JP2004002295A - Body weight increase inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a body weight increase inhibitor and its screening method. <P>SOLUTION: The body weight increase inhibitor, a body weight decreasing agent, a fat amount increasing agent and a hyperphagia inhibitor comprise a polypeptide containing a specific amino acid sequence (23 amino acids) or an amide, an ester or a salt thereof. The polypeptide and the like are useful for screening and the like of the body weight increase inhibitor and the like. <P>COPYRIGHT: (C)2004,JPO

Description

【００７８】
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〕
ヒトＧＰＲ８タンパク質をコードするｃＤＮＡのスクリーニングに使用した合成ＤＮＡの塩基配列を示す。
〔配列番号：２〕
ヒトＧＰＲ８タンパク質をコードするｃＤＮＡのスクリーニングに使用した合成ＤＮＡの塩基配列を示す。
〔配列番号：３〕
５’側に制限酵素ＣｌａＩの認識する塩基配列が付加され、また３’側に制限酵素ＳｐｅＩの認識する塩基配列が付加されたヒトＧＰＲ８タンパク質ｃＤＮＡの全塩基配列を示す。
〔配列番号：４〕
ヒトＧＰＲ８タンパク質の全アミノ酸配列を示す。
〔配列番号：５〕
ＧＰＲ８発現ＣＨＯ細胞株の各クローンにおけるＧＰＲ８タンパク質ｍＲＮＡの発現量を測定するために使用したｒｉｂｏｐｒｏｂｅの配列を示す。
〔配列番号：６〕
ブタ視床下部から精製されたＧＰＲ８に対するリガンドペプチドのアミノ末端アミノ酸配列解析の結果から得られたアミノ酸配列を示す。
〔配列番号：７〕
相補鎖がＧＰＲ８リガンドペプチドのヒトホモログの前駆体タンパク質の一部をコードしていると推定されるＥＳＴ配列（アクセッション番号：ＡＷ００７５３１）を示す。
〔配列番号：８〕
相補鎖がＧＰＲ８リガンドペプチドのヒトホモログの前駆体タンパク質の一部をコードしていると推定されるＥＳＴ配列（アクセッション番号：ＡＩ５００３０３）を示す。
〔配列番号：９〕
相補鎖がＧＰＲ８リガンドペプチドのヒトホモログの前駆体タンパク質の一部をコードしていると推定されるＥＳＴ配列（アクセッション番号：ＡＩ９９０９６４）を示す。
〔配列番号：１０〕
相補鎖がＧＰＲ８リガンドペプチドのヒトホモログの前駆体タンパク質の一部をコードしていると推定されるＥＳＴ配列（アクセッション番号：ＡＡ７４４８０４）を示す。
〔配列番号：１１〕
ＧＰＲ８リガンドペプチドのラットホモログの前駆体タンパク質の一部をコードしていると推定されるＥＳＴ配列（アクセッション番号：Ｈ３１５９８）を示す。
〔配列番号：１２〕
ＧＰＲ８に対するリガンドペプチドのヒトホモログの前駆体タンパク質の一部をコードするｃＤＮＡのスクリーニングに使用した合成ＤＮＡの塩基配列を示す。
〔配列番号：１３〕
ＧＰＲ８に対するリガンドペプチドのヒトホモログの前駆体タンパク質の一部をコードするｃＤＮＡのスクリーニングに使用した合成ＤＮＡの塩基配列を示す。
〔配列番号：１４〕
ヒト脳由来ｃＤＮＡから増幅されたＧＰＲ８に対するリガンドペプチドのヒトホモログの前駆体タンパク質の一部をコードするＤＮＡの塩基配列を示す。
〔配列番号：１５〕
ＧＰＲ８に対するリガンドペプチドのヒトホモログの前駆体タンパク質の一部のアミノ酸配列を示す。
〔配列番号：１６〕
配列番号：１５から推定されたＧＰＲ８に対するリガンドペプチドのヒトホモログのアミノ酸配列を示す。
〔配列番号：１７〕
配列番号：１５から推定されたＧＰＲ８に対するリガンドペプチドのヒトホモログのアミノ酸配列を示す。
〔配列番号：１８〕
配列番号：１６で表されるアミノ酸配列をコードする塩基配列を示す。
〔配列番号：１９〕
配列番号：１７で表されるアミノ酸配列をコードする塩基配列を示す。
〔配列番号：２０〕
後述の参考例１４で合成されたヒトＧＰＲ８リガンド（１−２９）のアミノ酸配列を示す。
〔配列番号：２１〕
後述の参考例１５で合成されたヒトＧＰＲ８リガンド（１−２８）のアミノ酸配列を示す。
〔配列番号：２２〕
後述の参考例１６で合成されたヒトＧＰＲ８リガンド（１−２７）のアミノ酸配列を示す。
〔配列番号：２３〕
後述の参考例１７で合成されたヒトＧＰＲ８リガンド（１−２６）のアミノ酸配列を示す。
〔配列番号：２４〕
後述の参考例１８で合成されたヒトＧＰＲ８リガンド（１−２５）のアミノ酸配列を示す。
〔配列番号：２５〕
後述の参考例１９で合成されたヒトＧＰＲ８リガンド（１−２４）のアミノ酸配列を示す。
〔配列番号：２６〕
配列番号：２０で表されるアミノ酸配列をコードする塩基配列を示す。
〔配列番号：２７〕
配列番号：２１で表されるアミノ酸配列をコードする塩基配列を示す。
〔配列番号：２８〕
配列番号：２２で表されるアミノ酸配列をコードする塩基配列を示す。
〔配列番号：２９〕
配列番号：２３で表されるアミノ酸配列をコードする塩基配列を示す。
〔配列番号：３０〕
配列番号：２４で表されるアミノ酸配列をコードする塩基配列を示す。
〔配列番号：３１〕
配列番号：２５で表されるアミノ酸配列をコードする塩基配列を示す。
〔配列番号：３２〕
配列番号：４で表されるアミノ酸配列をコードする塩基配列を示す。
〔配列番号：３３〕
ＧＰＲ８に対するリガンドペプチドのヒトホモログの前駆体タンパク質をコードするｃＤＮＡの５’上流側配列を得るのに使用した合成ＤＮＡの塩基配列を示す。
〔配列番号：３４〕
ＧＰＲ８に対するリガンドペプチドのヒトホモログの前駆体タンパク質をコードするｃＤＮＡの５’上流側配列を得るのに使用した合成ＤＮＡの塩基配列を示す。
〔配列番号：３５〕
ＧＰＲ８に対するリガンドペプチドのヒトホモログの前駆体ッタンパク質をコードするｃＤＮＡの５’上流側ＤＮＡの塩基配列を示す。
〔配列番号：３６〕
ＧＰＲ８に対するリガンドペプチドのヒトホモログの前駆体タンパク質をコードするｃＤＮＡの３’下流側配列を得るのに使用した合成ＤＮＡの塩基配列を示す。
〔配列番号：３７〕
ＧＰＲ８に対するリガンドペプチドのヒトホモログの前駆体タンパク質をコードするｃＤＮＡの３’下流側配列を得るのに使用した合成ＤＮＡの塩基配列を示す。
〔配列番号：３８〕
ＧＰＲ８に対するリガンドペプチドのヒトホモログの前駆体タンパク質をコードするｃＤＮＡの３’下流側ＤＮＡの塩基配列を示す。
〔配列番号：３９〕
ＧＰＲ８に対するリガンドペプチドのヒトホモログの前駆体タンパク質をコードするｃＤＮＡを得るのに使用した合成ＤＮＡの塩基配列を示す。
〔配列番号：４０〕
ＧＰＲ８に対するリガンドペプチドのヒトホモログの前駆体タンパク質をコードするｃＤＮＡを得るのに使用した合成ＤＮＡの塩基配列を示す。
〔配列番号：４１〕
ＧＰＲ８に対するリガンドペプチドのヒトホモログの前駆体タンパク質をコードするｃＤＮＡの配列を示す。
〔配列番号：４２〕
ＧＰＲ８に対するリガンドペプチドのヒトホモログの前駆体タンパク質のアミノ酸配列を示す。
〔配列番号：４３〕
ＧＰＲ８に対するリガンドペプチドのブタホモログの前駆体タンパク質をコードするｃＤＮＡの５’上流側配列を得るのに使用した合成ＤＮＡの塩基配列を示す。
〔配列番号：４４〕
ＧＰＲ８に対するリガンドペプチドのブタホモログの前駆体タンパク質をコードするｃＤＮＡの５’上流側配列を得るのに使用した合成ＤＮＡの塩基配列を示す。
〔配列番号：４５〕
ＧＰＲ８に対するリガンドペプチドのブタホモログの前駆体タンパク質をコードするｃＤＮＡの５’上流側ＤＮＡの塩基配列を示す。
〔配列番号：４６〕
ＧＰＲ８に対するリガンドペプチドのブタホモログの前駆体タンパク質をコードするｃＤＮＡの５’上流側配列を得るのに使用した合成ＤＮＡの塩基配列を示す。
〔配列番号：４７〕
ＧＰＲ８に対するリガンドペプチドのブタホモログの前駆体タンパク質をコードするｃＤＮＡの５’上流側配列を得るのに使用した合成ＤＮＡの塩基配列を示す。
〔配列番号：４８〕
ＧＰＲ８に対するリガンドペプチドのブタホモログの前駆体タンパク質をコードするｃＤＮＡの５’上流側ＤＮＡの塩基配列を示す。
〔配列番号：４９〕
ＧＰＲ８に対するリガンドペプチドのブタホモログの前駆体タンパク質をコードするｃＤＮＡの３’下流側配列を得るのに使用した合成ＤＮＡの塩基配列を示す。
〔配列番号：５０〕
ＧＰＲ８に対するリガンドペプチドのブタホモログの前駆体タンパク質をコードするｃＤＮＡの３’下流側配列を得るのに使用した合成ＤＮＡの塩基配列を示す。
【００７９】
〔配列番号：５１〕
ＧＰＲ８に対するリガンドペプチドのブタホモログの前駆体タンパク質をコードするｃＤＮＡの３’下流側ＤＮＡの塩基配列を示す。
〔配列番号：５２〕
ＧＰＲ８に対するリガンドペプチドのブタホモログの前駆体タンパク質をコードするｃＤＮＡを得るのに使用した合成ＤＮＡの塩基配列を示す。
〔配列番号：５３〕
ＧＰＲ８に対するリガンドペプチドのブタホモログの前駆体タンパク質をコードするｃＤＮＡを得るのに使用した合成ＤＮＡの塩基配列を示す。
〔配列番号：５４〕
ＧＰＲ８に対するリガンドペプチドのブタホモログの前駆体タンパク質をコードするｃＤＮＡの配列を示す。
〔配列番号：５５〕
ＧＰＲ８に対するリガンドペプチドのブタホモログの前駆体タンパク質のアミノ酸配列を示す。
〔配列番号：５６〕
配列番号：５５から推定されたＧＰＲ８に対するリガンドペプチドのブタホモログのアミノ酸配列を示す。
〔配列番号：５７〕
配列番号：５５から推定されたＧＰＲ８に対するリガンドペプチドのブタホモログのアミノ酸配列を示す。
〔配列番号：５８〕
配列番号：５６で表されるアミノ酸配列をコードする塩基配列を示す。
〔配列番号：５９〕
配列番号：５７で表されるアミノ酸配列をコードする塩基配列を示す。
〔配列番号：６０〕
ＧＰＲ８に対するリガンドペプチドのラットホモログの前駆体タンパク質の一部をコードするｃＤＮＡを得るのに使用した合成ＤＮＡの塩基配列を示す。
〔配列番号：６１〕
ＧＰＲ８に対するリガンドペプチドのラットホモログの前駆体タンパク質の一部をコードするｃＤＮＡを得るのに使用した合成ＤＮＡの塩基配列を示す。
〔配列番号：６２〕
ＧＰＲ８に対するリガンドペプチドのラットホモログの前駆体タンパク質の一部をコードするｃＤＮＡの配列を示す。
〔配列番号：６３〕
ＧＰＲ８に対するリガンドペプチドのラットホモログの前駆体タンパク質をコードするｃＤＮＡの５’上流側配列を得るのに使用した合成ＤＮＡの塩基配列を示す。
〔配列番号：６４〕
ＧＰＲ８に対するリガンドペプチドのラットホモログの前駆体タンパク質をコードするｃＤＮＡの５’上流側配列を得るのに使用した合成ＤＮＡの塩基配列を示す。
〔配列番号：６５〕
ＧＰＲ８に対するリガンドペプチドのラットホモログの前駆体タンパク質をコードするｃＤＮＡの５’上流側ＤＮＡの塩基配列を示す。
〔配列番号：６６〕
ＧＰＲ８に対するリガンドペプチドのラットホモログの前駆体タンパク質をコードするｃＤＮＡの３’下流側配列を得るのに使用した合成ＤＮＡの塩基配列を示す。
〔配列番号：６７〕
ＧＰＲ８に対するリガンドペプチドのラットホモログの前駆体タンパク質をコードするｃＤＮＡの３’下流側配列を得るのに使用した合成ＤＮＡの塩基配列を示す。
〔配列番号：６８〕
ＧＰＲ８に対するリガンドペプチドのラットホモログの前駆体タンパク質をコードするｃＤＮＡの３’下流側ＤＮＡの塩基配列を示す。
〔配列番号：６９〕
ＧＰＲ８に対するリガンドペプチドのラットホモログの前駆体タンパク質をコードするｃＤＮＡを得るのに使用した合成ＤＮＡの塩基配列を示す。
〔配列番号：７０〕
ＧＰＲ８に対するリガンドペプチドのラットホモログの前駆体タンパク質をコードするｃＤＮＡを得るのに使用した合成ＤＮＡの塩基配列を示す。
〔配列番号：７１〕
ＧＰＲ８に対するリガンドペプチドのラットホモログの前駆体タンパク質をコードするｃＤＮＡの配列を示す。
〔配列番号：７２〕
ＧＰＲ８に対するリガンドペプチドのラットホモログの前駆体タンパク質のアミノ酸配列を示す。
〔配列番号：７３〕
配列番号：７２から推定されたＧＰＲ８に対するリガンドペプチドのラットホモログのアミノ酸配列を示す。
〔配列番号：７４〕
配列番号：７２から推定されたＧＰＲ８に対するリガンドペプチドのラットホモログのアミノ酸配列を示す。
〔配列番号：７５〕
配列番号：７３で表されるアミノ酸配列をコードする塩基配列を示す。
〔配列番号：７６〕
配列番号：７４で表されるアミノ酸配列をコードする塩基配列を示す。
〔配列番号：７７〕
ヒトＧＰＲ７発現ＣＨＯ細胞のＧＰＲ７遺伝子発現量を測定するのにプローブとして用いた合成ＤＮＡの配列を示す。
〔配列番号：７８〕
ＧＰＲ８に対するリガンドペプチドのマウスホモログの前駆体タンパク質の一部をコードするｃＤＮＡのスクリーニングに使用した合成ＤＮＡの塩基配列を示す。
〔配列番号：７９〕
ＧＰＲ８に対するリガンドペプチドのマウスホモログの前駆体タンパク質の一部をコードするｃＤＮＡのスクリーニングに使用した合成ＤＮＡの塩基配列を示す。
〔配列番号：８０〕
マウス精巣由来ｃＤＮＡから増幅されたＧＰＲ８に対するリガンドペプチドのヒトホモログの前駆体タンパク質の一部をコードするＤＮＡの塩基配列を示す。
〔配列番号：８１〕
ＧＰＲ８に対するリガンドペプチドのマウスホモログの前駆体タンパク質をコードするｃＤＮＡの５’上流側配列を得るのに使用した合成ＤＮＡの塩基配列を示す。
〔配列番号：８２〕
ＧＰＲ８に対するリガンドペプチドのマウスホモログの前駆体タンパク質をコードするｃＤＮＡの５’上流側配列を得るのに使用した合成ＤＮＡの塩基配列を示す。
〔配列番号：８３〕
ＧＰＲ８に対するリガンドペプチドのマウスホモログの前駆体タンパク質をコードするｃＤＮＡの５’上流側ＤＮＡの塩基配列を示す。
〔配列番号：８４〕
ＧＰＲ８に対するリガンドペプチドのマウスホモログの前駆体タンパク質をコードするｃＤＮＡの３’下流側配列を得るのに使用した合成ＤＮＡの塩基配列を示す。
〔配列番号：８５〕
ＧＰＲ８に対するリガンドペプチドのマウスホモログの前駆体タンパク質をコードするｃＤＮＡの３’下流側配列を得るのに使用した合成ＤＮＡの塩基配列を示す。
〔配列番号：８６〕
ＧＰＲ８に対するリガンドペプチドのマウスホモログの前駆体タンパク質をコードするｃＤＮＡの３’下流側ＤＮＡの塩基配列を示す。
〔配列番号：８７〕
ＧＰＲ８に対するリガンドペプチドのマウスホモログの前駆体タンパク質をコードするｃＤＮＡを得るのに使用した合成ＤＮＡの塩基配列を示す。
〔配列番号：８８〕
ＧＰＲ８に対するリガンドペプチドのマウスホモログの前駆体タンパク質をコードするｃＤＮＡを得るのに使用した合成ＤＮＡの塩基配列を示す。
〔配列番号：８９〕
ＧＰＲ８に対するリガンドペプチドのマウスホモログの前駆体タンパク質をコードするｃＤＮＡの配列を示す。
〔配列番号：９０〕
ＧＰＲ８に対するリガンドペプチドのマウスホモログの前駆体タンパク質のアミノ酸配列を示す。
〔配列番号：９１〕
配列番号：９０から推定されたＧＰＲ８に対するリガンドペプチドのマウスホモログのアミノ酸配列を示す。
〔配列番号：９２〕
配列番号：９０から推定されたＧＰＲ８に対するリガンドペプチドのマウスホモログのアミノ酸配列を示す。
〔配列番号：９３〕
配列番号：９１で表されるアミノ酸配列をコードする塩基配列を示す。
〔配列番号：９４〕
配列番号：９２で表されるアミノ酸配列をコードする塩基配列を示す。
〔配列番号：９５〕
後述の参考例４４で合成されたヒトＧＰＲ８リガンド（１−２３）酸化体のアミノ酸配列を示す。
〔配列番号：９６〕
後述の参考例４５で合成されたヒトＧＰＲ８リガンド（１−２２）のアミノ酸配列を示す。
〔配列番号：９７〕
後述の参考例４６で合成されたヒトＧＰＲ８リガンド（１−２１）のアミノ酸配列を示す。
〔配列番号：９８〕
後述の参考例４７で合成されたヒトＧＰＲ８リガンド（１−２０）のアミノ酸配列を示す。
〔配列番号：９９〕
後述の参考例４８で合成されたヒトＧＰＲ８リガンド（１−１９）のアミノ酸配列を示す。
〔配列番号：１００〕
後述の参考例４９で合成されたヒトＧＰＲ８リガンド（１−１８）のアミノ酸配列を示す。
【００８０】
〔配列番号：１０１〕
後述の参考例５０で合成されたヒトＧＰＲ８リガンド（１−１７）のアミノ酸配列を示す。
〔配列番号：１０２〕
後述の参考例５１で合成されたヒトＧＰＲ８リガンド（１−１６）のアミノ酸配列を示す。
〔配列番号：１０３〕
後述の参考例５４で合成されたＧＰＲ８リガンド（１−２３）酸化体のアミノ酸配列を示す。
〔配列番号：１０４〕
後述の参考例５５で合成されたラットあるいはマウスＧＰＲ８リガンド（１−２３）酸化体のアミノ酸配列を示す。
〔配列番号：１０５〕
後述の参考例１２で合成されたＦｍｏｃ化ヒトＧＰＲ８リガンド（１−２３）のアミノ酸配列を示す。
〔配列番号：１０６〕
後述の参考例５６で合成された［Ｎα−Ａｃｅｔｙｌ−Ｔｒｐ^１］−ヒトＧＰＲ８リガンド（１−２３）のアミノ酸配列を示す。
〔配列番号：１０７〕
後述の参考例５７で合成されたヒトＧＰＲ８リガンド（２−２３）のアミノ酸配列を示す。
〔配列番号：１０８〕
後述の参考例５８で合成されたヒトＧＰＲ８リガンド（４−２３）のアミノ酸配列を示す。
〔配列番号：１０９〕
後述の参考例５９で合成されたヒトＧＰＲ８リガンド（９−２３）のアミノ酸配列を示す。
〔配列番号：１１０〕
後述の参考例６０で合成されたヒトＧＰＲ８リガンド（１５−２３）のアミノ酸配列を示す。
〔配列番号：１１１〕
後述の参考例６１で合成された［Ｎ−Ａｃｅｔｙｌ−Ｔｙｒ^２］−ヒトＧＰＲ８リガンド（２−２３）のアミノ酸配列を示す。
〔配列番号：１１２〕
後述の参考例６２で合成された［Ｄ−Ｔｒｐ^１］−ヒトＧＰＲ８リガンド（１−２３）のアミノ酸配列を示す。
〔配列番号：１１３〕
後述の参考例６３で合成された［Ｎ−３−Ｉｎｄｏｌｅｐｒｏｐａｎｏｙｌ−Ｔｙｒ^２］−ヒトＧＰＲ８リガンド（２−２３）のアミノ酸配列を示す。
〔配列番号：１１４〕
配列番号：９６で表されるアミノ酸配列をコードする塩基配列を示す。
〔配列番号：１１５〕
配列番号：９７で表されるアミノ酸配列をコードする塩基配列を示す。
〔配列番号：１１６〕
配列番号：９８で表されるアミノ酸配列をコードする塩基配列を示す。
〔配列番号：１１７〕
配列番号：９９で表されるアミノ酸配列をコードする塩基配列を示す。
〔配列番号：１１８〕
配列番号：１００で表されるアミノ酸配列をコードする塩基配列を示す。
〔配列番号：１１９〕
配列番号：１０１で表されるアミノ酸配列をコードする塩基配列を示す。
〔配列番号：１２０〕
配列番号：１０２で表されるアミノ酸配列をコードする塩基配列を示す。
〔配列番号：１２１〕
配列番号：１０７で表されるアミノ酸配列をコードする塩基配列を示す。
〔配列番号：１２２〕
配列番号：１０８で表されるアミノ酸配列をコードする塩基配列を示す。
〔配列番号：１２３〕
配列番号：１０９で表されるアミノ酸配列をコードする塩基配列を示す。
〔配列番号：１２４〕
配列番号：１１０で表されるアミノ酸配列をコードする塩基配列を示す。
〔配列番号：１２５〕
配列番号：６で表されるアミノ酸配列をコードする塩基配列を示す。
〔配列番号：１２６〕
本発明のラット由来新規Ｇタンパク質共役型レセプタータンパク質ＴＧＲ２６のアミノ酸配列を示す。
〔配列番号：１２７〕
本発明のラット由来新規Ｇタンパク質共役型レセプタータンパク質ＴＧＲ２６をコードするｃＤＮＡの塩基配列を示す。
〔配列番号：１２８〕
以下の参考例６７におけるＰＣＲ反応で使用したプライマー１の塩基配列を示す。
〔配列番号：１２９〕
以下の参考例６７におけるＰＣＲ反応で使用したプライマー２の塩基配列を示す。
〔配列番号：１３０〕
以下の参考例６８におけるＴＧＲ２６発現ＣＨＯ細胞のＴＧＲ２６レセプター遺伝子発現量を測定するのに使用したプライマーの塩基配列を示す。
〔配列番号：１３１〕
以下の参考例６８におけるＴＧＲ２６発現ＣＨＯ細胞のＴＧＲ２６遺伝子発現量を測定するのに使用したプライマーの塩基配列を示す。
〔配列番号：１３２〕
以下の参考例７５におけるＰＣＲ反応で使用したプライマーの塩基配列を示す。
〔配列番号：１３３〕
以下の参考例７５におけるＰＣＲ反応で使用したプライマーの塩基配列を示す。
〔配列番号：１３４〕
以下の参考例７５で得られたマウスＴＧＲ２６をコードするＤＮＡの５’上流端部分の塩基配列を示す。
〔配列番号：１３５〕
以下の参考例２４におけるＰＣＲ反応で使用したプライマーの塩基配列を示す。
〔配列番号：１３６〕
以下の参考例２４におけるＰＣＲ反応で使用したプライマーの塩基配列を示す。
〔配列番号：１３７〕
以下の参考例２４で得られたマウスＴＧＲ２６をコードするｃＤＮＡの塩基配列を示す。
〔配列番号：１３８〕
マウス由来ＴＧＲ２６のアミノ酸配列を示す。
〔配列番号：１３９〕
マウス由来ＴＧＲ２６をコードするｃＤＮＡの塩基配列を示す。
〔配列番号：１４０〕
以下の参考例６８におけるＴＧＲ２６発現ＣＨＯ細胞のＴＧＲ２６遺伝子発現量を測定するのに使用したプローブの塩基配列を示す。
〔配列番号：１４１〕
ヒトＧＰＲ７をコードするｃＤＮＡのスクリーニングに使用した合成ＤＮＡの塩基配列を示す。
〔配列番号：１４２〕
ヒトＧＰＲ７をコードするｃＤＮＡのスクリーニングに使用した合成ＤＮＡの塩基配列を示す。
〔配列番号：１４３〕
５’側に制限酵素ＣｌａＩの認識する塩基配列が付加され、また３’側に制限酵素ＳｐｅＩの認識する塩基配列が付加されたヒトＧＰＲ７タンパク質ｃＤＮＡの全塩基配列を示す。
〔配列番号：１４４〕
ヒトＧＰＲ７の全アミノ酸配列を示す。
〔配列番号：１４５〕
標準ヒトＧＰＲ７　ＤＮＡを増幅するのにプライマーとして使用したＤＮＡ塩基配列を示す。
〔配列番号：１４６〕
標準ヒトＧＰＲ７　ＤＮＡを増幅するのにプライマーとして使用したＤＮＡ塩基配列を示す。
〔配列番号：１４７〕
ヒトＧＰＲ７発現ＣＨＯ細胞のＧＰＲ７遺伝子発現量を測定するのにプライマーとして用いた合成ＤＮＡの塩基配列を示す。
〔配列番号：１４８〕
ヒトＧＰＲ７発現ＣＨＯ細胞のＧＰＲ７遺伝子発現量を測定するのにプライマーとして用いた合成ＤＮＡの塩基配列を示す。
〔配列番号：１４９〕
［Ｐｈｅ^２］ヒトＧＰＲ８リガンド（１−２０）のアミノ酸配列を示す。
〔配列番号：１５０〕
配列番号：１４９で表されるアミノ酸配列をコードする塩基配列を示す。
【００８１】
後述の参考例３で得られた形質転換体Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ　ＤＨ５α／ｐＡＫＫＯ−ＧＰＲ８は、大阪府大阪市淀川区十三本町２丁目１７番８５号（郵便番号５３２−８６８６）、財団法人発酵研究所（ＩＦＯ）に２００１年２月２７日から寄託番号ＩＦＯ１６５６４として、茨城県つくば市東１丁目１番地１　中央第６（郵便番号３０５−８５６６）、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２００１年４月１１日から受託番号ＦＥＲＭ　ＢＰ−７５４０として、それぞれ寄託されている。
後述の参考例２８で得られた形質転換体Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ　ＴＯＰ１０／ｐＣＲ２．１−ＴＯＰＯＨｕｍａｎ　ＧＰＲ８　Ｌｉｇａｎｄ　Ｐｒｅｃｕｒｓｏｒは、大阪府大阪市淀川区十三本町２丁目１７番８５号（郵便番号５３２−８６８６）、財団法人発酵研究所（ＩＦＯ）に２００１年２月２７日から寄託番号ＩＦＯ　１６５６８として、茨城県つくば市東１丁目１番地１　中央第６（郵便番号３０５−８５６６）、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２００１年４月１１日から受託番号ＦＥＲＭ　ＢＰ−７５４４として、それぞれ寄託されている。
後述の参考例３２で得られた形質転換体Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ　ＴＯＰ１０／ｐＣＲ２．１−ＴＯＰＯＰｏｒｃｉｎｅ　ＧＰＲ８　Ｌｉｇａｎｄ　Ｐｒｅｃｕｒｓｏｒは、大阪府大阪市淀川区十三本町２丁目１７番８５号（郵便番号５３２−８６８６）、財団法人発酵研究所（ＩＦＯ）に２００１年２月２７日から寄託番号ＩＦＯ　１６５６５として、茨城県つくば市東１丁目１番地１　中央第６（郵便番号３０５−８５６６）、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２００１年４月１１日から受託番号ＦＥＲＭ
ＢＰ−７５４１として、それぞれ寄託されている。
後述の参考例３６で得られた形質転換体Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ　ＴＯＰ１０／ｐＣＲ２．１−ＴＯＰＯＲａｔ　ＧＰＲ８　Ｌｉｇａｎｄ　Ｐｒｅｃｕｒｓｏｒは、大阪府大阪市淀川区十三本町２丁目１７番８５号（郵便番号５３２−８６８６）、財団法人発酵研究所（ＩＦＯ）に２００１年２月２７日から寄託番号ＩＦＯ　１６５６７、茨城県つくば市東１丁目１番地１中央第６（郵便番号３０５−８５６６）、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２００１年４月１１日から受託番号ＦＥＲＭ　ＢＰ−７５４３として、それぞれ寄託されている。
後述の参考例４１で得られた形質転換体Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ　ＴＯＰ１０／ｐＣＲ２．１−ＴＯＰＯＭｏｕｓｅ　ＧＰＲ８　Ｌｉｇａｎｄ　Ｐｒｅｃｕｒｓｏｒは、大阪府大阪市淀川区十三本町２丁目１７番８５号（郵便番号５３２−８６８６）、財団法人発酵研究所（ＩＦＯ）に２００１年２月２７日から寄託番号ＩＦＯ　１６５６６として、茨城県つくば市東１丁目１番地１　中央第６（郵便番号３０５−８５６６）、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２００１年４月１１日から受託番号ＦＥＲＭ　ＢＰ−７５４２として、それぞれ寄託されている。
後述の参考例６７で得られた形質転換体　大腸菌（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）ＤＨ１０Ｂ／ｐＡＫ−ｒＧＰＲ７は、２０００年１０月３１日から大阪府大阪市淀川区十三本町２丁目１７番８５号（郵便番号５３２−８６８６）、財団法人・発酵研究所（ＩＦＯ）に受託番号ＩＦＯ　１６４９６として、２０００年１１月１３日から茨城県つくば市東１丁目１番地１　中央第６（郵便番号３０５−８５６６）、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（旧通商産業省工業技術院生命工学工業技術研究所（ＮＩＢＨ））に受託番号ＦＥＲＭ　ＢＰ−７３６５としてそれぞれ寄託されている。
後述の参考例２４で得られた形質転換体　大腸菌（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）ＴＯＰ１０／ｐＣＲ２．１−ＴＯＰＯＭｏｕｓｅＧＰＲ７は、２００１年９月２０日から大阪府大阪市淀川区十三本町２丁目１７番８５号（郵便番号５３２−８６８６）、財団法人・発酵研究所（ＩＦＯ）に受託番号ＩＦＯ　１６７０４として、２００１年１０月１５日から茨城県つくば市東１丁目１番地１　中央第６（郵便番号３０５−８５６６）、独立行政法人産業技術総合研究所　特許生物寄託センターに受託番号ＦＥＲＭ　ＢＰ−７７７５としてそれぞれ寄託されている。
【００８２】
【実施例】
以下に参考例および実施例を示して、本発明をより詳細に説明するが、これらは本発明の範囲を限定するものではない。
【００８３】
参考例１　ヒト脳由来ｃＤＮＡを用いたＰＣＲ法によるヒトＧＰＲ８　ｃＤＮＡの増幅
ヒト脳由来ｐｏｌｙ　（Ａ）　^＋ＲＮＡ（クローンテック）を鋳型として、ランダムプライマーを用いて逆転写反応を行なった。逆転写は、タカラＲＮＡ　ＰＣＲ　ｖｅｒ　２．１キット試薬を使用した。次にこの逆転写生成物を鋳型として用い、配列番号：１および配列番号：２で表される合成プライマーを用いてＰＣＲ法による増幅を行なった。合成プライマーは受容体タンパク質に翻訳される領域の遺伝子が増幅されるように構築したが、その際に遺伝子の５’側に制限酵素ＣｌａＩの認識する塩基配列が付加され、３’側に制限酵素ＳｐｅＩの認識する塩基配列が付加されるように、５’側および３’側にそれぞれの制限酵素の認識配列を付加した。反応液の組成は、ｃＤＮＡ鋳型５μｌ，合成ＤＮＡプライマー各０．４μＭ、０．８ｍＭ　ｄＮＴＰｓ、Ｐｆｕポリメラーゼ（ストラタジーン）０．５μｌおよび酵素に付属のバッファーで、総反応量は５０μｌとした。増幅のためのサイクルはサーマルサイクラー（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）を用い、９４℃・６０秒の加熱の後、９４℃・６０秒、６５℃・６０秒、７２℃・１５０秒のサイクルを３５回繰り返した。増幅産物の確認は、０．８％アガロースゲル電気泳動の後、エチジウムブロマイド染色によって行なった。
【００８４】
参考例２　ＰＣＲ産物のプラスミドベクターへのサブクローニングおよび挿入ｃＤＮＡ部分の塩基配列の解読による増幅ｃＤＮＡ配列の確認
参考例１で行なったＰＣＲ反応液を０．８％の低融点アガロースゲル電気泳動により分離し、バンドの部分をかみそりで切り出した後、細片化、フェノール抽出、フェノール・クロロホルム抽出、エタノール沈殿の操作を行なってＤＮＡを回収した。ＰＣＲ−Ｓｃｒｉｐｔ^ＴＭ　Ａｍｐ　ＳＫ（＋）クローニングキット（ストラタジーン）の処方に従い、回収したＤＮＡをプラスミドベクターｐＣＲ−Ｓｃｒｉｐｔ　Ａｍｐ　ＳＫ（＋）へサブクローニングした。これをエシェリヒアコリ（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）　ＤＨ５αｃｏｍｐｅｔｅｎｔ　ｃｅｌｌ（東洋紡）に導入して形質転換した後、ｃＤＮＡ挿入断片を持つクローンをアンピシリン、ＩＰＴＧおよびＸ−ｇａｌを含むＬＢ寒天培地で選択し、白色を呈するクローンのみを滅菌したつま楊枝を用いて分離し、形質転換体Ｅ．　ｃｏｌｉ　ＤＨ５α／ＧＰＲ８を得た。個々のクローンをアンピシリンを含むＬＢ培地で一晩培養し、ＱＩＡｗｅｌｌ　８　Ｐｌａｓｍｉｄ　Ｋｉｔ（キアゲン）を用いてプラスミドＤＮＡを調製した。調製したＤＮＡの一部に対して制限酵素ＣｌａＩおよびＳｐｅＩによる切断を行ない、挿入されている受容体ｃＤＮＡ断片の大きさを確認した。塩基配列の決定のための反応はＤｙｅＤｅｏｘｙＴｅｒｍｉｎａｔｏｒ　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｅ　Ｋｉｔ（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）を用いて行ない、蛍光式自動シーケンサーを用いて解読した（配列番号：３）。ヒトＧＰＲ８受容体タンパク質ｃＤＮＡの全塩基配列が配列番号：３に、およびそれから翻訳されるヒトＧＰＲ８受容体タンパク質の全アミノ酸配列が配列番号：４に示されている。
【００８５】
参考例３　ＧＰＲ８発現ＣＨＯ細胞の作製
参考例２で配列が確認されたヒト脳由来のＧＰＲ８の全長アミノ酸配列をコードし５’側にＣｌａＩ認識配列を付加し、また３’側にＳｐｅＩ認識配列を付加した遺伝子が導入されたプラスミドによって形質転換されたＥ．　ｃｏｌｉのクローンからＰｌａｓｍｉｄ　Ｍｉｄｉ　ＫＩｔ（キアゲン）を用いてプラスミドＤＮＡを調製し、これを制限酵素ＣｌａＩおよびＳｐｅＩで消化してインサートＤＮＡを切り出した。インサートＤＮＡは電気泳動後、アガロースゲルからカミソリで切り出し、次に細片化、フェノール抽出、フェノール・クロロホルム抽出、エタノール沈殿の操作により回収された。このインサートＤＮＡをＣｌａＩおよびＳｐｅＩで切断した動物細胞発現用ベクタープラスミドｐＡＫＫＯ−１１１Ｈ（Ｓ．　Ｈｉｎｕｍａ　ｅｔ　ａｌ．、Ｂｉｏｃｈｉｍ．　Ｂｉｏｐｈｙｓ．　Ａｃｔａ、１２１９巻、２５１−２５９頁、１９９４年、記載のｐＡＫＫＯ１．１１Ｈと同一のベクタープラスミド）に加え、Ｔ４リガーゼ（宝酒造）を用いてライゲーションを行ない、タンパク質発現用プラスミドｐＡＫＫＯ−ＧＰＲ８を構築した。このプラスミドｐＡＫＫＯ−ＧＰＲ８で形質転換した大腸菌をＤＨ５α／ｐＡＫＫＯ−ＧＰＲ８（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ　ＤＨ５α／ｐＡＫＫＯ−ＧＰＲ８）と命名した。
ｐＡＫＫＯ−ＧＰＲ８で形質転換したＥ．　ｃｏｌｉ　ＤＨ５α（東洋紡）を培養後、Ｐｌａｓｍｉｄ　Ｍｉｄｉ　Ｋｉｔ（キアゲン）を用いてｐＡＫＫＯ−ＧＰＲ８プラスミドＤＮＡを調製した。これをＣｅｌｌＰｈｅｃｔ　Ｔｒａｎｓｆｅｃｔｉｏｎ　Ｋｉｔ（アマシャムファルマシアバイオテク）を用いて、添付のプロトコールに従ってＣＨＯ　ｄｈｆｒ⁻細胞に導入した。４．５μｇのＤＮＡをリン酸カルシウムとの共沈懸濁液とし、２４時間前に５　ｘ　１０^５または１　ｘ　１０^６個のＣＨＯ　ｄｈｆｒ⁻細胞を播種した直径６ｃｍシャーレに添加した。１０％ウシ胎児血清を含むＭＥＭα培地で１日間培養した後、継代し、選択培地である１０％透析ウシ胎児血清を含む核酸不含ＭＥＭα培地で培養した。選択培地中に増殖してくるＧＰＲ８発現ＣＨＯ細胞である形質転換細胞のコロニー４７クローンを選択した。
【００８６】
参考例４　全長ヒトＧＰＲ８タンパク質ｍＲＮＡの発現量の高いＣＨＯ／ＧＰＲ８細胞株の選択
参考例３で樹立されたＣＨＯ／ＧＰＲ８細胞株４７クローンの全長ＧＰＲ８タンパク質ｍＲＮＡの発現量をＣｙｔｏｓｔａｒ　Ｔ　Ｐｌａｔｅ（アマシャムファルマシアバイオテク）を用い、添付のプロトコールに従って以下のように測定した。ＣＨＯ／ＧＰＲ８細胞株の各クローンをＣｙｔｏｓｔａｒ　Ｔ　Ｐｌａｔｅに１ｗｅｌｌ当たり２．５　ｘ　１０^４個ずつ播種して２４時間培養した後、１０％ホルマリンによって細胞を固定した。各ｗｅｌｌに０．２５％　Ｔｒｉｔｏｎ　Ｘ−１００を添加して細胞の透過性をあげた後、^３５Ｓラベルした配列番号：５のｒｉｂｏｐｒｏｂｅを加えてハイブリダイズさせた。２０μｇ／ｍｌのＲＮａｓｅ　Ａを各ｗｅｌｌに加えて遊離のｒｉｂｏｐｒｏｂｅを消化し、プレートをよく洗浄した後、ハイブリダイズしたｒｉｂｏｐｒｏｂｅの放射活性をＴｏｐｃｏｕｎｔｅｒで測定した。放射活性の高い細胞株は、ｍＲＮＡ発現量が高い。ｍＲＮＡ発現量の高い３クローン（＃１７，４１および４６）を以下に実験に用いたが、特にクローン番号１７を用いた。
【００８７】
参考例５　ＧＰＲ８発現ＣＨＯ細胞を用いた細胞内ｃＡＭＰ産生量の測定
参考例４で作製したＣＨＯ／ＧＰＲ８細胞およびｍｏｃｋ　ＣＨＯ細胞を２４穴プレートに５　ｘ　１０^４　ｃｅｌｌ／ｗｅｌｌで播種し、４８時間培養した。細胞を０．２ｍＭ　３−イソブチル−メチルキサンチンと０．０５％　ＢＳＡと２０ｍＭ　ＨＥＰＳを含むハンクスバッファー（ｐＨ７．４）で洗浄した（以下、０．２ｍＭ　３−イソブチル−メチルキサンチンと０．０５％　ＢＳＡと２０ｍＭ　ＨＥＰＳを含むハンクスバッファー（ｐＨ７．４）を、反応用バッファーと呼ぶ）。その後０．５ｍｌの反応用バッファーを加えて３０分間培養器で保温した。反応用バッファーを除き、新たに０．２５ｍｌの反応用バッファーを細胞に加えた後、試料と２μＭフォルスコリンを含む０．２５ｍｌの反応用バッファーを細胞に加え、３７℃で２４分間反応させた。１００μｌの２０％過塩素酸を加えて反応を停止させ、次に氷上で１時間置くことにより細胞内ｃＡＭＰを抽出した。抽出液中のｃＡＭＰ量は、ｃＡＭＰ　ＥＩＡキット（アマシャムファルマシアバイオテク）を用いて測定した。
【００８８】
参考例６　ＧＰＲ８発現ＣＨＯ細胞の膜画分を用いたＧＴＰγＳ結合活性の測定
ＧＰＲ８発現ＣＨＯ細胞膜画分に対する　［^３５Ｓ］−Ｇｕａｎｏｓｉｎｅ　５’−（γ−ｔｈｉｏ）ｔｒｉｐｈｏｓｐｈａｔｅの結合促進活性を以下の方法により測定した。最初に膜画分の調製法を記載する。１　ｘ　１０^８個のＣＨＯ／ＧＰＲ８細胞に１０ｍｌのホモジネートバッファー（１０ｍＭ　ＮａＨＣＯ_３，　５ｍＭ　ＥＤＴＡ，　０．５ｍＭ　ＰＭＳＦ，　１μｇ／ｍｌ　ｐｅｐｓｔａｔｉｎ，　４μｇ／ｍｌ　Ｅ６４，　２０μｇ／ｍｌ　ｌｅｕｐｅｐｔｉｎ）添加し、ポリトロン（１２，０００　ｒｐｍ、１分間）を用いて破砕した。細胞破砕液を遠心（１，０００　ｇ，　１５分間）して上清を得た。次にこの上清を超遠心分離（Ｂｅｃｋｍａｎ　ｔｙｐｅ　３０ローター、３０，０００　ｒｐｍ，　１時間）し、得られた沈殿物をＧＰＲ８発現ＣＨＯ細胞膜画分とした。
ＧＴＰγＳ結合活性の測定は以下の通りである。ＧＰＲ８発現ＣＨＯ細胞膜画分を膜希釈緩衝液（５０ｍＭトリス塩酸緩衝液（ｐＨ　７．４），　５ｍＭ　ＭｇＣｌ_２，　１５０ｍＭ　ＮａＣｌ，　１μＭ　ＧＤＰ）で希釈して、タンパク質濃度３０　ｍｇ／ｍｌのアッセイ用細胞膜画分溶液を調製した。アッセイ用膜画分溶液２００μｌに、５１．５ｎＭ濃度の［^３５Ｓ］−Ｇｕａｎｏｓｉｎｅ　５’−（γ−ｔｈｉｏ）ｔｒｉｐｈｏｓｐｈａｔｅ（ＮＥＮ社）を２μｌと試料を添加し、この混合液を２５℃で一時間保温した。混合液をフィルター濾過し、さらにフィルターを洗浄用バッファー（５０ｍＭトリス塩酸緩衝液（ｐＨ　７．４），　５ｍＭ　ＭｇＣｌ_２，　１ｍＭ　ＥＤＴＡ，　０．１％　ＢＳＡ）１．５ｍｌで２回洗浄した後、フィルターの放射活性を液体シンチレーションカウンターで測定した。
【００８９】
参考例７　ブタ視床下部抽出物に含まれ、ＣＨＯ／ＧＰＲ８細胞株に対して特異的にｃＡＭＰ産生抑制およびＧＴＰγＳ結合促進を示す活性の検出
ブタ視床下部抽出物の高速液体クロマトグラフィー（ＨＰＬＣ）フラクションを以下に述べる方法で調製した。東京芝浦臓器（株）より購入した、処理当日に屠殺して摘出後は氷冷保存したブタ視床下部５００　ｇ（３０頭分）を細かく切断し、直ぐに沸騰した蒸留水２．０　ｌに投じて１０分間煮沸した。煮沸後、直ちに氷冷し、１２０　ｍｌの酢酸を加えて終濃度１．０　Ｍとし、ポリトロン（２０，０００　ｒｐｍ、６分間）を用いて破砕した。破砕液を遠心（８，０００　ｒｐｍ、３０分）して上清を取り、沈殿には１．０Ｍ酢酸２．０　ｌを加えて再度ポリトロンによって破砕し、一晩攪拌した後、遠心（８，０００　ｒｐｍ、３０分）して上清を得た。上清に２倍量の冷アセトンを４℃でゆっくり滴下した後、１回目の遠心によって得た上清については一晩攪拌し、２回目の遠心によって得た上清については４時間攪拌した。アセトンを加えた抽出液は遠心（８，０００　ｒｐｍ、３０分）して沈殿を除き、得られた上清からエバポレーターによって減圧下にアセトンを溜去した。アセトンを除いた抽出液に等量のジエチルエーテルを加え、分液ロートを使って脂質を含むエーテル層を分離して水層を回収した。エーテル脱脂した抽出液はエバポレーターによって減圧下に濃縮してエーテルを完全に除去した。濃縮液をガラス繊維濾紙（アドバンテック、ＤＰ７０　（９０　ｍｍφ））で濾過し、濾液をガラス製カラム（３０φ　ｘ　２４０　ｍｍ）に充填したＣ１８（ワイエムシー、ＹＭＣｇｅｌ　ＯＤＳ−ＡＭ　１２０−Ｓ５０）カラムに添加した。カラムを１．０　Ｍ酢酸４００　ｍｌで洗浄後、０．１％トリフルオロ酢酸を含む６０％アセトニトリル５００　ｍｌで溶出した。溶出液を減圧下に濃縮して溶媒を溜去した後、濃縮液を凍結乾燥した。凍結乾燥品約０．５　ｇを３０　ｍｌの０．１％トリフルオロ酢酸を含む１０％アセトニトリルに溶解し、１０　ｍｌずつをＣ１８カラム（トーソー、ＴＳＫｇｅｌ　ＯＤＳ−８０ＴＳ　（２１．５φ　ｘ　３００　ｍｍ））を用いた１０％から６０％の０．１％トリフルオロ酢酸を含むアセトニトリルの濃度勾配溶出法によるＨＰＬＣにかけた。ＨＰＬＣは３回行なった。溶出液は６０分画に分取し、３回分の溶出液をまとめた。各分画を減圧下に濃縮・乾固し、残渣を０．５　ｍｌのジメチルスルフオキシド（ＤＭＳＯ）で溶解した。
上記によって得られたＨＰＬＣフラクションのＤＭＳＯ溶液を参考例５に示した方法によってＣＨＯ／ＧＰＲ８細胞に投与し、細胞内ｃＡＭＰ産生量の測定を行なった結果、分画番号３０に顕著なｃＡＭＰ産生抑制活性が認められた。また同様な試料についてＧＰＲ８発現ＣＨＯ細胞用いてＧＴＰγＳ結合促進活性を調べたところ、やはり分画番号３０付近に顕著な活性が確認された。これらの活性は他の受容体発現細胞では認められなかったことからブタ視床下部抽出物にＧＰＲ８特異的なリガンド活性物質が存在することが示された。
【００９０】
参考例８　ブタ視床下部抽出物中のＧＰＲ８発現ＣＨＯ細胞に対して特異的に細胞内ｃＡＭＰ産生抑制活性を示す活性物質のプロナーゼによる失活
参考例７でＧＰＲ８発現ＣＨＯ細胞に対して細胞内ｃＡＭＰ産生抑制活性を示したＨＰＬＣ分画３０をタンパク質分解酵素であるプロナーゼ（Ｓｉｇｍａ，　ｐｒｏｔｅａｓｅ　Ｔｙｐｅ　ＸＩＶ　（Ｐ５１４７））で処理し、活性物質が蛋白性であるかを調べた。上記視床下部抽出物ＨＰＬＣ分画（＃３０）２μｌを０．２　Ｍ酢酸アンモニウム２００μｌに加え、これにプロナーゼ３μｇを添加して３７℃で２時間インキュベートした後、沸騰水中で１０分間加熱してプロナーゼを失活させた。これにＢＳＡ　０．０５ｍｇおよびＣＨＡＰＳ　０．０５　ｍｇを含む蒸留水２　ｍｌを加えてから凍結乾燥した。プロナーゼそのものあるいは加熱および凍結乾燥の影響を調べるため、プロナーゼのみ、ＨＰＬＣ分画のみおよびプロナーゼのみを加熱処理した後にＨＰＬＣ分画を加えたものについても同様に処理して凍結乾燥した。凍結乾燥した各試料を参考例５に示す方法によってＧＰＲ８発現ＣＨＯ細胞に添加して細胞内ｃＡＭＰ産生抑制活性を測定した。ブタ視床下部抽出物中のＧＰＲ８発現ＣＨＯ細胞に対する細胞内ｃＡＭＰ産生抑制活性を示す活性物質はプロナーゼによって完全に失活したことからこの物質がタンパク質もしくはペプチドであることが示された。
【００９１】
参考例９　ブタ視床下部からのＧＰＲ８発現ＣＨＯ細胞膜画分に対して特異的にＧＴＰγＳ結合促進活性を示す活性物質の精製
ＧＰＲ８に特異的なリガンド活性を示す物質をＧＰＲ８発現ＣＨＯ細胞膜画分に対するＧＴＰγＳ結合促進活性を指標としてブタ視床下部から精製した代表例を以下に具体的に述べる。参考例７に述べた方法と全く同一の方法により、ブタ視床下部５００　ｇ（３０頭分）を１．０　Ｍ酢酸で抽出し、アセトン沈殿およびエーテル脱脂をした後、Ｃ１８（ワイエムシー、ＹＭＣｇｅｌ　ＯＤＳ−ＡＭ　１２０−Ｓ５０）カラムに吸着させ、０．１％トリフルオロ酢酸を含む６０％アセトニトリルで溶出した。溶出液を濃縮し、凍結乾燥した後、Ｃ１８カラム（トーソー、ＴＳＫｇｅｌ　ＯＤＳ−８０ＴＳ　（２１．５φ　ｘ　３００　ｍｍ））を用いたＨＰＬＣによって活性分画を得た。活性は分画番号３０に回収された。これを以下の方法によってさらに精製した。
この分画を１０％アセトニトリルを含む１０　ｍＭギ酸アンモニウム１０　ｍｌに溶解し、陽イオン交換カラム（トーソー、ＴＳＫｇｅｌ　ＳＰ−５ＰＷ　（２０　ｍｍφ　ｘ　１５０　ｍｍ））に添加した後、１０％アセトニトリルを含む１０　ｍＭから２．０　Ｍのギ酸アンモニウムの濃度勾配によってカラムを溶出した。活性はギ酸アンモニウム０．８　Ｍ付近に回収された。活性分画を凍結乾燥し、０．１％トリフルオロ酢酸を含む１０％アセトニトリル１．０　ｍｌに溶解し、ＣＮカラム（野村化学、Ｄｅｖｅｌｏｓｉｌ　ＣＮ−ＵＧ−５　（４．６　ｍｍφｘ　２５０　ｍｍ））に添加した後、０．１％トリフルオロ酢酸を含む２１％から２６％のアセトニトリルの濃度勾配によって溶出した。活性はアセトニトリル２２．１％付近に出現した。活性分画を凍結乾燥し、０．１　ｍｌのＤＭＳＯで溶解し、さらに０．４　ｍｌの０．１％トリフルオロ酢酸を含む１０％アセトニトリルを加えてＯＤＳカラム（和光純薬、Ｗａｋｏｓｉｌ−ＩＩ　３Ｃ１８ＨＧ（２．０　ｍｍφｘ　１５０　ｍｍ））に添加した後、０．１％トリフルオ酢酸を含む２２．５％から３２．５％のアセトニトリルの濃度勾配によって溶出した。活性はアセトニトリル２６．５％付近に単一ピークとして出現した（図１）。
【００９２】
参考例１０　ブタ視床下部から精製されたＧＰＲ８発現ＣＨＯ細胞膜画分に対して特異的にＧＴＰγＳ結合促進活性を示す活性物質のアミノ末端アミノ酸配列の解析およびＧＰＲ８リガンドのヒトおよびラットホモログペプチドの前駆体タンパク質の一部をコードしていると推定されるＥＳＴ配列
参考例９で精製されたＧＰＲ８発現ＣＨＯ細胞膜画分に対して特異的にＧＴＰγＳ結合促進活性を示す活性物質のアミノ末端アミノ酸配列解析を行なった。本活性物質は参考例８に示すようにタンパク質またはペプチドであることが推定されていたので、活性ピークを含む溶出液を用いてパーキンエルマー社Ｐｒｏｃｉｓｅ　４９４　Ｐｒｏｔｅｉｎ　Ｓｅｑｕｅｎｃｅｒによってアミノ末端アミノ酸配列分析を行なった。その結果、アミノ末端から１７残基までに配列番号：６に示す配列が得られた。この配列はリガンドペプチドの一部であると考えられた。
【００９３】
この配列に基づいて遺伝子データベースの検索を行なったところ、そのものもしくはその相補鎖がこのペプチドの前駆体タンパク質の一部をコードしていると推定される幾つかのＥＳＴ（Ｅｘｐｒｅｓｓｅｄ　Ｓｅｑｕｅｎｃｅ　Ｔａｇ）配列が見出された。それらのアクセッション番号、ｃＤＮＡの由来、配列の長さおよび配列番号は次の通りである。ＡＷ００７５３１　（ａｎａｐｌａｓｔｉｃ　ｏｌｉｇｏｄｅｎｄｒｏｇｌｉｏｍａ、４３８　ｂａｓｅ、配列番号：７）、ＡＩ５００３０３　（ａｎａｐｌａｓｔｉｃ　ｏｌｉｇｏｄｅｎｄｒｏｇｌｉｏｍａ、　２６４　ｂａｓｅ、配列番号：８）、ＡＩ９９０９６４　（ｃｏｌｏｎｉｃ　ｍｕｃｏｓａ　ｆｒｏｍ　ｐａｔｉｅｎｔ　ｏｆ　Ｃｒｏｈｎ’ｓ　ｄｉｓｅａｓｅ、４２４　ｂａｓｅ、配列番号：９）、ＡＡ７４４８０４　（ｇｅｒｍｉｎａｌ　ｃｅｎｔｅｒ　Ｂ　ｃｅｌｌ、３７５　ｂａｓｅ、配列番号：１０）、Ｈ３１５９８　（ＰＣ１２　ｃｅｌｌｓ、２６０　ｂａｓｅ、配列番号：１１）。初めの４つはヒト由来であり、最後の１つはラット由来である。これらのＥＳＴのＤＮＡ配列はブタ視床下部より単離した活性ペプチドの配列に相当するアミノ酸配列をコードする領域では極めてよく一致してしており、また翻訳されたアミノ酸配列はブタ視床下部より単離され明らかとなったペプチドの配列とは５残基目のＴｈｒがＶａｌであること以外はほぼ一致した。以上からこれらのＥＳＴはＧＰＲ８のリガンドペプチドのヒトおよびラットホモログの前駆体タンパク質の一部をコードしているものと推定した。
【００９４】
参考例１１　ＧＰＲ８リガンドペプチド前駆体の一部をコードするヒトｃＤＮＡの増幅と増幅ｃＤＮＡ配列の解読
参考例１０に述べたＧＰＲ８リガンドペプチドの前駆体タンパク質の一部をコードすると推定されたＥＳＴ配列に基づいてプライマーを設計し、ヒト脳由来ｃＤＮＡよりＰＣＲによってＧＰＲ８リガンドペプチド前駆体の一部をコードするｃＤＮＡを増幅した。
ヒト脳由来ｐｏｌｙ　（Ａ）　＋ＲＮＡ（クローンテック）を鋳型として、ランダムプライマーを用いて逆転写反応を行なった。逆転写反応には、ＲＮａｓｅ　Ｈ活性を欠失させたＭＭＬＶ由来の逆転写酵素であるＲｅｖｅｒＴｒａ　Ａｃｅ（東洋紡）を使用した。続いて参考例１０に述べたＥＳＴ配列に基づいて設計した配列番号：１２および配列番号：１３の合成ＤＮＡプライマーを用いてＰＣＲ法による増幅を行なった。反応液の組成は、ｃＤＮＡ鋳型２μｌ、合成ＤＮＡプライマー各０．５μＭ、１．６ｍＭ　ｄＮＴＰｓ、ＬＡＴａｑ（宝酒造）０．２μｌおよび酵素に付属のバッファーで、総反応量は２０μｌとした。増幅のためのサイクルはサーマルサイクラー（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）を用い、９６℃・１２０秒の加熱の後、９６℃・３０秒、７２℃・４５秒のサイクルを４回、９６℃・３０秒、７０℃・４５秒のサイクルを４回、９６℃・３０秒、６８℃・４５秒のサイクルを４回、９６℃・３０秒、６４℃・３０秒、７２℃・４５秒のサイクルを５回、９６℃・３０秒、６０℃・３０秒、７２℃・４５秒のサイクルを２０回繰り返し、最後に７２℃で１０分間保温した。増幅産物の確認は、３％アガロースゲル電気泳動の後、エチジウムブロマイド染色によって行なった。
ＰＣＲ反応液を３％の低融点アガロースゲル電気泳動により分離し、バンドの部分をかみそりで切り出した後、ＱＩＡｑｕｉｃｋ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　Ｋｉｔ（キアゲン）を用いてＤＮＡを回収した。ＴＯＰＯ　ＴＡクローニングキット（インビトロゲン）の処方に従い、回収したＤＮＡをプラスミドベクターｐＣＲ２．１−ＴＯＰＯへサブクローニングした。これをＥｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ　ＴＯＰ１０（インビトロゲン）に導入して形質転換した後、ｃＤＮＡ挿入断片を持つクローンをアンピシリンおよびＸ−ｇａｌを含むＬＢ寒天培地で選択し、白色を呈するクローンのみを滅菌したつま楊枝を用いて分離し、形質転換体を得た。個々のクローンをアンピシリンを含むＬＢ培地で一晩培養し、ＱＩＡｗｅｌｌ　８　Ｐｌａｓｍｉｄ　ＫＩｔ（キアゲン）を用いてプラスミドＤＮＡを調製した。塩基配列の決定のための反応はＤｙｅＤｅｏｘｙＴｅｒｍｉｎａｔｏｒ　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｅ　Ｋｉｔ（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）を用いて行ない、蛍光式自動シーケンサーを用いて解読し、配列番号：１４に示すＤＮＡ配列を得た。このこの配列から翻訳されるＧＰＲ８リガンドぺプチド前駆体タンパク質の一部（配列番号：１５）には予想どおりブタ視床下部より単離されて配列が明らかになった活性ペプチドに相当するペプチド配列が存在した。さらに、そのＣ末側には通常の生理活性ペプチドが切り出されると考えられるＡｒｇ−Ａｒｇの配列（Ｓｅｉｄａｈ，　Ｎ．　Ｇ．　ｅｔ　ａｌ．、Ａｎｎ．　Ｎ．　Ｙ．　Ａｃａｄ．　Ｓｃｉ．、８３９巻、９−２４頁、１９９８年）が２ヶ所存在した。このことから、ＧＰＲ８のリガンドペプチドのヒトホモログのアミノ酸配列は配列番号：１６および１７のいずれかもしくは両方であると推定された。
【００９５】
参考例１２　Ｆｍｏｃ化ヒトＧＰＲ８　ｌｉｇａｎｄ　（１−２３）：Ｆｍｏｃ−Ｔｒｐ−Ｔｙｒ−Ｌｙｓ−Ｈｉｓ−Ｖａｌ−Ａｌａ−Ｓｅｒ−Ｐｒｏ−Ａｒｇ−Ｔｙｒ−Ｈｉｓ−Ｔｈｒ−Ｖａｌ−Ｇｌｙ−Ａｒｇ−Ａｌａ−Ａｌａ−Ｇｌｙ−Ｌｅｕ−Ｌｅｕ−Ｍｅｔ−Ｇｌｙ−Ｌｅｕ（配列番号：１０５）およびヒトＧＰＲ８　ｌｉｇａｎｄ　（１−２３）：Ｔｒｐ−Ｔｙｒ−Ｌｙｓ−Ｈｉｓ−Ｖａｌ−Ａｌａ−Ｓｅｒ−Ｐｒｏ−Ａｒｇ−Ｔｙｒ−Ｈｉｓ−Ｔｈｒ−Ｖａｌ−Ｇｌｙ−Ａｒｇ−Ａｌａ−Ａｌａ−Ｇｌｙ−Ｌｅｕ−Ｌｅｕ−Ｍｅｔ−Ｇｌｙ−Ｌｅｕ（配列番号：１６）の製造
市販　２−ｃｈｌｏｒｏｔｒｉｔｙｌ　ｒｅｓｉｎ　（Ｃｌｔ　ｒｅｓｉｎ，１．３３ｍｍｏｌ／ｇ）にＦｍｏｃ−Ｌｅｕを導入したＦｍｏｃ−Ｌｅｕ−Ｏ−Ｃｌｔ　ｒｅｓｉｎ（０．７６ｍｍｏｌ／ｇ）　０．２５ｍｍｏｌを出発原料とし、ペプチド合成機ＡＢＩ　４３３Ａを用い　Ｆｍｏｃ／　ＤＣＣ／　ＨＯＢｔ法により、Ｆｍｏｃ−Ｇｌｙ，　Ｆｍｏｃ−Ｍｅｔ，　Ｆｍｏｃ−Ｌｅｕ，　Ｆｍｏｃ−Ｌｅｕ，　Ｆｍｏｃ−Ｇｌｙ，　Ｆｍｏｃ−Ａｌａ，　Ｆｍｏｃ−Ａｌａ，　Ｆｍｏｃ−Ａｒｇ（Ｐｂｆ），　Ｆｍｏｃ−Ｇｌｙ，　Ｆｍｏｃ−Ｖａｌ，　Ｆｍｏｃ−Ｔｈｒ（Ｂｕ^ｔ），　Ｆｍｏｃ−Ｈｉｓ（Ｔｒｔ），　Ｆｍｏｃ−Ｔｙｒ（Ｂｕ^ｔ），　Ｆｍｏｃ−Ａｒｇ（Ｐｂｆ），　Ｆｍｏｃ−Ｐｒｏ，　Ｆｍｏｃ−Ｓｅｒ（Ｂｕ^ｔ），　Ｆｍｏｃ−Ａｌａ，　Ｆｍｏｃ−Ｖａｌ，　Ｆｍｏｃ−Ｈｉｓ（Ｔｒｔ），　Ｆｍｏｃ−Ｌｙｓ（Ｂｏｃ），　Ｆｍｏｃ−Ｔｙｒ（Ｂｕ^ｔ），　Ｆｍｏｃ−Ｔｒｐ（Ｂｏｃ）の順に縮合を行い、Ｆｍｏｃ−Ｔｒｐ（Ｂｏｃ）−Ｔｙｒ（Ｂｕ^ｔ）−Ｌｙｓ（Ｂｏｃ）−Ｈｉｓ（Ｔｒｔ）−Ｖａｌ−Ａｌａ−Ｓｅｒ（Ｂｕ^ｔ）−Ｐｒｏ−Ａｒｇ（Ｐｂｆ）−Ｔｙｒ（Ｂｕ^ｔ）−Ｈｉｓ（Ｔｒｔ）−Ｔｈｒ（Ｂｕ^ｔ）−Ｖａｌ−Ｇｌｙ−Ａｒｇ（Ｐｂｆ）−Ａｌａ−Ａｌａ−Ｇｌｙ−Ｌｅｕ−Ｌｅｕ−Ｍｅｔ−Ｇｌｙ−Ｌｅｕ−Ｏ−Ｃｌｔ　ｒｅｓｉｎ　８３０　ｍｇを得た。　この樹脂１５０ｍｇに　ＴＦＡ　／　ｔｈｉｏａｎｉｓｏｌｅ　／　ｍ−ｃｒｅｓｏｌ　／　ｔｒｉｉｓｏｐｒｏｐｙｌｓｉｌａｎｅ　／　ｅｔｈａｎｅｄｉｔｈｉｏｌ　　（８５　／　５　／　５　／　２．５　／　２．５）　　５　ｍｌを加え、室温にて　２時間振盪した後樹脂をろ去し、溶媒を濃縮後エーテルを加え、粗　Ｆｍｏｃ−Ｔｒｐ−Ｔｙｒ−Ｌｙｓ−Ｈｉｓ−Ｖａｌ−Ａｌａ−Ｓｅｒ−Ｐｒｏ−Ａｒｇ−Ｔｙｒ−Ｈｉｓ−Ｔｈｒ−Ｖａｌ−Ｇｌｙ−Ａｒｇ−Ａｌａ−Ａｌａ−Ｇｌｙ−Ｌｅｕ−Ｌｅｕ−Ｍｅｔ−Ｇｌｙ−Ｌｅｕを沈殿として得た。これを　ＹＭＣ　Ｄ−ＯＤＳ−５−ＳＴ　Ｓ−５　１２０Ａカラム（２０　ｘ　１５０ｍｍ）を用いた分取ＨＰＬＣで、Ａ液：　０．１％ＴＦＡ−水、Ｂ液：　０．１％ＴＦＡ含有アセトニトリルによるＡ／Ｂ：　７２／２８〜５２／４８への直線型濃度勾配溶出（６０分）を行い、目的物を含む分画を集め凍結乾燥し白色粉末９．７ｍｇを得た。
【００９６】
質量分析による（Ｍ＋Ｈ）^＋　　　２８０５．７　（計算値２８０５．４）
ＨＰＬＣ溶出時間　２５．１分
溶出条件
カラム　Ｗａｋｏｓｉｌ−ＩＩ　５Ｃ１８　ＨＧ　　　（４．６　ｘ　１００ｍｍ）
溶離液　Ａ液：　０．１％ＴＦＡ−水、Ｂ液：　０．１％ＴＦＡ含有アセトニトリルを用い、　Ａ／Ｂ：　１００　／　０〜　３０　／　７０へ　直線型濃度勾配溶出（３５分）
流速　１．０ｍｌ／分　得られた　Ｆｍｏｃ−Ｔｒｐ−Ｔｙｒ−Ｌｙｓ−Ｈｉｓ−Ｖａｌ−Ａｌａ−Ｓｅｒ−Ｐｒｏ−Ａｒｇ−Ｔｙｒ−Ｈｉｓ−Ｔｈｒ−Ｖａｌ−Ｇｌｙ−Ａｒｇ−Ａｌａ−Ａｌａ−Ｇｌｙ−Ｌｅｕ−Ｌｅｕ−Ｍｅｔ−Ｇｌｙ−Ｌｅｕ　　５　ｍｇに　２０％　ｄｉｅｔｈｙｌａｍｉｎｅ　／　ＤＭＦ　１　ｍＬを加え室温にて　２時間撹拌した。溶媒を留去後　ＹＭＣ　Ｄ−ＯＤＳ−５−ＳＴ　Ｓ−５　１２０Ａカラム（２０　ｘ　１５０ｍｍ）を用いた分取ＨＰＬＣで、Ａ液：　０．１％ＴＦＡ−水、Ｂ液：　０．１％ＴＦＡ含有アセトニトリルによるＡ／Ｂ：　７４／２６〜６４／３６への直線型濃度勾配溶出（６０分）を行い、目的物を含む分画を集め凍結乾燥し白色粉末１．２ｍｇを得た。
質量分析による（Ｍ＋Ｈ）^＋　２５８３．６　（計算値２５８３．４）
ＨＰＬＣ溶出時間　２０．４分
溶出条件
カラム　Ｗａｋｏｓｉｌ−ＩＩ　５Ｃ１８　ＨＧ　　　（４．６　ｘ　１００ｍｍ）
溶離液　Ａ液：　０．１％ＴＦＡ−水、Ｂ液：　０．１％ＴＦＡ含有アセトニトリルを用い、　Ａ／Ｂ：　１００　／　０〜３０　／　７０へ　直線型濃度勾配溶出（３５分）
流速　１．０ｍｌ／分
【００９７】
参考例１３　ヒトＧＰＲ８　ｌｉｇａｎｄ　（１−３０）：Ｔｒｐ−Ｔｙｒ−Ｌｙｓ−Ｈｉｓ−Ｖａｌ−Ａｌａ−Ｓｅｒ−Ｐｒｏ−Ａｒｇ−Ｔｙｒ−Ｈｉｓ−Ｔｈｒ−Ｖａｌ−Ｇｌｙ−Ａｒｇ−Ａｌａ−Ａｌａ−Ｇｌｙ−Ｌｅｕ−Ｌｅｕ−Ｍｅｔ−Ｇｌｙ−Ｌｅｕ−Ａｒｇ−Ａｒｇ−Ｓｅｒ−Ｐｒｏ−Ｔｙｒ−Ｌｅｕ−Ｔｒｐ（配列番号：１７）の製造
市販　２−ｃｈｌｏｒｏｔｒｉｔｙｌ　ｒｅｓｉｎ　（Ｃｌｔ　ｒｅｓｉｎ，１．３３ｍｍｏｌ／ｇ）にＦｍｏｃ−Ｔｒｐ（Ｂｏｃ）を導入したＦｍｏｃ−Ｔｒｐ（Ｂｏｃ）−Ｏ−Ｃｌｔ　ｒｅｓｉｎ（０．６４ｍｍｏｌ／ｇ）　０．２５ｍｍｏｌを出発原料として参考例１２と同様に配列順通りにアミノ酸を縮合、最後のＴｒｐを導入後樹脂から切り出す前にＦｍｏｃ基を樹脂上で除去したのち、　ＴＦＡ　／　ｔｈｉｏａｎｉｓｏｌｅ　／　ｍ−ｃｒｅｓｏｌ　／　ｔｒｉｉｓｏｐｒｏｐｙｌｓｉｌａｎｅ　／　ｅｔｈａｎｅｄｉｔｈｉｏｌ　　（８５　／　５　／　５　／　２．５　／　２．５）で処理し樹脂からの切り出しと側鎖保護基の除去を同時に行った。粗ペプチドを参考例１２と同様の方法で精製しＴｒｐ−Ｔｙｒ−Ｌｙｓ−Ｈｉｓ−Ｖａｌ−Ａｌａ−Ｓｅｒ−Ｐｒｏ−Ａｒｇ−Ｔｙｒ−Ｈｉｓ−Ｔｈｒ−Ｖａｌ−Ｇｌｙ−Ａｒｇ−Ａｌａ−Ａｌａ−Ｇｌｙ−Ｌｅｕ−Ｌｅｕ−Ｍｅｔ−Ｇｌｙ−Ｌｅｕ−Ａｒｇ−Ａｒｇ−Ｓｅｒ−Ｐｒｏ−Ｔｙｒ−Ｌｅｕ−Ｔｒｐを得た。
質量分析による（Ｍ＋Ｈ）^＋　　　３５４３．４　（計算値３５４４．２）
ＨＰＬＣ溶出時間　２１．５分
溶出条件
カラム　Ｗａｋｏｓｉｌ−ＩＩ　５Ｃ１８　ＨＧ　　　（４．６　ｘ　１００ｍｍ）
溶離液　Ａ液：　０．１％ＴＦＡ−水、Ｂ液：　０．１％ＴＦＡ含有アセトニトリルを用い、　Ａ／Ｂ：　１００　／　０〜　３０　／　７０へ　直線型濃度勾配溶出（３５分）
流速　１．０ｍｌ／分
【００９８】
参考例１４　ヒトＧＰＲ８　ｌｉｇａｎｄ　（１−２９）：Ｔｒｐ−Ｔｙｒ−Ｌｙｓ−Ｈｉｓ−Ｖａｌ−Ａｌａ−Ｓｅｒ−Ｐｒｏ−Ａｒｇ−Ｔｙｒ−Ｈｉｓ−Ｔｈｒ−Ｖａｌ−Ｇｌｙ−Ａｒｇ−Ａｌａ−Ａｌａ−Ｇｌｙ−Ｌｅｕ−Ｌｅｕ−Ｍｅｔ−Ｇｌｙ−Ｌｅｕ−Ａｒｇ−Ａｒｇ−Ｓｅｒ−Ｐｒｏ−Ｔｙｒ−Ｌｅｕ（配列番号：２０）の製造
参考例１２の樹脂を用い参考例１３と同様に配列順にアミノ酸を縮合したのち樹脂からの切り出しと精製を行いＴｒｐ−Ｔｙｒ−Ｌｙｓ−Ｈｉｓ−Ｖａｌ−Ａｌａ−Ｓｅｒ−Ｐｒｏ−Ａｒｇ−Ｔｙｒ−Ｈｉｓ−Ｔｈｒ−Ｖａｌ−Ｇｌｙ−Ａｒｇ−Ａｌａ−Ａｌａ−Ｇｌｙ−Ｌｅｕ−Ｌｅｕ−Ｍｅｔ−Ｇｌｙ−Ｌｅｕ−Ａｒｇ−Ａｒｇ−Ｓｅｒ−Ｐｒｏ−Ｔｙｒ−Ｌｅｕを得る。
【００９９】
参考例１５　ヒトＧＰＲ８　ｌｉｇａｎｄ　（１−２８）：Ｔｒｐ−Ｔｙｒ−Ｌｙｓ−Ｈｉｓ−Ｖａｌ−Ａｌａ−Ｓｅｒ−Ｐｒｏ−Ａｒｇ−Ｔｙｒ−Ｈｉｓ−Ｔｈｒ−Ｖａｌ−Ｇｌｙ−Ａｒｇ−Ａｌａ−Ａｌａ−Ｇｌｙ−Ｌｅｕ−Ｌｅｕ−Ｍｅｔ−Ｇｌｙ−Ｌｅｕ−Ａｒｇ−Ａｒｇ−Ｓｅｒ−Ｐｒｏ−Ｔｙｒ（配列番号：２１）の製造
市販　２−ｃｈｌｏｒｏｔｒｉｔｙｌ　ｒｅｓｉｎ　（Ｃｌｔ　ｒｅｓｉｎ，１．３３ｍｍｏｌ／ｇ）にＦｍｏｃ−Ｔｙｒ（Ｂｕ^ｔ）を導入したのち参考例１３と同様に配列順にアミノ酸の縮合と樹脂からの切り出し、精製を行いＴｒｐ−Ｔｙｒ−Ｌｙｓ−Ｈｉｓ−Ｖａｌ−Ａｌａ−Ｓｅｒ−Ｐｒｏ−Ａｒｇ−Ｔｙｒ−Ｈｉｓ−Ｔｈｒ−Ｖａｌ−Ｇｌｙ−Ａｒｇ−Ａｌａ−Ａｌａ−Ｇｌｙ−Ｌｅｕ−Ｌｅｕ−Ｍｅｔ−Ｇｌｙ−Ｌｅｕ−Ａｒｇ−Ａｒｇ−Ｓｅｒ−Ｐｒｏ−Ｔｙｒを得る。
【０１００】
参考例１６　ヒトＧＰＲ８　ｌｉｇａｎｄ　（１−２７）：Ｔｒｐ−Ｔｙｒ−Ｌｙｓ−Ｈｉｓ−Ｖａｌ−Ａｌａ−Ｓｅｒ−Ｐｒｏ−Ａｒｇ−Ｔｙｒ−Ｈｉｓ−Ｔｈｒ−Ｖａｌ−Ｇｌｙ−Ａｒｇ−Ａｌａ−Ａｌａ−Ｇｌｙ−Ｌｅｕ−Ｌｅｕ−Ｍｅｔ−Ｇｌｙ−Ｌｅｕ−Ａｒｇ−Ａｒｇ−Ｓｅｒ−Ｐｒｏ（配列番号：２２）の製造
市販　２−ｃｈｌｏｒｏｔｒｉｔｙｌ　ｒｅｓｉｎ　（Ｃｌｔ　ｒｅｓｉｎ，１．３３ｍｍｏｌ／ｇ）にＦｍｏｃ−Ｐｒｏを導入したのち参考例１３と同様に配列順にアミノ酸の縮合と樹脂からの切り出し、精製を行いＴｒｐ−Ｔｙｒ−Ｌｙｓ−Ｈｉｓ−Ｖａｌ−Ａｌａ−Ｓｅｒ−Ｐｒｏ−Ａｒｇ−Ｔｙｒ−Ｈｉｓ−Ｔｈｒ−Ｖａｌ−Ｇｌｙ−Ａｒｇ−Ａｌａ−Ａｌａ−Ｇｌｙ−Ｌｅｕ−Ｌｅｕ−Ｍｅｔ−Ｇｌｙ−Ｌｅｕ−Ａｒｇ−Ａｒｇ−Ｓｅｒ−Ｐｒｏを得る。
【０１０１】
参考例１７　ヒトＧＰＲ８　ｌｉｇａｎｄ　（１−２６）：Ｔｒｐ−Ｔｙｒ−Ｌｙｓ−Ｈｉｓ−Ｖａｌ−Ａｌａ−Ｓｅｒ−Ｐｒｏ−Ａｒｇ−Ｔｙｒ−Ｈｉｓ−Ｔｈｒ−Ｖａｌ−Ｇｌｙ−Ａｒｇ−Ａｌａ−Ａｌａ−Ｇｌｙ−Ｌｅｕ−Ｌｅｕ−Ｍｅｔ−Ｇｌｙ−Ｌｅｕ−Ａｒｇ−Ａｒｇ−Ｓｅｒ（配列番号：２３）の製造
市販　２−ｃｈｌｏｒｏｔｒｉｔｙｌ　ｒｅｓｉｎ　（Ｃｌｔ　ｒｅｓｉｎ，１．３３ｍｍｏｌ／ｇ）にＦｍｏｃ−Ｓｅｒ（Ｂｕ^ｔ）を導入したのち参考例１３と同様に配列順にアミノ酸の縮合と樹脂からの切り出し、精製を行いＴｒｐ−Ｔｙｒ−Ｌｙｓ−Ｈｉｓ−Ｖａｌ−Ａｌａ−Ｓｅｒ−Ｐｒｏ−Ａｒｇ−Ｔｙｒ−Ｈｉｓ−Ｔｈｒ−Ｖａｌ−Ｇｌｙ−Ａｒｇ−Ａｌａ−Ａｌａ−Ｇｌｙ−Ｌｅｕ−Ｌｅｕ−Ｍｅｔ−Ｇｌｙ−Ｌｅｕ−Ａｒｇ−Ａｒｇ−Ｓｅｒを得る。
【０１０２】
