ｐＴＡＲＧＥＴ　Ｖｅｃｔｏｒ（プロメガ社）の　ＢａｍＨ　ＩおよびＸｈｏ　Ｉ間隙に挿入し、ｍＮＨＥＳ強制発現プラスミドを作製した。作製した強制発現プラスミドをｍＮＨＥＳ１−１と命名した。ｍＮＨＥＳ１−１およびコントロールとしてのｐＴＡＲＧＥＴ　Ｖｅｃｔｏｒを、それぞれＦｕｇｅｎｅ６トランスフェクション試薬（ロシュ・ダイアグノスティックス社）を用いて１０％　ＦＢＳ（Ｉｎｖｉｔｒｏｇｅｎ社）を含むα−ＭＥＭ培地（Ｉｎｖｉｔｒｏｇｅｎ社）で培養中のＰ１９細胞に遺伝子導入した。その後、Ｐ１９細胞を、５ｘ１０^５ｃｅｌｌ／ｍｌの細胞濃度で、５ｘ１０^−７Ｍ　レチノイン酸（和光純薬工業）および１０％　ＦＢＳ（Ｉｎｖｉｔｒｏｇｅｎ社）を含むα−ＭＥＭ培地（Ｉｎｖｉｔｒｏｇｅｎ社）に縣濁した。その細胞縣濁液　３ｍｌを、３．５ｃｍ浮遊培養用プレート（住友ベークライト社）を用い、２日間培養した。ついでトリプシンで消化し、５ｘ１０^４ｃｅｌｌ／ｍｌの細胞濃度で、１％　Ｎ−２　ｓｕｐｐｌｉｍｅｎｔ（Ｉｎｖｉｔｒｏｇｅｎ社）および１μｇ／μｌ　ファイブロネクチン（Ｉｎｖｉｔｒｏｇｅｎ社）を含むＤＭＥＭ／Ｆ１２培地（Ｉｎｖｉｔｒｏｇｅｎ社）に縣濁した。この細胞縣濁液　０．５ｍｌを、４８ｗｅｌｌ　ｐｏｌｙ−Ｄ−Ｌｙｓｉｎｅコートプレート（ベクトンデッキソン社）を用いて培養し、以下の実験に供した。培養開始後５日目に、ＲＮｅａｓｙ　９６ｋｉｔ（ＱＩＡＧＥＮ社）を用いて、細胞からｔｏｔａｌ　ＲＮＡを抽出した。得られたｔｏｔａｌ　ＲＮＡから、ＴａｑＭａｎ　Ｒｅｖｅｒｓｅ　Ｔｒａｎｓｃｒｉｐｔｉｏｎ　Ｒｅａｇｅｎｔｓ（ＰＥアプライドバイオシステムズ社製）を用いてｃＤＮＡを合成した。
コリン作動性神経マーカーであるコリンアセチルトランスフェラーゼ遺伝子を特異的に増幅、検出できる２種の合成プライマー（配列番号：２７および配列番号：２８）およびＴａｑＭａｎプローブ（配列番号：２９；Ｆａｍ−　ｃｃａａｔｇａｃｃａｇｃｔａａｇｇｔｔｔｇｃａｇｃｃ−Ｔａｍｒａ；配列中、Ｆａｍは６−ｃａｒｂｏｘｙ−ｆｌｕｏｒｅｓｃｅｉｎを、Ｔａｍｒａは６−ｃａｒｂｏｘｙ−ｔｅｔｒａｍｅｔｈｙｌ−ｒｈｏｄａｍｉｎｅ　をそれぞれ示す）を用い、ＰＣＲによるコリンアセチルトランスフェラーゼ遺伝子のコピー数の定量を、ＡＢＩ　Ｐｒｉｓｍ　７９００　ｓｅｑｕｅｎｃｅ　Ｄｅｔｅｃｔｉｏｎ　Ｓｙｓｔｅｍを用いて行った。ＰＣＲ反応は、ＴａｑＭａｎ　ｕｎｉｖｅｒｓａｌ　ＰＣＲ　Ｍａｓｔｅｒ　Ｍｉｘ（ＰＥアプライドバイオシステムズ社製）を使用し、条件は添付されている説明書に従って行った。算出されたコピー数を内部コントロールとして、ｍＮＨＥＳ遺伝子のコピー数と同様にＴａｑＭａｎ　Ｒｏｄｅｎｔ　ＧＡＰＤＨ　Ｃｏｎｔｒｏｌ　ｒｅｇｅｎｔｓ　ＶＩＣ（ＰＥアプライドバイオシステムズ社製）を用いて算出したグリセロール３リン酸脱水酵素のコピー数で補正した後、値を発現量とした。
その結果、ｍＮＨＥＳ１−１導入細胞でのコリンアセチルトランスフェラーゼ遺伝子発現量は、コントロールのｐＴＡＲＧＥＴ　Ｖｅｃｔｏｒ導入細胞の、５２％の発現量であることが明らかとなった。
これよりｍＮＨＥＳ遺伝子は、コリン作動性神経の分化を抑制すると考えられる。
【００８１】
実施例８
ｈＮＨＥＳ遺伝子と神経変性疾患との関連を明らかにするため、ヒトアルツハイマー脳組織（海馬）由来ｃＤＮＡ（Ｂｉｏｃｈａｉｎ社）およびヒト健常人脳組織（海馬）由来ｃＤＮＡ（Ｂｉｏｃｈａｉｎ社）を材料とし、ｈＮＨＥＳ遺伝子発現量の比較を行った。
ｈＮＨＥＳ遺伝子を特異的に増幅、検出できる２種の合成プライマー（配列番号：２１および配列番号：２２）およびＴａｑＭａｎプローブ（配列番号：２３）を用い、ＰＣＲによるｈＮＨＥＳ遺伝子のコピー数の定量を、ＡＢＩ　Ｐｒｉｓｍ　７９００　ｓｅｑｕｅｎｃｅ　Ｄｅｔｅｃｔｉｏｎ　Ｓｙｓｔｅｍを用いて行った。ＰＣＲ反応は、ＴａｑＭａｎ　ｕｎｉｖｅｒｓａｌ　ＰＣＲ　Ｍａｓｔｅｒ　Ｍｉｘ（ＰＥアプライドバイオシステムズ社製）を使用し、条件は添付されている説明書に従って行った。算出されたコピー数を、内部コントロールとしてｈＮＨＥＳ遺伝子のコピー数と同様にＴａｑＭａｎ　ＧＡＰＤＨ　Ｃｏｎｔｒｏｌ　Ｒｅａｇｅｎｔｓ（ＰＥアプライドバイオシステムズ社製）を用いて算出したグリセロール３リン酸脱水酵素のコピー数で補正した後、値を発現量として算出した。
その結果、アルツハイマー病患者の海馬でのｈＮＨＥＳ遺伝子の発現は、健常人の海馬での発現に比べ、３５８倍高いことが明らかになった。
これよりｈＮＨＥＳ遺伝子遺伝子は、アルツハイマー病などの神経変性疾患に関与すると考えられる。
【００８２】
実施例９
ｍＮＨＥＳ遺伝子と神経分化との関連を明らかにするため、マウス胎児神経幹細胞を培養、神経分化させ、その発現プロファイルについて検討を行った。
神経幹細胞として、妊娠１４〜１５日マウス胎児から、ｎｅｕｒｏｓｐｈｅｒｅ法（Ｅｘｐｅｒｉｍｅｎｔａｌ　Ｎｅｕｒｏｌｏｇｙ，１４４巻，３５０−３６０頁，１９９７年）により取得した神経幹細胞を用いた。
神経幹細胞を、１５０ｃｍ^２ｆｌａｓｋ（ＩＷＡＫＩ住友ベークライト社）を用い、５×１０^５ｃｅｌｌ／ｍｌの細胞濃度で、５０ｍｌ　２０ｎｇ／ｍｌのｂＦＧＦ（ｇｅｎｚｙｍｅ　ｔｅｃｈｎｅ社）およびＮ_２ｓｕｐｐｌｅｍｅｎｔ（Ｉｎｖｉｔｒｏｇｅｎ社）を含むＤＭＥＭ／Ｆ１２培地（Ｉｎｖｉｔｒｏｇｅｎ社）中、ニューロスフェアとして維持した。ついで、トリプシン消化し、ＥＣＬ（ＵＰＳＴＡＴＥ　ｂｉｏｔｅｃｈｎｏｌｏｇｙ）コートを行った２４ｗｅｌｌプレート（ベクトンデッキソン社）を用い、２×１０^５ｃｅｌｌ／ｍｌの細胞濃度で、０．５ｍｌの１００ｎｇ／ｍｌのＮＧＦ（ｇｅｎｚｙｍｅ　ｔｅｃｈｎｅ社）およびＮ_２ｓｕｐｐｌｅｍｅｎｔ（ｇｉｂｃｏ社）を含むＤＭＥＭ／Ｆ１２培地（ｇｉｂｃｏＩｎｖｉｔｒｏｇｅｎ社）中で分化培養を行った。
分化培養前、および分化誘導後１、３ならびに５日目に、ＲＮｅａｓｙ　９６ｋｉｔ（ＱＩＡＧＥＮ社）を用い、細胞からｔｏｔａｌ　ＲＮＡを回収した。得られたｔｏｔａｌ　ＲＮＡから、ＴａｑＭａｎ　Ｒｅｖｅｒｓｅ　Ｔｒａｎｓｃｒｉｐｔｉｏｎ　Ｒｅａｇｅｎｔｓ（ＰＥアプライドバイオシステムズ社製）を用いてｃＤＮＡを合成した。
ｍＮＨＥＳ遺伝子を特異的に増幅、検出できる２種の合成プライマー（配列番号：１８および配列番号：１９）およびＴａｑＭａｎプローブ（配列番号：２０）を用い、ＰＣＲによるｍＮＨＥＳ遺伝子のコピー数の定量を、ＡＢＩ　Ｐｒｉｓｍ　７９００　ｓｅｑｕｅｎｃｅ　Ｄｅｔｅｃｔｉｏｎ　Ｓｙｓｔｅｍを用いて行った。ＰＣＲ反応は、ＴａｑＭａｎ　ｕｎｉｖｅｒｓａｌ　ＰＣＲ　Ｍａｓｔｅｒ　Ｍｉｘ（ＰＥアプライドバイオシステムズ社製）を使用し、条件は添付されている説明書に従って行った。算出されたコピー数を内部コントロールとして、ｍＮＨＥＳ遺伝子のコピー数と同様にＴａｑＭａｎ　Ｒｏｄｅｎｔ　ＧＡＰＤＨ　Ｃｏｎｔｒｏｌ　ｒｅｇｅｎｔｓ　ＶＩＣ（ＰＥアプライドバイオシステムズ社製）を用いて算出したグリセロール３リン酸脱水酵素のコピー数で補正した後、値を発現量とし、分化誘導前の発現量を１として、ｍＮＨＥＳ遺伝子発現量の変動率を算出した。
その結果、ｍＮＨＥＳ遺伝子は分化誘導後１、３および５日目で、それぞれ、３０．６、９．８および１．３となり、分化誘導後、一過性に発現が増加することが明らかとなった。
これより、ｍＮＨＥＳ遺伝子は、神経幹細胞で神経分化と共に発現誘導されることが明らかとなった。
【００８３】
【発明の効果】
本発明のタンパク質は、神経系疾患の診断マーカーであり、コリン作動性神経分化抑制作用を有する。したがって、該タンパク質の活性または該タンパク質の遺伝子の発現を調節（好ましくは阻害）する化合物またはその塩、本発明の抗体（好ましくは中和抗体）、本発明のアンチセンスポリヌクレオチドなどは、例えばコリン作動性神経分化促進剤として、例えば神経系疾患〔例、神経変性疾患（例、アルツハイマー病、パーキンソン症候群など）、脊髄損傷、てんかん、脳虚血性障害、脳血管性痴呆など〕、精神系疾患（例、精神分裂病、うつ病など）、腎疾患（例、腎不全、腎炎、尿毒症など）、生殖器疾患（例、前立腺肥大症、前立腺癌炎、精巣神経症など）、骨・軟骨疾患（例、骨そしょう症、変形性関節症、慢性関節リウマチなど）、筋疾患（例、筋ジストロフィー症など）、心疾患（例、心筋症、心筋梗塞、心不全、狭心症など）、自己免疫性疾患（例、全身性エリテマトーデス、免疫関連糸球体腎炎など）、癌（例、腎臓癌、肺癌、肝臓癌、非小細胞肺癌、卵巣癌、前立腺癌、胃癌、膀胱癌、乳癌、子宮頚部癌、結腸癌、直腸癌、膵臓癌など）、高血圧などの予防・治療剤、好ましくは神経変性疾患（好ましくはアルツハイマー病など）などの予防・治療剤として安全に使用することができる。
【００８４】
【配列表】

【図面の簡単な説明】
【図１】ｍＮＨＥＳのマウスブレインｃＤＮＡパネルでの発現量の結果を表す図である。図中、縦軸は、ｍＮＨＥＳの発現量を内部コントロールとして用いたグリセロール３リン酸脱水酵素の発現量で補正した値を示す。横軸は、Ｅ１２は胎齢１２日を、Ｅ１４は胎齢１４日を、Ｅ１６は胎齢１６日を、Ｅ１８は胎齢１８日を、Ｐ１は生後１日を、Ｐ４は生後４日を、Ｐ７は生後７日を、Ｐ１４は生後１４日を、Ｐ２１は生後２１日を、Ｐ６０は生後６０日をそれぞれ示す。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a novel transcription factor useful for a preventive / therapeutic agent and a diagnostic agent for a nervous system disease, a cholinergic neuronal differentiation promoter, and the like.
[0002]
[Prior art]
In eukaryotic cells, the amount of transcription of genes is controlled by the interaction between chromosomal sequences (cis-elements) such as promoters and enhancers and transcription factors binding thereto. The promoter comprises a core (basic) sequence composed of a TATA box and a transcription initiation site, and a gene-specific expression control region. The transcription regulatory factor bound to the expression control region activates RNA polymerase II holoenzyme (a complex of RNA polymerase II and a group of basic transcription factors) bound to the core sequence, and promotes gene transcription. Basic transcription factors that bind to the core sequence are involved in the transcription of many genes and have no gene specificity.
On the other hand, a transcriptional regulator that binds to the expression control region is activated when cells proliferate or differentiate or responds to external stimuli such as hormones and cytokines, and promotes transcription of defined genes. For this reason, transcription regulatory factors are sometimes referred to as gene-specific transcription factors. It is expected that about 3,000 gene-specific transcription factors exist in humans, and they are expected to be drug targets as well as cell surface receptors (Mol. Med. Today, 358, 1998).
The basic helix-loop-helix (bHLH) motif is a motif structure abundant in transcription factors. The bHLH family transcription factors include MyoD, dHAND, eHAND, HES1, HES5, Mash1, Math3, Neurogenin1, Neurogenin2, Hesr-1 and the like. Regarding its function, Myo D controls myoblast determination, differentiation into myotube cells, and has the ability to differentiate fibroblasts into myocytes by overexpression. dHAND and eHAND play an important role in the formation of hearts in which their knockout mice become defective in cardiac development at the stage of development (Experimental Medicine, Vol. 17, pp. 1298-34). It has been found that HES1 and HES5 can maintain neural stem cells in an undifferentiated state when forcedly expressed, and can inhibit maintenance of an undifferentiated state when both genes are deleted. HES1 and HES5 can be used to maintain neural stem cells. It has been shown to have an essential role (J Biol Chem, 276, 30467-74, 2001). It has been shown that Mash1 and Math3 can induce differentiation of neural stem cells into neurons when forcedly expressed, and can inhibit differentiation into neurons when both genes are deleted, and that Mash1 and Math3 are neuronal differentiation determinants. It is functioning (EMBO J, 19, 5460-72, 2000). In neurogenin knockout mice, cerebral ganglion neurons do not differentiate, and Neurogenin plays an important role in neuronal differentiation (Neuron, 20, 469-82, 1998, Neuron, 20, 483-94). , 1998). Hesr-1 is also called HERP-2 or Hey1, and is activated by a Notch signal that is important for neuronal differentiation (Mol Cell Biol., 21, 6080-9, 2001). Thus, the transcription factor of the bHLH family plays an important role in the maintenance, differentiation and development of stem cells (Experimental Medicine, Vol. 18, pp. 1252-1256, 2000). It is known that the transcription factor of the bHLH family forms a dimer and has an activity. For example, Mash1 binds to E12 and E47 to form a heterodimer and has a transcription promoting activity (Development 127, 2933-43). P., 2000).