参考例１８　ヒトＧＰＲ８　ｌｉｇａｎｄ　（１−２５）：Ｔｒｐ−Ｔｙｒ−Ｌｙｓ−Ｈｉｓ−Ｖａｌ−Ａｌａ−Ｓｅｒ−Ｐｒｏ−Ａｒｇ−Ｔｙｒ−Ｈｉｓ−Ｔｈｒ−Ｖａｌ−Ｇｌｙ−Ａｒｇ−Ａｌａ−Ａｌａ−Ｇｌｙ−Ｌｅｕ−Ｌｅｕ−Ｍｅｔ−Ｇｌｙ−Ｌｅｕ−Ａｒｇ−Ａｒｇ（配列番号：２４）の製造
市販　２−ｃｈｌｏｒｏｔｒｉｔｙｌ　ｒｅｓｉｎ　（Ｃｌｔ　ｒｅｓｉｎ，１．３３ｍｍｏｌ／ｇ）にＦｍｏｃ−Ａｒｇ（Ｐｂｆ）を導入したのち参考例１３と同様に配列順にアミノ酸の縮合と樹脂からの切り出し、精製を行いＴｒｐ−Ｔｙｒ−Ｌｙｓ−Ｈｉｓ−Ｖａｌ−Ａｌａ−Ｓｅｒ−Ｐｒｏ−Ａｒｇ−Ｔｙｒ−Ｈｉｓ−Ｔｈｒ−Ｖａｌ−Ｇｌｙ−Ａｒｇ−Ａｌａ−Ａｌａ−Ｇｌｙ−Ｌｅｕ−Ｌｅｕ−Ｍｅｔ−Ｇｌｙ−Ｌｅｕ−Ａｒｇ−Ａｒｇを得る。
【０１０３】
参考例１９　ヒトＧＰＲ８　ｌｉｇａｎｄ　（１−２４）：Ｔｒｐ−Ｔｙｒ−Ｌｙｓ−Ｈｉｓ−Ｖａｌ−Ａｌａ−Ｓｅｒ−Ｐｒｏ−Ａｒｇ−Ｔｙｒ−Ｈｉｓ−Ｔｈｒ−Ｖａｌ−Ｇｌｙ−Ａｒｇ−Ａｌａ−Ａｌａ−Ｇｌｙ−Ｌｅｕ−Ｌｅｕ−Ｍｅｔ−Ｇｌｙ−Ｌｅｕ−Ａｒｇ（配列番号：２５）の製造
市販　２−ｃｈｌｏｒｏｔｒｉｔｙｌ　ｒｅｓｉｎ　（Ｃｌｔ　ｒｅｓｉｎ，１．３３ｍｍｏｌ／ｇ）にＦｍｏｃ−Ａｒｇ（Ｐｂｆ）を導入したのち参考例１３と同様に配列順にアミノ酸の縮合と樹脂からの切り出し、精製を行いＴｒｐ−Ｔｙｒ−Ｌｙｓ−Ｈｉｓ−Ｖａｌ−Ａｌａ−Ｓｅｒ−Ｐｒｏ−Ａｒｇ−Ｔｙｒ−Ｈｉｓ−Ｔｈｒ−Ｖａｌ−Ｇｌｙ−Ａｒｇ−Ａｌａ−Ａｌａ−Ｇｌｙ−Ｌｅｕ−Ｌｅｕ−Ｍｅｔ−Ｇｌｙ−Ｌｅｕ−Ａｒｇを得る。
【０１０４】
参考例２０　ＧＰＲ８発現ＣＨＯ細胞膜画分を用いて測定した２３残基のＧＰＲ８リガンドペプチドのヒトホモログのＧＴＰγＳ結合促進活性
参考例１２で合成した２３残基のＧＰＲ８リガンドペプチドのヒトホモログ（以下、ｈＧＰＲ８Ｌ（１−２３）と記載することがある）を種々の濃度で参考例６に記載した方法でＧＰＲ８発現ＣＨＯ細胞膜画分に投与してＧＴＰγＳ結合促進活性を測定した。結果を図２に示した。明らかにｈＧＰＲ８Ｌ（１−２３）は濃度依存的にＧＰＲ８発現ＣＨＯ細胞膜画分のＧＴＰγＳ結合を促進した。このことから配列番号：１６の構造を有するペプチドがＧＰＲ８に対するリガンドであることが明らかとなった。
【０１０５】
参考例２１　ＧＰＲ８発現ＣＨＯ細胞膜画分を用いて測定した３０残基のＧＰＲ８リガンドペプチドのヒトホモログのＧＴＰγＳ結合促進活性
参考例１３で合成した３０残基のＧＰＲ８リガンドペプチドのヒトホモログ（以下、ｈＧＰＲ８Ｌ（１−３０）と記載することがある）を種々の濃度で参考例６に記載した方法でＧＰＲ８発現ＣＨＯ細胞膜画分に投与してＧＴＰγＳ結合促進活性を測定した。結果を図３に示した。明らかにｈＧＰＲ８Ｌ（１−３０）は濃度依存的にＧＰＲ８発現ＣＨＯ細胞膜画分のＧＴＰγＳ結合を促進した。このことから配列番号：１７の構造を有するペプチドがＧＰＲ８に対するリガンドであることが明らかとなった。
【０１０６】
参考例２２　ＧＰＲ８発現ＣＨＯ細胞を用いて測定した２３残基のＧＰＲ８リガンドペプチドのヒトホモログの細胞内ｃＡＭＰ産生抑制活性
参考例１２で合成したｈＧＰＲ８Ｌ（１−２３）を種々の濃度で参考例５に記載した方法でＧＰＲ８発現ＣＨＯ細胞に接触させて細胞内ｃＡＭＰ産生抑制活性を測定した。結果を図４に示した。明らかにｈＧＰＲ８Ｌ（１−２３）は濃度依存的にＧＰＲ８発現ＣＨＯ細胞に対して細胞内ｃＡＭＰの産生を抑制した。図中、ｃＡＭＰ合成抑制活性は、フォルスコリンを含む反応用バッファーを添加したときの細胞内ｃＡＭＰ量から反応用バッファーを添加したときの細胞内ｃＡＭＰ量を減じた量を１００％として、ｈＧＰＲ８Ｌ（１−２３）を加えたときの細胞内ｃＡＭＰ量から反応用バッファーを添加したときの細胞内ｃＡＭＰ量を減じた量を％として表わした。
【０１０７】
参考例２３　ＧＰＲ８発現ＣＨＯ細胞を用いて測定した３０残基のＧＰＲ８リガンドペプチドのヒトホモログの細胞内ｃＡＭＰ産生抑制活性
参考例１３で合成したｈＧＰＲ８Ｌ（１−３０）を種々の濃度で参考例５に記載した方法でＧＰＲ８発現ＣＨＯ細胞に接触させて細胞内ｃＡＭＰ産生抑制活性を測定した。結果を図５に示した。明らかにｈＧＰＲ８Ｌ（１−３０）は濃度依存的にＧＰＲ８発現ＣＨＯ細胞に対して細胞内ｃＡＭＰの産生を抑制した。図５中、ｃＡＭＰ合成抑制活性は、フォルスコリンを含む反応用バッファーを添加したときの細胞内ｃＡＭＰ量から反応用バッファーを添加したときの細胞内ｃＡＭＰ量を減じた量を１００％として、ｈＧＰＲ８Ｌ（１−３０）を加えたときの細胞内ｃＡＭＰ量から反応用バッファーを添加したときの細胞内ｃＡＭＰ量を減じた量を％として表わした。
【０１０８】
参考例２４　マウスＴＧＲ２６をコードするｃＤＮＡのクローニング
マウス脳ｃＤＮＡを鋳型として配列番号：１３５の合成プライマーと配列番号：１３６の合成プライマーを用いたＰＣＲ法によりマウスＴＧＲ２６ＤＮＡの増幅を行なった。
反応液の組成は、マウス脳ｃＤＮＡ１μｌ、合成プライマー各０．２μＭ、０．８　ｍＭ　ｄＮＴＰｓ、Ａｄｖａｎｔａｇｅ　ｃＤＮＡ　Ｐｏｌｙｍｅｒａｓｅ　Ｍｉｘ　（ＣＬＯＮＴＥＣＨ）０．４μｌおよび酵素に付属のバッファーで、総反応量を２０μｌとした。サーマルサイクラー　（Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍｓ社）を用い、９６℃で２分の加熱した後、９６℃で３０秒、６４℃で３０秒、７２℃で１分の加熱サイクルを３０回繰り返し、最後に７２℃で１０分間保温した。ＰＣＲ反応液中の約１１００塩基長の増幅ＤＮＡ断片を、ＴＯＰＯ　ＴＡ　Ｃｌｏｎｉｎｇ　Ｋｉｔ　（Ｉｎｖｉｔｒｏｇｅｎ）のプロトコールに従い、ｐＣＲ　２．１−ＴＯＰＯへクローニングした。これをエシェリヒア・コリ（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）ＴＯＰ１０　ｃｏｍｐｅｔｅｎｔ　ｃｅｌｌ（Ｉｎｖｉｔｒｏｇｅｎ）に導入して形質転換した後、ｃＤＮＡ挿入断片を持つクローンをアンピシリンおよびＸ−ｇａｌを含むＬＢ寒天培地で選択し、白色を呈するクローンのみを滅菌したつま楊枝を用いて分離し、形質転換体を得た。個々のクローンをアンピシリンを含むＬＢ培地で一晩培養し、ＱＩＡｗｅｌｌ　８　Ｐｌａｓｍｉｄ　Ｋｉｔ（キアゲン）を用いてプラスミドＤＮＡを調製した。塩基配列の決定のための反応はＢｉｇＤｙｅ　Ｔｅｒｍｉｎａｔｏｒ　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　Ｒｅａｄｙ　Ｒｅａｃｔｉｏｎ　Ｋｉｔ　（ＰＥバイオシステムズ）を用いて行ない、蛍光式自動シーケンサーを用いて解読し、配列番号：１３７で表される塩基配列を得た。
配列番号：１３７で表される塩基配列をアミノ酸配列に翻訳したものをマウスＴＧＲ２６アミノ酸配列とし、配列番号：１３８として示す。
配列番号：１３８をラット由来のＴＧＲ２６のアミノ酸配列と比較したところ、９６．０％のアミノ酸の一致が認められた。
配列番号：１３７で表される塩基配列を有するＤＮＡを含むプラスミドで形質転換した大腸菌を、大腸菌（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）ＴＯＰ１０／ｐＣＲ２．１−ＴＯＰＯマウスＧＰＲ７と命名した。
【０１０９】
参考例２５　ヒトＧＰＲ８リガンド前駆体タンパク質をコードするｃＤＮＡの５’上流端のクローニング
参考例１１に記載したＧＰＲ８のリガンドペプチドのヒトホモログ（以下、ヒトＧＰＲ８リガンドと記載することがある）の前駆体タンパク質の一部をコードするヒトｃＤＮＡ配列（配列番号：１４）を基に作製したプライマーでヒト視床下部ｃＤＮＡを鋳型とした５’　ＲＡＣＥ　ＰＣＲを行ない、ヒトＧＰＲ８リガンド前駆体タンパク質をコードするｃＤＮＡの５’上流塩基配列を明らかにした。５’ＲＡＣＥ　ＰＣＲクローニングは、ヒト視床下部Ｍａｒａｔｈｏｎ−Ｒｅａｄｙ　ｃＤＮＡ　（ＣＬＯＮＴＥＣＨ）を鋳型としてキットに添付のＡＰ１プライマーと配列番号：３３の合成プライマーでＰＣＲ反応を行ない、次にこのＰＣＲ反応液を鋳型としてキットに添付のＡＰ２プライマーと配列番号：３４の合成プライマーでＰＣＲ反応を行なうことにより達成された。ＰＣＲの反応液組成と反応条件は以下のとおりである。反応液はヒト視床下部ｃＤＮＡ　４μｌ、ＡＰ１プライマー０．５μＭ、配列番号：３３の合成ＤＮＡプライマー０．５μＭ、０．４　ｍＭ　ｄＮＴＰｓ、ＬＡＴａｑポリメラーゼ（宝酒造）０．２μｌおよび酵素に付属のＧＣ（Ｉ）バッファーで総反応量を２０μｌとし、サーマルサイクラー（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）を用い、９６℃・１２０秒の加熱の後、９６℃・３０秒、６８℃・２４０秒のサイクルを３０回繰り返し、さらに７２℃で１０分間保温した。次に、キットに添付のＴｒｉｃｉｎｅ−ＥＤＴＡ　Ｂｕｆｆｅｒで５０倍希釈したＰＣＲ反応液２μｌ、ＡＰ２プライマー０．５μＭ、配列番号：３４の合成ＤＮＡプライマー０．５μＭ、０．４　ｍＭ　ｄＮＴＰｓ、ＬＡＴａｑポリメラーゼ（宝酒造）０．２μｌおよび酵素に付属のＧＣ（Ｉ）バッファーで総反応量を２０μｌとし、サーマルサイクラー（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）を用い、９６℃・１２０秒の加熱の後、９６℃・３０秒、７２℃・１８０秒のサイクルを４回、次に９６℃・３０秒、７０℃・１８０秒のサイクルを４回、次に９６℃・３０秒、６８℃・１８０秒のサイクルを１７回、最後に７２℃で１０分間保温した。増幅したＤＮＡを１．２％のアガロースゲル電気泳動により分離した後、約１，２００塩基長のＤＮＡをカミソリで切り出し、ＤＮＡをＱＩＡｑｕｉｃｋ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　Ｋｉｔ（キアゲン）を用いて回収した。このＤＮＡを、ＴＯＰＯ　ＴＡ　Ｃｌｏｎｉｎｇ　Ｋｉｔ　（Ｉｎｖｉｔｒｏｇｅｎ）のプロトコールに従ってｐＣＲ２．１−ＴＯＰＯベクターへクローニングした。これをエシェリヒアコリ（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）　ＴＯＰ１０　ｃｏｍｐｅｔｅｎｔ　ｃｅｌｌ（Ｉｎｖｉｔｒｏｇｅｎ）に導入して形質転換した後、ｃＤＮＡ挿入断片を持つクローンをアンピシリンおよびＸ−ｇａｌを含むＬＢ寒天培地で選択し、白色を呈するクローンのみを滅菌したつま楊枝を用いて分離し、形質転換体を得た。個々のクローンをアンピシリンを含むＬＢ培地で一晩培養し、ＱＩＡｗｅｌｌ　８　Ｐｌａｓｍｉｄ　Ｋｉｔ（キアゲン）を用いてプラスミドＤＮＡを調製した。塩基配列の決定のための反応はＢｉｇＤｙｅ　Ｔｅｒｍｉｎａｔｏｒ　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　Ｒｅａｄｙ　Ｒｅａｃｔｉｏｎ　Ｋｉｔ　（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）を用いて行ない、蛍光式自動シーケンサーを用いて解読し、配列番号：３５に示すＤＮＡ配列を得た。
【０１１０】
参考例２６　ヒト脳ｃＤＮＡの作製
ヒト脳ｃＤＮＡは、Ｍａｒａｔｈｏｎ　^ＴＭ　ｃＤＮＡ　Ａｍｐｌｉｆｉｃａｔｉｏｎ　Ｋｉｔ　（ＣＬＯＮＴＥＣＨ）を用いてヒト脳ｐｏｌｙ　Ａ（＋）　ＲＮＡ　（ＣＬＯＮＴＥＣＨ）から作製した。ＲＡＣＥ　ＰＣＲに供されるｃＤＮＡは１ｓｔ　ｓｔｒａｎｄ　ｃＤＮＡ合成を除き、キットに添付のプロトコールに従って作製した。１ｓｔ　ｓｔｒａｎｄ　ｃＤＮＡは、キットに添付の逆転写酵素ＡＭＶの代わりに逆転写酵素ＭＭＬＶ　（−ＲＮＡｓｅ　Ｈ）（ＲｅｖＴｒａＡｃｅ，東洋紡）を用いて、１μｇのヒト脳ｐｏｌｙ　Ａ（＋）　ＲＮＡから合成した。
【０１１１】
参考例２７　ヒトＧＰＲ８リガンド前駆体タンパク質をコードするｃＤＮＡの３’下流端のクローニング
参考例１１に記載のヒトＧＰＲ８リガンド前駆体タンパク質の一部をコードするヒトｃＤＮＡ配列（配列番号：１４）を基に作製したプライマーでヒト脳ｃＤＮＡを鋳型とした３’　ＲＡＣＥ　ＰＣＲを行ない、ヒトＧＰＲ８リガンドをコードするｃＤＮＡの３’下流塩基配列を明らかにした。３’ＲＡＣＥ　ＰＣＲクローニングは、ヒト脳ｃＤＮＡを鋳型としてキットに添付のＡＰ１プライマーと配列番号：３６の合成プライマーでＰＣＲ反応を行ない、次にこのＰＣＲ反応液を鋳型としてキットに添付のＡＰ２プライマーと配列番号：３７の合成プライマーでＰＣＲ反応を行なうことにより達成された。ＰＣＲの反応液組成と反応条件は以下のとおりである。反応液はキットに添付のＴｒｉｃｉｎｅ−ＥＤＴＡ　Ｂｕｆｆｅｒで５０倍希釈したヒト脳ｃＤＮＡ　１μｌ、ＡＰ１プライマー０．５μＭ、配列番号：３６の合成ＤＮＡプライマー０．５μＭ、０．４　ｍＭ　ｄＮＴＰｓ、ＬＡＴａｑポリメラーゼ（宝酒造）０．２μｌおよび酵素に付属のＧＣ（Ｉ）バッファーで総反応量を２０μｌとし、サーマルサイクラー（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）を用い、９６℃・１２０秒の加熱の後、９６℃・３０秒、６８℃・２４０秒のサイクルを３０回繰り返し、さらに７２℃で１０分間保温した。次に、キットに添付のＴｒｉｃｉｎｅ−ＥＤＴＡ　Ｂｕｆｆｅｒで５０倍希釈したＰＣＲ反応液１μｌ、ＡＰ２プライマー０．５μＭ、配列番号：３７の合成ＤＮＡプライマー０．５μＭ、０．４　ｍＭ　ｄＮＴＰｓ、ＬＡＴａｑポリメラーゼ（宝酒造）０．２μｌおよび酵素に付属のＧＣ（Ｉ）バッファーで総反応量を２０μｌとし、サーマルサイクラー（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）を用い、９６℃・１２０秒の加熱の後、９６℃・３０秒、７２℃・１８０秒のサイクルを４回、次に９６℃・３０秒、７０℃・１８０秒のサイクルを４回、次に９６℃・３０秒、６８℃・１８０秒のサイクルを１７回、最後に７２℃で１０分間保温した。増幅したＤＮＡを１．５％のアガロースゲル電気泳動により分離した後、約６００塩基長のＤＮＡをカミソリで切り出し、ＤＮＡをＱＩＡｑｕｉｃｋ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　Ｋｉｔ（キアゲン）を用いて回収した。このＤＮＡを、ＴＯＰＯ　ＴＡ　Ｃｌｏｎｉｎｇ　Ｋｉｔ　（Ｉｎｖｉｔｒｏｇｅｎ）のプロトコールに従ってｐＣＲ２．１−ＴＯＰＯベクターへクローニングした。これをエシェリヒア　コリ（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）　ＴＯＰ１０　ｃｏｍｐｅｔｅｎｔ　ｃｅｌｌ（Ｉｎｖｉｔｒｏｇｅｎ）に導入して形質転換した後、ｃＤＮＡ挿入断片を持つクローンをアンピシリンおよびＸ−ｇａｌを含むＬＢ寒天培地で選択し、白色を呈するクローンのみを滅菌したつま楊枝を用いて分離し、形質転換体を得た。個々のクローンをアンピシリンを含むＬＢ培地で一晩培養し、ＱＩＡｗｅｌｌ　８　Ｐｌａｓｍｉｄ　Ｋｉｔ（キアゲン）を用いてプラスミドＤＮＡを調製した。塩基配列の決定のための反応はＢｉｇＤｙｅ　Ｔｅｒｍｉｎａｔｏｒ　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　Ｒｅａｄｙ　Ｒｅａｃｔｉｏｎ　Ｋｉｔ　（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）を用いて行ない、蛍光式自動シーケンサーを用いて解読し、配列番号：３８に示すＤＮＡ配列を得た。
【０１１２】
参考例２８　ヒトＧＰＲ８リガンド前駆体タンパク質をコードするｃＤＮＡのクローニング
ヒト視床下部ｃＤＮＡを鋳型として、ヒトＧＰＲ８リガンド前駆体タンパク質をコードするｃＤＮＡの５’上流塩基配列を基に作製したプライマーとヒトＧＰＲ８リガンド前駆体タンパク質をコードするｃＤＮＡの３’下流塩基配列を基に作製したプライマーでＰＣＲ増幅を行なうことにより、ヒトＧＰＲ８リガンド前駆体タンパク質をコードするｃＤＮＡをクローニングした。ＰＣＲの反応液組成と反応条件は以下のとおりである。反応液は、ヒト視床下部Ｍａｒａｔｈｏｎ−Ｒｅａｄｙ　ｃＤＮＡ　（ＣＬＯＮＴＥＣＨ）　１μｌ、配列番号：３９の合成ＤＮＡプライマー０．５μＭ、配列番号：４０の合成ＤＮＡプライマー０．５μＭ、０．４　ｍＭ　ｄＮＴＰｓ、２．５　ｍＭ　ＭｇＣｌ_２、５％　ＤＭＳＯ、ＬＡＴａｑポリメラーゼ（宝酒造）０．２μｌおよび酵素に付属のバッファーで総反応量を２０μｌとし、サーマルサイクラー（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）を用い、９６℃・６０秒の加熱の後、９６℃・３０秒、６４℃・３０秒、７２℃・１２０秒のサイクルを３５回、最後に７２℃で１０分間保温した。増幅したＤＮＡを１．５％のアガロースゲル電気泳動により分離した後、約７００塩基長のＤＮＡをカミソリで切り出し、ＤＮＡをＱＩＡｑｕｉｃｋ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　Ｋｉｔ（キアゲン）を用いて回収した。このＤＮＡを、ＴＯＰＯ　ＴＡ　Ｃｌｏｎｉｎｇ　Ｋｉｔ　（Ｉｎｖｉｔｒｏｇｅｎ）のプロトコールに従ってｐＣＲ２．１−ＴＯＰＯベクターへクローニングした。これをエシェリヒア　コリ（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）　ＴＯＰ１０　ｃｏｍｐｅｔｅｎｔ　ｃｅｌｌ（Ｉｎｖｉｔｒｏｇｅｎ）に導入して形質転換した後、ｃＤＮＡ挿入断片を持つクローンをアンピシリンおよびＸ−ｇａｌを含むＬＢ寒天培地で選択し、白色を呈するクローンのみを滅菌したつま楊枝を用いて分離し、形質転換体を得た。個々のクローンをアンピシリンを含むＬＢ培地で一晩培養し、ＱＩＡｗｅｌｌ　８　Ｐｌａｓｍｉｄ　Ｋｉｔ（キアゲン）を用いてプラスミドＤＮＡを調製した。塩基配列の決定のための反応はＢｉｇＤｙｅ　Ｔｅｒｍｉｎａｔｏｒ　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　Ｒｅａｄｙ　Ｒｅａｃｔｉｏｎ　Ｋｉｔ　（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）を用いて行ない、蛍光式自動シーケンサーを用いて解読し、配列番号：４１に示すＤＮＡ配列を得た。
この配列（配列番号：４１）はヒトＧＰＲ８リガンド前駆体タンパク質をコードするためこのＤＮＡを含むプラスミドで形質転換した大腸菌をＴＯＰ１０／ｐＣＲ２．１−ＴＯＰＯヒトＧＰＲ８リガンド前駆体（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ　ＴＯＰ１０／ｐＣＲ２．１−ＴＯＰＯ　Ｈｕｍａｎ　ＧＰＲ８　Ｌｉｇａｎｄ　Ｐｒｅｃｕｒｓｏｒ）と命名した。
配列番号：４１に示すＤＮＡ配列には、参考例１１に記載したヒトＧＰＲ８リガンドペプチドのアミノ酸配列をコードするようなフレームが存在するが、その５’上流側にはタンパク質翻訳の開始コドンであると予想されるＡＴＧが存在しない。しかし、これまでに幾つかのタンパク質でＡＴＧ以外のコドンを開始コドンとすることが予想されている例が報告されている（ヒト塩基性線維芽細胞増殖因子（Ｈ．　Ｐｒａｔｓ　ｅｔ　ａｌ．、Ｐｒｏｃ．　Ｎａｔｌ．　Ａｃａｄ．　Ｓｃｉ．　ＵＳＡ、８６巻、１８３６−１８４０頁、１９８９年、Ｒ．　Ｚ．　ＦｌｏｒｋｉｅｗｉｃｚおよびＡ．　Ｓｏｍｍｅｒ、Ｐｒｏｃ．　Ｎａｔｌ．　Ａｃａｄ．　Ｓｃｉ．　ＵＳＡ、８６巻、３９７８−３９８１頁、１９８９年）、マウスレチノイン酸受容体β４（Ｓ．　Ｎａｇｐａｌ　ｅｔ　ａｌ．、Ｐｒｏｃ．　Ｎａｔｌ．　Ａｃａｄ．　Ｓｃｉ．　ＵＳＡ、８９巻、２７１８頁、１９９２年）、ヒトホスホリボシルピロリン酸合成酵素（Ｍ．　Ｔａｉｒａ　ｅｔ　ａｌ．、Ｊ．　Ｂｉｏｌ．　Ｃｈｅｍ．、２６５巻、１６４９１−１６４９７頁、１９９０年）、ショウジョウバエコリンアセチル転移酵素（Ｈ．　Ｓｕｇｉｈａｒａ　ｅｔ　ａｌ．、Ｊ．　Ｂｉｏｌ．　Ｃｈｅｍ．、２６５巻、２１７１４−２１７１９頁、１９９０年））。
これらの例ではＡＴＧに代わりＬｅｕをコードするＣＴＧが開始コドンとして仮定されていることが多い。ヒトＧＰＲ８リガンド前駆体タンパク質においても同様であると考え、後述のブタあるいはラットのＧＰＲ８リガンドホモログの前駆体タンパク質との比較からこれらの前駆体タンパク質における開始コドンと予想されるＡＴＧにほぼ対応する位置に存在するＣＴＧコドンを開始コドンと仮定し、前駆体タンパク質の配列を推定した。この仮想的ヒトＧＰＲ８リガンド前駆体タンパク質のアミノ酸配列を配列番号：４２に示した。また、仮想的ヒトＧＰＲ８リガンド前駆体タンパク質のアミノ酸配列およびＤＮＡ配列を図６に示した。
【０１１３】
参考例２９　ブタ脊髄ｃＤＮＡの作製
ブタ脊髄ｃＤＮＡは、Ｍａｒａｔｈｏｎ　^ＴＭ　ｃＤＮＡ　Ａｍｐｌｉｆｉｃａｔｉｏｎ　Ｋｉｔ　（ＣＬＯＮＴＥＣＨ）を用いてブタ脊髄ｐｏｌｙ　Ａ（＋）　ＲＮＡから作製した。ブタ脊髄ｐｏｌｙ　Ａ（＋）　ＲＮＡは、ブタ脊髄から以下のように調製した。ブタ脊髄を、ＩＳＯＧＥＮ（日本ジーン）中にてポリトロンホモゲナイザーで完全に破砕し、この破砕溶液からＩＳＯＧＥＮ溶液を用いたｔｏｔａｌ　ＲＮＡ調製法に従ってブタ脊髄ｔｏｔａｌ　ＲＮＡを得た。次に、ブタ脊髄ｔｏｔａｌ　ＲＮＡからｍＲＮＡ　Ｐｕｒｉｆｉｃａｔｉｏｎ　Ｋｉｔ（Ａｍｅｒｓｈａｍ　Ｐｈａｒｍａｃｉａ　Ｂｉｏｔｅｃｈ）に添付のｏｌｉｇｏ　ｄＴ　ｃｅｌｌｕｌｏｓｅカラムを用いたクロマトグラフィーを２回行なうことにより、７μｇのブタ脊髄ｐｏｌｙ　Ａ（＋）　ＲＮＡを得た。ＲＡＣＥ　ＰＣＲに供したｃＤＮＡは１ｓｔ　ｓｔｒａｎｄ　ｃＤＮＡ合成を除き、キットに添付のプロトコールに従って作製した。１ｓｔ　ｓｔｒａｎｄ　ｃＤＮＡは、キットに添付の逆転写酵素ＡＭＶの代わりに逆転写酵素ＭＭＬＶ　（−ＲＮＡｓｅ　Ｈ）（ＲｅｖＴｒａＡｃｅ，東洋紡）を用いて、１μｇのブタ脊髄ｐｏｌｙ　Ａ（＋）　ＲＮＡから合成した。
【０１１４】
参考例３０　ブタＧＰＲ８リガンド前駆体タンパク質をコードするｃＤＮＡの５’上流端のクローニング
１回目の５’ＲＡＣＥ　ＰＣＲクローニングと、そのＰＣＲ増幅ＤＮＡの塩基配列を利用した２回目の５’ＲＡＣＥ　ＰＣＲクローニングにより、ＧＰＲ８のリガンドペプチドのブタホモログ（以下、ブタＧＰＲ８リガンドと記載することがある）の前駆体タンパク質をコードするｃＤＮＡの５’上流塩基配列を明らかにした。
１回目の５’ＲＡＣＥ　ＰＣＲクローニングは、上記ブタ脊髄ｃＤＮＡを鋳型としてキットに添付のＡＰ１プライマーと配列番号：４３の合成プライマーでＰＣＲ反応を行ない、次にこのＰＣＲ反応液を鋳型としてキットに添付のＡＰ２プライマーと配列番号：４４の合成プライマーでＰＣＲ反応を行なうことにより達成された。ＰＣＲの反応液組成と反応条件は以下のとおりである。反応液はキットに添付のＴｒｉｃｉｎｅ−ＥＤＴＡ　Ｂｕｆｆｅｒで５０倍希釈したブタ脊髄ｃＤＮＡ　４μｌ、ＡＰ１プライマー０．５μＭ、配列番号：４３の合成ＤＮＡプライマー０．５μＭ、０．４　ｍＭ　ｄＮＴＰｓ、ＬＡＴａｑポリメラーゼ（宝酒造）０．２μｌおよび酵素に付属のＧＣ（Ｉ）バッファーで総反応量を２０μｌとし、サーマルサイクラー（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）を用い、９６℃・１２０秒の加熱の後、９６℃・３０秒、６８℃・１８０秒のサイクルを３０回繰り返し、さらに７２℃で１０分間保温した。次に、キットに添付のＴｒｉｃｉｎｅ−ＥＤＴＡ　Ｂｕｆｆｅｒで１００倍希釈したＰＣＲ反応液１μｌ、ＡＰ２プライマー０．５μＭ、配列番号：４４の合成ＤＮＡプライマー０．５μＭ、０．４　ｍＭ　ｄＮＴＰｓ、Ａｄｖａｎｔａｇｅ−ＧＣ　２ポリメラーゼ（ＣＬＯＮＴＥＣＨ）　０．２μｌおよび酵素に付属のバッファーで総反応量を２０μｌとし、サーマルサイクラー（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）を用い、９６℃・６０秒の加熱の後、９６℃・３０秒、７２℃・１８０秒のサイクルを３回、次に９６℃・３０秒、７０℃・１８０秒のサイクルを３回、次に９６℃・３０秒、６８℃・１８０秒のサイクルを４回、次に９６℃・３０秒、６４℃・３０秒、７２℃・１８０秒のサイクルを１５回繰り返し、最後に７２℃で１０分間保温した。増幅したＤＮＡを１．２％のアガロースゲル電気泳動により分離した後、約３００塩基長のＤＮＡをカミソリで切り出し、ＤＮＡをＱＩＡｑｕｉｃｋ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　Ｋｉｔ（キアゲン）を用いて回収した。このＤＮＡを、ＴＯＰＯ　ＴＡ　Ｃｌｏｎｉｎｇ　Ｋｉｔ　（Ｉｎｖｉｔｒｏｇｅｎ）のプロトコールに従ってｐＣＲ２．１−ＴＯＰＯベクターへクローニングした。これをエシェリヒア　コリ（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）　ＴＯＰ１０Ｆ’　ｃｏｍｐｅｔｅｎｔ　ｃｅｌｌ（Ｉｎｖｉｔｒｏｇｅｎ）に導入して形質転換した後、ｃＤＮＡ挿入断片を持つクローンをアンピシリン、ＩＰＴＧおよびＸ−ｇａｌを含むＬＢ寒天培地で選択し、白色を呈するクローンのみを滅菌したつま楊枝を用いて分離し、形質転換体を得た。個々のクローンをアンピシリンを含むＬＢ培地で一晩培養し、ＱＩＡｗｅｌｌ　８　Ｐｌａｓｍｉｄ　Ｋｉｔ（キアゲン）を用いてプラスミドＤＮＡを調製した。塩基配列の決定のための反応はＢｉｇＤｙｅ　Ｔｅｒｍｉｎａｔｏｒ　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　Ｒｅａｄｙ　Ｒｅａｃｔｉｏｎ　Ｋｉｔ　（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）を用いて行ない、蛍光式自動シーケンサーを用いて解読し、配列番号：４５に示すＤＮＡ配列を得た。
２回目の５’ＲＡＣＥ　ＰＣＲクローニングは、ブタ脊髄ｃＤＮＡを鋳型としてキットに添付のＡＰ１プライマーと配列番号：４６の合成プライマーでＰＣＲ反応を行ない、次にこのＰＣＲ反応液を鋳型としてキットに添付のＡＰ２プライマーと配列番号：４７の合成プライマーでＰＣＲ反応を行なうことにより達成された。ＰＣＲの反応液組成と反応条件は以下のとおりである。反応液は、キットに添付のＴｒｉｃｉｎｅ−ＥＤＴＡ　Ｂｕｆｆｅｒで５０倍希釈したブタ脊髄ｃＤＮＡ　１μｌ、ＡＰ１プライマー０．５μＭ、配列番号：４６の合成ＤＮＡプライマー０．５μＭ、０．４　ｍＭ　ｄＮＴＰｓ、Ａｄｖａｎｔａｇｅ−ＧＣ　２ポリメラーゼ（ＣＬＯＮＴＥＣＨ）　０．２μｌおよび酵素に付属のバッファーで総反応量を２０μｌとし、サーマルサイクラー（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）を用い、９６℃・６０秒の加熱の後、９６℃・３０秒、７２℃・１８０秒のサイクルを５回、次に９６℃・３０秒、７０℃・１８０秒のサイクルを５回、次に９６℃・３０秒、６８℃・１８０秒のサイクルを２０回繰り返し、最後に７２℃で１０分間保温した。次に、キットに添付のＴｒｉｃｉｎｅ−ＥＤＴＡ　Ｂｕｆｆｅｒで１００倍希釈したＰＣＲ反応液１μｌ、ＡＰ２プライマー０．５μＭ、配列番号：４７の合成ＤＮＡプライマー０．５μＭ、０．４　ｍＭ　ｄＮＴＰｓ、Ａｄｖａｎｔａｇｅ−ＧＣ　２ポリメラーゼ（ＣＬＯＮＴＥＣＨ）　０．２μｌおよび酵素に付属のバッファーで総反応量を２０μｌとし、サーマルサイクラー（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）を用い、９６℃・６０秒の加熱の後、９６℃・３０秒、６８℃・１８０秒のサイクルを３１回繰り返し、最後に７２℃で１０分間保温した。増幅したＤＮＡを２．０％のアガロースゲル電気泳動により分離した後、約２００塩基長のＤＮＡをカミソリで切り出し、ＤＮＡをＱＩＡｑｕｉｃｋ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　Ｋｉｔ（キアゲン）を用いて回収した。このＤＮＡを、ＴＯＰＯ　ＴＡ　Ｃｌｏｎｉｎｇ　Ｋｉｔ　（Ｉｎｖｉｔｒｏｇｅｎ）のプロトコールに従ってｐＣＲ２．１−ＴＯＰＯベクターへクローニングした。これをエシェリヒアコリ（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）　ＴＯＰ１０Ｆ’　ｃｏｍｐｅｔｅｎｔ　ｃｅｌｌ（Ｉｎｖｉｔｒｏｇｅｎ）に導入して形質転換した後、ｃＤＮＡ挿入断片を持つクローンをアンピシリン、ＩＰＴＧおよびＸ−ｇａｌを含むＬＢ寒天培地で選択し、白色を呈するクローンのみを滅菌したつま楊枝を用いて分離し、形質転換体を得た。個々のクローンをアンピシリンを含むＬＢ培地で一晩培養し、ＱＩＡｗｅｌｌ　８　Ｐｌａｓｍｉｄ　Ｋｉｔ（キアゲン）を用いてプラスミドＤＮＡを調製した。塩基配列の決定のための反応はＢｉｇＤｙｅＴｅｒｍｉｎａｔｏｒ　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　Ｒｅａｄｙ　Ｒｅａｃｔｉｏｎ　Ｋｉｔ（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）を用いて行ない、蛍光式自動シーケンサーを用いて解読し、配列番号：４８に示すＤＮＡ配列を得た。
【０１１５】
参考例３１　ブタＧＰＲ８リガンド前駆体タンパク質をコードするｃＤＮＡの３’下流端のクローニング
ブタＧＰＲ８リガンド前駆体タンパク質をコードするｃＤＮＡの５’上流端の塩基配列を基に作製したプライマーを用いた３’ＲＡＣＥ　ＰＣＲクローニングにより、ブタＧＰＲ８リガンドペプチドの前駆体タンパク質をコードするｃＤＮＡの３’下流塩基配列を明らかにした。３’ＲＡＣＥ　ＰＣＲクローニングは、ブタ脊髄ｃＤＮＡを鋳型としてキットに添付のＡＰ１プライマーと配列番号：４９の合成プライマーでＰＣＲ反応を行ない、次にこのＰＣＲ反応液を鋳型としてキットに添付のＡＰ２プライマーと配列番号：５０の合成プライマーでＰＣＲ反応を行なうことにより達成された。ＰＣＲの反応液組成と反応条件は以下のとおりである。反応液は、キットに添付のＴｒｉｃｉｎｅ−ＥＤＴＡ　Ｂｕｆｆｅｒで５０倍希釈したブタ脊髄ｃＤＮＡ　１μｌ、ＡＰ１プライマー０．５μＭ、配列番号：４９の合成ＤＮＡプライマー０．５μＭ、０．４　ｍＭｄＮＴＰｓ、Ａｄｖａｎｔａｇｅ−ＧＣ　２ポリメラーゼ（ＣＬＯＮＴＥＣＨ）　０．２μｌおよび酵素に付属のバッファーで総反応量を２０μｌとし、サーマルサイクラー（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）を用い、９６℃・６０秒の加熱の後、９６℃・３０秒、７２℃・１２０秒のサイクルを５回、次に９６℃・３０秒、７０℃・１２０秒のサイクルを５回、次に９６℃・３０秒、６８℃・１２０秒のサイクルを２０回繰り返し、最後に７２℃で１０分間保温した。次に、キットに添付のＴｒｉｃｉｎｅ−ＥＤＴＡ　Ｂｕｆｆｅｒで１００倍希釈したＰＣＲ反応液１μｌ、ＡＰ２プライマー０．５μＭ、配列番号：５０の合成ＤＮＡプライマー０．５μＭ、０．４　ｍＭ　ｄＮＴＰｓ、Ａｄｖａｎｔａｇｅ−ＧＣ　２ポリメラーゼ（ＣＬＯＮＴＥＣＨ）　０．２μｌおよび酵素に付属のバッファーで総反応量を２０μｌとし、サーマルサイクラー（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）を用い、９６℃・１２０秒の加熱の後、９６℃・３０秒、６８℃・１２０秒のサイクルを３１回繰り返し、最後に７２℃で１０分間保温した。増幅したＤＮＡを２．０％のアガロースゲル電気泳動により分離した後、約６５０塩基長のＤＮＡをカミソリで切り出し、ＤＮＡをＱＩＡｑｕｉｃｋ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　Ｋｉｔ（キアゲン）を用いて回収した。このＤＮＡを、ＴＯＰＯ　ＴＡ　Ｃｌｏｎｉｎｇ　Ｋｉｔ　（Ｉｎｖｉｔｒｏｇｅｎ）のプロトコールに従ってｐＣＲ２．１−ＴＯＰＯベクターへクローニングした。これをエシェリヒアコリ（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）　ＴＯＰ１０Ｆ’　ｃｏｍｐｅｔｅｎｔ　ｃｅｌｌ（Ｉｎｖｉｔｒｏｇｅｎ）に導入して形質転換した後、ｃＤＮＡ挿入断片を持つクローンをアンピシリンおよびＸ−ｇａｌ、ＩＰＴＧを含むＬＢ寒天培地で選択し、白色を呈するクローンのみを滅菌したつま楊枝を用いて分離し、形質転換体を得た。個々のクローンをアンピシリンを含むＬＢ培地で一晩培養し、ＱＩＡｗｅｌｌ　８　Ｐｌａｓｍｉｄ　Ｋｉｔ（キアゲン）を用いてプラスミドＤＮＡを調製した。塩基配列の決定のための反応はＢｉｇＤｙｅ　Ｔｅｒｍｉｎａｔｏｒ　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　Ｒｅａｄｙ　Ｒｅａｃｔｉｏｎ　Ｋｉｔ　（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）を用いて行ない、蛍光式自動シーケンサーを用いて解読し、配列番号：５１に示すＤＮＡ配列を得た。
【０１１６】
参考例３２　ブタＧＰＲ８リガンド前駆体タンパク質をコードするｃＤＮＡのクローニング
ブタ脊髄ｃＤＮＡを鋳型として、ブタＧＰＲ８リガンド前駆体タンパク質をコードするｃＤＮＡの５’上流塩基配列を基に作製したプライマーとブタＧＰＲ８リガンド前駆体タンパク質をコードするｃＤＮＡの３’下流塩基配列を基に作製したプライマーでＰＣＲ増幅を行なうことにより、ブタＧＰＲ８リガンド前駆体タンパク質をコードするｃＤＮＡをクローニングした。ＰＣＲの反応液組成と反応条件は以下のとおりである。反応液は、キットに添付のＴｒｉｃｉｎｅ−ＥＤＴＡ　Ｂｕｆｆｅｒで５０倍希釈したブタ脊髄ｃＤＮＡ　１μｌ、配列番号：５２の合成ＤＮＡプライマー０．５μＭ、配列番号：５３の合成ＤＮＡプライマー０．５μＭ、０．４　ｍＭ　ｄＮＴＰｓ、Ａｄｖａｎｔａｇｅ　２ポリメラーゼ（ＣＬＯＮＴＥＣＨ）　０．２μｌおよび酵素に付属のバッファーで総反応量を２０μｌとし、サーマルサイクラー（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）を用い、９６℃・６０秒の加熱の後、９６℃・３０秒、７２℃・７５秒のサイクルを４回、次に９６℃・３０秒、７０℃・７５秒のサイクルを４回、次に９６℃・３０秒、６８℃・７５秒のサイクルを４回、次に９６℃・３０秒、６４℃・３０秒、７２℃・４５秒のサイクルを５回、次に９６℃・３０秒、６０℃・３０秒、７２℃・４５秒のサイクルを２０回繰り返し、最後に７２℃で１０分間保温した。増幅したＤＮＡを１．２％のアガロースゲル電気泳動により分離した後、約６００塩基長のＤＮＡをカミソリで切り出し、ＤＮＡをＱＩＡｑｕｉｃｋ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　Ｋｉｔ（キアゲン）を用いて回収した。このＤＮＡを、ＴＯＰＯ　ＴＡ　Ｃｌｏｎｉｎｇ　Ｋｉｔ　（Ｉｎｖｉｔｒｏｇｅｎ）のプロトコールに従ってｐＣＲ２．１−ＴＯＰＯベクターへクローニングした。これをエシェリヒアコリ（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）　ＴＯＰ１０　ｃｏｍｐｅｔｅｎｔ　ｃｅｌｌ（Ｉｎｖｉｔｒｏｇｅｎ）に導入して形質転換した後、ｃＤＮＡ挿入断片を持つクローンをアンピシリンおよびＸ−ｇａｌを含むＬＢ寒天培地で選択し、白色を呈するクローンのみを滅菌したつま楊枝を用いて分離し、形質転換体を得た。個々のクローンをアンピシリンを含むＬＢ培地で一晩培養し、ＱＩＡｗｅｌｌ　８　Ｐｌａｓｍｉｄ　Ｋｉｔ（キアゲン）を用いてプラスミドＤＮＡを調製した。塩基配列の決定のための反応はＢｉｇＤｙｅ　Ｔｅｒｍｉｎａｔｏｒ　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　Ｒｅａｄｙ　Ｒｅａｃｔｉｏｎ　Ｋｉｔ（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）を用いて行ない、蛍光式自動シーケンサーを用いて解読し、配列番号：５４に示すＤＮＡ配列を得た。この配列（配列番号：５４）はブタＧＰＲ８リガンド前駆体タンパク質をコードするためこのＤＮＡを含むプラスミドで形質転換した大腸菌をＴＯＰ１０／ｐＣＲ２．１−ＴＯＰＯブタＧＰＲ８リガンド前駆体（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ　ＴＯＰ１０／ｐＣＲ２．１−ＴＯＰＯ　Ｐｏｒｃｉｎｅ　ＧＰＲ８　Ｌｉｇａｎｄ　Ｐｒｅｃｕｒｓｏｒ）と命名した。
配列番号：５４のＤＮＡ配列がコードするブタＧＰＲ８リガンド前駆体タンパク質のアミノ酸配列を配列番号：５５に示した。この前駆体タンパク質のアミノ酸配列には参考例１０に記載のＧＰＲ８発現細胞膜画分に対するＧＴＰγＳ結合活性を指標にブタ視床下部より単離されたＧＰＲ８リガンドペプチドのアミノ酸配列分析によって明らかにされたアミノ末端から１７残基までの配列が存在した。さらにその配列のカルボキシ末端側にはＧＰＲ８リガンドペプチドのヒトホモログ前駆体タンパク質の場合と同様に、通常の生理活性ペプチドが切り出されると考えられるＡｒｇ−Ａｒｇの配列（Ｓｅｉｄａｈ，　Ｎ．　Ｇ．　ｅｔ　ａｌ．、Ａｎｎ．　Ｎ．　Ｙ．　Ａｃａｄ．Ｓｃｉ．、８３９巻、９−２４頁、１９９８年）が２ヶ所存在した。このことから、ＧＰＲ８リガンドペプチドのブタホモログのアミノ酸配列は配列番号：５６および５７のいずれかもしくは両方であると推定された。ブタＧＰＲ８リガンド前駆体タンパク質のアミノ酸配列およびＤＮＡ配列を図７に示した。
【０１１７】
参考例３３　ラットＧＰＲ８リガンド前駆体タンパク質の一部をコードするｃＤＮＡ断片のクローニング
参考例１０に記載したように、ＧＰＲ８発現細胞膜画分に対するＧＴＰγＳ結合活性を指標にブタ視床下部から精製したペプチドのアミノ末端から１７アミノ酸配列（配列番号：６）に基づいてデータベース検索を行なったところ、配列番号：１１の塩基配列に合致するラットＥＳＴ塩基配列（アクセッション番号：Ｈ３１５９８）が見出された。このＤＮＡ配列は１５アミノ酸の配列がブタ視床下部から精製したペプチドのアミノ酸配列（配列番号：６）と同一となる翻訳枠を持っていた。このＨ３１５９８は、ラットＰＣ１２細胞から作製したｃＤＮＡライブラリー由来のＥＳＴ配列であり、未確定な７塩基を含む２６０塩基からなる。このＨ３１５９８はＧＰＲ８リガンドのラットホモログペプチド（以下、ラットＧＰＲ８リガンドと記載することがある）の前駆体タンパク質の一部をコードしていると推定されたのでその正確な塩基配列を決定するため、Ｈ３１５９８の５’塩基配列と３’塩基配列を基に作製したそれぞれのプライマーでラット脳Ｍａｒａｔｈｏｎ−Ｒｅａｄｙ　ｃＤＮＡ　（ＣＬＯＮＴＥＣＨ）を鋳型としたＰＣＲクローニングを行なった。ＰＣＲの反応液組成と反応条件は以下のとおりである。ラット脳Ｍａｒａｔｈｏｎ　ｃＤＮＡ　（ＣＬＯＮＴＥＣＨ）　２μｌ、配列番号：６０の合成ＤＮＡプライマー０．５μＭ、配列番号：６１の合成ＤＮＡプライマー０．５μＭ、０．４　ｍＭ　ｄＮＴＰｓ、Ａｄｖａｎｔａｇｅ−ＧＣ　２ポリメラーゼ（ＣＬＯＮＴＥＣＨ）　０．２μｌおよび酵素に付属のバッファーで総反応量を２０μｌとし、サーマルサイクラー（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）を用い、９６℃・６０秒の加熱の後、次に９６℃・３０秒、６０℃・３０秒、７２℃・６０秒のサイクルを３５回繰り返し、最後に７２℃で１０分間保温した。増幅したＤＮＡを４．０％のアガロースゲル電気泳動により分離した後、約２５０塩基長のＤＮＡをカミソリで切り出し、ＤＮＡをＱＩＡｑｕｉｃｋ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　Ｋｉｔ（キアゲン）を用いて回収した。このＤＮＡを、ＴＯＰＯ　ＴＡ　Ｃｌｏｎｉｎｇ　Ｋｉｔ　（Ｉｎｖｉｔｒｏｇｅｎ）のプロトコールに従ってｐＣＲ２．１−ＴＯＰＯベクターへクローニングした。これをエシェリヒアコリ（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）　ＴＯＰ１０　ｃｏｍｐｅｔｅｎｔ　ｃｅｌｌ（Ｉｎｖｉｔｒｏｇｅｎ）に導入して形質転換した後、ｃＤＮＡ挿入断片を持つクローンをアンピシリンおよびＸ−ｇａｌを含むＬＢ寒天培地で選択し、白色を呈するクローンのみを滅菌したつま楊枝を用いて分離し、形質転換体を得た。個々のクローンをアンピシリンを含むＬＢ培地で一晩培養し、ＱＩＡｗｅｌｌ　８　Ｐｌａｓｍｉｄ　Ｋｉｔ（キアゲン）を用いてプラスミドＤＮＡを調製した。塩基配列の決定のための反応はＢｉｇＤｙｅ　Ｔｅｒｍｉｎａｔｏｒ　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　Ｒｅａｄｙ　Ｒｅａｃｔｉｏｎ　Ｋｉｔ　（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）を用いて行ない、蛍光式自動シーケンサーを用いて解読し、配列番号：６２に示すＤＮＡ配列を得た。ＰＣＲクローニングしたＤＮＡの塩基配列（配列番号：６２）とＨ３１５９８の塩基配列との比較により、１塩基欠失の読み誤りがＨ３１５９８の塩基配列にあることが明らかになった。
【０１１８】
参考例３４　ラットＧＰＲ８リガンド前駆体タンパク質をコードするｃＤＮＡの５’上流端のクローニング
５’ＲＡＣＥＰＣＲクローニングによりラットＧＰＲ８リガンド前駆体タンパク質をコードするｃＤＮＡの５’上流塩基配列を明らかにした。５’ＲＡＣＥ　ＰＣＲクローニングは、ラット脳Ｍａｒａｔｈｏｎ−Ｒｅａｄｙ　ｃＤＮＡ　（ＣＬＯＮＴＥＣＨ）を鋳型としてキットに添付のＡＰ１プライマーと配列番号：６３の合成プライマーでＰＣＲ反応を行ない、次にこのＰＣＲ反応液を鋳型としてキットに添付のＡＰ２プライマーと配列番号：６４の合成プライマーでＰＣＲ反応を行なうことにより達成された。ＰＣＲの反応液組成と反応条件は以下のとおりである。反応液はラット脳Ｍａｒａｔｈｏｎ　ｃＤＮＡ　２μｌ、ＡＰ１プライマー０．５μＭ、配列番号：６３の合成ＤＮＡプライマー０．５μＭ、０．４　ｍＭ　ｄＮＴＰｓ、ＬＡＴａｑポリメラーゼ（宝酒造）０．２μｌおよび酵素に付属のＧＣ（Ｉ）バッファーで総反応量を２０μｌとし、サーマルサイクラー（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）を用い、９６℃・　６０秒の加熱の後、９６℃・３０秒、６８℃・１２０秒のサイクルを３０回繰り返し、さらに７２℃で１０分間保温した。次に、キットに添付のＴｒｉｃｉｎｅ−ＥＤＴＡ　Ｂｕｆｆｅｒで２００倍希釈したＰＣＲ反応液２μｌ、ＡＰ２プライマー０．５μＭ、配列番号：６４の合成ＤＮＡプライマー０．５μＭ、０．４　ｍＭ　ｄＮＴＰｓ、Ａｄｖａｎｔａｇｅ−ＧＣ　２ポリメラーゼ（ＣＬＯＮＴＥＣＨ）　０．２μｌおよび酵素に付属のバッファーで総反応量を２０μｌとし、サーマルサイクラー（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）を用い、９６℃・６０秒の加熱の後、９６℃・３０秒、６８℃・１２０秒のサイクルを３１回、最後に７２℃で１０分間保温した。増幅したＤＮＡを１．２％のアガロースゲル電気泳動により分離した後、約６００塩基長のＤＮＡをカミソリで切り出し、ＤＮＡをＱＩＡｑｕｉｃｋ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　Ｋｉｔ（キアゲン）を用いて回収した。このＤＮＡを、ＴＯＰＯ　ＴＡ　Ｃｌｏｎｉｎｇ　Ｋｉｔ　（Ｉｎｖｉｔｒｏｇｅｎ）のプロトコールに従ってｐＣＲ２．１−ＴＯＰＯベクターへクローニングした。これをエシェリヒアコリ（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）　ＴＯＰ１０　ｃｏｍｐｅｔｅｎｔ　ｃｅｌｌ（Ｉｎｖｉｔｒｏｇｅｎ）に導入して形質転換した後、ｃＤＮＡ挿入断片を持つクローンをアンピシリンおよびＸ−ｇａｌを含むＬＢ寒天培地で選択し、白色を呈するクローンのみを滅菌したつま楊枝を用いて分離し、形質転換体を得た。個々のクローンをアンピシリンを含むＬＢ培地で一晩培養し、ＱＩＡｗｅｌｌ　８　Ｐｌａｓｍｉｄ　Ｋｉｔ（キアゲン）を用いてプラスミドＤＮＡを調製した。塩基配列の決定のための反応はＢｉｇＤｙｅ　Ｔｅｒｍｉｎａｔｏｒ　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　Ｒｅａｄｙ　Ｒｅａｃｔｉｏｎ　Ｋｉｔ　（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）を用いて行ない、蛍光式自動シーケンサーを用いて解読し、配列番号：６５に示すＤＮＡ配列を得た。
【０１１９】
参考例３５　ラットＧＰＲ８リガンド前駆体タンパク質をコードするｃＤＮＡの３’下流端のクローニング
ラットＧＰＲ８リガンド前駆体タンパク質をコードするｃＤＮＡの５’上流端の塩基配列を基に作製したプライマーおよびラットＧＰＲ８リガンド前駆体タンパク質の一部をコードするｃＤＮＡ断片配列を基に作製したプライマーを用いた３’ＲＡＣＥ　ＰＣＲクローニングにより、ラットＧＰＲ８リガンド前駆体タンパク質をコードするｃＤＮＡの３’下流塩基配列を明らかにした。３’ＲＡＣＥ　ＰＣＲクローニングは、ラット脳Ｍａｒａｔｈｏｎ−Ｒｅａｄｙ　ｃＤＮＡ　（ＣＬＯＮＴＥＣＨ）を鋳型としてキットに添付のＡＰ１プライマーと配列番号：６６の合成プライマーでＰＣＲ反応を行ない、次にこのＰＣＲ反応液を鋳型としてキットに添付のＡＰ２プライマーと配列番号：６７の合成プライマーでＰＣＲ反応を行なうことにより達成された。ＰＣＲの反応液組成と反応条件は以下のとおりである。反応液は、ラット脳Ｍａｒａｔｈｏｎ−Ｒｅａｄｙ　ｃＤＮＡ　２μｌ、ＡＰ１プライマー０．５μＭ、配列番号：６６の合成ＤＮＡプライマー０．５μＭ、０．４　ｍＭ　ｄＮＴＰｓ、Ａｄｖａｎｔａｇｅ−ＧＣ　２ポリメラーゼ（ＣＬＯＮＴＥＣＨ）　０．４μｌおよび酵素に付属のバッファーで総反応量を２０μｌとし、サーマルサイクラー（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）を用い、９６℃・６０秒の加熱の後、９６℃・３０秒、６８℃・１８０秒のサイクルを３０回繰り返し、最後に７２℃で１０分間保温した。次に、キットに添付のＴｒｉｃｉｎｅ−ＥＤＴＡ　Ｂｕｆｆｅｒで２００倍希釈したＰＣＲ反応液２μｌ、ＡＰ２プライマー０．５μＭ、配列番号：６７の合成ＤＮＡプライマー０．５μＭ、０．４　ｍＭ　ｄＮＴＰｓ、Ａｄｖａｎｔａｇｅ−ＧＣ　２ポリメラーゼ（ＣＬＯＮＴＥＣＨ）　０．４μｌおよび酵素に付属のバッファーで総反応量を２０μｌとし、サーマルサイクラー（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）を用い、９６℃・６０秒の加熱の後、９６℃・３０秒、６８℃・１８０秒のサイクルを３０回繰り返し、最後に７２℃で１０分間保温した。増幅したＤＮＡを１．２％のアガロースゲル電気泳動により分離した後、約６００塩基長のＤＮＡをカミソリで切り出し、ＤＮＡをＱＩＡｑｕｉｃｋ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　Ｋｉｔ（キアゲン）を用いて回収した。このＤＮＡを、ＴＯＰＯ　ＴＡ　Ｃｌｏｎｉｎｇ　Ｋｉｔ　（Ｉｎｖｉｔｒｏｇｅｎ）のプロトコールに従ってｐＣＲ２．１−ＴＯＰＯベクターへクローニングした。これをエシェリヒア　コリ（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）　ＴＯＰ１０　ｃｏｍｐｅｔｅｎｔ　ｃｅｌｌ（Ｉｎｖｉｔｒｏｇｅｎ）に導入して形質転換した後、ｃＤＮＡ挿入断片を持つクローンをアンピシリンおよびＸ−ｇａｌを含むＬＢ寒天培地で選択し、白色を呈するクローンのみを滅菌したつま楊枝を用いて分離し、形質転換体を得た。個々のクローンをアンピシリンを含むＬＢ培地で一晩培養し、ＱＩＡｗｅｌｌ　８　Ｐｌａｓｍｉｄ　Ｋｉｔ（キアゲン）を用いてプラスミドＤＮＡを調製した。塩基配列の決定のための反応はＢｉｇＤｙｅＴｅｒｍｉｎａｔｏｒ　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　Ｒｅａｄｙ　Ｒｅａｃｔｉｏｎ　Ｋｉｔ　（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）を用いて行ない、蛍光式自動シーケンサーを用いて解読し、配列番号：６８に示すＤＮＡ配列を得た。
【０１２０】
参考例３６　ラットＧＰＲ８リガンド前駆体タンパク質をコードするｃＤＮＡのクローニング
ラット脳ｃＤＮＡを鋳型として、ラットＧＰＲ８リガンド前駆体タンパク質をコードするｃＤＮＡの５’上流塩基配列を基にしたプライマーとラットＧＰＲ８リガンド前駆体タンパク質をコードするｃＤＮＡの３’下流塩基配列を基にしたプライマーでＰＣＲ増幅を行なうことにより、ラットＧＰＲ８リガンド前駆体タンパク質をコードするｃＤＮＡをクローニングした。ＰＣＲの反応液組成と反応条件は以下のとおりである。反応液は、ラット脳Ｍａｒａｔｈｏｎ−Ｒｅａｄｙ　ｃＤＮＡ　１μｌ、配列番号：６９の合成ＤＮＡプライマー０．５μＭ、配列番号：７０の合成ＤＮＡプライマー０．５μＭ、０．４　ｍＭ　ｄＮＴＰｓ、Ａｄｖａｎｔａｇｅ　２ポリメラーゼ（ＣＬＯＮＴＥＣＨ）　０．４μｌおよび酵素に付属のバッファーで総反応量を２０μｌとし、サーマルサイクラー（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）を用い、９６℃・６０秒の加熱の後、９６℃・３０秒、６０℃・３０秒、７２℃・６０秒のサイクルを３５回繰り返し、最後に７２℃で１０分間保温した。増幅したＤＮＡを１．２％のアガロースゲル電気泳動により分離した後、約７５０塩基長のＤＮＡをカミソリで切り出し、ＤＮＡをＱＩＡｑｕｉｃｋ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　Ｋｉｔ（キアゲン）を用いて回収した。このＤＮＡを、ＴＯＰＯ　ＴＡ　Ｃｌｏｎｉｎｇ　Ｋｉｔ　（Ｉｎｖｉｔｒｏｇｅｎ）のプロトコールに従ってｐＣＲ２．１−ＴＯＰＯベクターへクローニングした。これをエシェリヒア　コリ（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）　ＴＯＰ１０　ｃｏｍｐｅｔｅｎｔ　ｃｅｌｌ（Ｉｎｖｉｔｒｏｇｅｎ）に導入して形質転換した後、ｃＤＮＡ挿入断片を持つクローンをアンピシリンおよびＸ−ｇａｌを含むＬＢ寒天培地で選択し、白色を呈するクローンのみを滅菌したつま楊枝を用いて分離し、形質転換体を得た。個々のクローンをアンピシリンを含むＬＢ培地で一晩培養し、ＱＩＡｗｅｌｌ　８　Ｐｌａｓｍｉｄ　Ｋｉｔ（キアゲン）を用いてプラスミドＤＮＡを調製した。塩基配列の決定のための反応はＢｉｇＤｙｅ　Ｔｅｒｍｉｎａｔｏｒ　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　Ｒｅａｄｙ　Ｒｅａｃｔｉｏｎ　Ｋｉｔ（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）を用いて行ない、蛍光式自動シーケンサーを用いて解読し、配列番号：７１に示すＤＮＡ配列を得た。この配列（配列番号：７１）は、ラットＧＰＲ８リガンド前駆体タンパク質をコードするためこのＤＮＡを含むプラスミドで形質転換した大腸菌をＴＯＰ１０／ｐＣＲ２．１−ＴＯＰＯラットＧＰＲ８リガンド前駆体（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ　ＴＯＰ１０／ｐＣＲ２．１−ＴＯＰＯ　Ｒａｔ　ＧＰＲ８　Ｌｉｇａｎｄ　Ｐｒｅｃｕｒｓｏｒ）と命名した。
配列番号：７１のＤＮＡ配列がコードするラットＧＰＲ８リガンド前駆体タンパク質のアミノ酸配列を配列番号：７２に示した。この前駆体タンパク質のアミノ酸配列には参考例１０に記載のＧＰＲ８発現細胞膜画分に対するＧＴＰγＳ結合活性を指標にブタ視床下部より単離されたＧＰＲ８リガンドペプチドのアミノ酸配列分析によって明らかにされたアミノ末端から１７残基までの配列と５残基目および１７残基目のアミノ酸のみが異なる類似した配列が存在した。さらにその配列のカルボキシ末端側にはＧＰＲ８リガンドペプチドのヒトあるいはブタホモログ前駆体タンパク質の場合と同様に、通常の生理活性ペプチドが切り出されると考えられるＡｒｇ−Ａｒｇの配列（Ｓｅｉｄａｈ，　Ｎ．　Ｇ．　ｅｔ　ａｌ．、Ａｎｎ．　Ｎ．　Ｙ．　Ａｃａｄ．　Ｓｃｉ．、８３９巻、９−２４頁、１９９８年）が２ヶ所存在した。これらのことから、ＧＰＲ８リガンドペプチドのラットホモログのアミノ酸配列は配列番号：７３および７４のいずれかもしくは両方であると推定された。ラットＧＰＲ８リガンド前駆体タンパク質のアミノ酸配列およびＤＮＡ配列を図８に示した。
【０１２１】
参考例３７　マウスＧＰＲ８リガンド前駆体タンパク質の一部をコードするｃＤＮＡ断片のクローニング
ＧＰＲ８リガンドペプチドのマウスホモログ（以下、マウスＧＰＲ８リガンドと記載することがある）の前駆体タンパク質を取得するため、マウス精巣ｃＤＮＡを鋳型として、ＰＣＲ増幅を行ない、増幅ＤＮＡの塩基配列を決定した。ＰＣＲの反応液組成と反応条件は以下のとおりである。マウス精巣ｃＤＮＡ　（ＣＬＯＮＴＥＣＨ）　１μｌ、配列番号：７８の合成ＤＮＡプライマー０．５μＭ、配列番号：７９の合成ＤＮＡプライマー０．５μＭ、０．４　ｍＭ　ｄＮＴＰｓ、ＬＡＴａｑポリメラーゼ（宝酒造）０．２μｌおよび酵素に付属のＧＣ（Ｉ）バッファーで総反応量を２０μｌとし、サーマルサイクラー（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）を用い、９６℃・１２０秒の加熱の後、９６℃・３０秒、６８℃・１２０秒のサイクルを１０回繰り返し、次に９６℃・３０秒、６４℃・３０秒、７２℃・１２０秒のサイクルを２５回繰り返し、最後に７２℃で１０分間保温した。増幅したＤＮＡを１．５％のアガロースゲル電気泳動により分離した後、約３５０塩基長のＤＮＡをカミソリで切り出し、ＤＮＡをＱＩＡｑｕｉｃｋ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　Ｋｉｔ（キアゲン）を用いて回収した。このＤＮＡを、ＴＯＰＯ　ＴＡ　Ｃｌｏｎｉｎｇ　Ｋｉｔ　（Ｉｎｖｉｔｒｏｇｅｎ）のプロトコールに従ってｐＣＲ２．１−ＴＯＰＯベクターへクローニングした。これをエシェリヒアコリ（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）　ＴＯＰ１０　ｃｏｍｐｅｔｅｎｔ　ｃｅｌｌ（Ｉｎｖｉｔｒｏｇｅｎ）に導入して形質転換した後、ｃＤＮＡ挿入断片を持つクローンをアンピシリンおよびＸ−ｇａｌを含むＬＢ寒天培地で選択し、白色を呈するクローンのみを滅菌したつま楊枝を用いて分離し、形質転換体を得た。個々のクローンをアンピシリンを含むＬＢ培地で一晩培養し、ＱＩＡｗｅｌｌ　８　Ｐｌａｓｍｉｄ　Ｋｉｔ（キアゲン）を用いてプラスミドＤＮＡを調製した。塩基配列の決定のための反応はＢｉｇＤｙｅ　Ｔｅｒｍｉｎａｔｏｒ　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　ＲｅａｄｙＲｅａｃｔｉｏｎ　Ｋｉｔ　（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）を用いて行ない、蛍光式自動シーケンサーを用いて解読した（配列番号：８０）。
【０１２２】
参考例３８　マウス脳ｃＤＮＡの作製
マウス脳ｃＤＮＡは、ＳＭＡＲＴ^ＴＭ　ＲＡＣＥｃＤＮＡ　Ａｍｐｌｉｆｉｃａｔｉｏｎ　Ｋｉｔ　（ＣＬＯＮＴＥＣＨ）を用いてマウス脳ｐｏｌｙ　Ａ（＋）　ＲＮＡ　（ＣＬＯＮＴＥＣＨ）から、キットに添付のプロトコールに従って作製した。合成した１ｓｔ　ｓｔｒａｎｄ　ｃＤＮＡ溶液を、キットに添付のＴｒｉｃｉｎｅ−ＥＤＴＡ　Ｂｕｆｆｅｒで１０倍に希釈し、この溶液をＲＡＣＥ　ＰＣＲ反応に供した。
【０１２３】
参考例３９　マウスＧＰＲ８リガンド前駆体タンパク質をコードするｃＤＮＡの５’上流端のクローニング
５’ＲＡＣＥ　ＰＣＲクローニングにより、マウスＧＰＲ８リガンド前駆体タンパク質をコードするｃＤＮＡの５’上流塩基配列を明らかにした。５’ＲＡＣＥ　ＰＣＲクローニングは、マウス脳ｃＤＮＡを鋳型としてＳＭＡＲＴ^ＴＭ　ＲＡＣＥｃＤＮＡ　ＡｍｐｌｉｆｉｃａｔｉｏｎＫｉｔに添付のＵｎｉｖｅｒｓａｌ　Ｐｒｉｍｅｒ　Ｍｉｘと配列番号：８１の合成プライマーでＰＣＲ反応を行ない、次にこのＰＣＲ反応液を鋳型としてキットに添付のＮｅｓｔｅｄ　Ｕｎｉｖｅｒｓａｌ　Ｐｒｉｍｅｒと配列番号：８２の合成プライマーでＰＣＲ反応を行なうことにより達成された。ＰＣＲの反応液組成と反応条件は以下のとおりである。反応液はマウス脳ｃＤＮＡ　１μｌ、Ｕｎｉｖｅｒｓａｌ　Ｐｒｉｍｅｒ　Ｍｉｘ　２μｌ、配列番号：８１の合成ＤＮＡプライマー０．２μＭ、０．８　ｍＭ　ｄＮＴＰｓ、Ａｄｖａｎｔａｇｅ−ＧＣ　２ポリメラーゼ（ＣＬＯＮＴＥＣＨ）０．４μｌおよび酵素に付属のバッファーで総反応量を２０μｌとし、サーマルサイクラー（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）を用い、９６℃・１２０秒の加熱の後、９６℃・３０秒、６８℃・１２０秒のサイクルを３０回繰り返し、さらに７２℃で１０分間保温した。次に、キットに添付のＴｒｉｃｉｎｅ−ＥＤＴＡ　Ｂｕｆｆｅｒで５０倍希釈したＰＣＲ反応液０．５μｌ、Ｎｅｓｔｅｄ　Ｕｎｉｖｅｒｓａｌ　Ｐｒｉｍｅｒ　０．５μＭ、配列番号：８２の合成ＤＮＡプライマー０．５μＭ、０．８　ｍＭ　ｄＮＴＰｓ、Ａｄｖａｎｔａｇｅ−ＧＣ　２ポリメラーゼ（ＣＬＯＮＴＥＣＨ）　０．４μｌおよび酵素に付属のバッファーで総反応量を２０μｌとし、サーマルサイクラー（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）を用い、９６℃・１２０秒の加熱の後、９６℃・３０秒、６０℃・３０秒、７２℃・１２０秒のサイクルを３０回繰り返し、さらに７２℃で１０分間保温した。増幅したＤＮＡを１．５％のアガロースゲル電気泳動により分離した後、約３００塩基長のＤＮＡをカミソリで切り出し、ＤＮＡをＱＩＡｑｕｉｃｋ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　Ｋｉｔ（キアゲン）を用いて回収した。このＤＮＡを、ＴＯＰＯ　ＴＡ　Ｃｌｏｎｉｎｇ　Ｋｉｔ　（Ｉｎｖｉｔｒｏｇｅｎ）のプロトコールに従ってｐＣＲ２．１−ＴＯＰＯベクターへクローニングした。これをエシェリヒア　コリ（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）　ＴＯＰ１０　ｃｏｍｐｅｔｅｎｔ　ｃｅｌｌ（Ｉｎｖｉｔｒｏｇｅｎ）に導入して形質転換した後、ｃＤＮＡ挿入断片を持つクローンをアンピシリンおよびＸ−ｇａｌを含むＬＢ寒天培地で選択し、白色を呈するクローンのみを滅菌したつま楊枝を用いて分離し、形質転換体を得た。個々のクローンをアンピシリンを含むＬＢ培地で一晩培養し、ＱＩＡｗｅｌｌ　８　Ｐｌａｓｍｉｄ　Ｋｉｔ（キアゲン）を用いてプラスミドＤＮＡを調製した。塩基配列の決定のための反応はＢｉｇＤｙｅ　Ｔｅｒｍｉｎａｔｏｒ　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　Ｒｅａｄｙ　Ｒｅａｃｔｉｏｎ　Ｋｉｔ　（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）を用いて行ない、蛍光式自動シーケンサーを用いて解読し、配列番号：８３に示すＤＮＡ配列を得た。
【０１２４】
参考例４０　マウスＧＰＲ８リガンド前駆体タンパク質をコードするｃＤＮＡの３’下流端のクローニング
３’ＲＡＣＥ　ＰＣＲクローニングにより、マウスＧＰＲ８リガンド前駆体タンパク質をコードするｃＤＮＡの３’下流塩基配列を明らかにした。３’ＲＡＣＥ　ＰＣＲクローニングは、マウス脳ｃＤＮＡを鋳型としてＳＭＡＲＴ^ＴＭ　ＲＡＣＥｃＤＮＡ　Ａｍｐｌｉｆｉｃａｔｉｏｎ　Ｋｉｔに添付のＵｎｉｖｅｒｓａｌ　Ｐｒｉｍｅｒ　Ｍｉｘと配列番号：８４の合成プライマーでＰＣＲ反応を行ない、次にこのＰＣＲ反応液を鋳型としてキットに添付のＮｅｓｔｅｄ　ＵｎｉｖｅｒｓａｌＰｒｉｍｅｒと配列番号：８５の合成プライマーでＰＣＲ反応を行なうことにより達成された。ＰＣＲの反応液組成と反応条件は以下のとおりである。反応液は、マウス脳ｃＤＮＡ　１μｌ、Ｕｎｉｖｅｒｓａｌ　Ｐｒｉｍｅｒ　Ｍｉｘ　２μｌ、配列番号：８４の合成ＤＮＡプライマー０．５μＭ、０．８　ｍＭ　ｄＮＴＰｓ、Ａｄｖａｎｔａｇｅ−ＧＣ　２ポリメラーゼ（ＣＬＯＮＴＥＣＨ）　０．４μｌおよび酵素に付属のバッファーで総反応量を２０μｌとし、サーマルサイクラー（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）を用い、９６℃・１２０秒の加熱の後、９６℃・３０秒、６８℃・１２０秒のサイクルを３０回繰り返し、最後に７２℃で１０分間保温した。次に、キットに添付のＴｒｉｃｉｎｅ−ＥＤＴＡ　Ｂｕｆｆｅｒで５０倍希釈したＰＣＲ反応液０．５μｌ、Ｎｅｓｔｅｄ　Ｕｎｉｖｅｒｓａｌ　Ｐｒｉｍｅｒ　０．５μＭ、配列番号：８５の合成ＤＮＡプライマー０．５μＭ、０．８　ｍＭｄＮＴＰｓ、Ａｄｖａｎｔａｇｅ−ＧＣ　２ポリメラーゼ（ＣＬＯＮＴＥＣＨ）　０．４μｌおよび酵素に付属のバッファーで総反応量を２０μｌとし、サーマルサイクラー（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）を用い、９６℃・１２０秒の加熱の後、９６℃・３０秒、６４℃・３０秒、７２℃・１２０秒のサイクルを３０回繰り返し、最後に７２℃で１０分間保温した。増幅したＤＮＡを１．５％のアガロースゲル電気泳動により分離した後、約７００塩基長のＤＮＡをカミソリで切り出し、ＤＮＡをＱＩＡｑｕｉｃｋ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　Ｋｉｔ（キアゲン）を用いて回収した。このＤＮＡを、ＴＯＰＯ　ＴＡ　Ｃｌｏｎｉｎｇ　Ｋｉｔ　（Ｉｎｖｉｔｒｏｇｅｎ）のプロトコールに従ってｐＣＲ２．１−ＴＯＰＯベクターへクローニングした。これをエシェリヒア　コリ（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）　ＴＯＰ１０　ｃｏｍｐｅｔｅｎｔ　ｃｅｌｌ（Ｉｎｖｉｔｒｏｇｅｎ）に導入して形質転換した後、ｃＤＮＡ挿入断片を持つクローンをアンピシリンおよびＸ−ｇａｌを含むＬＢ寒天培地で選択し、白色を呈するクローンのみを滅菌したつま楊枝を用いて分離し、形質転換体を得た。個々のクローンをアンピシリンを含むＬＢ培地で一晩培養し、ＱＩＡｗｅｌｌ　８　Ｐｌａｓｍｉｄ　Ｋｉｔ（キアゲン）を用いてプラスミドＤＮＡを調製した。塩基配列の決定のための反応はＢｉｇＤｙｅ　Ｔｅｒｍｉｎａｔｏｒ　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　Ｒｅａｄｙ　Ｒｅａｃｔｉｏｎ　Ｋｉｔ　（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）を用いて行ない、蛍光式自動シーケンサーを用いて解読し、配列番号：８６に示すＤＮＡ配列を得た。
【０１２５】
参考例４１　マウスＧＰＲ８リガンド前駆体タンパク質をコードするｃＤＮＡのクローニング
マウス脳ｃＤＮＡを鋳型として、マウスＧＰＲ８リガンド前駆体タンパク質をコードするｃＤＮＡの５’上流塩基配列を基にしたプライマーとマウスＧＰＲ８リガンド前駆体タンパク質をコードするｃＤＮＡの３’下流塩基配列を基にしたプライマーでＰＣＲ増幅を行なうことにより、マウスＧＰＲ８リガンド前駆体タンパク質をコードするｃＤＮＡをクローニングした。ＰＣＲの反応液組成と反応条件は以下のとおりである。反応液は、マウス脳ｃＤＮＡ　０．５μｌ、配列番号：８７の合成ＤＮＡプライマー０．５μＭ、配列番号：８８の合成ＤＮＡプライマー０．５μＭ、１．６　ｍＭ　ｄＮＴＰｓ、ＬＡＴａｑポリメラーゼ（宝酒造）０．２μｌおよび酵素に付属のＧＣ（Ｉ）バッファーで総反応量を２０μｌとし、サーマルサイクラー（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）を用い、９６℃・１２０秒の加熱の後、９６℃・３０秒、６４℃・３０秒、７２℃・１２０秒のサイクルを４０回繰り返し、最後に７２℃で１０分間保温した。増幅したＤＮＡを１．５％のアガロースゲル電気泳動により分離した後、約７００塩基長のＤＮＡをカミソリで切り出し、ＤＮＡをＱＩＡｑｕｉｃｋ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　Ｋｉｔ（キアゲン）を用いて回収した。このＤＮＡを、ＴＯＰＯ　ＴＡ　Ｃｌｏｎｉｎｇ　Ｋｉｔ　（Ｉｎｖｉｔｒｏｇｅｎ）のプロトコールに従ってｐＣＲ２．１−ＴＯＰＯベクターへクローニングした。これをエシェリヒア　コリ（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）　ＴＯＰ１０　ｃｏｍｐｅｔｅｎｔ　ｃｅｌｌ（Ｉｎｖｉｔｒｏｇｅｎ）に導入して形質転換した後、ｃＤＮＡ挿入断片を持つクローンをアンピシリンおよびＸ−ｇａｌを含むＬＢ寒天培地で選択し、白色を呈するクローンのみを滅菌したつま楊枝を用いて分離し、形質転換体を得た。個々のクローンをアンピシリンを含むＬＢ培地で一晩培養し、ＱＩＡｗｅｌｌ　８　Ｐｌａｓｍｉｄ　Ｋｉｔ（キアゲン）を用いてプラスミドＤＮＡを調製した。塩基配列の決定のための反応はＢｉｇＤｙｅ　Ｔｅｒｍｉｎａｔｏｒ　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　ＲｅａｄｙＲｅａｃｔｉｏｎ　Ｋｉｔ　（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）を用いて行ない、蛍光式自動シーケンサーを用いて解読し、配列番号：８９に示すＤＮＡ配列を得た。この配列（配列番号：８９）は，マウスＧＰＲ８リガンド前駆体タンパク質をコードするため、このＤＮＡを含むプラスミドで形質転換した大腸菌をＴＯＰ１０／ｐＣＲ２．１−ＴＯＰＯマウスＧＰＲ８リガンド前駆体（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ　ＴＯＰ１０／ｐＣＲ２．１−ＴＯＰＯ　Ｍｏｕｓｅ　ＧＰＲ８　Ｌｉｇａｎｄ　Ｐｒｅｃｕｒｓｏｒ）と命名した。
配列番号：８９に示すＤＮＡ配列には、参考例１０に記載のＧＰＲ８発現細胞膜画分に対するＧＴＰγＳ結合活性を指標にブタ視床下部より単離されたＧＰＲ８リガンドペプチドのアミノ酸配列分析によって明らかにされたアミノ末端から１７残基までの配列と５残基目および１７残基目のアミノ酸のみが異なる類似したアミノ酸配列をコードするようなフレームが存在する。しかし、ヒトＧＰＲ８リガンド前駆体の場合と同様に、その５’上流側にはタンパク質翻訳の開始コドンであると予想されるＡＴＧが存在しない。しかし、ヒトＧＰＲ８リガンド前駆体タンパク質において推測されたように、ブタあるいはラットのＧＰＲ８リガンドホモログの前駆体タンパク質との比較からこれらの前駆体タンパク質における開始コドンと予想されるＡＴＧにほぼ対応する位置に存在するＣＴＧコドンを開始コドンと仮定し、マウスＧＰＲ８リガンド前駆体タンパク質の配列を推定した。この仮想的マウスＧＰＲ８リガンド前駆体タンパク質のアミノ酸配列を配列番号：９０に示した。マウスＧＰＲ８リガンドのアミノ酸配列と予想される配列のカルボキシ末端側にはＧＰＲ８リガンドペプチドのヒト、ブタあるいはラットホモログ前駆体タンパク質の場合と同様に、通常の生理活性ペプチドが切り出されると考えられるＡｒｇ−Ａｒｇの配列（Ｓｅｉｄａｈ，　Ｎ．　Ｇ．　ｅｔ　ａｌ．、Ａｎｎ．　Ｎ．　Ｙ．　Ａｃａｄ．　Ｓｃｉ．、８３９巻、９−２４頁、１９９８年）が２ヶ所存在した。これらのことから、ＧＰＲ８リガンドペプチドのマウスホモログのアミノ酸配列は配列番号：９１および９２のいずれかもしくは両方であると推定された。なお、配列番号：９１の２３残基型マウスＧＰＲ８リガンドのアミノ酸配列は、２３残基型ラットＧＰＲ８リガンドのアミノ酸配列（配列番号：７３）と一致している。仮想的マウスＧＰＲ８リガンド前駆体タンパク質のアミノ酸配列およびＤＮＡ配列を図９に示した。
【０１２６】
参考例４２　ラクトパーオキシダーゼ法を用いた　［^１２５Ｉ−Ｔｙｒ^２］−ｈＧＰＲ８Ｌ（１−２３）および［^１２５Ｉ−Ｔｙｒ^１０］−ｈＧＰＲ８Ｌ（１−２３）の作製
ＤＭＳＯ　５μｌに溶かしたｈＧＰＲ８Ｌ（１−２３）（配列番号：１６で表されるアミノ酸配列からなるポリペプチド）１　ｎｍｏｌを０．１　Ｍ塩化ニッケル５μｌと混合し、　０．１　Ｍ　ＨＥＰＥＳ　（ｐＨ　７）に溶かした　０．００１％過酸化水素水　１０μｌ、０．１　Ｍ　ＨＥＰＥＳ　（ｐＨ　７）に溶かしたラクトパーオキシダーゼ　（シグマ社）　１０μｇ／ｍｌを１０μｌおよび　［^１２５Ｉ］　ＮａＩ　３７　ＭＢｑ　（ＮＥＮライフサイエンスプロダクツ社）　１０μｌを混合後、室温で６０分反応し、以下の条件でＨＰＬＣ分取した。
用いたカラムは、ＯＤＳ−８０ＴＭ　（４．６　ｍｍ　ｘ　１５　ｃｍ）（トーソー社）、溶出液Ａとして１０％アセトニトリル／０．１％　ＴＦＡ、溶出液Ｂとして６０％アセトニトリル／０．１％　ＴＦＡを用い、０−０　（２　ｍｉｎ）、０−３０　（３　ｍｉｎ）、３０−３８　（５　ｍｉｎ）、３８−４３　（５５　ｍｉｎ）％Ｂ／Ａ＋Ｂのグラディエント溶出法を行なった。流速は１　ｍＬ／ｍｉｎ、カラム温度は２５℃、検出は２２０ｎｍの吸光度を用いて行った。
ｈＧＰＲ８Ｌ（１−２３）には、チロシン残基が２つ存在するので、ヨード化によって、［^１２５Ｉ−Ｔｙｒ^２］−ｈＧＰＲ８Ｌ（１−２３）および　［^１２５Ｉ−Ｔｙｒ^１０］−ｈＧＰＲ８Ｌ（１−２３）が生成する。このＨＰＬＣ条件では、ｈＧＰＲ８Ｌ（１−２３）が２４分、［^１２５Ｉ−Ｔｙｒ^２］−ｈＧＰＲ８Ｌ（１−２３）が３０分、［^１２５Ｉ−Ｔｙｒ^１０］−ｈＧＰＲ８Ｌ（１−２３）が３２分付近に溶出した。
【０１２７】
参考例４３　［^１２５Ｉ−Ｔｙｒ^１０］−ｈＧＰＲ８Ｌ（１−２３）を用いた受容体結合実験
参考例４２に記載したように作製した［^１２５Ｉ］−標識ｈＧＰＲ８Ｌ（１−２３）および参考例６に記載した方法と同様にしてヒトＧＰＲ８発現ＣＨＯ細胞から調製した細胞膜画分を用いて受容体結合実験を行なった。
ヒトＧＰＲ８発現ＣＨＯ細胞から調製した細胞膜画分を、アッセイ用バッファー（２５　ｍＭ　Ｔｒｉｓ−ＨＣｌ、５　ｍＭ　ＥＤＴＡ（エチレンジアミン四酢酸）、０．０５％　ＣＨＡＰＳ（３−〔（３−コラミドプロピル）ジメチルアンモニオ〕−１−プロパン硫酸）、０．１％　ＢＳＡ（ウシ血清アルブミン）、０．２５　ｍＭ　ＰＭＳＦ（フェニルメタンスルホニルフルオライド）、１μｇ／ｍｌペプスタチン、２０μｇ／ｍｌロイペプチン、ｐＨ　７．４）で各種濃度に希釈後、ポリプロピレン製試験管（Ｆａｌｃｏｎ　２０５３）に２００μｌずつ分注した。最大結合量（ＴＢ）を測定するために２μｌのＤＭＳＯと７　ｎＭの［^１２５Ｉ−Ｔｙｒ^２］−ｈＧＰＲ８Ｌ（１−２３）または［^１２５Ｉ−Ｔｙｒ^１０］−ｈＧＰＲ８Ｌ（１−２３）　２μｌを膜画分溶液に添加した。また、非特異的結合（ＮＳＢ）を測定するために１００μＭ　ｈＧＰＲ８Ｌ（１−２３）のＤＭＳＯ溶液２μｌと７　ｎＭの［^１２５Ｉ−Ｔｙｒ^２］−ｈＧＰＲ８Ｌ（１−２３）または［^１２５Ｉ−Ｔｙｒ^１０］−ｈＧＰＲ８Ｌ（１−２３）　２μｌを膜画分溶液に添加した。２５℃で６０分間反応させた後、ポリエチレンイミン処理したワットマングラスフィルター（ＧＦ−Ｆ）を用いて反応液を吸引ろ過した。ろ過後、γ−カウンターを用いてろ紙上に残った放射活性を測定し、最大結合量から非特異的結合量を引いて特異的結合量（ＳＢ）を見積もった。［^１２５Ｉ−Ｔｙｒ^２］−ｈＧＰＲ８Ｌ（１−２３）を用いた場合に比べて［^１２５Ｉ−Ｔｙｒ^１０］−ｈＧＰＲ８Ｌ（１−２３）を用いた方が、特異的結合量が２倍多かったので実際の結合実験には［^１２５Ｉ−Ｔｙｒ^１０］−ｈＧＰＲ８Ｌ（１−２３）を用いた。膜画分の濃度を変化させると膜画分の濃度に依存した［^１２５Ｉ−Ｔｙｒ^１０］−ｈＧＰＲ８Ｌ（１−２３）の特異的な結合が認められた。また、膜画分濃度を５μｇ／ｍｌに設定して阻害率（％）からｈＧＰＲ８Ｌ（１−２３）の５０％阻害濃度（ＩＣ_５０値）を算出したところ、ＩＣ_５０値は０．２５　ｎＭであった。図１０に種々の濃度におけるｈＧＰＲＬ（１−２３）の結合阻害を示す。
【０１２８】
参考例４４　ヒトＧＰＲ８　ｌｉｇａｎｄ　（１−２３）酸化体：Ｔｒｐ−Ｔｙｒ−Ｌｙｓ−Ｈｉｓ−Ｖａｌ−Ａｌａ−Ｓｅｒ−Ｐｒｏ−Ａｒｇ−Ｔｙｒ−Ｈｉｓ−Ｔｈｒ−Ｖａｌ−Ｇｌｙ−Ａｒｇ−Ａｌａ−Ａｌａ−Ｇｌｙ−Ｌｅｕ−Ｌｅｕ−Ｍｅｔ（Ｏ）−Ｇｌｙ−Ｌｅｕ（配列番号：９５）の製造
参考例１２の化合物０．４５　ｍｇを　５０％酢酸水０．５　ｍｌに溶解後、０．３％過酸化水素水０．０５　ｍｌを加え、室温にて８時間放置した。減圧濃縮後ＳｅｐＰａｋにより精製し、白色粉末０．４４３　ｍｇを得た。
質量分析による（Ｍ＋Ｈ）^＋：２５９９．２　（計算値２５９９．４）
ＨＰＬＣ溶出時間：１９．１分
溶出条件
カラム：Ｗａｋｏｓｉｌ−ＩＩ　５Ｃ１８ＨＧ　（４．６　ｘ　１００　ｍｍ）
溶離液：Ａ液：　０．１％ＴＦＡ−水、Ｂ液：　０．１％ＴＦＡ含有アセトニトリルを用い、Ａ／Ｂ：　１００／０〜０／７０へ直線型濃度勾配溶出（３５分）
流速：１．０　ｍｌ／分
【０１２９】
参考例４５　ヒトＧＰＲ８　ｌｉｇａｎｄ　（１−２２）：Ｔｒｐ−Ｔｙｒ−Ｌｙｓ−Ｈｉｓ−Ｖａｌ−Ａｌａ−Ｓｅｒ−Ｐｒｏ−Ａｒｇ−Ｔｙｒ−Ｈｉｓ−Ｔｈｒ−Ｖａｌ−Ｇｌｙ−Ａｒｇ−Ａｌａ−Ａｌａ−Ｇｌｙ−Ｌｅｕ−Ｌｅｕ−Ｍｅｔ−Ｇｌｙ（配列番号：９６）の製造
市販　２−ｃｈｌｏｒｏｔｒｉｔｙｌ　ｒｅｓｉｎ　（Ｃｌｔ　ｒｅｓｉｎ、１．３３　ｍｍｏｌ／ｇ）にＦｍｏｃ−Ｇｌｙを導入したのち参考例１３と同様に配列順にアミノ酸の縮合と樹脂からの切り出し、精製を行ない目的物を得た。
【０１３０】
参考例４６　ヒトＧＰＲ８　ｌｉｇａｎｄ　（１−２１）：Ｔｒｐ−Ｔｙｒ−Ｌｙｓ−Ｈｉｓ−Ｖａｌ−Ａｌａ−Ｓｅｒ−Ｐｒｏ−Ａｒｇ−Ｔｙｒ−Ｈｉｓ−Ｔｈｒ−Ｖａｌ−Ｇｌｙ−Ａｒｇ−Ａｌａ−Ａｌａ−Ｇｌｙ−Ｌｅｕ−Ｌｅｕ−Ｍｅｔ（配列番号：９７）の製造市販２−ｃｈｌｏｒｏｔｒｉｔｙｌ　ｒｅｓｉｎ　（Ｃｌｔ　ｒｅｓｉｎ，１．３３ｍｍｏｌ／ｇ）にＦｍｏｃ−Ｍｅｔを導入したのち参考例−１３と同様に配列順にアミノ酸の縮合と樹脂からの切り出し、精製を行ない目的物を得た。
【０１３１】
参考例４７　ヒトＧＰＲ８　ｌｉｇａｎｄ　（１−２０）：Ｔｒｐ−Ｔｙｒ−Ｌｙｓ−Ｈｉｓ−Ｖａｌ−Ａｌａ−Ｓｅｒ−Ｐｒｏ−Ａｒｇ−Ｔｙｒ−Ｈｉｓ−Ｔｈｒ−Ｖａｌ−Ｇｌｙ−Ａｒｇ−Ａｌａ−Ａｌａ−Ｇｌｙ−Ｌｅｕ−Ｌｅｕ（配列番号：９８）の製造
市販２−ｃｈｌｏｒｏｔｒｉｔｙｌ　ｒｅｓｉｎ　（Ｃｌｔ　ｒｅｓｉｎ、１．３３ｍｍｏｌ／ｇ）にＦｍｏｃ−Ｌｅｕを導入したのち参考例１３と同様に配列順にアミノ酸の縮合と樹脂からの切り出し、精製を行ない目的物を得た。
質量分析によるＭ^＋：２２８２．８　（計算値２２８２．６）
ＨＰＬＣ溶出時間：１７．２分
溶出条件
カラム：Ｗａｋｏｓｉｌ−ＩＩ　５Ｃ１８ＨＧ　（４．６　ｘ　１００　ｍｍ）
溶離液：Ａ液：　０．１％ＴＦＡ−水、Ｂ液：　０．１％ＴＦＡ含有アセトニトリルを用い、Ａ／Ｂ：　１００／０〜０／７０へ直線型濃度勾配溶出（３５分）
流速：１．０　ｍｌ／分
【０１３２】
参考例４８　ヒトＧＰＲ８　ｌｉｇａｎｄ　（１−１９）：Ｔｒｐ−Ｔｙｒ−Ｌｙｓ−Ｈｉｓ−Ｖａｌ−Ａｌａ−Ｓｅｒ−Ｐｒｏ−Ａｒｇ−Ｔｙｒ−Ｈｉｓ−Ｔｈｒ−Ｖａｌ−Ｇｌｙ−Ａｒｇ−Ａｌａ−Ａｌａ−Ｇｌｙ−Ｌｅｕ（配列番号：９９）の製造
市販２−ｃｈｌｏｒｏｔｒｉｔｙｌ　ｒｅｓｉｎ　（Ｃｌｔ　ｒｅｓｉｎ、１．３３ｍｍｏｌ／ｇ）にＦｍｏｃ−Ｌｅｕを導入したのち参考例１３と同様に配列順にアミノ酸の縮合と樹脂からの切り出し、精製を行ない目的物を得た。
質量分析によるＭ^＋：２１６９．６（計算値２１６９．５）
ＨＰＬＣ溶出時間：１６．４分
溶出条件
カラム：Ｗａｋｏｓｉｌ−ＩＩ　５Ｃ１８ＨＧ　（４．６　ｘ　１００　ｍｍ）
溶離液：Ａ液：　０．１％ＴＦＡ−水、Ｂ液：　０．１％ＴＦＡ含有アセトニトリルを用い、Ａ／Ｂ：　１００／０〜０／７０へ直線型濃度勾配溶出（３５分）
流速：１．０　ｍｌ／分
【０１３３】
参考例４９　ヒトＧＰＲ８　ｌｉｇａｎｄ　（１−１８）：Ｔｒｐ−Ｔｙｒ−Ｌｙｓ−Ｈｉｓ−Ｖａｌ−Ａｌａ−Ｓｅｒ−Ｐｒｏ−Ａｒｇ−Ｔｙｒ−Ｈｉｓ−Ｔｈｒ−Ｖａｌ−Ｇｌｙ−Ａｒｇ−Ａｌａ−Ａｌａ−Ｇｌｙ（配列番号：１００）の製造
市販２−ｃｈｌｏｒｏｔｒｉｔｙｌ　ｒｅｓｉｎ　（Ｃｌｔ　ｒｅｓｉｎ、１．３３ｍｍｏｌ／ｇ）にＦｍｏｃ−Ｇｌｙを導入したのち参考例１３と同様に配列順にアミノ酸の縮合と樹脂からの切り出し、精製を行ない目的物を得た。
質量分析によるＭ^＋：２０５６．８（計算値２０５６．３）
ＨＰＬＣ溶出時間：１４．２分
溶出条件
カラム：Ｗａｋｏｓｉｌ−ＩＩ　５Ｃ１８ＨＧ　（４．６　ｘ　１００　ｍｍ）
溶離液：Ａ液：　０．１％ＴＦＡ−水、Ｂ液：　０．１％ＴＦＡ含有アセトニトリルを用い、Ａ／Ｂ：　１００／０〜０／７０へ直線型濃度勾配溶出（３５分）
流速：１．０　ｍｌ／分
【０１３４】
参考例５０　ヒトＧＰＲ８　ｌｉｇａｎｄ　（１−１７）：Ｔｒｐ−Ｔｙｒ−Ｌｙｓ−Ｈｉｓ−Ｖａｌ−Ａｌａ−Ｓｅｒ−Ｐｒｏ−Ａｒｇ−Ｔｙｒ−Ｈｉｓ−Ｔｈｒ−Ｖａｌ−Ｇｌｙ−Ａｒｇ−Ａｌａ−Ａｌａ（配列番号：１０１）の製造
市販２−ｃｈｌｏｒｏｔｒｉｔｙｌ　ｒｅｓｉｎ　（Ｃｌｔ　ｒｅｓｉｎ、１．３３ｍｍｏｌ／ｇ）にＦｍｏｃ−Ａｌａを導入したのち参考例１３と同様に配列順にアミノ酸の縮合と樹脂からの切り出し、精製を行ない目的物を得た。
【０１３５】
参考例５１　ヒトＧＰＲ８　ｌｉｇａｎｄ　（１−１６）：Ｔｒｐ−Ｔｙｒ−Ｌｙｓ−Ｈｉｓ−Ｖａｌ−Ａｌａ−Ｓｅｒ−Ｐｒｏ−Ａｒｇ−Ｔｙｒ−Ｈｉｓ−Ｔｈｒ−Ｖａｌ−Ｇｌｙ−Ａｒｇ−Ａｌａ（配列番号：１０２）の製造
市販２−ｃｈｌｏｒｏｔｒｉｔｙｌ　ｒｅｓｉｎ　（Ｃｌｔ　ｒｅｓｉｎ、１．３３ｍｍｏｌ／ｇ）にＦｍｏｃ−Ａｌａを導入したのち参考例１３と同様に配列順にアミノ酸の縮合と樹脂からの切り出し、精製を行ない目的物を得た。
【０１３６】
参考例５２　ブタＧＰＲ８　ｌｉｇａｎｄ　（１−２３）：Ｔｒｐ−Ｔｙｒ−Ｌｙｓ−Ｈｉｓ−Ｔｈｒ−Ａｌａ−Ｓｅｒ−Ｐｒｏ−Ａｒｇ−Ｔｙｒ−Ｈｉｓ−Ｔｈｒ−Ｖａｌ−Ｇｌｙ−Ａｒｇ−Ａｌａ−Ａｌａ−Ｇｌｙ−Ｌｅｕ−Ｌｅｕ−Ｍｅｔ−Ｇｌｙ−Ｌｅｕ（配列番号：５６）の製造
市販２−ｃｈｌｏｒｏｔｒｉｔｙｌ　ｒｅｓｉｎ　（Ｃｌｔ　ｒｅｓｉｎ，１．３３ｍｍｏｌ／ｇ）にＦｍｏｃ−Ｌｅｕを導入したのち参考例１３と同様に配列順にアミノ酸の縮合と樹脂からの切り出し、精製を行ない目的物を得た。
質量分析による（Ｍ＋Ｈ）^＋：２５８５．２　（計算値２５８５．４）
ＨＰＬＣ溶出時間：２０．２分
溶出条件
カラム：Ｗａｋｏｓｉｌ−ＩＩ　５Ｃ１８ＨＧ　（４．６　ｘ　１００　ｍｍ）
溶離液：Ａ液：　０．１％ＴＦＡ−水、Ｂ液：　０．１％ＴＦＡ含有アセトニトリルを用い、Ａ／Ｂ：　１００／０〜０／７０へ直線型濃度勾配溶出（３５分）
流速：１．０　ｍｌ／分
【０１３７】
参考例５３　ラット／マウスＧＰＲ８　ｌｉｇａｎｄ　（１−２３）：Ｔｒｐ−Ｔｙｒ−Ｌｙｓ−Ｈｉｓ−Ｖａｌ−Ａｌａ−Ｓｅｒ−Ｐｒｏ−Ａｒｇ−Ｔｙｒ−Ｈｉｓ−Ｔｈｒ−Ｖａｌ−Ｇｌｙ−Ａｒｇ−Ａｌａ−Ｓｅｒ−Ｇｌｙ−Ｌｅｕ−Ｌｅｕ−Ｍｅｔ−Ｇｌｙ−Ｌｅｕの製造（配列番号：７３および配列番号：９１）
参考例５２と同様に配列順にアミノ酸の縮合と樹脂からの切り出し、精製を行ない目的物を得ることができる。
【０１３８】
参考例５４　ブタＧＰＲ８　ｌｉｇａｎｄ　（１−２３）酸化体：Ｔｒｐ−Ｔｙｒ−Ｌｙｓ−Ｈｉｓ−Ｔｈｒ−Ａｌａ−Ｓｅｒ−Ｐｒｏ−Ａｒｇ−Ｔｙｒ−Ｈｉｓ−Ｔｈｒ−Ｖａｌ−Ｇｌｙ−Ａｒｇ−Ａｌａ−Ａｌａ−Ｇｌｙ−Ｌｅｕ−Ｌｅｕ−Ｍｅｔ（Ｏ）−Ｇｌｙ−Ｌｅｕ（配列番号：１０３）の製造
参考例５２の化合物を用い参考例４４と同様に酸化して目的物を得た。
質量分析による（Ｍ＋Ｈ）^＋：２６０１．３　（計算値２６０１．４）
ＨＰＬＣ溶出時間：１８．９分
溶出条件
カラム：Ｗａｋｏｓｉｌ−ＩＩ　５Ｃ１８ＨＧ　（４．６　ｘ　１００　ｍｍ）
溶離液：Ａ液：　０．１％ＴＦＡ−水、Ｂ液：　０．１％ＴＦＡ含有アセトニトリルを用い、Ａ／Ｂ：　１００／０〜０／７０へ直線型濃度勾配溶出（３５分）
流速：１．０　ｍｌ／分
【０１３９】
参考例５５　ラット／マウスＧＰＲ８　ｌｉｇａｎｄ　（１−２３）酸化体：Ｔｒｐ−Ｔｙｒ−Ｌｙｓ−Ｈｉｓ−Ｖａｌ−Ａｌａ−Ｓｅｒ−Ｐｒｏ−Ａｒｇ−Ｔｙｒ−Ｈｉｓ−Ｔｈｒ−Ｖａｌ−Ｇｌｙ−Ａｒｇ−Ａｌａ−Ｓｅｒ−Ｇｌｙ−Ｌｅｕ−Ｌｅｕ−Ｍｅｔ（Ｏ）−Ｇｌｙ−Ｌｅｕ（配列番号：１０４）の製造
参考例５３の化合物を用い参考例４４と同様に酸化して目的物を得ることができる。
【０１４０】
参考例５６　［Ｎα−Ａｃｅｔｙｌ−Ｔｒｐ^１］−ヒトＧＰＲ８　ｌｉｇａｎｄ　（１−２３）：Ａｃ−Ｔｒｐ−Ｔｙｒ−Ｌｙｓ−Ｈｉｓ−Ｖａｌ−Ａｌａ−Ｓｅｒ−Ｐｒｏ−Ａｒｇ−Ｔｙｒ−Ｈｉｓ−Ｔｈｒ−Ｖａｌ−Ｇｌｙ−Ａｒｇ−Ａｌａ−Ａｌａ−Ｇｌｙ−Ｌｅｕ−Ｌｅｕ−Ｍｅｔ−Ｇｌｙ−Ｌｅｕ（配列番号：１０６）の製造
参考例１２で調製した樹脂のＦｍｏｃ基を除去、無水酢酸でアセチル化した後、ＴＦＡ　／　ｔｈｉｏａｎｉｓｏｌｅ　／　ｍ−ｃｒｅｓｏｌ　／　ｔｒｉｉｓｏｐｒｏｐｙｌｓｉｌａｎｅ　／　ｅｔｈａｎｅｄｉｔｈｉｏｌ　　（８５／　５　／　５　／　２．５　／　２．５）で処理し樹脂からの切り出しと側鎖保護基の除去を同時に行った。粗ペプチドを参考例１２と同様の方法で精製し目的物を得た。
質量分析による　（Ｍ＋Ｈ）^＋　　　２６２６．１２６２５．８　（計算値２６２７．１２６２６．１）
ＨＰＬＣ溶出時間　２１．４分
溶出条件
カラム　Ｗａｋｏｓｉｌ−ＩＩ　５Ｃ１８　ＨＧ　　　（４．６　ｘ　１００ｍｍ）
溶離液　Ａ液：　０．１％ＴＦＡ−水、Ｂ液：　０．１％ＴＦＡ含有アセトニトリルを用い、　Ａ／Ｂ：　１００　／　０〜　３０　／　７０へ　直線型濃度勾配溶出（３５分）
流速　１．０ｍｌ／分
【０１４１】
参考例５７　ヒトＧＰＲ８　ｌｉｇａｎｄ　（２−２３）：　Ｔｙｒ−Ｌｙｓ−Ｈｉｓ−Ｖａｌ−Ａｌａ−Ｓｅｒ−Ｐｒｏ−Ａｒｇ−Ｔｙｒ−Ｈｉｓ−Ｔｈｒ−Ｖａｌ−Ｇｌｙ−Ａｒｇ−Ａｌａ−Ａｌａ−Ｇｌｙ−Ｌｅｕ−Ｌｅｕ−Ｍｅｔ−Ｇｌｙ−Ｌｅｕ（配列番号：１０７）の製造
参考例１２と同様に所望のアミノ酸配列を樹脂に導入した。最後のＴｙｒを導入後樹脂から切り出す前にＦｍｏｃ基を樹脂上で除去したのち、　ＴＦＡ　／　ｔｈｉｏａｎｉｓｏｌｅ　／ｍ−ｃｒｅｓｏｌ　／　ｔｒｉｉｓｏｐｒｏｐｙｌｓｉｌａｎｅ　／　ｅｔｈａｎｅｄｉｔｈｉｏｌ　　（８５　／　５　／　５　／　２．５　／　２．５）で処理し樹脂からの切り出しと側鎖保護基の除去を同時に行った。粗ペプチドを参考例１２と同様の方法で精製し目的物を得た。
質量分析による（Ｍ＋Ｈ）＋　　　２３９７．１　（計算値２３９７．３）
ＨＰＬＣ溶出時間　１９．９分
溶出条件
カラム　Ｗａｋｏｓｉｌ−ＩＩ　５Ｃ１８　ＨＧ　　　（４．６　ｘ　１００ｍｍ）
溶離液　Ａ液：　０．１％ＴＦＡ−水、Ｂ液：　０．１％ＴＦＡ含有アセトニトリルを用い、　Ａ／Ｂ：　１００　／　０〜　３０　／　７０へ　直線型濃度勾配溶出（３５分）
流速　１．０ｍｌ／分
【０１４２】
参考例５８　ヒトＧＰＲ８　ｌｉｇａｎｄ　（４−２３）：　Ｈｉｓ−Ｖａｌ−Ａｌａ−Ｓｅｒ−Ｐｒｏ−Ａｒｇ−Ｔｙｒ−Ｈｉｓ−Ｔｈｒ−Ｖａｌ−Ｇｌｙ−Ａｒｇ−Ａｌａ−Ａｌａ−Ｇｌｙ−Ｌｅｕ−Ｌｅｕ−Ｍｅｔ−Ｇｌｙ−Ｌｅｕ（配列番号：１０８）の製造
参考例１２と同様に所望のアミノ酸配列を樹脂に導入した。最後のＨｉｓを導入後樹脂から切り出す前にＦｍｏｃ基を樹脂上で除去したのち、　ＴＦＡ　／　ｔｈｉｏａｎｉｓｏｌｅ　／ｍ−ｃｒｅｓｏｌ　／　ｔｒｉｉｓｏｐｒｏｐｙｌｓｉｌａｎｅ　／　ｅｔｈａｎｅｄｉｔｈｉｏｌ　　（８５　／　５　／　５　／　２．５　／　２．５）で処理し樹脂からの切り出しと側鎖保護基の除去を同時に行った。粗ペプチドを参考例１２と同様の方法で精製し目的物を得た。
質量分析による（Ｍ＋Ｈ）＋　　　２１０６．０　（計算値２１０６．１）
ＨＰＬＣ溶出時間　２０．０分
溶出条件
カラム　Ｗａｋｏｓｉｌ−ＩＩ　５Ｃ１８　ＨＧ　　　（４．６　ｘ　１００ｍｍ）
溶離液　Ａ液：　０．１％ＴＦＡ−水、Ｂ液：　０．１％ＴＦＡ含有アセトニトリルを用い、　Ａ／Ｂ：　１００　／　０〜　３０　／　７０へ　直線型濃度勾配溶出（３５分）
流速　１．０ｍｌ／分
【０１４３】
参考例５９　ヒトＧＰＲ８　ｌｉｇａｎｄ　（９−２３）：　Ａｒｇ−Ｔｙｒ−Ｈｉｓ−Ｔｈｒ−Ｖａｌ−Ｇｌｙ−Ａｒｇ−Ａｌａ−Ａｌａ−Ｇｌｙ−Ｌｅｕ−Ｌｅｕ−Ｍｅｔ−Ｇｌｙ−Ｌｅｕ（配列番号：１０９）の製造
参考例１２と同様に所望のアミノ酸配列を樹脂に導入した。最後のＡｒｇを導入後樹脂から切り出す前にＦｍｏｃ基を樹脂上で除去したのち、　ＴＦＡ　／　ｔｈｉｏａｎｉｓｏｌｅ　／ｍ−ｃｒｅｓｏｌ　／　ｔｒｉｉｓｏｐｒｏｐｙｌｓｉｌａｎｅ　／　ｅｔｈａｎｅｄｉｔｈｉｏｌ　　（８５　／　５　／　５　／　２．５　／　２．５）で処理し樹脂からの切り出しと側鎖保護基の除去を同時に行った。粗ペプチドを参考例１２と同様の方法で精製し目的物を得た。
質量分析による（Ｍ＋Ｈ）＋　　　１６１５．０　（計算値１６１４．９）
ＨＰＬＣ溶出時間　２０．２分
溶出条件
カラム　Ｗａｋｏｓｉｌ−ＩＩ　５Ｃ１８　ＨＧ　　　（４．６　ｘ　１００ｍｍ）
溶離液　Ａ液：　０．１％ＴＦＡ−水、Ｂ液：　０．１％ＴＦＡ含有アセトニトリルを用い、　Ａ／Ｂ：　１００　／　０〜　３０　／　７０へ　直線型濃度勾配溶出（３５分）
流速　１．０ｍｌ／分
【０１４４】
参考例６０　ヒトＧＰＲ８　ｌｉｇａｎｄ　（１５−２３）：　Ａｒｇ−Ａｌａ−Ａｌａ−Ｇｌｙ−Ｌｅｕ−Ｌｅｕ−Ｍｅｔ−Ｇｌｙ−Ｌｅｕ（配列番号：１１０）の製造
参考例１２と同様に所望のアミノ酸配列を樹脂に導入した。最後のＡｒｇを導入後樹脂から切り出す前にＦｍｏｃ基を樹脂上で除去したのち、　ＴＦＡ　／　ｔｈｉｏａｎｉｓｏｌｅ　／ｍ−ｃｒｅｓｏｌ　／　ｔｒｉｉｓｏｐｒｏｐｙｌｓｉｌａｎｅ　／　ｅｔｈａｎｅｄｉｔｈｉｏｌ　　（８５　／　５　／　５　／　２．５　／　２．５）で処理し樹脂からの切り出しと側鎖保護基の除去を同時に行った。粗ペプチドを参考例１２と同様の方法で精製し目的物を得た。
質量分析による（Ｍ＋Ｈ）＋　　　９０１．４　（計算値９０１．５）
ＨＰＬＣ溶出時間　２０．２分
溶出条件
カラム　Ｗａｋｏｓｉｌ−ＩＩ　５Ｃ１８　ＨＧ　　　（４．６　ｘ　１００ｍｍ）
溶離液　Ａ液：　０．１％ＴＦＡ−水、Ｂ液：　０．１％ＴＦＡ含有アセトニトリルを用い、　Ａ／Ｂ：　１００　／　０〜　３０　／　７０へ　直線型濃度勾配溶出（３５分）
流速　１．０ｍｌ／分
【０１４５】
参考例６１　［Ｎ−Ａｃｅｔｙｌ−Ｔｙｒ^２］−ヒトＧＰＲ８　ｌｉｇａｎｄ　（２−２３）：Ａｃ−Ｔｙｒ−Ｌｙｓ−Ｈｉｓ−Ｖａｌ−Ａｌａ−Ｓｅｒ−Ｐｒｏ−Ａｒｇ−Ｔｙｒ−Ｈｉｓ−Ｔｈｒ−Ｖａｌ−Ｇｌｙ−Ａｒｇ−Ａｌａ−Ａｌａ−Ｇｌｙ−Ｌｅｕ−Ｌｅｕ−Ｍｅｔ−Ｇｌｙ−Ｌｅｕ（配列番号：１１１）の製造
参考例５７で調製した樹脂を無水酢酸でアセチル化した後、参考例５７と同様に処理、精製し目的物を得た。
質量分析による（Ｍ＋Ｈ）＋　　　２４３９．３　（計算値２４３９．３）
ＨＰＬＣ溶出時間　２０．２分
溶出条件
カラム　Ｗａｋｏｓｉｌ−ＩＩ　５Ｃ１８　ＨＧ　　　（４．６　ｘ　１００ｍｍ）
溶離液　Ａ液：　０．１％ＴＦＡ−水、Ｂ液：　０．１％ＴＦＡ含有アセトニトリルを用い、　Ａ／Ｂ：　１００　／　０〜　３０　／　７０へ　直線型濃度勾配溶出（３５分）
流速　１．０ｍｌ／分
【０１４６】
参考例６２　［Ｄ−Ｔｒｐ^１］−ヒトＧＰＲ８　ｌｉｇａｎｄ　（１−２３）：Ｄ−Ｔｒｐ−Ｔｙｒ−Ｌｙｓ−Ｈｉｓ−Ｖａｌ−Ａｌａ−Ｓｅｒ−Ｐｒｏ−Ａｒｇ−Ｔｙｒ−Ｈｉｓ−Ｔｈｒ−Ｖａｌ−Ｇｌｙ−Ａｒｇ−Ａｌａ−Ａｌａ−Ｇｌｙ−Ｌｅｕ−Ｌｅｕ−Ｍｅｔ−Ｇｌｙ−Ｌｅｕ（配列番号：１１２）の製造
参考例１２のＦｍｏｃ−Ｔｒｐ（Ｂｏｃ）の代りにＦｍｏｃ−Ｄ−Ｔｒｐ（Ｂｏｃ）を用い同様に目的物を得た。
質量分析による（Ｍ＋Ｈ）＋　　　２５８３．４　（計算値２５８３．４）
ＨＰＬＣ溶出時間　２０．６分
溶出条件
カラム　Ｗａｋｏｓｉｌ−ＩＩ　５Ｃ１８　ＨＧ　　　（４．６　ｘ　１００ｍｍ）
溶離液　Ａ液：　０．１％ＴＦＡ−水、Ｂ液：　０．１％ＴＦＡ含有アセトニトリルを用い、　Ａ／Ｂ：　１００　／　０〜　３０　／　７０へ　直線型濃度勾配溶出（３５分）
流速　１．０ｍｌ／分
【０１４７】
参考例６３　［Ｎ−３−Ｉｎｄｏｌｅｐｒｏｐａｎｏｙｌ−Ｔｙｒ^２］−ヒトＧＰＲ８　ｌｉｇａｎｄ　（２−２３）：３−Ｉｎｄｏｌｅｐｒｏｐａｎｏｙｌ−Ｔｙｒ−Ｌｙｓ−Ｈｉｓ−Ｖａｌ−Ａｌａ−Ｓｅｒ−Ｐｒｏ−Ａｒｇ−Ｔｙｒ−Ｈｉｓ−Ｔｈｒ−Ｖａｌ−Ｇｌｙ−Ａｒｇ−Ａｌａ−Ａｌａ−Ｇｌｙ−Ｌｅｕ−Ｌｅｕ−Ｍｅｔ−Ｇｌｙ−Ｌｅｕ（配列番号：１１３）の製造
参考例１２のＦｍｏｃ−Ｔｒｐ（Ｂｏｃ）の代りに３−Ｉｎｄｏｌｅｐｒｏｐｉｏｎｉｃ　ａｃｉｄを用い所望の樹脂を得、これをＴＦＡ　／　ｔｈｉｏａｎｉｓｏｌｅ　／　ｍ−ｃｒｅｓｏｌ　／　ｔｒｉｉｓｏｐｒｏｐｙｌｓｉｌａｎｅ　／　ｅｔｈａｎｅｄｉｔｈｉｏｌ　　（８５　／　５　／　５　／　２．５　／　２．５）で処理し樹脂からの切り出しと側鎖保護基の除去を同時に行った。粗ペプチドを参考例１２と同様の方法で精製し目的物を得た。
質量分析による（Ｍ＋Ｈ）＋　　　２５６８．４　（計算値２５６８．４）
ＨＰＬＣ溶出時間　２１．７分
溶出条件
カラム　Ｗａｋｏｓｉｌ−ＩＩ　５Ｃ１８　ＨＧ　　　（４．６　ｘ　１００ｍｍ）
溶離液　Ａ液：　０．１％ＴＦＡ−水、Ｂ液：　０．１％ＴＦＡ含有アセトニトリルを用い、　Ａ／Ｂ：　１００　／　０〜　３０　／　７０へ　直線型濃度勾配溶出（３５分）
流速　１．０ｍｌ／分
【０１４８】
参考例６４　ＧＰＲ８発現ＣＨＯ細胞膜画分を用いて測定したＧＰＲ８リガンドペプチドのヒトおよびブタホモログの誘導体のＧＴＰγＳ結合促進活性
本明細書に合成法を記載したＧＰＲ８リガンドペプチドのヒトおよびブタホモログの誘導体を種々の濃度で参考例６に記載した方法でＧＰＲ８発現ＣＨＯ細胞膜画分に投与してＧＴＰγＳ結合促進活性を測定した。測定した誘導体の配列番号とＧＴＰγＳ結合促進活性を表１に示した。なお、活性は５０％有効濃度（ＥＣ_５０値）で示した。また、参考例２０および２１に記載のｈＧＰＲ８Ｌ（１−２３）およびｈＧＰＲ８Ｌ（１−３０）のＧＴＰγＳ結合促進活性も合わせて記載した。
【０１４９】
参考例６５　ＧＰＲ８発現ＣＨＯ細胞膜画分および［^１２５Ｉ−Ｔｙｒ^１０］−ｈＧＰＲ８Ｌ（１−２３）を用いて測定したＧＰＲ８リガンドペプチドのヒトおよびブタホモログの誘導体の受容体結合活性
本明細書に合成法を記載したＧＰＲ８リガンドペプチドのヒトおよびブタホモログの誘導体の受容体結合活性を参考例４３に記載した方法でＧＰＲ８発現ＣＨＯ細胞膜画分および［^１２５Ｉ−Ｔｙｒ^１０］−ｈＧＰＲ８Ｌ（１−２３）を用いて測定した。測定した誘導体の配列番号と受容体結合活性を表１に示した。なお、受容体結合活性は５０％結合阻害濃度（ＩＣ_５０値）で示した。また、参考例４３に記載のｈＧＰＲ８Ｌ（１−２３）の受容体結合活性も合わせて記載した。
【０１５０】
【表１】

【０１５１】
参考例６６　ラット全脳由来Ｇタンパク質共役型レセプタータンパク質をコードするｃＤＮＡのクローニングと塩基配列の決定
ラット全脳ｃＤＮＡ（ＣＬＯＮＴＥＣＨ）を鋳型とし、ヒトＧＰＲ８をコードするＤＮＡの塩基配列を元に設計した２個のプライマー、プライマー１（配列番号：１２８）およびプライマー２（配列番号：１２９）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を１０分の１量鋳型として使用し、Ａｄｖａｎｔａｇｅ−２　ｃＤＮＡ　Ｐｏｌｙｍｅｒａｓｅ　Ｍｉｘ（ＣＬＯＮＴＥＣＨ）１／５０量、プライマー３　０．２μＭ、プライマー２　０．２μＭ、ｄＮＴＰｓ　２００μＭ、および酵素に添付のバッファーを加え、２５μｌの液量とした。ＰＣＲ反応は、（ｉ）９４℃・２分の後、（ｉｉ）９４℃・２０秒、７２℃・２分のサイクルを３回、（ｉｉｉ）９４℃・２０秒、６６℃・２０秒、６８℃・２分のサイクルを３回、（ｉｖ）９４℃・２０秒、６０℃・２０秒、６８℃・２分のサイクルを３６回繰り返し、最後に６８℃・７分の伸長反応を行った。該ＰＣＲ反応後の反応産物を、ＴＡクローニングキット（Ｉｎｖｉｔｒｏｇｅｎ）の処方に従い、プラスミドベクターｐＣＲ２．１−ＴＯＰＯ（Ｉｎｖｉｔｒｏｇｅｎ）へサブクローニングした。これを大腸菌ＤＨ５αに導入し、アンピシリンを含むＬＢ寒天培地中で、ｃＤＮＡを持つクローンを選択した。個々のクローンの配列を解析した結果、新規Ｇタンパク質共役型レセプタータンパク質をコードするｃＤＮＡの塩基配列（配列番号：１２７）を得た。このＤＮＡの塩基配列がコードするアミノ酸配列（配列番号：１２６）を含有する新規Ｇタンパク質共役型レセプタータンパク質をＴＧＲ２６と命名した（本明細書中、ラットＴＧＲ２６とも称する）。
配列番号：１２６で表されるアミノ酸配列は、既知のヒトＧタンパク質共役型レセプタータンパク質であるＧＰＲ７〔ゲノミクス（Ｇｅｎｏｍｉｃｓ），２８巻，８４−９１頁，１９９５年〕との間に８４．８％の相同性を有していた。
ＴＧＲ２６をコードするＤＮＡを挿入したプラスミドを有する前述した形質転換体から１クローンを選択し、アンピシリンを含むＬＢ培地で振とう培養し、プラスミドを得た。これを制限酵素ＣｌａＩおよびＳｐｅＩで処理し、ＴＧＲ２６をコードするインサート部分を切り出した。同様に制限酵素ＣｌａＩおよびＳｐｅＩで処理したｐＡＫＫＯ−１．１１ＨおよびＬｉｇａｔｉｏｎ　Ｅｘｐｒｅｓｓ　Ｋｉｔ（ＣＬＯＮＴＥＣＨ）を用いて連結し、大腸菌ＤＨ１０Ｂにエレクトロポーレーション法にて導入した。得られたクローンについては、有する発現細胞構築用プラスミドの構造を、制限酵素処理および配列解析で確認したうえ、大腸菌（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）ＤＨ１０Ｂ／ｐＡＫ−ｒＧＰＲ７と命名した。
ＴＧＲ２６の疎水性プロット図を〔図１１〕に示す。
【０１５２】
参考例６７　ＴＧＲ２６発現ＣＨＯ細胞の作製
参考例６６に記載の発現プラスミドｐＡＫ−ｒＧＰＲ７で形質転換したＥｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ　ＤＨ５α（東洋紡）を培養後、Ｐｌａｓｍｉｄ　Ｍｉｄｉ　Ｋｉｔ（キアゲン）を用いてｐＡＫ−ｒＧＰＲ７プラスミドＤＮＡを調製した。これをＣｅｌｌＰｈｅｃｔ　Ｔｒａｎｓｆｅｃｔｉｏｎ　Ｋｉｔ（アマシャムファルマシアバイオテク）を用いて、添付のプロトコールに従ってＣＨＯ　ｄｈｆｒ⁻細胞に導入した。５μｇのＤＮＡをリン酸カルシウムとの共沈懸濁液とし、４８時間前に３×１０^５個のＣＨＯ　ｄｈｆｒ⁻細胞を播種した直径６ｃｍシャーレ２枚に添加した。１０％ウシ胎児血清を含むＭＥＭα培地で１日間培養した後、継代し、選択培地である１０％透析ウシ胎児血清を含む核酸不含ＭＥＭα培地で培養した。選択培地中に増殖してくるＴＧＲ２６発現ＣＨＯ細胞である形質転換細胞のコロニー４４クローンを選択した。
【０１５３】
参考例６８　ＴａｑＭａｎＰＣＲ法を用いたＴＧＲ２６発現ＣＨＯ細胞株のＴＧＲ２６発現量の定量
参考例６７で得たＴＧＲ２６発現ＣＨＯ細胞株４４クローンを、各２５ｃｍ^２フラスコに培養し、ＲＮｅａｓｙ　Ｍｉｎｉ　Ｋｉｔｓ（キアゲン）を用いてｔｏｔａｌ　ＲＮＡを調製した後、ＲＮａｓｅ−ｆｒｅｅ　ＤＮａｓｅ　Ｓｅｔ（キアゲン）を用いてＤＮａｓｅ処理をした。得られたｔｏｔａｌ　ＲＮＡ　４μｇをランダムプライマー（宝酒造）５００ｐｍｏｌを含む溶液１２μｌとして７０℃で１０分間処理した後氷冷し、さらに１ｘＦｉｒｓｔ　Ｓｔｒａｎｄ　Ｂｕｆｆｅｒ、１０　ｍＭ　ＤＴＴ、５００μＭ　ｄＡ／ｄＣ／ｄＧ／ｄＴＴＰおよび２００　ｕｎｉｔｓ　ＳＵＰＥＲＳＣＲＩＰＴ　ＩＩ（ギブコ）を添加し、混合液２０μｌを、３０℃・１０分、４２℃・６０分、５１℃・３０分、７０℃・１５分で処理することにより逆転写反応を行なった。得られたｔｏｔａｌ　ＲＮＡ　５ｎｇ相当の逆転写産物、または後に述べるようにして作製した１０〜１×１０^７コピーの標準ｃＤＮＡ、１ｘＵｎｉｖｅｒｓａｌ　ＰＣＲ　Ｍａｓｔｅｒ　Ｍｉｘ（ＰＥバイオシステムズ）、配列番号：１３０で表されるプライマーおよび配列番号：１３１で表されるプライマー各１００ｎＭ、および配列番号：１４０（Ｆａｍ−ｔｃｃｔｃｔｇｃｔｇ　ｇａｃａｃｃｇｔａｃ　ｃａｃｃｔｇａ−Ｔａｍｒａ；配列中、Ｆａｍは６−ｃａｒｂｏｘｙ−ｆｌｕｏｒｅｓｃｅｉｎを、Ｔａｍｒａは６−ｃａｒｂｏｘｙ−ｔｅｔｒａｍｅｔｈｙｌ−ｒｈｏｄａｍｉｎｅを、それぞれ示す。）で表されるＴａｑＭａｎプローブ　１００ｎＭを含む反応混合液２５μｌについてＡＢＩ　ＰＲＩＳＭ　７７００　Ｓｅｑｕｅｎｃｅ　Ｄｅｔｅｃｔｏｒ（ＰＥバイオシステムズ）を用いてＰＣＲを行なった。ＰＣＲは、５０℃・２分、９５℃・１０分で処理後、９５℃・１５秒、６０℃・６０秒のサイクルを４０回繰り返すことにより行なった。
標準ｃＤＮＡは、１００ｐｇのＴＧＲ２６発現プラスミドＤＮＡ（ｐＡＫ−ｒＧＰＲ７）、配列番号：１３０で表されるプライマーおよび配列番号：１３１で表されるプライマー各５００ｎＭ、１ｘＰＣＲ　Ｇｏｌｄ　Ｂｕｆｆｅｒ、２．５ｍＭ　ＭｇＣｌ_２、２００μＭ　ｄＡ／ｄＣ／ｄＧ／ｄＴＴＰおよび２０ｕｎｉｔｓ　ＡｍｐｌｉＴａｑ　Ｇｏｌｄ（ＰＥバイオシステムズ）を含む反応混合液２００μｌを、ＧｅｎｅＡｍｐＰＣＲ　Ｓｙｓｔｅｍ　９７００（ＰＥバイオシステムズ）を用いて、９５℃・１０分で処理後、９５℃・１０秒、６３℃・１５秒、７２℃・１０秒のサイクルを４０回繰り返す条件のＰＣＲを行なって増幅して調製した。ＱＩＡｑｕｉｃｋ　ＰＣＲ　Ｐｕｒｉｆｉｃａｔｉｏｎ　Ｋｉｔ（キアゲン）を用いて精製した合成ｃＤＮＡの２６０ｎｍの吸光度を測定して濃度を算出し、さらに標準ｃＤＮＡの正確なコピー数を算出した後、１ｍＭ　ＥＤＴＡを含む１０ｍＭ　Ｔｒｉｓ−ＨＣｌ（ｐＨ８．０）溶液で希釈し、１ｘ１０^８コピー／μｌの濃度の標準ｃＤＮＡ溶液を調製した。また、ＴａｑＭａｎ　ＰＣＲ用プローブおよびプライマーはＰｒｉｍｅｒ　Ｅｘｐｒｅｓｓ（Ｖｅｒｓｉｏｎ１．０）（ＰＥバイオシステムズ）により設計した。
発現量はＡＢＩ　ＰＲＩＳＭ　７７００　ＳＤＳソフトウェアによって算出した。リポーターの蛍光強度が設定された値に達した瞬間のサイクル数を縦軸にとり、標準ｃＤＮＡの初期濃度の対数値を横軸にとり、標準曲線を作成した。標準曲線より各逆転写産物の初期濃度を算出し、各クローンのｔｏｔａｌ　ＲＮＡ当たりのＴＧＲ２６遺伝子発現量を求めた。その結果、ＴＧＲ２６発現ＣＨＯ細胞株クローン番号１８および２８が高い発現量を示すことがわかった。以後の実験にはこれら２つのクローンの発現細胞を用いた。
【０１５４】
参考例６９　ＴＧＲ２６発現ＣＨＯ細胞を用いた細胞内ｃＡＭＰ産生量の測定
参考例６８で作製したＴＧＲ２６発現ＣＨＯ細胞を２４穴プレートに５ｘ１０^４ｃｅｌｌ／ｗｅｌｌで播種し、４８時間培養した。細胞を０．２ｍＭ　３−イソブチル−メチルキサンチン、０．０５％　ＢＳＡ（ウシ血清アルブミン）および２０ｍＭ　ＨＥＰＥＳを含むＭＥＭαバッファー（ｐＨ７．４）で洗浄した〔以下、０．２ｍＭ　３−イソブチル−メチルキサンチン、０．０５％　ＢＳＡおよび２０ｍＭ　ＨＥＰＥＳを含むＭＥＭαバッファー（ｐＨ７．４）を、反応用バッファーと呼ぶ〕。その後、０．５ｍｌの反応用バッファーを加えて３０分間培養器で保温した。反応用バッファーを除き、新たに０．２５ｍｌの反応用バッファーを細胞に加えた後、適当な濃度のＤＭＳＯ溶液とした試料と２μＭフォルスコリンを含む０．２５ｍｌの反応用バッファーを細胞に加え、３７℃で３０分間反応させた。１００μｌの２０％過塩素酸を加えて反応を停止させ、次に氷上で１時間置くことにより細胞内ｃＡＭＰを抽出した。抽出液中のｃＡＭＰ量はｃＡＭＰ　ＥＩＡキット（アマシャムファルマシアバイオテク）を用いて測定した。
【０１５５】
参考例７０　ＴＧＲ２６発現ＣＨＯ細胞膜画分を用いて測定した２３残基または３０残基のＧＰＲ８リガンドペプチドヒトホモログの細胞内ｃＡＭＰ産生抑制活性
参考例で得られた２３残基のＧＰＲ８リガンドペプチドヒトホモログ（以下、ｈＧＰＲ８Ｌ（１−２３）と記載することがある）または参考例で得られた３０残基のＧＰＲ８リガンドペプチドヒトホモログ（以下、ｈＧＰＲ８Ｌ（１−３０）と記載することがある）を、種々の濃度で参考例６９に記載した方法でＴＧＲ２６発現ＣＨＯ細胞膜画分に投与し、細胞内ｃＡＭＰ産生抑制活性を測定した。
結果を〔図１２〕に示す。
これより明らかに、ｈＧＰＲ８Ｌ（１−２３）およびｈＧＰＲ８Ｌ（１−３０）は濃度依存的にＴＧＲ２６発現ＣＨＯ細胞細胞内ｃＡＭＰの産生を抑制した。
図中、ｃＡＭＰ合成抑制活性は、フォルスコリンを含む反応用バッファーを添加したときの細胞内ｃＡＭＰ量から反応用バッファーを添加したときの細胞内ｃＡＭＰ量を減じた量を１００％として、ｈＧＰＲ８Ｌ（１−２３）またはｈＧＰＲ８Ｌ（１−３０）を加えたときの細胞内ｃＡＭＰ量から反応用バッファーを添加したときの細胞内ｃＡＭＰ量を減じた量を％として表わした。
これより、ｈＧＰＲ８Ｌ（１−２３）またはｈＧＰＲ８Ｌ（１−３０）が、ＴＧＲ２６に対するリガンドであることが明らかとなった。
ｈＧＰＲ８Ｌ（１−２３）のブタ、ラットおよびマウスのホモログおよびｈＧＰＲ８Ｌ（１−３０）のブタ、ラットおよびマウスのホモログを用いても、上記と同様に、濃度依存的にＴＧＲ２６発現ＣＨＯ細胞細胞内ｃＡＭＰの産生抑制を確認できる。
【０１５６】
参考例７１　ＴＧＲ２６発現ＣＨＯ細胞の膜画分を用いたＧＴＰγＳ結合活性の測定
ＴＧＲ２６発現ＣＨＯ細胞膜画分に対する［^３５Ｓ］−ｇｕａｎｏｓｉｎｅ　５’−（γ−ｔｈｉｏ）ｔｒｉｐｈｏｓｐｈａｔｅ（ＧＴＰγＳ）の結合促進活性を以下の方法により測定した。
１）膜画分の調製法
１ｘ１０^８個のＴＧＲ２６発現ＣＨＯ細胞に１０ｍｌのホモジネートバッファー（１０ｍＭ　ＮａＨＣＯ_３、５ｍＭ　ＥＤＴＡ、０．５ｍＭ　ＰＭＳＦ（フェニルメチルスルホニルフルオライド）、１μｇ／ｍｌペプスタチン、４μｇ／ｍｌ　Ｅ−６４、２０μｇ／ｍｌロイペプチン）を添加し、ポリトロン（１２，０００ｒｐｍ、１分間）を用いて破砕した。細胞破砕液を遠心（１，０００ｇ、１５分間）して上清を得た。次にこの上清を超遠心分離（Ｂｅｃｋｍａｎ　ｔｙｐｅ　３０ローター、３０，０００ｒｐｍ、１時間）し、得られた沈殿物をＴＧＲ２６発現ＣＨＯ細胞膜画分とした。
２）ＧＴＰγＳ結合活性の測定
ＴＧＲ２６発現ＣＨＯ細胞膜画分を膜希釈緩衝液（５０ｍＭトリス塩酸緩衝液（ｐＨ７．４）、５　ｍＭ　ＭｇＣｌ_２、１５０ｍＭ　ＮａＣｌ、１μＭ　ＧＤＰ、０．１％　ＢＳＡ）で希釈して、タンパク質濃度３０μｇ／ｍｌのアッセイ用細胞膜画分溶液を調製した。アッセイ用膜画分溶液２００μｌに、５０ｎＭ濃度の［^３５Ｓ］−ｇｕａｎｏｓｉｎｅ　５’−（γ−ｔｈｉｏ）ｔｒｉｐｈｏｓｐｈａｔｅ（ＮＥＮ社）を２μｌと適当な濃度のＤＭＳＯ溶液とした試料２μｌとを添加し、この混合液を２５℃で１時間保温した。混合液をフィルター濾過し、さらにフィルターを洗浄用バッファー（５０ｍＭトリス塩酸緩衝液（ｐＨ７．４）、５ｍＭ　ＭｇＣｌ_２、１ｍＭ　ＥＤＴＡ、０．１％　ＢＳＡ）１．５ｍｌで２回洗浄した後、フィルターの放射活性を液体シンチレーションカウンターで測定した。
【０１５７】
参考例７２　ＴＧＲ２６発現ＣＨＯ細胞膜画分を用いて測定した２３残基または３０残基のＧＰＲ８リガンドペプチドヒトホモログのＧＴＰγＳ結合促進活性
ｈＧＰＲ８Ｌ（１−２３）またはｈＧＰＲ８Ｌ（１−３０）を種々の濃度で、参考例７１に記載した方法に従い、ＴＴＧＲ２６発現ＣＨＯ細胞膜画分と混合し、ＧＴＰγＳ結合促進活性を測定した。
結果を〔図１３〕に示す。
これより明らかに、ｈＧＰＲ８Ｌ（１−２３）およびｈＧＰＲ８Ｌ（１−３０）は濃度依存的にＴＧＲ２６発現ＣＨＯ細胞膜画分のＧＴＰγＳ結合を促進した。
ｈＧＰＲ８Ｌ（１−２３）のブタ、ラットおよびマウスのホモログおよびｈＧＰＲ８Ｌ（１−３０）のブタ、ラットおよびマウスのホモログを用いても、上記と同様に、濃度依存的にＴＧＲ２６発現ＣＨＯ細胞膜画分のＧＴＰγＳ結合促進を確認できる。
【０１５８】
参考例７３　［^１２５Ｉ−Ｔｙｒ^１０］−ｈＧＰＲ８Ｌ（１−２３）を用いたレセプター結合実験
参考例４２に記載した方法により作製した［^１２５Ｉ−Ｔｙｒ^１０］−ｈＧＰＲ８Ｌ（１−２３）および参考例７１に記載したＴＴＧＲ２６発現ＣＨＯ細胞から調製した細胞膜画分を用いて以下のようにしてレセプター結合実験を行なった。
ＴＧＲ２６発現ＣＨＯ細胞から調製した細胞膜画分を、アッセイ用バッファー（２５　ｍＭ　Ｔｒｉｓ−ＨＣｌ、５　ｍＭ　ＥＤＴＡ、０．０５％　ＣＨＡＰＳ（３−［（３−コラミドプロピル）ジメチルアンモニオ］−１−プロパン硫酸）、０．１％　ＢＳＡ、０．５　ｍＭ　ＰＭＳＦ、１μｇ／ｍｌペプスタチン、２０μｇ／ｍｌロイペプチン、４μｇ／ｍｌ　Ｅ−６４、ｐＨ　７．４）で各種濃度に希釈後、ポリプロピレン製試験管（Ｆａｌｃｏｎ　２０５３）に２００μｌずつ分注した。最大結合量を測定するために２μｌのＤＭＳＯと７ｎＭの［^１２５Ｉ−Ｔｙｒ^１０］−ｈＧＰＲ８Ｌ（１−２３）２μｌを膜画分溶液に添加した。また、非特異的結合を測定するために１００μＭ　ｈＧＰＲ８Ｌ（１−２３）のＤＭＳＯ溶液２μｌと７ｎＭの［^１２５Ｉ−Ｔｙｒ^１０］−ｈＧＰＲ８Ｌ（１−２３）２μｌを膜画分溶液に添加した。２５℃で７５分間反応させた後、ポリエチレンイミン処理したワットマングラスフィルター（ＧＦ−Ｆ）を用いて反応液を吸引ろ過しさらにフィルターを洗浄用バッファー（２５　ｍＭ　Ｔｒｉｓ−ＨＣｌ、５　ｍＭ　ＥＤＴＡ、０．０５％　ＣＨＡＰＳ、０．１％　ＢＳＡ、ｐＨ　７．４）１．５　ｍｌで２回洗浄した。ろ過後、γ−カウンターを用いてろ紙上に残った放射活性を測定し、最大結合量から非特異的結合量を引いて特異的結合量を見積もった。
膜画分の濃度を変化させると膜画分の濃度に依存した［^１２５Ｉ−Ｔｙｒ^１０］−ｈＧＰＲ８Ｌ（１−２３）の特異的な結合が認められた。膜画分濃度を３μｇ／ｍｌに設定してｈＧＰＲ８Ｌ（１−２３）およびｈＧＰＲ８Ｌ（１−３０）による［^１２５Ｉ−Ｔｙｒ^１０］−ｈＧＰＲ８Ｌ（１−２３）のＴＧＲ２６発現細胞膜画分への特異的結合に対する結合阻害を調べた。阻害率から５０％阻害濃度（ＩＣ_５０値）を算出したところ、ｈＧＰＲ８Ｌ（１−２３）のＩＣ_５０値は０．１２ｎＭであった。また、ｈＧＰＲ８Ｌ（１−３０）のＩＣ_５０値は０．０２８ｎＭであった。
これより、ｈＧＰＲ８Ｌ（１−２３）およびｈＧＰＲ８Ｌ（１−３０）がＴＧＲ２６発現細胞膜画分に対して高い親和性を有することが示された。このことは、ｈＧＰＲ８Ｌ（１−２３）およびｈＧＰＲ８Ｌ（１−３０）がＴＧＲ２６レセプターの高親和性リガンドであることを意味するものである。
〔図１４〕に種々の濃度におけるｈＧＰＲ８Ｌ（１−２３）およびｈＧＰＲ８Ｌ（１−３０）の結合阻害を示す。
ｈＧＰＲ８Ｌ（１−２３）のラットおよびマウスのホモログおよびｈＧＰＲ８Ｌ（１−３０）のブタ、ラットおよびマウスのホモログを用いても、上記と同様に、［^１２５Ｉ−Ｔｙｒ^１０］−ｈＧＰＲ８Ｌ（１−２３）のＴＧＲ２６発現細胞膜画分への特異的結合に対する結合阻害を確認できる。
【０１５９】
参考例７４　ＴＧＲ２６発現ＣＨＯ細胞膜画分および［^１２５Ｉ−Ｔｙｒ^１０］−ｈＧＰＲ８Ｌ（１−２３）を用いて測定したＧＰＲ８リガンドペプチドのヒトおよびブタホモログの誘導体のレセプター結合活性
参考例で得られたＧＰＲ８リガンドペプチドのヒトおよびブタホモログの誘導体のレセプター結合活性を、参考例７３に記載した方法でＴＧＲ２６発現ＣＨＯ細胞膜画分および［^１２５Ｉ−Ｔｙｒ^１０］−ｈＧＰＲ８Ｌ（１−２３）を用いて測定した。測定した誘導体とレセプター結合活性を表２に示す。レセプター結合活性は、５０％結合阻害濃度（ＩＣ_５０値）で示した。
【０１６０】
【表２】

【０１６１】
参考例７５　マウスＴＧＲ２６をコードするｃＤＮＡの５’上流端のクローニング
５’ＲＡＣＥ　ＰＣＲクローニングによりマウスＴＧＲ２６をコードするｃＤＮＡの５’上流塩基配列を明らかにした。
５’ＲＡＣＥ　ＰＣＲクローニングは、参考例に記載のマウス脳ｃＤＮＡを鋳型としてＳＭＡＲＴ^ＴＭ　ＲＡＣＥｃＤＮＡ　Ａｍｐｌｉｆｉｃａｔｉｏｎ　Ｋｉｔに添付のＵｎｉｖｅｒｓａｌ　Ｐｒｉｍｅｒ　Ｍｉｘと配列番号：１３２の合成プライマーでＰＣＲ反応を行ない、次にこのＰＣＲ反応液を鋳型としてキットに添付のＮｅｓｔｅｄ　Ｕｎｉｖｅｒｓａｌ　Ｐｒｉｍｅｒと配列番号：１３３の合成プライマーでＰＣＲ反応を行なうことにより達成された。配列番号：１３２および配列番号：１３３のプライマーは、Ｇｅｎｂａｎｋに登録のマウスＧＰＲ７ｃＤＮＡ断片配列（Ａｃｃｅｓｓｉｏｎ：Ｕ２３８０７）を基に設計した。ＰＣＲの反応液組成と反応条件は以下のとおりである。反応液はマウス脳ｃＤＮＡ１μｌ、Ｕｎｉｖｅｒｓａｌ　Ｐｒｉｍｅｒ　Ｍｉｘ２μｌ、配列番号：１３２の合成ＤＮＡプライマー０．２μＭ、０．８ｍＭ　ｄＮＴＰｓ、Ａｄｖａｎｔａｇｅ−ＧＣ　２ポリメラーゼ（ＣＬＯＮＴＥＣＨ）０．４μｌおよび酵素に付属のバッファーで総反応量を２０μｌとし、サーマルサイクラー（ＰＥバイオシステムズ）を用い、９６℃・１２０秒の加熱の後、９６℃・３０秒、６８℃・１２０秒のサイクルを３０回繰り返し、さらに７２℃で１０分間保温した。次に、キットに添付のＴｒｉｃｉｎｅ−ＥＤＴＡ　Ｂｕｆｆｅｒで５０倍希釈したＰＣＲ反応液０．５μｌ、Ｎｅｓｔｅｄ　Ｕｎｉｖｅｒｓａｌ　Ｐｒｉｍｅｒ０．５μＭ、配列番号：１３３の合成ＤＮＡプライマー０．５μＭ、０．８ｍＭ　ｄＮＴＰｓ、Ａｄｖａｎｔａｇｅ−ＧＣ　２ポリメラーゼ（ＣＬＯＮＴＥＣＨ）０．４μｌおよび酵素に付属のバッファーで総反応量を２０μｌとし、サーマルサイクラー（ＰＥバイオシステムズ）を用い、９６℃・１２０秒の加熱の後、９６℃・３０秒、６０℃・３０秒、７２℃・６０秒のサイクルを３０回繰り返し、さらに７２℃で１０分間保温した。増幅したＤＮＡを１．５％のアガロースゲル電気泳動により分離した後、約４５０塩基長のＤＮＡをカミソリで切り出し、ＤＮＡをＱＩＡｑｕｉｃｋ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　Ｋｉｔ（キアゲン）を用いて回収した。このＤＮＡをＴＯＰＯ　ＴＡ　Ｃｌｏｎｉｎｇ　Ｋｉｔ　（Ｉｎｖｉｔｒｏｇｅｎ）のプロトコールに従ってｐＣＲ２．１−ＴＯＰＯベクターへクローニングした。これをエシェリヒア・コリ（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）　ＴＯＰ１０　ｃｏｍｐｅｔｅｎｔ　ｃｅｌｌ（Ｉｎｖｉｔｒｏｇｅｎ）に導入して形質転換した後、ｃＤＮＡ挿入断片を持つクローンをアンピシリンおよびＸ−ｇａｌを含むＬＢ寒天培地で選択し、白色を呈するクローンのみを滅菌したつま楊枝を用いて分離し、形質転換体を得た。個々のクローンをアンピシリンを含むＬＢ培地で一晩培養し、ＱＩＡｗｅｌｌ　８　Ｐｌａｓｍｉｄ　Ｋｉｔ（キアゲン）を用いてプラスミドＤＮＡを調製した。塩基配列の決定のための反応はＢｉｇＤｙｅＴｅｒｍｉｎａｔｏｒ　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　Ｒｅａｄｙ　Ｒｅａｃｔｉｏｎ　Ｋｉｔ（ＰＥバイオシステムズ）を用いて行ない、蛍光式自動シーケンサーを用いて解読し、配列番号：１３４で表される塩基配列を得た。
【０１６２】
参考例７６　ヒト染色体ＤＮＡを用いたＰＣＲ法によるヒトＧＰＲ７　ＤＮＡの増幅
ヒト染色体ＤＮＡを鋳型として、２種の合成プライマー（配列番号：１４１および配列番号：１４２）を用いたＰＣＲ法によるＤＮＡ増幅を行なった。合成プライマーは受容体タンパク質に翻訳される領域の遺伝子が増幅されるように構築したが、その際に遺伝子の５’側に制限酵素Ｃｌａ　Ｉの認識する塩基配列が付加され、３’側に制限酵素Ｓｐｅ　Ｉの認識する塩基配列が付加されるように、５’側および３’側にそれぞれの制限酵素の認識配列を付加した。反応液の組成は、ヒト染色体ＤＮＡ（タカラ）０．５μｇ、合成ＤＮＡプライマー各１μＭ、０．８　ｍＭ　ｄＮＴＰｓ、１　ｍＭ　ＭｇＣｌ_２、ＫＯＤポリメラーゼ（トーヨーボー）１μｌおよび酵素に付属のバッファーで、総反応量は５０μｌとした。増幅のためのサイクルはサーマルサイクラー（タカラ）を用い、９４℃・６０秒の加熱の後、９８℃・１５秒、６５℃・２秒、７４℃・３０秒のサイクルを３５回繰り返した。増幅産物の確認は、０．８％アガロースゲル電気泳動の後、エチジウムブロマイド染色によって行なった。
【０１６３】
参考例７７　ＰＣＲ産物のプラスミドベクターへのサブクローニングおよび挿入ＤＮＡ部分の塩基配列の解読による増幅ＤＮＡ配列の確認
参考例７６で行なったＰＣＲ反応液を０．８％の低融点アガロースゲル電気泳動により分離し、バンドの部分をかみそりで切り出した後、細片化、フェノール抽出、フェノール・クロロホルム抽出、エタノール沈殿の操作を行なってＤＮＡを回収した。ｐＣＲ−Ｓｃｒｉｐｔ^ＴＭ　Ａｍｐ　ＳＫ（＋）クローニングキット（ストラタジーン）の処方に従い、回収したＤＮＡをプラスミドベクターｐＣＲ−Ｓｃｒｉｐｔ　Ａｍｐ　ＳＫ（＋）へサブクローニングした。これをエシェリヒア　コリ（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）　ＤＨ５αｃｏｍｐｅｔｅｎｔ　ｃｅｌｌ（トーヨーボー）に導入して形質転換した後、ＤＮＡ挿入断片を持つクローンをアンピシリン、ＩＰＴＧおよびＸ−ｇａｌを含むＬＢ寒天培地で選択し、白色を呈するクローンのみを滅菌したつま楊枝を用いて分離し、形質転換体Ｅ．　ｃｏｌｉ　ＤＨ５α／ＧＰＲ７を得た。個々のクローンをアンピシリンを含むＬＢ培地で一晩培養し、ＱＩＡｗｅｌｌ　８　Ｐｌａｓｍｉｄ　Ｋｉｔ（キアゲン）を用いてプラスミドＤＮＡを調製した。調製したＤＮＡの一部に対して制限酵素Ｃｌａ　ＩおよびＳｐｅ　Ｉによる切断を行ない、挿入されている受容体ＤＮＡ断片の大きさを確認した。塩基配列の決定のための反応はＤｙｅＤｅｏｘｙＴｅｒｍｉｎａｔｏｒ　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｅ　Ｋｉｔ　（Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍｓ社）を用いて行ない、蛍光式自動シーケンサーを用いて解読した（配列番号：１４３）。配列番号：１４３で表される塩基配列を有するＤＮＡを保持するｐＣＲ−Ｓｃｒｉｐｔ　Ａｍｐ　ＳＫ（＋）プラスミドを、ｐＣＲ−ＳｃｒｉｐｔヒトＧＰＲ７と命名した。配列番号：１４３で表される塩基配列を有するＤＮＡがコードするヒトＧＰＲ７のアミノ酸配列を配列番号：１４４に示した。ここで配列を決定したヒトＧＰＲ７のＤＮＡ配列はＯ’Ｄｏｗｄらの報告（Ｏ’Ｄｏｗｄ，　Ｂ．　Ｆ．　ｅｔ　ａｌ．、Ｇｅｎｏｍｉｃｓ、２８巻、８４−９１頁、１９９５年）にあるＤＮＡ配列とは２塩基が異なっていた。これらは配列番号：１４３の８９３番目および８９４番目に当たり、Ｏ’Ｄｏｗｄらの報告ではそれぞれＣおよびＧであるが、本参考例ではＧおよびＣであった。これにより、翻訳されるアミノ酸配列において配列番号：１４４の２９６番目のアミノ酸が、Ｏ’Ｄｏｗｄらの報告のＴｈｒが本参考例ではＳｅｒとなる。
【０１６４】
参考例７８　ヒトＧＰＲ７発現ＣＨＯ細胞の作製
参考例７７で配列が確認されたヒトＧＰＲ７の全長アミノ酸配列をコードし５’側にＣｌａ　Ｉ認識配列を付加し、また３’側にＳｐｅ　Ｉ認識配列を付加した遺伝子が導入されたプラスミドによって形質転換されたＥ．　ｃｏｌｉのクローンからＰｌａｓｍｉｄ　Ｍｉｄｉ　ＫＩｔ（キアゲン）を用いてプラスミドＤＮＡを調製し、これを制限酵素ＣｌａＩおよびＳｐｅ　Ｉで消化してインサートＤＮＡを切り出した。インサートＤＮＡは電気泳動後、アガロースゲルからカミソリで切り出し、次に細片化、フェノール抽出、フェノール・クロロホルム抽出、エタノール沈殿の操作により回収された。このインサートＤＮＡをＣｌａＩおよびＳｐｅ　Ｉで切断した動物細胞発現用ベクタープラスミドｐＡＫＫＯ−１１１Ｈ（Ｈｉｎｕｍａ，　Ｓ．　ｅｔ　ａｌ．　Ｂｉｏｃｈｉｍ．　Ｂｉｏｐｈｙｓ．　Ａｃｔａ、１２１９巻、２５１−２５９頁、１９９４年、記載のｐＡＫＫＯ１．１１Ｈと同一のベクタープラスミド）に加え、Ｔ４ライゲース（タカラ）を用いてライゲーションを行ない、タンパク質発現用プラスミドｐＡＫＫＯ−Ｈｕｍａｎ　ＧＰＲ７を構築した。このプラスミドｐＡＫＫＯ−Ｈｕｍａｎ　ＧＰＲ７で形質転換した大腸菌をＤＨ５α／ｐＡＫＫＯ−Ｈｕｍａｎ　ＧＰＲ７と命名した。
ｐＡＫＫＯ−Ｈｕｍａｎ　ＧＰＲ７で形質転換したＥ．　ｃｏｌｉ　ＤＨ５α（トーヨーボー）を培養後、Ｐｌａｓｍｉｄ　Ｍｉｄｉ　Ｋｉｔ（キアゲン）を用いてｐＡＫＫＯ−Ｈｕｍａｎ　ＧＰＲ７プラスミドＤＮＡを調製した。これをＣｅｌｌＰｈｅｃｔ　Ｔｒａｎｓｆｅｃｔｉｏｎ　Ｋｉｔ（アマシャムファルマシアバイオテク）を用いて、添付のプロトコールに従ってＣＨＯ　ｄｈｆｒ⁻細胞に導入した。３μｇのＤＮＡをリン酸カルシウムとの共沈懸濁液とし、２４時間前に５　ｘ　１０^５または１　ｘ　１０^６個のＣＨＯ　ｄｈｆｒ⁻細胞を播種した直径６　ｃｍシャーレに添加した。１０％ウシ胎児血清を含むＭＥＭα培地で１日間培養した後、継代し、選択培地である１０％透析ウシ胎児血清を含む核酸不含ＭＥＭα培地で培養した。選択培地中に増殖してくるＧＰＲ８発現ＣＨＯ細胞である形質転換細胞のコロニー２４クローンを選択した。
【０１６５】
参考例７９　ＴａｑＭａｎ　ＰＣＲ法を用いたヒトＧＰＲ７発現ＣＨＯ細胞株のヒトＧＰＲ７遺伝子発現量の測定
参考例７８に従って作製したヒトＧＰＲ７発現ＣＨＯ細胞株２４クローンを各２５　ｃｍ^２フラスコに培養し、増殖した細胞からＩＳＯＧＥＮ（ニッポンジーン社）を用いてｔｏｔａｌ　ＲＮＡ画分を調製した。このｔｏｔａｌ　ＲＮＡ画分に対してＭｅｓｓａｇｅＣｌｅａｎ　（Ｇｅｎ　Ｈｕｎｔｅｒ社）キットを用いたＤＮａｓｅ　Ｉ処理を行ない、ＤＮＡを含まないｔｏｔａｌ　ＲＮＡを得た。
Ｔｏｔａｌ　ＲＮＡを鋳型としたｃＤＮＡ合成は、ＴａｑＭａｎ　Ｒｅｖｅｒｓｅ　Ｔｒａｎｓｃｒｉｐｔｉｏｎ　Ｒｅａｇｅｎｔｓ　（Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍｓ社）キットを用いて行なった。反応液の組成は、ＤＮａｓｅ　Ｉ処理したｔｏｔａｌ　ＲＮＡ　４μｇ、ランダムプライマー１μｌ、２５　ｍＭ　ＭｇＣｌ_２溶液４．４μｌ、１０　ｍＭ　ｄＮＴＰ　ｍｉｘ　２μｌ、ＲＮａｓｅ　Ｉｎｈｉｂｉｔｏｒ　０．４μｌ、逆転写酵素０．５μｌおよびキットに付属の反応バッファーで、総反応量を２０μｌとした。逆転写反応はサーマルサイクラー（タカラ）を用いて、２５℃・１０分、４８℃・３０分、９５℃・５分の条件で行なった。
標準ヒトＧＰＲ７　ＤＮＡは、全長ヒトＧＰＲ７　ＤＮＡを鋳型としたＰＣＲ増幅ＤＮＡを精製することにより調製された。ＰＣＲ反応液の組成は、参考例７７に記載のｐＣＲ−ＳｃｒｉｐｔヒトＧＰＲ７　５ｐｇ、合成ＤＮＡプライマー（配列番号：１４５）０．５μＭ、合成ＤＮＡプライマー（配列番号：１４６）０．５μＭ、１．６　ｍＭ　ｄＮＴＰｓ、２．５　ｍＭ　ＭｇＣｌ_２、ＬＡＴａｑポリメラーゼ（タカラ）０．５μｌおよび酵素に付属のバッファーで、総反応量は５０μｌとした。増幅のための反応はサーマルサイクラー（Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍｓ社）を用い、９４℃・１２０秒の加熱の後、９４℃・３０秒、６０℃・３０秒、７２℃・６０秒のサイクルを２５回繰り返し、最後に７２℃・１０分間保温した。ＰＣＲ反応液を０．８％のアガロースゲル電気泳動により分離し、バンドの部分をかみそりで切り出した後、ＱＩＡｑｕｉｃｋ　ＰＣＲ　Ｐｕｒｉｆｉｃａｔｉｏｎ　Ｋｉｔ（キアゲン）を用いてＰＣＲ増幅ＤＮＡを回収した。このＰＣＲ増幅ＤＮＡ溶液に混入しているプライマーＤＮＡおよびｄＮＴＰｓを取り除くため、このＤＮＡ溶液をクロモスピンカラム４００（ＣＬＯＮＴＥＣＨ社）ゲルクロマトグラフィーに供し、増幅ヒトＧＰＲ７　ＤＮＡ溶出画分を得た。この増幅ヒトＧＰＲ７　ＤＮＡ溶液の２６０　ｎｍの吸収から計算されたＤＮＡ量と増幅ヒトＧＰＲ７　ＤＮＡ塩基組成から、本増幅ヒトＧＰＲ７　ＤＮＡ溶液に含まれるＤＮＡのコピー数が算出された。このＤＮＡコピー数が明らかとなった増幅ヒトＧＰＲ７　ＤＮＡを標準ヒトＧＰＲ７　ＤＮＡとして、定量を目的としたＴａｑＭａｎ　ＰＣＲに用いることにした。