Recently, it has been reported that Medane, a transcription factor of the bHLH family, is involved in the differentiation of dopaminergic neurons (Patent Document 1 WO 02/60928).
Cholinergic nerves are important for the cognitive function of the brain, and it is known that their shedding is remarkable in Alzheimer's disease patients (protein nucleic acid enzyme, 10, 1722, 2000).
[Patent Document 1]
WO 02/60928
[0003]
[Problems to be solved by the invention]
In recent years, the accumulation of sequence information in the random sequencing of human genomic DNA or cDNA derived from various human tissues and rapid advances in gene technology have accelerated the elucidation of human genes. Along with this, the existence of many genes predicted to encode proteins of unknown function has been revealed. These are expected to contain many novel transcription factors belonging to the bHLH family. To find a new transcription factor of the bHLH family that has a novel action in the maintenance, differentiation and development of neural stem cells, the transcription factor is directly used or a drug screening system using the transcription factor is used. This enables the development of completely new and safer and superior agents for preventing / treating neurological diseases, and therapeutic methods. Therefore, in this field, it has been desired to find a new transcription regulatory factor and to develop a method for mass production thereof.
[0004]
[Means for Solving the Problems]
The present inventors have conducted intensive studies to solve the above problems, and as a result, have found a transcription factor gene that is expressed in the mouse embryo brain, and furthermore that this transcribed gene has a cholinergic neuronal differentiation inhibitory action. The present inventor has further found that the present invention has been completed as a result of further study based on these findings.
[0005]
That is, the present invention
(1) 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: 5, or SEQ ID NO: 26, 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 comprising an amino acid sequence represented by SEQ ID NO: 5 or a salt thereof;
(4) a protein comprising an amino acid sequence represented by SEQ ID NO: 26 or a salt thereof;
(5) a partial peptide of the protein according to (1) or a salt thereof;
(6) a polynucleotide containing a polynucleotide encoding the protein of (1) or the partial peptide of (5),
(7) the polynucleotide according to (6), which is a DNA;
(8) a polynucleotide having a base sequence represented by SEQ ID NO: 2, SEQ ID NO: 6, or SEQ ID NO: 17,
(9) a recombinant vector containing the polynucleotide according to (6),
(10) a transformant transformed with the recombinant vector according to (9),
(11) culturing the transformant according to (10), producing and accumulating the protein according to (1) or the partial peptide according to (5), and collecting the protein; A) a partial peptide according to (5) or a salt thereof,
(12) a medicine comprising the protein of (1) or the partial peptide of (5) or a salt thereof,
(13) a medicine comprising the polynucleotide according to (6),
(14) a diagnostic agent comprising the polynucleotide of (6) above,
(15) an antibody against the protein according to (1) or the partial peptide according to (5) or a salt thereof,
(16) a medicament comprising the antibody according to the above (15),
(17) a diagnostic agent comprising the antibody according to the above (15),
(18) a base sequence complementary or substantially complementary to the base sequence of a DNA having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof, An antisense polynucleotide containing a portion thereof,
(19) a medicine comprising the antisense polynucleotide according to (18),
(20) a diagnostic agent comprising the antisense polynucleotide according to (18),
(21) The protein according to (1), the partial peptide according to (5), or a salt thereof, wherein the protein according to (1), the partial peptide according to (5), or a salt thereof is used. A method for screening a compound or a salt thereof that promotes or inhibits the activity,
(22) The activity of the protein of (1) or the partial peptide of (5) or a salt thereof, comprising the protein of (1) or the partial peptide of (5) or a salt thereof. A kit for screening for a compound that promotes or inhibits or a salt thereof,
(23) Promotes the activity of the protein of (1), the partial peptide of (5) or a salt thereof obtained by using the screening method of (21) or the screening kit of (22). Or a compound that inhibits or a salt thereof,
(24) a medicament comprising the compound according to (23) or a salt thereof,
(25) A method for screening a compound or a salt thereof that promotes or inhibits the expression of the protein gene according to (1), which comprises using the polynucleotide according to (6).
(26) a kit for screening a compound or a salt thereof that promotes or inhibits the expression of the protein gene according to (1), which comprises the polynucleotide according to (6);
(27) A compound or a salt thereof that promotes or inhibits the expression of the protein gene according to (1), obtained by using the screening method according to (25) or the screening kit according to (26);
(28) a medicament comprising the compound according to (27) or a salt thereof,
(28a) The medicament according to the above (12), (13), (16), (19), (24) or (28), which is a preventive / therapeutic agent for a nervous system disease.
(29) The medicament according to the above (12), (13), (16), (19), (24) or (28), which is a prophylactic / therapeutic agent for a neurodegenerative disease.
(29a) The prophylactic / therapeutic agent according to the above (29), wherein the neurodegenerative disease is Alzheimer's disease or Parkinson's syndrome,
(29b) The diagnostic agent according to the above (14), (17) or (20), which is a diagnostic agent for a nervous system disease.
(30) The diagnostic agent according to the above (14), (17) or (20), which is a diagnostic agent for a neurodegenerative disease,
(30a) The diagnostic agent according to the above (30), wherein the neurodegenerative disease is Alzheimer's disease or Parkinson's syndrome,
(31) the prophylactic / therapeutic agent according to the above (29), which is a prophylactic / therapeutic agent for Alzheimer's disease;
(32) The diagnostic agent according to (30) above, which is a diagnostic agent for Alzheimer's disease.
(33) a method for screening a cholinergic neuronal differentiation promoting agent, comprising using the protein according to (1) or the partial peptide according to (5) or a salt thereof;
(34) a kit for screening a cholinergic neuronal differentiation promoting agent, comprising the protein according to (1) or the partial peptide according to (5) or a salt thereof;
(35) a method for screening a cholinergic neuronal differentiation promoting agent, comprising using the polynucleotide according to (6);
(36) a kit for screening a cholinergic neuronal differentiation promoting agent, which comprises the polynucleotide according to (6);
(37) a cholinergic neuronal differentiation promoting agent obtainable by using the screening method according to (33) or (35) or the screening kit according to (34) or (36);
(38) A cholinergic neuronal differentiation promoter comprising a compound or a salt thereof that inhibits the activity of the protein of (1) or the partial peptide of (5) or a salt thereof,
(39) A cholinergic neuronal differentiation promoting agent comprising a compound or a salt thereof that inhibits the expression of the protein gene according to (1),
(40) a cholinergic neuronal differentiation promoter comprising the antibody according to (15), (41) a cholinergic neuronal differentiation promoter comprising the antisense polynucleotide according to (18),
(41a) The cholinergic neuronal differentiation promoter according to the above (38) to (41), which is a prophylactic / therapeutic agent for Alzheimer's disease,
(42) A method for preventing or treating a neurodegenerative disease, which comprises administering to a mammal an effective amount of the compound according to (23) or (27) or a salt thereof,
(43) Use of the compound or the salt thereof according to (23) or (27) for producing a prophylactic / therapeutic agent for a neurodegenerative disease,
(44) a multimer of the protein or a salt thereof according to (1),
(45) the multimer according to the above (44), which is a heterodimer;
(46) The multimer as described in (45) above, which is a heterodimer with a basic helix-loop-helix family protein.
[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: 5 or SEQ ID NO: 26 (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, hepatocytes Or stromal cells, or precursor cells of these cells, stem cells or cancer cells) or any tissue where these 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, colon, Small intestine), blood vessels, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testis, ovary, placenta, uterus, bone, joint, skeletal muscle, etc. Is also good.
[0007]
As the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1, about 50% or more, preferably about 60% or more, and more preferably about 70% Above, more preferably about 80% or more, particularly preferably about 90% or more, most preferably about 95% or more amino acid sequence having homology.
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 proteins having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 1. The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 5 is at least 75%, preferably at least about 80%, more preferably at least about 90%, the amino acid sequence represented by SEQ ID NO: 5. And most preferably an amino acid sequence having about 95% or more homology.
Examples of the protein having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 5 include, for example, a protein having the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 5 described above. And a protein having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 5. Examples of the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 26 include an amino acid sequence having 98% or more, preferably about 99% or more homology with the amino acid sequence represented by SEQ ID NO: 26. Is mentioned.
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 containing the amino acid sequence represented by SEQ ID NO: 26.
[0008]
Examples of substantially equivalent activities include, for example, acting as a transcription factor (transcription regulating activity), cholinergic nerve differentiation inhibitory action, and the like. Substantially homogenous indicates that the properties are homogenous (eg, physiologically or pharmacologically). Therefore, the transcription regulatory activity or cholinergic neuronal differentiation inhibitory activity may be equivalent (eg, about 0.01 to 100 times, preferably about 0.1 to 10 times, more preferably 0.5 to 2 times). Although preferred, quantitative factors such as the degree of these activities and the molecular weight of the protein may be different.
The measurement of the transcription regulation activity is carried out by a method known per se, for example, a CAT assay (Mol. Cell. Biol., 2, 1044-11051, 1982), a luciferase assay (Mol. Cell Biol. 7, 725-75). 737, 1987), a co-transfection method (EMBO J., vol. 7, pp. 3771-3778, 1988) or a method similar thereto. As a specific example, an expression vector incorporating a chimeric gene in which a sequence of a cis-element region capable of binding to a transcription factor of interest is bound upstream of a reporter gene, and an expression vector incorporating the transcription factor gene are incorporated into cells. Then, the expression level of the reporter gene is measured by a method known per se.
The binding sequence of the transcription factor is identified by a method known per se, for example, a target detection assay (Andrew, H., J. Biol. Chem., 276, 18282-18289, 2001), a gel shift method (Tokyo) The University of Medical Science, Cancer Research Division, New Cell Engineering Experimental Method, p. 375), Dnase I Footprint Method (The University of Tokyo Institute of Medical Science, Cancer Research Division, New Cell Engineering Experimental Method, p. 385), Southwestern The method is described by the method described in Japanese Patent Application Laid-open No. H10 (963), the New Cell Engineering Experimental Method, edited by the Cancer Research Division of the Institute of Medical Science, The University of Tokyo, p. 396, or a method analogous thereto. As a specific example, in the target detection assay, an oligomer is prepared in which both sides of a random 12 base sequence are sandwiched between base sequences consisting of 22 specific sequences, and this is used as a template to primer the specific sequence. And a double strand is formed. In addition, a transcription factor gene is expressed as a recombinant protein in cells derived from Escherichia coli, animal cells or human cancer cells. A tag such as His may be added to the recombinant protein. After reacting the double-stranded oligomer with the protein expressed in the recombinant protein, the double-stranded oligomer is filtered through a nitrocellulose filter, and the base sequence of DNA binding to the target protein is separated and identified. In the gel shift method, etc., ³² A method in which a DNA labeled with P and a target protein (transcription factor) are bound and then separated by gel electrophoresis or the like to identify a base sequence of the DNA bound to the target protein, or the like.
The cholinergic neuronal differentiation inhibitory action is measured by, for example, RT-PCR, in situ hybridization, immunohistochemistry, immunocytochemistry, acetylcholine content measurement, and acetylcholine synthase activity measurement. Can be measured. For example, in the RT-PCR method, the copy number of the choline acetyltransferase gene is quantified by RT-PCR using a primer capable of specifically amplifying and detecting a cholinergic neuronal marker such as a choline acetyltransferase gene (eg, described later). Example 7).
In the in situ hybridization method, an oligonucleotide probe or riboprobe of a cholinergic neuronal marker such as the choline acetyltransferase gene is prepared, and a known method is used, for example, an introductory brain / neurochemistry introductory lecture / the first volume immunohistochemistry. And the immunocytochemistry method, pages 130-133, 2002 (Yodosha).
In the immunohistochemistry and immunocytochemistry methods, antibodies using cholinergic neuronal markers such as the choline acetyltransferase gene are used, and known methods, for example, introductory lectures on brain / neurochemistry, upper volume immunohistochemistry, immunocytochemistry Method 134-136, 2002 (Yodosha) and the like.
The acetylcholine content can be measured by extracting acetylcholine in a tissue or a cell by a known method, for example, the method described in Biochem Pharmacol Vol. 57, pp. 1321-1329, 1999, and quantifying it by HPLC.
In the method for measuring the activity of acetylcholine synthase, the activity of choline acetyltransferase can be measured using a tritium-labeled acetyl-CoA and choline, and the amount of acetylcholine synthesized can be determined by a known method, for example, Brain Res. 395, 47-56, 1986.
[0009]
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 100, preferably about 1 to 30, An amino acid sequence in which about 1 to 10 amino acids have been deleted, more preferably about 1 to 5 amino acids, and (ii) one or more amino acids (for example, 1 to 10) in the amino acid sequence represented by SEQ ID NO: 1. An amino acid sequence to which about 100, preferably about 1 to 30, preferably about 1 to 10, and more preferably several (1 to 5) amino acids have been added; (iii) represented by SEQ ID NO: 1 One or two or more amino acids (for example, about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, and more preferably a number (1 to 5)) Amino acid sequence, ( v) 1 or 2 or more (for example, about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, more preferably about 1 to 10 in the amino acid sequence represented by SEQ ID NO: 1; So-called muteins such as (5) an amino acid sequence in which one amino acid is substituted with another amino acid, or (v) a protein containing an amino acid sequence obtained by combining them; (2) (i) represented by SEQ ID NO: 5 One or two or more amino acids in the amino acid sequence (for example, about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, and more preferably several (1 to 5) amino acids) The amino acid sequence, (ii) one or more amino acid sequences represented by SEQ ID NO: 5 (for example, about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, more preferably Or (iii) one or more amino acids (for example, about 1 to 100, preferably 1 to 30) in the amino acid sequence represented by SEQ ID NO: 5. Amino acid sequence into which about, preferably about 1 to 10, and more preferably number (1 to 5) amino acids have been inserted; (iv) one or two amino acids in the amino acid sequence represented by SEQ ID NO: 5 An amino acid sequence in which the above (for example, about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, and more preferably number (1 to 5) amino acids have been substituted with other amino acids; or (V) so-called muteins such as proteins containing an amino acid sequence obtained by combining them; (3) (i) one or more (for example, 1 to 4) amino acids in the amino acid sequence represented by SEQ ID NO: 26; (Ii) one or more amino acids (for example, about 1 to 100, preferably about 1 to 30, and preferably about 1 to 10) And more preferably an amino acid sequence to which a number (1 to 5) of amino acids have been added, and (iii) one or more amino acids (for example, about 1 to 100, preferably 1) to the amino acid sequence represented by SEQ ID NO: 26. About 30 amino acids, preferably about 1 to 10 amino acids, more preferably about 1 to 10 amino acids, and (iv) one or more amino acids in the amino acid sequence represented by SEQ ID NO: 26. An amino acid sequence in which two or more (eg, about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, and more preferably number (1 to 5) amino acids) have been substituted with other amino acids , Others include so-called muteins such as proteins containing the amino acid sequence comprising a combination thereof (v).
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.
[0010]
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, SEQ ID NO: 5 or SEQ ID NO: 26, 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 mouse-derived protein containing the amino acid sequence represented by SEQ ID NO: 1, a human-derived protein containing the amino acid sequence represented by SEQ ID NO: 5, and a SEQ ID NO: : 26, and a rat-derived protein containing the amino acid sequence represented by SEQ ID NO: 26.
[0011]
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.
Specifically, for the purpose of preparing the antibody of the present invention described below, a peptide having the amino acid sequence at positions 8 to 67 and 68 to 240 in the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: A peptide having the amino acid sequence at positions 8 to 67 and 68 to 241 in the amino acid sequence represented by No. 5, and the amino acid at positions 8 to 67 and 68 to 241 in the amino acid sequence represented by SEQ ID NO: 26 Examples include a peptide having a sequence. For example, peptides having at least 20 or more, preferably 50 or more, more preferably 70 or more, more preferably 100 or more, and most preferably 200 or more amino acid sequences among the constituent amino acid sequences of the protein of the present invention. Is used.
In the partial peptide used in the present invention, one or more (preferably about 1 to 10, more preferably a number (1 to 5)) amino acids in the amino acid sequence are deleted, or 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 added to the amino acid sequence, or the amino acid One or more (preferably about 1 to 20, more preferably about 1 to 10, and still more preferably, about 1 to 5) amino acids are inserted into the sequence, or One or more (preferably about 1 to 10, more preferably several, and still more preferably about 1 to 5) amino acids may be substituted with another amino acid.
[0012]
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.
The proteins of the present invention may form multimers (eg, dimers, etc.). Examples of the multimer include basic helix-loop-helix family proteins (eg, MyoD, dHAND, eHAND, HES1, HES5, Mash1, Math3, Neurogenin1, Neurogenin2, Hesr-1, E12, E47, etc.). Heteromultimers, preferably heterodimers and the like.