【０１６６】
ヒトＧＰＲ７ＣＨＯ細胞株の発現ヒトＧＰＲ７遺伝子コピー数は、ＴａｑＭａｎ　ＰＣＲ法により決定された。ＴａｑＭａｎ　ＰＣＲ反応液の組成は、蒸留水で１００倍希釈した逆転写ｃＤＮＡ溶液　１μｌまたは種々のコピー数の標準ヒトＧＰＲ７　ＤＮＡ溶液　１μｌ、合成ＤＮＡプライマー（配列番号：１４７）０．２μＭ、合成ＤＮＡプライマー（配列番号：１４８）０．２μＭ、ヒトＧＰＲ７　ＴａｑＭａｎプローブ〔配列番号：７７（Ｆａｍ−ＴＴＣＡＴＣＣＴＣＡ　ＡＣＣＴＧＧＣＣＡＴ　ＣＧＣ−Ｔａｍｒａ；配列中、Ｆａｍは６−ｃａｒｂｏｘｙ−ｆｌｕｏｒｅｓｃｅｉｎを、Ｔａｍｒａは６−ｃａｒｂｏｘｙ−ｔｅｔｒａｍｅｔｈｙｌ−ｒｈｏｄａｍｉｎｅを、それぞれ示す。）で表される塩基配列を有するプローブ〕０．２μＭおよびＴａｑＭａｎ　Ｕｎｉｖｅｒｓａｌ　ＰＣＲ　Ｍａｓｔｅｒ　Ｍｉｘ（Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍｓ社）で、総反応量を２５μｌとした。ＰＣＲ反応は、ＡＢＩ　ＰＲＩＳＭ　７７００　Ｓｅｑｕｅｎｃｅ　Ｄｅｔｅｃｔｏｒ　Ｓｙｓｔｅｍ　（Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍｓ社）を用い、５０℃・２分、９５℃・１０分で保温し、次に９５℃・１５秒、６０℃・６０秒のサイクルを４０回繰り返すことにより行なった。ヒトＧＰＲ７遺伝子発現量はＡＢＩ　ＰＲＩＳＭ　７７００　ＳＤＳソフトウェアによって算出した。リポーターの蛍光強度が、設定された値に達した瞬間のサイクル数を縦軸にとり、種々の標準ヒトＧＰＲ７　ＤＮＡのコピー数の対数値を横軸にとって標準曲線を作成した。標準曲線より逆転写ｃＤＮＡに含まれるヒトＧＰＲ７　ｃＤＮＡのコピー数を算出し、ｔｏｔａｌ　ＲＮＡ１ｎｇ当たりのヒトＧＰＲ７遺伝子発現量を決定した。ヒトＧＰＲ７遺伝子発現量の高いクローンＮｏ．　７，　８および　１４を、ヒトＧＰＲ７遺伝子高発現細胞株として選択した。
【０１６７】
参考例８０　ヒトＧＰＲ７発現ＣＨＯ細胞を用いた細胞内ｃＡＭＰ産生量の測定
参考例７８で作製し、参考例７９に記載したようにして選択したヒトＧＰＲ７発現ＣＨＯ細胞を２４穴プレートに５　ｘ　１０^４　ｃｅｌｌ／ｗｅｌｌで播種し、４８時間培養した。細胞を０．２　ｍＭ　３−イソブチル−メチルキサンチン、０．０５％　　ＢＳＡ（ウシ血清アルブミン）および２０　ｍＭ　ＨＥＰＥＳを含むＭＥＭαバッファー（ｐＨ７．４）で洗浄した（以下、０．２ｍＭ　３−イソブチル−メチルキサンチン、０．０５％　　ＢＳＡおよび２０　ｍＭ　ＨＥＰＥＳを含むＭＥＭαバッファー（ｐＨ７．４）を、反応用バッファーと呼ぶ）。その後０．５　ｍｌの反応用バッファーを加えて３０分間培養器で保温した。反応用バッファーを除き、新たに０．２５　ｍｌの反応用バッファーを細胞に加えた後、適当な濃度のＤＭＳＯ溶液とした試料と２μＭフォルスコリンを含む０．２５　ｍｌの反応用バッファーを細胞に加え、３７℃で３０分間反応させた。１００μｌの２０％過塩素酸を加えて反応を停止させ、次に氷上で１時間置くことにより細胞内ｃＡＭＰを抽出した。抽出液中のｃＡＭＰ量は、　ｃＡＭＰ　ＥＩＡキット（アマシャムファルマシアバイオテク）を用いて測定した。
【０１６８】
参考例８１　ヒトＧＰＲ７発現ＣＨＯ細胞を用いて測定した２３残基または３０残基のＧＰＲ８リガンドペプチドヒトホモログの細胞内ｃＡＭＰ産生抑制活性
ｈＧＰＲ８Ｌ（１−２３）またはｈＧＰＲ８Ｌ（１−３０）を種々の濃度で、参考例８０に記載した方法に従い、ヒトＧＰＲ７発現ＣＨＯ細胞に投与して細胞内ｃＡＭＰ産生抑制活性を測定した。結果を図１５に示す。図中、ｃＡＭＰ合成抑制活性は、フォルスコリンを含む反応用バッファーを添加したときの細胞内ｃＡＭＰ量から反応用バッファーを添加したときの細胞内ｃＡＭＰ量を減じた量を１００％として、ｈＧＰＲ８Ｌ（１−２３）またはｈＧＰＲ８Ｌ（１−３０）を加えたときの細胞内ｃＡＭＰ量から反応用バッファーを添加したときの細胞内ｃＡＭＰ量を減じた量を％として表わした。
明らかにｈＧＰＲ８Ｌ（１−２３）およびｈＧＰＲ８Ｌ（１−３０）は濃度依存的にヒトＧＰＲ７発現ＣＨＯ細胞細胞内ｃＡＭＰの産生を抑制した。このことからｈＧＰＲ８Ｌ（１−２３）およびｈＧＰＲ８Ｌ（１−３０）が、ヒトＧＰＲ７に対するリガンドであることが明らかとなった。ｃＡＭＰ産生量から５０％阻害濃度（ＩＣ_５０値）を算出したところ、ｈＧＰＲ８Ｌ（１−２３）のＩＣ_５０値は０．０２５　ｎＭであった。また、ｈＧＰＲ８Ｌ（１−３０）のＩＣ_５０値は０．１３ｎＭであった。
ｈＧＰＲ８Ｌ（１−２３）のブタ、ラットおよびマウスのホモログおよびｈＧＰＲ８Ｌ（１−３０）のブタ、ラットおよびマウスのホモログを用いても、上記と同様にヒトＧＰＲ７発現ＣＨＯ細胞の反応を確認できる。
【０１６９】
参考例８２　［^１２５Ｉ−Ｔｙｒ^１０］−ｈＧＰＲ８Ｌ（１−２３）を用いた受容体結合実験
参考例４２に記載した方法により作製した［^１２５Ｉ−Ｔｙｒ^１０］−ｈＧＰＲ８Ｌ（１−２３）およびヒトＧＰＲ７発現ＣＨＯ細胞から調製した細胞膜画分を用いて受容体結合実験を行なった。
最初に膜画分の調製法を以下に記載する。
１ｘ１０^８個のヒトＧＰＲ７発現ＣＨＯ細胞に１０ｍｌのホモジネートバッファー（１０ｍＭ　ＮａＨＣＯ_３、５ｍＭ　ＥＤＴＡ（エチレンジアミン四酢酸）、０．５ｍＭ　ＰＭＳＦ（フェニルメタンスルホニルフルオライド）、１μｇ／ｍｌペプスタチン、４μｇ／ｍｌ　Ｅ６４、２０μｇ／ｍｌ　ロイペプチン）添加し、ポリトロン（１２，０００ｒｐｍ、１分間）を用いて破砕した。細胞破砕液を遠心（１，０００ｇ、１５分間）して上清を得た。次にこの上清を超遠心分離（Ｂｅｃｋｍａｎ　ｔｙｐｅ　３０ローター、３０，０００ｒｐｍ，１時間）し、得られた沈殿物をヒトＧＰＲ７発現ＣＨＯ細胞膜画分とした。
かくして調製された細胞膜画分を、アッセイ用バッファー（２５ｍＭ　Ｔｒｉｓ−ＨＣｌ、５ｍＭ　ＥＤＴＡ、０．０５％　ＣＨＡＰＳ（３−〔（３−コラミドプロピル）ジメチルアンモニオ〕−１−プロパン硫酸）、０．１％　ＢＳＡ、０．５ｍＭ　ＰＭＳＦ、１μｇ／ｍｌ　ペプスタチン、２０μｇ／ｍｌ　ロイペプチン、４μｇ／ｍｌ　Ｅ−６４、ｐＨ７．４）で各種濃度に希釈後、ポリプロピレン製試験管（Ｆａｌｃｏｎ　２０５３）に２００μｌずつ分注した。最大結合量を測定するために２μｌのＤＭＳＯと８　ｎＭの［^１２５Ｉ−Ｔｙｒ^１０］−ｈＧＰＲ８Ｌ（１−２３）　２μｌを膜画分溶液に添加した。また、非特異的結合を測定するために１ｍＭ　ｈＧＰＲ８Ｌ（１−２３）のＤＭＳＯ溶液２μｌと８ｎＭの　［^１２５Ｉ−Ｔｙｒ^１０］−ｈＧＰＲ８Ｌ（１−２３）　２μｌを膜画分溶液に添加した。２５℃で７５分間反応させた後、ポリエチレンイミン処理したワットマングラスフィルター（ＧＦ−Ｆ）を用いて反応液を吸引ろ過しさらにフィルターを洗浄用バッファー（２５ｍＭ　Ｔｒｉｓ−ＨＣｌ、５ｍＭ　ＥＤＴＡ、０．０５％　ＣＨＡＰＳ、０．１％　ＢＳＡ、ｐＨ７．４）１．５ｍｌで２回洗浄した。ろ過後、γ−カウンターを用いてろ紙上に残った放射活性を測定し、最大結合量から非特異的結合量を引いて特異的結合量を見積もった。
膜画分の濃度を変化させると膜画分の濃度に依存した［^１２５Ｉ−Ｔｙｒ^１０］−ｈＧＰＲ８Ｌ（１−２３）の特異的な結合が認められた。膜画分濃度を１０μｇ／ｍｌに設定してｈＧＰＲ８Ｌ（１−２３）およびｈＧＰＲ８Ｌ（１−３０）による［^１２５Ｉ−Ｔｙｒ^１０］−ｈＧＰＲ８Ｌ（１−２３）のヒトＧＰＲ７発現細胞膜画分への特異的結合に対する結合阻害を調べた。阻害率から５０％阻害濃度（ＩＣ_５０値）を算出したところ、ｈＧＰＲ８Ｌ（１−２３）のＩＣ_５０値は０．０９９ｎＭであった。また、ｈＧＰＲ８Ｌ（１−３０）のＩＣ_５０値は０．０２５ｎＭであった。
これより、ｈＧＰＲ８Ｌ（１−２３）およびｈＧＰＲ８Ｌ（１−３０）がヒトＧＰＲ７発現細胞膜画分に対して高い親和性を有することが示された。このことは、ｈＧＰＲ８Ｌ（１−２３）およびｈＧＰＲ８Ｌ（１−３０）がヒトＧＰＲ７受容体の高親和性リガンドであることを意味するものである。図１６に、種々の濃度におけるｈＧＰＲ８Ｌ（１−２３）およびｈＧＰＲ８Ｌ（１−３０）の結合阻害を示す。
ｈＧＰＲ８Ｌ（１−２３）のラットおよびマウスのホモログおよびｈＧＰＲ８Ｌ（１−３０）のブタ、ラットおよびマウスのホモログを用いても、上記と同様に［^１２５Ｉ−Ｔｙｒ^１０］−ｈＧＰＲ８Ｌ（１−２３）のヒトＧＰＲ７発現細胞膜画分への特異的結合に対する結合阻害を確認できる。
【０１７０】
参考例８３
１）ＧＰＲ７発現ＣＨＯ細胞の膜画分を用いたＧＴＰγＳ結合活性の測定
ＧＰＲ７発現ＣＨＯ細胞膜画分に対する［^３５Ｓ］−ｇｕａｎｏｓｉｎｅ　５’−（γ−ｔｈｉｏ）ｔｒｉｐｈｏｓｐｈａｔｅ（ＧＴＰγＳ）の結合促進活性を以下の方法により測定した。
参考例８２に記載の方法により調製したＧＰＲ７発現ＣＨＯ細胞膜画分を膜希釈緩衝液（５０ｍＭトリス塩酸緩衝液（ｐＨ７．４）、５ｍＭ　ＭｇＣｌ_２、１５０　ｍＭ　ＮａＣｌ、１μＭ　ＧＤＰ、０．１％　ＢＳＡ）で希釈して、タンパク質濃度３０μｇ／ｍｌのアッセイ用細胞膜画分溶液を調製した。アッセイ用膜画分溶液２００μｌに、５０　ｎＭ濃度の［^３５Ｓ］−ｇｕａｎｏｓｉｎｅ　５’−（γ−ｔｈｉｏ）ｔｒｉｐｈｏｓｐｈａｔｅ（ＮＥＮ社）を２μｌと適当な濃度のＤＭＳＯ溶液とした試料２μｌとを添加し、この混合液を２５℃で１時間保温した。混合液をフィルター濾過し、さらにフィルターを洗浄用バッファー（５０ｍＭトリス塩酸緩衝液（ｐＨ７．４）、５ｍＭ　ＭｇＣｌ_２、１ｍＭ　ＥＤＴＡ、０．１％　ＢＳＡ）１．５ｍｌで２回洗浄した後、フィルターの放射活性を液体シンチレーションカウンターで測定した。
２）ＧＰＲ７発現ＣＨＯ細胞膜画分を用いて測定したｈＧＰＲ８Ｌ（１−２３）またはｈＧＰＲ８Ｌ（１−３０）のＧＴＰγＳ結合促進活性
ｈＧＰＲ８Ｌ（１−２３）またはｈＧＰＲ８Ｌ（１−３０）を種々の濃度で、上記１）に記載した方法に従い、ＧＰＲ７発現ＣＨＯ細胞膜画分に投与し、ＧＴＰγＳ結合促進活性を測定した。
結果を図１７に示す。
これより明らかに、ｈＧＰＲ８Ｌ（１−２３）およびｈＧＰＲ８Ｌ（１−３０）は濃度依存的にＧＰＲ７発現ＣＨＯ細胞膜画分のＧＴＰγＳ結合を促進した。
ＧＴＰγＳ結合促進活性から５０％有効濃度（ＥＣ_５０値）を算出したところ、ｈＧＰＲ８Ｌ（１−２３）のＥＣ_５０値は０．７４ｎＭであった。また、ｈＧＰＲ８Ｌ（１−３０）のＥＣ_５０値は０．６７ｎＭであった（表３）。
ｈＧＰＲ８Ｌ（１−２３）のラットおよびマウスのホモログおよびｈＧＰＲ８Ｌ（１−３０）のブタ、ラットおよびマウスのホモログを用いても、上記と同様にヒトＧＰＲ７発現ＣＨＯ細胞の反応を確認できる。
【０１７１】
参考例８４　ＧＰＲ７発現ＣＨＯ細胞膜画分を用いて測定したＧＰＲ８リガンドペプチドのヒトおよびブタホモログの誘導体のＧＴＰγＳ結合促進活性
参考例で得られたＧＰＲ８リガンドペプチドのヒトおよびブタホモログの誘導体を、種々の濃度で、参考例８３に記載した方法でＧＰＲ７発現ＣＨＯ細胞膜画分に投与し、ＧＴＰγＳ結合促進活性を測定した。
測定した誘導体およびＧＴＰγＳ結合促進活性を表３に示す。活性は、５０％有効濃度（ＥＣ_５０値）で示した。
【０１７２】
【表３】

【０１７３】
参考例８５　ＧＰＲ７発現ＣＨＯ細胞膜画分および［^１２５Ｉ−Ｔｙｒ^１０］−ｈＧＰＲ８Ｌ（１−２３）を用いて測定したＧＰＲ８リガンドペプチドのヒトおよびブタホモログの誘導体の受容体結合活性
参考例で得られたＧＰＲ８リガンドペプチドのヒトおよびブタホモログの誘導体の受容体結合活性を、参考例８２に記載した方法でＧＰＲ７発現ＣＨＯ細胞膜画分および［^１２５Ｉ−Ｔｙｒ^１０］−ｈＧＰＲ８Ｌ（１−２３）を用いて測定した。
測定した誘導体のおよび受容体結合活性を表３に示す。受容体結合活性は５０％結合阻害濃度（ＩＣ_５０値）で示した。
【０１７４】
実施例１
ｈＧＰＲ８Ｌ（１−２３）の皮下への持続投与によるラット摂餌量および体重増加に対する作用
（１）摂餌量および体重増加に対する作用
Ｗｉｓｔａｒ雄性ラット（８週齢、日本チャールズリバー）を１週間ほどＭＦ粉末食（オリエンタル酵母（株））で馴化した。生理食塩水に１　ｍＭ濃度で溶解したｈＧＰＲ８Ｌ（１−２３）〔ヒトＧＰＲ　８リガンド（１−２３）〕またはｖｅｈｉｃｌｅ群として生理食塩水を２００μｌ充填した浸透圧ポンプ（ａｌｚｅｔ，　ＭＩＮＩ−ＯＳＭＯＴＩＣ　ＰＵＭＰ　Ｍｏｄｅｌ　２００１，放出速度；２４μｌ　／ｄａｙ）をペントバルビタール麻酔下の上記ラットの皮下（背中中央）に装着した（各ｎ＝６）。装着日の翌日を０日目とし、ＭＦ粉末食を自由摂食下８日目まで毎日８時と２０時に餌の量と体重を測定した。８時から２０時を明期、２０時から翌朝８時を暗期とした。８日目の８時に測定後断頭屠殺し、肝臓、腎臓、心臓、脾臓、精巣、腎臓周囲の脂肪、性器周囲の脂肪および褐色脂肪の重量を測定した。動物は全て明期周期１２時間（明：８時から２０時）で飼育した。
摂餌量は、明期では両群での差が見られなかったが、暗期ではｈＧＰＲ８Ｌ（１−２３）投与群はｖｅｈｉｃｌｅ群に比較していずれの測定時点においても減少傾向が見られ（図１８および１９）、１日の摂餌量の総量で減少が見られた（図２０）。体重（平均値±標準誤差）は、投与２日目以降から徐々に差が広がり投与７日目においてｖｅｈｉｃｌｅ群　３７８．４±　５．８　ｇに対してｈＧＰＲ８Ｌ（１−２３）投与群は３６４．６±　６．０　ｇであった（図２１）。また、０日目から７日目までの体重の増加量はｖｅｈｉｃｌｅ群　３９．６±　４．１　ｇに対してｈＧＰＲ８Ｌ（１−２３）投与群は２８．９±　３．２　ｇであり、ｈＧＰＲ８Ｌ（１−２３）の１週間の皮下の持続投与により体重の増加が約１０．７ｇ抑制された（図２２）。
投与８日目の臓器重量を比較すると、ｖｅｈｉｃｌｅ群と比較してｈＧＰＲ８Ｌ（１−２３）投与群では、肝臓（１．６　ｇの減少）ならびに白色脂肪組織である腎臓周囲脂肪（１．２　ｇの減少）および性器周囲脂肪（０．７　ｇの減少）においてそれぞれ０．５　ｇ以上の減少がみられた　（表４）。
【表４】

以上の結果からｈＧＰＲ８Ｌ（１−２３）の皮下投与によって摂餌量の減少および脂肪重量の低下を伴う体重増加抑制効果が認められた。
（２）血中グルコース、総コレステロールおよびトリグリセリドに対する作用
上記（１）のｈＧＰＲ８Ｌ（１−２３）投与群およびｖｅｈｉｃｌｅ群の投与８日目の血中グルコース、総コレステロールおよびトリグリセリドを富士ドライケムを用いて測定した。血中グルコース濃度（ｖｅｈｉｃｌｅ群；　１５０±４．９　ｍｇ／ｄｌ，　ｈＧＰＲ８Ｌ（１−２３）投与群；１５５±４．５　ｍｇ／ｄｌ）および血中総コレステロール濃度（ｖｅｈｉｃｌｅ群；　７３．６±２．４　ｍｇ／ｄｌ，ｈＧＰＲ８Ｌ（１−２３）投与群；６９．１±１．８　ｍｇ／ｄｌ）に両群の差は認められなかったが、血中トリグリセリド濃度（ｖｅｈｉｃｌｅ群；　１６８±８．９　　ｍｇ／ｄｌ，　ｈＧＰＲ８Ｌ（１−２３）投与群；１３４±２８　ｍｇ／ｄｌ）は、ｈＧＰＲ８Ｌ（１−２３）投与群において３０　ｍｇ／ｄｌ以上の低値を示した（図２３）。
以上の結果から、ｈＧＰＲ８Ｌ（１−２３）の皮下投与によって血中トリグリセリド濃度の低下が認められた。
【０１７５】
実施例２
ｈＧＰＲ８Ｌ（１−２３）の腹腔内投与によるラットの摂餌量および体重に対する作用
実施例１と同様にＷｉｓｔａｒ雄性ラット（８週齢、日本チャールスリバー）を１週間ほどＭＦ粉末食（オリエンタル酵母（株））で馴化した。生理食塩水に０．２　ｍｇ／ｍｌおよび２　ｍｇ／ｍｌ濃度で溶解したｈＧＰＲ８Ｌ（１−２３）〔ヒトＧＰＲ　８リガンド（１−２３）〕またはｖｅｈｉｃｌｅ群として生理食塩水を１　ｍｌ／ｋｇの割合で暗期の開始直前（１９時４５分から２０時）に腹腔内投与を３日間行なった（各ｎ＝１０）。投与翌日の朝８時に摂餌量および体重を測定した。動物は全て８時から２０時を明期、２０時から翌朝８時を暗期とした明期周期１２時間で飼育した。
摂餌量は、ｈＧＰＲ８Ｌ（１−２３）投与群において用量依存的に減少傾向を示し、２　ｍｇ／ｋｇ投与群では、投与後３日ともｖｅｈｉｃｌｅ群と比較して有意な（ｐ＜＝０．０５またはｐ＜＝０．０１）減少を示した　（図２４）。
体重増加量は、２　ｍｇ／ｋｇ投与群において３日ともｖｅｈｉｃｌｅ群と比較して減少傾向を示した（平均値の差；１日目；　１．２　ｇ，　　２日目；　３．１　ｇ，　　３日目；　２．３　ｇ）（図２５）。
以上の結果から、ｈＧＰＲ８Ｌ（１−２３）は、腹腔内投与により暗期での摂餌量の減少および体重増加抑制効果が認められた。
【０１７６】
実施例３
［Ｐｈｅ^２］ヒトＧＰＲ８リガンド（１−２０）：　Ｔｒｐ−Ｐｈｅ−Ｌｙｓ−Ｈｉｓ−Ｖａｌ−Ａｌａ−Ｓｅｒ−Ｐｒｏ−Ａｒｇ−Ｔｙｒ−Ｈｉｓ−Ｔｈｒ−Ｖａｌ−Ｇｌｙ−Ａｒｇ−Ａｌａ−Ａｌａ−Ｇｌｙ−Ｌｅｕ−Ｌｅｕ（配列番号：１４９）の製造
アプライドバイオシステムズ社のペプチド自動合成機（ＡＢＩ４３３モデル）を使用し、プログラムに従ってＣ端より逐次Ｆｍｏｃ法によりペプチド鎖を延長し目的の保護ペプチド樹脂の合成を行った。
出発アミノ酸樹脂担体はＷａｎｇ（ｐ−ｂｅｎｚｙｌｏｘｙｂｅｎｚｙｌ　ａｌｃｏｈｏｌ）樹脂（０．２５　ｍｍｏｌ）を使用し、Ｆｍｏｃ−Ｌｅｕ、Ｆｍｏｃ−Ｇｌｙ、Ｆｍｏｃ−Ａｌａ、Ｆｍｏｃ−Ａｒｇ（Ｐｂｆ）、Ｆｍｏｃ−Ｖａｌ、Ｆｍｏｃ−Ｔｈｒ（Ｂｕ^ｔ）、Ｆｍｏｃ−Ｈｉｓ（Ｔｒｔ）、Ｆｍｏｃ−Ｔｙｒ（Ｂｕ^ｔ）、Ｆｍｏｃ−Ｐｒｏ、Ｆｍｏｃ−Ｓｅｒ（Ｂｕ^ｔ）、Ｆｍｏｃ−Ｌｙｓ（Ｂｏｃ）、Ｆｍｏｃ−Ｐｈｅ、Ｆｍｏｃ−Ｔｒｐ（Ｂｏｃ）のＦｍｏｃアミノ酸誘導体をＨＢＴＵ（２−（１Ｈ−ベンゾトリアゾール−１−イル）−１，１，３，３−テトラメチルウロニウム　ヘキサフルオロホスフェート）によりシークエンスにしたがって逐次縮合した。
樹脂上へのペプチドの構築が終了後、保護ペプチド樹脂を乾燥した。得られた保護ペプチドの脱保護処理とペプチドの樹脂担体からの切り離しはＴＦＡ処理によって行なった。得られた粗ペプチドは０．１％　ＴＦＡ水によって抽出し、凍結乾燥により粉末固体として得た。続いて、粗ペプチドを逆相高速クロマトグラフィー（島津製作所、分取装置：モデルＬＣ８Ａ）によりアセトニトリル−０．１％　ＴＦＡ水の系（１５−３５％、８０分）を用いて分取精製を行なって目的とする精製ペプチド３５　ｍｇを得た。
精製物を０．２％３−（２−アミノエチル）インドールを含む４Ｎメタンスルホン酸によって１１０℃・２２時間の条件で加水分解して得た加水分解物のアミノ酸分析値は（括弧内は理論値）以下のとおり。
Ｔｈｒ　（１）　０．９３，　Ｓｅｒ　（１）　０．９２，　Ｇｌｙ　（２）　２．０３，　Ａｌａ　（３）　３．０９，　Ｖａｌ　（２）　１．９０，　Ｌｅｕ　（２）　２．０２，　Ｔｙｒ　（１）　１．０２，　Ｐｈｅ　（１）　１．００，　Ｈｉｓ　（２）　１．９１，　Ｌｙｓ　（１）　０．９８，　Ｔｒｐ　（１）　０．８８，　Ａｒｇ　（２）　２．０６，　Ｐｒｏ　（１）　１．０２
純度はＨＰＬＣにより９８．８％と算出された。また、質量分析値は２２６６．６（理論値２２６６．６）であった。
【０１７７】
実施例４
ラクトパーオキシダーゼ法を用いた［Ｐｈｅ^２，^１２５Ｉ−Ｔｙｒ^１０］ヒトＧＰＲ８リガンド（１−２０）の作製
ＤＭＳＯ　１０μｌに溶かした、実施例３に記載した製法に準じて得られた［Ｐｈｅ^２］ヒトＧＰＲ８リガンド（１−２０）（配列番号：１４９）１０　ｎｍｏｌを、０．１　Ｍ塩化ニッケル水溶液１０μｌ、０．１　Ｍ　ＨＥＰＥＳ　（ｐＨ　７．６）に溶かした０．００１％過酸化水素水１０μｌ、０．１　Ｍ　ＨＥＰＥＳ　（ｐＨ　７．６）に溶かしたラクトパーオキシダーゼ　（シグマ社）　１０μｇ／ｍｌを１０μｌ、および［^１２５Ｉ］ＮａＩ　４０　ＭＢｑ（ＮＥＮライフサイエンスプロダクツ社）１０μｌを混合して室温で５０分間反応させた後、生成した［Ｐｈｅ^２，　^１２５Ｉ−Ｔｙｒ^１０］ヒトＧＰＲ８リガンド（１−２０）を以下の条件のＨＰＬＣにより分取した。
用いたカラムは、ＯＤＳ−８０ＴＭ　（４．６　ｍｍ　ｘ　１５　ｃｍ）（トーソー社）、溶出液Ａとして１０％アセトニトリル／０．１％　ＴＦＡ、溶出液Ｂとして６０％アセトニトリル／０．１％　ＴＦＡを用い、０−０　（２　ｍｉｎ）、０−２７　（５　ｍｉｎ）、２７−３２　（４０　ｍｉｎ）　％Ｂ／Ａ＋Ｂのグラディエント溶出法を行なった。流速は１　ｍＬ／ｍｉｎ、カラム温度は４０℃、検出は２１５　ｎｍとした。このＨＰＬＣ条件では、［Ｐｈｅ^２，　^１２５Ｉ−Ｔｙｒ^１０］ヒトＧＰＲ８リガンド（１−２０）は２５分付近に溶出した。
【０１７８】
実施例５
［Ｐｈｅ^２，^１２５Ｉ−Ｔｙｒ^１０］ヒトＧＰＲ８リガンド（１−２０）を用いた受容体結合実験
実施例４に記載した方法によって作製した［Ｐｈｅ^２，　^１２５Ｉ−Ｔｙｒ^１０］ヒトＧＰＲ８リガンド（１−２０）、参考例８２に記載した方法により調製されたＧＰＲ７発現ＣＨＯ細胞膜画分および参考例６に記載した方法により調製されたＧＰＲ８発現ＣＨＯ細胞膜画分を用いて受容体結合実験を行なった。
ＧＰＲ７発現ＣＨＯ細胞およびＧＰＲ８発現ＣＨＯ細胞から調製した細胞膜画分をアッセイ用バッファー（２５　ｍＭ　Ｔｒｉｓ−ＨＣｌ、５　ｍＭ　ＥＤＴＡ、０．０５％　ＣＨＡＰＳ、０．１％　ＢＳＡ、０．５　ｍＭ　ＰＭＳＦ、１μｇ／ｍｌペプスタチン、４μｇ／ｍｌ　Ｅ−６４、２０μｇ／ｍｌロイペプチン、ｐＨ　７．４）で各種濃度に希釈後、ポリプロピレン製試験管（Ｆａｌｃｏｎ　２０５３）に２００μｌずつ分注した。最大結合量を測定するために、２μｌのＤＭＳＯおよび７　ｎＭの［Ｐｈｅ^２，　^１２５Ｉ−Ｔｙｒ^１０］ヒトＧＰＲ８リガンド（１−２０）　２μｌを膜画分溶液に添加した。また、非特異的結合を測定するために１００μＭヒトＧＰＲ８リガンド（１−２３）のＤＭＳＯ溶液２μｌおよび７　ｎＭの［Ｐｈｅ^２，　^１２５Ｉ−Ｔｙｒ^１０］ヒトＧＰＲ８リガンド（１−２０）　２μｌを膜画分溶液に添加した。２５℃で７５分間反応させた後、ポリエチレンイミン処理したワットマングラスフィルター（ＧＦ−Ｆ）を用いて反応液を吸引ろ過した。ろ過後、γ−カウンターを用いて濾紙上に残った放射活性を測定し、最大結合量から非特異的結合量を引いて特異的結合量を見積もった。膜画分の濃度を変化させると膜画分の濃度に依存した［Ｐｈｅ^２，　^１２５Ｉ−Ｔｙｒ^１０］ヒトＧＰＲ８リガンド（１−２０）の特異的な結合が認められた。
被験試料のＧＰＲ７受容体またはＧＰＲ８受容体に対する結合阻害活性（阻害率（％））は、最大結合量（ＴＢ）から被検試料および［Ｐｈｅ^２，　^１２５Ｉ−Ｔｙｒ^１０］ヒトＧＰＲ８リガンド（１−２０）を加えたときに濾紙上に残った放射活性（Ｘ）を減じた値の特異的結合量（ＳＢ）に対する比率（（ＴＢ−Ｘ）／ＳＢ　ｘ　１００　（％））で示される。
ＧＰＲ７発現ＣＨＯ細胞から調製した膜画分について膜画分濃度を１５μｇ／ｍｌに設定して阻害率からヒトＧＰＲ８リガンド（１−２３）およびヒトＧＰＲ８リガンド（１−３０）の５０％阻害濃度（ＩＣ_５０値）を算出したところ、ＩＣ_５０値はそれぞれ０．１３　ｎＭおよび０．０３９　ｎＭであった。図２６に種々の濃度におけるヒトＧＰＲ８リガンド（１−２３）およびヒトＧＰＲ８リガンド（１−３０）の結合阻害活性を示す。ＧＰＲ８発現ＣＨＯ細胞から調製した膜画分について膜画分濃度を５μｇ／ｍｌに設定して阻害率からヒトＧＰＲ８リガンド（１−２３）およびヒトＧＰＲ８リガンド（１−３０）の５０％阻害濃度（ＩＣ_５０値）を算出したところ、ＩＣ_５０値はそれぞれ０．１９　ｎＭおよび０．０３７　ｎＭであった。図２７に種々の濃度におけるヒトＧＰＲ８リガンド（１−２３）およびヒトＧＰＲ８リガンド（１−３０）の結合阻害活性を示す。
【０１７９】
実施例６
［Ｐｈｅ^２，^１２５Ｉ−Ｔｙｒ^１０］ヒトＧＰＲ８リガンド（１−２０）およびヒトＧＰＲ７発現ＣＨＯ細胞膜画分を用いたＧＰＲ７とＧＰＲ８リガンドの結合性を変化させる化合物のスクリーニング方法
実施例４に記載した方法によって作製した［Ｐｈｅ^２，　^１２５Ｉ−Ｔｙｒ^１０］ヒトＧＰＲ８リガンド（１−２０）および参考例８２に記載した方法により調製されたＧＰＲ７発現ＣＨＯ細胞膜画分を用いた受容体結合実験によってＧＰＲ７とＧＰＲ８リガンドの結合性を変化させる化合物のスクリーニングを実施した。
ＧＰＲ７発現ＣＨＯ細胞から調製した細胞膜画分を用いた上記の実施例５において、２μｌの試験化合物のＤＭＳＯ溶液および７　ｎＭの［Ｐｈｅ^２，　^１２５Ｉ−Ｔｙｒ^１０］ヒトＧＰＲ８リガンド（１−２０）　２μｌを膜画分溶液に添加した。２５℃で７５分間反応させた後、ポリエチレンイミン処理したワットマングラスフィルター（ＧＦ−Ｆ）を用いて反応液を吸引ろ過した。ろ過後、γ−カウンターを用いて濾紙上に残った放射活性を測定し、試験化合物添加時の結合量を求めた。最大結合量からこの試験化合物添加時の結合量を減じた値を特異的結合量によって除した百分率を求め、試験化合物の結合阻害活性（％）を算出した。
結合阻害活性を示した試験化合物について、種々の濃度に調製した試験化合物を用いて結合阻害活性を測定し、５０％阻害濃度（ＩＣ_５０値）を求めた。より低いＩＣ_５０値を与える化合物をＧＰＲ７とＧＰＲ８リガンドとの結合をより強く阻害する化合物として選択した。
一方、結合阻害活性の値として負の値が得られる化合物をＧＰＲ７とＧＰＲ８リガンドとの結合を促進する化合物として選択した。
【０１８０】
【発明の効果】
本発明のＤＮＡまたは本発明のポリペプチドは、例えば体重増加抑制剤、体重減少剤、脂肪量増加抑制剤、摂食抑制剤または体重増加薬などのスクリーニングに、あるいは、体重増加抑制剤、体重減少剤、脂肪量増加抑制剤、摂食抑制剤、体重増加剤として有用である。本発明のポリペプチドを用いるスクリーニング、好ましくは本発明の受容体も用いるスクリーニングで得られる体重増加抑制剤、体重減少剤、脂肪量増加抑制剤、摂食抑制剤は、安全で優れた医薬として用いられる。
【０１８１】
【配列表】

【図面の簡単な説明】
【図１】Ｗａｋｏｓｉｌ−ＩＩ　３Ｃ１８ＨＧカラムを用いたＧＰＲ８リガンドの最終段階の精製におけるＨＰＬＣのＵＶ吸収（ａ）と各ピークのＧＴＰγＳ活性（ｂ）を示す。活性は矢印に示すピークに回収された。
【図２】種々の濃度の２３残基のＧＰＲ８リガンドペプチドのヒトホモログのＣＨＯ／ＧＰＲ８細胞膜画分に対するＧＴＰγＳ結合促進活性を示す。
【図３】種々の濃度の３０残基のＧＰＲ８リガンドペプチドのヒトホモログのＣＨＯ／ＧＰＲ８細胞膜画分に対するＧＴＰγＳ結合促進活性を示す。
【図４】種々の濃度の２３残基のＧＰＲ８リガンドペプチドのヒトホモログのＣＨＯ／ＧＰＲ８細胞に対するｃＡＭＰ産生抑制活性を示す。
【図５】種々の濃度の３０残基のＧＰＲ８リガンドペプチドのヒトホモログのＣＨＯ／ＧＰＲ８細胞に対するｃＡＭＰ産生抑制活性を示す。
【図６】ＧＰＲ８リガンドペプチドのヒトホモログ前駆体タンパク質ｃＤＮＡの全塩基配列およびそれから翻訳されるＧＰＲ８リガンドペプチドのヒトホモログ前駆体受容体タンパク質の全アミノ酸配列を示す。予想される２３残基のＧＰＲ８リガンドのヒトホモログペプチドの配列を四角で示す。
【図７】ＧＰＲ８リガンドペプチドのブタホモログ前駆体タンパク質ｃＤＮＡの全塩基配列およびそれから翻訳されるＧＰＲ８リガンドペプチドのブタホモログ前駆体受容体タンパク質の全アミノ酸配列を示す。予想される２３残基のＧＰＲ８リガンドのブタホモログペプチドの配列を四角で示す。
【図８】ＧＰＲ８リガンドペプチドのラットホモログ前駆体タンパク質ｃＤＮＡの全塩基配列およびそれから翻訳されるＧＰＲ８リガンドペプチドのラットホモログ前駆体受容体タンパク質の全アミノ酸配列を示す。予想される２３残基のＧＰＲ８リガンドのラットホモログペプチドの配列を四角で示す。
【図９】ＧＰＲ８リガンドペプチドのマウスホモログ前駆体タンパク質ｃＤＮＡの全塩基配列およびそれから翻訳されるＧＰＲ８リガンドペプチドのマウスホモログ前駆体受容体タンパク質の全アミノ酸配列を示す。予想される２３残基のＧＰＲ８リガンドのマウスホモログペプチドの配列を四角で示す。
【図１０】ヒトＧＰＲ８発現ＣＨＯ細胞から調製した細胞膜画分を用いた、［^１２５Ｉ］で標識した２３残基のヒトＧＰＲ８リガンドに対する２３残基のヒトＧＰＲ８リガンドの結合阻害活性を示す図を示す。
【図１１】ＴＧＲ２６の疎水性プロット図である。
【図１２】ｈＧＰＲ８Ｌ（１−２３）およびｈＧＰＲ８Ｌ（１−３０）のＣＨＯ／ＴＧＲ２６細胞に対するｃＡＭＰ産生抑制活性を示す図である。図中、−○−は、ｈＧＰＲ８Ｌ（１−２３）を投与した場合を、−△−は、ｈＧＰＲ８Ｌ（１−３０）を投与した場合を示す。
【図１３】ｈＧＰＲ８Ｌ（１−２３）およびｈＧＰＲ８Ｌ（１−３０）のＣＨＯ／ＴＧＲ２６細胞膜画分に対するＧＴＰγＳ結合促進活性を示す図である。図中、−○−は、ｈＧＰＲ８Ｌ（１−２３）を投与した場合を、−△−は、ｈＧＰＲ８Ｌ（１−３０）を混合した場合を示す。
【図１４】［^１２５Ｉ］で標識した２３残基のヒトＧＰＲ８リガンドのＴＴＧＲ２６発現ＣＨＯ細胞から調製した細胞膜画分への結合に対する種々の濃度のｈＧＰＲ８Ｌ（１−２３）およびｈＧＰＲ８Ｌ（１−３０）の結合阻害活性を示す図を示す。図中、−○−は、ｈＧＰＲ８Ｌ（１−２３）を投与した場合を、−△−は、ｈＧＰＲ８Ｌ（１−３０）を投与した場合を示す。
【図１５】種々の濃度の２３残基および３０残基のＧＰＲ８リガンドペプチドヒトホモログのＣＨＯ／ＧＰＲ７細胞に対するｃＡＭＰ産生抑制活性を示す。図中、−●−は、ｈＧＰＲ８Ｌ（１−２３）を投与した場合を、−黒四角−は、ｈＧＰＲ８Ｌ（１−３０）を投与した場合を示す。
【図１６】［^１２５Ｉ］で標識した２３残基のヒトＧＰＲ８リガンドのヒトＧＰＲ７発現ＣＨＯ細胞から調製した細胞膜画分への結合に対する種々の濃度のｈＧＰＲ８Ｌ（１−２３）およびｈＧＰＲ８Ｌ（１−３０）の結合阻害活性を示す図を示す。図中、−●−は、ｈＧＰＲ８Ｌ（１−２３）を投与した場合を、−黒四角−は、ｈＧＰＲ８Ｌ（１−３０）を投与した場合を示す。
【図１７】ｈＧＰＲ８Ｌ（１−２３）およびｈＧＰＲ８Ｌ（１−３０）のＣＨＯ／ＧＰＲ７細胞膜画分に対するＧＴＰγＳ結合促進活性を示す図である。図中、−●−は、ｈＧＰＲ８Ｌ（１−２３）を投与した場合を、−黒四角−は、ｈＧＰＲ８Ｌ（１−３０）を投与した場合を示す。
【図１８】皮下に持続投与したｈＧＰＲ８Ｌ（１−２３）のラットの明期での摂餌量に対する作用を示す。図中、□はｖｅｈｉｃｌｅ群を、黒四角は、ｈＧＰＲ８Ｌ（１−２３）群を示す。
【図１９】皮下に持続投与したｈＧＰＲ８Ｌ（１−２３）のラットの暗期での摂餌量に対する作用を示す。図中、□はｖｅｈｉｃｌｅ群を、黒四角は、ｈＧＰＲ８Ｌ（１−２３）群を示す。
【図２０】皮下に持続投与したｈＧＰＲ８Ｌ（１−２３）のラットの１日の摂餌量に対する作用を示す。図中、□はｖｅｈｉｃｌｅ群を、黒四角はｈＧＰＲ８Ｌ（１−２３）群を示す。
【図２１】皮下に持続投与したｈＧＰＲ８Ｌ（１−２３）のラットの体重増加に対する作用を示す。図中、−○−はｖｅｈｉｃｌｅ群を、−□−はｈＧＰＲ８Ｌ（１−２３）群を示す。
【図２２】皮下に持続投与したｈＧＰＲ８Ｌ（１−２３）のラットの体重増加に対する作用を示す。図中、−○−はｖｅｈｉｃｌｅ群を、−□−はｈＧＰＲ８Ｌ（１−２３）群を示す。
【図２３】皮下に持続投与したｈＧＰＲ８Ｌ（１−２３）のラットの血中グルコース量（ａ）、血中総コレステロール量（ｂ）および血中トリグリセリド濃度（ｃ）に対する作用を示す。○は各個体の数値、−は平均値を示す。
【図２４】腹腔内投与したｈＧＰＲ８Ｌ（１−２３）のラットの摂餌量に対する作用を示す。図中、白はｖｅｈｉｃｌｅ群を、灰色は０．２ｍｇ投与群を、黒は２ｍｇ投与群を示す。また、＊は危険率５％で有意、＊＊は危険率１％で有意であることを示す。
【図２５】腹腔内投与したｈＧＰＲ８Ｌ（１−２３）のラットの体重増加量に対する作用を示す。図中、白はｖｅｈｉｃｌｅ群を、灰色は０．２ｍｇ投与群を、黒は２ｍｇ投与群を示す。
【図２６】［Ｐｈｅ^２，^１２５Ｉ−Ｔｙｒ^１０］ヒトＧＰＲ８リガンド（１−２０）のヒトＧＰＲ７発現ＣＨＯ細胞から調製した細胞膜画分への結合に対する種々の濃度のｈＧＰＲ８Ｌ（１−２３）およびｈＧＰＲ８Ｌ（１−３０）の結合阻害活性を示す図を示す。図中、−○−は、ｈＧＰＲ８Ｌ（１−２３）を投与した場合を、−□−は、ｈＧＰＲ８Ｌ（１−３０）を投与した場合を示す。
【図２７】［Ｐｈｅ^２，^１２５Ｉ−Ｔｙｒ^１０］ヒトＧＰＲ８リガンド（１−２０）のヒトＧＰＲ８発現ＣＨＯ細胞から調製した細胞膜画分への結合に対する種々の濃度のｈＧＰＲ８Ｌ（１−２３）およびｈＧＰＲ８Ｌ（１−３０）の結合阻害活性を示す図を示す。図中、−○−は、ｈＧＰＲ８Ｌ（１−２３）を投与した場合を、−□−は、ｈＧＰＲ８Ｌ（１−３０）を投与した場合を示す。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a weight gain inhibitor, a weight loss agent, a fat mass increase inhibitor, an eating inhibitor, and the like.
[0002]
[Prior art]
The increase in diabetes and other lifestyle-related diseases is due to the fact that the directional biological function of accumulating fat in preparation for a starvation state typified by so-called frugal genes such as PPARγ, has led to a diet that has been focused on modern high-fat diets. The main cause is the inability to adapt to the living environment such as lack of living or exercise. As a result, obesity not only causes diabetes but also becomes a risk factor such as hypertension, so the development of safe anti-obesity drugs with few side effects leads to prevention of the development of many lifestyle-related diseases, It is one of the most demanding medical economics. As such antiobesity agents, sibutramine, a catecholamine / serotonin reuptake inhibitor, and orlistat, a fat absorption inhibitor, are currently used. In addition, β3 agonists of thermogenesis promoters, central antifeedant neuropeptide Y antagonists, melanocortin receptor subtype 4 agonists and the like are under development or research (J. @C. @Clapham et al., Pharmacol. @Ther., 89, 81-121, 2001, M. Chiesi et al., Trends Pharmacol. Sci. 22, 247-54, 2001).
[Non-patent document 1]
Genomics, Vol. 28, pp. 84-91, 1995
[Patent Document 1]
WO 01/98494
[0003]
[Problems to be solved by the invention]
However, there has been a demand for the development of a safe weight gain inhibitor and a weight loss agent that are powerful and have few side effects based on a new mechanism.
[0004]
[Means for Solving the Problems]
The present inventors have conducted intensive studies in order to solve the above problems, and as a result, an endogenous ligand (WO 01/98494) binding to GPR8 (Genomics, 28, 84-91, 1995) has been developed. The present inventors have found that they have a weight gain suppressing activity, and have completed the present invention.
[0005]
That is, the present invention
(1) a weight gain inhibitor comprising a polypeptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 16, or an amide or ester thereof, or a salt thereof;
(2) a weight loss agent comprising a polypeptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 16, or an amide or ester thereof, or a salt thereof;
(3) A fat mass increase inhibitor comprising a polypeptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 16, or an amide or ester thereof, or a salt thereof,
(4) an antifeedant comprising a polypeptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 16, or an amide or ester or salt thereof,
(5) A weight gain inhibitor characterized by using a polypeptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 16, or an amide or ester thereof, or a salt thereof, Screening method of a reducing agent, a fat mass increase inhibitor or an antifeedant,
(6) Further, a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 126, SEQ ID NO: 138 or SEQ ID NO: 144, or a partial peptide thereof, or a salt thereof The screening method according to the above (5), wherein
(7) a weight gain inhibitor comprising a polypeptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 16, or an amide or ester or salt thereof, A kit for screening a weight loss agent, a fat mass increase inhibitor or an antifeedant,
(8) Further, a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 126, SEQ ID NO: 138 or SEQ ID NO: 144, or a partial peptide thereof, or a salt thereof The screening kit according to the above (7), comprising:
(9) a weight gain inhibitor, a weight loss agent, a fat mass increase inhibitor or an antifeedant obtained by using the screening method according to (5) or the screening kit according to (7);
(10) A polypeptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 16, a compound having the activity of an amide or an ester thereof, or a salt thereof, or a salt thereof. Weight gain inhibitors, weight loss agents, fat gain inhibitors or food intake inhibitors,
(11) Agonists of a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 126, SEQ ID NO: 138 or SEQ ID NO: 144, a partial peptide thereof, or a salt thereof A weight gain inhibitor, a weight loss agent, a fat mass increase inhibitor or an antifeedant comprising,
(12) a polynucleotide containing a polypeptide containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 16, or a polynucleotide containing a base sequence encoding an amide or an ester or a salt thereof Weight gain inhibitor, weight loss agent, fat mass increase inhibitor or food intake inhibitor,
(13) Use of a polynucleotide containing a nucleotide sequence encoding a polypeptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 16, or an amide or ester thereof, or a salt thereof A method for screening a weight gain inhibitor, a weight loss agent, a fat mass increase inhibitor or an antifeedant, characterized by the characteristics of:
(14) A polynucleotide containing a polypeptide containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 16, or a polynucleotide containing a base sequence encoding an amide or an ester or a salt thereof A weight gain inhibitor, a weight loss agent, a kit for screening a fat mass increase inhibitor or an antifeedant,
(15) a weight gain inhibitor, a weight loss agent, a fat mass increase inhibitor or an antifeedant obtained by using the screening method according to (13) or the screening kit according to (14);
(16) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 149,
(17) a labeled polypeptide according to the above (16),
(18) The screening method according to (5) using the polypeptide according to (16),
(19) (i) the polypeptide according to (17) above, and (ii) the same or substantially the same as the amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 126, SEQ ID NO: 138 or SEQ ID NO: 144 The screening method according to the above (6), wherein a protein containing the amino acid sequence of
(20) (i) the polypeptide described in (17) above, and (ii) a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 4 or SEQ ID NO: 144, or a portion thereof The screening method according to the above (19), wherein the peptide or a salt thereof is used,
(21) The screening method according to (20), wherein the polypeptide according to (17) and a protein containing the amino acid sequence represented by SEQ ID NO: 144, a partial peptide thereof, or a salt thereof,
(22) {For a mammal, (i) a polypeptide containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 16, or an amide or ester thereof, or a salt thereof, (ii) A compound having the activity of the polypeptide or its amide or its ester or its salt or its salt, or (iii) an amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 126, SEQ ID NO: 138 or SEQ ID NO: 144; A method for suppressing weight gain, a method for suppressing weight loss, a method for suppressing fat mass increase, or eating, comprising administering an effective amount of an agonist of a protein or a partial peptide thereof or a salt thereof containing the same or substantially the same amino acid sequence. Suppression method,
(23) (i) an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 16 for producing a weight gain inhibitor, a weight loss agent, a fat mass increase inhibitor or an antifeedant; Or a amide or an ester thereof or a salt thereof, (ii) a compound having the activity of the polypeptide or an amide or an ester thereof or a salt thereof, or (iii) SEQ ID NO: 4, SEQ ID NO: : 126, SEQ ID NO: 138 or SEQ ID NO: 144. Use of an agonist of a protein or a partial peptide thereof or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 144 is provided.
[0006]
BEST MODE FOR CARRYING OUT THE INVENTION
A polypeptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 16 used in the present invention (hereinafter, sometimes referred to as the polypeptide of the present invention) may be a human or a warm-blooded animal ( For example, cells of guinea pigs, rats, mice, chickens, rabbits, pigs, sheep, cows, monkeys, etc. (eg, retinal cells, hepatocytes, splenocytes, nerve cells, glial cells, pancreatic β cells, bone marrow cells, mesangial cells) , Langerhans cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts, fibrocytes, muscle cells, adipocytes, immune cells (eg, macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, Basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts, mammary cells, hepatocytes or stromal cells, Are precursor cells of these cells, stem cells or cancer cells, etc.) or any tissue in which those cells are present, such as the brain, various parts of the brain (eg, retina, olfactory bulb, amygdala, basal sphere, hippocampus, thalamus, thalamus Lower, cerebral cortex, medulla, cerebellum), spinal cord, pituitary, stomach, pancreas, kidney, liver, gonads, thyroid, gall bladder, bone marrow, adrenal gland, skin, muscle, lung, digestive tract (eg, large intestine, small intestine), blood vessels , Heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testis, ovary, placenta, uterus, bone, joint, skeletal muscle, etc., or cells of the blood cell system or cultured cells thereof (eg, MEL, M1, CTLL- 2, HT-2, WEHI-3, HL-60, JOSK-1, K562, ML-1, MOLT-3, MOLT-4, MOLT-10, CCRF-CEM, TALL-1, Jurkat, CCRT-HSB- 2, KE-37, SKW-3, HUT-78, HUT-102, H9, U937, THP-1, HEL, JK-1, CMK, KO-812, MEG-01) Alternatively, it may be a synthetic polypeptide.
[0007]
The amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 16 includes, for example, about 70% or more, preferably about 80% or more, preferably about 90% or more, Preferably, the amino acid sequence has about 95% or more, more preferably about 98% or more homology.
In particular, the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 16 includes, in addition to the above amino acid sequence,
(I) an amino acid sequence in which 1 to 5 (preferably 1 to 3, more preferably 1 to 2, and more preferably 1) amino acids in the amino acid sequence represented by SEQ ID NO: 16 have been deleted ,
(Ii) an amino acid sequence obtained by adding 1 to 5 (preferably 1 to 3, more preferably 1 to 2, and more preferably 1) amino acids to the amino acid sequence represented by SEQ ID NO: 16,
(Iii) an amino acid sequence in which 1 to 5 (preferably 1 to 3, more preferably 1 to 2, and more preferably 1) amino acids have been inserted into the amino acid sequence represented by SEQ ID NO: 16;
(Iv) 1 to 5 (preferably 1 to 3, more preferably 1 to 2, more preferably 1) amino acids in the amino acid sequence represented by SEQ ID NO: 16 are substituted with other amino acids Amino acid sequence,
(V) An amino acid sequence obtained by combining the above (i) to (iv).
[0008]
Examples of the polypeptide having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 16 include, for example, a polypeptide having an amino acid sequence substantially identical to the amino acid sequence represented by the aforementioned SEQ ID NO: 16; A polypeptide having substantially the same activity as the polypeptide having the amino acid sequence represented by No. 16 is preferred.
Examples of the substantially equivalent activity include, for example, the activity of the polypeptide of the present invention (eg, a weight gain suppressing action, a weight loss reducing action, a fat mass increase suppressing action, a food intake suppressing action) and the like.
Substantially equivalent activities indicate that those activities are qualitatively (eg, physiochemically or pharmacologically) equivalent.
Specific examples of the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 16 include, for example, SEQ ID NO: 6, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 , SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 95 , SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105 SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113 or SEQ ID NO: 149 Such as amino acid sequence, and the like.