[0013]
As salts of the protein or partial peptide of the present invention, salts with physiologically acceptable acids (eg, inorganic acids, organic acids) and bases (eg, alkali metals) are used, and especially physiologically acceptable salts. Acid addition salts are preferred. 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 or a partial peptide thereof or a salt thereof can be produced from the above-described method of purifying a protein from human or warm-blooded animal cells or tissues, or can be transformed using a DNA encoding the protein. It can also be produced by culturing the body. 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.
[0014]
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.
[0015]
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.
[0016]
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.
[0017]
Examples of the activated carboxyl group of the raw material include, for example, a corresponding acid anhydride, azide, active ester [alcohol (eg, pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, Cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, and an ester with HOBt). As the activated amino group of the raw material, for example, a corresponding phosphoric amide is used.
As a method for removing (eliminating) the protecting group, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, or hydrogen fluoride anhydride, methanesulfonic acid, trifluoromethane, etc. Acid treatment with dichloromethane, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., reduction with sodium in liquid ammonia, and the like are also used. The elimination reaction by the above acid treatment is generally performed at a temperature of about -20 ° C to 40 ° C. In the acid treatment, for example, anisole, phenol, thioanisole, metacresol, paracresol, dimethylsulfide, 1,4 -Addition of a cation scavenger such as butanedithiol, 1,2-ethanedithiol and the like is effective. Further, the 2,4-dinitrophenyl group used as an imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as an indole protecting group of tryptophan is the above 1,2-ethanedithiol, 1,4-butane. In addition to deprotection by acid treatment in the presence of dithiol or the like, it is also removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia or the like.
[0018]
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.
[0019]
The partial peptide or a salt thereof used in the present invention can be produced according to a peptide synthesis method known per se 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. Known condensation methods and elimination of protecting groups include, for example, the methods described in the following (i) to (v).
(I) M.I. Bodanszky and M.A. A. Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966) (ii) Schroeder and Luebke, The Peptide (The Peptide, Academic Year, 65 years).
(Iii) Nobuo Izumiya et al., Basics and Experiments of Peptide Synthesis, Maruzen Co., Ltd. (1975)
(Iv) Haruaki Yajima and Shunpei Sakakibara, Laboratory of Biochemistry 1, Protein Chemistry IV, 205, (1977)
(V) Supervisor of Haruaki Yajima, Development of Continuing Drugs, Volume 14, Peptide Synthesis, Hirokawa Shoten
After the reaction, the partial peptide 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.
[0020]
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 carried out directly by Reverse Transcriptase Polymerase Chain Reaction (hereinafter abbreviated as RT-PCR method) using a total RNA or mRNA fraction prepared from the above-mentioned cells / tissues.
The DNA encoding the protein of the present invention includes, for example, (1) a DNA containing the nucleotide sequence represented by SEQ ID NO: 2 or a nucleotide sequence represented by SEQ ID NO: 2 under high stringent conditions. DNA encoding a protein containing a hybridizing base sequence and having substantially the same properties as the protein having the amino acid sequence represented by SEQ ID NO: 1 described above; (2) a DNA represented by SEQ ID NO: 6 DNA containing the base sequence represented by SEQ ID NO: 6 or a base sequence hybridizable under high stringent conditions to the base sequence represented by SEQ ID NO: 6, and containing the amino acid sequence represented by SEQ ID NO: 5 described above. DNA encoding a protein having substantially the same properties as the protein to be synthesized, (3) DNA containing the base sequence represented by SEQ ID NO: 17, Or a nucleotide sequence that hybridizes with the nucleotide sequence represented by SEQ ID NO: 17 under high stringency conditions, and is substantially the same as the protein containing the amino acid sequence represented by SEQ ID NO: 26 described above. Any DNA may be used as long as it encodes a protein having the above properties.
[0021]
Examples of the DNA that can hybridize with the base sequence represented by SEQ ID NO: 2 under high stringency conditions include, for example, about 50% or more, preferably about 60% or more with the base sequence represented by SEQ ID NO: 2. More preferably, a DNA containing a base sequence having a homology of about 70% or more, more preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more is used.
Examples of the DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 6 under high stringency conditions include, for example, 75% or more, preferably about 80% or more, and more preferably the nucleotide sequence represented by SEQ ID NO: 6 Preferably, a DNA containing a base sequence having a homology of about 90% or more, most preferably about 95% or more is used. Examples of the DNA containing the base sequence represented by SEQ ID NO: 6 include a DNA having the base sequence represented by SEQ ID NO: 7, a DNA having the base sequence represented by SEQ ID NO: 16, and the like. .
Examples of the DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 17 under high stringency conditions include, for example, 98% or more, preferably about 99% or more homology with the nucleotide sequence represented by SEQ ID NO: 17 For example, DNA containing a base sequence having a property is used. Examples of the DNA containing the nucleotide sequence represented by SEQ ID NO: 17 include DNA having the nucleotide sequence represented by SEQ ID NO: 25.
Hybridization can be performed according to a method known per se or a method analogous thereto, for example, the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. More preferably, it can be performed under high stringent conditions.
The high stringent conditions are, for example, conditions in which the sodium concentration is about 19 to 40 mM, preferably about 19 to 20 mM, and the temperature is about 50 to 70 ° C, preferably about 60 to 65 ° C. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C. is most preferable.
More specifically, as the DNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 1, a DNA having the base sequence represented by SEQ ID NO: 2 or the like is represented by SEQ ID NO: 5. Examples of the DNA encoding the protein having the amino acid sequence include a DNA having a base sequence represented by SEQ ID NO: 6, a DNA having a base sequence represented by SEQ ID NO: 7, and a base sequence represented by SEQ ID NO: 16. The DNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 26 includes the DNA having the nucleotide sequence represented by SEQ ID NO: 17 and the DNA having the nucleotide sequence represented by SEQ ID NO: 25. Or the like having DNA.
[0022]
The DNA encoding the partial peptide used in the present invention may be any DNA 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 a DNA having a part of the DNA containing the base sequence represented by SEQ ID NO: 2, SEQ ID NO: 6, or SEQ ID NO: 17, or SEQ ID NO: 2. A protein containing a nucleotide sequence that hybridizes under high stringent conditions with the nucleotide sequence represented by SEQ ID NO: 6 or SEQ ID NO: 17, and encodes a protein having substantially the same activity as the protein of the present invention. DNA containing a part of the DNA to be used is used.
DNAs capable of hybridizing to the nucleotide sequence represented by SEQ ID NO: 2, SEQ ID NO: 6, or SEQ ID NO: 17 have the same significance as described above.
The same hybridization method and high stringency conditions as described above are used.
[0023]
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, 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 method known per se such as the ODA-LA PCR method, the Gapped Duplex method, the Kunkel method, or a method similar 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.
[0024]
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, adenovirus, 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 Escherichia, trp promoter, lac promoter, recA promoter, λP _L Promoter, lpp promoter, T7 promoter, etc., when the host is Bacillus, SPO1 promoter, SPO2 promoter, penP promoter, etc., and when the host is yeast, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, etc. Is preferred. When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable.
[0025]
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. can be used, 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.
[0026]
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 [Journalo] 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.
[0027]
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), MG1 cell derived from the midgut of Trichoplusia ni, and Hig derived from Trichoplusia ni egg. ^TM Cells, cells derived from Mamestra brassicae, cells derived from Estigmena acrea, and the like are used. When the virus is BmNPV, a silkworm-derived cell line (Bombyx mori N cell; BmN cell) or the like is used. As the Sf cells, for example, Sf9 cells (ATCC CRL 1711), Sf21 cells (Vaughn, JL, et al., In Vivo, 13, 213-217, (1977)) and the like are used. .
As insects, for example, silkworm larvae are used [Maeda et al., Nature, 315, 592 (1985)].
Examples of animal cells include monkey cells COS-7, Vero, Chinese hamster cells CHO (hereinafter abbreviated as CHO cells), dhfr gene-deficient Chinese hamster cells CHO (hereinafter CHO (dhfr). ⁻ ) Cells), mouse L cells, mouse AtT-20, mouse myeloma cells, mouse ATDC5 cells, rat GH3, human FL cells, and the like. Human cancer cell-derived cell lines (DLD-1, HCT-15, SW-480, LoVo, HCT-116, WiDr, HT-29, LS-174T, SNU-C1, SNU- C4 cells, SNU-C2A cells, CX-1 cells, GI-112 cells, HL-60 cells, Raji cells, G361 cells, S3 cells) and the like are also used.
[0028]
For transformation of Escherichia, for example, Proc. Natl. Acad. Sci. USA, 69, 2110 (1972), and Gene, 17, 107 (1982).
Bacillus spp. Can be transformed, for example, according to the method described in Molecular & General Genetics, vol. 168, 111 (1979).
To transform yeast, 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.
[0029]
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 19] 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 whose host is yeast, for example, a Burkholder minimum medium [Proc. Natl. Acad. Sci. USA, vol. 77, 4505 (1980)] or an SD medium containing 0.5% casamino acid [Proc. Natl. Acad. Sci. USA, 81, 5330 (1984)]. Preferably, the pH of the medium is adjusted to about 5-8. The cultivation is usually performed at about 20 ° C. to 35 ° C. for about 24 to 72 hours, and if necessary, aeration and stirring are added.
When culturing a transformant 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)], and 199 medium [Proceeding of the Society for Biotechnology, etc., 73, etc. Is used. Preferably, the pH is between about 6 and 8. The cultivation is usually performed at about 30 ° C. to 40 ° C. for about 15 to 60 hours, and if necessary, aeration and stirring are added.
As described above, the protein of the present invention can be produced in cells, cell membranes or extracellular cells of the transformant.
[0030]
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 is completed, the supernatant is separated from the cells or cells by a method known per se, and the supernatant is collected.
The protein contained in the culture supernatant or extract obtained in this manner 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.
[0031]
When the protein thus obtained is obtained in a free form, it can be converted to a salt by a method known per se or a method analogous thereto, and conversely, when it is obtained as a salt, a method known per se or analogous thereto Can be converted to a free form or another salt.
The protein produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by the action of 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.
[0032]
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) is a known antibody using the protein of the present invention as an antigen. Alternatively, it can be produced according to a method for producing an 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 Koehler and Milstein [Nature, 256, 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used.
[0033]
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.
Selection of the monoclonal antibody can be performed according to a method known per se 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.
[0034]
(B) Purification of monoclonal antibody
The monoclonal antibody can be separated and purified by a method known per se, for example, an immunoglobulin separation and purification method [eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, DEAE)] Adsorption-desorption method, ultracentrifugation method, gel filtration method, antigen-binding solid phase or specific purification method of collecting antibody only with an active adsorbent such as protein A or protein G and dissociating the bond to obtain the antibody] Can be.
(Preparation of polyclonal antibody)
The polyclonal antibody of the present invention can be produced according to a method known per se or a method analogous thereto. For example, a immunizing antigen (protein antigen) itself or a complex thereof with a carrier protein is formed, and a warm-blooded animal is immunized in the same manner as in the above-described method for producing a monoclonal antibody. It can be produced by collecting a substance and separating and purifying the antibody.
Regarding a complex of an immunizing antigen and a carrier protein used to immunize a warm-blooded animal, the type of 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. As long as it can be efficiently carried out, any material may be crosslinked at any ratio.For example, bovine serum albumin, bovine thyroglobulin, hemocyanin and the like are preferably used in a weight ratio of about 0.1 to 20 with respect to hapten 1 and preferably about 0.1 to 20. A method of coupling at a rate of about 1 to 5 is used.
Various coupling agents can be used for coupling the hapten and the carrier, and glutaraldehyde, carbodiimide, a maleimide active ester, an active ester reagent containing a thiol group or a dithioviridyl group, or the like is used.
The condensation product is administered to a warm-blooded animal itself or together with a carrier or diluent at a site where antibody production is possible. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. The administration is usually performed once every about 2 to 6 weeks, about 3 to 10 times in total.
The polyclonal antibody can be collected from the blood, ascites, or the like, preferably from the blood of a warm-blooded animal immunized by the above method.
The measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the antiserum described above. The 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, in the case of an antisense polynucleotide directed to translation inhibition, 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 site of the protein of the present invention (for example, An antisense polynucleotide having a homology of about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more with a complementary strand of a base sequence near a codon, etc. B) In the case of an antisense polynucleotide that directs RNA degradation by RNase H, it is about 70% or more, preferably about 80% or more, more preferably about 90% with the complementary strand of the entire base sequence of the DNA of the present invention including introns. As described above, most preferably, antisense polynucleotides having about 95% or more homology are suitable.
Specifically, a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of the DNA containing the nucleotide sequence represented by SEQ ID NO: 2, SEQ ID NO: 6, or SEQ ID NO: 17, or a part thereof Having a base sequence complementary to the base sequence of DNA containing the base sequence represented by SEQ ID NO: 2, SEQ ID NO: 6, or SEQ ID NO: 17, or a part thereof. Antisense polynucleotide (more preferably, an antisense having a base sequence complementary to the base sequence of DNA containing the base sequence represented by SEQ ID NO: 2, SEQ ID NO: 6 or SEQ ID NO: 17, or a part thereof) Polynucleotide).
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, the phosphate residue (phosphate) of each nucleotide constituting the antisense DNA is replaced with, for example, a chemically modified phosphate residue such as phosphorothioate, methylphosphonate or phosphorodithionate. It may be substituted. In addition, the sugar (deoxyribose) of each nucleotide may be substituted with a chemically modified sugar structure such as 2′-O-methylation, and the base portion (pyrimidine, purine) may also be chemically modified. And any one that hybridizes to the DNA having the base sequence represented by SEQ ID NO: 2 or SEQ ID NO: 6. These antisense polynucleotides can be produced using a known DNA synthesizer or the like.
[0036]
According to the present invention, an antisense polynucleotide (nucleic acid) capable of inhibiting the replication or expression of the protein gene of the present invention is designed based on the cloned or determined nucleotide sequence information of the DNA encoding the protein. And can be synthesized. Such a polynucleotide (nucleic acid) can hybridize with the RNA of the protein gene of the present invention and inhibit the synthesis or function of the RNA, or through the interaction with the protein-related RNA of the present invention. Thus, the expression of the protein gene of the present invention can be regulated and controlled. Polynucleotides that are complementary to a selected sequence of the protein-associated RNA of the present invention, and that can specifically hybridize with the protein-associated RNA of the present invention, can be used in vivo and in vitro to produce the protein gene of the present invention. It is useful for regulating and controlling the expression of, and also useful for treating or diagnosing diseases and the like. The term “corresponding” means having homology or being complementary to a specific sequence of nucleotides, base sequences or nucleic acids including genes. "Corresponding" between a nucleotide, base sequence or nucleic acid and a peptide (protein) usually refers to the amino acid of the peptide (protein) as directed by the nucleotide (nucleic acid) sequence or its complement. . 5 'end hairpin loop of protein gene, 5' end 6-base pair repeat, 5 'end untranslated region, polypeptide translation start codon, protein coding region, ORF translation stop codon, 3' end untranslated region, 3 ' The terminal palindrome region and the 3'-end hairpin loop may be selected as preferred regions of interest, but any region within the protein gene may be selected as a target.
The relationship between the target nucleic acid and a polynucleotide complementary to at least a part of the target region can be said to be "antisense" if the relationship between the target nucleic acid and a polynucleotide capable of hybridizing with the target is. Antisense polynucleotides include polynucleotides containing 2-deoxy-D-ribose, polynucleotides containing D-ribose, other types of polynucleotides that are N-glycosides of purine or pyrimidine bases, Alternatively, other polymers having a non-nucleotide backbone (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special linkages, such as those found in DNA or RNA Base pairing and nucleotides having a configuration that allows base attachment). They can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and also DNA: RNA hybrids, and can be unmodified polynucleotides (or unmodified oligonucleotides), or even known. Such as labeled, capped, methylated, one or more naturally-occurring nucleotides replaced by analogs, intramolecular nucleotides Modified, eg, those having an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.), charged or sulfur-containing bonds (eg, phosphorothioate, phosphorodithioate, etc.) ), Such as proteins (nucleases, nuclease inhibitors, One having a side chain group such as syn, antibody, signal peptide, poly-L-lysine or sugar (for example, monosaccharide), one having an interactive compound (for example, acridine, psoralen, etc.), Those containing chelating compounds (eg, metals, radioactive metals, boron, oxidizing metals, etc.), those containing alkylating agents, those with modified bonds (eg, α-anomeric nucleic acids, etc.) ). Here, "nucleoside", "nucleotide", and "nucleic acid" may include not only those containing purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, e.g., where one or more hydroxyl groups have been replaced by halogens, aliphatic groups, etc., or by functional groups such as ethers, amines, etc. It may have been converted.
The antisense polynucleotide (nucleic acid) of the present invention is RNA, DNA, or a modified nucleic acid (RNA, DNA). Specific examples of the modified nucleic acid include, but are not limited to, sulfur derivatives and thiophosphate derivatives of nucleic acids, and those that are resistant to degradation of polynucleoside amides and oligonucleoside amides. The antisense nucleic acid of the present invention can be preferably designed according to the following policy. That is, to make the antisense nucleic acid more stable in the cell, to make the antisense nucleic acid more cell permeable, to have a greater affinity for the target sense strand, and to be more toxic if it is toxic. Minimize the toxicity of sense nucleic acids.
Thus, many modifications are known in the art, for example, Kawakami et al. , Pharm Tech Japan, Vol. 8, pp. 247, 1992; Vol. 8, pp. 395, 1992; T. Crooke et al. ed. , Antisense Research and Applications, CRC Press, 1993.
The antisense nucleic acid of the present invention may contain altered or modified sugars, bases or bonds, may be provided in a special form such as liposomes or microspheres, may be applied by gene therapy, It could be provided in an added form. Thus, in the form of addition, polycations such as polylysine which acts to neutralize the charge of the phosphate skeleton, lipids which enhance the interaction with the cell membrane or increase the uptake of nucleic acids ( For example, hydrophobic substances such as phospholipid and cholesterol can be mentioned. Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chloroformate, cholic acid, etc.). These can be attached to the 3 'end or 5' end of the nucleic acid, and can be attached via a base, sugar, or intramolecular nucleoside bond. Other groups include cap groups specifically arranged at the 3 ′ end or 5 ′ end of a nucleic acid for preventing degradation by nucleases such as exonuclease and RNase. Such capping groups include, but are not limited to, hydroxyl-protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.
The inhibitory activity of the antisense nucleic acid can be examined using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention, or the in vivo or in vitro translation system of the protein of the present invention. The nucleic acid can be applied to cells by various methods known per se.
[0037]
Hereinafter, the protein or partial peptide of the present invention or a salt thereof (hereinafter sometimes abbreviated as the protein of the present invention), the DNA encoding the protein or partial peptide of the present invention (hereinafter referred to as the DNA of the present invention) ), 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 antisense polynucleotide of the present invention). (May be abbreviated as nucleotide).
[0038]
The protein of the present invention can be used as a disease marker because its expression is frequently found in nerve tissue and hippocampus of Alzheimer's disease patients. That is, it is useful as a marker for early diagnosis of nervous system diseases (preferably Alzheimer's disease), determination of symptom severity, and prediction of disease progression. Furthermore, since the protein of the present invention has a cholinergic nerve differentiation inhibitory action, it inhibits the regeneration of cholinergic nerves.
Thus, a compound or a salt thereof that inhibits the activity of the protein of the present invention, a compound or a salt thereof that inhibits the expression of the protein gene of the present invention, the antisense polynucleotide of the present invention, and the antibody of the present invention include, for example, choline Examples of the agonist for promoting neuronal differentiation include nervous system diseases (eg, neurodegenerative diseases (eg, Alzheimer's disease, Parkinson's syndrome, etc.), spinal cord injury, epilepsy, cerebral ischemic disorder, cerebrovascular dementia, etc.), psychiatric diseases ( E.g., schizophrenia, depression, etc.), renal disease (e.g., renal failure, nephritis, uremic disease, etc.), genital disease (e.g., benign prostatic hyperplasia, prostate cancer, testicular neurosis, etc.), bone / cartilage disease (e.g., Eg, osteoporosis, osteoarthritis, rheumatoid arthritis), muscular disease (eg, muscular dystrophy), heart disease (eg, cardiomyopathy, myocardial infarction, heart failure, angina), Autoimmune diseases (eg, systemic lupus erythematosus, immune-associated glomerulonephritis, etc.), cancer (eg, kidney cancer, lung cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, uterus Cervical cancer, colon cancer, rectal cancer, pancreatic cancer, etc.), and hypertension. Preferably, it is an agent for preventing or treating Alzheimer's disease.
[0039]
(1) Agent for preventing or treating various diseases related to the protein of the present invention
The protein of the present invention functions as a transcription factor, is frequently expressed in nervous tissue and the hippocampus of Alzheimer's disease patients, and has a cholinergic neuronal differentiation inhibitory action. 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, a nervous system disease (eg, a neurodegenerative disease (eg, E.g., Alzheimer's disease, Parkinson's syndrome), spinal cord injury, epilepsy, cerebral ischemic injury, cerebrovascular dementia, etc.), psychiatric disorders (e.g., schizophrenia, depression, etc.), renal diseases (e.g., renal failure, Nephritis, uremic disease, etc., genital disease (eg, prostatic hypertrophy, prostate carcinitis, testicular neurosis, etc.), bone / cartilage disease (eg, osteoporosis, osteoarthritis, rheumatoid arthritis, etc.), muscle Diseases (eg, muscular dystrophy, etc.), heart diseases (eg, cardiomyopathy, myocardial infarction, heart failure, angina pectoris, etc.), autoimmune diseases (eg, systemic lupus erythematosus, immune-associated glomerulonephritis, etc.), cancer (eg, , Kidney cancer Lung cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, stomach cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, pancreatic cancer, etc.), such as hypertension develops.
Therefore, the protein of the present invention and the DNA of the present invention can be used, for example, for nervous system diseases (eg, neurodegenerative diseases (eg, Alzheimer's disease, Parkinson's syndrome, etc.), spinal cord injury, epilepsy, cerebral ischemic disorder, cerebral vascular dementia, etc.). ], Psychiatric disorders (eg, schizophrenia, depression, etc.), renal disorders (eg, renal failure, nephritis, uremics, etc.), genital disorders (eg, prostatic hyperplasia, prostate carcinitis, testicular neurosis, etc.) , Bone and cartilage diseases (eg, osteoporosis, osteoarthritis, rheumatoid arthritis, etc.), muscular diseases (eg, muscular dystrophy, etc.), heart diseases (eg, cardiomyopathy, myocardial infarction, heart failure, angina pectoris) ), Autoimmune diseases (eg, systemic lupus erythematosus, immune-associated glomerulonephritis, etc.), cancer (eg, kidney cancer, lung 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, etc.), can be used as medicaments, such as prophylactic or therapeutic agent such as hypertension. Preferably, it is a preventive or therapeutic agent for a neurodegenerative disease, more preferably Alzheimer's disease.
For example, when there is a patient whose transcription regulation is not sufficiently or normally exerted because the protein of the present invention is reduced or deleted in a living body, (a) administering the DNA of the present invention to the patient, (B) by inserting the DNA of the present invention into cells, expressing the protein of the present invention, and then transplanting the cells into a patient by expressing the protein of the present invention in vivo, or (c) By administering the protein of the present invention to the patient, the role of the protein of the present invention in the patient can be sufficiently or normally exerted.
When the DNA of the present invention is used as the above-mentioned prophylactic / therapeutic agent, the DNA of the present invention is used alone or after being inserted into an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, etc. Can be administered to humans or other warm-blooded animals according to 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.
[0040]
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.
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 of the present invention is orally administered for the treatment of Alzheimer's disease, it is generally adult (60 kg body weight). )), About 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg of the protein is administered per day. When administered parenterally, the single dose of the protein or the like varies depending on the subject to be administered, the target disease, and the like. For example, the protein of the present invention is injected into an adult (body weight) for the purpose of treating Alzheimer's disease. (As 60 kg), it can be 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 convenient. 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 functions as a transcription factor, is frequently expressed in nerve tissue and in the hippocampus of Alzheimer's disease patients, further has a cholinergic nerve differentiation inhibitory action, and inhibits cholinergic nerve regeneration.
Accordingly, compounds or salts thereof that regulate (preferably inhibit) the activity of the protein of the present invention can be used, for example, as cholinergic neuronal differentiation promoters, for example, nervous system diseases [eg, neurodegenerative diseases (eg, Alzheimer's disease, Parkinson's disease, etc.). Syndrome, etc.), spinal cord injury, epilepsy, cerebral ischemic disorder, cerebrovascular dementia, etc.), psychiatric disorders (eg, schizophrenia, depression, etc.), renal diseases (eg, renal failure, nephritis, uremics, etc.) , Reproductive disorders (eg, prostatic hyperplasia, prostate carcinitis, testicular neurosis, etc.), bone and cartilage diseases (eg, osteoporosis, osteoarthritis, rheumatoid arthritis, etc.), muscular disorders (eg, muscular dystrophy) ), Heart disease (eg, cardiomyopathy, myocardial infarction, heart failure, angina), autoimmune disease (eg, systemic lupus erythematosus, immune-associated glomerulonephritis, etc.), cancer (eg, kidney cancer, lung cancer, Liver cancer, non Cell lung cancer, ovarian cancer, prostate cancer, stomach cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, pancreatic cancer, etc.), can be used as a prophylactic or therapeutic agent for hypertension. Preferably, it is a preventive or therapeutic agent for a neurodegenerative disease, more preferably Alzheimer's disease.
Therefore, the protein of the present invention is useful as a reagent for screening a compound or a salt thereof that regulates the activity of the protein of the present invention.
That is, the present invention provides a method for screening for a compound or a salt thereof that regulates the activity of the protein of the present invention (eg, transcriptional regulatory activity, cholinergic neuronal differentiation inhibitory activity, etc.), characterized by using the protein of the present invention. provide.
Specifically, for example, (i) transcriptional regulation activity of a cell capable of producing the protein of the present invention, and (ii) transcriptional regulation of a cell capable of producing the protein of the present invention in the presence of a test compound. A method for screening a compound or a salt thereof that regulates (promotes or inhibits) the activity of the protein of the present invention, which is characterized by comparing the activities, is used.
In the above screening method, for example, in the cases of (i) and (ii), the transcription regulation activity is determined by a method known per se, for example, a CAT assay (Mol. Cel. Biol., 2, 1044-11051, 1982). ), Luciferase assay method (Mol. Cell Biol. 7, 725-737, 1987), co-transfection method (EMBO J., 7, 3777-1778, 1988) and the like, or in order therefor. Measure and compare by shearing method.
Specifically, (i) an expression vector incorporating a chimeric gene in which a sequence containing a cis-element region to which the protein (transcription factor) of the present invention can bind upstream of a reporter gene; And (ii) expression incorporating a chimeric gene in which a sequence containing a cis-element region capable of binding to the protein (transcription factor) of the present invention is linked upstream of a reporter gene. The vector and an expression vector incorporating the transcription factor gene are incorporated into cells, and the expression level of the reporter when cultured in the presence of a test compound is measured and compared. The expression level of the reporter is measured by a method known per se, and a compound or a salt thereof that regulates (promotes or inhibits) the activity of the protein of the present invention is screened.
The binding sequence (cis-element) of the protein (transcription factor) of the present invention can be identified by using a target detection assay, a gel shift method, or a method analogous thereto. For example, in the target detection assay method, an oligomer is prepared in which both sides of a random 12 base sequence are sandwiched between base sequences consisting of 22 specific sequences, and this is used as a template to perform PCR using the specific sequence as a primer. To form a double strand. The protein (transcription factor) of the present invention is expressed as a recombinant protein in Escherichia coli, animal cells, or cells derived from human cancer cells. A tag such as His may be added to the recombinant protein. After reacting the double-stranded oligomer with the protein expressed in the recombinant protein, the double-stranded oligomer is filtered through a nitrocellulose filter, and the nucleotide sequence of the DNA binding to the target protein is separated and identified. In the gel shift method, ³² After binding the DNA labeled with P to the target protein (transcription factor), the DNA is separated by gel electrophoresis or the like to identify the base sequence of the DNA that binds to the target protein.
Specifically, for example, (i ′) the cholinergic neuronal differentiation inhibitory action of cells having the ability to produce the protein of the present invention, and (ii ′) the ability to produce the protein of the present invention in the presence of a test compound A method for screening for a compound or a salt thereof that regulates (promotes or inhibits) the activity of the protein of the present invention, which is characterized by comparing the inhibitory action of cells having the above on cholinergic neuronal differentiation.
In the above screening method, for example, when cells are cultured in (i ′) and (ii ′), respectively, the action of inhibiting cholinergic neuronal differentiation can be determined by, for example, RT-PCR, in situ hybridization, immunohistochemistry, and the like. It is measured using an immunocytochemical method, a method for measuring acetylcholine content, and a method for measuring acetylcholine synthase activity.
For example, in the RT-PCR method, the copy number of the choline acetyltransferase gene is quantified by RT-PCR using a primer capable of specifically amplifying and detecting a cholinergic neuronal marker such as a choline acetyltransferase gene (eg, described later). Example 7).
In the in situ hybridization method, an oligonucleotide probe or riboprobe of a cholinergic neuronal marker such as the choline acetyltransferase gene is prepared, and a known method is used, for example, an introductory brain / neurochemistry introductory lecture / the first volume immunohistochemistry. And the immunocytochemistry method, pages 130-133, 2002 (Yodosha).
In the immunohistochemistry and immunocytochemistry methods, antibodies using cholinergic neuronal markers such as the choline acetyltransferase gene are used, and known methods, for example, introductory lectures on brain / neurochemistry, upper volume immunohistochemistry, immunocytochemistry Method 134-136, 2002 (Yodosha) and the like.
The acetylcholine content can be measured by extracting acetylcholine in a tissue or a cell by a known method, for example, the method described in Biochem Pharmacol Vol. 57, pp. 1321-1329, 1999, and quantifying it by HPLC.
In the method for measuring the activity of acetylcholine synthase, the activity of choline acetyltransferase can be measured using a tritium-labeled acetyl-CoA and choline, and the amount of acetylcholine synthesized can be determined by a known method, for example, Brain Res. 395, 47-56, 1986.
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 COS7 cells, CHO cells, and HEK293 cells are preferably used. For the screening, for example, a transformant in which the protein of the present invention is expressed in cells by culturing by the above-described method is preferably used. The method for culturing cells capable of expressing the protein of the present invention is the same as the above-described method for culturing the transformant of the present invention.
Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like.
For example, a test compound which increases the transcription control activity in the case (ii) by about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the case in the above (i), by using the present invention As a compound that promotes the activity of a protein, the transcription control activity in the case of (ii) is reduced by about 20% or more, preferably 30% or more, more preferably about 50% or more, as compared with the case of (i). Test compounds can be selected as compounds that inhibit the activity of the protein of the invention.
For example, a test in which the action of inhibiting cholinergic neuronal differentiation in the case of (ii ') is increased by about 20% or more, preferably 30% or more, more preferably about 50% or more, as compared with the case of (i'). When the compound is a compound that promotes the activity of the protein of the present invention, the inhibitory effect on cholinergic neuronal differentiation in the case (ii ′) is about 20% or more, preferably 30%, as compared with the case (i). As described above, a test compound that reduces the activity of the protein of the present invention more preferably by about 50% or more can be selected as a compound that inhibits the activity of the protein of the present invention.
Among them, a compound having an activity of inhibiting the activity of the protein of the present invention or a salt thereof is preferable.
[0042]
The compound having the activity of promoting the activity of the protein of the present invention is useful as a safe and low-toxic drug for enhancing the action of the protein of the present invention.
The compound having the activity of inhibiting the activity of the protein of the present invention is useful as a safe and low-toxic drug for suppressing the physiological activity of the protein of the present invention.
Compounds or salts thereof obtained using the screening method or screening kit of the present invention include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts And compounds selected from plasma and the like. As the salt of the compound, those similar to the aforementioned salts of the protein of the present invention are used.
Furthermore, the gene encoding the protein of the present invention is also frequently expressed in nerve tissue and the hippocampus of Alzheimer's disease patients.Therefore, compounds that regulate the expression of the gene encoding the protein of the present invention or salts thereof include, for example, choline. Examples of the agonist for promoting neuronal differentiation include nervous system diseases (eg, neurodegenerative diseases (eg, Alzheimer's disease, Parkinson's syndrome, etc.), spinal cord injury, epilepsy, cerebral ischemic disorder, cerebrovascular dementia, etc.), psychiatric diseases ( E.g., schizophrenia, depression, etc.), renal disease (e.g., renal failure, nephritis, uremic disease, etc.), genital disease (e.g., benign prostatic hyperplasia, prostate cancer, testicular neurosis, etc.), bone / cartilage disease (e.g., Eg, osteoporosis, osteoarthritis, rheumatoid arthritis, etc., muscular disease (eg, muscular dystrophy, etc.), heart disease (eg, cardiomyopathy, myocardial infarction, heart failure, angina) ), Autoimmune diseases (eg, systemic lupus erythematosus, immune-associated glomerulonephritis, etc.), cancer (eg, kidney cancer, lung 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, etc.) and hypertension. Preferably, it is a preventive or therapeutic agent for a neurodegenerative disease, more preferably Alzheimer's disease.