[0009]
Specific examples of the polypeptide of the present invention include, for example, a polypeptide having an amino acid sequence represented by SEQ ID NO: 16, a polypeptide having an amino acid sequence represented by SEQ ID NO: 6, and a polypeptide having SEQ ID NO: 17. Polypeptide having an amino acid sequence represented by SEQ ID NO: 20; polypeptide having an amino acid sequence represented by SEQ ID NO: 21; polypeptide having an amino acid sequence represented by SEQ ID NO: 22 Peptide, polypeptide having the amino acid sequence represented by SEQ ID NO: 23, polypeptide having the amino acid sequence represented by SEQ ID NO: 24, polypeptide having the amino acid sequence represented by SEQ ID NO: 25, SEQ ID NO: A polypeptide having an amino acid sequence represented by SEQ ID NO: 57; Polypeptide, a polypeptide having the amino acid sequence represented by SEQ ID NO: 73, a polypeptide having the amino acid sequence represented by SEQ ID NO: 74, a polypeptide having the amino acid sequence represented by SEQ ID NO: 91, sequence Polypeptide having the amino acid sequence represented by SEQ ID NO: 92, polypeptide having the amino acid sequence represented by SEQ ID NO: 95, polypeptide having the amino acid sequence represented by SEQ ID NO: 96, represented by SEQ ID NO: 97 A polypeptide having an amino acid sequence represented by SEQ ID NO: 98, a polypeptide having an amino acid sequence represented by SEQ ID NO: 99, an amino acid sequence represented by SEQ ID NO: 100 Polypeptide having the amino acid sequence represented by SEQ ID NO: 101, SEQ ID NO: : A polypeptide having the amino acid sequence represented by 102, a polypeptide having the amino acid sequence represented by SEQ ID NO: 103, a polypeptide having the amino acid sequence represented by SEQ ID NO: 104, represented by SEQ ID NO: 105 A polypeptide having the amino acid sequence represented by SEQ ID NO: 106, a polypeptide having the amino acid sequence represented by SEQ ID NO: 107, and a polypeptide having the amino acid sequence represented by SEQ ID NO: 108 Polypeptide, polypeptide having the amino acid sequence represented by SEQ ID NO: 109, polypeptide having the amino acid sequence represented by SEQ ID NO: 110, polypeptide having the amino acid sequence represented by SEQ ID NO: 111, SEQ ID NO: : Polypeptide having the amino acid sequence represented by 112 and SEQ ID NO: Polypeptides having the ability to specifically bind to GPR8, such as a polypeptide having the amino acid sequence represented by 113 and a polypeptide having the amino acid sequence represented by SEQ ID NO: 149.
Further, the polypeptide of the present invention is used in a sense that also encompasses a precursor polypeptide of the polypeptide of the present invention.
Specific examples of the precursor polypeptide include, for example, a polypeptide characterized by containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 15.
More specifically,
The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 15 includes about 80% or more, preferably about 90% or more, more preferably about 95% or more of the amino acid sequence represented by SEQ ID NO: 15. Examples include amino acid sequences having homology.
In particular, the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 15 includes, in addition to the above amino acid sequence,
(I) 1 to 15 (preferably 1 to 10, more preferably 1 to 5, and more preferably 1 to 3) amino acids in the amino acid sequence represented by SEQ ID NO: 15 have been deleted Amino acid sequence,
(Ii) an amino acid sequence obtained by adding 1 to 100 (preferably 1 to 50, more preferably 1 to 5, and more preferably 1 to 3) amino acids to the amino acid sequence represented by SEQ ID NO: 15 ,
(Iii) an amino acid in which 1 to 15 (preferably 1 to 10, more preferably 1 to 5, and more preferably 1 to 3) amino acids have been inserted into the amino acid sequence represented by SEQ ID NO: 15 Array,
(Iv) 1 to 15 (preferably 1 to 10, more preferably 1 to 5, and more preferably 1 to 3) amino acids in the amino acid sequence represented by SEQ ID NO: 15 are other amino acids An amino acid sequence substituted with
(V) An amino acid sequence obtained by combining the above (i) to (iv).
Specific examples of the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 15 include, for example, the amino acid represented by SEQ ID NO: 42, SEQ ID NO: 55, SEQ ID NO: 72 or SEQ ID NO: 90 Arrays and the like.
Specific examples of the precursor polypeptide include, for example, a polypeptide having an amino acid sequence represented by SEQ ID NO: 15, a polypeptide having an amino acid sequence represented by SEQ ID NO: 42, and a polypeptide having SEQ ID NO: 55. A polypeptide having an amino acid sequence represented by SEQ ID NO: 72; a polypeptide having an amino acid sequence represented by SEQ ID NO: 90;
[0010]
Among the various receptors, the receptor for the polypeptide of the present invention has a binding activity with the polypeptide of the present invention, and the polypeptide of the present invention stimulates the cell stimulating activity of cells expressing the receptor (for example, arachidonic acid release). , Acetylcholine release, intracellular Ca²⁺Release, Intracellular cAMP production / suppression, Intracellular cGMP production, Inositol phosphate production, Cell membrane potential fluctuation, Intracellular protein phosphorylation, c-fos activation, pH reduction, GTPγS binding activity, etc. ) Is observed.
Specifically, (1) GPR8 (SEQ ID NO: 4; Genomics, 28, 84-91, 1995), or an amino acid sequence substantially identical to GPR8 (amino acid sequence represented by SEQ ID NO: 4) (2) a receptor containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 126, (3) a SEQ ID NO: 138 And (4) GPR7 (SEQ ID NO: 144; Genomics, Vol. 28, pp. 84-91, 1995) and the like.
A protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 4, a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 126, SEQ ID NO: 138 A protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 144, and a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 144 (hereinafter referred to as the receptor of the present invention; Cells) (eg, retinal cells, hepatocytes, spleen cells, nerve cells) of humans or warm-blooded animals (eg, guinea pigs, rats, mice, chickens, rabbits, pigs, sheep, cows, monkeys, etc.) , Glial cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, tables Cells, epithelial cells, endothelial cells, fibroblasts, fibrocytes, muscle cells, adipocytes, immune cells (eg, macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, Acidocytes, monocytes), megakaryocytes, synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts, mammary cells, hepatocytes or stromal cells, or precursors, stem cells or cancer cells of these cells, etc. ) Or any tissue where these cells are present, such as the brain, various parts of the brain (eg, retina, olfactory bulb, amygdala, basal sphere, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla, cerebellum), spinal cord, Pituitary, stomach, pancreas, kidney, liver, gonad, thyroid, gall bladder, bone marrow, adrenal gland, skin, muscle, lung, digestive tract (eg, large intestine, small intestine), blood vessels, heart, thymus, spleen, submandibular gland, peripheral Blood, prostate, testicle, ovary, placenta, uterus, bone, joint , Skeletal muscle, etc., or blood cell line or cultured cells thereof (for example, MEL, M1, CTLL-2, HT-2, WEHI-3, HL-60, JOSK-1, K562, ML-1, MOLT-3) , MOLT-4, MOLT-10, CCRF-CEM, TALL-1, Jurkat, CCRT-HSB-2, KE-37, SKW-3, HUT-78, HUT-102, H9, U937, THP-1, HEL , JK-1, CMK, KO-812, MEG-01, etc.) or a synthetic protein.
[0011]
The amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 4 includes about 70% or more, preferably about 80% or more, more preferably about 90% or more of the amino acid sequence represented by SEQ ID NO: 4. Examples include amino acid sequences having homology.
The amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 126 includes, for example, 85% or more, preferably about 90% or more, more preferably about 95% or more of the amino acid sequence represented by SEQ ID NO: 126. And amino acid sequences having at least% homology.
Examples of the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 138 include, for example, 86% or more, preferably about 90% or more, more preferably about 95% or more of the amino acid sequence represented by SEQ ID NO: 138. And amino acid sequences having at least% homology.
As the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 144, about 70% or more, preferably about 80% or more, more preferably about 90% or more of the amino acid sequence represented by SEQ ID NO: 144 Examples include amino acid sequences having homology.
Examples of the protein containing an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 126, SEQ ID NO: 138 or SEQ ID NO: 144 include, for example, SEQ ID NO: 4, SEQ ID NO: 126, comprising an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 138 or SEQ ID NO: 144, and represented by SEQ ID NO: 4, SEQ ID NO: 126, SEQ ID NO: 138 or SEQ ID NO: 144. Preferred are proteins having substantially the same activity as the protein having the amino acid sequence to be obtained.
The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 126, SEQ ID NO: 138 or SEQ ID NO: 144 includes (i) SEQ ID NO: 4, SEQ ID NO: 126, SEQ ID NO: : 138 or 1 to 15 (preferably 1 to 10, more preferably 1 to 5, and more preferably 1 to 3) amino acids in the amino acid sequence represented by SEQ ID NO: 144 have been deleted. The amino acid sequence represented by (ii) the amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 126, SEQ ID NO: 138 or SEQ ID NO: 144, preferably 1 to 15 (preferably 1 to 10, more preferably 1 to 5) (Preferably, 1 to 3 amino acids), and (iii) represented by SEQ ID NO: 4, SEQ ID NO: 126, SEQ ID NO: 138, or SEQ ID NO: 144. An amino acid sequence in which 1 to 15 (preferably 1 to 10, more preferably 1 to 5, and more preferably 1 to 3) amino acids have been inserted into the amino acid sequence, (iv) SEQ ID NO: 4, 1 to 15 (preferably 1 to 10, more preferably 1 to 5, and more preferably 1 to 3) in the amino acid sequence represented by SEQ ID NO: 126, SEQ ID NO: 138 or SEQ ID NO: 144 And (v) an amino acid sequence obtained by combining the above (i) to (iv).
Specific examples of the receptor of the present invention include, for example, a protein containing the amino acid sequence represented by SEQ ID NO: 4; a protein containing the amino acid sequence represented by SEQ ID NO: 126; And a protein containing the amino acid sequence represented by SEQ ID NO: 144, and the like.
[0012]
As the partial peptide of the receptor of the present invention (hereinafter sometimes referred to as the partial peptide of the present invention), any peptide can be used as long as it can be used in a method for screening a drug or the like described below. Often, preferably, a partial peptide having an ability to bind to the polypeptide of the present invention, a partial peptide containing an amino acid sequence corresponding to an extracellular region, and the like are used. Peptides having an amino acid sequence of 20 or more, preferably 50 or more, more preferably 100 or more of the constituent amino acid sequences of the receptor of the present invention are preferred. (I) one or two or more (preferably about 1 to 10, more preferably several (1 to 5)) amino acids in the amino acid sequence are deleted; Or two or more (preferably about 1 to 20, more preferably about 1 to 10, and even more preferably several (1 to 5)) amino acids are added, or (iii) the amino acid sequence 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. As a specific example, (a) a partial amino acid sequence consisting of the 1st (Met) to 123rd (Phe) amino acid residues in the amino acid sequence represented by SEQ ID NO: 4 or a part thereof, and the 301st (Asn) A partial amino acid sequence consisting of the 358th amino acid residue (Lys) or a part thereof; a partial amino acid sequence consisting of the 548th amino acid residue (Tyr) to the 593th amino acid residue (Arg); or a 843th amino acid sequence (Ala) A) a partial peptide comprising one or more partial amino acid sequences selected from the partial amino acid sequence consisting of the amino acid residues at positions 895 to 895 (Ile) or a part thereof; (b) the amino acid represented by SEQ ID NO: 126 A partial amino acid sequence consisting of the 1st (Met) to 85th (Asp) amino acid residues or a part thereof, Such as 222 th (Cys) ~329 th peptide containing a partial amino acid sequence or a portion thereof comprising the amino acid residues (Ala) and the like.
[0013]
The left end of the polypeptide, receptor or partial peptide thereof of the present invention is the N-terminal (amino terminal) and the right end is the C-terminal (carboxyl terminal) according to the convention of peptide labeling. The polypeptide, receptor or partial peptide thereof of the present invention has a carboxyl group (-COOH), a carboxylate (-COO) at the C-terminus.⁻), Amide (-CONH₂)) Or an ester (—COOR).
Here, R in the ester is, for example, C, such as methyl, ethyl, n-propyl, isopropyl or n-butyl._1-6Alkyl groups such as C, cyclopentyl, cyclohexyl, etc._3-8Cycloalkyl groups such as phenyl, α-naphthyl and the like;_6-12Aryl groups, for example phenyl-C such as benzyl, phenethyl, etc._1-2An alkyl group or α-naphthyl-C such as α-naphthylmethyl_1-2C such as an alkyl group_7-14In addition to aralkyl groups, pivaloyloxymethyl groups commonly used as oral esters are used.
When the polypeptide, the receptor or the partial peptide thereof of the present invention has a carboxyl group (or carboxylate) other than the C-terminus, those in which the carboxyl group is amidated or esterified are also included in the polypeptide of the present invention. included. As the ester in this case, for example, the above-mentioned C-terminal ester and the like are used. Furthermore, in the polypeptide, receptor or partial peptide thereof according to 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.)._1-6C such as alkanoyl_1-6Acyl 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.)._1-6C 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.
[0014]
As the salt of the polypeptide, receptor or partial peptide thereof of the present invention, salts with physiologically acceptable acids (eg, inorganic acids, organic acids) and bases (eg, alkali metal salts) are used. Particularly preferred are physiologically acceptable acid addition salts. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) 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 polypeptide, receptor or partial peptide thereof of the present invention can be produced by a known polypeptide purification method from human or warm-blooded animal cells or tissues described above, or can be a DNA encoding the polypeptide described below. It can also be produced by culturing the transformed transformant. 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.
[0015]
For the synthesis of the polypeptide, receptor, partial peptide thereof, or salt or amide thereof of the present invention, a commercially available resin for polypeptide synthesis can be usually 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. Examples include 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 polypeptide according to various known condensation methods. At the end of the reaction, the polypeptide is cleaved from the resin, and at the same time, various protecting groups are removed.In addition, an intramolecular disulfide bond formation reaction is carried out in a highly diluted solution, and the desired polypeptide, receptor, partial peptide or amide thereof is obtained. To get.
For the condensation of the protected amino acids described above, various activating reagents that can be used for polypeptide 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 a HOBt ester or a HOOBt ester. Later it can be added to the resin.
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 polypeptide 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. Examples thereof include 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. The reaction temperature is appropriately selected from a range known to be usable for a polypeptide 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, the unreacted amino acid can be acetylated using acetic anhydride or acetylimidazole, so that the subsequent reaction is not 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_1-6A) 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, Cl₂-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.
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.
[0018]
As another method for obtaining the amide form of the polypeptide, receptor or partial peptide thereof of the present invention, for example, firstly, the α-carboxyl group of the carboxy terminal amino acid is protected by amidation and then the peptide (poly) After extending the peptide) chain to a desired chain length, a polypeptide obtained by removing only the protecting group of the N-terminal α-amino group of the peptide chain and a polypeptide obtained by removing only the protecting group of the C-terminal carboxyl group are obtained. It is prepared and both polypeptides are condensed in a mixed solvent as described above. Details of the condensation reaction are the same as described above. After purifying the protected polypeptide obtained by the condensation, all the protecting groups are removed by the above-mentioned method to obtain a desired crude polypeptide. This crude polypeptide is purified by various known purification means, and the main fraction is freeze-dried to obtain an amide of the desired polypeptide, receptor or its partial peptide.
In order to obtain an ester of the polypeptide, the receptor or a partial peptide thereof of the present invention, 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 polypeptide, the receptor or In the same manner as the amide of the partial peptide, a desired polypeptide, receptor or ester of the partial peptide can be obtained.
[0019]
The polypeptide, receptor or partial peptide thereof of the present invention can be produced according to a known peptide synthesis method, or, for a partial peptide of the receptor, by cleaving the receptor 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 polypeptide of the present invention, the target peptide by condensing the partial peptide or amino acid that can constitute the receptor or its partial peptide and the remaining portion, and if the product has a protecting group, removing the protecting group Can be manufactured. Examples of known condensation methods and elimination of protecting groups include the methods described in the following (a) to (e).
(A) M.I. Bodanszky and M. A. Ondetti, Peptide Synthesis, Interscience ｌｉPublishers, New York (1966)
(B) Schroeder and Luebke, The Peptide, Academic Press, New York (1965)
(C) Nobuo Izumiya et al., Basics and Experiments of Peptide Synthesis, Maruzen Co., Ltd. (1975)
(D) Haruaki Yajima and Shunpei Sakakibara, Biochemistry Laboratory Course No. 1, Protein Chemistry IV, No. 205, (1977)
(E) Supervised by Haruaki Yajima, Development of Continuing Drugs, Volume 14, Peptide Synthesis, Hirokawa Shoten
After the reaction, the polypeptide, receptor or its partial peptide of the present invention can be purified and isolated by a combination of ordinary purification methods, for example, solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization and the like. it can. When the polypeptide, receptor or its 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, Can be converted to a free form or another salt by a known method or a method analogous thereto.
[0020]
The DNA encoding the polypeptide, receptor or partial peptide thereof of the present invention may be any DNA containing the above-described nucleotide sequence encoding the polypeptide, receptor or partial peptide thereof of the present invention. Is also good. Further, any of genomic DNA, genomic DNA library, cDNA derived from the above-mentioned cells / tissues, cDNA library derived from the aforementioned cells / tissues, and synthetic DNA may be used.
The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Alternatively, amplification can be directly performed by using Reverse RNA / Transscriptase / Polymerase / Chain / Reaction (hereinafter abbreviated as RT-PCR) using a total RNA or mRNA fraction prepared from the cells and tissues described above.
[0021]
Examples of the DNA encoding the polypeptide of the present invention include (a) SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 , SEQ ID NO: 31, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116 , SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125 or SEQ ID NO: 150 (B) SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: : 30, SEQ ID NO: 31, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: : 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125 or SEQ ID NO: No .: DNA encoding a polypeptide having a nucleotide sequence that hybridizes under high stringent conditions with the nucleotide sequence represented by 150, and encoding a polypeptide having substantially the same activity as the polypeptide of the present invention; (c) SEQ ID NO: No .: 14, DNA having the base sequence represented by SEQ ID NO: 41, SEQ ID NO: 54, SEQ ID NO: 71 or SEQ ID NO: 89, or (d) sequence No. 14, SEQ ID NO: 41, SEQ ID NO: 54, SEQ ID NO: 71 or SEQ ID NO: 89, as long as it has a base sequence which hybridizes under high stringent conditions to a base sequence. It may be something.
(I) SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 58, SEQ ID NO : 59, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125 or SEQ ID NO: 150, or (ii) SEQ ID NO: 14, a DNA which can hybridize with the base sequence represented by SEQ ID NO: 41, SEQ ID NO: 54, SEQ ID NO: 71 or SEQ ID NO: 89 under high stringent conditions. Are, for example, (i) SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: : 58, SEQ ID NO: 59, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: : 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125 or SEQ ID NO: 150, or (Ii) about 70% or more, preferably about 80% or more, of the nucleotide sequence represented by SEQ ID NO: 14, SEQ ID NO: 41, SEQ ID NO: 54, SEQ ID NO: 71 or SEQ ID NO: 89. Preferably about 90% or more, more preferably a DNA containing the base sequence having about 95% homology are used.
[0022]
Hybridization can be performed according to a known method or a method analogous thereto, for example, the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. More preferably, it can be performed under high stringent conditions.
The high stringent conditions are, for example, conditions in which the sodium concentration is about 19 to 40 mM, preferably about 19 to 20 mM, and the temperature is about 50 to 70 ° C, preferably about 60 to 65 ° C. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C. is most preferable.
More specifically,
(I) As the DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 16, a DNA containing the base sequence represented by SEQ ID NO: 18 or the like is used,
(Ii) As the DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 17, a DNA containing the base sequence represented by SEQ ID NO: 19, or the like is used,
(Iii) As the DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 20, a DNA containing the base sequence represented by SEQ ID NO: 26 or the like is used,
(Iv) As the DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 21, a DNA containing the base sequence represented by SEQ ID NO: 27 or the like is used,
(V) As the DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 22, a DNA containing the base sequence represented by SEQ ID NO: 28 or the like is used,
(Vi) As the DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 23, a DNA containing the base sequence represented by SEQ ID NO: 29 or the like is used,
(Vii) As the DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 24, a DNA containing the base sequence represented by SEQ ID NO: 30 or the like is used,
(Viii) As the DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 25, a DNA containing the base sequence represented by SEQ ID NO: 31 or the like is used,
(Ix) As the DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 56, a DNA containing the base sequence represented by SEQ ID NO: 58 or the like is used,
(X) As the DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 57, a DNA containing the base sequence represented by SEQ ID NO: 59 or the like is used,
(Xi) As the DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 73, a DNA containing the base sequence represented by SEQ ID NO: 75 or the like is used,
(Xii) As the DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 74, a DNA containing the base sequence represented by SEQ ID NO: 76 or the like is used,
(Xiii) As the DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 91, a DNA containing the base sequence represented by SEQ ID NO: 93 or the like is used,
(Xiv) As the DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 92, a DNA containing the base sequence represented by SEQ ID NO: 94 or the like is used,
(Xv) As the DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 95, a DNA containing the base sequence represented by SEQ ID NO: 18 or the like is used,
(Xvi) As the DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 96, a DNA containing the base sequence represented by SEQ ID NO: 114 or the like is used,
(Xvii) As the DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 97, a DNA containing the base sequence represented by SEQ ID NO: 115 or the like is used,
(Xviii) As the DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 98, a DNA containing the base sequence represented by SEQ ID NO: 116 or the like is used,
(Xix) As the DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 99, a DNA containing the base sequence represented by SEQ ID NO: 117, or the like is used,
(Xx) As the DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 100, a DNA containing the base sequence represented by SEQ ID NO: 118 or the like is used,
(Xxi) As the DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 101, a DNA containing the base sequence represented by SEQ ID NO: 119 or the like is used,
(Xxii) As the DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 102, a DNA containing the base sequence represented by SEQ ID NO: 120 or the like is used,
(Xxiii) As the DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 103, a DNA containing the base sequence represented by SEQ ID NO: 58 or the like is used,
(Xxiv) As the DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 104, a DNA containing the base sequence represented by SEQ ID NO: 75 or the like is used,
(Xxv) As the DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 105, a DNA containing the base sequence represented by SEQ ID NO: 18 or the like is used,
(Xxvi) As the DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 106, a DNA containing the base sequence represented by SEQ ID NO: 18 or the like is used,
(Xxvii) As the DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 107, a DNA containing the base sequence represented by SEQ ID NO: 121 or the like is used,
(Xxviii) As the DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 108, a DNA containing the base sequence represented by SEQ ID NO: 122 or the like is used,
(Xxix) As the DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 109, a DNA containing the base sequence represented by SEQ ID NO: 123 or the like is used,
(Xxx) As the DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 110, a DNA containing the base sequence represented by SEQ ID NO: 124 or the like is used,
(Xxxi) As the DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 6, a DNA containing the base sequence represented by SEQ ID NO: 125 or the like is used,
(Xxxii) As the DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 111, a DNA containing the base sequence represented by SEQ ID NO: 121 or the like is used,
(Xxxiii) As the DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 112, a DNA containing the base sequence represented by SEQ ID NO: 18 or the like is used,
(Xxxiv) As the DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 113, a DNA containing the base sequence represented by SEQ ID NO: 121 or the like is used,
(Xxxv) As the DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 149, a DNA containing the base sequence represented by SEQ ID NO: 150 is used.
[0023]
As the DNA encoding the receptor of the present invention, for example, (1) a DNA containing the nucleotide sequence represented by SEQ ID NO: 32 or the nucleotide sequence represented by SEQ ID NO: 32 under high stringent conditions DNA encoding a protein having a hybridizing nucleotide sequence and having substantially the same activity as the protein containing the amino acid represented by SEQ ID NO: 4, (2) the nucleotide sequence represented by SEQ ID NO: 127 Or a nucleotide sequence that hybridizes under high stringent conditions with the nucleotide sequence represented by SEQ ID NO: 32 and is substantially the same as the protein containing the amino acid represented by SEQ ID NO: 126 DNA encoding a protein having the activity of SEQ ID NO: 139, (3) DNA containing the nucleotide sequence represented by SEQ ID NO: 139, or SEQ ID NO: : Encoding a protein having a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence represented by 139 and having substantially the same activity as the protein containing the amino acid represented by SEQ ID NO: 138 DNA, (4) a DNA containing the nucleotide sequence represented by SEQ ID NO: 143, or a nucleotide sequence that hybridizes with the nucleotide sequence represented by SEQ ID NO: 143 under high stringent conditions; Any DNA may be used as long as it encodes a protein having substantially the same activity as the protein containing the amino acid represented by 143.
Examples of DNAs that can hybridize to the base sequence represented by SEQ ID NO: 32, SEQ ID NO: 127, SEQ ID NO: 139, or SEQ ID NO: 143 under high stringent conditions include, for example, SEQ ID NO: 32 and SEQ ID NO: Base sequence having about 70% or more, preferably about 80% or more, more preferably about 90% or more, and still more preferably about 95% or more homology with the base sequence represented by SEQ ID NO: 127, SEQ ID NO: 139 or SEQ ID NO: 143. DNA containing a sequence or the like is used.
[0024]
Hybridization can be performed according to a known method or a method analogous thereto, for example, the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. More preferably, it can be performed under high stringent conditions.
The high stringent conditions are, for example, conditions in which the sodium concentration is about 19 to 40 mM, preferably about 19 to 20 mM, and the temperature is about 50 to 70 ° C, preferably about 60 to 65 ° C. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C. is most preferable.
More specifically, the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 4 includes a DNA containing the base sequence represented by SEQ ID NO: 32, and an amino acid sequence represented by SEQ ID NO: 126. The DNA encoding the protein containing the nucleic acid is a DNA containing the base sequence represented by SEQ ID NO: 127, and the DNA encoding the protein containing the amino acid sequence represented by the SEQ ID NO: 138 is SEQ ID NO: 139. As the DNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 144, or the DNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 144, a DNA having the nucleotide sequence represented by SEQ ID NO: 143 is used.
[0025]
The DNA encoding the partial peptide of the receptor of the present invention may be any DNA containing the above-described nucleotide sequence encoding the partial peptide of the receptor of the present invention. Further, any of genomic DNA, genomic DNA library, cDNA derived from the above-mentioned cells / tissues, cDNA library derived from the aforementioned cells / tissues, and synthetic DNA may be used.
Examples of the DNA encoding the partial peptide of the receptor of the present invention include (1) a DNA having a partial nucleotide sequence of a DNA having a nucleotide sequence represented by SEQ ID NO: 32, or a nucleotide sequence represented by SEQ ID NO: 32 And a partial nucleotide sequence of DNA encoding a protein having substantially the same activity as the protein containing the amino acid represented by SEQ ID NO: 4. DNA, (2) a DNA having a partial base sequence of the DNA having the base sequence of SEQ ID NO: 127, or a base sequence which hybridizes with the base sequence of SEQ ID NO: 127 under high stringent conditions And has substantially the same activity as the protein containing the amino acid represented by SEQ ID NO: 126. DNA having a partial base sequence of DNA encoding a protein, (3) DNA having a partial base sequence of DNA having a base sequence represented by SEQ ID NO: 139, or a base sequence represented by SEQ ID NO: 138 and a high string A DNA having a partial nucleotide sequence of a DNA having a nucleotide sequence that hybridizes under gentle conditions and encoding a protein having substantially the same activity as the protein containing the amino acid represented by SEQ ID NO: 126; 4) a DNA having a partial base sequence of a DNA having the base sequence of SEQ ID NO: 143 or a base sequence which hybridizes with the base sequence of SEQ ID NO: 143 under high stringent conditions; Has substantially the same activity as the protein containing the amino acid represented by No. 144 A DNA having a partial nucleotide sequence of the DNA encoding the protein is used.
DNAs that can hybridize to the base sequence represented by SEQ ID NO: 32, SEQ ID NO: 127, SEQ ID NO: 139 or SEQ ID NO: 143 have the same significance as described above.
The same hybridization method and high stringency conditions as described above are used.
More specifically, as the DNA encoding the partial peptide of the receptor of the present invention, specifically, in the amino acid sequence represented by SEQ ID NO: 4, the 1st (Met) to 43rd (Phe) and the 101st (Asn) One or two or more partial amino acid sequences selected from the partial amino acid sequences represented by the amino acid sequence represented by the amino acid sequence of positions １１８118 to (Lys), 188 (Tyr) to 213 (Arg), and 283 (Ala) to 295 (Ile) Examples of such a DNA include a DNA containing a DNA having a nucleotide sequence encoding a partial peptide contained therein, and a DNA containing a DNA having a nucleotide sequence hybridizing with these under high stringency conditions.
[0026]
The DNA encoding the polypeptide, receptor or partial peptide thereof of the present invention may be labeled by a known method, and specifically, isotope-labeled or fluorescently-labeled (for example, fluorescein) And the like, biotinylated or enzyme-labeled. Preferably, an isotope-labeled polypeptide of the present invention is used.
The polypeptide, the receptor or its partial peptide of the present invention (hereinafter, in the description of the cloning and expression of DNAs encoding these polypeptides and the like, these polypeptides and the like may be simply abbreviated as the polypeptide of the present invention). As a means for cloning a DNA that completely encodes the DNA of the present invention, the DNA of the present invention is amplified by a known PCR method using a synthetic DNA primer having a partial base sequence of the polypeptide of the present invention, or the DNA of the present invention is incorporated into an appropriate vector. Can be selected by hybridization with a DNA fragment coding for a part or the entire region of the polypeptide or labeled with a 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.
[0027]
Conversion of the DNA base sequence can be performed by a known kit, for example, Mutan.^TM-Super @ Express @ Km (Takara Shuzo Co., Ltd.), Mutan^TMUsing -K (Takara Shuzo Co., Ltd.) or the like, it can be carried out according to a known method such as the ODA-LA @ PCR method, the Gapped @ duplex method, the Kunkel method, or a method analogous thereto.
The DNA encoding the cloned polypeptide can be used as it is depending on the purpose, or can be used after digesting with a restriction enzyme or adding 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 stop codon at the 3' end. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter.
[0028]
The expression vector for the polypeptide of the present invention can be prepared, for example, by (A) cutting out a DNA fragment of interest from DNA encoding the polypeptide of the present invention, and (B) placing the DNA fragment downstream of a promoter in an appropriate expression vector. It can be manufactured by connecting.
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, etc. In addition to animal viruses such as bacteriophage, retrovirus, vaccinia virus, and baculovirus, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo, and the like are used.
The promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression. For example, when animal cells are used as hosts, SRα promoter, SV40 promoter, HIV / LTR promoter, CMV promoter, HSV-TK promoter and the like can be mentioned.
Of these, it is preferable to use a CMV (cytomegalovirus) promoter, SRα promoter, or the like. When the host is a bacterium belonging to the genus Escherichia, trp promoter, lac promoter, recA promoter, λPL promoter, lpp promoter, T7 promoter, etc., and when the host is a bacterium belonging to the genus Bacillus, SPO1 promoter, SPO2 promoter, penP promoter, When the host is yeast, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable. When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable. 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. 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).^rNeomycin resistance gene (hereinafter Neo).^rG418 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 polypeptide of the present invention. When the host is a bacterium belonging to the genus Escherichia, a PhoA signal sequence, an OmpA signal sequence, or the like is used. When the host is a bacterium belonging to the genus Bacillus, an α-amylase signal sequence, a subtilisin signal sequence, or the like is used. In some cases, MFα signal sequence, SUC2 signal sequence, etc., and when the host is an animal cell, insulin signal sequence, α-interferon signal sequence, antibody molecule, signal sequence, etc. can be used.
Using the vector containing the DNA encoding the polypeptide of the present invention thus constructed, a transformant can be produced.
As the host, for example, Escherichia, Bacillus, yeast, insect cells, insects, animal cells and the like are used.
[0029]
As a specific example of the genus Escherichia, for example, Escherichia coli (Escherichia coli) K12.DH1 [Processings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), Vol. 60, 160 (1968)], JM103 [Nucleic Acids Research, Vol. 9, 309 (1981)], JA221 [Journal of Molecular Biology (Journal of Molecular Biology)]. Biology)], 120, 517 (1978)], HB101 [Journal of Molecular Biology, 41, 459 (1969)], C600 [ Enetikkusu (Genetics), 39 vol., 440 (1954)], etc..
Examples of Bacillus bacteria include, for example, Bacillus subtilis MI114 [Gene, 24, 255 (1983)], 207-21 [Journal of Biochemistry, 95, 87 (1984)]. )] Is used.
Examples of yeast include, for example, Saccharomyces cerevisiae AH22, AH22R⁻, NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036, Pichia pastoris KM71 and the like.
[0030]
As the insect cells, for example, when the virus is AcNPV, a cell line derived from the larva of Spodoptera litura (Spodoptera frugiperda cell; Sf cell), MG1 cell derived from the midgut of Trichoplusia ni, and the egg derived from the egg of Trichoplusia ni ni^TMA cell, a cell derived from Maestrabrassicae, a cell derived from Estigmena acrea, or the like is 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 @ CRL1711), Sf21 cells (above, Vaughn, @JL, et al., In Vivo, 13, 213-217, (1977)) and the like are used. .
As insects, for example, silkworm larvae are used [Maeda et al., Nature, 315, 592 (1985)].
Examples of animal cells include monkey cells COS-7, Vero, Chinese hamster cells CHO (hereinafter abbreviated as CHO cells), dhfr gene-deficient Chinese hamster cells CHO (hereinafter CHO (dhfr).⁻) Cells), mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells, and the like.
In order to transform Escherichia sp., For example, Procesings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 69, 2110 ( 1972) and Gene, Vol. 17, 107 (1982).
Transformation of Bacillus spp. Can be performed, for example, according to the method described in Molecular & General Genetics, Vol. 168, 111 (1979).
In order to transform yeast, for example, Methods in Enzymology, 194, 182-187 (1991), Processings of the National Academy of Sciences of Science -The USA (Proc. Natl. Acad. Sci. USA), Vol. 75, 1929 (1978).
[0031]
Insect cells or insects can be transformed, for example, according to the method described in Bio / Technology (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 polypeptide 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.
Examples of a medium for culturing Escherichia include, for example, an M9 medium containing glucose and casamino acid [Miller, Journal of Experiments in Molecular Genetics (Journal of Experimentals in in Molecular Genetics)]. 431-433, Cold Spring Harbor Laboratory, New York 1972]. If necessary, an agent such as 3β-indolylacrylic acid can be added in order to make the promoter work efficiently.
[0032]
When the host is a bacterium belonging to the genus Escherichia, the cultivation is usually performed at about 15 to 43 ° C. for about 3 to 24 hours, and if necessary, aeration and stirring can be added.
When the host is a bacterium belonging to the genus Bacillus, the cultivation is usually carried out at about 30 to 40 ° C. for about 6 to 24 hours, and if necessary, aeration and stirring may be added.
When culturing a transformant in which the host is yeast, as a medium, for example, a Burkholder minimum medium [Bostian, ΔK. L. Procurings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 77, 4505 (1980)] and 0.5% casamino acid. Medium [Bitter, @G. A. Procurings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 81, 5330 (1984)]. Preferably, the pH of the medium is adjusted to about 5-8. The cultivation is usually performed at about 20 ° C. to 35 ° C. for about 24 to 72 hours, and if necessary, aeration and stirring are added.
When culturing a transformant whose host is an insect cell or an insect, the culture medium is immobilized as Grace's {Insect} Medium (Grace, @TCC, Nature, 195, 788 (1962)). For example, those to which an additive such as 10% bovine serum or the like is appropriately added are used. The pH of the medium is preferably adjusted to about 6.2 to 6.4. The cultivation is usually performed at about 27 ° C. for about 3 to 5 days, and if necessary, aeration or stirring is added.
When culturing a transformant in which the host is an animal cell, examples of the medium include a MEM medium containing about 5 to 20% fetal bovine serum [Science, 122, 501 (1952)], a DMEM medium. [Virology, 8, 396 (1959)], RPMI 1640 medium [Journal of the American Medical Association (The Journal of the American Medical Association) Volume 199, 519 (1967)], 199 medium Proceeding of the Society for the Biological Medicine, 73, Proceding of the Society for the Biological Medicine, 73 And 1 (1950)] 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 polypeptide of the present invention can be produced in cells, cell membranes or extracellular cells of the transformant.
The polypeptide of the present invention can be separated and purified from the culture by, for example, the following method.
[0033]
When the polypeptide of the present invention is extracted from cultured cells or cells, the cells or cells are collected by a known method after culture, suspended in an appropriate buffer, and subjected to ultrasonication, lysozyme and / or freeze-thawing. After the cells or cells are destroyed by, for example, a method of obtaining a crude extract of the polypeptide by centrifugation or filtration, etc. are used as appropriate. In a buffer, a protein denaturant such as urea or guanidine hydrochloride, or Triton X-100 is used.^TMAnd the like. When the polypeptide is secreted into the culture solution, after the culture, the supernatant is separated from the cells or cells by a known method, and the supernatant is collected.
Purification of the polypeptide contained in the thus obtained culture supernatant or extract can be carried out by appropriately combining known separation and purification methods. These known separation and purification methods include methods utilizing solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, mainly molecular weight. Using differences in charge, methods using charge differences such as ion exchange chromatography, methods using specific affinity such as affinity chromatography, and using differences in hydrophobicity such as reversed-phase high-performance liquid chromatography A method utilizing a difference between isoelectric points, such as an isoelectric focusing method, is used.
When the polypeptide thus obtained is obtained in a free form, it can be converted to a salt by a known method or a method analogous thereto, and conversely, when the polypeptide is obtained as a salt, a known method or a method analogous thereto , Free form or other salts.
The polypeptide produced by the recombinant can be arbitrarily modified, or the polypeptide can be partially removed, by applying an appropriate protein modifying enzyme before or after purification. As the protein modifying enzyme, for example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used.
[0034]
An antibody against the polypeptide of the present invention (hereinafter sometimes simply referred to as the antibody of the present invention) is a polyclonal antibody as long as it can recognize an antibody against the polypeptide of the present invention or an amide or an ester or a salt thereof. And monoclonal antibodies.
An antibody against the polypeptide of the present invention can be produced by using the polypeptide of the present invention as an antigen according to a known antibody or antiserum production method.
[Preparation of monoclonal antibody]
(A) Preparation of monoclonal antibody-producing cells
The polypeptide of the present invention is administered to a warm-blooded animal itself or together with a carrier or 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 producing 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 therein. 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 polypeptide 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, and PEG is preferably used.
Examples of myeloma cells include myeloma cells of warm-blooded animals such as NS-1, P3U1, SP2 / 0, and AP-1, but P3U1 is preferably used. The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG1000 to PEG6000) is added at a concentration of about 10 to 80%. By incubating at 20 to 40 ° C, preferably 30 to 37 ° C for 1 to 10 minutes, cell fusion can be carried out efficiently. Various methods can be used to screen for monoclonal antibody-producing hybridomas. For example, a hybridoma culture supernatant is added to a solid phase (eg, a microplate) on which a polypeptide (protein) antigen is adsorbed directly or together with a carrier. Anti-immunoglobulin antibody (anti-mouse immunoglobulin antibody is used if the cell used for cell fusion is mouse) or protein A, and monoclonal antibody bound to the solid phase is detected. Method of adding a hybridoma culture supernatant to a solid phase to which an anti-immunoglobulin antibody or protein A has been adsorbed, adding a polypeptide labeled with a radioactive substance, an enzyme, or the like, and detecting a monoclonal antibody bound to the solid phase And so on.
The selection of the monoclonal antibody can be performed according to a known method or a method analogous thereto. Usually, it can be performed in a medium for animal cells to which HAT (hypoxanthine, aminopterin, thymidine) is added. As a selection and breeding medium, any medium can be used as long as a hybridoma can grow. For example, an RPMI 1640 medium containing 1 to 20%, preferably 10 to 20% fetal bovine serum, a GIT medium (Wako Pure Chemical Industries, Ltd.) containing 1 to 10% fetal bovine serum, or a serum-free medium for hybridoma culture (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.
(B) Purification of monoclonal antibody
Monoclonal antibodies can be separated and purified by known methods, for example, immunoglobulin separation and purification methods [eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, absorption by ion exchanger (eg, DEAE)]. Desorption method, ultracentrifugation method, gel filtration method, antigen-binding solid phase or specific purification method of collecting only the antibody with an active adsorbent such as protein A or protein G and dissociating the bond to obtain the antibody] it can.
[0035]
(Preparation of polyclonal antibody)
The polyclonal antibody of the present invention can be produced according to a known method or a method analogous thereto. For example, an immunizing antigen (polypeptide 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. The antibody can be produced by collecting an antibody-containing substance and separating and purifying the antibody.
Regarding a complex of an immunizing antigen and a carrier protein used to immunize a warm-blooded animal, the type of carrier protein and the mixing ratio of the carrier and the hapten are such that the antibody is efficiently cross-linked to the hapten immunized by cross-linking with the carrier. If possible, any material may be cross-linked at any ratio. For example, bovine serum albumin, bovine thyroglobulin, hemocyanin and the like are used in a weight ratio of about 0.1 to 20, preferably about 1 to 20, relative to hapten 1. A method of coupling at a rate of ~ 5 is used.
Various coupling agents can be used for coupling the hapten and the carrier, and glutaraldehyde, carbodiimide, a maleimide active ester, an active ester reagent containing a thiol group or a dithioviridyl group, or the like is used.
The condensation product is administered to a warm-blooded animal itself or together with a carrier or diluent at a site where antibody production is possible. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. The administration is usually performed once every about 2 to 6 weeks, about 3 to 10 times in total.
The polyclonal antibody can be collected from the blood, ascites, or the like, preferably from the blood of a warm-blooded animal immunized by the above method.
The measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the antiserum described above. The 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.
[0036]
A nucleotide sequence complementary to or substantially complementary to DNA encoding the polypeptide, receptor or partial peptide thereof of the present invention (hereinafter, these DNAs may be abbreviated as the DNA of the present invention), or The antisense polynucleotide (preferably DNA (hereinafter, these DNAs may be abbreviated as the antisense DNA of the present invention) sometimes) containing a part thereof is complementary to or substantially complementary to the DNA of the present invention. Any antisense polynucleotide (preferably, antisense DNA) may be used as long as it contains a nucleotide sequence complementary to or a part thereof and has an effect of suppressing the expression of the DNA.
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 the antisense nucleotide directed to translation inhibition in 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, initiation codon) An antisense nucleotide 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 the complementary strand of In the case of an antisense nucleotide directing RNA degradation by RNase H, it is at least about 70%, preferably at least about 80%, more preferably at least about 90%, most preferably the complementary strand of the entire nucleotide sequence of the DNA of the present invention including introns. Preferably, antisense nucleotides having about 95% or more homology are each suitable. For example, an antisense nucleotide (DNA) having a base sequence complementary to or substantially complementary to a base sequence of a DNA containing a base sequence encoding the polypeptide or the receptor of the present invention, or a part thereof, may be mentioned. Can be
The antisense nucleotide is usually composed of about 10 to 40, preferably about 15 to 30 bases.
The antisense DNA of the present invention can be produced using a known DNA synthesizer or the like.
The use of (a) the polypeptide of the present invention, (b) the DNA of the present invention, (c) the antibody of the present invention, and (d) the antisense DNA of the present invention will be described below.
[0037]
(1) An agent for treating or preventing various diseases related to the polypeptide of the present invention
The polypeptide of the present invention has a cell stimulating activity on cells expressing the receptor of the present invention (eg, GPR8, GPR7, rat TGR26, mouse TGR26, etc.) and is an endogenous ligand of the receptor of the present invention.
Therefore, when the polypeptide of the present invention or the DNA of the present invention is abnormal or defective, or when the receptor of the present invention or the DNA encoding the receptor is abnormal or defective, For example, there is a high possibility of weight gain. Therefore, the polypeptide of the present invention and the DNA of the present invention can be used, for example, as a weight gain inhibitor, a weight loss agent, a fat mass increase inhibitor, and a food intake inhibitor. In addition, for example, obesity [eg, malignant mastocytosis, exogenous obesity, hyperinsulin obesity, hyperplasmic obesity, hypoplasmic obesity, obesity exogenous obesity, hyperinsulin obesity, hyperplasmic obesity adipotency, hypoplasmic obesity, hypothyroid obesity, hypothalamic obesity, symptomatic obesity, pediatric obesity, obesity obesity, obesity obesity (Upper @ body @ obesity), Adipose obesity (hypogonadal obesity), systemic mastocytosis (simple obesity), simple obesity (central obesity treatment), etc.] Can be used as
[0038]
The polypeptide of the present invention and the DNA of the present invention can be obtained, for example, by administering the DNA of the present invention to a patient when the polypeptide of the present invention is reduced or deficient in a living body. (B) by inserting the DNA of the present invention into cells, expressing the polypeptide of the present invention, and then transplanting the cells into a patient by expressing the polypeptide of the present invention in the body; ) By administering the polypeptide of the present invention to the patient, the role of the polypeptide 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 therapeutic / prophylactic agent, the DNA may be inserted alone or into a suitable vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, etc. It can be administered to humans or warm-blooded animals. The DNA of the present invention can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as an auxiliary agent for promoting uptake, using a gene gun or a catheter such as a hydrogel catheter.
When the polypeptide of the present invention is used as the above-mentioned therapeutic / prophylactic agent, it should be purified to at least 90%, preferably 95% or more, more preferably 98% or more, and still more preferably 99% or more. Is preferred.
The polypeptide of the present invention can be, for example, sterilized with tablets or capsules, capsules, elixirs, microcapsules, or the like, optionally coated with sugar, or with water or other pharmaceutically acceptable liquids. It can be used parenterally (preferably subcutaneous administration) in the form of injections such as aqueous solutions or suspensions. For example, the polypeptides of the present invention can be combined with physiologically acceptable carriers, flavors, excipients, vehicles, preservatives, stabilizers, binders, and the like, in the form of unit dosages generally required for the practice of accepted pharmaceutical formulations. 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.
[0039]
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.
Examples of aqueous liquids for injection include 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 preparations obtained in this way are safe and have low toxicity, for example, human or warm-blooded animals (eg, rat, mouse, guinea pig, rabbit, bird, sheep, pig, cow, horse, cat, dog, monkey). , Etc.).
[0040]
The dose of the polypeptide of the present invention varies depending on the target disease, the subject of administration, the administration route, and the like. For example, when the polypeptide of the present invention is subcutaneously administered for the purpose of suppressing weight gain, generally the adult (60 kg) is used. )), The polypeptide is administered at about 0.1 mg to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day. In the case of other animals, the amount can be administered in terms of 60 kg.
[0041]
(2) Screening of drug candidate compounds for disease
Since the polypeptide of the present invention has a function as a ligand of the receptor of the present invention and the like, a compound or a salt thereof that promotes the activity or function of the polypeptide of the present invention includes, for example, a weight-gain suppressing effect, a weight-reducing effect, It has an action of suppressing an increase in fat mass, an effect of suppressing eating, and the like, and can be used as a low-toxicity and safe weight gain inhibitor, a weight loss agent, a fat mass increase inhibitor, an eating inhibitor, and the like.
On the other hand, a compound or a salt thereof that inhibits the activity or function of the polypeptide of the present invention has, for example, a body weight increasing effect, and is useful as a low toxicity and safe weight gaining agent. Screening for a compound or a salt thereof that promotes or inhibits the activity or function of the polypeptide of the present invention uses the polypeptide of the present invention or constructs a recombinant expression system of the polypeptide of the present invention. A receptor binding assay system using is used. By this screening, a compound that changes the binding property between the polypeptide of the present invention and its receptor (a compound that promotes or inhibits the activity of the polypeptide of the present invention) (for example, a peptide, protein, non-peptidic compound, synthetic compound) , Fermentation products, etc.) or salts thereof can also be screened. Such compounds may have cell stimulating activity (eg, arachidonic acid release, acetylcholine release, intracellular Ca²⁺Release, Intracellular cAMP production / suppression, Intracellular cGMP production, Inositol phosphate production, Cell membrane potential fluctuation, Intracellular protein phosphorylation, c-fos activation, pH reduction, GTPγS binding activity, etc. ), The compound having no cell stimulating activity (ie, the receptor antagonist of the present invention), and the like. “Altering the binding to the polypeptide of the present invention” includes both cases of inhibiting the binding to the polypeptide of the present invention and promoting the binding to the polypeptide of the present invention. .
[0042]
Specific examples of the method for screening a compound or a salt thereof that promotes or inhibits the activity of the polypeptide of the present invention, which comprises using the polypeptide of the present invention, include (i) the receptor of the present invention or a partial peptide thereof ( Hereinafter, these may be simply referred to as the receptor of the present invention) and the polypeptide of the present invention in contact with (ii) the receptor of the present invention and the test compound of the present invention. A compound that changes the binding property between the polypeptide of the present invention and the receptor of the present invention (compound that promotes or inhibits the activity of the polypeptide of the present invention) or a compound thereof. A method for screening a salt may be used.
In the above-mentioned screening method, the polypeptide of the present invention and the test compound are used in (i) the case where the polypeptide of the present invention is brought into contact with the above-mentioned receptor of the present invention and (ii) the above-mentioned receptor of the present invention. For example, the amount of ligand binding to the receptor of the present invention, the cell stimulating activity, and the like in the case of contact are measured and compared.
[0043]
As further specific examples of the above screening method,
(A) When the labeled polypeptide of the present invention is brought into contact with the receptor of the present invention described above, and when the labeled polypeptide of the present invention and the test compound are brought into contact with the receptor of the present invention, Measuring the amount of binding of the labeled polypeptide of the present invention to the receptor of the present invention, and comparing the measured amount with the compound of the present invention which alters the binding between the polypeptide of the present invention and the receptor of the present invention (the present invention). Screening method for a compound that promotes or inhibits the activity of the polypeptide of the invention) or a salt thereof,
(B) When the labeled polypeptide of the present invention is brought into contact with a cell containing the receptor of the present invention or a membrane fraction of the cell, the labeled polypeptide of the present invention and the test compound are compared with the present invention. Measuring the amount of binding of the labeled polypeptide of the present invention to the cell or the membrane fraction when the cell is contacted with a cell containing the receptor of the present invention or the membrane fraction of the cell, and comparing the measured amounts. A method for screening a compound that changes the binding property between the polypeptide of the present invention and the receptor of the present invention (a compound that promotes or inhibits the activity of the polypeptide of the present invention) or a salt thereof,
(C) contacting the labeled polypeptide of the present invention with a receptor of the present invention expressed on a cell membrane by culturing a transformant containing a DNA encoding the receptor of the present invention; When the labeled polypeptide of the present invention and a test compound are brought into contact with the receptor of the polypeptide of the present invention expressed on the cell membrane by culturing a transformant containing the DNA encoding the receptor of the present invention. Measuring the amount of binding of the labeled polypeptide of the present invention to the receptor of the present invention, and comparing the measured results with the compound that alters the binding between the polypeptide of the present invention and the receptor of the present invention ( Screening method for a compound that promotes or inhibits the activity of the polypeptide of the present invention) or a salt thereof,
[0044]
(D) when a compound that activates the receptor of the present invention (for example, the polypeptide of the present invention) is brought into contact with a cell containing the receptor of the present invention, a compound that activates the receptor of the present invention, and When a test compound is brought into contact with a cell containing the receptor of the present invention, cell stimulating activity via the receptor of the present invention (for example, arachidonic acid release, acetylcholine release, intracellular Ca²⁺Activity to promote release, intracellular cAMP production / inhibition, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH reduction, GTPγS binding activity, etc. A compound that changes the binding property between the polypeptide of the present invention and the receptor of the present invention (a compound that promotes or inhibits the activity of the polypeptide of the present invention), which is characterized by measuring and comparing the inhibitory activity and the like. Or a method for screening for a salt thereof, and
(E) a book expressing a compound that activates the receptor of the present invention (eg, a polypeptide of the present invention) on a cell membrane by culturing a transformant containing a DNA encoding the receptor of the present invention; When brought into contact with the receptor of the present invention, a compound that activates the receptor of the present invention and a test compound are expressed on a cell membrane by culturing a transformant containing a DNA encoding the receptor of the present invention. Cell contact stimulating activity (e.g., arachidonic acid release, acetylcholine release, intracellular Ca²⁺Activity to promote release, intracellular cAMP production / inhibition, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH reduction, GTPγS binding activity, etc. A compound that changes the binding property between the polypeptide of the present invention and the receptor of the present invention (a compound that promotes or inhibits the activity of the polypeptide of the present invention), which is characterized by measuring and comparing the inhibitory activity and the like. Or a method for screening a salt thereof.
Preferred specific examples of the labeled polypeptide of the present invention include radioisotopes (eg, [¹²⁵I], [¹³¹I], [³H], [¹⁴C], etc.), fluorescent substances [eg, cyanine fluorescent dyes (eg, Cy2, Cy3, Cy5, Cy5.5, Cy7 (manufactured by Amersham Bioscience)), enzymes such as fluorescamine, fluorescein isothiocyanate, etc. Example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase, etc., luminescent substance (eg, luminol, luminol derivative, luciferin, lucigenin, etc.) or a sequence number labeled with a lanthanide element or the like 149, SEQ ID NO: 16, SEQ ID NO: 6, SEQ ID NO: 17, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 91 or SEQ ID NO: 92 And a polypeptide having the same amino acid sequence. Preferably〔¹²⁵I], a polypeptide having the amino acid sequence represented by SEQ ID NO: 149, and the like.
[0045]
The specific description of the above screening method is as follows.
First, the receptor of the present invention used in the screening method of the present invention may be any receptor as long as it recognizes the polypeptide of the present invention as a ligand. Fractions and the like are preferred. However, since it is particularly difficult to obtain human-derived organs, the receptor of the present invention and the like, which is expressed in large amounts using recombinants, is suitable for screening.
In order to produce the receptor of the present invention, the aforementioned method for producing the polypeptide of the present invention and the like are used.
When using the cells containing the receptor of the present invention or the cell membrane fraction in the screening method of the present invention, the preparation method described below may be used.
When a cell containing the receptor of the present invention is used, the cell may be fixed with glutaraldehyde, formalin, or the like. The immobilization method can be performed according to a known method.
The cell containing the receptor of the present invention refers to a host cell expressing the receptor of the present invention. Examples of the host cell include the aforementioned Escherichia coli, Bacillus subtilis, yeast, insect cells, animal cells, and the like. . The host cells expressing the receptor of the present invention include the same method as the above-mentioned method for producing a transformant transformed with the expression vector containing the polypeptide of the present invention.
The membrane fraction refers to a fraction containing a large amount of cell membrane obtained by a known method after crushing cells. As a method for crushing the cells, a method of crushing the cells with a Potter-Elvehjem type homogenizer, crushing with a Waring blender or a polytron (manufactured by Kinematica), crushing with an ultrasonic wave, or ejecting the cells from a thin nozzle while applying pressure by a French press or the like. Crushing and the like. For fractionation of cell membranes, fractionation by centrifugal force such as fractionation centrifugation or density gradient centrifugation is mainly used. For example, the cell lysate is centrifuged at a low speed (500 rpm to 3000 rpm) for a short time (usually about 1 minute to 10 minutes), and the supernatant is further centrifuged at a high speed (15000 rpm to 30000 rpm) for usually 30 minutes to 2 hours. The precipitate is the membrane fraction. The membrane fraction is rich in the expressed receptor of the present invention and membrane components such as cell-derived phospholipids and membrane proteins.
The amount of the receptor of the present invention in the cell or membrane fraction containing the receptor of the present invention is 10 per cell.³-10⁸Preferably a molecule⁵-10⁷Preferably it is a molecule. The higher the expression level, the higher the ligand binding activity (specific activity) per membrane fraction, which makes it possible not only to construct a highly sensitive screening system, but also to measure a large number of samples in the same lot. .
[0046]
To carry out the above (a) to (c) for screening a compound that changes the binding property between the polypeptide of the present invention and the receptor of the present invention (a compound that promotes or inhibits the activity of the polypeptide of the present invention) For this, an appropriate receptor fraction of the present invention and a labeled polypeptide of the present invention are used. The receptor fraction of the present invention is preferably a natural receptor fraction of the present invention or a recombinant receptor fraction of the present invention having an activity equivalent thereto. Here, the equivalent activity indicates an equivalent ligand binding activity and the like. Labeled polypeptides include, for example, radioisotopes (eg, [¹²⁵I], [¹³¹I], [³H], [¹⁴C], etc.), fluorescent substances [eg, cyanine fluorescent dyes (eg, Cy2, Cy3, Cy5, Cy5.5, Cy7 (manufactured by Amersham Bioscience)], fluorescamine, fluorescein isothiocyanate, etc.), fluoresca Min, fluorescein isothiocyanate, etc.), enzymes (eg, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase, etc.), luminescent substances (eg, luminol, luminol derivatives, luciferin, lucigenin, etc.) Or a polypeptide labeled with a lanthanide element or the like. Of these, [¹²⁵I].
Specifically, to screen for a compound that alters the binding between the polypeptide of the present invention and the receptor of the present invention, first, a cell or a membrane fraction of the cell containing the receptor of the present invention is screened. A sample of the receptor is prepared by suspending in a buffer suitable for the above. The buffer may be any buffer such as a phosphate buffer having a pH of 4 to 10 (preferably pH 6 to 8) or a buffer such as Tris-HCl buffer, which does not inhibit the binding between the ligand and the receptor. Further, in order to reduce non-specific binding, CHAPS, Tween-80 are used.^TMSurfactants such as (Kao-Atlas), digitonin and deoxycholate can also be added to the buffer. Furthermore, a protease inhibitor such as PMSF, leupeptin, E-64 (manufactured by Peptide Research Institute), pepstatin and the like can be added for the purpose of suppressing the degradation of the receptor of the present invention and the polypeptide of the present invention by the protease. A fixed amount (5000 cpm to 500,000 cpm) of the labeled polypeptide of the present invention is added to 0.01 ml to 10 ml of the receptor solution, and 10^-10-10^-7M test compounds are allowed to coexist. A reaction tube containing a large excess of the unlabeled polypeptide of the present invention is also prepared to determine the non-specific binding amount (NSB). The reaction is carried out at 0 ° C. to 50 ° C., preferably 4 ° C. to 37 ° C., for 20 minutes to 24 hours, preferably 30 minutes to 3 hours. After the reaction, the mixture is filtered with a glass fiber filter or the like, washed with an appropriate amount of the same buffer, and the radioactivity remaining on the glass fiber filter is measured with a liquid scintillation counter or a γ-counter. Count when there is no antagonist (B₀) Minus the amount of non-specific binding (NSB) (B₀-NSB) as 100%, a test compound having a specific binding amount (B-NSB) of, for example, 50% or less can be selected as a candidate substance having competitive inhibitory ability.
[0047]
The above-mentioned methods (d) to (e) for screening a compound that changes the binding property between the polypeptide of the present invention and the receptor of the present invention (a compound that promotes or inhibits the activity of the polypeptide of the present invention) are performed. To do so, the cell stimulating activity via the receptor of the present invention (eg, arachidonic acid release, acetylcholine release, intracellular Ca²⁺Activity to promote release, intracellular cAMP production / inhibition, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH reduction, GTPγS binding activity, etc. Inhibitory activity) can be measured using a known method or a commercially available measurement kit. Specifically, first, cells containing the receptor of the present invention are cultured in a multiwell plate or the like. Prior to screening, the cells were exchanged with a fresh medium or an appropriate buffer that was not toxic to the cells in advance, and the test compounds were added and incubated for a certain period of time. The product is quantified according to the respective method. When the production of a substance (for example, arachidonic acid) as an indicator of cell stimulating activity is difficult due to a degrading enzyme contained in a cell, an assay may be performed by adding an inhibitor to the degrading enzyme. In addition, the activity such as cAMP production suppression can be detected as a production suppression effect on cells whose basic production has been increased by forskolin or the like.
In order to perform screening by measuring cell stimulating activity, cells expressing an appropriate receptor of the present invention are required. As the cells expressing the receptor of the present invention, the above-mentioned cell lines expressing the receptor of the present invention are desirable.
Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like.
[0048]
A kit for screening a compound that changes the binding property between the polypeptide of the present invention and the receptor of the present invention (a compound that promotes or inhibits the activity of the polypeptide of the present invention) or a salt thereof is provided by the receptor of the present invention or a salt thereof. A salt, a partial peptide of the receptor of the present invention or a salt thereof, a cell containing the receptor of the present invention, or a membrane fraction of a cell containing the receptor of the present invention, and a polypeptide containing the polypeptide of the present invention. is there.
Examples of the screening kit of the present invention include the following.
1. Screening reagent
(A) Measurement buffer and washing buffer
Hanks' Balanced Salt Solution (manufactured by Gibco) plus 0.05% bovine serum albumin (manufactured by Sigma).
The solution may be sterilized by filtration through a 0.45 μm filter and stored at 4 ° C., or may be prepared at use.
(B) Receptor preparation of the present invention
CHO cells expressing the receptor of the present invention were placed in a 12-well plate at 5 × 10 5⁵Passage at 37 ° C, 5% CO₂, Cultured at 95% air for 2 days.
(C) labeled polypeptide of the invention
[³H], [¹²⁵I], [¹⁴C], [³⁵S] or the like, and the polypeptide of the present invention dissolved in an appropriate solvent or buffer is stored at 4 ° C. or −20 ° C., and diluted to 1 μM with a measuring buffer before use.
(D) Standard polypeptide solution of the present invention
The polypeptide of the present invention is dissolved to a concentration of 1 mM in PBS containing 0.1% bovine serum albumin (manufactured by Sigma) and stored at -20 ° C.
[0049]
2. Measurement method
(A) The cells expressing the receptor of the present invention cultured on a 12-well tissue culture plate are washed twice with 1 ml of the measurement buffer, and then 490 μl of the measurement buffer is added to each well.
(B) 10^-3-10^-10After adding 5 μl of the M test compound solution, 5 μl of the labeled peptide of the present invention is added, and reacted at room temperature for 1 hour. To determine the amount of non-specific binding, 10^-3Add 5 μl of M polypeptide of the invention.
(C) Remove the reaction solution and wash three times with 1 ml of washing buffer. The labeled polypeptide of the present invention bound to the cells is dissolved in 0.2N NaOH-1% SDS, and mixed with 4 ml of liquid scintillator A (manufactured by Wako Pure Chemical Industries, Ltd.).
(D) The radioactivity is measured using a liquid scintillation counter (manufactured by Beckman), and Percent {Maximum} Binding (PMB) is determined by the following equation (Equation 1).
[Equation 1]
PMB = [(B-NSB) / (B₀−NSB)] × 100
PMB: Percent Maximum Maximum Binding
B: Value when the sample is added
NSB: Non-specific binding (non-specific binding amount)
B₀: Maximum binding amount
[0050]
A compound or a salt thereof obtained by using the above-described screening method or screening kit is a compound that changes (inhibits or promotes the binding) the binding of the polypeptide of the present invention to the receptor of the present invention (the polypeptide of the present invention). A compound that promotes or inhibits the activity of the compound of the present invention, specifically, a compound having a cell stimulating activity via the receptor of the present invention or a salt thereof (a so-called receptor agonist of the present invention), or does not have the stimulating activity. A compound (a so-called receptor antagonist of the present invention). Examples of the compound include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, and the like, and these compounds may be novel compounds or known compounds.
The specific method for evaluating whether the receptor agonist or antagonist of the present invention is described above may be in accordance with the following (i) or (ii).
(I) A compound that changes the binding (particularly, inhibits the binding) between the polypeptide of the present invention and the receptor of the present invention by performing the binding assay shown in the screening methods (a) to (c) above. After obtaining the above, it is determined whether or not the compound has the above-described receptor-mediated cell stimulating activity of the present invention. A compound having a cell stimulating activity or a salt thereof is a receptor agonist of the present invention, and a compound having no such activity or a salt thereof is a receptor antagonist of the present invention.
(Ii) (a) A test compound is brought into contact with a cell containing the receptor of the present invention, and the cell stimulating activity via the receptor of the present invention is measured. The compound having a cell stimulating activity or a salt thereof is the receptor agonist of the present invention.
(B) a case where a compound that activates the receptor of the present invention (for example, the polypeptide of the present invention or the receptor agonist of the present invention) is brought into contact with cells containing the receptor of the present invention; When a compound that activates the receptor and a test compound are brought into contact with cells containing the receptor of the present invention, the cell-stimulating activities mediated by the receptor of the present invention are measured and compared. A compound capable of decreasing the cell stimulating activity of the compound activating the receptor of the present invention or a salt thereof is the receptor antagonist of the present invention.
Since the above-mentioned receptor agonist of the present invention has the same activity as the physiological activity of the polypeptide of the present invention on the receptor of the present invention, it has the same effect as the polypeptide of the present invention in suppressing weight gain and weight loss. It has an action, an effect of suppressing an increase in fat mass, an effect of suppressing eating, and the like, and can be used as a safe and low toxic body weight increase inhibitor, a weight loss agent, a fat mass increase inhibitor, an eating inhibitor, and the like.
Since the above-mentioned receptor antagonist of the present invention can suppress the physiological activity of the polypeptide of the present invention for the receptor of the present invention, it can be used as a safe and low-toxic body weight increasing agent.
[0051]
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 , Plasma, etc., which promote or inhibit the function of the polypeptide of the present invention.
As the salt of the compound, those similar to the aforementioned salts of the polypeptide of the present invention are used.
When a compound obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned therapeutic / prophylactic agent, it can be carried out according to conventional means. For example, tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions, and the like can be prepared in the same manner as in the above-mentioned drug containing the polypeptide of the present invention.
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.
The dose of the compound or a salt thereof varies depending on its action, target disease, subject of administration, administration route, and the like.For example, a compound that promotes the activity and function of the polypeptide of the present invention for the purpose of suppressing weight gain may be used. When administered subcutaneously, generally for an adult (per 60 kg body weight), the compound is administered in an amount of about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day. . In the case of other animals, the amount can be administered in terms of 60 kg.
In addition, for example, when a compound that inhibits the activity or function of the polypeptide of the present invention is administered subcutaneously for the purpose of increasing body weight, generally, in an adult (per 60 kg body weight), the compound is administered in an amount of about 0.1 per day. -100 mg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg. In the case of other animals, the amount can be administered in terms of 60 kg.
[0052]
(3) Quantification of the polypeptide of the present invention
An antibody against the polypeptide of the present invention (hereinafter sometimes abbreviated as the antibody of the present invention) can specifically recognize the polypeptide of the present invention. It can be used for quantification, particularly for quantification by sandwich immunoassay.
That is, the present invention
(I) reacting the antibody of the present invention with a test solution and a labeled polypeptide of the present invention competitively, and measuring the ratio of the labeled polypeptide of the present invention bound to the antibody; A method for quantifying the polypeptide of the present invention in a test solution,
(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 polypeptide of the present invention in a test solution is provided.
In the quantification method (ii), one antibody may be an antibody that recognizes the N-terminal of the polypeptide of the present invention, and the other antibody may be an antibody that reacts with the C-terminal of the polypeptide of the present invention. desirable.
In addition, the polypeptide of the present invention can be quantified using a monoclonal antibody against the polypeptide 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 polypeptide of the present invention using the antibody of the present invention is not particularly limited, and may be an antibody, an antigen, or an antibody-antigen complex corresponding to the amount of the antigen (eg, the amount of the polypeptide) in the liquid to be measured. Any measurement method may be used as long as the amount of the body 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.
[0053]
As a labeling agent used in a measuring method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, a lanthanide element and the like are used. As the radioisotope, for example, [¹²⁵I], [¹³¹I], [³H], [¹⁴C] is used. As the above-mentioned enzyme, a stable enzyme having a large specific activity is preferable. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, a luminol derivative, luciferin, lucigenin and the like are used. Further, a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.
For the insolubilization of an antigen or an antibody, physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing and immobilizing a polypeptide 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 polypeptide 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.
[0054]
In the method for measuring the polypeptide of the present invention by the sandwich method of the present invention, the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction is preferably an antibody having a different binding site to the polypeptide of the present invention. Can be That is, the antibody used in the primary reaction and the secondary reaction is preferably used, for example, when the antibody used in the secondary reaction recognizes the C-terminal of the polypeptide of the present invention. Is an antibody that recognizes other than the C-terminal, for example, the N-terminal.
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.
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. What is necessary is just to construct the measuring system of the polypeptide of the present invention 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, "Radio immunoassay" edited by Hiroshi Irie (Kodansha, published in 1974), "Radio immunoassay continued" edited by Hiroshi Irie (published in 1974), "Enzyme immunoassay" edited by Eiji Ishikawa et al. (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 ENZY MOLOGY" Vol. 70 (Immunochemical Techniques (Part A)), ibid. Vol. # 73 (Immunochemical @ Techniques (Part @ B)), ibid., Vol. $ 74 (Immunochemical @ Techniques (Part @ C)), ibid., Vol. 84 (Immunochemical Techniques (Part D): Selected 同 Immunoassays), ibid. No. 92 (Immunochemical Techniques (Part E): Monoclonal Antibodies and General Immunoassay Methods), ibid. Vol. $ 121 (Immunochemical Techniques (Part I): Hybridoma Technology and Monoclonal Antibodies) (above, published by Academic Press) can be referred to.
As described above, the polypeptide of the present invention can be quantified with high sensitivity by using the antibody of the present invention.
[0055]
Furthermore, when a decrease in the concentration of the polypeptide of the present invention is detected by quantifying the concentration of the polypeptide of the present invention using the antibody of the present invention, for example, weight gain or fat mass increase, or It can be diagnosed that there is a high possibility that weight or fat mass will increase in the future.
In addition, when an increase in the concentration of the polypeptide of the present invention is detected, for example, it can be diagnosed that the disease is a disease such as weight loss or the likelihood of becoming ill in the future is high.
Further, the antibody of the present invention can be used for detecting the polypeptide of the present invention present in a subject such as a body fluid or a tissue. In addition, preparation of an antibody column used for purifying the polypeptide of the present invention, detection of the polypeptide of the present invention in each fraction at the time of purification, analysis of the behavior of the polypeptide of the present invention in test cells, etc. Can be used for
[0056]
(4) Gene diagnostics
The DNA of the present invention can be used, for example, in a human or warm-blooded animal (eg, rat, mouse, guinea pig, rabbit, bird, sheep, pig, cow, horse, cat, dog, monkey, etc.) by using it as a probe. Since abnormality (gene abnormality) of DNA or mRNA encoding the polypeptide of the present invention can be detected, for example, damage, mutation or decreased expression of the DNA or mRNA, and increase or excessive expression of the DNA or mRNA It is useful as a diagnostic agent for genes.
The above-described genetic diagnosis using the DNA of the present invention can be carried out, for example, by the known Northern hybridization or PCR-SSCP method (Genomics, Vol. 5, pp. 874-879 (1989), Processings of the The National Academy of Sciences of USA (Proceedings of the National Academy of Sciences of the United States States of America), Vol. 86, pp. 2766-2770 (1989), and the like.
For example, when a decrease in expression is detected by Northern hybridization, for example, it can be diagnosed that there is a high possibility that weight gain or fat mass is increased or that there is a high possibility that weight gain or fat mass will increase in the future. .
When excessive expression is detected by Northern hybridization, for example, it can be diagnosed that the possibility of weight loss or the like or the possibility of weight loss in the future is high.
[0057]
(5) A medicine containing the antisense DNA of the present invention
The antisense DNA of the present invention can suppress the expression of the polypeptide or the receptor of the present invention, and can be used, for example, as a weight gain agent.
For example, when using the antisense DNA, the antisense DNA can be used alone or after being inserted into an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, and the like, followed by a conventional method. The antisense DNA can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as an adjuvant for promoting uptake, and can be administered using a gene gun or a catheter such as a hydrogel catheter.
Furthermore, the antisense DNA can also be used as a diagnostic oligonucleotide probe for examining the presence of the DNA of the present invention in tissues or cells and the state of expression thereof.
[0058]
(6) A medicine containing the antibody of the present invention
The antibody of the present invention having the action of neutralizing the polypeptide of the present invention can be used, for example, as a weight gain agent.
The agent for the above-mentioned diseases containing the antibody of the present invention may be used as it is as a liquid or as a pharmaceutical composition in an appropriate dosage form, in humans or mammals (eg, rat, rabbit, sheep, pig, cow, cat, dog, monkey). Etc.) can be administered orally or parenterally. The dose varies depending on the administration subject, target disease, symptoms, administration route and the like. For example, when used for suppressing weight gain in adults, the antibody of the present invention is usually used in a dose of 0.01%. About 20 to about 20 mg / kg body weight, preferably about 0.1 to 10 mg / kg body weight, more preferably about 0.1 to 5 mg / kg body weight, about 1 to 5 times a day, preferably about 1 to 3 times a day. It is conveniently administered by intravenous injection. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If the symptoms are particularly severe, the dose may be increased according to the symptoms.
The antibodies of the present invention can be administered as such or as a suitable pharmaceutical composition. The pharmaceutical composition used for the above administration contains the above or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient. Such compositions are provided in dosage forms suitable for oral or parenteral administration.
That is, for example, compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (soft capsules and the like). Syrup, emulsion, suspension and the like. Such a composition is produced by a known method and contains a carrier, diluent or excipient commonly used in the field of pharmaceuticals. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.