The polynucleotide (eg, DNA) encoding the protein of the present invention is a polynucleotide encoding the protein of the present invention, since the gene encoding the protein of the present invention is frequently expressed in neural tissues and the hippocampus of Alzheimer's disease patients. (Eg, DNA) is useful as a reagent for screening a compound or a salt thereof that regulates the expression of a gene encoding the protein of the present invention.
The screening methods include (iii) culturing cells capable of producing the protein of the present invention, and (iv) culturing cells capable of producing the protein of the present invention in the presence of the test compound. And a screening method characterized by performing a comparison.
In the above method, in the cases (iii) and (iv), the expression level of the gene (specifically, the amount of the protein of the present invention or the amount of the mRNA encoding the protein) is measured and compared.
Examples of the test compound and cells having the ability to produce the protein of the present invention include those described above.
The protein amount is measured by a known method, for example, using an antibody recognizing the protein of the present invention, and measuring the protein present in a cell extract or the like according to a method such as Western analysis or ELISA method or a method analogous thereto. can do.
The amount of mRNA can be measured by a known method, for example, Northern hybridization using a nucleic acid containing SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 26 or a part thereof as a probe, or SEQ ID NO: 2 as a primer. It can be measured according to a PCR method using a nucleic acid containing SEQ ID NO: 5 or SEQ ID NO: 26 or a part thereof, or a method analogous thereto.
For example, a test compound that increases the gene expression level in the case of the above (iv) by about 20% or more, preferably 30% or more, and more preferably about 50% or more as compared with the case of the above (iii), according to the present invention As a compound that promotes the expression of the gene encoding the protein of the present invention, a test compound that inhibits about 20% or more, preferably 30% or more, more preferably about 50% or more suppresses the expression of the gene encoding the protein of the present invention. Can be selected as the compound.
[0043]
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 include the test compounds described above, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, and plant extracts. A compound selected from animal tissue extract, plasma, or the like, or a salt thereof, and a compound or a salt thereof that regulates the activity of the protein of the present invention (eg, transcription regulation activity and the like).
As the salt of the compound, those similar to the aforementioned salts of the protein of the present invention are used.
A compound or a salt thereof that regulates (preferably inhibits) the activity of the protein of the present invention and a compound or a salt thereof that regulates (preferably inhibits) the expression of a gene encoding the protein of the present invention promote cholinergic neuronal differentiation. Examples of the agent include nervous system diseases (eg, neurodegenerative diseases (eg, Alzheimer's disease, Parkinson's syndrome, etc.), spinal cord injury, epilepsy, cerebral ischemic disorder, cerebrovascular dementia, etc.), psychiatric diseases (eg, schizophrenia) Disease, depression, etc.), renal disease (eg, renal failure, nephritis, uremia, etc.), genital disease (eg, prostatic hypertrophy, prostate carcinitis, testicular neurosis, etc.), bone / cartilage disease (eg, osteotomy) Sclerosis, osteoarthritis, rheumatoid arthritis, etc.), muscular disease (eg, muscular dystrophy, etc.), heart disease (eg, cardiomyopathy, myocardial infarction, heart failure, angina, etc.), autoimmune disease (eg, Somatic lupus erythematosus, immune-associated glomerulonephritis, etc., cancer (eg, kidney cancer, lung 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, etc.) and hypertension. Preferably, it is a preventive or therapeutic agent for a neurodegenerative disease, more preferably Alzheimer's disease.
When a 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 agent, it can be formulated according to a conventional method.
For example, compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, and capsules (including soft capsules). Syrup, emulsion, suspension and the like. Such a composition is produced by a method known per se and contains a carrier, diluent or excipient commonly used in the field of formulation. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.
As a composition for parenteral administration, for example, injections, suppositories, etc. are used. Injections are intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, intravenous injections, intraarticular injections. Dosage forms such as agents. Such injections are prepared according to a method known per se, for example, by dissolving, suspending or emulsifying the antibody or a salt thereof in a sterile aqueous or oily liquid commonly used for injections. As an aqueous liquid for injection, for example, physiological saline, isotonic solution containing glucose and other auxiliary agents and the like are used, and suitable solubilizing agents, for example, alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) add-on of hydro- genated castor oil)) and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil, and the like are used, and benzyl benzoate, benzyl alcohol, and the like may be used in combination as a solubilizing agent. The prepared injection is usually filled in an appropriate ampoule. Suppositories to be used for rectal administration are prepared by mixing the above antibody or a salt thereof with a conventional suppository base.
[0044]
The above-mentioned oral or parenteral pharmaceutical composition is conveniently prepared in a unit dosage form so as to be compatible with the dosage of the active ingredient. Examples of such dosage unit dosage forms include tablets, pills, capsules, injections (ampoules), suppositories, and the like. Usually, each dosage unit dosage form has a dosage of 5 to 500 mg, especially 5 to 100 mg for an injection. Other dosage forms preferably contain 10 to 250 mg of the above compound.
Each of the above-mentioned compositions may contain other active ingredients as long as the composition does not cause an undesirable interaction with the compound.
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, subject to be administered, administration route, and the like. When a compound or a salt thereof is orally administered, the compound or a salt thereof is generally used in an adult (with a body weight of 60 kg) per day 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. Is administered at about 1.0-20 mg. When administered parenterally, the single dose of the compound or a salt thereof varies depending on the subject to be administered, the target disease, and the like. For example, the activity of the protein of the present invention is regulated (preferably When a compound or a salt thereof 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 per day. And more preferably about 0.1 to 10 mg is administered to the cancerous lesion by injection. In the case of other animals, the amount can be administered in terms of the weight per 60 kg.
[0045]
(3) Quantification of the protein of the present invention, its partial peptide or its salt
An antibody against the protein of the present invention (hereinafter sometimes abbreviated as the antibody of the present invention) can specifically recognize the protein of the present invention. It can be used for quantification by sandwich immunoassay and the like.
That is, the present invention
(I) reacting the antibody of the present invention with a test solution and a labeled protein of the present invention competitively, and measuring the ratio of the labeled protein of the present invention bound to the antibody; A method for quantifying the protein of the present invention in a test solution, and
(Ii) After reacting a test solution with the antibody of the present invention immobilized on a carrier and another labeled antibody of the present invention simultaneously or continuously, the activity of the labeling agent on the insolubilized carrier is measured. A method for quantifying the protein of the present invention in a test solution is provided.
In the quantitative method (ii), it is desirable that one antibody is an antibody that recognizes the N-terminal of the protein of the present invention and the other antibody is an antibody that reacts with the C-terminal of the protein of the present invention.
[0046]
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.
[0047]
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.
[0048]
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.
[0049]
In applying these individual immunological measurement methods to the quantification method of the present invention, no special conditions, operations, and the like need to be set. The protein measurement system of the present invention may be constructed by adding ordinary technical considerations of those skilled in the art to ordinary conditions and operation methods in each method. For details of these general technical means, reference can be made to reviews, books, and the like.
For example, Hiroe Irie, "Radio Immunoassay" (Kodansha, published in 1974), Hiroshi Irie, "Radio Immunoassay" (Kodansha, published in 1974), Eiji Ishikawa et al., "Enzyme Immunoassay" (Medical Shoin, Showa Ed. Ishikawa et al., “Enzyme Immunoassay” (2nd edition) (Issue Shoin, 1982), Eiji Ishikawa et al. “Enzyme Immunoassay” (Third Edition), 3rd edition (Medical Shoin, Showa) 62)), "Methods in ENZYMOLOGY" Vol. 70 (Immunochemical Techniques (Part A)), ibid., Vol. 73 (Immunochemical Technologies (Part B)), ibid., Vol. 74 (Immunochemical Technologies (Part C)), ibid., Vol. 84 (Immunochemical Technologies (Part D: Selected Immunoassays)), ibid., Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)), ibid., Vol. 121 (Immunochemical Technologies (Part I: Hybridoma Technology and Monoclonal Antibodies)) (all published by Academic Press) can be referred to.
As described above, the protein of the present invention can be quantified with high sensitivity by using the antibody of the present invention.
Furthermore, when an increase or 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 nervous system disease (eg, a neurodegenerative disease (eg, a neurodegenerative disease ( E.g., Alzheimer's disease, Parkinson's syndrome), spinal cord injury, epilepsy, cerebral ischemic injury, cerebrovascular dementia, etc.), psychiatric disorders (e.g., schizophrenia, depression, etc.), renal diseases (e.g., renal failure, Nephritis, uremic disease, etc., genital disease (eg, prostatic hypertrophy, prostate carcinitis, testicular neurosis, etc.), bone / cartilage disease (eg, osteoporosis, osteoarthritis, rheumatoid arthritis, etc.), muscle Diseases (eg, muscular dystrophy, etc.), heart diseases (eg, cardiomyopathy, myocardial infarction, heart failure, angina pectoris, etc.), autoimmune diseases (eg, systemic lupus erythematosus, immune-associated glomerulonephritis, etc.), cancer (eg, , Kidney cancer, lung , Liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, stomach cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, pancreatic cancer, etc.), high blood pressure, etc. or are likely to suffer in the future Can be diagnosed.
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.
[0050]
(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 Science of the). USA), Vol. 86, pp. 2766-2770 (1989)).
For example, when overexpression or decrease is detected by Northern hybridization, or when DNA mutation is detected by PCR-SSCP method, for example, nervous system diseases [eg, neurodegenerative diseases (eg, Alzheimer's disease, Parkinson's disease, etc.) Syndrome, etc.), spinal cord injury, epilepsy, cerebral ischemic disorder, cerebrovascular dementia, etc.), psychiatric disorders (eg, schizophrenia, depression, etc.), renal diseases (eg, renal failure, nephritis, uremics, etc.) , Reproductive disorders (eg, prostatic hyperplasia, prostate carcinitis, testicular neurosis, etc.), bone and cartilage diseases (eg, osteoporosis, osteoarthritis, rheumatoid arthritis, etc.), muscular disorders (eg, muscular dystrophy) ), Heart disease (eg, cardiomyopathy, myocardial infarction, heart failure, angina), autoimmune disease (eg, systemic lupus erythematosus, immune-related glomerulonephritis, etc.), cancer (eg, kidney cancer) Lung 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, etc.) Can be.
[0051]
(5) Drug containing antisense polynucleotide
The antisense polynucleotide of the present invention, which can complementarily bind to the DNA of the present invention and suppress 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, as a cholinergic nerve differentiation promoting agent, for example, nervous system diseases [eg, neurodegenerative diseases (eg, Alzheimer's disease, Parkinson's syndrome) ), Spinal cord injury, epilepsy, cerebral ischemic disorder, cerebral vascular dementia, etc.), psychiatric disorders (eg, schizophrenia, depression, etc.), renal diseases (eg, renal failure, nephritis, uremics, etc.), Genital disorders (eg, benign prostatic hyperplasia, prostate cancer, testicular neurosis, etc.), bone and cartilage disorders (eg, osteoporosis, osteoarthritis, rheumatoid arthritis, etc.), muscular disorders (eg, muscular disorders) Trophy, etc.), heart disease (eg, cardiomyopathy, myocardial infarction, heart failure, angina), autoimmune disease (eg, systemic lupus erythematosus, immune-associated glomerulonephritis, etc.), cancer (eg, kidney cancer, Lung cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, stomach cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, pancreatic cancer, etc.) it can. Preferably, it is a prophylactic / therapeutic agent for a neurodegenerative disease, more preferably Alzheimer's disease.
When the antisense polynucleotide is used as the prophylactic / therapeutic agent, it can be formulated and administered according to a method known per se.
Also, for example, after inserting the antisense polynucleotide alone or into an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, and the like, a human or mammal (eg, rat, Rabbits, sheep, pigs, cows, cats, dogs, monkeys, and the like). 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.
Furthermore, for the purpose of improving pharmacokinetics, prolonging the half-life, and improving the efficiency of cellular uptake, the above antisense polynucleotide is formulated alone or together with a carrier such as liposome (injection), and is used for intravenous, subcutaneous, or joint cavities. In addition, it may be administered to a cancer lesion.
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 administered for the purpose of treating Alzheimer's disease, it is generally used in adults. (60 kg body weight), 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.
[0052]
Similarly to the above-mentioned antisense polynucleotide, a double-stranded RNA containing a part of the RNA encoding the protein of the present invention, a ribozyme containing a part of the RNA encoding the protein of the present invention, and a protein of the present invention are bound. A decoy oligonucleotide for a DNA sequence that inhibits the expression of the gene of the present invention can also suppress the function of the protein of the present invention or the DNA used in the present invention in vivo (eg, transcription regulatory activity, cholinergic neuronal differentiation). For example, as a cholinergic neuronal differentiation promoter, for example, nervous system diseases (eg, neurodegenerative diseases (eg, Alzheimer's disease, Parkinson's syndrome, etc.), spinal cord injury, epilepsy, brain ischemia) Blood disorder, cerebrovascular dementia, etc.), psychiatric disorders (eg, schizophrenia, depression, etc.), kidney disease (Eg, renal failure, nephritis, uremic disease, etc.), genital diseases (eg, prostatic hypertrophy, prostate carcinitis, testicular neurosis, etc.), bone and cartilage diseases (eg, osteoporosis, osteoarthritis, chronic Rheumatoid arthritis), muscular disease (eg, muscular dystrophy), heart disease (eg, cardiomyopathy, myocardial infarction, heart failure, angina), autoimmune disease (eg, systemic lupus erythematosus, immune-associated glomerulonephritis) ), Cancer (eg, kidney cancer, lung 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, etc.), high blood pressure, etc. Can be used as a prophylactic / therapeutic agent. Preferably, it is a preventive or therapeutic agent for a neurodegenerative disease, more preferably Alzheimer's disease.
The double-stranded RNA can be produced by designing based on the sequence of the polynucleotide of the present invention according to a known method (eg, Nature, 411, 494, 2001).
A ribozyme can be designed and produced based on the sequence of the polynucleotide of the present invention according to a known method (eg, TRENDS in Molecular Medicine, Vol. 7, pp. 221, 2001). For example, it can be produced by linking a known ribozyme to a part of RNA encoding the protein of the present invention. As a part of the RNA encoding the protein of the present invention, a portion (RNA fragment) close to a cleavage site on the RNA of the present invention, which can be cleaved by a known ribozyme, can be mentioned.
The decoy oligonucleotide can be produced by designing based on a DNA sequence to which the protein of the present invention binds, according to a known method (eg, The Journal of Clinical Investigation, 106, 1071, 2000). . Specifically, the decoy oligonucleotide has a base sequence that hybridizes under high stringent conditions with the sequence of the DNA to which the protein of the present invention binds, as long as the protein of the present invention can bind thereto. Any one may be used. The base sequence capable of hybridizing with the sequence of the DNA to which the protein of the present invention binds may be, for example, about 70% or more, preferably about 80% or more, more preferably about 90% with the sequence of the DNA to which the protein of the present invention binds. As described above, most preferably, a nucleotide sequence having about 95% or more homology is used.
When the above double-stranded RNA, ribozyme or decoy oligonucleotide is used as the prophylactic / therapeutic agent, it can be formulated and administered in the same manner as the antisense polynucleotide.
[0053]
(6) A medicine 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 (eg, transcriptional regulation activity, cholinergic neuronal differentiation inhibitory action, etc.), is used as a cholinergic neuronal differentiation promoter, for example, as a nervous system disease [ Eg, neurodegenerative diseases (eg, Alzheimer's disease, Parkinson's syndrome, etc.), spinal cord injury, epilepsy, cerebral ischemic disorder, cerebrovascular dementia, etc.), psychiatric diseases (eg, schizophrenia, depression, etc.), kidney disease (Eg, renal failure, nephritis, uremic disease, etc.), genital diseases (eg, prostatic hypertrophy, prostate carcinitis, testicular neurosis, etc.), bone and cartilage diseases (eg, osteoporosis, osteoarthritis, chronic Rheumatoid arthritis), muscular disease (eg, muscular dystrophy), heart disease (eg, cardiomyopathy, myocardial infarction, heart failure, angina), autoimmune disease (eg, systemic lupus erythematosus, immune-associated glomerulonephritis) Such), (Eg, kidney cancer, lung cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, stomach cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, pancreatic cancer, etc.), prevention and treatment of hypertension It can be used as a medicine such as an agent. Preferably, it is a prophylactic / therapeutic agent for a neurodegenerative disease, more preferably Alzheimer's disease. 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, intravenous, intraarticular) to cattle, cats, dogs, monkeys, and the like. The dosage varies depending on the administration subject, target disease, symptoms, administration route, and the like. For example, when used for treatment or prevention of Alzheimer's disease, the antibody of the present invention is usually administered in a dose of 0.1 mg. About 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 3 times a day To the extent it is convenient to administer intravenously. 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 a composition is provided in a dosage form suitable for oral or parenteral administration (eg, intravenous administration, intraarticular 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.
[0054]
(7) DNA transfer animal
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), 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) It is intended to provide a recombinant vector containing the exogenous DNA of the present invention or its mutant DNA and capable of being expressed in mammals.
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 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 transferring a target DNA by a microinjection method, a particle gun method, a DEAE-dextran method, or the like. Further, by the DNA transfer method, the exogenous DNA of the present invention can be transferred to somatic cells, organs of living organisms, tissue cells, and the like, and can be used for cell culture, tissue culture, and the like. The DNA-transferred animal of the present invention can also be produced by fusing the above-mentioned germ cells with a cell fusion method known per se.
As the non-human mammal, for example, cow, pig, sheep, goat, rabbit, dog, cat, guinea pig, hamster, mouse, rat and the like are used. Among them, rodents, which are relatively short in ontogeny and biological cycle in terms of preparation of disease animal model systems, and are easy to breed, especially mice (for example, hybrids such as C57BL / 6 strain and DBA2 strain as pure strains) B6C3F as a system ₁ System, BDF ₁ System, B6D2F ₁ Strain, BALB / c strain, ICR strain, etc.) or rat (eg, Wistar, SD etc.) is preferred.
"Mammals" in a recombinant vector that can be expressed in mammals include humans in addition to the above-mentioned non-human mammals.
[0055]
The exogenous DNA of the present invention refers to the DNA of the present invention once isolated and extracted from a mammal, not the DNA of the present invention originally possessed by a non-human mammal.
As the mutant DNA of the present invention, those having a mutation (for example, mutation) in the base sequence of the original DNA of the present invention, specifically, base addition, deletion, substitution with another base, etc. The resulting DNA or the like is used, and also includes abnormal DNA.
The abnormal DNA means a DNA that expresses an abnormal protein of the present invention, and for example, a DNA that expresses a protein that suppresses the function of the normal protein of the present invention is used.
The exogenous DNA of the present invention may be derived from a mammal that is the same or different from the animal of interest. In transferring the DNA of the present invention to 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 transferred, DNA 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 is 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-transferring mammals can be created.
[0056]
Examples of the expression vector of the protein of the present invention include plasmids derived from Escherichia coli, plasmids derived from Bacillus subtilis, plasmids derived from yeast, bacteriophages such as λ phage, retroviruses such as Moloney leukemia virus, animal viruses such as vaccinia virus or baculovirus. Are used. Among them, a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis or a plasmid derived from yeast is preferably used.
Examples of the promoter for controlling the DNA expression include, but are not limited to, promoters for DNAs derived from (1) viruses (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), peptide chain elongation factor 1α (EF-1α), β actin , Α and β myosin heavy chain, myosin light chain 1 and 2, myelin basic protein, thyroglobulin, Thy-1, immunoglobulin, heavy chain variable region (VNP), serum amyloid P component, myoglobin, troponin C, smooth muscle α Kuching, pre-pro-enkephalin A, such as a promoter, such as vasopressin is used. Among them, a cytomegalovirus promoter capable of high expression throughout the body, a human peptide chain elongation factor 1α (EF-1α) promoter, a human and chicken β-actin promoter, and the like are preferable.
The vector preferably has a sequence that terminates transcription of the target messenger RNA in a DNA-transferred mammal (generally called a terminator). For example, a sequence of each DNA derived from a virus and various mammals can be used. A simian virus SV40 terminator or the like is preferably used.
[0057]
In addition, a splicing signal of each DNA, an enhancer region, a part of an intron of a eukaryotic DNA, and the like are placed 5 ′ upstream of the promoter region, between the promoter region and the translation region, or between the translation region for the purpose of further expressing the desired foreign DNA. It is also possible to connect 3 ′ downstream of the above depending on the purpose.
The normal translation region of the protein of the present invention can be obtained from liver, kidney, thyroid cells, fibroblast-derived DNA derived from humans or various mammals (eg, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.) and commercially available DNAs. From a variety of genomic DNA libraries, or as a starting material, a complementary DNA prepared by known methods from RNA derived from liver, kidney, thyroid cells, and fibroblasts. In addition, a translation region obtained by mutating a translation region of a normal protein obtained from the above-described cells or tissues by a point mutagenesis method can be used for the exogenous abnormal DNA.
The translation region can be prepared as a DNA construct that can be expressed in a transposed animal by a conventional DNA engineering technique in which it is ligated downstream of the above-mentioned promoter and, if desired, upstream of the transcription termination site.
Transfer 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 DNA transfer means that all the progeny of the produced animal retain the exogenous DNA of the present invention in all of the germinal cells and somatic cells. means. The offspring of such animals that have inherited the exogenous DNA of the present invention have the exogenous DNA of the present invention in all of their germ cells and somatic cells.
The non-human mammal to which the exogenous normal DNA of the present invention has been transferred can be subcultured in a normal breeding environment as an animal having the DNA after confirming that the exogenous DNA is stably retained by mating. .
Transfer 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 germ cells of a produced animal after DNA transfer means that all offspring of the produced animal have an excessive amount of the exogenous DNA of the present invention in all of their germ cells and somatic cells. Means Progeny of such animals that have inherited the exogenous DNA of the present invention have an excess of the exogenous DNA of the present invention in all of their germinal and somatic cells.
By obtaining a homozygous animal having the introduced DNA on both homologous chromosomes and mating the male and female animals, all offspring can be bred to have the DNA in excess.
[0058]
The non-human mammal having the normal DNA of the present invention expresses the normal DNA of the present invention at a high level and promotes the function of the endogenous normal DNA to eventually cause hyperactivity of the protein of the present invention. Occasionally, it can be used as a disease model animal. For example, using the normal DNA-transferred animal of the present invention, it is possible to elucidate the pathological mechanism of 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 transferred has an increased symptom of the free protein of the present invention, a preventive / therapeutic agent for a disease related to the protein of the present invention, for example, a nervous system disease [ Eg, neurodegenerative diseases (eg, Alzheimer's disease, Parkinson's syndrome, etc.), spinal cord injury, epilepsy, cerebral ischemic disorder, cerebrovascular dementia, etc.), psychiatric diseases (eg, schizophrenia, depression, etc.), kidney disease (Eg, renal failure, nephritis, uremic disease, etc.), genital diseases (eg, prostatic hypertrophy, prostate carcinitis, testicular neurosis, etc.), bone and cartilage diseases (eg, osteoporosis, osteoarthritis, chronic Rheumatoid arthritis), muscular disease (eg, muscular dystrophy), heart disease (eg, cardiomyopathy, myocardial infarction, heart failure, angina), autoimmune disease (eg, systemic lupus erythematosus, immune-associated glomerulonephritis) What ), Cancer (eg, kidney cancer, lung 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, etc.), hypertension, etc. It can also be used for screening tests for preventive and therapeutic agents. It is preferably a screening test for a prophylactic / therapeutic agent for a neurodegenerative disease, more preferably an Alzheimer's disease.
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. The transfer 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.
[0059]
The non-human mammal having the abnormal DNA of the present invention, in which the abnormal DNA of the present invention is highly expressed, inhibits the function of endogenous normal DNA, and finally, the functionally inactive form of the protein of the present invention. It can be refractory and can be used as a model animal for the disease. For example, using the abnormal DNA-transferred 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 transferred has an increased symptom of the free protein of the present invention, a prophylactic / therapeutic agent for the protein of the present invention or a functionally inactive refractory disease, for example, a nervous system Diseases (eg, neurodegenerative diseases (eg, Alzheimer's disease, Parkinson's syndrome, etc.), spinal cord injury, epilepsy, cerebral ischemic disorder, cerebrovascular dementia, etc.), psychiatric diseases (eg, schizophrenia, depression, etc.), Renal disease (eg, renal failure, nephritis, uremic disease, etc.), genital disease (eg, prostatic hypertrophy, prostate carcinitis, testicular neurosis, etc.), bone / cartilage disease (eg, osteoporosis, osteoarthritis) , Rheumatoid arthritis), muscular disease (eg, muscular dystrophy), heart disease (eg, cardiomyopathy, myocardial infarction, heart failure, angina), autoimmune disease (eg, systemic lupus erythematosus, immune-related thread) ball Nephritis), cancer (eg, kidney cancer, lung 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, etc.), high blood pressure It can also be used for screening tests for prophylactic and therapeutic agents such as Preferably, it is a screening test for a prophylactic / therapeutic agent for a neurodegenerative disease, more preferably Alzheimer's disease.
In addition, other possible uses of the above two DNA-transferred animals of the present invention include, for example,
(1) Use as a cell source for tissue culture,
(2) A peptide specifically expressed or activated by the protein of the present invention by directly analyzing DNA or RNA in the tissue of the DNA-transferred animal of the present invention or analyzing a peptide tissue expressed by the DNA Analysis of the relationship with
{Circle around (3)} The cells of a tissue having DNA are cultured by standard tissue culture techniques, and these are used to study the function of cells from tissues that are generally difficult to culture.
(4) screening for a drug that enhances the function of the cell by using the cell described in (3) above, and
{Circle around (5)} Isolation and purification of the mutant protein of the present invention and production of its antibody can be considered.
[0060]
Furthermore, using the DNA-transferred animal of the present invention, it is possible to examine clinical symptoms of a disease associated with the protein of the present invention, including a functionally inactive refractory disease of the protein of the present invention, and the like. More detailed pathological findings in each organ of the 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-transferred animal of the present invention, cut it into small pieces, and then use a proteolytic enzyme such as trypsin to obtain the released DNA-transferred 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 disease of the protein of the present invention, using the DNA-transferred 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 transgenic 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 diseases associated with the protein of the present invention.
[0061]
(8) Knockout animal
The present invention provides a non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated, and a non-human mammal deficient in expression of the DNA of the present invention.
That is, the present invention
(1) a non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated,
(2) The embryonic stem cell according to (1), wherein the DNA is inactivated by introducing a reporter gene (eg, a β-galactosidase gene derived from E. 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 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 (6). The non-human mammal according to the item,
(8) the non-human mammal according to (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 expression of a reporter gene. I will provide a.
[0062]
A non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated is a DNA which is artificially mutated to the DNA of the present invention possessed by the non-human mammal, thereby suppressing 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.
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.
[0063]
Specific examples of the non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated (hereinafter abbreviated as the DNA-inactivated ES cells of the present invention or the knockout ES cells of the present invention) include, for example, The DNA of the present invention possessed by a non-human mammal is isolated and its exon portion is a drug resistance gene typified by a neomycin resistance gene or a hygromycin resistance gene, or lacZ (β-galactosidase gene), cat (chloramphenicol acetyl). A reporter gene or the like typified by a transferase gene) to disrupt the exon function, or insert a DNA sequence that terminates gene transcription into an intron between exons (for example, a polyA addition signal); Inability to synthesize complete messenger RNA Thus, a DNA strand having a DNA sequence constructed so as to eventually destroy the gene (hereinafter abbreviated as a targeting vector) is introduced into the chromosome of the animal by, for example, homologous recombination, and the resulting ES cells PCR using the DNA sequence on or near the DNA of the present invention as a probe, and using the DNA sequence on the targeting vector and the DNA sequence in the neighboring region other than the DNA of the present invention used for the preparation of the targeting vector as primers And selecting the knockout ES cells of the present invention.
As the ES cells from which the DNA of the present invention is inactivated by the homologous recombination method or the like, for example, those already established as described above may be used, or according to the known Evans and Kaufma method. It may be a newly established one. 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.
[0064]
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.
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. [Nature 292, 154, 1981; Proc. Natl. Acad. Sci. U. S. A. 78, 7634, 1981; Journal of Embryology and Experimental Morphology, 87, 27, 1985], which is obtained by differentiating the ES cell of the present invention. Cells deficient in DNA expression are useful in cell biology studies of the proteins of the invention in vitro.
[0065]
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.
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.
[0066]
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 / preventive effect on a disease, for example, a nervous system 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.
[0068]
Nervous system diseases (eg, neurodegenerative diseases (eg, Alzheimer's disease, Parkinson's syndrome, etc.), spinal cord injury, epilepsy, cerebral ischemic disorders, cerebrovascular dementia, etc.), psychiatric diseases (eg, schizophrenia, depression, etc.) ), Renal diseases (eg, renal failure, nephritis, uremics, etc.), genital diseases (eg, prostatic hypertrophy, prostate carcinitis, testicular neurosis, etc.), bone and cartilage diseases (eg, osteoporosis, deformity) Arthropathy, rheumatoid arthritis, etc., muscular disease (eg, muscular dystrophy, etc.), heart disease (eg, cardiomyopathy, myocardial infarction, heart failure, angina pectoris, etc.), autoimmune disease (eg, systemic lupus erythematosus, immunity Related glomerulonephritis, etc., cancer (eg, kidney cancer, lung cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, stomach cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectum cancer, pancreatic cancer, etc. ), Hypertension, etc., preferably Alzheimer's disease When screening for a compound having a prophylactic / therapeutic effect, a test compound is administered to a non-human mammal deficient in expressing the DNA of the present invention. The difference in the degree of healing is observed over time in the tissue.
In the screening method, when the test compound is administered to a test animal, if the disease symptom of the test animal is improved by about 10% or more, preferably about 30% or more, more preferably about 50% or more, the test compound is administered. It can be selected as a compound having a therapeutic / preventive effect on the above diseases.
The compound obtained by using the screening method is a compound selected from the test compounds described above, and has a therapeutic / preventive effect on 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.
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.
Since the preparation thus obtained is safe and low toxic, it can be used, for example, in humans or mammals (eg, rat, mouse, guinea pig, rabbit, sheep, pig, cow, horse, cat, dog, monkey, etc.). Can be administered.
The dose of the compound or a salt thereof varies depending on the target disease, the subject of administration, the administration route, and the like. For example, when the compound is orally administered, it is generally used in an adult (assuming a body weight of 60 kg) Alzheimer's disease patient. 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 administered in the form of an injection to an adult (with a body weight of 60 kg) Alzheimer's disease patient. When administered, 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) Method for screening for 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.
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.
The compound obtained by the screening method may form a salt, and the salt of the compound may include a physiologically acceptable acid (eg, an inorganic acid) and a base (eg, an organic acid). And particularly preferably a physiologically acceptable acid addition salt. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, For example, salts with succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and the like are used.
[0070]
The compound or a salt thereof that promotes or inhibits the promoter activity of the DNA of the present invention regulates the expression of the protein of the present invention and regulates the function of the protein (eg, transcriptional regulatory activity, cholinergic neuronal differentiation inhibitory effect, etc.) As a cholinergic neuronal differentiation promoter, for example, nervous system diseases [eg, neurodegenerative diseases (eg, Alzheimer's disease, Parkinson's syndrome, etc.), spinal cord injury, epilepsy, cerebral ischemic disorder, cerebrovascular dementia, etc. ], Psychiatric disorders (eg, schizophrenia, depression, etc.), renal disorders (eg, renal failure, nephritis, uremics, etc.), genital disorders (eg, prostatic hyperplasia, prostate carcinitis, testicular neurosis, etc.) , Bone and cartilage diseases (eg, osteoporosis, osteoarthritis, rheumatoid arthritis, etc.), muscle diseases (eg, muscular dystrophy, etc.), heart diseases (eg, cardiomyopathy, myocardial infarction, heart All, angina, etc.), autoimmune diseases (eg, systemic lupus erythematosus, immune-associated glomerulonephritis, etc.), cancer (eg, kidney cancer, lung 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, etc.), a prophylactic / therapeutic agent for hypertension, etc., preferably a neurodegenerative disease, more preferably a prophylactic / therapeutic agent for Alzheimer's disease. .
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.
Since the preparation thus obtained is safe and low toxic, it can be used, for example, in humans or mammals (eg, rat, mouse, guinea pig, rabbit, sheep, pig, cow, horse, cat, dog, monkey, etc.). Can be administered.