[0059]
As the composition for parenteral administration, for example, injections, suppositories, etc. are used, and the injections are in the form of intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, intravenous injections and the like. Is included. Such injections are prepared according to a known method, for example, by dissolving, suspending or emulsifying the antibody or a salt thereof in a sterile aqueous or oily liquid commonly used for injections. As an aqueous liquid for injection, for example, physiological saline, isotonic solution containing glucose and other auxiliary agents and the like are used, and suitable solubilizing agents, for example, alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) add-on of hydrogenated castor oil)) and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil, and the like are used, and benzyl benzoate, benzyl alcohol, and the like may be used in combination as a solubilizing agent. The prepared injection is usually filled in an appropriate ampoule. Suppositories to be used for rectal administration are prepared by mixing the above antibody or a salt thereof with a conventional suppository base.
The above-mentioned oral or parenteral pharmaceutical composition is conveniently prepared in a unit dosage form so as to be compatible with the dosage of the active ingredient. Examples of such dosage unit dosage forms include tablets, pills, capsules, injections (ampoules), suppositories, and the like. Usually, each dosage unit dosage form has a dosage of 5 to 500 mg, especially 5 to 100 mg for an injection. Other dosage forms preferably contain 10 to 250 mg of the above antibody.
Each of the above-mentioned compositions may contain another active ingredient as long as the composition does not cause an undesirable interaction with the antibody.
[0060]
(7) DNA transfer animal
Non-human mammals having a foreign DNA encoding the polypeptide of the present invention (hereinafter abbreviated as the foreign DNA of the present invention) or a mutant DNA thereof (sometimes abbreviated as the foreign mutant DNA of the present invention) They are also used for screening weight gain inhibitors, weight loss agents, fat gain inhibitors, food intake inhibitors and the like.
Non-human mammals having the exogenous DNA of the present invention or the mutant DNA thereof (hereinafter abbreviated as the DNA-transferred animal of the present invention) can be used for non-fertilized eggs, fertilized eggs, germ cells including spermatozoa and their progenitor cells, and the like. And preferably at the stage of embryonic development in non-human mammal development (more preferably at the stage of single cells or fertilized eggs and generally before the 8-cell stage), the calcium phosphate method, the electric pulse method, the lipofection method, the agglutination method, It can be produced by 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 known cell fusion method.
As the non-human mammal, for example, cow, pig, sheep, goat, rabbit, dog, cat, guinea pig, hamster, mouse, rat and the like are used. Among them, rodents, which are relatively short in ontogeny and biological cycle in terms of preparation of disease animal model systems, and are easy to breed, especially mice (for example, hybrids such as C57BL / 6 strain and DBA2 strain as pure strains) B6C3F as a system₁System, BDF₁System, B6D2F₁Strain, 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.
[0061]
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 refers to a DNA that expresses an abnormal polypeptide of the present invention, and for example, a DNA that expresses a polypeptide that suppresses the function of the normal polypeptide 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.
[0062]
Examples of the expression vector of the polypeptide 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, animals such as vaccinia virus or baculovirus. A virus or the like is used. Among them, a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis or a plasmid derived from yeast is preferably used.
Examples of the promoter for controlling the DNA expression include, for example, (a) a promoter of a DNA derived from a virus (eg, simian virus, cytomegalovirus, Moloney leukemia virus, JC virus, breast cancer virus, poliovirus, etc.); ) Promoters derived from various mammals (human, rabbit, dog, cat, guinea pig, hamster, rat, mouse, etc.), for example, albumin, insulin II, uroplakin II, elastase, erythropoietin, endothelin, muscle creatine kinase, glial fibrillary acid Protein, glutathione S-transferase, platelet-derived growth factor β, keratins K1, K10 and K14, collagen type I and type II, cyclic AMP-dependent protein kinase βI subunit, dystrophy Fin, tartrate-resistant alkaline phosphatase, atrial natriuretic factor, endothelial receptor tyrosine kinase (commonly abbreviated as Tie2), sodium potassium adenosine 3 kinase (Na, K-ATPase), neurofilament light chain, metallothionein I and IIA, metalloproteinase 1 tissue inhibitor, MHC class I antigen (H-2L), H-ras, renin, dopamine β-hydroxylase, thyroid peroxidase (TPO), polypeptide chain elongation factor 1α (EF-1α), β Actin, α and β myosin heavy chain, myosin light chain 1 and 2, myelin basic protein, thyroglobulin, Thy-1, immunoglobulin, heavy chain variable region (VNP), serum amyloid P component, myoglobin, troponin C, smooth muscle Actin, pre-pro-enkephalin A, such as a promoter, such as vasopressin is used. Among them, a cytomegalovirus promoter capable of high expression throughout the body, a promoter of human polypeptide chain elongation factor 1α (EF-1α), human and chicken β-actin promoters, and the like are preferable.
The vector preferably has a sequence that terminates transcription of 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.
[0063]
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 polypeptide of the present invention includes DNA derived from liver, kidney, thyroid cell, fibroblast derived from human or various mammals (eg, rabbit, dog, cat, guinea pig, hamster, rat, mouse, etc.) and All or part of the genomic DNA can be obtained from various commercially available genomic DNA libraries, or complementary DNA prepared by known methods from liver, kidney, thyroid cells, and fibroblast-derived RNA can be obtained as a raw material. In addition, a translation region obtained by mutating a translation region of a normal polypeptide obtained from the above-mentioned cells or tissues by a point mutagenesis method can be used for the exogenous abnormal DNA.
The translation region can be prepared as a DNA construct that can be expressed in a 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.
[0064]
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 eventually promotes the function of the endogenous normal DNA, thereby ultimately resulting in hyperactivity of the polypeptide of the present invention. May be developed and can be used as a disease model animal. For example, using the normal DNA transgenic animal of the present invention, elucidation of the hyperfunction of the polypeptide of the present invention and the pathological mechanism of a disease associated with the polypeptide of the present invention, and examination of a method for treating these diseases. It is possible.
In addition, since the mammal into which the exogenous normal DNA of the present invention has been transferred has an increased symptom of the released polypeptide of the present invention, it is also used for screening tests for therapeutic drugs for diseases related to the polypeptide of the present invention. It is possible.
On the other hand, a non-human mammal having the exogenous abnormal DNA of the present invention can be subcultured in an ordinary breeding environment as an animal having the DNA after confirming that the exogenous DNA is stably maintained by mating. Furthermore, the desired foreign DNA can be incorporated into the above-described plasmid and used as a source material. The DNA construct with the promoter can be prepared by a usual DNA engineering technique. 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.
[0065]
The non-human mammal having the abnormal DNA of the present invention has a high level of expression of the abnormal DNA of the present invention, and eventually inhibits the function of the polypeptide of the present invention by inhibiting the function of endogenous normal DNA. It may cause type refractory disease and can be used as a disease state model animal. 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 polypeptide of the present invention and to examine a method for treating this disease.
In addition, as a specific possibility, an animal in which the abnormal DNA of the present invention is highly expressed is characterized in that the abnormal polypeptide of the present invention inhibits the function of a normal polypeptide by the abnormal polypeptide of the present invention (dominant negative) Action).
In addition, since the mammal to which the foreign abnormal DNA of the present invention has been transferred has an increased symptom of the released polypeptide of the present invention, it can be used in a therapeutic drug screening test for the polypeptide of the present invention or a functionally inactive type refractory disease. Is also available.
In addition, other possible uses of the above two DNA-transferred animals of the present invention include, for example,
(A) use as a cell source for tissue culture;
(B) Expression or activation specifically by the polypeptide of the present invention by directly analyzing DNA or RNA in the tissue of the DNA-transferred animal of the present invention or by analyzing the polypeptide tissue expressed by the DNA Analysis of the relationship with the polypeptide
(C) culturing cells of DNA-bearing tissue by standard tissue culture techniques and using them to study the function of cells from tissues that are generally difficult to culture;
(D) screening for an agent that enhances the function of the cell by using the cell described in (c) above, and
(E) Isolation and purification of the mutant polypeptide of the present invention and production of its antibody can be considered.
Furthermore, using the DNA-transferred animal of the present invention, it is possible to examine clinical symptoms of a disease associated with the polypeptide of the present invention, including a functionally inactive refractory disease of the polypeptide of the present invention. More detailed pathological findings in each organ of the disease model related to the polypeptide of the present invention 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 polypeptide-producing cell of the present invention, its relationship with apoptosis, differentiation or proliferation, or the mechanism of signal transduction in them, to investigate their abnormalities, etc., and the polypeptide of the present invention and its action. It is an effective research material for elucidation.
Furthermore, in order to develop a therapeutic agent for a disease associated with the polypeptide of the present invention, including a functionally inactive refractory type of the polypeptide 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 the therapeutic agent for the disease by using the method and the quantitative method. 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 a disease associated with the polypeptide of the present invention.
[0066]
(8) Knockout animal
The non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated and the non-human mammal deficient in expressing the DNA of the present invention are also screened for weight gain inhibitors, weight loss agents, fat mass increase inhibitors, and food intake inhibitors. Used to
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 to suppress the DNA expression ability, or By substantially eliminating the activity of the polypeptide of the present invention encoded by the DNA, the DNA does not substantially have the ability to express the polypeptide of the present invention (hereinafter referred to as the knockout DNA of the present invention). A) embryonic stem cells of non-human mammals (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.
[0067]
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). The exon function is destroyed by inserting a reporter gene or the like represented by a transferase gene), or a DNA sequence (for example, a poly-A addition signal) that terminates transcription of the gene is inserted into an intron between exons. Inability to synthesize complete messenger RNA Thus, a DNA strand having a DNA sequence constructed so as to eventually disrupt the gene (hereinafter abbreviated as a targeting vector) is introduced into the chromosome of the animal by, for example, homologous recombination, and the obtained ES cell PCR using a DNA sequence on or near the DNA of the present invention as a probe and a DNA sequence on a targeting vector and PCR using a DNA sequence of a nearby region other than the DNA of the present invention used for the preparation of the targeting vector as a primer It can be obtained by analyzing by the method and selecting the knockout ES cells of the present invention.
As the original ES cells for inactivating the DNA of the present invention by the homologous recombination method or the like, for example, those already established as described above may be used, or according to the known method of Evans and Kaufman. May be newly established. For example, in the case of mouse ES cells, currently, 129-line ES cells are generally used. For example, for the purpose of obtaining ES cells in which BDF is clearly known, for example, BDF in which the number of eggs collected by C57BL / 6 mice or C57BL / 6 has been improved by hybridization with DBA / 2₁Mouse (F of C57BL / 6 and DBA / 2)₁) Can also be used favorably. BDF₁In 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.
[0068]
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.⁶Since 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 (1-10000 U / ml) in a carbon dioxide incubator (preferably, 5% carbon dioxide, 95% air or 5% oxygen, 5% The cells are cultured by a method such as culturing at about 37 ° C. in carbon dioxide gas and 90% air. At the time of subculture, for example, a trypsin / EDTA solution (usually 0.001-0.5% trypsin / 0.1-5 mM EDTA, preferably Is performed by treating the cells into single cells by treatment with about 0.1% trypsin / 1 mM EDTA, and seeding the cells on newly prepared feeder cells. Such subculture is usually performed every 1 to 3 days. At this time, it is desirable to observe the cells and, if any morphologically abnormal cells are found, discard the cultured cells.
ES cells are differentiated into various types of cells, such as parietal muscle, visceral muscle, and cardiac muscle, by monolayer culture up to high density or suspension culture until cell clumps are formed under appropriate conditions. [M. {J. {Evans and M.A. H. Kaufman, Nature, 292, 154, 1981; R. {Martin} Proceedings of National Academy of Science USA (Proc. Natl. Acad. Sci. USA) 78, 7634, 1981; C. Doetschman et al., Journal of Embryology and Experimental Morphology, Vol. 87, p. 27, 1985], that the DNA-deficient cells of the present invention obtained by differentiating the ES cells of the present invention are in vitro. The present invention is useful in the cell biological examination of the polypeptide of the present invention or the receptor of the present invention.
[0069]
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 obtained in this manner is usually an individual heterozygously deficient in expression of the polypeptide of the present invention, and mated with an individual heterozygously deficient in expression of the polypeptide of the present invention or the receptor of the present invention. Thus, an individual deficient in homoexpression of the polypeptide of the present invention or the receptor of the present invention can be obtained.
[0070]
When an egg cell is used, 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 one normal individual and a plurality of homozygous animals are obtained. 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 expressing the DNA of the present invention lacks various biological activities that can be induced by the polypeptide of the present invention or the receptor of the present invention, the polypeptide of the present invention or the receptor of the present invention It can be used as a model for diseases caused by inactivation of the biological activity of E. coli, and is useful for investigating the causes of these diseases and examining treatment methods.
[0071]
(8a) A method for screening a compound having a therapeutic / preventive effect on diseases caused by DNA deficiency or damage of the present invention
The non-human mammal deficient in expression of the DNA of the present invention can be used for screening for a compound having a therapeutic / preventive effect against diseases caused by deficiency or damage of the DNA of the present invention.
That is, the present invention is characterized by administering a test compound to a non-human mammal deficient in expressing the DNA of the present invention, and observing and measuring a change in the animal. Provided is a method for screening a compound or a salt thereof having a therapeutic / prophylactic effect against a disease.
Examples of the non-human mammal deficient in expressing DNA of the present invention used in the screening method include the same as described above.
Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma, and the like. These compounds are novel compounds. Or a known compound.
Specifically, a non-human mammal deficient in expression of the DNA of the present invention is treated with a test compound, compared with an untreated control animal, and tested using changes in each organ, tissue, disease symptoms, etc. of the animal as an index. The therapeutic / preventive effects of the compounds can be tested.
As a method of treating a test animal with a test compound, for example, oral administration, intravenous injection and the like are used, and it can be appropriately selected according to the symptoms of the test animal, the properties of the test compound, and the like. The dose of the test compound can be appropriately selected according to the administration method, the properties of the test compound, and the like.
[0072]
For example, when screening for a weight gain inhibitor, a non-human mammal deficient in expressing the DNA of the present invention is subjected to glucose loading treatment, and a test compound is administered before or after the glucose loading treatment, and the blood glucose level and weight change of the animal are measured. Is measured over time.
[0073]
In the screening method, when a test compound is administered to a test animal and the blood glucose level of the test animal decreases by about 10% or more, preferably about 30% or more, more preferably about 50% or more, the test compound Can be selected as compounds 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 against a disease caused by deficiency or damage of the polypeptide of the present invention. It can be used as a medicament such as a safe and low toxic therapeutic and / or prophylactic agent. 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, 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 drug containing the polypeptide of the present invention described above.
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 administered subcutaneously, it is generally one dose for an adult patient (with a body weight of 60 kg). About 0.1-100 mg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg of the compound is administered per day. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, and the like. For example, the compound of the present invention that promotes the promoter activity for DNA is usually administered in the form of an injection to an adult ( (As 60 kg) to anorexia nervosa patient, about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg of the compound per day by intravenous injection. It is convenient to administer. In the case of other animals, the amount can be administered in terms of 60 kg.
[0074]
(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 polypeptide of the present invention is replaced with a β-galactosidase gene (lacZ) derived from Escherichia coli, the tissue expressing the polypeptide of the present invention originally should Β-galactosidase is expressed instead of the peptide. 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). Of the polypeptide in vivo in an animal can be observed. Specifically, the polypeptide-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 at 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 the 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, Salts with succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and the like are used.
[0075]
The compound or a salt thereof that promotes the promoter activity for the DNA of the present invention can promote the expression of the polypeptide of the present invention and promote the function of the polypeptide. , Can be used as a fat increase inhibitor, an eating inhibitor, and the like.
In addition, a compound or a salt thereof that inhibits the promoter activity of the DNA of the present invention can inhibit the expression of the polypeptide of the present invention and inhibit the function of the polypeptide. be able to.
Furthermore, compounds derived from the compounds obtained by the above screening can be used in the same manner.
[0076]
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 polypeptide 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 disease to be treated, the subject of administration, the administration route, and the like. For example, when the compound of the present invention that promotes the promoter activity for DNA is orally administered, generally the adult (body weight) is used. For patients weighing 60 kg (as 60 kg), about 0.1-100 mg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg of the compound are administered per day. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, and the like. For example, the compound of the present invention that promotes the promoter activity for DNA is usually administered in the form of an injection to an adult ( When administered to a patient (as 60 kg), about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg of the compound is administered intravenously per day. It is convenient. In the case of other animals, the amount can be administered in terms of 60 kg.
On the other hand, for example, when the compound of the present invention that inhibits the promoter activity for DNA is orally administered, generally, in an adult patient (with a body weight of 60 kg), about 0.1 to 100 mg, preferably, 0.1 to 100 mg of the compound per day is preferably used. About 1.0 to 50 mg, more preferably about 1.0 to 20 mg is administered. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, and the like. For example, a compound that inhibits the promoter activity of the DNA of the present invention is usually administered to adults ( When administered to a patient (as 60 kg), about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg of the compound is administered intravenously per day. It is convenient. In the case of other animals, the amount can be administered in terms of 60 kg.
As described above, the non-human mammal deficient in expression of the DNA of the present invention is extremely useful for screening for a compound or a salt thereof that promotes or inhibits the activity of the promoter for the DNA of the present invention, It can greatly contribute to investigating the cause of various diseases caused by it or to develop preventive and therapeutic drugs.
Further, using a DNA containing the promoter region of the polypeptide of the present invention, genes encoding various proteins are ligated downstream thereof and injected into egg cells of an animal to produce a so-called transgenic animal (transgenic animal). By preparing, it is possible to specifically synthesize the polypeptide and examine its action in a living body. Further, by binding an appropriate reporter gene to the above promoter portion and establishing a cell line that expresses the same, a low molecule having an action of specifically promoting or suppressing the in vivo production of the polypeptide of the present invention itself. It can be used as a compound search system.
[0077]
In the present specification and drawings, bases, amino acids, and the like are indicated by abbreviations based on the abbreviations by IUPAC-IUB \ Commission \ on \ Biochemical \ Nomenclature or commonly used abbreviations 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.

[0078]
The sequence numbers in the sequence listing in the present specification indicate the following sequences.
[SEQ ID NO: 1]
1 shows the base sequence of a synthetic DNA used for screening of cDNA encoding human GPR8 protein.
[SEQ ID NO: 2]
1 shows the base sequence of a synthetic DNA used for screening of cDNA encoding human GPR8 protein.
[SEQ ID NO: 3]
The entire nucleotide sequence of human GPR8 protein cDNA having a nucleotide sequence recognized by restriction enzyme ClaI added to the 5 ′ side and a nucleotide sequence recognized by restriction enzyme SpeI added to the 3 ′ side is shown.
[SEQ ID NO: 4]
1 shows the entire amino acid sequence of human GPR8 protein.
[SEQ ID NO: 5]
The sequence of riboprobe used to measure the expression level of GPR8 protein mRNA in each clone of the GPR8-expressing CHO cell line is shown.
[SEQ ID NO: 6]
2 shows an amino acid sequence obtained from the result of amino-terminal amino acid sequence analysis of a ligand peptide for GPR8 purified from pig hypothalamus.
[SEQ ID NO: 7]
The EST sequence (accession number: AW007531), whose complementary chain is presumed to encode a part of the precursor protein of the human homolog of the GPR8 ligand peptide, is shown.
[SEQ ID NO: 8]
The EST sequence (accession number: AI500303) in which the complementary chain is presumed to encode a part of the precursor protein of the human homolog of the GPR8 ligand peptide is shown.
[SEQ ID NO: 9]
The EST sequence (accession number: AI990964) in which the complementary chain is presumed to encode a part of the precursor protein of the human homolog of the GPR8 ligand peptide is shown.
[SEQ ID NO: 10]
The EST sequence (accession number: AA744804) in which the complementary chain is presumed to encode a part of the precursor protein of the human homolog of the GPR8 ligand peptide is shown.
[SEQ ID NO: 11]
The EST sequence (accession number: H31598) presumed to encode a part of the precursor protein of the rat homolog of the GPR8 ligand peptide is shown.
[SEQ ID NO: 12]
1 shows the nucleotide sequence of a synthetic DNA used for screening cDNA encoding a part of the precursor protein of a human homolog of a ligand peptide to GPR8.
[SEQ ID NO: 13]
1 shows the nucleotide sequence of a synthetic DNA used for screening cDNA encoding a part of the precursor protein of a human homolog of a ligand peptide to GPR8.
[SEQ ID NO: 14]
1 shows a nucleotide sequence of a DNA encoding a part of a precursor protein of a human homolog of a ligand peptide for GPR8 amplified from cDNA derived from human brain.
[SEQ ID NO: 15]
2 shows a partial amino acid sequence of a precursor protein of a human homolog of a ligand peptide for GPR8.
[SEQ ID NO: 16]
The amino acid sequence of a human homolog of a ligand peptide for GPR8 deduced from SEQ ID NO: 15 is shown.
[SEQ ID NO: 17]
The amino acid sequence of a human homolog of a ligand peptide for GPR8 deduced from SEQ ID NO: 15 is shown.
[SEQ ID NO: 18]
This shows the base sequence encoding the amino acid sequence represented by SEQ ID NO: 16.
[SEQ ID NO: 19]
This shows the base sequence encoding the amino acid sequence represented by SEQ ID NO: 17.
[SEQ ID NO: 20]
14 shows the amino acid sequence of human GPR8 ligand (1-29) synthesized in Reference Example 14 described later.
[SEQ ID NO: 21]
14 shows the amino acid sequence of human GPR8 ligand (1-28) synthesized in Reference Example 15 described later.
[SEQ ID NO: 22]
14 shows the amino acid sequence of human GPR8 ligand (1-27) synthesized in Reference Example 16 described later.
[SEQ ID NO: 23]
17 shows the amino acid sequence of human GPR8 ligand (1-26) synthesized in Reference Example 17 described later.
[SEQ ID NO: 24]
17 shows the amino acid sequence of human GPR8 ligand (1-25) synthesized in Reference Example 18 described later.
[SEQ ID NO: 25]
19 shows the amino acid sequence of human GPR8 ligand (1-24) synthesized in Reference Example 19 described later.
[SEQ ID NO: 26]
This shows the base sequence encoding the amino acid sequence represented by SEQ ID NO: 20.
[SEQ ID NO: 27]
This shows the base sequence encoding the amino acid sequence represented by SEQ ID NO: 21.
[SEQ ID NO: 28]
This shows the base sequence encoding the amino acid sequence represented by SEQ ID NO: 22.
[SEQ ID NO: 29]
This shows the base sequence encoding the amino acid sequence represented by SEQ ID NO: 23.
[SEQ ID NO: 30]
This shows the base sequence encoding the amino acid sequence represented by SEQ ID NO: 24.
[SEQ ID NO: 31]
This shows the base sequence encoding the amino acid sequence represented by SEQ ID NO: 25.
[SEQ ID NO: 32]
This shows the base sequence encoding the amino acid sequence represented by SEQ ID NO: 4.
[SEQ ID NO: 33]
The base sequence of the synthetic DNA used to obtain the 5 'upstream sequence of the cDNA encoding the precursor protein of the human homolog of the ligand peptide to GPR8 is shown.
[SEQ ID NO: 34]
The base sequence of the synthetic DNA used to obtain the 5 'upstream sequence of the cDNA encoding the precursor protein of the human homolog of the ligand peptide to GPR8 is shown.
[SEQ ID NO: 35]
The base sequence of the 5 'upstream DNA of cDNA encoding the precursor protein of the human homolog of the ligand peptide to GPR8 is shown.
[SEQ ID NO: 36]
The base sequence of the synthetic DNA used to obtain the 3 'downstream sequence of the cDNA encoding the precursor protein of the human homolog of the ligand peptide to GPR8 is shown.
[SEQ ID NO: 37]
The base sequence of the synthetic DNA used to obtain the 3 'downstream sequence of the cDNA encoding the precursor protein of the human homolog of the ligand peptide to GPR8 is shown.
[SEQ ID NO: 38]
The base sequence of the 3 'downstream DNA of the cDNA encoding the precursor protein of the human homolog of the ligand peptide for GPR8 is shown.
[SEQ ID NO: 39]
1 shows the nucleotide sequence of a synthetic DNA used to obtain cDNA encoding a precursor protein of a human homolog of a ligand peptide to GPR8.
[SEQ ID NO: 40]
1 shows the nucleotide sequence of a synthetic DNA used to obtain cDNA encoding a precursor protein of a human homolog of a ligand peptide to GPR8.
[SEQ ID NO: 41]
[Fig. 4] Fig. 4 shows a sequence of a cDNA encoding a precursor protein of a human homolog of a ligand peptide for GPR8.
[SEQ ID NO: 42]
Fig. 4 shows the amino acid sequence of the precursor protein of a human homolog of a ligand peptide for GPR8.
[SEQ ID NO: 43]
The base sequence of the synthetic DNA used to obtain the 5 'upstream sequence of the cDNA encoding the precursor protein of the pig homolog of the ligand peptide to GPR8 is shown.
[SEQ ID NO: 44]
The base sequence of the synthetic DNA used to obtain the 5 'upstream sequence of the cDNA encoding the precursor protein of the pig homolog of the ligand peptide to GPR8 is shown.
[SEQ ID NO: 45]
The base sequence of the 5 'upstream DNA of cDNA encoding the precursor protein of the pig homolog of the ligand peptide to GPR8 is shown.
[SEQ ID NO: 46]
The base sequence of the synthetic DNA used to obtain the 5 'upstream sequence of the cDNA encoding the precursor protein of the pig homolog of the ligand peptide to GPR8 is shown.
[SEQ ID NO: 47]
The base sequence of the synthetic DNA used to obtain the 5 'upstream sequence of the cDNA encoding the precursor protein of the pig homolog of the ligand peptide to GPR8 is shown.
[SEQ ID NO: 48]
The base sequence of the 5 'upstream DNA of cDNA encoding the precursor protein of the pig homolog of the ligand peptide to GPR8 is shown.
[SEQ ID NO: 49]
The base sequence of the synthetic DNA used to obtain the 3 'downstream sequence of the cDNA encoding the precursor protein of the porcine homolog of the ligand peptide to GPR8 is shown.
[SEQ ID NO: 50]
The base sequence of the synthetic DNA used to obtain the 3 'downstream sequence of the cDNA encoding the precursor protein of the porcine homolog of the ligand peptide to GPR8 is shown.
[0079]
[SEQ ID NO: 51]
The base sequence of the 3 'downstream DNA of cDNA encoding the precursor protein of the porcine homolog of the ligand peptide to GPR8 is shown.
[SEQ ID NO: 52]
1 shows the nucleotide sequence of a synthetic DNA used to obtain cDNA encoding a precursor protein of a porcine homolog of a ligand peptide to GPR8.
[SEQ ID NO: 53]
1 shows the nucleotide sequence of a synthetic DNA used to obtain cDNA encoding a precursor protein of a porcine homolog of a ligand peptide to GPR8.
[SEQ ID NO: 54]
[Fig. 4] Fig. 4 shows a sequence of a cDNA encoding a precursor protein of a pig homolog of a ligand peptide for GPR8.
[SEQ ID NO: 55]
Fig. 4 shows the amino acid sequence of a precursor protein of a pig homolog of a ligand peptide for GPR8.
[SEQ ID NO: 56]
The amino acid sequence of a pig homolog of a ligand peptide to GPR8 deduced from SEQ ID NO: 55 is shown.
[SEQ ID NO: 57]
The amino acid sequence of a pig homolog of a ligand peptide to GPR8 deduced from SEQ ID NO: 55 is shown.
[SEQ ID NO: 58]
The base sequence encoding the amino acid sequence represented by SEQ ID NO: 56 is shown.
[SEQ ID NO: 59]
This shows the base sequence encoding the amino acid sequence represented by SEQ ID NO: 57.
[SEQ ID NO: 60]
1 shows the nucleotide sequence of a synthetic DNA used to obtain cDNA encoding a part of the precursor protein of a rat homolog of a ligand peptide to GPR8.
[SEQ ID NO: 61]
1 shows the nucleotide sequence of a synthetic DNA used to obtain cDNA encoding a part of the precursor protein of a rat homolog of a ligand peptide to GPR8.
[SEQ ID NO: 62]
2 shows the sequence of a cDNA encoding a part of the precursor protein of a rat homolog of a ligand peptide for GPR8.
[SEQ ID NO: 63]
The base sequence of the synthetic DNA used to obtain the 5 'upstream sequence of the cDNA encoding the precursor protein of the rat homolog of the ligand peptide to GPR8 is shown.
[SEQ ID NO: 64]
The base sequence of the synthetic DNA used to obtain the 5 'upstream sequence of the cDNA encoding the precursor protein of the rat homolog of the ligand peptide to GPR8 is shown.
[SEQ ID NO: 65]
The nucleotide sequence of the 5 'upstream DNA of the cDNA encoding the precursor protein of the rat homolog of the ligand peptide to GPR8 is shown.
[SEQ ID NO: 66]
The base sequence of the synthetic DNA used to obtain the 3 'downstream sequence of the cDNA encoding the precursor protein of the rat homolog of the ligand peptide to GPR8 is shown.
[SEQ ID NO: 67]
The base sequence of the synthetic DNA used to obtain the 3 'downstream sequence of the cDNA encoding the precursor protein of the rat homolog of the ligand peptide to GPR8 is shown.
[SEQ ID NO: 68]
The nucleotide sequence of the 3 'downstream DNA of the cDNA encoding the precursor protein of the rat homolog of the ligand peptide to GPR8 is shown.
[SEQ ID NO: 69]
1 shows the nucleotide sequence of a synthetic DNA used to obtain cDNA encoding a precursor protein of a rat homolog of a ligand peptide to GPR8.
[SEQ ID NO: 70]
1 shows the nucleotide sequence of a synthetic DNA used to obtain cDNA encoding a precursor protein of a rat homolog of a ligand peptide to GPR8.
[SEQ ID NO: 71]
[Fig. 4] Fig. 4 shows a sequence of a cDNA encoding a precursor protein of a rat homolog of a ligand peptide for GPR8.
[SEQ ID NO: 72]
2 shows the amino acid sequence of a rat homolog precursor protein of a ligand peptide for GPR8.
[SEQ ID NO: 73]
The amino acid sequence of a rat homolog of a ligand peptide to GPR8 deduced from SEQ ID NO: 72 is shown.
[SEQ ID NO: 74]
The amino acid sequence of a rat homolog of a ligand peptide to GPR8 deduced from SEQ ID NO: 72 is shown.
[SEQ ID NO: 75]
This shows the base sequence encoding the amino acid sequence represented by SEQ ID NO: 73.
[SEQ ID NO: 76]
This shows the base sequence encoding the amino acid sequence represented by SEQ ID NO: 74.
[SEQ ID NO: 77]
The sequence of a synthetic DNA used as a probe to measure the GPR7 gene expression level in human GPR7-expressing CHO cells is shown.
[SEQ ID NO: 78]
The base sequence of a synthetic DNA used for screening a cDNA encoding a part of the precursor protein of a mouse homolog of a ligand peptide to GPR8 is shown.
[SEQ ID NO: 79]
The base sequence of a synthetic DNA used for screening a cDNA encoding a part of the precursor protein of a mouse homolog of a ligand peptide to GPR8 is shown.
[SEQ ID NO: 80]
1 shows a nucleotide sequence of a DNA encoding a part of a precursor protein of a human homolog of a ligand peptide to GPR8 amplified from mouse testis-derived cDNA.
[SEQ ID NO: 81]
The nucleotide sequence of the synthetic DNA used to obtain the 5 'upstream sequence of the cDNA encoding the precursor protein of the mouse homolog of the ligand peptide to GPR8 is shown.
[SEQ ID NO: 82]
The nucleotide sequence of the synthetic DNA used to obtain the 5 'upstream sequence of the cDNA encoding the precursor protein of the mouse homolog of the ligand peptide to GPR8 is shown.
[SEQ ID NO: 83]
The nucleotide sequence of the 5 'upstream DNA of the cDNA encoding the precursor protein of the mouse homolog of the ligand peptide to GPR8 is shown.
[SEQ ID NO: 84]
The base sequence of the synthetic DNA used to obtain the 3 'downstream sequence of the cDNA encoding the precursor protein of the mouse homolog of the ligand peptide to GPR8 is shown.
[SEQ ID NO: 85]
The base sequence of the synthetic DNA used to obtain the 3 'downstream sequence of the cDNA encoding the precursor protein of the mouse homolog of the ligand peptide to GPR8 is shown.
[SEQ ID NO: 86]
The base sequence of the 3 'downstream DNA of the cDNA encoding the precursor protein of the mouse homolog of the ligand peptide to GPR8 is shown.
[SEQ ID NO: 87]
1 shows the nucleotide sequence of a synthetic DNA used to obtain cDNA encoding a precursor protein of a mouse homolog of a ligand peptide to GPR8.
[SEQ ID NO: 88]
1 shows the nucleotide sequence of a synthetic DNA used to obtain cDNA encoding a precursor protein of a mouse homolog of a ligand peptide to GPR8.
[SEQ ID NO: 89]
2 shows the sequence of a cDNA encoding a precursor protein of a mouse homolog of a ligand peptide for GPR8.
[SEQ ID NO: 90]
2 shows the amino acid sequence of a mouse homolog precursor protein of a ligand peptide for GPR8.
[SEQ ID NO: 91]
The amino acid sequence of a mouse homolog of a ligand peptide for GPR8 deduced from SEQ ID NO: 90 is shown.
[SEQ ID NO: 92]
The amino acid sequence of a mouse homolog of a ligand peptide for GPR8 deduced from SEQ ID NO: 90 is shown.
[SEQ ID NO: 93]
The base sequence encoding the amino acid sequence represented by SEQ ID NO: 91 is shown.
[SEQ ID NO: 94]
This shows the base sequence encoding the amino acid sequence represented by SEQ ID NO: 92.
[SEQ ID NO: 95]
26 shows the amino acid sequence of an oxidized human GPR8 ligand (1-23) synthesized in Reference Example 44 described later.
[SEQ ID NO: 96]
14 shows the amino acid sequence of human GPR8 ligand (1-22) synthesized in Reference Example 45 described below.
[SEQ ID NO: 97]
26 shows the amino acid sequence of human GPR8 ligand (1-21) synthesized in Reference Example 46 described later.
[SEQ ID NO: 98]
26 shows the amino acid sequence of human GPR8 ligand (1-20) synthesized in Reference Example 47 described later.
[SEQ ID NO: 99]
19 shows the amino acid sequence of human GPR8 ligand (1-19) synthesized in Reference Example 48 described later.
[SEQ ID NO: 100]
26 shows the amino acid sequence of human GPR8 ligand (1-18) synthesized in Reference Example 49 described later.
[0080]
[SEQ ID NO: 101]
26 shows the amino acid sequence of human GPR8 ligand (1-17) synthesized in Reference Example 50 described later.
[SEQ ID NO: 102]
26 shows the amino acid sequence of human GPR8 ligand (1-16) synthesized in Reference Example 51 described later.
[SEQ ID NO: 103]
The amino acid sequence of the oxidized GPR8 ligand (1-23) synthesized in Reference Example 54 described later is shown.
[SEQ ID NO: 104]
The amino acid sequence of an oxidized rat or mouse GPR8 ligand (1-23) synthesized in Reference Example 55 described below is shown.
[SEQ ID NO: 105]
13 shows the amino acid sequence of Fmoc-modified human GPR8 ligand (1-23) synthesized in Reference Example 12 described later.
[SEQ ID NO: 106]
[Nα-Acetyl-Trp synthesized in Reference Example 56 described below.¹-Shows the amino acid sequence of human GPR8 ligand (1-23).
[SEQ ID NO: 107]
26 shows the amino acid sequence of human GPR8 ligand (2-23) synthesized in Reference Example 57 described below.
[SEQ ID NO: 108]
26 shows the amino acid sequence of human GPR8 ligand (4-23) synthesized in Reference Example 58 described later.
[SEQ ID NO: 109]
26 shows the amino acid sequence of human GPR8 ligand (9-23) synthesized in Reference Example 59 described later.
[SEQ ID NO: 110]
26 shows the amino acid sequence of human GPR8 ligand (15-23) synthesized in Reference Example 60 described later.
[SEQ ID NO: 111]
[N-Acetyl-Tyr synthesized in Reference Example 61 described later²-Shows the amino acid sequence of human GPR8 ligand (2-23).
[SEQ ID NO: 112]
[D-Trp synthesized in Reference Example 62 described later¹-Shows the amino acid sequence of human GPR8 ligand (1-23).
[SEQ ID NO: 113]
[N-3-Indolepropanoyl-Tyr synthesized in Reference Example 63 described later.²-Shows the amino acid sequence of human GPR8 ligand (2-23).
[SEQ ID NO: 114]
The nucleotide sequence coding for the amino acid sequence represented by SEQ ID NO: 96 is shown.
[SEQ ID NO: 115]
This shows a base sequence encoding the amino acid sequence represented by SEQ ID NO: 97.
[SEQ ID NO: 116]
The base sequence encoding the amino acid sequence represented by SEQ ID NO: 98 is shown.
[SEQ ID NO: 117]
The base sequence encoding the amino acid sequence represented by SEQ ID NO: 99 is shown.
[SEQ ID NO: 118]
This shows the base sequence encoding the amino acid sequence represented by SEQ ID NO: 100.
[SEQ ID NO: 119]
This shows the base sequence encoding the amino acid sequence represented by SEQ ID NO: 101.
[SEQ ID NO: 120]
This shows the base sequence encoding the amino acid sequence represented by SEQ ID NO: 102.
[SEQ ID NO: 121]
The nucleotide sequence coding for the amino acid sequence represented by SEQ ID NO: 107 is shown.
[SEQ ID NO: 122]
The nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 108 is shown.
[SEQ ID NO: 123]
The nucleotide sequence coding for the amino acid sequence represented by SEQ ID NO: 109 is shown.
[SEQ ID NO: 124]
The nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 110 is shown.
[SEQ ID NO: 125]
This shows the base sequence encoding the amino acid sequence represented by SEQ ID NO: 6.
[SEQ ID NO: 126]
1 shows the amino acid sequence of a rat-derived novel G protein-coupled receptor protein TGR26 of the present invention.
[SEQ ID NO: 127]
1 shows the nucleotide sequence of cDNA encoding the novel rat G protein-coupled receptor protein TGR26 of the present invention.
[SEQ ID NO: 128]
The base sequence of primer 1 used in the PCR reaction in Reference Example 67 below is shown.
[SEQ ID NO: 129]
The base sequence of primer 2 used in the PCR reaction in Reference Example 67 below is shown.
[SEQ ID NO: 130]
The base sequence of the primer used for measuring the expression level of the TGR26 receptor gene in TGR26-expressing CHO cells in Reference Example 68 below is shown.
[SEQ ID NO: 131]
The base sequence of the primer used for measuring the TGR26 gene expression level of the TGR26-expressing CHO cells in Reference Example 68 below is shown.
[SEQ ID NO: 132]
The base sequence of the primer used in the PCR reaction in Reference Example 75 below is shown.
[SEQ ID NO: 133]
The base sequence of the primer used in the PCR reaction in Reference Example 75 below is shown.
[SEQ ID NO: 134]
The base sequence of the 5 'upstream end of the DNA encoding mouse TGR26 obtained in Reference Example 75 below is shown.
[SEQ ID NO: 135]
The base sequence of the primer used in the PCR reaction in Reference Example 24 below is shown.
[SEQ ID NO: 136]
The base sequence of the primer used in the PCR reaction in Reference Example 24 below is shown.
[SEQ ID NO: 137]
The base sequence of the cDNA encoding mouse TGR26 obtained in Reference Example 24 below is shown.
[SEQ ID NO: 138]
2 shows the amino acid sequence of mouse-derived TGR26.
[SEQ ID NO: 139]
2 shows the nucleotide sequence of cDNA encoding mouse-derived TGR26.
[SEQ ID NO: 140]
The base sequence of a probe used for measuring the expression level of the TGR26 gene in TGR26-expressing CHO cells in Reference Example 68 below is shown.
[SEQ ID NO: 141]
1 shows the nucleotide sequence of a synthetic DNA used for screening of cDNA encoding human GPR7.
[SEQ ID NO: 142]
1 shows the nucleotide sequence of a synthetic DNA used for screening of cDNA encoding human GPR7.
[SEQ ID NO: 143]
The entire nucleotide sequence of the human GPR7 protein cDNA having a nucleotide sequence recognized by the restriction enzyme ClaI added to the 5 ′ side and a nucleotide sequence recognized by the restriction enzyme SpeI added to the 3 ′ side.
[SEQ ID NO: 144]
1 shows the entire amino acid sequence of human GPR7.
[SEQ ID NO: 145]
The DNA base sequence used as a primer for amplifying the standard human GPR7 DNA is shown.
[SEQ ID NO: 146]
The DNA base sequence used as a primer for amplifying the standard human GPR7 DNA is shown.
[SEQ ID NO: 147]
The base sequence of synthetic DNA used as a primer to measure the expression level of the GPR7 gene in human GPR7-expressing CHO cells is shown.
[SEQ ID NO: 148]
The base sequence of synthetic DNA used as a primer to measure the expression level of the GPR7 gene in human GPR7-expressing CHO cells is shown.
[SEQ ID NO: 149]
[Phe²1] shows the amino acid sequence of human GPR8 ligand (1-20).
[SEQ ID NO: 150]
This shows a base sequence encoding the amino acid sequence represented by SEQ ID NO: 149.
[0081]
The transformant Escherichia coli DH5α / pAKKO-GPR8 obtained in Reference Example 3 to be described later is 2-17-85, Jusanhoncho, Yodogawa-ku, Osaka-shi, Osaka (postal code 532-8686), and the Fermentation Research Institute ( IFO) from February 27, 2001 as deposit number IFO16564, 1-1 1-1 Higashi, Tsukuba City, Ibaraki Prefecture, Chuo No. 6 (Postal Code 305-8566), National Institute of Advanced Industrial Science and Technology @ Patent Organism Depositary, 2001 They have been deposited under the accession number FERM @ BP-7540 since April 11th.
The transformant Escherichia coli E. coli TOP10 / pCR2.1-TOPOHMan GPR8 Ligand Precursor obtained in Reference Example 28 to be described later is 2-17-85, Jusanhoncho, Yodogawa-ku, Osaka-shi, Osaka-shi (postal code 532-8686), Foundation From the Institute of Fermentation (IFO) on February 27, 2001, accession number IFO # 16568, 1-1 1-1 Higashi, Tsukuba, Ibaraki, Chuo No. 6 (Zip code 305-8566), National Institute of Advanced Industrial Science and Technology, Patent They have been deposited with the Organism Depositary under the Accession No. FERM @ BP-7544 since April 11, 2001.
The transformant Escherichia coli E. coli TOP10 / pCR2.1-TOPOPorcine GPR8 Ligand Precursor obtained in Reference Example 32 described below is available at 2-17-1885 Jusanhoncho, Yodogawa-ku, Osaka-shi, Osaka-shi (Zip code 532-8686), Foundation From the Institute of Fermentation (IFO) on February 27, 2001, deposit number IFO 16565, 1-1 1-1 Higashi, Tsukuba City, Ibaraki Prefecture, Chuo No. 6 (zip code 305-8566), National Institute of Advanced Industrial Science and Technology Accession No. FERM to the Biological Depositary Center from April 11, 2001
BP-7541, respectively.
The transformant Escherichia coli TOP10 / pCR2.1-TOPORat GPR8 Ligand Precursor obtained in Reference Example 36 described later is 2- 17-85, Jusanhoncho, Yodogawa-ku, Osaka-shi, Osaka-shi (postal code 532-8686), Foundation Deposited from the Institute of Fermentation (IFO) on February 27, 2001, Deposit No. IFO @ 16567, 1-1-1 Higashi, Tsukuba, Ibaraki, Chuo No.6 (Postal Code 305-8566), National Institute of Advanced Industrial Science and Technology, Patent Organism They have been deposited at the Depositary Center under the accession number FERM @ BP-7543 since April 11, 2001.
The transformant Escherichia coli TOP10 / pCR2.1-TOPOMuse GPR8 Ligand Precursor obtained in Reference Example 41 described later is 2- 17-85, Jusanhoncho, Yodogawa-ku, Osaka-shi, Osaka-shi, Japan (postal code 532-8686). From the Institute of Fermentation (IFO) on February 27, 2001, accession number IFO # 16566, 1-1 1-1 Higashi, Tsukuba, Ibaraki Prefecture, Chuo No. 6 (zip code 305-8566), National Institute of Advanced Industrial Science and Technology, Patent They have been deposited with the Organism Depositary under the Accession No. FERM @ BP-7542 from April 11, 2001.
The transformant Escherichia coli DH10B / pAK-rGPR7 obtained in Reference Example 67 described below has been used since October 31, 2000, at 2-173-17, Jusanhoncho, Yodogawa-ku, Osaka-shi, Osaka, Japan (postal code 532). -8686), the fermentation research institute (IFO), accession number IFO 16496, from November 13, 2000, 1-1 1-1 Higashi, Tsukuba City, Ibaraki Prefecture, Chuo No. 6 (zip code 305-8566), independent administrative agency They have been deposited with the National Institute of Advanced Industrial Science and Technology at the Patent Organism Depositary Center (formerly Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology (NIBH)) under the accession number FERM @ BP-7365.
The transformant E. coli (Escherichia coli) TOP10 / pCR2.1-TOPOMuseGPR7 obtained in Reference Example 24 described below has been available from September 20, 2001 at 2-17-85, Jusanhoncho, Yodogawa-ku, Osaka-shi, Osaka, Japan (postal service). No. 532-8686) and the fermentation research institute (IFO) under the accession number IFO 16704, from October 15, 2001, 1-1 1-1 Higashi, Tsukuba City, Ibaraki Prefecture, Chuo No. 6 (zip code 305-8566), independent They have been deposited with the National Institute of Advanced Industrial Science and Technology at the Patent Organism Depositary under the accession number FERM BP-7775.
[0082]
【Example】
Hereinafter, the present invention will be described in more detail with reference to Reference Examples and Examples, but these examples do not limit the scope of the present invention.
[0083]
Reference Example 1 Amplification of human GPR8 cDNA by PCR using cDNA derived from human brain
Poly (A) from human brain⁺A reverse transcription reaction was performed using RNA (Clontech) as a template and random primers. For reverse transcription, Takara RNA PCR ver 2.1 kit reagent was used. Next, using the reverse transcription product as a template, amplification by the PCR method was performed using synthetic primers represented by SEQ ID NO: 1 and SEQ ID NO: 2. The synthetic primer was constructed so that the gene in the region translated into the receptor protein was amplified. At that time, a nucleotide sequence recognized by the restriction enzyme ClaI was added to the 5 ′ side of the gene, and the restriction enzyme was added to the 3 ′ side. Recognition sequences for the respective restriction enzymes were added to the 5 ′ and 3 ′ sides so that the base sequence recognized by SpeI was added. The composition of the reaction solution was 5 μl of cDNA template, 0.4 μM each of synthetic DNA primers, 0.8 mM ΔdNTPs, 0.5 μl of Pfu polymerase (Stratagene) and a buffer attached to the enzyme, and the total reaction volume was 50 μl. The cycle for amplification was performed using a thermal cycler (PE Biosystems), and after heating at 94 ° C for 60 seconds, a cycle of 94 ° C for 60 seconds, 65 ° C for 60 seconds, and 72 ° C for 150 seconds was repeated 35 times. Confirmation of the amplification product was performed by ethidium bromide staining after 0.8% agarose gel electrophoresis.
[0084]
Reference Example 2 Confirmation of amplified cDNA sequence by subcloning PCR product into plasmid vector and decoding base sequence of inserted cDNA portion
The PCR reaction solution performed in Reference Example 1 was separated by electrophoresis on a 0.8% low melting point agarose gel, and the band was cut out with a razor, followed by fragmentation, phenol extraction, phenol / chloroform extraction, and ethanol precipitation. The operation was performed to recover the DNA. PCR-Script^TMThe recovered DNA was subcloned into a plasmid vector pCR-Script {Amp} SK (+) according to the recipe of the {Amp} SK (+) cloning kit (Stratagene). After introducing this into Escherichia coli (DH5α competent) cell (Toyobo) and transforming it, a clone having a cDNA insert was selected on an LB agar medium containing ampicillin, IPTG and X-gal, and only clones exhibiting white color were selected. Was isolated using a sterilized toothpick and the transformant E. E. coli DH5α / GPR8 was obtained. Individual clones were cultured overnight in LB medium containing ampicillin, and plasmid DNA was prepared using QIAwell \ 8 \ Plasmid \ Kit (Qiagen). A portion of the prepared DNA was cleaved with restriction enzymes ClaI and SpeI to confirm the size of the inserted receptor cDNA fragment. The reaction for determining the nucleotide sequence was performed using DyeDeoxyTerminator \ Cycle \ Sequence \ Kit (PE \ Biosystems) and decoded using a fluorescent automatic sequencer (SEQ ID NO: 3). The entire nucleotide sequence of the human GPR8 receptor protein cDNA is shown in SEQ ID NO: 3, and the entire amino acid sequence of the human GPR8 receptor protein translated therefrom is shown in SEQ ID NO: 4.
[0085]
Reference Example 3 Preparation of GPR8-expressing CHO cells
A plasmid into which a gene encoding the full-length amino acid sequence of GPR8 derived from human brain whose sequence was confirmed in Reference Example 2 and having a ClaI recognition sequence added to the 5 ′ side and a SpeI recognition sequence added to the 3 ′ side was introduced. The transformed E. coli Plasmid DNA was prepared from the E. coli clone using Plasmid \ Midi \ KIt (Qiagen) and digested with restriction enzymes ClaI and SpeI to cut out insert DNA. After the electrophoresis, the insert DNA was cut out from the agarose gel with a razor, and then recovered by the operations of fragmentation, phenol extraction, phenol / chloroform extraction, and ethanol precipitation. This insert DNA was digested with ClaI and SpeI, and vector plasmid pAKKO-111H for expression of animal cells (pAKKO1.11H described in S. \ Hinuma \ et \ al., Biochim. \ Biophys. \ Acta, Vol. Ligation using T4 ligase (Takara Shuzo) in addition to the same vector plasmid to construct a protein expression plasmid pAKKO-GPR8. Escherichia coli transformed with the plasmid pAKKO-GPR8 was designated as DH5α / pAKKO-GPR8 (Escherichia coli DH5α / pAKKO-GPR8).
E. coli transformed with pAKKO-GPR8. After cultivation of E. coli DH5α (Toyobo), pAKKO-GPR8 plasmid DNA was prepared using Plasmid Midi Kit (Qiagen). This was purified using CellPhect \ Transfection \ Kit (Amersham Pharmacia Biotech) according to the attached protocol and CHO \ dhfr.⁻The cells were introduced. 4.5 μg of DNA was made into a coprecipitation suspension with calcium phosphate.⁵Or 1 x 10⁶CHO @ dhfr⁻The cells were added to a 6 cm diameter petri dish inoculated. After culturing for 1 day in MEMα medium containing 10% fetal calf serum, the cells were subcultured and cultured in nucleic acid-free MEMα medium containing 10% dialyzed fetal bovine serum as a selective medium. Forty-seven clones of transformed cell colonies, which are GPR8-expressing CHO cells, growing in the selection medium were selected.
[0086]
Reference Example 4 Selection of CHO / GPR8 Cell Line with High Expression of Full-length Human GPR8 Protein mRNA
The expression level of full-length GPR8 protein mRNA of 47 clones of the CHO / GPR8 cell line established in Reference Example 3 was measured using Cytostar @ T @ Plate (Amersham Pharmacia Biotech) according to the attached protocol as follows. Each clone of the CHO / GPR8 cell line was placed on a Cytostar T Plate at 2.5 x 10 per well.⁴After seeding each and culturing for 24 hours, the cells were fixed with 10% formalin. After adding 0.25% {Triton} X-100 to each well to increase cell permeability,³⁵The S-labeled riboprobe of SEQ ID NO: 5 was added and hybridized. 20 μg / ml of RNaseΔA was added to each well to digest free riboprobe, and the plate was thoroughly washed. The radioactivity of the hybridized riboprobe was measured with a Topcounter. Cell lines with high radioactivity have high mRNA expression levels. Three clones with high mRNA expression levels (# 17, 41 and 46) were used in the experiments below, and in particular, clone number 17 was used.
[0087]
Reference Example 5 Measurement of intracellular cAMP production using GPR8-expressing CHO cells
The CHO / GPR8 cells and mock @ CHO cells prepared in Reference Example 4 were placed in a 24-well plate at 5 × 10⁴The cells were seeded at cell / well and cultured for 48 hours. The cells were washed with Hanks buffer (pH 7.4) containing 0.2 mM {3-isobutyl-methylxanthine, 0.05%} BSA and 20 mM HEPS (hereinafter referred to as 0.2 mM {3-isobutyl-methylxanthine and 0.05%} BSA). And a Hanks buffer (pH 7.4) containing 20 mM @HEPS is referred to as a reaction buffer). Thereafter, 0.5 ml of a reaction buffer was added, and the mixture was kept warm in an incubator for 30 minutes. After removing the reaction buffer, 0.25 ml of the reaction buffer was newly added to the cells, and then 0.25 ml of the reaction buffer containing the sample and 2 μM forskolin was added to the cells, followed by reaction at 37 ° C. for 24 minutes. The reaction was stopped by adding 100 μl of 20% perchloric acid, and then placed on ice for 1 hour to extract intracellular cAMP. The amount of cAMP in the extract was measured using a cAMP @ EIA kit (Amersham Pharmacia Biotech).
[0088]
Reference Example 6 Measurement of GTPγS binding activity using membrane fraction of GPR8-expressing CHO cells
[For GPR8 expressing CHO cell membrane fraction³⁵The binding promoting activity of [S] -Guanosine '5'-([gamma] -thio) triphosphate was measured by the following method. First, the method for preparing the membrane fraction will be described. 1 x 10⁸Of CHO / GPR8 cells into 10 ml of a homogenate buffer (10 mM NaHCO3).₃, 5 mM EDTA, 0.5 mM PMSF, 1 μg / ml pepstatin, 4 μg / ml E64, 20 μg / ml leupeptin, and crushed using a Polytron (12,000 rpm, 1 minute). The cell lysate was centrifuged (1,000 g, @ 15 minutes) to obtain a supernatant. Next, the supernatant was subjected to ultracentrifugation (Beckman type 30 rotor, 30,000 rpm, 1 hour), and the obtained precipitate was used as a GPR8-expressing CHO cell membrane fraction.
The measurement of GTPγS binding activity is as follows. The GPR8-expressing CHO cell membrane fraction was treated with a membrane dilution buffer (50 mM Tris-HCl buffer (pH 7.4), {5 mM} MgCl₂, 150 mM NaCl, 1 μM GDP) to prepare a cell membrane fraction solution for assay with a protein concentration of 30 mg / ml. 200 μl of the membrane fraction solution for assay was used at a concentration of 51.5 nM [³⁵2 μl of [S] -Guanosine 5 ′-(γ-thio) triphosphate (NEN) and a sample were added, and the mixture was kept at 25 ° C. for 1 hour. The mixture was filtered through a filter, and the filter was further washed with a washing buffer (50 mM Tris-HCl buffer (pH 7.4), 5 mM MgCl 2).₂, {1 mM} EDTA, {0.1%} BSA) twice, and then the radioactivity of the filter was measured with a liquid scintillation counter.
[0089]
Reference Example 7: Detection of an activity contained in the porcine hypothalamus extract that specifically inhibits cAMP production and promotes GTPγS binding to the CHO / GPR8 cell line
A high performance liquid chromatography (HPLC) fraction of the porcine hypothalamic extract was prepared by the method described below. After being slaughtered on the day of treatment and extirpated, purchased from Tokyo Shibaura Organs Co., Ltd., 500 g (30 cats) of the hypothalamus of the pig, which had been ice-cooled and preserved, were cut into small pieces and immediately poured into 2.0 ml of boiling distilled water. Boil for 10 minutes. Immediately after boiling, the mixture was ice-cooled, 120 ml of acetic acid was added to a final concentration of 1.0 M, and the mixture was crushed using a polytron (20,000 rpm, 6 minutes). The crushed liquid was centrifuged (8,000 rpm, 30 minutes), the supernatant was collected, and 2.0 μl of 1.0 M acetic acid was added to the precipitate, crushed again with a polytron, stirred overnight, and then centrifuged (8, 8 rpm). 000 rpm for 30 minutes) to obtain a supernatant. After slowly dropping twice the amount of cold acetone at 4 ° C. into the supernatant, the supernatant obtained by the first centrifugation was stirred overnight, and the supernatant obtained by the second centrifugation was stirred for 4 hours. The extract to which acetone was added was centrifuged (8,000 rpm, 30 minutes) to remove the precipitate, and the acetone was distilled off from the obtained supernatant under reduced pressure using an evaporator. An equal amount of diethyl ether was added to the extract from which acetone was removed, and the lipid-containing ether layer was separated using a separating funnel, and the aqueous layer was collected. The extract defatted with ether was concentrated under reduced pressure by an evaporator to completely remove ether. The concentrate was filtered through a glass fiber filter paper (Advantech, DP70 (90 mm)), and the filtrate was added to a C18 (YMC gel, YMCgel ODS-AM 120-S50) column packed in a glass column (30 x240 mm). . The column was washed with 400% of 1.0M acetic acid and then eluted with 500% of 60% acetonitrile containing 0.1% trifluoroacetic acid. After the eluate was concentrated under reduced pressure to distill off the solvent, the concentrate was freeze-dried. About 0.5 mg of the lyophilized product is dissolved in 30 ml of 10% acetonitrile containing 0.1% trifluoroacetic acid, and 10 ml each is dissolved in a C18 column (TOSO, TSKgel ODS-80TS II (21.5 φ x 300 mm)). HPLC was performed by gradient elution of acetonitrile containing 10% to 60% of 0.1% trifluoroacetic acid using HPLC. HPLC was performed three times. The eluate was collected in 60 fractions, and the eluates for three times were collected. Each fraction was concentrated and dried under reduced pressure, and the residue was dissolved in 0.5 ml of dimethyl sulfoxide (DMSO).
A DMSO solution of the HPLC fraction obtained above was administered to CHO / GPR8 cells by the method described in Reference Example 5, and the amount of intracellular cAMP production was measured. Was observed. When GTPγS binding promoting activity was examined using GPR8-expressing CHO cells for a similar sample, a remarkable activity was also confirmed near fraction No. 30. These activities were not observed in other receptor-expressing cells, indicating that a GPR8-specific ligand active substance was present in the swine hypothalamus extract.
[0090]
Reference Example 8 Inactivation by pronase of an active substance that specifically inhibits intracellular cAMP production in GPR8-expressing CHO cells in porcine hypothalamic extract
The HPLC fraction 30 which showed the activity of suppressing intracellular cAMP production on GPR8-expressing CHO cells in Reference Example 7 was treated with pronase (pronase, Sigma, {protease} Type {XIV} (P5147)), and the active substance was proteinaceous. Was checked. 2 μl of the above hypothalamic extract HPLC fraction (# 30) was added to 200 μl of 0.2 M ammonium acetate, 3 μg of pronase was added thereto, and the mixture was incubated at 37 ° C. for 2 hours, and then heated in boiling water for 10 minutes to obtain pronase. Was deactivated. 2 ml of distilled water containing 0.05 mg of BSA and 0.05 mg of CHAPS was added thereto, followed by freeze-drying. In order to examine the effects of pronase itself or heating and freeze-drying, pronase alone, HPLC fraction only, and pronase alone were subjected to heat treatment, followed by HPLC fractionation and lyophilized. Each of the freeze-dried samples was added to GPR8-expressing CHO cells by the method shown in Reference Example 5, and the activity of inhibiting intracellular cAMP production was measured. The active substance showing the intracellular cAMP production inhibitory activity against GPR8-expressing CHO cells in the porcine hypothalamus extract was completely inactivated by pronase, indicating that this substance was a protein or peptide.
[0091]
Reference Example 9 Purification of an active substance that specifically exhibits GTPγS binding promoting activity with respect to the GPR8-expressing CHO cell membrane fraction from porcine hypothalamus
Representative examples in which a substance exhibiting a ligand activity specific to GPR8 is purified from porcine hypothalamus using GTPγS binding promoting activity on a GPR8-expressing CHO cell membrane fraction as an index will be specifically described below. By exactly the same method as described in Reference Example 7, 500 g (30 cats) of swine hypothalamus was extracted with 1.0 M acetic acid, acetone precipitated and ether degreased, and then C18 (YMC gel, YMCgel ODS). -AM @ 120-S50) column and eluted with 60% acetonitrile containing 0.1% trifluoroacetic acid. After the eluate was concentrated and lyophilized, an active fraction was obtained by HPLC using a C18 column (Tosoh, TSKgel {ODS-80TS} (21.5φx300mm)). The activity was recovered in fraction no. This was further purified by the following method.
This fraction was dissolved in 10 ml of 10 mM ammonium formate containing 10% acetonitrile, added to a cation exchange column (Tosoh, TSKgel SP-5PW (20 mm mm x x 150 mm)), and then dissolved in 10 mM containing 10% acetonitrile. The column was eluted with a concentration gradient of from 2.0 to 2.0 M ammonium formate. Activity was recovered around 0.8 ア ン モ ニ ウ ム M ammonium formate. The active fraction was lyophilized, dissolved in 1.0 ml of 10% acetonitrile containing 0.1% trifluoroacetic acid, and applied to a CN column (Nomura Chemical, Develosil CN-UG-5} (4.6 mm mm x 250 mm)). After addition, elution was performed with a gradient of 21% to 26% acetonitrile containing 0.1% trifluoroacetic acid. Activity appeared around 22.1% acetonitrile. The active fraction was lyophilized, dissolved in 0.1 ml of DMSO, further added with 0.4 ml of 10% acetonitrile containing 0.1% trifluoroacetic acid, and added to an ODS column (Wako Pure Chemical Industries, Wakosil-II @ 3C18HG). (2.0 mm x x 150 mm)) and eluted with a 22.5% to 32.5% acetonitrile gradient containing 0.1% trifluoroacetic acid. The activity appeared as a single peak around 26.5% acetonitrile (FIG. 1).
[0092]
Reference Example 10 Analysis of the amino-terminal amino acid sequence of an active substance that specifically exhibits GTPγS binding promoting activity on the GPR8-expressing CHO cell membrane fraction purified from porcine hypothalamus and the precursor protein of human and rat homolog peptides of GPR8 ligand EST sequence presumed to encode part of
The amino-terminal amino acid sequence analysis of an active substance that specifically exhibits GTPγS binding promoting activity was performed on the GPR8-expressing CHO cell membrane fraction purified in Reference Example 9. Since this active substance was presumed to be a protein or peptide as shown in Reference Example 8, amino-terminal amino acid sequence analysis was performed using Prokinse 494 Protein Protein Sequencer by Perkin Elmer, Inc. using an eluate containing an activity peak. As a result, the sequence shown in SEQ ID NO: 6 was obtained up to 17 residues from the amino terminal. This sequence was considered to be part of the ligand peptide.
[0093]
A search of the gene database based on this sequence revealed several EST (Expressed \ Sequence \ Tag) sequences which are presumed to encode themselves or part of the precursor protein of this peptide, either themselves or their complementary chains. Was issued. The accession number, the origin of the cDNA, the sequence length and the sequence number are as follows. AW007531 (anaplastic oligodendroglioma, 438 base, SEQ ID NO: 7), AI500303 (anaplastic oligodendroglioma, 264 base, SEQ ID NO: 8), AI990964 (colonic mucosa from patient of Crohn's disease, 424 base, SEQ ID NO: 9), AA744804 (Germinal center B cell, 375 base, SEQ ID NO: 10), H31598} (PC12 cells, 260 base, SEQ ID NO: 11). The first four are from humans and the last one is from rats. The DNA sequences of these ESTs correspond very well in the region encoding the amino acid sequence corresponding to the sequence of the active peptide isolated from the porcine hypothalamus, and the translated amino acid sequence was isolated from the porcine hypothalamus. The sequence of the peptide was almost identical to that of the peptide except that Thr at the fifth residue was Val. From the above, it was presumed that these ESTs encode a part of the precursor protein of the human and rat homologs of the ligand peptide of GPR8.
[0094]
Reference Example 11 Amplification of human cDNA encoding a part of GPR8 ligand peptide precursor and decoding of amplified cDNA sequence
Primers are designed based on the EST sequence presumed to encode a part of the GPR8 ligand peptide precursor protein described in Reference Example 10, and the human brain-derived cDNA encodes a part of the GPR8 ligand peptide precursor by PCR. The cDNA was amplified.
Reverse transcription reaction was performed using human brain-derived poly {(A)} + RNA (Clontech) as a template and random primers. For the reverse transcription reaction, ReverseTra @ Ace (Toyobo), which is a MMLV-derived reverse transcriptase lacking RNase @ H activity, was used. Subsequently, amplification was performed by PCR using synthetic DNA primers of SEQ ID NO: 12 and SEQ ID NO: 13 designed based on the EST sequence described in Reference Example 10. The composition of the reaction solution was 2 μl of cDNA template, 0.5 μM each of synthetic DNA primers, 1.6 mM ΔdNTPs, 0.2 μl of LATaq (Takara Shuzo) and a buffer attached to the enzyme, and the total reaction volume was 20 μl. A cycle for amplification was performed using a thermal cycler (PE Biosystems), and after heating at 96 ° C for 120 seconds, four cycles of 96 ° C for 30 seconds, 72 ° C for 45 seconds, 96 ° C for 30 seconds, and 70 ° C. 4 cycles of 45 seconds, 96 ° C. 30 seconds, 4 cycles of 68 ° C. 45 seconds, 96 ° C. 30 seconds, 64 ° C. 30 seconds, 5 cycles of 72 ° C. 45 seconds, 96 A cycle of 30 ° C. for 30 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 45 seconds was repeated 20 times, and finally the temperature was kept at 72 ° C. for 10 minutes. Confirmation of the amplification product was performed by ethidium bromide staining after 3% agarose gel electrophoresis.
The PCR reaction solution was separated by 3% low-melting-point agarose gel electrophoresis, the band was cut out with a razor, and the DNA was recovered using QIAquick \ Gel \ Extraction \ Kit (Qiagen). The recovered DNA was subcloned into the plasmid vector pCR2.1-TOPO according to the instructions of the TOPO @ TA cloning kit (Invitrogen). This was introduced into Escherichia coli TOP10 (Invitrogen) for transformation, and clones having cDNA inserts were selected on LB agar medium containing ampicillin and X-gal, and only white clones were sterilized using a toothpick. To obtain a transformant. Individual clones were cultured overnight in LB medium containing ampicillin, and plasmid DNA was prepared using QIAwell \ 8 \ Plasmid \ KIt (Qiagen). The reaction for determining the nucleotide sequence was performed using DyeDeoxyTerminator \ Cycle \ Sequence \ Kit (PE \ Biosystems), and the DNA sequence was decoded using a fluorescent automatic sequencer to obtain the DNA sequence shown in SEQ ID NO: 14. As expected, a part of the GPR8 ligand peptide precursor protein translated from this sequence (SEQ ID NO: 15) has a peptide sequence corresponding to the active peptide isolated from porcine hypothalamus and identified. did. Further, an Arg-Arg sequence (Seidah, N. G. et al., Ann. N. Y. Acad. Sci., Vol. 839, 9) from which a normal bioactive peptide is considered to be cut out at the C-terminal side. -24, 1998). From this, it was estimated that the amino acid sequence of the human homolog of the ligand peptide of GPR8 was either or both of SEQ ID NOs: 16 and 17.
[0095]
Reference Example 12 {Fmoc-modified human GPR8 {ligand} (1-23): Fmoc-Trp-Tyr-Lys-His-Val-Ala-Ser-Pro-Arg-Tyr-His-Thr-Val-Gly-Arg-Ala-Ala- Gly-Leu-Leu-Met-Gly-Leu (SEQ ID NO: 105) and human GPR8 {ligand} (1-23): Trp-Tyr-Lys-His-Val-Ala-Ser-Pro-Arg-Tyr-His-Thr Production of -Val-Gly-Arg-Ala-Ala-Gly-Leu-Leu-Met-Gly-Leu (SEQ ID NO: 16)
Fmoc-Leu-O-Clt-resin (0.76 mmol / g) obtained by introducing Fmoc-Leu into commercially available {2-chlorotrityl-resin} (Clt-resin, 1.33 mmol / g) {0.25 mmol} as a starting material, and a peptide synthesizer ABI 433A Fmoc- Gly, −Fmoc-Met, Fmoc-Leu, Fmoc-Leu, Fmoc-Gly, Fmoc-Ala, Fmoc-Ala, Fmoc-Fmoc-Fmoc-Fmoc-Fmoc-Fmoc-Fmoc-Fmoc-Fmoc-Fmoc-Fmoc-Fmoc-Fmoc-Fmoc-Fmoc-Fmoc-Fmoc-Fmoc-Fmoc-Fmoc-Fmoc-Fmoc-Fmoc-Fmoc-Fmoc-Fmoc-Fmog-Fmoc-Fmoc-Fmoc-Fmoc-Fmoc-Fmoc-Fmoc-Fmoc-Fmoc-Fmog-Fmoc-Fmoc-Fmog-Fmoc-Fmoc-Fmog-Fmoc-Fmog-Fmoc-Fmog-Fmoc-Fmog-Fmoc-Fmoc-Fmog-Fmoc-Fmoc-Fmoc-Fgmo-Fgmo). −Val, ΔFmoc-Thr (Bu^t), Fmoc-His (Trt), Fmoc-Tyr (Bu^t), Fmoc-Arg (Pbf), Fmoc-Pro, Fmoc-Ser (Bu^t), Fmoc-Ala, Fmoc-Val, Fmoc-His (Trt), Fmoc-Lys (Boc), Fmoc-Tyr (Bu)^t), Fmoc-Trp (Boc) and then Fmoc-Trp (Boc) -Tyr (Bu^t) -Lys (Boc) -His (Trt) -Val-Ala-Ser (Bu^t) -Pro-Arg (Pbf) -Tyr (Bu^t) -His (Trt) -Thr (Bu^t) -Val-Gly-Arg (Pbf) -Ala-Ala-Gly-Leu-Leu-Met-Gly-Leu-O-Clt {resin} 830 mg. To 150 mg of this resin was added {TFA} / {thioanisole} / {m-cresol} / {triisopropylsilane} / {ethanethithiol} (85/5/5 / 2.5 / 2.5) 5 ml. After concentration, ether was added thereto and crude Fmoc-Trp-Tyr-Lys-His-Val-Ala-Ser-Pro-Arg-Tyr-His-Thr-Val-Gly-Arg-Ala-Ala-Gly-Leu-Leu- Met-Gly-Leu was obtained as a precipitate. This was subjected to preparative HPLC using a {YMC} D-ODS-5-ST {S-5} 120A column (20 mm x 150 mm), and solution A: 0.1% TFA-water, solution B: acetonitrile containing 0.1% TFA. A / B: A linear concentration gradient elution was performed from # 72/28 to 52/48 (60 minutes), and the fractions containing the desired product were collected and freeze-dried to obtain 9.7 mg of a white powder.
[0096]
By mass spectrometry (M + H)⁺{2805.7} (calculated value 2805.4)
HPLC elution time 25.1 minutes
Elution conditions
Column Wakosil-II 5C18 HG (4.6 x 100 mm)
Eluent: Solution A: {0.1% TFA-water, Solution B: Using acetonitrile containing 0.1% TFA, {A / B: {100} / {0} to {30} / {70} linear concentration gradient elution (35 minutes)
Flow rate {1.0 ml / min} obtained {Fmoc-Trp-Tyr-Lys-His-Val-Ala-Ser-Pro-Arg-Tyr-His-Thr-Val-Gly-Arg-Ala-Ala-Gly-Leu-Leu {20% {diethylamine} / {DMF} 1} mL was added to -Met-Gly-Leu 5mg, and the mixture was stirred at room temperature for 2 hours. After evaporating the solvent, preparative HPLC using a YMC D-ODS-5-ST S-5 120A column (20 x 150 mm), liquid A: 0.1% TFA-water, liquid B: 0.1% TFA The linear concentration gradient elution was performed (60 minutes) to A / B: 74/26 to 64/36 with the contained acetonitrile, and the fractions containing the target compound were collected and freeze-dried to obtain 1.2 mg of a white powder.
By mass spectrometry (M + H)⁺{2583.6} (calculated value 2583.4)
HPLC elution time 20.4 minutes
Elution conditions
Column Wakosil-II 5C18 HG (4.6 x 100 mm)
Eluent: A solution: {0.1% TFA-water, B solution: Using acetonitrile containing 0.1% TFA, and {A / B: {100} / {0-30} / 70} linear concentration gradient elution (35 minutes)
Flow rate 1.0 ml / min
[0097]
Reference Example 13 {human GPR8 ligand} (1-30): Trp-Tyr-Lys-His-Val-Ala-Ser-Pro-Arg-Tyr-His-Thr-Val-Gly-Arg-Ala-Ala-Gly-Leu- Production of Leu-Met-Gly-Leu-Arg-Arg-Ser-Pro-Tyr-Leu-Trp (SEQ ID NO: 17)
Starting from Fmoc-Trp (Boc) -O-Clt @ resin (0.64 mmol / g) 0.25 mmol obtained by introducing Fmoc-Trp (Boc) into commercially available {2-chlorotrityl @ resin} (Clt @ resin, 1.33 mmol / g). Amino acids were condensed in the same sequence as in Reference Example 12, and after the last Trp was introduced and before the Fmoc group was removed from the resin, the Fmoc group was removed on the resin. /{5}/{2.5}/{2.5) to simultaneously cut out from the resin and remove the side-chain protecting group. The crude peptide was purified in the same manner as in Reference Example 12, and Trp-Tyr-Lys-His-Val-Ala-Ser-Pro-Arg-Tyr-His-Thr-Val-Gly-Arg-Ala-Ala-Gly-Leu. -Leu-Met-Gly-Leu-Arg-Arg-Ser-Pro-Tyr-Leu-Trp was obtained.