The dose of the compound or a salt thereof varies depending on the target disease, the subject to be administered, the administration route, and the like. For example, when the compound of the present invention that inhibits the promoter activity for DNA is orally administered, generally the adult (body weight) is used. For an Alzheimer's disease patient (as 60 kg), about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg of the compound is administered per day. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, and the like. For example, the compound 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 an Alzheimer's disease patient (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 intravenously 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 or developing preventive and therapeutic agents.
Further, using a DNA containing the promoter region of the protein of the present invention, genes encoding various proteins are ligated downstream thereof and injected into egg cells of an animal to produce a so-called transgenic animal (gene-transferred animal). Once created, the protein can be specifically synthesized and its action in the living body can be examined. 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.
[0071]
In the present specification, bases, amino acids, and the like are represented by abbreviations based on the abbreviations by IUPAC-IUB Commission on Biochemical Nomenclature or abbreviations commonly used in the art, and examples thereof are described below. When an amino acid can have an optical isomer, the L-form is indicated unless otherwise specified.
DNA: deoxyribonucleic acid
cDNA: complementary deoxyribonucleic acid
A: Adenine
T: Thymine
G: Guanine
C: Cytosine
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
Sec: selenocysteine
[0072]
Further, substituents, protecting groups and reagents frequently used in the present specification are represented by the following symbols.

[0073]
The sequence numbers in the sequence listing in the present specification indicate the following sequences.
[SEQ ID NO: 1]
1 shows the amino acid sequence of the mouse-derived protein mNHES obtained in Example 1.
[SEQ ID NO: 2]
This shows the base sequence of DNA encoding the protein mNHES having the amino acid sequence represented by SEQ ID NO: 1.
[SEQ ID NO: 3]
1 shows the nucleotide sequence of DNA containing the full-length gene encoding the protein obtained in Example 1.
[SEQ ID NO: 4]
1 shows the base sequence obtained in Example 1.
[SEQ ID NO: 5]
3 shows the amino acid sequence of human-derived protein hNHES obtained in Example 3. [SEQ ID NO: 6]
This shows the base sequence of DNA encoding the protein hNHES having the amino acid sequence represented by SEQ ID NO: 5.
[SEQ ID NO: 7]
3 shows the base sequence obtained in Example 3.
[SEQ ID NO: 8]
1 shows the nucleotide sequence of primer 1 used in Examples 1 to 5.
[SEQ ID NO: 9]
The base sequence of the primer 2 used in Example 1, Example 3 and Example 4 is shown.
[SEQ ID NO: 10]
3 shows the nucleotide sequence of primer 3 used in Examples 1 and 5.
[SEQ ID NO: 11]
2 shows the base sequence of primer 4 used in Examples 1 and 2.
[SEQ ID NO: 12]
3 shows the base sequence of primer 5 used in Example 3.
[SEQ ID NO: 13]
7 shows the base sequence of primer 6 used in Example 3.
[SEQ ID NO: 14]
7 shows the base sequence of primer 7 used in Example 4.
[SEQ ID NO: 15]
7 shows the base sequence of primer 8 used in Example 4.
[SEQ ID NO: 16]
6 shows the nucleotide sequence of the DNA obtained in Example 4.
[SEQ ID NO: 17]
This shows the base sequence of DNA encoding the protein rNHES having the amino acid sequence represented by SEQ ID NO: 26.
[SEQ ID NO: 18]
14 shows the base sequence of a primer used in Example 6.
[SEQ ID NO: 19]
14 shows the base sequence of a primer used in Example 6.
[SEQ ID NO: 20]
14 shows the base sequence of a TaqMan probe used in Example 6.
[SEQ ID NO: 21]
14 shows the base sequence of a primer used in Example 8.
[SEQ ID NO: 22]
14 shows the base sequence of a primer used in Example 8.
[SEQ ID NO: 23]
14 shows the base sequence of a TaqMan probe used in Example 8.
[SEQ ID NO: 24]
14 shows the base sequence of primer 9 used in Example 5.
[SEQ ID NO: 25]
6 shows the nucleotide sequence of the DNA obtained in Example 5.
[SEQ ID NO: 26]
7 shows the amino acid sequence of the rat-derived protein rNHES obtained in Example 5.
[SEQ ID NO: 27]
14 shows the base sequence of a primer used in Example 7.
[SEQ ID NO: 28]
14 shows the base sequence of a primer used in Example 7.
[SEQ ID NO: 29]
14 shows the base sequence of a probe used in Example 7. At the 5 'end, FAM (6-carboxy-fluorescein) was labeled as a reporter dye, and at the 3' end, TAMRA (6-carboxy-tetramethyl-rhodamine) was labeled as a quencher.
Escherichia coli JM109 / pTB2264, which was obtained in Example 1 described below, was obtained from April 1-1, 2002, by the 1-1 of Higashi 1-1-1, Tsukuba, Ibaraki, Japan, Central No. 6 (zip code 305-8566). The National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary, deposited as FERM BP-7992, from March 19, 2002, 2-17-85, Jusanhoncho, Yodogawa-ku, Osaka-shi, Osaka (postal code 532-8686). And deposited with the Fermentation Research Institute (IFO) under the accession number IFO 16779.
Escherichia coli JM109 / pTB2274 obtained in Example 4, which will be described later, has been obtained from July 1-1, 2002 by the independent of Chuo No. 6-1-1, Higashi 1-1-1, Tsukuba, Ibaraki, Japan (Zip code 305-8566). Deposited at the Patent Organism Depositary, the National Institute of Advanced Industrial Science and Technology under the deposit number FERM BP-8111.
Escherichia coli JM109 / rNHESTOPO obtained in Example 5 described below was obtained from February 1-1, 2002 by the 1-1, Higashi 1-1-1, Tsukuba, Ibaraki, Japan, central sixth (postal code 305-8566). Deposited at the Patent Organism Depositary, the National Institute of Advanced Industrial Science and Technology under the deposit number FERM BP-8299.
[0074]
Hereinafter, the present invention will be more specifically described with reference to examples, but the present invention is not limited thereto.
Example 1
A PCR reaction was performed using Marathon-Ready cDNA (Clontech) derived from mouse brain as a template and two primers, Primer 1 (SEQ ID NO: 8) and Primer 2 (SEQ ID NO: 9). The composition of the reaction solution in the reaction was such that 1 μl of the above cDNA was used as a template, 10 μl of 2 × SYBER Green Master Mix of QuantiTect SYBR Green PCR kit (QIAGEN), 0.4 μM each of primer 1 and primer 2, 0.4 μM of sterilized distilled water, and 7 μl of sterilized distilled water. In addition, a liquid volume of 20 μl was obtained. In the PCR reaction, a cycle of 95 ° C. for 20 seconds, 60 ° C. for 20 seconds, and 72 ° C. for 1 minute was repeated 40 times after 95 ° C. for 15 minutes, and finally an extension reaction at 72 ° C. for 4 minutes was performed. The PCR reaction product was again subjected to a PCR reaction using Primer 3 (SEQ ID NO: 10) and Primer 2. The composition of the reaction solution in the reaction was as follows: 1 μl of the above PCR reaction product was used as a template, 10 μl of 2X SYBER Green Master Mix of QuantiTect SYBR Green PCR kit (QIAGEN), 0.4 μM each of primer 2 and primer 3 and sterile distillation. 7 μl of water was added to make a liquid volume of 20 μl. In the PCR reaction, a cycle of 95 ° C. for 20 seconds, 60 ° C. for 20 seconds, 72 ° C. for 1 minute was repeated 10 times after 95 ° C. for 15 minutes, and finally an extension reaction at 72 ° C. for 4 minutes was performed. It was subcloned into the plasmid vector pCRII-TOPO vector (Invitrogen) according to the prescription of TOPO TA Cloning Kit (Invitrogen). This was introduced into Escherichia coli TOP10F ', and a clone having cDNA was selected on an LB agar medium containing ampicillin. As a result of analyzing the sequence of each clone, a cDNA sequence (SEQ ID NO: 3) encoding a basic helix-loop-helix-type transcription factor protein was obtained. In addition, a PCR reaction was performed using Marathon-Ready cDNA (Clontech) derived from mouse brain as a template and using two primers, Primer 1 and Primer 4 (SEQ ID NO: 11). In the reaction, the composition of the reaction solution was prepared by using 1 μl of the above cDNA as a template, 10 μl of 2X SYBER Green Master Mix of QuantiTect SYBR Green PCR kit (QIAGEN), 0.4 μM each of primer 1 and primer 4, and 7 μl of sterile distilled water. In addition, a liquid volume of 20 μl was obtained. In the PCR reaction, a cycle of 95 ° C. for 20 seconds, 60 ° C. for 20 seconds, and 72 ° C. for 1 minute was repeated 40 times after 95 ° C. for 15 minutes, and finally an extension reaction at 72 ° C. for 4 minutes was performed. The PCR reaction product was again subjected to a PCR reaction using Primer 1 and Primer 4. The composition of the reaction solution in this reaction was such that 1 μl of the PCR reaction product was used as a template, 10 μl of 2 × SYBER Green Master Mix of QuantiTect SYBR Green PCR kit (QIAGEN), 0.4 μM each of Primer 1 and Primer 4 and sterile distilled water. 7 μl was added to make a liquid volume of 20 μl. In the PCR reaction, a cycle of 95 ° C. for 20 seconds, 60 ° C. for 20 seconds, 72 ° C. for 1 minute was repeated 10 times after 95 ° C. for 15 minutes, and finally an extension reaction at 72 ° C. for 4 minutes was performed. As a result of analyzing the base sequence of the obtained DNA fragment, the obtained DNA fragment was a 437 bp fragment containing the same portion as the base sequence represented by SEQ ID NO: 3 (SEQ ID NO: 4). The cDNA fragment represented by SEQ ID NO: 3 encoded a 240 amino acid sequence (SEQ ID NO: 1) (SEQ ID NO: 2). The protein containing the amino acid sequence represented by SEQ ID NO: 1 was named mNHES. mNHES (SEQ ID NO: 1) and a basic helix-loop-helix transcription factor protein Hesr-1 (SWISS-PROT ID: Q9WV93; Biochem. Biophys. Res. Commun. 260, 459-465, 1999). Indicates 47% homology at the amino acid level. The plasmid having the nucleotide sequence of SEQ ID NO: 3 was named pTB2264, and the resulting transformant was named Escherichia coli JM109 / pTB2264.
[0075]
Example 2
To clarify the expression profile of the protein gene containing the amino acid sequence represented by SEQ ID NO: 1 in brain tissue, a mouse brain cDNA panel (Cemines) was used as a material, and the ABI Prism 7900 Sequence Detection System (Applied Biosystems) was used. ) Was used to perform a real-time PCR reaction.
The composition of the reaction solution in this reaction was such that 1 μl of each cDNA in the above cDNA panel was used as a template, and primers 1 and 4 were each added to 0.3 μM each, 10 μl of 2X SYBER Green Master Mix, 7 μl of sterile distilled water, and 20 μl of a solution was added. Amount. The PCR reaction was performed by repeating a cycle of 50 ° C. for 2 minutes-95 ° C. for 10 minutes and then 95 ° C. for 15 seconds-60 ° C. for 1 minute 40 times. The calculated copy number was corrected as an internal control with the copy number of glycerol triphosphate dehydratase calculated in the same manner as the copy number of the protein gene containing the amino acid sequence represented by SEQ ID NO: 1.
As a result, the expression of the protein gene obtained in Example 1 was higher in the fetal brain than after birth (FIG. 1).
This revealed that the gene of the mNHES protein obtained in Example 1 was highly expressed in the fetal brain.
[0076]
Example 3
A PCR was performed using Marathon-Ready cDNA derived from human kidney (Clontech) as a template and two primers, Primer 5 (SEQ ID NO: 12) and Primer 6 (SEQ ID NO: 13). The composition of the reaction solution in the reaction was such that 1 μl of the above cDNA was used as a template, 10 μl of 2 × SYBER Green Master Mix of QuantiTect SYBR Green PCR kit (QIAGEN), 0.4 μM each of primer 1 and primer 2, 0.4 μM of sterilized distilled water, and 7 μl of sterilized distilled water. In addition, a liquid volume of 20 μl was obtained. After the PCR reaction at 95 ° C for 15 minutes, a cycle of 95 ° C for 20 seconds-50 ° C for 20 seconds-72 ° C for 1 minute was repeated 50 times, and finally an extension reaction at 72 ° C for 4 minutes was performed. The obtained DNA fragment of about 900 bp was recovered, and the nucleotide sequence was analyzed. As a result, a sequence consisting of 919 bases was obtained (SEQ ID NO: 7). The cDNA fragment represented by SEQ ID NO: 7 encoded a 241 amino acid sequence (SEQ ID NO: 5) (SEQ ID NO: 6).
The protein containing the amino acid sequence represented by SEQ ID NO: 5 was designated as hNHES.
The nucleotide sequence represented by SEQ ID NO: 2 obtained in Example 1 and the nucleotide sequence represented by SEQ ID NO: 6 have 85% homology with the amino acid sequence represented by SEQ ID NO: 1, The amino acid sequence represented by SEQ ID NO: 5 had 91% homology.
hNHES (SEQ ID NO: 5) showed 73% homology at the amino acid level with a basic helix-loop-helix transcription factor protein (Genbank ID: XP 070283) reported as a predicted sequence. HNHES (SEQ ID NO: 5) is similar to the basic helix-loop-helix transcription factor protein Hey1 (Genbank ID: NP 036390; Genomics, 66, 195-203, 2000) at the amino acid level. It showed 26% homology.
[0077]
Example 4
A PCR reaction was performed using Marathon-Ready cDNA (Clontech) derived from human kidney as a template and two primers, Primer 7 (SEQ ID NO: 14) and Primer 8 (SEQ ID NO: 15). The composition of the reaction solution in the reaction was such that 1 μl of the above cDNA was used as a template, 10 μl of 2 × SYBER Green Master Mix of QuantiTect SYBR Green PCR kit (QIAGEN), 0.4 μM each of primer 1 and primer 2, 0.4 μM of sterilized distilled water, and 7 μl of sterilized distilled water. In addition, a liquid volume of 20 μl was obtained. After the PCR reaction at 95 ° C for 15 minutes, a cycle of 95 ° C for 20 seconds-50 ° C for 20 seconds-72 ° C for 1 minute was repeated 50 times, and finally an extension reaction at 72 ° C for 4 minutes was performed. As a result, the obtained about 800 bp DNA fragment was recovered. The recovered DNA fragment was amplified again using Primer 7 and Primer 8 using HotStarTaq DNA polymerase (QIAGEN) according to the instruction manual. The DNA fragment contained in the resulting reaction solution was cloned into pDrive cloning vector using QIAGEN PCR cloning kit (QIAGEN) according to the instruction manual. As a result of analysis, the nucleotide sequence of the cloned DNA fragment was a nucleotide sequence encoding hNHES consisting of 819 bases (SEQ ID NO: 16). The plasmid having the nucleotide sequence of SEQ ID NO: 16 was designated as pTB2274, and the resulting transformant was designated as Escherichia coli JM109 / pTB2274.
[0078]
Example 5
Using a rat brain-derived Marathon-Ready cDNA (Clontech) as a template, a PCR reaction was performed using two primers, primer 1 (SEQ ID NO: 8) and primer 9 (SEQ ID NO: 24). The composition of the reaction solution used in the reaction was such that 1 μl of the above cDNA was used as a template, 10 μl of 2X SYBER Green Master Mix of QuantiTect SYBR Green PCR kit (QIAGEN), primer 1 (SEQ ID NO: 8) and 0.4 μM each of primer 9 Then, 7 μl of sterilized distilled water was added to make a liquid volume of 20 μl. After the PCR reaction at 95 ° C for 15 minutes, a cycle of 95 ° C for 20 seconds, 50 ° C for 20 seconds, and 72 ° C for 1 minute was repeated 50 times, and finally an extension reaction at 72 ° C for 4 minutes was performed. The PCR reaction product was again subjected to a PCR reaction using Primer 1 and Primer 7. The composition of the reaction solution in this reaction was prepared by using 1 μl of the above PCR reaction product as a template, adding 10 μl of HotstarTaq Master Mix of HotstarTaq Master Mix kit (QIAGEN), 0.4 μM each of primer 1 and primer 79, and 7 μl of sterile distilled water. , 20 μl. In the PCR reaction, a cycle of 95 ° C. for 20 seconds, 60 ° C. for 20 seconds, 72 ° C. for 1 minute was repeated 10 times after 95 ° C. for 15 minutes, and finally an extension reaction at 72 ° C. for 4 minutes was performed. Subcloning into a plasmid vector pCRII-TOPO vector (Invitrogen) was performed according to the prescription of TOPO TA Cloning Kit (Invitrogen). This was introduced into Escherichia coli TOP10F ', and a clone having cDNA was selected on an LB agar medium containing ampicillin. As a result of analyzing the sequence of each clone, a cDNA sequence (SEQ ID NO: 25) encoding a basic helix-loop-helix-type transcription factor protein was obtained. The cDNA fragment represented by SEQ ID NO: 25 encoded a 241 amino acid sequence (SEQ ID NO: 26) (SEQ ID NO: 17). The protein containing the amino acid sequence represented by SEQ ID NO: 26 was designated as rNHES. rNHES (SEQ ID NO: 26) showed 97% homology at the amino acid level with the mouse-derived basic helix-loop-helix transcription factor protein Medane (WO 02/60928).
The plasmid having the nucleotide sequence of SEQ ID NO: 25 was designated as rNHESTOPO, and the obtained transformant was designated as Escherichia coli JM109 / rNHESTOPO.
rNHES and mNHES have 96% homology at the amino acid level and 94% homology at the base level.
rNHES and hNHES have 91% homology at the amino acid level and 85% homology at the base level.
[0079]
Example 6
In order to clarify the relationship between the mNHES gene and neuronal differentiation, mouse P19 embryonic tumor cells were cultured and differentiated into cholinergic neurons, and their expression profiles were examined.
P19 cells at 5 × 10 ⁵ 5x10 at a cell concentration of cell / ml ^-7 The suspension was suspended in an α-MEM medium (Invitrogen) containing M retinoic acid (Wako Pure Chemical Industries) and 10% FBS (Invitrogen). 3 ml of the cell suspension was cultured for 2 days using a 3.5 cm suspension culture plate (Sumitomo Bakelite). Then digest with trypsin, 5 × 10 ⁴ The cells were suspended in a DMEM / F12 medium (Invitrogen) containing 1% N-2 supplement (Invitrogen) and 1 μg / μl fibronectin (Invitrogen) at a cell concentration of cell / ml. 0.5 ml of the cell suspension was cultured using a 48-well poly-D-Lysine coated plate (Becton Deckson) and subjected to the following experiment. Three days after the start of the culture, cytosine β-D-arabinofuranoside (AraC) (Sigma) was added to the medium at a concentration of 5 μg / ml.
On the third, fourth, fifth and sixth days after the culturing, total RNA was extracted from the cells using RNeasy 96 kit (QIAGEN). From the obtained total RNA, cDNA was synthesized using TaqMan Reverse TranscriptionReagents (manufactured by PE Applied Biosystems). Using two kinds of synthetic primers (SEQ ID NO: 18 and SEQ ID NO: 19) capable of specifically amplifying and detecting the mNHES gene and a TaqMan probe (SEQ ID NO: 20), the copy number of the mNHES gene was quantified by PCR using ABI. This was performed using the Prism 7900 sequence Detection System. The PCR reaction used TaqMan universal PCR Master Mix (manufactured by PE Applied Biosystems) under the conditions according to the attached instructions. Using the calculated copy number as an internal control and correcting with the copy number of glycerol triphosphate dehydratase calculated using TaqMan Rodent GAPDH Control reagents VIC (manufactured by PE Applied Biosystems) in the same manner as the copy number of the mNHES gene , And the expression was used as the expression level, and the fluctuation rate was calculated with the expression level on the third day as 1.
As a result, the fluctuation rate of the mNHES gene expression was 3.0, 15.7 and 16.6 on the 4th, 5th and 6th days after the start of the culture, respectively. became.
This revealed that the expression of the mNHES gene was induced together with neural differentiation.
[0080]
Example 7
In order to clarify the relationship between the mNHES gene and cholinergic neuronal differentiation, the effect of mNHES gene forced expression on cholinergic neuronal differentiation of P19 embryonic tumor cells was examined.
The pTB2274 constructed in Example 1 was digested with restriction enzymes BamH I and Xho I (both from Takara Shuzo), a fragment of about 900 bp was recovered, and inserted into the gap between BamH I and Xho I of pTARGET Vector (Promega). MNHES forced expression plasmid was prepared. The prepared forced expression plasmid was designated as mNHES1-1. mNHES1-1 and pTARGET Vector as a control were each used for P19 in an α-MEM medium (Invitrogen) containing 10% FBS (Invitrogen) using Fugene6 transfection reagent (Roche Diagnostics). The cells were transfected. Thereafter, P19 cells were transferred to 5 × 10 ⁵ 5x10 at a cell concentration of cell / ml ^-7 The suspension was suspended in an α-MEM medium (Invitrogen) containing M retinoic acid (Wako Pure Chemical Industries) and 10% FBS (Invitrogen). 3 ml of the cell suspension was cultured for 2 days using a 3.5 cm suspension culture plate (Sumitomo Bakelite). Then digest with trypsin, 5 × 10 ⁴ The cells were suspended in a DMEM / F12 medium (Invitrogen) containing 1% N-2 supplement (Invitrogen) and 1 μg / μl fibronectin (Invitrogen) at a cell concentration of cell / ml. 0.5 ml of the cell suspension was cultured using a 48-well poly-D-Lysine coat plate (Becton Deckson) and subjected to the following experiment. Five days after the start of the culture, total RNA was extracted from the cells using RNeasy 96 kit (QIAGEN). From the obtained total RNA, cDNA was synthesized using TaqMan Reverse Transcription Reagents (manufactured by PE Applied Biosystems).
Two kinds of synthetic primers (SEQ ID NO: 27 and SEQ ID NO: 28) and a TaqMan probe (SEQ ID NO: 29; Fam-ccaatgaccagctagagttttgcagcc-Tamra) capable of specifically amplifying and detecting the cholinergic nervous marker choline acetyltransferase gene; In the sequence, Fam indicates 6-carboxy-fluorescein, and Tamra indicates 6-carboxy-tetramethyl-rhodamine), quantification of the copy number of the choline acetyltransferase gene by PCR, and ABI Prism 7900 sequence Detection System. went. The PCR reaction used TaqMan universal PCR Master Mix (manufactured by PE Applied Biosystems) under the conditions according to the attached instructions. Using the calculated copy number as an internal control and correcting with the copy number of glycerol triphosphate dehydratase calculated using TaqMan Rodent GAPDH Control reagents VIC (manufactured by PE Applied Biosystems) in the same manner as the copy number of the mNHES gene , And the value was used as the expression level.
As a result, it was revealed that the choline acetyltransferase gene expression level in the mNHES1-1-transfected cells was 52% of that of the control pTARGET Vector-transfected cells.
This suggests that the mNHES gene suppresses the differentiation of cholinergic nerves.
[0081]
Example 8
To clarify the relationship between the hNHES gene and the neurodegenerative disease, hNHES gene expression was performed using cDNA derived from human Alzheimer brain tissue (hippocampus) (Biochain) and cDNA derived from human healthy human brain tissue (hippocampus) (Biochain). A comparison of the amounts was made.
Using two kinds of synthetic primers (SEQ ID NO: 21 and SEQ ID NO: 22) capable of specifically amplifying and detecting the hNHES gene and a TaqMan probe (SEQ ID NO: 23), the copy number of the hNHES gene was quantified by PCR using ABI. This was performed using the Prism 7900 sequence Detection System. The PCR reaction used TaqMan universal PCR Master Mix (manufactured by PE Applied Biosystems) under the conditions according to the attached instructions. The calculated copy number was corrected with the copy number of glycerol triphosphate dehydratase calculated using TaqMan GAPDH Control Reagents (manufactured by PE Applied Biosystems) in the same manner as the hNHES gene copy number as an internal control. Was calculated as the expression level.
As a result, it was revealed that the expression of the hNHES gene in the hippocampus of Alzheimer's disease patients was 358 times higher than in the hippocampus of healthy subjects.
This suggests that the hNHES gene is involved in neurodegenerative diseases such as Alzheimer's disease.
[0082]
Example 9
In order to clarify the relationship between the mNHES gene and neural differentiation, mouse embryonic neural stem cells were cultured and differentiated into nerves, and their expression profiles were examined.
As the neural stem cells, neural stem cells obtained from the mouse fetus on the 15th to 15th days of gestation by the neurosphere method (Experimental Neurology, 144, 350-360, 1997) were used.
150 cm of neural stem cells ² 5 × 10 using flash (IWAKI Sumitomo Bakelite) ⁵ At a cell concentration of cell / ml, 50 ml 20 ng / ml bFGF (genzyme techne) and N ₂ It was maintained as a neurosphere in a DMEM / F12 medium (Invitrogen) containing the supplement (Invitrogen). Then, using a 24-well plate (Becton Deckson) coated with ECL (UPSATE biotechnology) digested with trypsin, 2 × 10 ⁵ At a cell concentration of cell / ml, 0.5 ml of 100 ng / ml NGF (genzyme techne) and N ₂ Differentiation culture was performed in a DMEM / F12 medium (gibcoInvitrogen) containing a supplement (gibco).
Total RNA was collected from cells using RNeasy 96 kit (QIAGEN) before differentiation culture and on days 1, 3 and 5 after differentiation induction. From the obtained total RNA, cDNA was synthesized using TaqMan Reverse Transcription Reagents (manufactured by PE Applied Biosystems).
Using two kinds of synthetic primers (SEQ ID NO: 18 and SEQ ID NO: 19) capable of specifically amplifying and detecting the mNHES gene and a TaqMan probe (SEQ ID NO: 20), the copy number of the mNHES gene was quantified by PCR using ABI. This was performed using the Prism 7900 sequence Detection System. The PCR reaction used TaqMan universal PCR Master Mix (manufactured by PE Applied Biosystems) under the conditions according to the attached instructions. Using the calculated copy number as an internal control and correcting with the copy number of glycerol triphosphate dehydratase calculated using TaqMan Rodent GAPDH Control reagents VIC (manufactured by PE Applied Biosystems) in the same manner as the copy number of the mNHES gene , And the expression level was set as the expression level, and the expression level before the induction of differentiation was set as 1, to calculate the fluctuation rate of the mNHES gene expression level.
As a result, the mNHES gene became 30.6, 9.8, and 1.3 on the 1st, 3rd, and 5th days after the differentiation induction, and it was revealed that the expression increased transiently after the differentiation induction. Was.
This revealed that the expression of the mNHES gene was induced in neural stem cells together with neural differentiation.
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【The invention's effect】
The protein of the present invention is a diagnostic marker for nervous system diseases, and has a cholinergic neuronal differentiation inhibitory action. Therefore, a compound or a salt thereof that regulates (preferably inhibits) the activity of the protein or the expression of a gene of the protein, the antibody of the present invention (preferably a neutralizing antibody), the antisense polynucleotide of the present invention, and the like include, for example, choline Examples of the agonist for promoting neuronal differentiation include nervous system diseases (eg, neurodegenerative diseases (eg, Alzheimer's disease, Parkinson's syndrome, etc.), spinal cord injury, epilepsy, cerebral ischemic disorder, cerebrovascular dementia, etc.), psychiatric diseases ( E.g., schizophrenia, depression, etc.), renal disease (e.g., renal failure, nephritis, uremic disease, etc.), genital disease (e.g., benign prostatic hyperplasia, prostate cancer, testicular neurosis, etc.), bone / cartilage disease (e.g., Eg, osteoporosis, osteoarthritis, rheumatoid arthritis, etc., muscular disease (eg, muscular dystrophy, etc.), heart disease (eg, cardiomyopathy, myocardial infarction, heart failure, angina, etc.) , Autoimmune diseases (eg, systemic lupus erythematosus, immune-associated glomerulonephritis, etc.), cancer (eg, kidney cancer, lung cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, It can be used safely as a prophylactic / therapeutic agent for cervical cancer, colon cancer, rectal cancer, pancreatic cancer, etc.) and hypertension, preferably for neurodegenerative diseases (preferably Alzheimer's disease etc.).
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[Sequence list]

[Brief description of the drawings]
FIG. 1 is a diagram showing the results of the expression level of mNHES in a mouse brain cDNA panel. In the figure, the vertical axis shows the value corrected for the expression level of glycerol triphosphate dehydratase using the expression level of mNHES as an internal control. On the horizontal axis, E12 is 12 days old, E14 is 14 days old, E16 is 16 days old, E18 is 18 days old, P1 is 1 day old, P4 is 4 days old, and P7 is 7 days old. Day, P14 indicates 14 days after birth, P21 indicates 21 days after birth, and P60 indicates 60 days after birth.

Claims (43)

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: 5, or SEQ ID NO: 26, or a salt thereof. A protein comprising an amino acid sequence represented by SEQ ID NO: 1 or a salt thereof. A protein consisting of the amino acid sequence represented by SEQ ID NO: 5, or a salt thereof. A protein consisting of the amino acid sequence represented by SEQ ID NO: 26, 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 5. The polynucleotide according to claim 6, which is DNA. A polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 2, SEQ ID NO: 6, or SEQ ID NO: 17. A recombinant vector containing the polynucleotide according to claim 6. A transformant transformed with the recombinant vector according to claim 9. The protein according to claim 1, wherein the transformant according to claim 10 is cultured to produce and accumulate the protein according to claim 1 or the partial peptide according to claim 5, and collect the protein. 5. The method for producing the partial peptide or a salt thereof according to 5. A medicament comprising the protein according to claim 1, the partial peptide according to claim 5, or a salt thereof. A medicament comprising the polynucleotide according to claim 6. A diagnostic agent comprising the polynucleotide according to claim 6. An antibody against the protein according to claim 1, the partial peptide according to claim 5, or a salt thereof. A medicament comprising the antibody according to claim 15. A diagnostic agent comprising the antibody according to claim 15. A base sequence complementary to or substantially complementary to a base sequence of a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof, or a part thereof; Antisense polynucleotide containing. A medicament comprising the antisense polynucleotide according to claim 18. A diagnostic agent comprising the antisense polynucleotide according to claim 18. A compound that promotes or inhibits the activity of the protein according to claim 1, the partial peptide according to claim 5, or a salt thereof, wherein the protein according to claim 1 or the partial peptide according to claim 5 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 5, or a salt thereof, or a compound thereof, comprising the protein according to claim 1, the partial peptide according to claim 5, 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 5 or a salt thereof, which is obtained by using the screening method of claim 21 or the screening kit of claim 22. . A medicament comprising the compound according to claim 23 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, wherein the polynucleotide according to claim 6 is used. 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 6. A compound or a salt thereof that promotes or inhibits the expression of the protein gene according to claim 1, which is obtained by using the screening method according to claim 25 or the screening kit according to claim 26. A medicament comprising the compound according to claim 27 or a salt thereof. The medicament according to claim 12, claim 13, claim 16, claim 19, claim 24 or claim 28, which is a prophylactic / therapeutic agent for a neurodegenerative disease. 21. The diagnostic agent according to claim 14, which is a diagnostic agent for a neurodegenerative disease. The prophylactic / therapeutic agent according to claim 29, which is a prophylactic / therapeutic agent for Alzheimer's disease. 31. The diagnostic agent according to claim 30, which is a diagnostic agent for Alzheimer's disease. A method for screening a cholinergic neuronal differentiation promoting agent, comprising using the protein according to claim 1, the partial peptide according to claim 5, or a salt thereof. A kit for screening a cholinergic neuronal differentiation promoting agent, comprising the protein according to claim 1, the partial peptide according to claim 5, or a salt thereof. A method for screening a cholinergic neuronal differentiation promoting agent, comprising using the polynucleotide according to claim 6. A kit for screening a cholinergic neuronal differentiation promoting agent, comprising the polynucleotide according to claim 6. A cholinergic neuronal differentiation promoting agent obtainable by using the screening method according to claim 33 or claim 35 or the screening kit according to claim 34 or claim 36. A cholinergic neuronal differentiation promoter comprising a compound or a salt thereof that inhibits the activity of the protein of claim 1 or the partial peptide of claim 5 or a salt thereof. An agent for promoting cholinergic neuronal differentiation, comprising a compound that inhibits expression of the protein gene according to claim 1 or a salt thereof. A cholinergic neuronal differentiation promoting agent comprising the antibody according to claim 15. A cholinergic neuronal differentiation promoter comprising the antisense polynucleotide according to claim 18. 28. A method for preventing or treating a neurodegenerative disease, comprising administering an effective amount of the compound according to claim 23 or 27 or a salt thereof to a mammal. 28. Use of the compound or a salt thereof according to claim 23 or 27 for producing a prophylactic / therapeutic agent for a neurodegenerative disease.

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