By mass spectrometry (M + H)⁺{3543.4} (calculated value 3544.2)
HPLC elution time 21.5 minutes
Elution conditions
Column Wakosil-II 5C18 HG (4.6 x 100 mm)
Eluent: Solution A: {0.1% TFA-water, Solution B: Using acetonitrile containing 0.1% TFA, {A / B: {100} / {0} to {30} / {70} linear concentration gradient elution (35 minutes)
Flow rate 1.0 ml / min
[0098]
Reference Example 14 {human GPR8 ligand} (1-29): Trp-Tyr-Lys-His-Val-Ala-Ser-Pro-Arg-Tyr-His-Thr-Val-Gly-Arg-Ala-Ala-Gly-Leu- Production of Leu-Met-Gly-Leu-Arg-Arg-Ser-Pro-Tyr-Leu (SEQ ID NO: 20)
Amino acids were condensed in the sequence in the same manner as in Reference Example 13 using the resin of Reference Example 12, cut out from the resin and purified, and then Trp-Tyr-Lys-His-Val-Ala-Ser-Pro-Arg-Tyr-His- Thr-Val-Gly-Arg-Ala-Ala-Gly-Leu-Leu-Met-Gly-Leu-Arg-Arg-Ser-Pro-Tyr-Leu is obtained.
[0099]
Reference Example 15 Human GPR8 ligand (1-28): Trp-Tyr-Lys-His-Val-Ala-Ser-Pro-Arg-Tyr-His-Thr-Val-Gly-Arg-Ala-Ala-Gly-Leu- Production of Leu-Met-Gly-Leu-Arg-Arg-Ser-Pro-Tyr (SEQ ID NO: 21)
Commercially available {2-chlorotrityl resin} (Clt resin, 1.33 mmol / g) and Fmoc-Tyr (Bu^t) Was introduced, amino acid was condensed in the sequence, cut out from the resin, purified and Trp-Tyr-Lys-His-Val-Ala-Ser-Pro-Arg-Tyr-His-Thr-Val in the same manner as in Reference Example 13. -Gly-Arg-Ala-Ala-Gly-Leu-Leu-Met-Gly-Leu-Arg-Arg-Ser-Pro-Tyr is obtained.
[0100]
Reference Example 16 {human GPR8 {ligand} (1-27): Trp-Tyr-Lys-His-Val-Ala-Ser-Pro-Arg-Tyr-His-Thr-Val-Gly-Arg-Ala-Ala-Gly-Leu- Production of Leu-Met-Gly-Leu-Arg-Arg-Ser-Pro (SEQ ID NO: 22)
After Fmoc-Pro was introduced into a commercially available {2-chlorotrityl resin} (Clt resin, 1.33 mmol / g), amino acids were condensed in the sequence and cut out from the resin in the same manner as in Reference Example 13, followed by purification, followed by Trp-Tyr-Lys-His. -Val-Ala-Ser-Pro-Arg-Tyr-His-Thr-Val-Gly-Arg-Ala-Ala-Gly-Leu-Leu-Met-Gly-Leu-Arg-Arg-Ser-Pro is obtained.
[0101]
Reference Example 17 human GPR8 ligand (1-26): Trp-Tyr-Lys-His-Val-Ala-Ser-Pro-Arg-Tyr-His-Thr-Val-Gly-Arg-Ala-Ala-Gly-Leu- Production of Leu-Met-Gly-Leu-Arg-Arg-Ser (SEQ ID NO: 23)
Commercially available {2-chlorotrityl resin} (Clt resin, 1.33 mmol / g) and Fmoc-Ser (Bu^t) Was introduced, amino acid was condensed in the sequence, cut out from the resin, purified and Trp-Tyr-Lys-His-Val-Ala-Ser-Pro-Arg-Tyr-His-Thr-Val in the same manner as in Reference Example 13. -Gly-Arg-Ala-Ala-Gly-Leu-Leu-Met-Gly-Leu-Arg-Arg-Ser is obtained.
[0102]
Reference Example 18 human GPR8 ligand (1-25): Trp-Tyr-Lys-His-Val-Ala-Ser-Pro-Arg-Tyr-His-Thr-Val-Gly-Arg-Ala-Ala-Gly-Leu- Production of Leu-Met-Gly-Leu-Arg-Arg (SEQ ID NO: 24)
After Fmoc-Arg (Pbf) was introduced into commercially available {2-chlorotrityl resin} (Clt resin, 1.33 mmol / g), the amino acid was condensed in the sequence and cut out from the resin in the same manner as in Reference Example 13, followed by purification, followed by Trp-Tyr-. Lys-His-Val-Ala-Ser-Pro-Arg-Tyr-His-Thr-Val-Gly-Arg-Ala-Ala-Gly-Leu-Leu-Met-Gly-Leu-Arg-Arg is obtained.
[0103]
Reference Example 19 {human GPR8 {ligand} (1-24): Trp-Tyr-Lys-His-Val-Ala-Ser-Pro-Arg-Tyr-His-Thr-Val-Gly-Arg-Ala-Ala-Gly-Leu- Production of Leu-Met-Gly-Leu-Arg (SEQ ID NO: 25)
After Fmoc-Arg (Pbf) was introduced into commercially available {2-chlorotrityl resin} (Clt resin, 1.33 mmol / g), the amino acid was condensed in the sequence and cut out from the resin in the same manner as in Reference Example 13, followed by purification, followed by Trp-Tyr-. Lys-His-Val-Ala-Ser-Pro-Arg-Tyr-His-Thr-Val-Gly-Arg-Ala-Ala-Gly-Leu-Leu-Met-Gly-Leu-Arg is obtained.
[0104]
Reference Example 20 GTPγS binding promoting activity of a human homolog of a 23-residue GPR8 ligand peptide measured using a GPR8-expressing CHO cell membrane fraction
The GPR8-expressing CHO cell membrane fraction was synthesized at the various concentrations of the human homologue of the GPR8 ligand peptide of 23 residues (hereinafter sometimes referred to as hGPR8L (1-23)) synthesized in Reference Example 12 at various concentrations. And the GTPγS binding promoting activity was measured. The results are shown in FIG. Clearly, hGPR8L (1-23) promoted GTPγS binding of the GPR8-expressing CHO cell membrane fraction in a concentration-dependent manner. This revealed that the peptide having the structure of SEQ ID NO: 16 was a ligand for GPR8.
[0105]
Reference Example 21 GTPγS binding promoting activity of a human homolog of a 30-residue GPR8 ligand peptide measured using a GPR8-expressing CHO cell membrane fraction
GPR8-expressing CHO cell membrane fractions obtained by the method described in Reference Example 6 at various concentrations of a human homolog of a 30-residue GPR8 ligand peptide synthesized in Reference Example 13 (hereinafter sometimes referred to as hGPR8L (1-30)). And the GTPγS binding promoting activity was measured. The results are shown in FIG. Clearly, hGPR8L (1-30) promoted GTPγS binding of GPR8-expressing CHO cell membrane fraction in a concentration-dependent manner. This revealed that the peptide having the structure of SEQ ID NO: 17 was a ligand for GPR8.
[0106]
Reference Example 22 Inhibitory activity of intracellular cAMP production of a human homologue of a GPR8 ligand peptide of 23 residues measured using GPR8-expressing CHO cells
HGPR8L (1-23) synthesized in Reference Example 12 was brought into contact with GPR8-expressing CHO cells at various concentrations by the method described in Reference Example 5, and the intracellular cAMP production inhibitory activity was measured. The results are shown in FIG. Clearly, hGPR8L (1-23) suppressed the production of intracellular cAMP in GPR8-expressing CHO cells in a concentration-dependent manner. In the figure, the cAMP synthesis inhibitory activity is defined as hGPR8L (1), where the amount obtained by subtracting the amount of intracellular cAMP when the reaction buffer is added from the amount of intracellular cAMP when the reaction buffer containing forskolin is added is 100%. The amount obtained by subtracting the amount of intracellular cAMP when the reaction buffer was added from the amount of intracellular cAMP when -23) was added was expressed as%.
[0107]
Reference Example 23 Activity for Inhibiting Intracellular cAMP Production of a 30-Residue GPR8 Ligand Peptide Human Homolog Measured Using GPR8-Expressing CHO Cells
HGPR8L (1-30) synthesized in Reference Example 13 was brought into contact with GPR8-expressing CHO cells at various concentrations by the method described in Reference Example 5, and the intracellular cAMP production inhibitory activity was measured. The results are shown in FIG. Apparently, hGPR8L (1-30) inhibited the production of intracellular cAMP in GPR8-expressing CHO cells in a concentration-dependent manner. In FIG. 5, cAMP synthesis inhibitory activity is calculated by subtracting the amount of intracellular cAMP when the reaction buffer is added from the amount of intracellular cAMP when the reaction buffer containing forskolin is added to 100% by using hGPR8L ( The amount obtained by subtracting the amount of intracellular cAMP when the reaction buffer was added from the amount of intracellular cAMP when 1-30) was added was expressed as%.
[0108]
Reference Example 24 Cloning of cDNA Encoding Mouse TGR26
The mouse TGR26 DNA was amplified by a PCR method using the synthetic primer of SEQ ID NO: 135 and the synthetic primer of SEQ ID NO: 136 using the mouse brain cDNA as a template.
The composition of the reaction solution was 1 μl of mouse brain cDNA, 0.2 μM each of synthetic primers, 0.8 μm dNTPs, Advantage cDNA cDNA Polymerase Mix (CLONTECH) 0.4 μl, and a buffer attached to the enzyme to make a total reaction volume of 20 μl. After heating at 96 ° C. for 2 minutes using a thermal cycler (Applied Biosystems), a heating cycle at 96 ° C. for 30 seconds, 64 ° C. for 30 seconds, and 72 ° C. for 1 minute was repeated 30 times, and finally at 72 ° C. It was kept warm for 10 minutes. The amplified DNA fragment of about 1100 bases in the PCR reaction solution was cloned into pCR 2.1-TOPO according to the protocol of TOPO TA Cloning Kit II (Invitrogen). This was introduced into Escherichia coli (Escherichia coli) TOP10 competent cell (Invitrogen) for transformation, and clones having cDNA inserts were selected on LB agar medium containing ampicillin and X-gal, and only white clones were obtained. Was isolated using a sterilized toothpick to obtain a transformant. Individual clones were cultured overnight in LB medium containing ampicillin, and plasmid DNA was prepared using QIAwell \ 8 \ Plasmid \ Kit (Qiagen). The reaction for determining the nucleotide sequence was carried out using BigDye Terminator Cycle Sequencing Ready Ready Kit (PE Biosystems), followed by decoding using a fluorescent automatic sequencer to obtain the nucleotide sequence represented by SEQ ID NO: 137.
A sequence obtained by translating the nucleotide sequence represented by SEQ ID NO: 137 into an amino acid sequence is referred to as a mouse TGR26 amino acid sequence, and is shown as SEQ ID NO: 138.
When SEQ ID NO: 138 was compared with the amino acid sequence of TGR26 derived from rat, a 96.0% amino acid identity was observed.
Escherichia coli transformed with a plasmid containing the DNA having the nucleotide sequence of SEQ ID NO: 137 was designated as Escherichia coli TOP10 / pCR2.1-TOPO mouse GPR7.
[0109]
Reference Example 25 Cloning of 5 'upstream end of cDNA encoding human GPR8 ligand precursor protein
A primer prepared based on a human cDNA sequence (SEQ ID NO: 14) encoding a part of a precursor protein of a human homolog of a ligand peptide of GPR8 described in Reference Example 11 (hereinafter sometimes referred to as human GPR8 ligand) 5′５RACE PCR was performed using human hypothalamus cDNA as a template to reveal the 5 ′ upstream nucleotide sequence of cDNA encoding human GPR8 ligand precursor protein. 5′RACE PCR cloning is performed by using the human hypothalamus Marathon-Ready cDNA (CLONTECH) as a template, performing a PCR reaction with the AP1 primer attached to the kit and the synthetic primer of SEQ ID NO: 33, and then using this PCR reaction solution as a template. Was achieved by performing a PCR reaction using the AP2 primer attached to the above and the synthetic primer of SEQ ID NO: 34. The reaction solution composition and reaction conditions for PCR are as follows. The reaction solution was 4 μl of the human hypothalamus cDNA, 0.5 μM of AP1 primer, 0.5 μM of synthetic DNA primer of SEQ ID NO: 33, 0.4 μm of dNTPs, 0.2 μl of LATaq polymerase (Takara Shuzo) and GC (I) attached to the enzyme. The total reaction volume was adjusted to 20 µl with a buffer, and after heating at 96 ° C for 120 seconds using a thermal cycler (PE Biosystems), a cycle of 96 ° C for 30 seconds and 68 ° C for 240 seconds was repeated 30 times. It was kept warm for 10 minutes. Next, 2 µl of a PCR reaction solution diluted 50-fold with Tricine-EDTA Buffer attached to the kit, 0.5 µM of AP2 primer, 0.5 µM of synthetic DNA primer of SEQ ID NO: 34, 0.4 µm dNTPs, LATaq polymerase (Takara Shuzo) The total reaction volume was adjusted to 20 μl with 0.2 μl and the GC (I) buffer attached to the enzyme, and after heating at 96 ° C. for 120 seconds using a thermal cycler (PE Biosystems), 96 ° C. for 30 seconds, 72 ° C. for 180 seconds. Cycle of 4 seconds, then 4 cycles of 96 ° C./30 seconds, 70 ° C./180 seconds, then 17 cycles of 96 ° C./30 seconds, 68 ° C./180 seconds, and finally at 72 ° C. It was kept warm for 10 minutes. After the amplified DNA was separated by 1.2% agarose gel electrophoresis, a DNA having a length of about 1,200 bases was cut out with a razor, and the DNA was recovered using QIAquick \ Gel \ Extraction \ Kit (Qiagen). This DNA was cloned into the pCR2.1-TOPO vector according to the protocol of TOPO \ TA \ Cloning \ Kit \ (Invitrogen). This was introduced into Escherichia coli (Escherichia coli) TOP10 competent cell (Invitrogen) for transformation, and clones having cDNA inserts were selected on LB agar medium containing ampicillin and X-gal, and only clones exhibiting white color were selected. Separation was performed using a sterilized toothpick to obtain a transformant. Individual clones were cultured overnight in LB medium containing ampicillin, and plasmid DNA was prepared using QIAwell \ 8 \ Plasmid \ Kit (Qiagen). The reaction for determining the base sequence was performed using BigDye \ Terminator \ Cycle \ Sequencing \ Ready \ Reaction \ Kit \ (PE \ Biosystems), followed by decoding using a fluorescent automatic sequencer to obtain the DNA sequence shown in SEQ ID NO: 35.
[0110]
Reference Example 26 Preparation of human brain cDNA
Human brain cDNA is available from Marathon®^TMIt was prepared from human brain poly {A (+)} RNA} (CLONTECH) by using cDNA {Amplification Kit} (CLONTECH). CDNA to be subjected to RACE @ PCR was prepared according to the protocol attached to the kit, except for the synthesis of 1st @ strand @ cDNA. 1st strand cDNA was synthesized from 1 μg of human brain poly A (+) RNA using reverse transcriptase MMLV (-RNAse H) (RevTraAce, Toyobo) instead of reverse transcriptase AMV attached to the kit.
[0111]
Reference Example 27 Cloning of 3 'Downstream End of cDNA Encoding Human GPR8 Ligand Precursor Protein
3 ′ RACE PCR using human brain cDNA as a template was performed with primers prepared based on the human cDNA sequence (SEQ ID NO: 14) encoding a part of the human GPR8 ligand precursor protein described in Reference Example 11 to obtain human GPR8 The 3 'downstream nucleotide sequence of the cDNA encoding the ligand was determined. 3′RACE @ PCR cloning is performed by using human brain cDNA as a template, performing a PCR reaction with the AP1 primer attached to the kit and the synthetic primer of SEQ ID NO: 36, and then using this PCR reaction solution as a template and the AP2 primer attached to the kit. This was achieved by performing a PCR reaction with the synthetic primer of No. 37. The reaction solution composition and reaction conditions for PCR are as follows. The reaction solution was 1 μl of human brain cDNA diluted 50-fold with Tricine-EDTA Buffer attached to the kit, 0.5 μM of AP1 primer, 0.5 μM of synthetic DNA primer of SEQ ID NO: 36, 0.4 μm dNTPs, LATaq polymerase (Takara Shuzo) The total reaction volume was adjusted to 20 μl with 0.2 μl and the GC (I) buffer attached to the enzyme, and after heating at 96 ° C. for 120 seconds using a thermal cycler (PE Biosystems), 96 ° C./30 seconds, 68 ° C./240 The second cycle was repeated 30 times, and the temperature was further kept at 72 ° C. for 10 minutes. Next, 1 μl of a PCR reaction solution diluted 50-fold with Tricine-EDTA Buffer attached to the kit, 0.5 μM of AP2 primer, 0.5 μM of synthetic DNA primer of SEQ ID NO: 37, 0.4 μm dNTPs, LATaq polymerase (Takara Shuzo) The total reaction volume was adjusted to 20 μl with 0.2 μl and the GC (I) buffer attached to the enzyme, and after heating at 96 ° C. for 120 seconds using a thermal cycler (PE Biosystems), 96 ° C. for 30 seconds, 72 ° C. for 180 seconds. Cycle of 4 seconds, then 4 cycles of 96 ° C./30 seconds, 70 ° C./180 seconds, then 17 cycles of 96 ° C./30 seconds, 68 ° C./180 seconds, and finally at 72 ° C. It was kept warm for 10 minutes. After the amplified DNA was separated by 1.5% agarose gel electrophoresis, a DNA of about 600 bases was cut out with a razor, and the DNA was recovered using QIAquick \ Gel \ Extraction \ Kit (Qiagen). This DNA was cloned into the pCR2.1-TOPO vector according to the protocol of TOPO \ TA \ Cloning \ Kit \ (Invitrogen). This was introduced into Escherichia coli (Escherichia coli) TOP10 competent cells (Invitrogen) and transformed, and clones having cDNA inserts were selected on LB agar medium containing ampicillin and X-gal, and only white clones were selected. Separation was performed using a sterilized toothpick to obtain a transformant. Individual clones were cultured overnight in LB medium containing ampicillin, and plasmid DNA was prepared using QIAwell \ 8 \ Plasmid \ Kit (Qiagen). The reaction for determining the nucleotide sequence was carried out using BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE Biosystems), followed by decoding using a fluorescent automatic sequencer to obtain the DNA sequence shown in SEQ ID NO: 38.
[0112]
Reference Example 28 Cloning of cDNA encoding human GPR8 ligand precursor protein
Using the human hypothalamus cDNA as a template, a primer prepared based on the 5 ′ upstream base sequence of the cDNA encoding the human GPR8 ligand precursor protein and the 3 ′ downstream base sequence of the cDNA encoding the human GPR8 ligand precursor protein By carrying out PCR amplification using the prepared primers, a cDNA encoding human GPR8 ligand precursor protein was cloned. The reaction solution composition and reaction conditions for PCR are as follows. The reaction solution was human hypothalamus Marathon-Ready {cDNA} (CLONTECH) 1 μl, a synthetic DNA primer of SEQ ID NO: 39 0.5 μM, a synthetic DNA primer of SEQ ID NO: 40 0.5 μM, 0.4 mM dNTPs, 2.5 mM MgCl₂5% 反 応 DMSO, 0.2 μl of LATaq polymerase (Takara Shuzo) and a buffer attached to the enzyme to make the total reaction volume 20 μl, and after heating at 96 ° C./60 seconds using a thermal cycler (PE @ Biosystems), 96 ° C./30 seconds The cycle of 30 seconds, 64 ° C. for 30 seconds, and 72 ° C. for 120 seconds was repeated 35 times, and finally, the temperature was kept at 72 ° C. for 10 minutes. After the amplified DNA was separated by 1.5% agarose gel electrophoresis, a DNA of about 700 bases was cut out with a razor, and the DNA was recovered using QIAquick \ Gel \ Extraction \ Kit (Qiagen). This DNA was cloned into the pCR2.1-TOPO vector according to the protocol of TOPO \ TA \ Cloning \ Kit \ (Invitrogen). This was introduced into Escherichia coli (Escherichia coli) TOP10 competent cells (Invitrogen) and transformed, and clones having cDNA inserts were selected on LB agar medium containing ampicillin and X-gal, and only white clones were selected. Separation was performed using a sterilized toothpick to obtain a transformant. Individual clones were cultured overnight in LB medium containing ampicillin, and plasmid DNA was prepared using QIAwell \ 8 \ Plasmid \ Kit (Qiagen). The reaction for determining the nucleotide sequence was carried out using BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE Biosystems), followed by decoding using a fluorescent automatic sequencer to obtain the DNA sequence shown in SEQ ID NO: 41.
This sequence (SEQ ID NO: 41) encodes human GPR8 ligand precursor protein and transforms E. coli transformed with a plasmid containing this DNA into TOP10 / pCR2.1-TOPO human GPR8 ligand precursor (Escherichia coli TOP10 / pCR2.1). -TOPO \ Human \ GPR8 \ Ligand \ Precursor).
In the DNA sequence shown in SEQ ID NO: 41, a frame encoding the amino acid sequence of the human GPR8 ligand peptide described in Reference Example 11 exists, but it is considered that the 5 'upstream side is the initiation codon for protein translation. There is no expected ATG. However, it has been reported that some proteins are predicted to use a codon other than ATG as an initiation codon in some proteins (human basic fibroblast growth factor (H. {Prats et al., Proc. Natl. @ Acad. @ Sci. @ USA, 86, pp. 1836-1840, 1989, R. @ Z.Flokiewicz and A. @ Somer, Proc. Natl. @ Acad. @ Sci. @ USA, 86, 3978-3981, 1989). Mouse retinoic acid receptor β4 (S. Nagpal et al., Proc. Natl. Acad. Sci. USA, 89, 2718, 1992), human phosphoribosyl pyrophosphate synthase (M. Taira et al., J. @Biol Chem., 265, pp. 16491-16497, 1990), Drosophila choline acetyltransferase (H. Sugihara et al., J. Biol. Chem., 265, pp. 21714-21719, 1990)).
In these examples, CTG encoding Leu is often assumed as the start codon instead of ATG. It is considered that the same is true for the human GPR8 ligand precursor protein. Based on a comparison with the precursor protein of the pig or rat GPR8 ligand homolog described below, a position almost corresponding to the ATG predicted as the initiation codon in these precursor proteins is considered. The sequence of the precursor protein was deduced, assuming the CTG codon present as the start codon. The amino acid sequence of this hypothetical human GPR8 ligand precursor protein is shown in SEQ ID NO: 42. In addition, FIG. 6 shows the amino acid sequence and DNA sequence of the hypothetical human GPR8 ligand precursor protein.
[0113]
Reference Example 29 Preparation of Porcine Spinal Cord cDNA
Porcine spinal cord cDNA was purchased from Marathon®^TMIt was prepared from porcine spinal cord poly A (+) RNA using cDNA Amplification Kit Kit (CLONTECH). Porcine spinal cord poly {A (+)} RNA was prepared from porcine spinal cord as follows. The porcine spinal cord was completely crushed with a polytron homogenizer in ISOGEN (Nippon Gene), and porcine spinal cord total RNA was obtained from the crushed solution according to a total RNA preparation method using an ISOGEN solution. Next, 7 μg of pig spinal cord poly (A) (+) RNA was obtained from the pig spinal cord total RNA by performing chromatography twice using an oligo (d) cellulose column attached to mRNA Purification Kit (Amersham Pharmacia Biotech). The cDNA subjected to RACE @ PCR was prepared according to the protocol attached to the kit, except for the synthesis of 1st @ strand @ cDNA. 1st strand cDNA was synthesized from 1 μg of porcine spinal cord poly A (+) RNA using reverse transcriptase MMLV (-RNAse H) (RevTraAce, Toyobo) instead of reverse transcriptase AMV attached to the kit.
[0114]
Reference Example 30 Cloning of 5 'upstream end of cDNA encoding porcine GPR8 ligand precursor protein
By the first 5′RACE PCR cloning and the second 5′RACE PCR cloning using the base sequence of the PCR-amplified DNA, the porcine homolog of the ligand peptide of GPR8 (hereinafter sometimes referred to as porcine GPR8 ligand) is obtained. The 5 'upstream nucleotide sequence of the cDNA encoding the precursor protein was revealed.
In the first 5′RACE PCR cloning, a PCR reaction was performed using the above-mentioned porcine spinal cord cDNA as a template and the AP1 primer attached to the kit and the synthetic primer of SEQ ID NO: 43. This was achieved by performing a PCR reaction with the AP2 primer and the synthetic primer of SEQ ID NO: 44. The reaction solution composition and reaction conditions for PCR are as follows. The reaction solution was 4 μl of swine spinal cord cDNA diluted 50-fold with Tricine-EDTA Buffer attached to the kit, AP1 primer 0.5 μM, synthetic DNA primer 0.5 μM of SEQ ID NO: 43, 0.4 μmM dNTPs, LATaq polymerase (Takara Shuzo) The total reaction volume was adjusted to 20 μl with 0.2 μl and the GC (I) buffer attached to the enzyme, and after heating at 96 ° C. for 120 seconds using a thermal cycler (PE Biosystems), 96 ° C. for 30 seconds, and 68 ° C. The second cycle was repeated 30 times, and the temperature was further kept at 72 ° C. for 10 minutes. Next, 1 μl of a PCR reaction solution diluted 100-fold with Tricine-EDTA Buffer attached to the kit, 0.5 μM of AP2 primer, 0.5 μM of synthetic DNA primer of SEQ ID NO: 44, 0.4 μm dNTPs, Advantage-GC 2 polymerase (CLONTECH) The total reaction volume was adjusted to 20 µl with 0.2 µl and the buffer attached to the enzyme, and after heating at 96 ° C for 60 seconds using a thermal cycler (PE Biosystems), 96 ° C for 30 seconds, 72 ° C for 180 seconds. Cycle, then three cycles of 96 ° C./30 seconds, 70 ° C./180 seconds, then four cycles of 96 ° C./30 seconds, 68 ° C./180 seconds, then 96 ° C./30 seconds Cycle of 30 seconds, 64 ° C for 30 seconds, and 72 ° C for 180 seconds is repeated 15 times, and finally kept at 72 ° C for 10 minutes did. After separating the amplified DNA by 1.2% agarose gel electrophoresis, a DNA having a length of about 300 bases was cut out with a razor, and the DNA was recovered using QIAquick \ Gel \ Extraction \ Kit (Qiagen). This DNA was cloned into the pCR2.1-TOPO vector according to the protocol of TOPO \ TA \ Cloning \ Kit \ (Invitrogen). This was introduced into Escherichia coli (Escherichia coli) TOP10F 'competent cell (Invitrogen) to transform the clone, and a clone having a cDNA insert was selected on an LB agar medium containing ampicillin, IPTG and X-gal to give a white color. Only the clone was separated using a sterilized toothpick to obtain a transformant. Individual clones were cultured overnight in LB medium containing ampicillin, and plasmid DNA was prepared using QIAwell \ 8 \ Plasmid \ Kit (Qiagen). The reaction for determining the base sequence was performed using BigDye \ Terminator \ Cycle \ Sequencing \ Ready \ Reaction \ Kit \ (PE \ Biosystems), followed by decoding using a fluorescent automatic sequencer to obtain the DNA sequence shown in SEQ ID NO: 45.
In the second 5′RACE PCR cloning, a PCR reaction was performed using the porcine spinal cord cDNA as a template and the AP1 primer attached to the kit and the synthetic primer of SEQ ID NO: 46, and then the PCR reaction solution was used as a template to prepare the AP2 attached to the kit. This was achieved by performing a PCR reaction with the primer and the synthetic primer of SEQ ID NO: 47. The reaction solution composition and reaction conditions for PCR are as follows. The reaction solution was 1 μl of porcine spinal cord cDNA diluted 50-fold with Tricine-EDTA buffer attached to the kit, 0.5 μM of AP1 primer, 0.5 μM of synthetic DNA primer of SEQ ID NO: 46, 0.4 mM of dNTPs, Advantage-GC 2 The total reaction volume was adjusted to 20 μl with 0.2 μl of polymerase (CLONTECH) and a buffer attached to the enzyme. After heating at 96 ° C. for 60 seconds using a thermal cycler (PE Biosystems), 96 ° C. for 30 seconds, 72 ° C. for 180 seconds 5 cycles of 96 seconds for 30 seconds and 70 degrees C for 180 seconds, then 20 cycles of 30 seconds for 96 degrees C and 68 seconds for 180 seconds, and finally 72 degrees C For 10 minutes. Next, 1 μl of a PCR reaction solution diluted 100-fold with Tricine-EDTA Buffer attached to the kit, 0.5 μM of AP2 primer, 0.5 μM of synthetic DNA primer of SEQ ID NO: 47, 0.4 μmｓdNTPs, Advantage-GC 2 polymerase (CLONTECH) Make the total reaction volume 20 μl with 0.2 μl and the buffer attached to the enzyme, use a thermal cycler (PE Biosystems), heat at 96 ° C. for 60 seconds, then 96 ° C. for 30 seconds, 68 ° C. for 180 seconds This cycle was repeated 31 times, and finally, the temperature was kept at 72 ° C. for 10 minutes. After the amplified DNA was separated by 2.0% agarose gel electrophoresis, a DNA having a length of about 200 bases was cut out with a razor, and the DNA was recovered using QIAquick \ Gel \ Extraction \ Kit (Qiagen). This DNA was cloned into the pCR2.1-TOPO vector according to the protocol of TOPO \ TA \ Cloning \ Kit \ (Invitrogen). This was introduced into Escherichia coli (Escherichia coli) TOP10F 'competent cell (Invitrogen) for transformation, and a clone having a cDNA insert was selected on an LB agar medium containing ampicillin, IPTG and X-gal, and exhibited white. Only the clone was separated using a sterilized toothpick to obtain a transformant. Individual clones were cultured overnight in LB medium containing ampicillin, and plasmid DNA was prepared using QIAwell \ 8 \ Plasmid \ Kit (Qiagen). The reaction for determining the nucleotide sequence was carried out using BigDyeTerminator \ Cycle \ Sequencing \ Ready \ Reaction \ Kit (PE \ Biosystems), followed by decoding using a fluorescent automatic sequencer to obtain the DNA sequence shown in SEQ ID NO: 48.
[0115]
Reference Example 31 Cloning of 3 'Downstream End of cDNA Encoding Porcine GPR8 Ligand Precursor Protein
3 ′ RACE PCR cloning using a primer prepared based on the base sequence at the 5 ′ upstream end of the cDNA encoding the porcine GPR8 ligand precursor protein, 3 ′ downstream of the cDNA encoding the porcine GPR8 ligand peptide precursor protein The nucleotide sequence was determined. 3′RACE PCR cloning is performed by performing a PCR reaction using the porcine spinal cord cDNA as a template and the AP1 primer attached to the kit and the synthetic primer of SEQ ID NO: 49, and then using this PCR reaction solution as a template and the AP2 primer attached to the kit. This was achieved by performing a PCR reaction with the synthetic primer of No. 50. The reaction solution composition and reaction conditions for PCR are as follows. The reaction solution was 1 μl of porcine spinal cord cDNA diluted 50-fold with Tricine-EDTA Buffer attached to the kit, 0.5 μM of AP1 primer, 0.5 μM of synthetic DNA primer of SEQ ID NO: 49, 0.4 μmM dNTPs, Advantage-GC ポ リ メ ラ ー ゼ 2 polymerase. (CLONTECH) Make the total reaction volume 20 μl with 0.2 μl and the buffer attached to the enzyme, use a thermal cycler (PE Biosystems), heat at 96 ° C. for 60 seconds, then 96 ° C. for 30 seconds, 72 ° C. for 120 seconds Cycle of 96 ° C./30 seconds, 70 ° C./120 seconds 5 times, then a cycle of 96 ° C./30 seconds, 68 ° C./120 seconds 20 times, and finally at 72 ° C. It was kept warm for 10 minutes. Next, 1 μl of a PCR reaction solution diluted 100-fold with Tricine-EDTA Buffer attached to the kit, 0.5 μM of AP2 primer, 0.5 μM of synthetic DNA primer of SEQ ID NO: 50, 0.4 μm dNTPs, Advantage-GC 2 polymerase (CLONTECH) The total reaction volume was adjusted to 20 µl with 0.2 µl and the buffer attached to the enzyme, and after heating at 96 ° C for 120 seconds using a thermal cycler (PE Biosystems), 96 ° C for 30 seconds and 68 ° C for 120 seconds. This cycle was repeated 31 times, and finally, the temperature was kept at 72 ° C. for 10 minutes. After the amplified DNA was separated by 2.0% agarose gel electrophoresis, a DNA of about 650 bases was cut out with a razor, and the DNA was recovered using QIAquick \ Gel \ Extraction \ Kit (Qiagen). This DNA was cloned into the pCR2.1-TOPO vector according to the protocol of TOPO \ TA \ Cloning \ Kit \ (Invitrogen). This was introduced into Escherichia coli (Escherichia coli) / TOP10F '/ competent / cell (Invitrogen) for transformation, and clones having the cDNA insert were selected on LB agar medium containing ampicillin, X-gal and IPTG, and appeared white. Only the clone was separated using a sterilized toothpick to obtain a transformant. Individual clones were cultured overnight in LB medium containing ampicillin, and plasmid DNA was prepared using QIAwell \ 8 \ Plasmid \ Kit (Qiagen). The reaction for determination of the base sequence was performed using BigDye \ Terminator \ Cycle \ Sequencing \ Ready \ Reaction \ Kit \ (PE \ Biosystems) and decoded using a fluorescent automatic sequencer to obtain the DNA sequence shown in SEQ ID NO: 51.
[0116]
Reference Example 32 Cloning of cDNA Encoding Porcine GPR8 Ligand Precursor Protein
Using the porcine spinal cord cDNA as a template, a primer prepared based on the 5 ′ upstream base sequence of the cDNA encoding the porcine GPR8 ligand precursor protein and the 3 ′ downstream base sequence of the cDNA encoding the porcine GPR8 ligand precursor protein By carrying out PCR amplification using the primers thus obtained, cDNA encoding the porcine GPR8 ligand precursor protein was cloned. The reaction solution composition and reaction conditions for PCR are as follows. The reaction solution was 1 μl of porcine spinal cord cDNA diluted 50-fold with Tricine-EDTAＥＤBuffer attached to the kit, 0.5 μM of synthetic DNA primer of SEQ ID NO: 52, 0.5 μM of synthetic DNA primer of SEQ ID NO: 53, 0.4 μmM The total reaction volume was adjusted to 20 μl with dNTPs, Advantageｌ2 polymerase (CLONTECH) 0.2 μl and the buffer attached to the enzyme, and after heating at 96 ° C. for 60 seconds, using a thermal cycler (PE Biosystems) at 96 ° C. for 30 seconds. Four cycles of 72 ° C / 75 seconds, then four cycles of 96 ° C / 30 seconds, 70 ° C / 75 seconds, then four cycles of 96 ° C / 30 seconds, 68 ° C / 75 seconds, then 5 cycles of 96 ° C for 30 seconds, 64 ° C for 30 seconds, 72 ° C for 45 seconds, then 96 ° C for 30 seconds, 6 The cycle of 0 ° C. for 30 seconds and 72 ° C. for 45 seconds was repeated 20 times, and finally the temperature was kept at 72 ° C. for 10 minutes. After the amplified DNA was separated by 1.2% agarose gel electrophoresis, a DNA having a length of about 600 bases was cut out with a razor, and the DNA was recovered using QIAquick \ Gel \ Extraction \ Kit (Qiagen). This DNA was cloned into the pCR2.1-TOPO vector according to the protocol of TOPO \ TA \ Cloning \ Kit \ (Invitrogen). This was introduced into Escherichia coli (Escherichia coli) TOP10 competent cell (Invitrogen) for transformation, and clones having cDNA inserts were selected on LB agar medium containing ampicillin and X-gal, and only clones exhibiting white color were selected. Separation was performed using a sterilized toothpick to obtain a transformant. Individual clones were cultured overnight in LB medium containing ampicillin, and plasmid DNA was prepared using QIAwell \ 8 \ Plasmid \ Kit (Qiagen). The reaction for determining the base sequence was performed using BigDye \ Terminator \ Cycle \ Sequencing \ Ready \ Reaction \ Kit (PE \ Biosystems), followed by decoding using a fluorescent automatic sequencer to obtain the DNA sequence shown in SEQ ID NO: 54. This sequence (SEQ ID NO: 54) encodes porcine GPR8 ligand precursor protein and transforms E. coli transformed with a plasmid containing this DNA into TOP10 / pCR2.1-TOPO porcine GPR8 ligand precursor (Escherichia coli TOP10 / pCR2.1). -TOPO \ Porcine \ GPR8 \ Ligand \ Precursor).
The amino acid sequence of the porcine GPR8 ligand precursor protein encoded by the DNA sequence of SEQ ID NO: 54 is shown in SEQ ID NO: 55. The amino acid sequence of this precursor protein was determined from the amino terminus determined by amino acid sequence analysis of the GPR8 ligand peptide isolated from the porcine hypothalamus using the GTPγS binding activity for the GPR8-expressing cell membrane fraction described in Reference Example 10 as an index. There were sequences up to 17 residues. Further, on the carboxy-terminal side of the sequence, an Arg-Arg sequence (Seidah, {N. @ G. @ et @ al., Supra), which is considered to be excised from a normal bioactive peptide, as in the case of the human homolog precursor protein of the GPR8 ligand peptide. Ann. @ N. @ Y. @ Acad.Sci., 839, 9-24, 1998). From this, it was deduced that the amino acid sequence of the pig homolog of the GPR8 ligand peptide was either or both of SEQ ID NOs: 56 and 57. The amino acid sequence and DNA sequence of the porcine GPR8 ligand precursor protein are shown in FIG.
[0117]
Reference Example 33 Cloning of cDNA fragment encoding a part of rat GPR8 ligand precursor protein
As described in Reference Example 10, a database search was performed using the GTPγS binding activity to the GPR8-expressing cell membrane fraction as an index based on the 17 amino acid sequence (SEQ ID NO: 6) from the amino terminus of the peptide purified from porcine hypothalamus. And a rat EST nucleotide sequence (Accession No .: H31598) that matches the nucleotide sequence of SEQ ID NO: 11. This DNA sequence had a translation frame in which the sequence of 15 amino acids was identical to the amino acid sequence of the peptide purified from pig hypothalamus (SEQ ID NO: 6). This H31598 is an EST sequence derived from a cDNA library prepared from rat PC12 cells, and consists of 260 bases including 7 undetermined bases. Since H31598 was presumed to encode a part of the precursor protein of the rat homolog peptide of GPR8 ligand (hereinafter sometimes referred to as rat GPR8 ligand), H31598 was used to determine the exact nucleotide sequence. PCR cloning was performed using rat brain Marathon-Ready {cDNA} (CLONTECH) as a template with the respective primers prepared based on the 5 ′ base sequence and the 3 ′ base sequence. The reaction solution composition and reaction conditions for PCR are as follows. Rat brain Marathon cDNA (CLONTECH) 2 μl, synthetic DNA primer of SEQ ID NO: 60 0.5 μM, synthetic DNA primer of SEQ ID NO: 61 0.5 μM, 0.4 mM dNTPs, Advantage-GC 2 polymerase (CLONTECH) 0.2 μl The total reaction volume was adjusted to 20 μl with a buffer attached to the enzyme, and after heating at 96 ° C. for 60 seconds using a thermal cycler (PE Biosystems), then at 96 ° C. for 30 seconds, then at 60 ° C. for 30 seconds, at 72 ° C. The cycle of 60 seconds was repeated 35 times, and finally, the temperature was kept at 72 ° C. for 10 minutes. After the amplified DNA was separated by 4.0% agarose gel electrophoresis, a DNA of about 250 bases was cut out with a razor, and the DNA was recovered using QIAquick \ Gel \ Extraction \ Kit (Qiagen). This DNA was cloned into the pCR2.1-TOPO vector according to the protocol of TOPO \ TA \ Cloning \ Kit \ (Invitrogen). This was introduced into Escherichia coli (Escherichia coli) TOP10 competent cell (Invitrogen) for transformation, and clones having cDNA inserts were selected on LB agar medium containing ampicillin and X-gal, and only clones exhibiting white color were selected. Separation was performed using a sterilized toothpick to obtain a transformant. Individual clones were cultured overnight in LB medium containing ampicillin, and plasmid DNA was prepared using QIAwell \ 8 \ Plasmid \ Kit (Qiagen). The reaction for determining the nucleotide sequence was carried out using BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE Biosystems), followed by decoding using a fluorescent automatic sequencer to obtain the DNA sequence shown in SEQ ID NO: 62. Comparison of the nucleotide sequence of the PCR-cloned DNA (SEQ ID NO: 62) with the nucleotide sequence of H31598 revealed that a reading error of a single nucleotide deletion was present in the nucleotide sequence of H31598.
[0118]
Reference Example 34 Cloning of 5 'upstream end of cDNA encoding rat GPR8 ligand precursor protein
5 'RACE PCR cloning revealed the 5' upstream base sequence of the cDNA encoding the rat GPR8 ligand precursor protein. 5′RACE PCR cloning is carried out by using the rat brain Marathon-Ready cDNA (CLONTECH) as a template, performing a PCR reaction with the AP1 primer attached to the kit and the synthetic primer of SEQ ID NO: 63, and then using this PCR reaction solution as a template for the kit. This was achieved by performing a PCR reaction using the attached AP2 primer and the synthetic primer of SEQ ID NO: 64. The reaction solution composition and reaction conditions for PCR are as follows. The reaction solution was 2 μl of rat brain Marathon cDNA, 0.5 μM of AP1 primer, 0.5 μM of synthetic DNA primer of SEQ ID NO: 63, 0.4 μm dNTPs, 0.2 μl of LATaq polymerase (Takara Shuzo) and GC (I) attached to the enzyme. The total reaction volume was adjusted to 20 μl with a buffer, and after heating at 96 ° C. for 60 seconds using a thermal cycler (PE Biosystems), a cycle of 96 ° C. for 30 seconds and 68 ° C. for 120 seconds was repeated 30 times. It was kept warm for 10 minutes. Next, 2 μl of a PCR reaction solution diluted 200-fold with Tricine-EDTA Buffer attached to the kit, 0.5 μM of AP2 primer, 0.5 μM of synthetic DNA primer of SEQ ID NO: 64, 0.4 μmM dNTPs, Advantage-GC 2 polymerase (CLONTECH) Make the total reaction volume 20 μl with 0.2 μl and the buffer attached to the enzyme, and use a thermal cycler (PE Biosystems) for heating at 96 ° C. for 60 seconds, then at 96 ° C. for 30 seconds and at 68 ° C. for 120 seconds. The cycle was repeated 31 times and finally kept at 72 ° C. for 10 minutes. After the amplified DNA was separated by 1.2% agarose gel electrophoresis, a DNA having a length of about 600 bases was cut out with a razor, and the DNA was recovered using QIAquick \ Gel \ Extraction \ Kit (Qiagen). This DNA was cloned into the pCR2.1-TOPO vector according to the protocol of TOPO \ TA \ Cloning \ Kit \ (Invitrogen). This was introduced into Escherichia coli (Escherichia coli) TOP10 competent cell (Invitrogen) for transformation, and clones having cDNA inserts were selected on LB agar medium containing ampicillin and X-gal, and only clones exhibiting white color were selected. Separation was performed using a sterilized toothpick to obtain a transformant. Individual clones were cultured overnight in LB medium containing ampicillin, and plasmid DNA was prepared using QIAwell \ 8 \ Plasmid \ Kit (Qiagen). The reaction for determining the base sequence was performed using BigDye \ Terminator \ Cycle \ Sequencing \ Ready \ Reaction \ Kit \ (PE \ Biosystems), followed by decoding using a fluorescent automatic sequencer to obtain the DNA sequence shown in SEQ ID NO: 65.
[0119]
Reference Example 35 Cloning of 3 'downstream end of cDNA encoding rat GPR8 ligand precursor protein
Using a primer prepared based on the base sequence at the 5 'upstream end of the cDNA encoding the rat GPR8 ligand precursor protein and a primer prepared based on the cDNA fragment sequence encoding a part of the rat GPR8 ligand precursor protein 'RACE PCR cloning revealed the 3' downstream nucleotide sequence of the cDNA encoding the rat GPR8 ligand precursor protein. 3′RACE PCR cloning involves performing a PCR reaction using the rat brain Marathon-Ready cDNA (CLONTECH) as a template and the AP1 primer attached to the kit and the synthetic primer of SEQ ID NO: 66, and then using this PCR reaction solution as a template for the kit. This was achieved by performing a PCR reaction using the attached AP2 primer and the synthetic primer of SEQ ID NO: 67. The reaction solution composition and reaction conditions for PCR are as follows. The reaction solution was composed of rat brain Marathon-Ready cDNA 2 μl, AP1 primer 0.5 μM, synthetic DNA primer 0.5 μM of SEQ ID NO: 66, 0.4 mM mM dNTPs, Advantage-GC 2 polymerase (CLONTECH) 0.4 μl and enzyme. After adjusting the total reaction volume to 20 µl with the attached buffer and heating at 96 ° C for 60 seconds using a thermal cycler (PE Biosystems), a cycle of 96 ° C for 30 seconds and 68 ° C for 180 seconds was repeated 30 times. Incubated at 72 ° C. for 10 minutes. Next, 2 µl of a PCR reaction solution diluted 200-fold with Tricine-EDTA Buffer attached to the kit, 0.5 µM of AP2 primer, 0.5 µM of synthetic DNA primer of SEQ ID NO: 67, 0.4 µm dNTPs, Advantage-GC 2 polymerase (CLONTECH) Make the total reaction volume 20 μl with 0.4 μl and the buffer attached to the enzyme, and use a thermal cycler (PE Biosystems) to heat at 96 ° C. for 60 seconds, then 96 ° C. for 30 seconds, 68 ° C. for 180 seconds. Was repeated 30 times, and finally, the mixture was kept at 72 ° C. for 10 minutes. After the amplified DNA was separated by 1.2% agarose gel electrophoresis, a DNA having a length of about 600 bases was cut out with a razor, and the DNA was recovered using QIAquick \ Gel \ Extraction \ Kit (Qiagen). This DNA was cloned into the pCR2.1-TOPO vector according to the protocol of TOPO \ TA \ Cloning \ Kit \ (Invitrogen). This was introduced into Escherichia coli (Escherichia coli) TOP10 competent cells (Invitrogen) and transformed, and clones having cDNA inserts were selected on LB agar medium containing ampicillin and X-gal, and only white clones were selected. Separation was performed using a sterilized toothpick to obtain a transformant. Individual clones were cultured overnight in LB medium containing ampicillin, and plasmid DNA was prepared using QIAwell \ 8 \ Plasmid \ Kit (Qiagen). The reaction for determining the nucleotide sequence was performed using BigDyeTerminator \ Cycle \ Sequencing \ Ready \ Reaction \ Kit \ (PE \ Biosystems), followed by decoding using a fluorescent automatic sequencer to obtain the DNA sequence shown in SEQ ID NO: 68.
[0120]
Reference Example 36 Cloning of cDNA encoding rat GPR8 ligand precursor protein
Using the rat brain cDNA as a template, a primer based on the 5 'upstream base sequence of the cDNA encoding the rat GPR8 ligand precursor protein and a primer based on the 3' downstream base sequence of the cDNA encoding the rat GPR8 ligand precursor protein The cDNA encoding the rat GPR8 ligand precursor protein was cloned by performing PCR amplification as described above. The reaction solution composition and reaction conditions for PCR are as follows. The reaction solution was 1 μl of rat brain Marathon-Ready cDNA, 0.5 μM of the synthetic DNA primer of SEQ ID NO: 69, 0.5 μM of the synthetic DNA primer of SEQ ID NO: 70, 0.4 μmM dNTPs, Advantage® 2 polymerase (CLONTECH) 0. The total reaction volume was adjusted to 20 μl with 4 μl and a buffer attached to the enzyme, and after heating at 96 ° C. for 60 seconds using a thermal cycler (PE Biosystems), 96 ° C. for 30 seconds, 60 ° C. for 30 seconds, 72 ° C. for 60 seconds. The second cycle was repeated 35 times, and finally the plate was incubated at 72 ° C. for 10 minutes. After separating the amplified DNA by 1.2% agarose gel electrophoresis, a DNA of about 750 bases was cut out with a razor, and the DNA was recovered using QIAquick \ Gel \ Extraction \ Kit (Qiagen). This DNA was cloned into the pCR2.1-TOPO vector according to the protocol of TOPO \ TA \ Cloning \ Kit \ (Invitrogen). This was introduced into Escherichia coli (Escherichia coli) TOP10 competent cells (Invitrogen) and transformed, and clones having cDNA inserts were selected on LB agar medium containing ampicillin and X-gal, and only white clones were selected. Separation was performed using a sterilized toothpick to obtain a transformant. Individual clones were cultured overnight in LB medium containing ampicillin, and plasmid DNA was prepared using QIAwell \ 8 \ Plasmid \ Kit (Qiagen). The reaction for determining the nucleotide sequence was performed using BigDye \ Terminator \ Cycle \ Sequencing \ Ready \ Reaction \ Kit (PE \ Biosystems), followed by decoding using a fluorescent automatic sequencer to obtain the DNA sequence shown in SEQ ID NO: 71. This sequence (SEQ ID NO: 71) is derived from TOP10 / pCR2.1-TOPO rat GPR8 ligand precursor (Escherichia coli TOP10 / pCR2.E.) Transformed with a plasmid containing this DNA to encode the rat GPR8 ligand precursor protein. 1-TOPO Rat GPR8 LigandｓPrecursor).
The amino acid sequence of rat GPR8 ligand precursor protein encoded by the DNA sequence of SEQ ID NO: 71 is shown in SEQ ID NO: 72. The amino acid sequence of this precursor protein was determined from the amino terminus determined by amino acid sequence analysis of the GPR8 ligand peptide isolated from the porcine hypothalamus using the GTPγS binding activity for the GPR8-expressing cell membrane fraction described in Reference Example 10 as an index. There were similar sequences that differed only in the sequence up to 17 residues from the amino acids at residues 5 and 17. Further, at the carboxy-terminal side of the sequence, an Arg-Arg sequence (Seidah, N. G. et al), which is considered to be excised from a normal bioactive peptide, as in the case of the human or porcine homolog precursor protein of the GPR8 ligand peptide. , Ann. N. Y. Acad. Sci., 839, 9-24, 1998). From these, the amino acid sequence of the rat homolog of the GPR8 ligand peptide was deduced to be either or both of SEQ ID NOs: 73 and 74. The amino acid sequence and DNA sequence of rat GPR8 ligand precursor protein are shown in FIG.
[0121]
Reference Example 37 Cloning of cDNA Fragment Encoding a Part of Mouse GPR8 Ligand Precursor Protein
In order to obtain a precursor protein of a mouse homolog of the GPR8 ligand peptide (hereinafter sometimes referred to as mouse GPR8 ligand), PCR amplification was performed using mouse testis cDNA as a template, and the nucleotide sequence of the amplified DNA was determined. The reaction solution composition and reaction conditions for PCR are as follows. Mouse testis cDNA {(CLONTECH)} 1 μl, synthetic DNA primer of SEQ ID NO: 78 0.5 μM, synthetic DNA primer of SEQ ID NO: 79 0.5 μM, 0.4 mM mM dNTPs, 0.2 μl of LATaq polymerase (Takara Shuzo) and enzymes included The total reaction volume was adjusted to 20 μl with the GC (I) buffer described above, and the cycle of 96 ° C./120 seconds, followed by the cycle of 96 ° C./30 seconds and 68 ° C./120 seconds using a thermal cycler (PE Biosystems) was repeated 10 times. Then, a cycle of 96 ° C. for 30 seconds, 64 ° C. for 30 seconds, and 72 ° C. for 120 seconds was repeated 25 times, and finally, the temperature was kept at 72 ° C. for 10 minutes. After separating the amplified DNA by 1.5% agarose gel electrophoresis, a DNA having a length of about 350 bases was cut out with a razor, and the DNA was recovered using QIAquick \ Gel \ Extraction \ Kit (Qiagen). This DNA was cloned into the pCR2.1-TOPO vector according to the protocol of TOPO \ TA \ Cloning \ Kit \ (Invitrogen). This was introduced into Escherichia coli (Escherichia coli) TOP10 competent cell (Invitrogen) for transformation, and clones having cDNA inserts were selected on LB agar medium containing ampicillin and X-gal, and only clones exhibiting white color were selected. Separation was performed using a sterilized toothpick to obtain a transformant. Individual clones were cultured overnight in LB medium containing ampicillin, and plasmid DNA was prepared using QIAwell \ 8 \ Plasmid \ Kit (Qiagen). The reaction for determining the base sequence was performed using BigDye Terminator Cycle Sequencing ReadyReaction Kit Kit (PE Biosystems) and decoded using a fluorescent automatic sequencer (SEQ ID NO: 80).
[0122]
Reference Example 38 Preparation of Mouse Brain cDNA
Mouse brain cDNA is SMART^TMRACEUsing cDNA {Amplification Kit} (CLONTECH), it was prepared from mouse brain poly {A (+)} RNA} (CLONTECH) according to the protocol attached to the kit. The synthesized 1st strand cDNA solution was diluted 10-fold with Tricine-EDTA Buffer attached to the kit, and this solution was subjected to a RACE PCR reaction.
[0123]
Reference Example 39 Cloning of 5 'upstream end of cDNA encoding mouse GPR8 ligand precursor protein
5'RACE @ PCR cloning revealed the 5 'upstream base sequence of the cDNA encoding the mouse GPR8 ligand precursor protein. 5′RACE PCR cloning is performed using SMART using mouse brain cDNA as a template.^TMRACEA PCR reaction is performed using the Universal Primer Mix attached to the cDNA Amplification Kit and the synthetic primer of SEQ ID NO: 81, and then a PCR reaction is performed using the PCR reaction solution as a template and the Nested Universal Universal Primer attached to the kit and the synthetic primer of SEQ ID NO: 82. Achieved by doing. The reaction solution composition and reaction conditions for PCR are as follows. The reaction solution was mouse brain cDNA 脳 1 μl, Universal Primer Mix 2 μl, synthetic DNA primer of SEQ ID NO: 81 0.2 μM, 0.8 mM dNTPs, Advantage-GC 2 polymerase (CLONTECH) 0.4 μl and a buffer attached to the enzyme. Using a thermal cycler (PE Biosystems), the reaction volume was 20 µl, and after heating at 96 ° C for 120 seconds, a cycle of 96 ° C for 30 seconds and 68 ° C for 120 seconds was repeated 30 times, and further kept at 72 ° C for 10 minutes. did. Next, 0.5 μl of a PCR reaction solution diluted 50-fold with Tricine-EDTA Buffer attached to the kit, Nested Universal Primer 0.5 μM, 0.5 μM of synthetic DNA primer of SEQ ID NO: 82, 0.8 μm dNTPs, Advantage- GC 2 polymerase (CLONTECH) 0.4 μl and a buffer attached to the enzyme to make the total reaction volume 20 μl, and using a thermal cycler (PE Biosystems), heating at 96 ° C. for 120 seconds, then 96 ° C. for 30 seconds, 60 ° C. A cycle of 30 seconds at 72 ° C. for 120 seconds was repeated 30 times, and the temperature was kept at 72 ° C. for 10 minutes. After separating the amplified DNA by 1.5% agarose gel electrophoresis, a DNA of about 300 bases was cut out with a razor, and the DNA was recovered using QIAquick \ Gel \ Extraction \ Kit (Qiagen). This DNA was cloned into the pCR2.1-TOPO vector according to the protocol of TOPO \ TA \ Cloning \ Kit \ (Invitrogen). This was introduced into Escherichia coli (Escherichia coli) TOP10 competent cells (Invitrogen) and transformed, and clones having cDNA inserts were selected on LB agar medium containing ampicillin and X-gal, and only white clones were selected. Separation was performed using a sterilized toothpick to obtain a transformant. Individual clones were cultured overnight in LB medium containing ampicillin, and plasmid DNA was prepared using QIAwell \ 8 \ Plasmid \ Kit (Qiagen). The reaction for determining the base sequence was performed using BigDye \ Terminator \ Cycle \ Sequencing \ Ready \ Reaction \ Kit \ (PE \ Biosystems) and decoded using a fluorescent automatic sequencer to obtain the DNA sequence shown in SEQ ID NO: 83.
[0124]
Reference Example 40 Cloning of 3 'Downstream End of cDNA Encoding Mouse GPR8 Ligand Precursor Protein
3'RACE @ PCR cloning revealed the 3 'downstream nucleotide sequence of the cDNA encoding the mouse GPR8 ligand precursor protein. 3′RACE PCR cloning is performed using SMART using mouse brain cDNA as a template.^TMRACEA PCR reaction is performed using the Universal Primer Mix attached to the cDNA amplification Kit and the synthetic primer of SEQ ID NO: 84, and then the PCR reaction is performed using the PCR reaction solution as a template and the Nested Universal Primer attached to the kit and the synthetic primer of SEQ ID NO: 85. Achieved by doing. The reaction solution composition and reaction conditions for PCR are as follows. The reaction solution was mouse brain cDNA {1 μl, Universal Primer Mix} 2 μl, synthetic DNA primer of SEQ ID NO: 84 0.5 μM, 0.8 mM dNTPs, Advantage-GC 2 polymerase (CLONTECH) 0.4 μl and a buffer attached to the enzyme. The total reaction volume was set to 20 μl, and using a thermal cycler (PE Biosystems), after heating at 96 ° C. for 120 seconds, a cycle of 96 ° C. for 30 seconds and 68 ° C. for 120 seconds was repeated 30 times. Incubated for minutes. Next, 0.5 μl of a PCR reaction solution diluted 50-fold with Tricine-EDTA Buffer attached to the kit, Nested Universal Primer 0.5 μM, 0.5 μM of synthetic DNA primer of SEQ ID NO: 85, 0.8 μm mM NTPs, Advantage-GC 2 The total reaction volume was adjusted to 20 μl with 0.4 μl of polymerase (CLONTECH) and a buffer attached to the enzyme, and after heating at 96 ° C. for 120 seconds using a thermal cycler (PE Biosystems), 96 ° C. for 30 seconds, 64 ° C. A cycle of 30 seconds and 72 ° C. for 120 seconds was repeated 30 times, and finally the temperature was kept at 72 ° C. for 10 minutes. After the amplified DNA was separated by 1.5% agarose gel electrophoresis, a DNA of about 700 bases was cut out with a razor, and the DNA was recovered using QIAquick \ Gel \ Extraction \ Kit (Qiagen). This DNA was cloned into the pCR2.1-TOPO vector according to the protocol of TOPO \ TA \ Cloning \ Kit \ (Invitrogen). This was introduced into Escherichia coli (Escherichia coli) TOP10 competent cells (Invitrogen) and transformed, and clones having cDNA inserts were selected on LB agar medium containing ampicillin and X-gal, and only white clones were selected. Separation was performed using a sterilized toothpick to obtain a transformant. Individual clones were cultured overnight in LB medium containing ampicillin, and plasmid DNA was prepared using QIAwell \ 8 \ Plasmid \ Kit (Qiagen). The reaction for determining the base sequence was performed using BigDye \ Terminator \ Cycle \ Sequencing \ Ready \ Reaction \ Kit \ (PE \ Biosystems), followed by decoding using a fluorescent automatic sequencer to obtain the DNA sequence shown in SEQ ID NO: 86.
[0125]
Reference Example 41 Cloning of cDNA encoding mouse GPR8 ligand precursor protein
Using mouse brain cDNA as a template, a primer based on the 5 ′ upstream nucleotide sequence of cDNA encoding mouse GPR8 ligand precursor protein and a primer based on 3 ′ downstream nucleotide sequence of cDNA encoding mouse GPR8 ligand precursor protein The cDNA encoding mouse GPR8 ligand precursor protein was cloned. The reaction solution composition and reaction conditions for PCR are as follows. The reaction solution was 0.5 μl of mouse brain cDNA, 0.5 μM of a synthetic DNA primer of SEQ ID NO: 87, 0.5 μM of a synthetic DNA primer of SEQ ID NO: 88, 1.6 μmM of dNTPs, 0.2 μl of LATaq polymerase (Takara Shuzo) and The total reaction volume was adjusted to 20 μl with the GC (I) buffer attached to the enzyme, and after heating at 96 ° C. for 120 seconds using a thermal cycler (PE Biosystems), 96 ° C. for 30 seconds, 64 ° C. for 30 seconds, 72 ° C. -A cycle of 120 seconds was repeated 40 times, and finally, the temperature was kept at 72 ° C for 10 minutes. After the amplified DNA was separated by 1.5% agarose gel electrophoresis, a DNA of about 700 bases was cut out with a razor, and the DNA was recovered using QIAquick \ Gel \ Extraction \ Kit (Qiagen). This DNA was cloned into the pCR2.1-TOPO vector according to the protocol of TOPO \ TA \ Cloning \ Kit \ (Invitrogen). This was introduced into Escherichia coli (Escherichia coli) TOP10 competent cells (Invitrogen) and transformed, and clones having cDNA inserts were selected on LB agar medium containing ampicillin and X-gal, and only white clones were selected. Separation was performed using a sterilized toothpick to obtain a transformant. Individual clones were cultured overnight in LB medium containing ampicillin, and plasmid DNA was prepared using QIAwell \ 8 \ Plasmid \ Kit (Qiagen). The reaction for determining the nucleotide sequence was performed using BigDye \ Terminator \ Cycle \ Sequencing \ ReadyReaction \ Kit \ (PE \ Biosystems), followed by decoding using a fluorescent automatic sequencer to obtain the DNA sequence shown in SEQ ID NO: 89. Since this sequence (SEQ ID NO: 89) encodes a mouse GPR8 ligand precursor protein, Escherichia coli transformed with a plasmid containing the DNA is used to transform TOP10 / pCR2.1-TOPO mouse GPR8 ligand precursor (Escherichia coli TOP10 / pCR2). .1-TOPO \ Mouse \ GPR8 \ Ligand \ Precursor).
The DNA sequence shown in SEQ ID NO: 89 has the amino acid sequence revealed by the amino acid sequence analysis of the GPR8 ligand peptide isolated from the porcine hypothalamus using the GTPγS binding activity for the GPR8-expressing cell membrane fraction described in Reference Example 10 as an index. There is a frame that encodes a similar amino acid sequence that differs only in the sequence from the terminal to the 17th residue and the amino acids at the 5th and 17th residues. However, as in the case of the human GPR8 ligand precursor, there is no ATG, which is predicted to be the initiation codon for protein translation, at the 5 'upstream side. However, as predicted from the human GPR8 ligand precursor protein, it is located at a position almost corresponding to the ATG which is expected to be the initiation codon in these precursor proteins from comparison with the precursor protein of the pig or rat GPR8 ligand homolog. The sequence of the mouse GPR8 ligand precursor protein was deduced, assuming that the CTG codon was the initiation codon. The amino acid sequence of this hypothetical mouse GPR8 ligand precursor protein is shown in SEQ ID NO: 90. Arg-Arg which is thought to be excised from the amino acid sequence predicted for the mouse GPR8 ligand at the carboxy terminus side in the same manner as in the case of the human, porcine or rat homolog precursor protein of the GPR8 ligand peptide. (Seidah, N. G. et al., Ann. N. Y. Acad. Sci., 839, 9-24, 1998). From these, the amino acid sequence of the mouse homolog of the GPR8 ligand peptide was deduced to be either or both of SEQ ID NOs: 91 and 92. The amino acid sequence of the 23-residue mouse GPR8 ligand of SEQ ID NO: 91 is identical to the amino acid sequence of the 23-residue rat GPR8 ligand (SEQ ID NO: 73). The amino acid sequence and DNA sequence of the hypothetical mouse GPR8 ligand precursor protein are shown in FIG.
[0126]
Reference Example 42 {Using lactoperoxidase method} [¹²⁵I-Tyr²] -HGPR8L (1-23) and [¹²⁵I-Tyr¹⁰Production of hGPR8L (1-23)
1 nmol of hGPR8L (1-23) (polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 16) dissolved in 5 μl of DMSO was mixed with 5 μl of 0.1 M nickel chloride, and the mixture was mixed with 0.1 M HEPES (pH 7). 10 μl of {0.001% aqueous hydrogen peroxide} 10 μl in water, lactoperoxidase {(Sigma)} 10 μg / ml in 0.1 μM HEPES (pH = 7) and {[¹²⁵I] {NaI} 37 {MBq} (NEN Life Science Products) {10 μl} was mixed, reacted at room temperature for 60 minutes, and subjected to HPLC fractionation under the following conditions.
The column used was ODS-80TM (4.6 mm x 15 cm) (Tosoh), 10% acetonitrile / 0.1% TFA as eluent A, and 60% acetonitrile / 0.1% TFA as eluate B. A gradient elution method of 0-0 ° (2 min), 0-30 ° (3 min), 30-38 ° (5 min), and 38-43 ° (55 min)% B / A + B was performed. The flow rate was 1 mL / min, the column temperature was 25 ° C, and the detection was performed using the absorbance at 220 nm.
Since hGPR8L (1-23) has two tyrosine residues, [iodination]¹²⁵I-Tyr²] -HGPR8L (1-23) and [¹²⁵I-Tyr¹⁰] -HGPR8L (1-23) is generated. Under these HPLC conditions, hGPR8L (1-23) was treated for 24 minutes, [¹²⁵I-Tyr²] -HGPR8L (1-23) for 30 minutes, [¹²⁵I-Tyr¹⁰] -HGPR8L (1-23) eluted around 32 minutes.
[0127]
Reference Example 43¹²⁵I-Tyr¹⁰] -HGPR8L (1-23) receptor binding experiment
Fabricated as described in Reference Example 42 [¹²⁵I] -labeled hGPR8L (1-23) and receptor binding experiments were performed using the cell membrane fraction prepared from human GPR8-expressing CHO cells in the same manner as described in Reference Example 6.
A cell membrane fraction prepared from human GPR8-expressing CHO cells was subjected to assay buffer (25 mM Tris-HCl, 5 mM EDTA (ethylenediaminetetraacetic acid), 0.05% CHAPS (3-[(3-cholamidopropyl) dimethyl ammonium). E) -1-propane sulfate), 0.1% BSA (bovine serum albumin), 0.25 mM PMSF (phenylmethanesulfonyl fluoride), 1 μg / ml pepstatin, 20 μg / ml leupeptin, pH 7.4 After dilution to a concentration, 200 μl was dispensed into polypropylene test tubes (Falcon # 2053). To determine the maximum binding (TB), 2 μl of DMSO and 7 nM [¹²⁵I-Tyr²] -HGPR8L (1-23) or [¹²⁵I-Tyr¹⁰] -HGPR8L (1-23) 2 μl was added to the membrane fraction solution. To measure non-specific binding (NSB), 2 μl of 100 μM @ hGPR8L (1-23) in DMSO and 7 μnM [¹²⁵I-Tyr²] -HGPR8L (1-23) or [¹²⁵I-Tyr¹⁰] -HGPR8L (1-23) 2 μl was added to the membrane fraction solution. After reacting at 25 ° C. for 60 minutes, the reaction solution was subjected to suction filtration using a polyethylene imine-treated Whatman glass filter (GF-F). After filtration, the radioactivity remaining on the filter paper was measured using a γ-counter, and the specific binding amount (SB) was estimated by subtracting the non-specific binding amount from the maximum binding amount. [¹²⁵I-Tyr²] -HGPR8L (1-23)¹²⁵I-Tyr¹⁰] -HGPR8L (1-23) had twice the amount of specific binding, so that in actual binding experiments [¹²⁵I-Tyr¹⁰] -HGPR8L (1-23) was used. When the concentration of the membrane fraction was changed, it was dependent on the concentration of the membrane fraction [¹²⁵I-Tyr¹⁰] -HGPR8L (1-23) specific binding was observed. Further, the concentration of the membrane fraction was set to 5 μg / ml, and the 50% inhibition concentration (IC₅₀Value), IC₅₀The value was 0.25 nM. FIG. 10 shows the inhibition of hGPRL (1-23) binding at various concentrations.
[0128]
Reference Example 44 {human GPR8 {ligand} (1-23) oxidized product: Trp-Tyr-Lys-His-Val-Ala-Ser-Pro-Arg-Tyr-His-Thr-Val-Gly-Arg-Ala-Aly-Gly- Production of Leu-Leu-Met (O) -Gly-Leu (SEQ ID NO: 95)
After dissolving 0.45 mg of the compound of Reference Example 12 in 0.5 ml of 50% aqueous acetic acid, 0.05 ml of 0.3% aqueous hydrogen peroxide was added and left at room temperature for 8 hours. After concentration under reduced pressure, purification was performed by SepPak to obtain 0.443 mg of white powder.
By mass spectrometry (M + H)⁺: 2599.2 (calculated value 2599.4)
HPLC elution time: 19.1 minutes
Elution conditions
Column: Wakosil-II {5C18HG} (4.6 x 100 mm)
Eluent: A solution: Acetonitrile containing 0.1% TFA-water and B solution: 0.1% TFA-containing acetonitrile, A / B: 100/0 to 0/70, linear concentration gradient elution (35 minutes)
Flow rate: 1.0 ml / min
[0129]
Reference Example 45 Human GPR8 ligand (1-22): Trp-Tyr-Lys-His-Val-Ala-Ser-Pro-Arg-Tyr-His-Thr-Val-Gly-Arg-Ala-Ala-Gly-Leu- Production of Leu-Met-Gly (SEQ ID NO: 96)
After Fmoc-Gly was introduced into commercially available {2-chlorotrityl resin} (Clt resin, 1.33 mmol / g), amino acids were condensed in the sequence and cut out from the resin in the same manner as in Reference Example 13 to obtain the desired product.
[0130]
Reference Example 46 human GPR8 ligand (1-21): Trp-Tyr-Lys-His-Val-Ala-Ser-Pro-Arg-Tyr-His-Thr-Val-Gly-Arg-Ala-Ala-Gly-Leu- Production of Leu-Met (SEQ ID NO: 97) Fmoc-Met was introduced into commercially available 2-chlorotrityl resin (Clt resin, 1.33 mmol / g), followed by amino acid condensation and resin separation from the resin in the same sequence as in Reference Example-13. It was cut out and purified to obtain the desired product.
[0131]
Reference Example 47 {human GPR8 {ligand} (1-20): Trp-Tyr-Lys-His-Val-Ala-Ser-Pro-Arg-Tyr-His-Thr-Val-Gly-Arg-Ala-Ala-Gly-Leu- Production of Leu (SEQ ID NO: 98)
After Fmoc-Leu was introduced into commercially available 2-chlorotrityl resin (Clt resin, 1.33 mmol / g), the amino acid was condensed in the order of arrangement and cut out from the resin in the same manner as in Reference Example 13 to obtain the desired product.
M by mass spectrometry⁺: 2282.8 (calculated value 2282.6)
HPLC elution time: 17.2 minutes
Elution conditions
Column: Wakosil-II {5C18HG} (4.6 x 100 mm)
Eluent: A solution: Acetonitrile containing 0.1% TFA-water and B solution: 0.1% TFA-containing acetonitrile, A / B: 100/0 to 0/70, linear concentration gradient elution (35 minutes)
Flow rate: 1.0 ml / min
[0132]
Reference Example 48 {human GPR8 {ligand} (1-19): Trp-Tyr-Lys-His-Val-Ala-Ser-Pro-Arg-Tyr-His-Thr-Val-Gly-Arg-Ala-Ala-Gly-Leu ( Production of SEQ ID NO: 99)
After Fmoc-Leu was introduced into commercially available 2-chlorotrityl resin (Clt resin, 1.33 mmol / g), the amino acid was condensed in the order of arrangement and cut out from the resin in the same manner as in Reference Example 13 to obtain the desired product.
M by mass spectrometry⁺: 2169.6 (calculated value 2169.5)
HPLC elution time: 16.4 minutes
Elution conditions
Column: Wakosil-II {5C18HG} (4.6 x 100 mm)
Eluent: A solution: Acetonitrile containing 0.1% TFA-water and B solution: 0.1% TFA-containing acetonitrile, A / B: 100/0 to 0/70, linear concentration gradient elution (35 minutes)
Flow rate: 1.0 ml / min
[0133]
Reference Example 49 {human GPR8 {ligand} (1-18): Trp-Tyr-Lys-His-Val-Ala-Ser-Pro-Arg-Tyr-His-Thr-Val-Gly-Arg-Ala-Ala-Gly (SEQ ID NO: : 100)
After Fmoc-Gly was introduced into commercially available 2-chlorotrityl resin (Clt resin, 1.33 mmol / g), the amino acid was condensed in the order of arrangement and cut out from the resin in the same manner as in Reference Example 13 to obtain the desired product.
M by mass spectrometry⁺: 2056.8 (calculated value 2056.3)
HPLC elution time: 14.2 minutes
Elution conditions
Column: Wakosil-II {5C18HG} (4.6 x 100 mm)
Eluent: A solution: Acetonitrile containing 0.1% TFA-water and B solution: 0.1% TFA-containing acetonitrile, A / B: 100/0 to 0/70, linear concentration gradient elution (35 minutes)
Flow rate: 1.0 ml / min
[0134]
Reference Example 50 {human GPR8 ligand} (1-17): Trp-Tyr-Lys-His-Val-Ala-Ser-Pro-Arg-Tyr-His-Thr-Val-Gly-Arg-Ala-Ala (SEQ ID NO: 101) )Manufacturing of
After Fmoc-Ala was introduced into commercially available 2-chlorotrityl resin (Clt resin, 1.33 mmol / g), the amino acid was condensed in the order of arrangement and cut out from the resin in the same manner as in Reference Example 13 to obtain the desired product.
[0135]
Reference Example 51 {human GPR8 {ligand} (1-16): of Trp-Tyr-Lys-His-Val-Ala-Ser-Pro-Arg-Tyr-His-Thr-Val-Gly-Arg-Ala (SEQ ID NO: 102) Manufacture
After Fmoc-Ala was introduced into commercially available 2-chlorotrityl resin (Clt resin, 1.33 mmol / g), the amino acid was condensed in the order of arrangement and cut out from the resin in the same manner as in Reference Example 13 to obtain the desired product.
[0136]
Reference Example 52 {porcine GPR8 ligand} (1-23): Trp-Tyr-Lys-His-Thr-Ala-Ser-Pro-Arg-Tyr-His-Thr-Val-Gly-Arg-Ala-Ala-Gly-Leu- Production of Leu-Met-Gly-Leu (SEQ ID NO: 56)
After Fmoc-Leu was introduced into commercially available 2-chlorotrityl resin (Clt resin, 1.33 mmol / g), the amino acid was condensed in the order of sequence and cut out from the resin in the same manner as in Reference Example 13 to obtain the desired product.
By mass spectrometry (M + H)⁺: 2585.2 (calculated value: 2585.4)
HPLC elution time: 20.2 minutes
Elution conditions
Column: Wakosil-II {5C18HG} (4.6 x 100 mm)
Eluent: A solution: Acetonitrile containing 0.1% TFA-water and B solution: 0.1% TFA-containing acetonitrile, A / B: 100/0 to 0/70, linear concentration gradient elution (35 minutes)
Flow rate: 1.0 ml / min
[0137]
Reference Example 53 {rat / mouse GPR8 {ligand} (1-23): Trp-Tyr-Lys-His-Val-Ala-Ser-Pro-Arg-Tyr-His-Thr-Val-Gly-Arg-Ala-Ser-Gly- Production of Leu-Leu-Met-Gly-Leu (SEQ ID NO: 73 and SEQ ID NO: 91)
As in Reference Example 52, the desired product can be obtained by condensing amino acids and cutting out from the resin in the order of the sequence, followed by purification.
[0138]
Reference Example 54 {porcine GPR8 {ligand} (1-23) oxidized product: Trp-Tyr-Lys-His-Thr-Ala-Ser-Pro-Arg-Tyr-His-Thr-Val-Gly-Arg-Ala-Ala-Gly- Production of Leu-Leu-Met (O) -Gly-Leu (SEQ ID NO: 103)
The compound of Reference Example 52 was oxidized in the same manner as in Reference Example 44 to obtain the desired product.
By mass spectrometry (M + H)⁺: 2601.3 (calculated value 2601.4)
HPLC elution time: 18.9 minutes
Elution conditions
Column: Wakosil-II {5C18HG} (4.6 x 100 mm)
Eluent: A solution: Acetonitrile containing 0.1% TFA-water and B solution: 0.1% TFA-containing acetonitrile, A / B: 100/0 to 0/70, linear concentration gradient elution (35 minutes)
Flow rate: 1.0 ml / min
[0139]
Reference Example 55 Rat / mouse GPR8 {ligand} (1-23) oxidized product: Trp-Tyr-Lys-His-Val-Ala-Ser-Pro-Arg-Tyr-His-Thr-Val-Gly-Arg-Ala-Ser- Production of Gly-Leu-Leu-Met (O) -Gly-Leu (SEQ ID NO: 104)
The target compound can be obtained by oxidizing the compound of Reference Example 53 in the same manner as in Reference Example 44.
[0140]
Reference Example 56 [Nα-Acetyl-Trp¹] -Human GPR8 {ligand} (1-23): Ac-Trp-Tyr-Lys-His-Val-Ala-Ser-Pro-Arg-Tyr-His-Thr-Val-Gly-Arg-Ala-Ala-Gly-Leu Production of Leu-Met-Gly-Leu (SEQ ID NO: 106)
After removing the Fmoc group of the resin prepared in Reference Example 12 and acetylating with acetic anhydride, the resin was treated with TFA / {thioanisole} / {m-cresol} / {triisopropylsilane} / {ethanedithiol} (85 / {5} / {5} / {2.5} / {2.5}). Excision from the resin and removal of the side chain protecting group were performed simultaneously. The crude peptide was purified in the same manner as in Reference Example 12 to obtain the desired product.
(M + H) by mass spectrometry⁺{262.12625.8} (calculated value: 2627.12626.1)
HPLC elution time 21.4 minutes
Elution conditions
Column Wakosil-II 5C18 HG (4.6 x 100 mm)
Eluent: Solution A: {0.1% TFA-water, Solution B: Using acetonitrile containing 0.1% TFA, {A / B: {100} / {0} to {30} / {70} linear concentration gradient elution (35 minutes)
Flow rate 1.0 ml / min
[0141]
Reference Example 57 {human GPR8 ligand} (2-23): {Tyr-Lys-His-Val-Ala-Ser-Pro-Arg-Tyr-His-Thr-Val-Gly-Arg-Ala-Aly-Gly-Leu-Leu- Production of Met-Gly-Leu (SEQ ID NO: 107)
As in Reference Example 12, the desired amino acid sequence was introduced into the resin. After the last Tyr was introduced and before the Fmoc group was removed from the resin before being cut out from the resin, treatment with {TFA} / {thioanisole} / m-cresol / triisopropylsilane / ethethanedithiol (85/5/5/5 / 2.5 / 2.5) was performed. Excision from the resin and removal of the side chain protecting group were performed simultaneously. The crude peptide was purified in the same manner as in Reference Example 12 to obtain the desired product.
(M + H) + {2397.1} by mass spectrometry (calculated value 2397.3)
HPLC elution time 19.9 minutes
Elution conditions
Column Wakosil-II 5C18 HG (4.6 x 100 mm)
Eluent: Solution A: {0.1% TFA-water, Solution B: Using acetonitrile containing 0.1% TFA, {A / B: {100} / {0} to {30} / {70} linear concentration gradient elution (35 minutes)
Flow rate 1.0 ml / min
[0142]
Reference Example 58 {human GPR8 ligand} (4-23): {His-Val-Ala-Ser-Pro-Arg-Tyr-His-Thr-Val-Gly-Arg-Ala-Ala-Gly-Leu-Leu-Met-Gly- Production of Leu (SEQ ID NO: 108)
As in Reference Example 12, the desired amino acid sequence was introduced into the resin. The Fmoc group is removed on the resin after the introduction of the last His and before it is cleaved from the resin, followed by treatment with {TFA} / {thioanisole} / m-cresol / triisopropylsilane / ethethanedithiol (85/5/5/5 / 2.5 / 2.5). Excision from the resin and removal of the side chain protecting group were performed simultaneously. The crude peptide was purified in the same manner as in Reference Example 12 to obtain the desired product.
(M + H) + {2106.0} by mass spectrometry (calculated value 2106.1)
HPLC elution time 20.0 minutes
Elution conditions
Column Wakosil-II 5C18 HG (4.6 x 100 mm)
Eluent: Solution A: {0.1% TFA-water, Solution B: Using acetonitrile containing 0.1% TFA, {A / B: {100} / {0} to {30} / {70} linear concentration gradient elution (35 minutes)
Flow rate 1.0 ml / min
[0143]
Reference Example 59 Production of {human GPR8 ligand} (9-23): {Arg-Tyr-His-Thr-Val-Gly-Arg-Ala-Ala-Gly-Leu-Leu-Met-Gly-Leu (SEQ ID NO: 109)
As in Reference Example 12, the desired amino acid sequence was introduced into the resin. The Fmoc group is removed on the resin after the introduction of the last Arg and before being cleaved from the resin, and then treated with {TFA} / {thioanisole} / m-cresol // triisopropylsilane // ethanedithiol (85/5/5 / 2.5 / 2.5). Excision from the resin and removal of the side chain protecting group were performed simultaneously. The crude peptide was purified in the same manner as in Reference Example 12 to obtain the desired product.
(M + H) + {1615.0} by mass spectrometry (calculated value 1614.9)
HPLC elution time 20.2 minutes
Elution conditions
Column Wakosil-II 5C18 HG (4.6 x 100 mm)
Eluent: Solution A: {0.1% TFA-water, Solution B: Using acetonitrile containing 0.1% TFA, {A / B: {100} / {0} to {30} / {70} linear concentration gradient elution (35 minutes)
Flow rate 1.0 ml / min
[0144]
Reference Example 60 Production of {human GPR8 ligand} (15-23): {Arg-Ala-Ala-Gly-Leu-Leu-Met-Gly-Leu (SEQ ID NO: 110)
As in Reference Example 12, the desired amino acid sequence was introduced into the resin. The Fmoc group is removed on the resin after the introduction of the last Arg and before being cleaved from the resin, and then treated with {TFA} / {thioanisole} / m-cresol // triisopropylsilane // ethanedithiol (85/5/5 / 2.5 / 2.5). Excision from the resin and removal of the side chain protecting group were performed simultaneously. The crude peptide was purified in the same manner as in Reference Example 12 to obtain the desired product.
(M + H) + {901.4} by mass spectrometry (calculated value 901.5)
HPLC elution time 20.2 minutes
Elution conditions
Column Wakosil-II 5C18 HG (4.6 x 100 mm)
Eluent: Solution A: {0.1% TFA-water, Solution B: Using acetonitrile containing 0.1% TFA, {A / B: {100} / {0} to {30} / {70} linear concentration gradient elution (35 minutes)
Flow rate 1.0 ml / min
[0145]
Reference Example 61 [N-Acetyl-Tyr²] -Human GPR8 {ligand} (2-23): Ac-Tyr-Lys-His-Val-Ala-Ser-Pro-Arg-Tyr-His-Thr-Val-Gly-Arg-Ala-Ala-Gly-Leu-Leu Production of -Met-Gly-Leu (SEQ ID NO: 111)
After acetylating the resin prepared in Reference Example 57 with acetic anhydride, it was treated and purified in the same manner as in Reference Example 57 to obtain the desired product.
(M + H) + {2439.3} by mass spectrometry (calculated value 2439.3)
HPLC elution time 20.2 minutes
Elution conditions
Column Wakosil-II 5C18 HG (4.6 x 100 mm)
Eluent: Solution A: {0.1% TFA-water, Solution B: Using acetonitrile containing 0.1% TFA, {A / B: {100} / {0} to {30} / {70} linear concentration gradient elution (35 minutes)
Flow rate 1.0 ml / min
[0146]
Reference Example 62 [D-Trp¹] -Human GPR8 {ligand} (1-23): D-Trp-Tyr-Lys-His-Val-Ala-Ser-Pro-Arg-Tyr-His-Thr-Val-Gly-Arg-Ala-Ala-Gly-Leu Production of -Leu-Met-Gly-Leu (SEQ ID NO: 112)
The target product was similarly obtained using Fmoc-D-Trp (Boc) instead of Fmoc-Trp (Boc) of Reference Example 12.
(M + H) + {2583.4} by mass spectrometry (calculated value 2583.4)
HPLC elution time 20.6 minutes
Elution conditions
Column Wakosil-II 5C18 HG (4.6 x 100 mm)
Eluent: Solution A: {0.1% TFA-water, Solution B: Using acetonitrile containing 0.1% TFA, {A / B: {100} / {0} to {30} / {70} linear concentration gradient elution (35 minutes)
Flow rate 1.0 ml / min
[0147]
Reference Example 63 [N-3-Indolepropanoyl-Tyr²] -Human GPR8 {ligand} (2-23): 3-Indolepropanoyl-Tyr-Lys-His-Val-Ala-Ser-Pro-Arg-Tyr-His-Thr-Val-Gly-Arg-Ala-Ala-Gly-Leu. Production of -Leu-Met-Gly-Leu (SEQ ID NO: 113)
3-Indolepropionic acid was used in place of Fmoc-Trp (Boc) in Reference Example 12 to obtain a desired resin, which was then TFA / thioanisole / m-cresol / triisopropysilane / ethanedithiol / 2.5 / 2.5 / 2/85 .5), and the cleavage from the resin and the removal of the side chain protecting group were performed simultaneously. The crude peptide was purified in the same manner as in Reference Example 12 to obtain the desired product.
(M + H) + {2568.4} by mass spectrometry (calculated value 2568.4)
HPLC elution time 21.7 minutes
Elution conditions
Column Wakosil-II 5C18 HG (4.6 x 100 mm)
Eluent: Solution A: {0.1% TFA-water, Solution B: Using acetonitrile containing 0.1% TFA, {A / B: {100} / {0} to {30} / {70} linear concentration gradient elution (35 minutes)
Flow rate 1.0 ml / min
[0148]
Reference Example 64 GTPγS binding promoting activity of human and porcine homolog derivatives of GPR8 ligand peptide measured using GPR8 expressing CHO cell membrane fraction
GTP8S binding promoting activity was measured by administering human and porcine homolog derivatives of the GPR8 ligand peptide described in the synthesis method at various concentrations to the GPR8-expressing CHO cell membrane fraction by the method described in Reference Example 6. Table 1 shows the measured SEQ ID NOs of the derivatives and the GTPγS binding promoting activity. The activity was measured at 50% effective concentration (EC₅₀Value). The GTPγS binding promoting activities of hGPR8L (1-23) and hGPR8L (1-30) described in Reference Examples 20 and 21 are also described.
[0149]
Reference Example 65 GPR8-expressing CHO cell membrane fraction and [¹²⁵I-Tyr¹⁰] -Receptor binding activity of human and porcine homolog derivatives of GPR8 ligand peptide measured using hGPR8L (1-23)
The receptor binding activity of the human and porcine homolog derivatives of the GPR8 ligand peptide described in the synthesis method herein was determined by the method described in Reference Example 43 using the GPR8-expressing CHO cell membrane fraction and [¹²⁵I-Tyr¹⁰] -HGPR8L (1-23). Table 1 shows the measured sequence numbers of the derivatives and the receptor binding activity. The receptor binding activity was measured at 50% binding inhibition concentration (IC₅₀Value). The receptor binding activity of hGPR8L (1-23) described in Reference Example 43 is also described.
[0150]
[Table 1]

[0151]
Reference Example 66 Cloning of cDNA encoding G protein-coupled receptor protein derived from whole rat brain and determination of nucleotide sequence
Using rat whole brain cDNA (CLONTECH) as a template, two primers, primer 1 (SEQ ID NO: 128) and primer 2 (SEQ ID NO: 129) designed based on the nucleotide sequence of DNA encoding human GPR8 A PCR reaction was performed. The composition of the reaction solution in the reaction was as follows: the cDNA was used as a 1/10 template, Advantage-2 cDNA <RTIgt; Polymerase </ RTI> Mix (CLONTECH) 1/50, primer 3 0.2 M, primer 2 0.2 M, dNTPs 200 M , And the enzyme were added to the attached buffer to make a liquid volume of 25 μl. In the PCR reaction, (i) after 94 ° C for 2 minutes, (ii) three cycles of 94 ° C for 20 seconds and 72 ° C for 2 minutes, (iii) 94 ° C for 20 seconds, 66 ° C for 20 seconds, A cycle of 68 ° C. for 2 minutes is repeated 3 times, and (iv) a cycle of 94 ° C. for 20 seconds, 60 ° C. for 20 seconds, and 68 ° C. for 2 minutes is repeated 36 times, and finally an extension reaction at 68 ° C. for 7 minutes is performed. Was. The reaction product after the PCR reaction was subcloned into a plasmid vector pCR2.1-TOPO (Invitrogen) according to the prescription of a TA cloning kit (Invitrogen). This was introduced into Escherichia coli DH5α, and a clone having cDNA was selected in an LB agar medium containing ampicillin. As a result of analyzing the sequence of each clone, a nucleotide sequence (SEQ ID NO: 127) of a cDNA encoding a novel G protein-coupled receptor protein was obtained. The novel G protein-coupled receptor protein containing the amino acid sequence (SEQ ID NO: 126) encoded by the nucleotide sequence of this DNA was named TGR26 (also referred to herein as rat TGR26).
The amino acid sequence represented by SEQ ID NO: 126 is 84.8% that of GPR7 (Genomics, 28, 84-91, 1995) which is a known human G protein-coupled receptor protein. Had homology.
One clone was selected from the above-mentioned transformants having the plasmid into which the DNA encoding TGR26 was inserted, and cultured with shaking in an LB medium containing ampicillin to obtain a plasmid. This was treated with restriction enzymes ClaI and SpeI to cut out the insert encoding TGR26. Similarly, pAKKO-1.11H treated with restriction enzymes ClaI and SpeI was ligated using Ligation Express Kit (CLONTECH), and introduced into Escherichia coli DH10B by electroporation. About the obtained clone, after confirming the structure of the plasmid for constructing an expression cell by restriction enzyme treatment and sequence analysis, the clone was named Escherichia coli DH10B / pAK-rGPR7.
The hydrophobicity plot of TGR26 is shown in FIG.
[0152]
Reference Example 67: Preparation of CHO cells expressing TGR26
After culturing Escherichia coli DH5α (Toyobo) transformed with the expression plasmid pAK-rGPR7 described in Reference Example 66, pAK-rGPR7 plasmid DNA was prepared using Plasmid \ Midi \ Kit (Qiagen). This was purified using CellPhect \ Transfection \ Kit (Amersham Pharmacia Biotech) according to the attached protocol and CHO \ dhfr.⁻The cells were introduced. 5 μg of DNA was made into a coprecipitation suspension with calcium phosphate, and⁵CHO @ dhfr⁻The cells were added to two seeded dishes of 6 cm in diameter. After culturing for 1 day in MEMα medium containing 10% fetal calf serum, the cells were subcultured and cultured in nucleic acid-free MEMα medium containing 10% dialyzed fetal bovine serum as a selective medium. Forty-four clones of transformed cell colonies, which are TGR26-expressing CHO cells, growing in the selection medium, were selected.
[0153]
Reference Example 68 Quantification of TGR26 expression level of TGR26-expressing CHO cell line using TaqMan PCR method
The 44 clones of the TGR26-expressing CHO cell line obtained in Reference Example 67 were²After culturing in a flask and preparing total RNA using RNeasy Mini Kits (Qiagen), the cells were treated with DNase using RNase-free DNase Set (Qiagen). 4 μg of the obtained total RNA was treated with 12 μl of a solution containing 500 pmol of random primer (Takara Shuzo) at 70 ° C. for 10 minutes after cooling at 10 ° C., and further cooled with 1 × First Strand Buffer, 10 mM DTT, 500 μM dA / dC / dG / dTTP and 200 units. SUPERSCRIPT II (Gibco) was added, and a reverse transcription reaction was performed by treating 20 μl of the mixture at 30 ° C. for 10 minutes, 42 ° C. for 60 minutes, 51 ° C. for 30 minutes, and 70 ° C. for 15 minutes. The resulting reverse transcript corresponding to 5 ng of total RNA or 10-1 × 10 5 prepared as described later⁷Copy standard cDNA, 1xUniversal @ PCR @ Master @ Mix (PE Biosystems), 100 nM each of the primer represented by SEQ ID NO: 130 and the primer represented by SEQ ID NO: 131, and SEQ ID NO: 140 (Fam-tcctctgctg @ gaccacctac @ acctga-Tamra) In the sequence, Fam represents 6-carboxy-fluorescein, and Tamra represents 6-carboxy-tetramethyl-rhodamine.) ABI {PRISM @ 7700 @ DeSequen (25 μl) of a reaction mixture containing TaqMan probe (100 nM) represented by TaqMan probe. The PCR was performed using Systems). The PCR was performed by repeating a cycle of 95 ° C. for 15 seconds and 60 ° C. for 60 seconds 40 times after treating at 50 ° C. for 2 minutes and 95 ° C. for 10 minutes.
The standard cDNA was 100 pg of TGR26 expression plasmid DNA (pAK-rGPR7), a primer represented by SEQ ID NO: 130 and a primer represented by SEQ ID NO: 131 each at 500 nM, 1 × PCR Gold Buffer, 2.5 mM MgCl₂, 200 μl of a reaction mixture containing 200 μM dA / dC / dG / dTTP and 20 units AmpliTaq Gold (PE Biosystems) at 95 ° C. for 10 minutes using GeneAmpPCR System 9700 (PE Biosystems) and then at 95 ° C. It was prepared by performing PCR under conditions where a cycle of 10 seconds, 63 ° C. for 15 seconds, and 72 ° C. for 10 seconds was repeated 40 times. The concentration was calculated by measuring the absorbance at 260 nm of the synthetic cDNA purified using QIAquick \ PCR \ Purification \ Kit (Qiagen), and the exact copy number of the standard cDNA was calculated. Then, 10 mM \ Tris-HCl (pH8) containing 1 mM \ EDTA was used. .0) diluted with solution, 1 × 10⁸A standard cDNA solution was prepared at a concentration of copy / μl. Further, TaqMan @ PCR probes and primers were designed by Primer @ Express (Version 1.0) (PE Biosystems).
The expression level was calculated using ABI PRISM 7700 SDS software. The number of cycles at the moment when the fluorescence intensity of the reporter reached the set value was plotted on the vertical axis, and the logarithmic value of the initial concentration of the standard cDNA was plotted on the horizontal axis to create a standard curve. The initial concentration of each reverse transcript was calculated from the standard curve, and the amount of TGR26 gene expression per total RNA of each clone was determined. As a result, it was found that the TGR26-expressing CHO cell line clones 18 and 28 showed a high expression level. Expression cells of these two clones were used in subsequent experiments.
[0154]
Reference Example 69 Measurement of intracellular cAMP production using TGR26-expressing CHO cells
The TGR26-expressing CHO cells prepared in Reference Example 68 were placed in a 24-well plate at 5 × 10 6⁴The cells were seeded at cell / well and cultured for 48 hours. The cells were washed with a MEMα buffer (pH 7.4) containing 0.2 mM 3-isobutyl-methylxanthine, 0.05% BSA (bovine serum albumin) and 20 mM HEPES [hereinafter, 0.2 mM 3-isobutyl-methylxanthine, A MEMα buffer (pH 7.4) containing 0.05% BSA and 20 mM HEPES is called a reaction buffer]. Thereafter, 0.5 ml of a reaction buffer was added, and the mixture was kept warm in an incubator for 30 minutes. After removing the reaction buffer, 0.25 ml of the reaction buffer was newly added to the cells, and a sample prepared as a DMSO solution having an appropriate concentration and 0.25 ml of the reaction buffer containing 2 μM forskolin were added to the cells. The reaction was carried out at 30 ° C for 30 minutes. The reaction was stopped by adding 100 μl of 20% perchloric acid, and then placed on ice for 1 hour to extract intracellular cAMP. The amount of cAMP in the extract was measured using a cAMP @ EIA kit (Amersham Pharmacia Biotech).
[0155]
Reference Example 70 Inhibitory activity of 23 or 30 residue GPR8 ligand peptide human homolog on intracellular cAMP production measured using TGR26-expressing CHO cell membrane fraction
GPR8 ligand peptide human homologue of 23 residues obtained in Reference Example (hereinafter sometimes referred to as hGPR8L (1-23)) or GPR8 ligand peptide human homologue of 30 residues obtained in Reference Example (hereinafter, referred to as hGPR8L (1-23)). hGPR8L (sometimes referred to as 1-30)) was administered at various concentrations to the TGR26-expressing CHO cell membrane fraction by the method described in Reference Example 69, and the intracellular cAMP production inhibitory activity was measured.
The results are shown in FIG.
Clearly, hGPR8L (1-23) and hGPR8L (1-30) inhibited the production of cAMP in TGR26-expressing CHO cells in a concentration-dependent manner.
In the figure, the cAMP synthesis inhibitory activity is defined as hGPR8L (1), where the amount obtained by subtracting the amount of intracellular cAMP when the reaction buffer is added from the amount of intracellular cAMP when the reaction buffer containing forskolin is added is 100%. -23) or the amount obtained by subtracting the amount of intracellular cAMP when the reaction buffer was added from the amount of intracellular cAMP when hGPR8L (1-30) was added was expressed as%.
This revealed that hGPR8L (1-23) or hGPR8L (1-30) is a ligand for TGR26.
Similar to the above, using the pig, rat, and mouse homologs of hGPR8L (1-23) and the pig, rat, and mouse homologs of hGPR8L (1-30), cAMP intracellular cAMP expressing TGR26 in a concentration-dependent manner. Can be confirmed.
[0156]
Reference Example 71 Measurement of GTPγS binding activity using membrane fraction of TGR26-expressing CHO cells
TGR26-expressing CHO cell membrane fraction [³⁵The binding promoting activity of [S] -guanosine 5 '-([gamma] -thio) triphosphate (GTP [gamma] S) was measured by the following method.
1) Preparation of membrane fraction
1x10⁸Of TGR26-expressing CHO cells was added to 10 ml of a homogenate buffer (10 mM NaHCO3).₃5 mM EDTA, 0.5 mM PMSF (phenylmethylsulfonyl fluoride), 1 μg / ml pepstatin, 4 μg / ml E-64, 20 μg / ml leupeptin), and crushed using Polytron (12,000 rpm, 1 minute). did. The cell lysate was centrifuged (1,000 g, 15 minutes) to obtain a supernatant. Next, this supernatant was subjected to ultracentrifugation (Beckman type 30 rotor, 30,000 rpm, 1 hour), and the obtained precipitate was used as a TGR26-expressing CHO cell membrane fraction.
2) Measurement of GTPγS binding activity
The CHO cell membrane fraction expressing TGR26 was treated with a membrane dilution buffer (50 mM Tris-HCl buffer (pH 7.4), 5 mM MgCl 2).₂, 150 mM NaCl, 1 μM GDP, 0.1% BSA) to prepare a cell membrane fraction solution for assay having a protein concentration of 30 μg / ml. 200 μl of the membrane fraction solution for assay was added to a 50 nM concentration of [³⁵2 μl of [S] -guanosine 5 ′-(γ-thio) triphosphate (NEN) and a 2 μl sample of a DMSO solution having an appropriate concentration were added, and the mixture was kept at 25 ° C. for 1 hour. The mixture was filtered with a filter, and the filter was further washed with a washing buffer (50 mM Tris-HCl buffer (pH 7.4), 5 mM MgCl 2).₂After washing twice with 1.5 ml of 1 mM (EDTA, 0.1% BSA), the radioactivity of the filter was measured with a liquid scintillation counter.
[0157]
Reference Example 72 GTPγS binding promoting activity of 23 or 30 residue GPR8 ligand peptide human homolog measured using TGR26-expressing CHO cell membrane fraction
hGPR8L (1-23) or hGPR8L (1-30) was mixed with TTGR26-expressing CHO cell membrane fraction at various concentrations according to the method described in Reference Example 71, and the GTPγS binding promoting activity was measured.
The results are shown in FIG.
Clearly, hGPR8L (1-23) and hGPR8L (1-30) promoted GTPγS binding of the TGR26-expressing CHO cell membrane fraction in a concentration-dependent manner.
In the same manner as described above, using the pig, rat, and mouse homologs of hGPR8L (1-23) and the pig, rat, and mouse homologs of hGPR8L (1-30), the TGR26-expressing CHO cell membrane fraction was determined in a concentration-dependent manner. GTPγS binding promotion can be confirmed.
[0158]
Reference Example 73¹²⁵I-Tyr¹⁰] -HGPR8L (1-23) receptor binding experiment
Produced by the method described in Reference Example 42 [¹²⁵I-Tyr¹⁰Using -hGPR8L (1-23) and the cell membrane fraction prepared from TTGR26-expressing CHO cells described in Reference Example 71, receptor binding experiments were performed as follows.
A cell membrane fraction prepared from TGR26-expressing CHO cells was subjected to assay buffer (25 mM Tris-HCl, 5 mM EDTA, 0.05% CHAPS (3-[(3-cholamidopropyl) dimethylammonio] -1-propane). Sulfuric acid), 0.1% BSA, 0.5 mM PMSF, 1 μg / ml pepstatin, 20 μg / ml leupeptin, 4 μg / ml E-64, pH 7.4), and then diluted to various concentrations with a polypropylene test tube (Falcon). 2053), 200 μl each. To determine the maximum binding, 2 μl of DMSO and 7 nM [¹²⁵I-Tyr¹⁰] -HGPR8L (1-23) was added to the membrane fraction solution. To measure non-specific binding, 2 μl of 100 μM @ hGPR8L (1-23) in DMSO and 7 nM [¹²⁵I-Tyr¹⁰] -HGPR8L (1-23) was added to the membrane fraction solution. After reacting at 25 ° C. for 75 minutes, the reaction solution was subjected to suction filtration using a polyethylene imine-treated Whatman glass filter (GF-F), and the filter was washed with a washing buffer (25 mM Tris-HCl, 5 mM EDTA, 0.1 mM EDTA). Washed twice with 1.5 ml of 05% CHAPS, 0.1% BSA, pH 7.4). After filtration, the radioactivity remaining on the filter paper was measured using a γ-counter, and the specific binding amount was estimated by subtracting the non-specific binding amount from the maximum binding amount.
When the concentration of the membrane fraction was changed, it was dependent on the concentration of the membrane fraction [¹²⁵I-Tyr¹⁰] -HGPR8L (1-23) specific binding was observed. The membrane fraction concentration was set to 3 μg / ml, and hGPR8L (1-23) and hGPR8L (1-30) [¹²⁵I-Tyr¹⁰] -HGPR8L (1-23) was examined for binding inhibition to specific binding to a TGR26-expressing cell membrane fraction. 50% inhibition concentration (IC₅₀Calculated), the IC of hGPR8L (1-23)₅₀The value was 0.12 nM. In addition, IC of hGPR8L (1-30)₅₀The value was 0.028 nM.
This indicated that hGPR8L (1-23) and hGPR8L (1-30) had high affinity for the TGR26-expressing cell membrane fraction. This means that hGPR8L (1-23) and hGPR8L (1-30) are high affinity ligands for the TGR26 receptor.
[FIG. 14] shows the inhibition of hGPR8L (1-23) and hGPR8L (1-30) binding at various concentrations.
Similar to the above, using the rat and mouse homologs of hGPR8L (1-23) and the pig, rat and mouse homologs of hGPR8L (1-30) [¹²⁵I-Tyr¹⁰] -HGPR8L (1-23) can be confirmed to inhibit the specific binding to the TGR26-expressing cell membrane fraction.
[0159]
Reference Example 74 TGR26-expressing CHO cell membrane fraction and [¹²⁵I-Tyr¹⁰Receptor binding activity of human and porcine homolog derivatives of GPR8 ligand peptide measured using -hGPR8L (1-23)
The receptor binding activity of the human and porcine homolog derivatives of the GPR8 ligand peptide obtained in Reference Example was determined by the method described in Reference Example 73 using the TGR26-expressing CHO cell membrane fraction and [¹²⁵I-Tyr¹⁰] -HGPR8L (1-23). Table 2 shows the measured derivatives and receptor binding activity. Receptor binding activity was measured at 50% binding inhibition concentration (IC₅₀Value).
[0160]
[Table 2]

[0161]
Reference Example 75 Cloning of 5 'upstream end of cDNA encoding mouse TGR26
5'RACE @ PCR cloning revealed the 5 'upstream base sequence of the cDNA encoding mouse TGR26.
5′RACE PCR cloning was performed using SMART using the mouse brain cDNA described in Reference Example as a template.^TMRACEA PCR reaction was performed using the Universal Primer Mix attached to the cDNA amplification Kit and the synthetic primer of SEQ ID NO: 132, and then using this PCR reaction solution as a template, a PCR reaction was performed using the Nested Universal Sal Primer attached to the kit and the synthetic primer of SEQ ID NO: 133. Was achieved. The primers of SEQ ID NO: 132 and SEQ ID NO: 133 were designed based on the mouse GPR7 cDNA fragment sequence registered in Genbank (Accession: U23807). The reaction solution composition and reaction conditions for PCR are as follows. The reaction mixture was 1 μl of mouse brain cDNA, 2 μl of Universal Primer Mix, 0.2 μM of synthetic DNA primer of SEQ ID NO: 132, 0.8 μm dNTPs, 0.4 μl of Advantage-GC II polymerase (CLONTECH), and the total reaction volume with a buffer attached to the enzyme. After heating at 96 ° C. for 120 seconds using a thermal cycler (PE Biosystems), a cycle of 96 ° C. for 30 seconds and 68 ° C. for 120 seconds was repeated 30 times, and further kept at 72 ° C. for 10 minutes. Next, 0.5 μl of a PCR reaction solution diluted 50-fold with Tricine-EDTA buffer attached to the kit, 0.5 μM of Nested Universal primer, 0.5 μM of synthetic DNA primer of SEQ ID NO: 133, 0.5 μM of 0.8 mM dNTPs, Advantage-GC 2 The total reaction volume was adjusted to 20 μl with 0.4 μl of polymerase (CLONTECH) and the buffer attached to the enzyme, and after heating at 96 ° C. for 120 seconds using a thermal cycler (PE Biosystems), 96 ° C. for 30 seconds, 60 ° C. A cycle of 30 seconds and 72 ° C. for 60 seconds was repeated 30 times, and further kept at 72 ° C. for 10 minutes. After the amplified DNA was separated by 1.5% agarose gel electrophoresis, a DNA having a length of about 450 bases was cut out with a razor, and the DNA was recovered using QIAquick \ Gel \ Extraction \ Kit (Qiagen). This DNA was cloned into the pCR2.1-TOPO vector according to the protocol of TOPO TA Cloning Kit (Invitrogen). After introducing this into Escherichia coli (Escherichia coli) TOP10 competent cell (Invitrogen) and transforming, a clone having a cDNA insert was selected on an LB agar medium containing ampicillin and X-gal, and only a clone exhibiting white color was selected. Was isolated using a sterilized toothpick to obtain a transformant. Individual clones were cultured overnight in LB medium containing ampicillin, and plasmid DNA was prepared using QIAwell \ 8 \ Plasmid \ Kit (Qiagen). The reaction for determining the nucleotide sequence was carried out using BigDyeTerminator \ Cycle \ Sequencing \ Ready \ Reaction \ Kit (PE Biosystems), followed by decoding using a fluorescent automatic sequencer to obtain the nucleotide sequence represented by SEQ ID NO: 134.
[0162]
Reference Example 76 Amplification of human GPR7 DNA by PCR using human chromosomal DNA
Using human chromosomal DNA as a template, DNA amplification was performed by PCR using two types of synthetic primers (SEQ ID NO: 141 and SEQ ID NO: 142). The synthetic primer was constructed so that the gene in the region translated into the receptor protein was amplified. At this time, a nucleotide sequence recognized by the restriction enzyme ClaII was added to the 5 'side of the gene, and the 3' Recognition sequences of the respective restriction enzymes were added to the 5 ′ and 3 ′ sides so that the base sequence recognized by the enzyme SpeΔI was added. The composition of the reaction solution was 0.5 μg of human chromosome DNA (Takara), 1 μM each of synthetic DNA primers, 0.8 mM dNTPs, 1 mM MgCl.₂, KOD polymerase (Toyo Bo) (1 μl) and the buffer attached to the enzyme, and the total reaction volume was 50 μl. The cycle for amplification was performed using a thermal cycler (Takara), and after heating at 94 ° C for 60 seconds, a cycle of 98 ° C for 15 seconds, 65 ° C for 2 seconds, and 74 ° C for 30 seconds was repeated 35 times. Confirmation of the amplification product was performed by ethidium bromide staining after 0.8% agarose gel electrophoresis.
[0163]
Reference Example 77 Confirmation of amplified DNA sequence by subcloning PCR product into plasmid vector and decoding base sequence of inserted DNA
The PCR reaction solution performed in Reference Example 76 was separated by 0.8% low-melting point agarose gel electrophoresis, and the band was cut out with a razor, followed by fragmentation, phenol extraction, phenol / chloroform extraction, and ethanol precipitation. The operation was performed to recover the DNA. pCR-Script^TMThe recovered DNA was subcloned into a plasmid vector pCR-Script {Amp} SK (+) according to the recipe of the {Amp} SK (+) cloning kit (Stratagene). This was introduced into Escherichia coli (DH) αDH competent cell (Toyo Bo) and transformed, and a clone having a DNA insert was selected on an LB agar medium containing ampicillin, IPTG and X-gal, and a white clone was obtained. Alone using a sterilized toothpick and transformants. E. coli DH5α / GPR7 was obtained. Individual clones were cultured overnight in LB medium containing ampicillin, and plasmid DNA was prepared using QIAwell \ 8 \ Plasmid \ Kit (Qiagen). A portion of the prepared DNA was cleaved with restriction enzymes ClaII and SpeII to confirm the size of the inserted receptor DNA fragment. The reaction for the determination of the nucleotide sequence was performed using DyeDeoxyTerminator \ Cycle \ Sequence \ Kit (Applied Biosystems) and decoded using a fluorescent automatic sequencer (SEQ ID NO: 143). The pCR-Script \ Amp \ SK (+) plasmid carrying the DNA having the nucleotide sequence of SEQ ID NO: 143 was designated as pCR-Script human GPR7. The amino acid sequence of human GPR7 encoded by the DNA having the nucleotide sequence of SEQ ID NO: 143 is shown in SEQ ID NO: 144. The DNA sequence of human GPR7 whose sequence was determined here is the DNA sequence described in O'Dowd et al. (O'Dowd, B. F. et al., Genomics, 28, 84-91, 1995). The two bases were different. These correspond to positions 893 and 894 of SEQ ID NO: 143, which were C and G in the report of O'Dowd et al., But were G and C in this reference example. Thus, in the translated amino acid sequence, the 296th amino acid of SEQ ID NO: 144 is Thr in O'Dowd et al.'S report in this reference example.
[0164]
Reference Example 78 Preparation of CHO Cells Expressing Human GPR7
A plasmid encoding the full-length amino acid sequence of human GPR7, the sequence of which was confirmed in Reference Example 77, having a ClaII recognition sequence added to the 5 'side and a SpeII recognition sequence added to the 3' side, was introduced into the plasmid. E. converted Plasmid DNA was prepared from the E. coli clone using Plasmid \ Midi \ KIt (Qiagen) and digested with restriction enzymes ClaI and Spe \ I to cut out insert DNA. After the electrophoresis, the insert DNA was cut out from the agarose gel with a razor, and then recovered by the operations of fragmentation, phenol extraction, phenol / chloroform extraction, and ethanol precipitation. The vector DNA pAKKO-111H for expression of animal cells obtained by digesting the insert DNA with ClaI and SpeII (pAKKO1.11H described in Hinuma, S. et al. Biochim. Biophys. Acta, Vol. Ligation using T4 ligase (Takara) to construct a protein expression plasmid pAKKO-Human @ GPR7. Escherichia coli transformed with this plasmid pAKKO-Human @ GPR7 was designated as DH5α / pAKKO-Human @ GPR7.
E. coli transformed with pAKKO-Human @ GPR7. After culturing E. coli DH5α (Toyo Bo), pAKKO-Human GPR7 plasmid DNA was prepared using Plasmid Midi Kit (Qiagen). This was purified using CellPhect \ Transfection \ Kit (Amersham Pharmacia Biotech) according to the attached protocol and CHO \ dhfr.⁻The cells were introduced. 3 μg of DNA was made into a coprecipitation suspension with calcium phosphate, and 24 hours before 5 x 10⁵Or 1 x 10⁶CHO @ dhfr⁻The cells were added to a 6-cm diameter petri dish in which the cells were seeded. After culturing for 1 day in MEMα medium containing 10% fetal calf serum, the cells were subcultured and cultured in nucleic acid-free MEMα medium containing 10% dialyzed fetal bovine serum as a selective medium. Twenty-four clones of colonies of transformed cells, which are GPR8-expressing CHO cells, growing in the selection medium were selected.
[0165]
Reference Example 79 Measurement of human GPR7 gene expression level in human GPR7-expressing CHO cell line using {TaqMan} PCR method
24 clones of the human GPR7-expressing CHO cell line prepared according to Reference Example 78 were each 25 cm in size.²The cells were cultured in a flask, and a total RNA fraction was prepared from the grown cells using ISOGEN (Nippon Gene). This total RNA fraction was treated with DNase I using a MessageClean (Gen Hunter) kit to obtain total RNA without DNA.
CDNA synthesis using Total RNA as a template was performed using a TaqMan Reverse Reverse Transcription Reagents (Applied Biosystems) kit. The composition of the reaction solution was as follows: DNase I-treated total RNA 4 μg, random primer 1 μl, 25 mM MgCl₂The total reaction volume was adjusted to 20 μl with 4.4 μl of solution, 2 μl of 10 mM dNTP mix, 0.4 μl of RNase Inhibitor, 0.5 μl of reverse transcriptase, and the reaction buffer attached to the kit. The reverse transcription reaction was performed using a thermal cycler (Takara) at 25 ° C. for 10 minutes, 48 ° C. for 30 minutes, and 95 ° C. for 5 minutes.
Standard human GPR7 DNA was prepared by purifying PCR-amplified DNA using full-length human GPR7 DNA as a template. The composition of the PCR reaction solution was as follows: pCR-Script human GPR7 5 pg described in Reference Example 77, synthetic DNA primer (SEQ ID NO: 145) 0.5 μM, synthetic DNA primer (SEQ ID NO: 146) 0.5 μM, 1.6 μmM dNTPs, 2.5 mM MgCl₂0.5 μl of LATaq polymerase (Takara) and a buffer attached to the enzyme, and the total reaction volume was 50 μl. The reaction for amplification was performed using a thermal cycler (Applied Biosystems), and after heating at 94 ° C for 120 seconds, a cycle of 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 60 seconds was repeated 25 times. Finally, the temperature was kept at 72 ° C. for 10 minutes. The PCR reaction solution was separated by 0.8% agarose gel electrophoresis, the band was cut out with a razor, and the PCR-amplified DNA was recovered using QIAquick \ PCR \ Purification \ Kit (Qiagen). In order to remove the primer DNA and dNTPs mixed in the PCR-amplified DNA solution, the DNA solution was subjected to chromospin column 400 (CLONTECH) gel chromatography to obtain an amplified human GPR7 DNA elution fraction. From the amount of DNA calculated from the absorption at 260 nm of the amplified human GPR7 DNA solution and the base composition of the amplified human GPR7 DNA, the copy number of the DNA contained in the amplified human GPR7 DNA solution was calculated. The amplified human GPR7 DNA whose DNA copy number became clear was used as a standard human GPR7 に DNA for TaqMan PCR for quantification.
[0166]
Expression of human GPR7 CHO cell line The copy number of the human GPR7 gene was determined by the TaqMan PCR method. The composition of the TaqMan {PCR reaction solution was 100 μl of a reverse transcribed cDNA solution diluted 100 times with distilled water or 1 μl of a standard human GPR7 of various copy numbers {DNA solution} 1 μl, 0.2 μM of synthetic DNA primer (SEQ ID NO: 147), 0.2 μM of synthetic DNA primer ( SEQ ID NO: 148) 0.2 μM, human GPR7ΔTaqMan probe [SEQ ID NO: 77 (Fam-TTCATCCTCAΔACCTGGCCAT @ CGC-Tamura; in the sequence, Fam represents 6-carboxy-fluorescein, Tamra represents 6-carboxy-tetramethyrate, Each having a nucleotide sequence represented by the following formula: 0.2 μM and TaqMan \ Universal \ PCR \ Master \ Mix (Applied Bios) system) to a total reaction volume of 25 μl. The PCR reaction was carried out using ABI PRISM 7700 Sequence Detector System (Applied Biosystems) at 50 ° C. for 2 minutes and 95 ° C. for 10 minutes, followed by a cycle of 95 ° C. for 15 seconds and 60 ° C. for 60 seconds. This was performed by repeating the procedure twice. The expression level of the human GPR7 gene was calculated using ABI PRISM 7700 SDS software. The number of cycles at the moment when the fluorescence intensity of the reporter reached the set value was plotted on the vertical axis, and the logarithm of the copy number of various standard human GPR7 DNAs was plotted on the horizontal axis to create a standard curve. From the standard curve, the copy number of the human GPR7 cDNA contained in the reverse transcribed cDNA was calculated, and the expression amount of the human GPR7 gene per 1 ng of the total RNA was determined. Clone No. having a high human GPR7 gene expression level. # 7, # 8, and # 14 were selected as human GPR7 gene high expression cell lines.
[0167]
Reference Example 80 Measurement of intracellular cAMP production using human GPR7-expressing CHO cells
Human GPR7-expressing CHO cells prepared in Reference Example 78 and selected as described in Reference Example 79 were placed in a 24-well plate at 5 x 10 cells.⁴The cells were seeded at cell / well and cultured for 48 hours. The cells were washed with a MEMα buffer (pH 7.4) containing 0.2 mM {3-isobutyl-methylxanthine, 0.05% BSA (bovine serum albumin) and 20 mM HEPES (hereinafter referred to as 0.2 mM 3-isobutyl-methyl). MEMα buffer (pH 7.4) containing xanthine, 0.05% BSA and 20 HEPES is called a reaction buffer). Thereafter, 0.5 μl of a reaction buffer was added, and the mixture was incubated in an incubator for 30 minutes. After removing the reaction buffer, a new 0.25 ml reaction buffer was added to the cells, and then a sample in an appropriate concentration of DMSO solution and a 0.25 ml reaction buffer containing 2 μM forskolin were added to the cells. At 37 ° C. for 30 minutes. The reaction was stopped by adding 100 μl of 20% perchloric acid, and then placed on ice for 1 hour to extract intracellular cAMP. The amount of cAMP in the extract was measured using a cAMP EIA kit (Amersham Pharmacia Biotech).
[0168]
Reference Example 81 Inhibitory activity of 23 or 30 residue GPR8 ligand peptide human homolog on intracellular cAMP production measured using human GPR7 expressing CHO cells
hGPR8L (1-23) or hGPR8L (1-30) was administered to human GPR7-expressing CHO cells at various concentrations according to the method described in Reference Example 80, and the intracellular cAMP production inhibitory activity was measured. The results are shown in FIG. In the figure, the cAMP synthesis inhibitory activity is defined as hGPR8L (1), where the amount obtained by subtracting the amount of intracellular cAMP when the reaction buffer is added from the amount of intracellular cAMP when the reaction buffer containing forskolin is added is 100%. -23) or the amount obtained by subtracting the amount of intracellular cAMP when the reaction buffer was added from the amount of intracellular cAMP when hGPR8L (1-30) was added was expressed as%.
Clearly, hGPR8L (1-23) and hGPR8L (1-30) suppressed the production of cAMP in human GPR7-expressing CHO cells in a concentration-dependent manner. This has revealed that hGPR8L (1-23) and hGPR8L (1-30) are ligands for human GPR7. 50% inhibitory concentration (IC₅₀Calculated), the IC of hGPR8L (1-23)₅₀The value was 0.025 nM. In addition, IC of hGPR8L (1-30)₅₀The value was 0.13 nM.
Using the pig, rat and mouse homologs of hGPR8L (1-23) and the pig, rat and mouse homologs of hGPR8L (1-30), the reaction of human GPR7-expressing CHO cells can be confirmed in the same manner as described above.
[0169]
Reference Example 82¹²⁵I-Tyr¹⁰] -HGPR8L (1-23) receptor binding experiment
Produced by the method described in Reference Example 42 [¹²⁵I-Tyr¹⁰] -HGPR8L (1-23) and a receptor binding experiment was performed using a cell membrane fraction prepared from human GPR7-expressing CHO cells.
First, the method for preparing the membrane fraction is described below.
1x10⁸CHO cells expressing human GPR7 were added to 10 ml of a homogenate buffer (10 mM NaHCO3).₃5 mM EDTA (ethylenediaminetetraacetic acid), 0.5 mM PMSF (phenylmethanesulfonylfluoride), 1 μg / ml pepstatin, 4 μg / ml E64, 20 μg / ml leupeptin) and use Polytron (12,000 rpm, 1 minute). Crushed. The cell lysate was centrifuged (1,000 g, 15 minutes) to obtain a supernatant. Next, the supernatant was subjected to ultracentrifugation (Beckman type 30 rotor, 30,000 rpm, 1 hour), and the obtained precipitate was used as a human GPR7-expressing CHO cell membrane fraction.
The cell membrane fraction thus prepared was subjected to assay buffer (25 mM Tris-HCl, 5 mM EDTA, 0.05% CHAPS (3-[(3-cholamidopropyl) dimethylammonio] -1-propane sulfate), 0.1% After dilution to various concentrations with 1% BSA, 0.5 mM PMSF, 1 μg / ml pepstatin, 20 μg / ml leupeptin, 4 μg / ml E-64, pH 7.4), 200 μl was dispensed into polypropylene test tubes (Falcon® 2053). did. To determine the maximum binding, 2 μl of DMSO and 8 nM [¹²⁵I-Tyr¹⁰] -HGPR8L (1-23) 2 μl was added to the membrane fraction solution. In addition, in order to measure non-specific binding, 2 μl of a 1 mM {hGPR8L (1-23) DMSO solution and 8 nM Δ [¹²⁵I-Tyr¹⁰] -HGPR8L (1-23) 2 μl was added to the membrane fraction solution. After reacting at 25 ° C. for 75 minutes, the reaction solution was subjected to suction filtration using a Whatman glass filter (GF-F) treated with polyethyleneimine, and the filter was further washed with a washing buffer (25 mM Tris-HCl, 5 mM EDTA, 0.05%). CHAPS, 0.1%） BSA, pH 7.4) was washed twice with 1.5 ml. After filtration, the radioactivity remaining on the filter paper was measured using a γ-counter, and the specific binding amount was estimated by subtracting the non-specific binding amount from the maximum binding amount.
When the concentration of the membrane fraction was changed, it was dependent on the concentration of the membrane fraction [¹²⁵I-Tyr¹⁰] -HGPR8L (1-23) specific binding was observed. The membrane fraction concentration was set to 10 μg / ml, and hGPR8L (1-23) and hGPR8L (1-30) [¹²⁵I-Tyr¹⁰] -HGPR8L (1-23) was examined for its inhibition of the specific binding to the human GPR7-expressing cell membrane fraction. 50% inhibition concentration (IC₅₀Calculated), the IC of hGPR8L (1-23)₅₀The value was 0.099 nM. In addition, IC of hGPR8L (1-30)₅₀The value was 0.025 nM.
This indicates that hGPR8L (1-23) and hGPR8L (1-30) have high affinity for human GPR7-expressing cell membrane fraction. This means that hGPR8L (1-23) and hGPR8L (1-30) are high affinity ligands for the human GPR7 receptor. FIG. 16 shows hGPR8L (1-23) and hGPR8L (1-30) binding inhibition at various concentrations.
Similarly, using the rat and mouse homolog of hGPR8L (1-23) and the pig, rat and mouse homolog of hGPR8L (1-30) [¹²⁵I-Tyr¹⁰] -HGPR8L (1-23) can be confirmed to inhibit the specific binding to the human GPR7-expressing cell membrane fraction.
[0170]
Reference Example 83
1) Measurement of GTPγS binding activity using membrane fraction of GPR7-expressing CHO cells
GPR7-expressing CHO cell membrane fraction [³⁵The binding promoting activity of [S] -guanosine 5 '-([gamma] -thio) triphosphate (GTP [gamma] S) was measured by the following method.
A GPR7-expressing CHO cell membrane fraction prepared by the method described in Reference Example 82 was treated with a membrane dilution buffer (50 mM Tris-HCl buffer (pH 7.4), 5 mM @MgCl₂, 150 mM NaCl, 1 μM GDP, 0.1% BSA) to prepare a cell membrane fraction solution for assay having a protein concentration of 30 μg / ml. To 200 μl of the membrane fraction solution for assay, add 50 nM concentration of [³⁵2 μl of [S] -guanosine 5 ′-(γ-thio) triphosphate (NEN) and a 2 μl sample of a DMSO solution having an appropriate concentration were added, and the mixture was kept at 25 ° C. for 1 hour. The mixture was filtered with a filter, and the filter was further washed with a washing buffer (50 mM Tris-HCl buffer (pH 7.4), 5 mM MgCl 2).₂After washing twice with 1.5 ml of 1 mM (EDTA, 0.1% BSA), the radioactivity of the filter was measured with a liquid scintillation counter.
2) GTPγS binding promoting activity of hGPR8L (1-23) or hGPR8L (1-30) measured using GPR7-expressing CHO cell membrane fraction
hGPR8L (1-23) or hGPR8L (1-30) was administered at various concentrations to the GPR7-expressing CHO cell membrane fraction according to the method described in 1) above, and the GTPγS binding promoting activity was measured.
The results are shown in FIG.
Clearly, hGPR8L (1-23) and hGPR8L (1-30) promoted GTPγS binding of GPR7-expressing CHO cell membrane fraction in a concentration-dependent manner.
50% effective concentration (EC₅₀Value), the EC of hGPR8L (1-23) was calculated.₅₀The value was 0.74 nM. In addition, EC of hGPR8L (1-30)₅₀The value was 0.67 nM (Table 3).
Using a rat and mouse homolog of hGPR8L (1-23) and a pig, rat and mouse homolog of hGPR8L (1-30), the reaction of human GPR7-expressing CHO cells can be confirmed in the same manner as described above.
[0171]
Reference Example 84 GTPγS binding promoting activity of human and porcine homolog derivatives of GPR8 ligand peptide measured using GPR7-expressing CHO cell membrane fraction
Human and porcine homolog derivatives of the GPR8 ligand peptide obtained in Reference Example were administered at various concentrations to the GPR7-expressing CHO cell membrane fraction by the method described in Reference Example 83, and the GTPγS binding promoting activity was measured.
Table 3 shows the measured derivative and GTPγS binding promoting activity. Activity was measured at 50% effective concentration (EC₅₀Value).
[0172]
[Table 3]

[0173]
Reference Example 85 GPR7-expressing CHO cell membrane fraction and [¹²⁵I-Tyr¹⁰] -Receptor binding activity of human and porcine homolog derivatives of GPR8 ligand peptide measured using hGPR8L (1-23)
The receptor binding activity of the human and porcine homolog derivatives of the GPR8 ligand peptide obtained in Reference Example was determined by the method described in Reference Example 82 using the GPR7-expressing CHO cell membrane fraction and [¹²⁵I-Tyr¹⁰] -HGPR8L (1-23).
Table 3 shows the measured derivative and receptor binding activities. Receptor binding activity was measured at 50% binding inhibition concentration (IC₅₀Value).
[0174]
Example 1
Effects of continuous administration of hGPR8L (1-23) subcutaneously on food intake and weight gain in rats
(1) Effects on food intake and weight gain
Wistar male rats (8 weeks old, Charles River Japan) were acclimated for about one week with MF powder diet (Oriental Yeast Co., Ltd.). HGPR8L (1-23) [human GPR-8 ligand (1-23)] dissolved in physiological saline at a concentration of 1 mM or an osmotic pump (alzet, MINI-OSMOTIC PUMP Model 2001) filled with 200 µl of physiological saline as a vehicle group. , Release rate; 24 μl / day) was subcutaneously (middle back) of the rat under pentobarbital anesthesia (n = 6 each). The day after the wearing day was set as day 0, the amount of food and body weight were measured at 8:00 and 20:00 every day until the 8th day under free feeding of MF powdered food. The light period was from 8:00 to 20:00, and the dark period from 20:00 to 8:00 the next morning. After the measurement at 8:00 on the 8th day, the animals were sacrificed by decapitation and the weights of liver, kidney, heart, spleen, testis, perirenal fat, perogenital fat and brown fat were measured. All animals were housed with a 12-hour light cycle (light: 8:00 to 20:00).
In the light period, there was no difference between the two groups in the light period, but in the dark period, the hGPR8L (1-23) administration group showed a decreasing tendency at any measurement time point as compared to the vehicle group ( 18 and 19) A decrease was observed in the total daily food consumption (FIG. 20). The body weight (mean value ± standard error) gradually increased from day 2 onward, and on day 7 of administration, vehicle group {378.4 ± {5.8} g vs. hGPR8L (1-23) administration group had 364. 6 ± {6.0} g (FIG. 21). In addition, the increase in body weight from day 0 to day 7 was 28.9 ± {3.2} g in the hGPR8L (1-23) -administered group compared to the vehicle group {39.6 ± 4.1%}, and hGPR8L. One week continuous subcutaneous administration of (1-23) suppressed an increase in body weight of about 10.7 g (FIG. 22).
Comparing the organ weight on day 8 of administration, the hGPR8L (1-23) administration group showed a decrease in liver (1.6 mg) and perirenal fat (1.2 mg) as white adipose tissue in the hGPR8L (1-23) administration group as compared with the vehicle group. Per genital fat (decrease of 0.7 g) was observed in each case by 0.5 g or more (Table 4).
[Table 4]

From the above results, it was confirmed that the subcutaneous administration of hGPR8L (1-23) had an effect of suppressing weight gain accompanied by a decrease in food intake and a decrease in fat weight.
(2) Effects on blood glucose, total cholesterol and triglycerides
Blood glucose, total cholesterol and triglyceride on day 8 of administration of the hGPR8L (1-23) administration group and the vehicle group of the above (1) were measured using Fuji Dry Chem. Blood glucose concentration (vehicle group; {150 ± 4.9} mg / dl, {hGPR8L (1-23) administration group; 155 ± 4.5} mg / dl) and blood total cholesterol concentration (vehicle group; {73.6 ± 2) There was no difference between the two groups in the 0.4 mg / dl, hGPR8L (1-23) administration group; 69.1 ± 1.8 mg / dl), but the blood triglyceride concentration (vehicle group; 168 ± 8. 9 mg / dl, (hGPR8L (1-23) administration group; 134 ± 28 mg / dl) showed a low value of 30 mg / dl or more in the hGPR8L (1-23) administration group (FIG. 23).
From the above results, a decrease in blood triglyceride concentration was observed by subcutaneous administration of hGPR8L (1-23).
[0175]
Example 2
Effect of intraperitoneal administration of hGPR8L (1-23) on food intake and body weight in rats
As in Example 1, Wistar male rats (8-week-old, Charles River Japan) were acclimated for about one week with an MF powder diet (Oriental Yeast Co., Ltd.). HGPR8L (1-23) [human GPR # 8 ligand (1-23)] dissolved in physiological saline at a concentration of 0.2 mg / ml and 2 mg / ml, or physiological saline at a concentration of 1 ml / kg as a vehicle group. Immediately before the start of the dark period (19:45 to 20:00), intraperitoneal administration was performed for 3 days (n = 10 each). The food intake and body weight were measured at 8:00 in the morning after the administration. All animals were housed on a 12 hour light cycle with a light period from 8:00 to 20:00 and a dark period from 20:00 to 8:00 the next morning.
The amount of food consumption decreased in a dose-dependent manner in the hGPR8L (1-23) administration group, and was significantly significant in the 2 mg / kg administration group on all three days after administration as compared with the vehicle group (p <= 0. 05 or p <= 0.01) showed a decrease (FIG. 24).
The body weight gain showed a decreasing tendency in the 2 mg / kg administration group on all three days compared with the vehicle group (difference in mean value; day 1; {1.2} g, day 2; {3.1} g, Day 3, {2.3} g) (FIG. 25).
From the above results, hGPR8L (1-23) was found to have an effect of reducing food intake and suppressing weight gain in the dark period by intraperitoneal administration.
[0176]
Example 3
[Phe²] Human GPR8 ligand (1-20): {Trp-Phe-Lys-His-Val-Ala-Ser-Pro-Arg-Tyr-His-Thr-Val-Gly-Arg-Ala-Ala-Gly-Leu-Leu ( Production of SEQ ID NO: 149)
Using an automatic peptide synthesizer (ABI433 model) manufactured by Applied Biosystems, the peptide chain was sequentially extended from the C-terminal by the Fmoc method according to the program to synthesize the target protected peptide resin.
As the starting amino acid resin carrier, Wang (p-benzyloxybenzyl alcohol) resin (0.25 mmol) is used, and Fmoc-Leu, Fmoc-Gly, Fmoc-Ala, Fmoc-Arg (Pbf), Fmoc-Val, Fmoc-Thr (Fmoc-Thr) Bu^t), Fmoc-His (Trt), Fmoc-Tyr (Bu^t), Fmoc-Pro, Fmoc-Ser (Bu^t), Fmoc-Lys (Boc), Fmoc-Phe, Fmoc amino acid derivatives of Fmoc-Trp (Boc) were converted to HBTU (2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluro). (Hexafluorophosphate) in accordance with the sequence.
After the construction of the peptide on the resin was completed, the protected peptide resin was dried. Deprotection of the resulting protected peptide and separation of the peptide from the resin carrier were performed by TFA treatment. The obtained crude peptide was extracted with 0.1% ΔTFA water and lyophilized to obtain a powder solid. Subsequently, the crude peptide was subjected to preparative purification by reverse-phase high-performance chromatography (Shimadzu Corporation, preparative apparatus: model LC8A) using acetonitrile-0.1% TFA water system (15-35%, 80 minutes). Thus, 35 mg of the desired purified peptide was obtained.
The amino acid analysis value of the hydrolyzate obtained by hydrolyzing the purified product with 4N methanesulfonic acid containing 0.2% 3- (2-aminoethyl) indole at 110 ° C. for 22 hours (the value in parentheses indicates the theory) Value) as follows.
Thr (1) 0.93 Ser (1) 0.92, Gly (2) 2.03, Ala (3) 3.09, Val (2) 1.90, Leu (2) 2.02, Tyr ( 1) 1.02, Phe (1) 1.00, His (2) 1.91, Lys (1) 0.98, Trp (1) 0.88, Arg (2) 2.06, Pro (1) 1.02
The purity was calculated to be 98.8% by HPLC. In addition, the mass spectrometry value was 2266.6 (theoretical value: 2266.6).
[0177]
Example 4
The lactoperoxidase method was used [Phe²,¹²⁵I-Tyr¹⁰Production of human GPR8 ligand (1-20)
[Phe was obtained according to the production method described in Example 3 and dissolved in 10 μl of DMSO.²] 0.001% hydrogen peroxide solution obtained by dissolving 10 nmol of human GPR8 ligand (1-20) (SEQ ID NO: 149) in 10 μl of 0.1 M nickel chloride aqueous solution and 0.1 M HEPES (pH 7.6) 10 μl, 10 μl of lactoperoxidase (Sigma) 10 μg / ml dissolved in 0.1 M HEPES (pH 7.6), and [¹²⁵I] NaI {40} MBq (NEN Life Science Products) was mixed, reacted at room temperature for 50 minutes, and then formed [Phe²,¹²⁵I-Tyr¹⁰] Human GPR8 ligand (1-20) was fractionated by HPLC under the following conditions.
The column used was ODS-80TM (4.6 mm x 15 cm) (Tosoh), 10% acetonitrile / 0.1% TFA as eluent A, and 60% acetonitrile / 0.1% TFA as eluate B. The gradient elution method of 0-0 (2 min), 0-27 (5 min), and 27-32 (40 min)% B / A + B was used. The flow rate was 1 mL / min, the column temperature was 40 ° C, and the detection was 215 nm. Under these HPLC conditions, [Phe²,¹²⁵I-Tyr¹⁰] The human GPR8 ligand (1-20) eluted around 25 minutes.
[0178]
Example 5
[Phe²,¹²⁵I-Tyr¹⁰] Receptor binding experiment using human GPR8 ligand (1-20)
[Phe prepared by the method described in Example 4.²,¹²⁵I-Tyr¹⁰Receptor using human GPR8 ligand (1-20), GPR7-expressing CHO cell membrane fraction prepared by the method described in Reference Example 82 and GPR8-expressing CHO cell membrane fraction prepared by the method described in Reference Example 6 A binding experiment was performed.
A cell membrane fraction prepared from GPR7-expressing CHO cells and GPR8-expressing CHO cells was subjected to assay buffer (25 mM Tris-HCl, 5 mM EDTA, 0.05% CHAPS, 0.1% BSA, 0.5 mM PMSF, 1 μg / After dilution to various concentrations with ml pepstatin, 4 μg / ml @ E-64, 20 μg / ml leupeptin, pH 7.4, 200 μl was dispensed into polypropylene test tubes (Falcon® 2053). To determine the maximum binding, 2 μl of DMSO and 7 nM [Phe²,¹²⁵I-Tyr¹⁰2 μl of human GPR8 ligand (1-20) was added to the membrane fraction solution. To measure non-specific binding, 2 μl of 100 μM human GPR8 ligand (1-23) in DMSO and 7 μM [Phe²,¹²⁵I-Tyr¹⁰2 μl of human GPR8 ligand (1-20) was added to the membrane fraction solution. After reacting at 25 ° C. for 75 minutes, the reaction solution was subjected to suction filtration using a polyethylene imine-treated Whatman glass filter (GF-F). After filtration, the radioactivity remaining on the filter paper was measured using a γ-counter, and the specific binding amount was estimated by subtracting the non-specific binding amount from the maximum binding amount. When the concentration of the membrane fraction was changed, it depended on the concentration of the membrane fraction [Phe²,¹²⁵I-Tyr¹⁰] Specific binding of human GPR8 ligand (1-20) was observed.
The binding inhibitory activity (inhibition rate (%)) of the test sample on the GPR7 receptor or GPR8 receptor was determined from the maximum binding amount (TB) based on the test sample and [Phe²,¹²⁵I-Tyr¹⁰] The ratio of the value obtained by subtracting the radioactivity (X) remaining on the filter paper when the human GPR8 ligand (1-20) was added to the specific binding amount (SB) ((TB-X) / SB {x} 100} (% )).
For the membrane fraction prepared from GPR7-expressing CHO cells, the concentration of the membrane fraction was set to 15 μg / ml, and the 50% inhibitory concentration of human GPR8 ligand (1-23) and human GPR8 ligand (1-30) (IC₅₀Value), IC₅₀The values were 0.13 nM and 0.039 nM, respectively. FIG. 26 shows the binding inhibitory activities of human GPR8 ligand (1-23) and human GPR8 ligand (1-30) at various concentrations. For the membrane fraction prepared from GPR8-expressing CHO cells, the concentration of the membrane fraction was set to 5 μg / ml, and the 50% inhibitory concentration of human GPR8 ligand (1-23) and human GPR8 ligand (1-30) (IC₅₀Value), IC₅₀The values were 0.19 nM and 0.037 nM, respectively. FIG. 27 shows the binding inhibitory activities of human GPR8 ligand (1-23) and human GPR8 ligand (1-30) at various concentrations.
[0179]
Example 6
[Phe²,¹²⁵I-Tyr¹⁰Screening method for compounds that alter the binding between GPR7 and GPR8 ligand using human GPR8 ligand (1-20) and human GPR7-expressing CHO cell membrane fraction
[Phe prepared by the method described in Example 4.²,¹²⁵I-Tyr¹⁰Screening for compounds that alter the binding between GPR7 and GPR8 ligand by receptor binding experiment using human GPR8 ligand (1-20) and GPR7-expressing CHO cell membrane fraction prepared by the method described in Reference Example 82 did.
In Example 5 above using cell membrane fractions prepared from GPR7 expressing CHO cells, 2 μl of test compound in DMSO and 7 μM [Phe²,¹²⁵I-Tyr¹⁰2 μl of human GPR8 ligand (1-20) was added to the membrane fraction solution. After reacting at 25 ° C. for 75 minutes, the reaction solution was subjected to suction filtration using a polyethylene imine-treated Whatman glass filter (GF-F). After the filtration, the radioactivity remaining on the filter paper was measured using a γ-counter to determine the amount of binding at the time of addition of the test compound. From the maximum binding amount, the value obtained by subtracting the binding amount at the time of adding the test compound was divided by the specific binding amount to obtain a percentage, and the binding inhibitory activity (%) of the test compound was calculated.
For the test compound showing the binding inhibitory activity, the binding inhibitory activity was measured using test compounds prepared at various concentrations, and the 50% inhibitory concentration (IC₅₀Value). Lower IC₅₀The compound giving the value was selected as the compound that more strongly inhibits the binding between GPR7 and GPR8 ligand.
On the other hand, a compound having a negative value of the binding inhibitory activity was selected as a compound that promotes the binding between GPR7 and a GPR8 ligand.
[0180]
【The invention's effect】
The DNA of the present invention or the polypeptide of the present invention can be used, for example, for screening a weight gain inhibitor, a weight loss agent, a fat mass increase inhibitor, a food intake inhibitor or a weight gain drug, or a weight gain inhibitor, a weight loss agent. It is useful as an agent, an inhibitor of increase in fat mass, an antifeedant, and an agent for weight gain. Screening using the polypeptide of the present invention, preferably a weight gain inhibitor obtained by screening using the receptor of the present invention, a weight loss agent, a fat mass increase inhibitor, and an antifeedant are used as safe and excellent medicaments. Can be
[0181]
[Sequence list]

[Brief description of the drawings]
FIG. 1 shows UV absorption by HPLC (a) and GTPγS activity (b) of each peak in purification of a GPR8 ligand at the final stage using a Wakosil-II @ 3C18HG column. Activity was recovered at the peak indicated by the arrow.
FIG. 2 shows the GTPγS binding promoting activity of human homologues of GPR8 ligand peptide of 23 residues at various concentrations on CHO / GPR8 cell membrane fraction.
FIG. 3 shows GTPγS binding promoting activity of human homologues of GPR8 ligand peptide having 30 residues at various concentrations on CHO / GPR8 cell membrane fraction.
FIG. 4 shows the activity of human homologs of GPR8 ligand peptide having 23 residues at various concentrations to suppress cAMP production on CHO / GPR8 cells.
FIG. 5 shows the cAMP production inhibitory activity of human homologs of GPR8 ligand peptide of 30 residues at various concentrations on CHO / GPR8 cells.
FIG. 6 shows the entire nucleotide sequence of the human homolog precursor protein cDNA of the GPR8 ligand peptide and the entire amino acid sequence of the human homolog precursor receptor protein of the GPR8 ligand peptide translated therefrom. The sequence of the predicted human homolog peptide of the GPR8 ligand of 23 residues is indicated by a square.
FIG. 7 shows the entire nucleotide sequence of the porcine homolog precursor protein of the GPR8 ligand peptide and the entire amino acid sequence of the porcine homolog precursor receptor protein of the GPR8 ligand peptide translated therefrom. The expected sequence of the 23 residue GPR8 ligand porcine homolog peptide is indicated by a square.
FIG. 8 shows the entire nucleotide sequence of the rat homolog precursor protein cDNA of the GPR8 ligand peptide and the entire amino acid sequence of the rat homolog precursor receptor protein of the GPR8 ligand peptide translated therefrom. The expected sequence of the 23 residue GPR8 ligand rat homolog peptide is indicated by a square.
FIG. 9 shows the entire nucleotide sequence of the mouse homolog precursor protein cDNA of the GPR8 ligand peptide and the entire amino acid sequence of the mouse homolog precursor receptor protein of the GPR8 ligand peptide translated therefrom. The expected sequence of the 23 residue GPR8 ligand mouse homolog peptide is indicated by a square.
FIG. 10 shows the results of using a cell membrane fraction prepared from human GPR8-expressing CHO cells.¹²⁵1] A figure showing the binding inhibitory activity of a 23-residue human GPR8 ligand to a 23-residue human GPR8 ligand labeled with [I].
FIG. 11 is a hydrophobicity plot of TGR26.
FIG. 12 is a graph showing the activity of hGPR8L (1-23) and hGPR8L (1-30) for suppressing cAMP production on CHO / TGR26 cells. In the figure,-○-indicates the case where hGPR8L (1-23) was administered, and-△-indicates the case where hGPR8L (1-30) was administered.
FIG. 13 is a graph showing the activity of hGPR8L (1-23) and hGPR8L (1-30) for promoting GTPγS binding to a CHO / TGR26 cell membrane fraction. In the figure,-○-indicates the case where hGPR8L (1-23) was administered, and-△-indicates the case where hGPR8L (1-30) was mixed.
FIG.¹²⁵I] shows the binding inhibitory activity of various concentrations of hGPR8L (1-23) and hGPR8L (1-30) on the binding of the 23-residue human GPR8 ligand to the cell membrane fraction prepared from TTGR26-expressing CHO cells. The figure is shown. In the figure,-○-indicates the case where hGPR8L (1-23) was administered, and-△-indicates the case where hGPR8L (1-30) was administered.
FIG. 15 shows cAMP production inhibitory activities of CHO / GPR7 cells of GPR8 ligand peptide human homologs of 23 and 30 residues at various concentrations. In the figure,-●-indicates the case where hGPR8L (1-23) was administered, and -solid square- indicates the case where hGPR8L (1-30) was administered.
FIG.¹²⁵I] shows the binding inhibitory activity of various concentrations of hGPR8L (1-23) and hGPR8L (1-30) on the binding of the 23-residue human GPR8 ligand to the cell membrane fraction prepared from human GPR7-expressing CHO cells. FIG. In the figure,-●-indicates the case where hGPR8L (1-23) was administered, and -solid square- indicates the case where hGPR8L (1-30) was administered.
FIG. 17 is a graph showing the activity of hGPR8L (1-23) and hGPR8L (1-30) for promoting GTPγS binding to a CHO / GPR7 cell membrane fraction. In the figure,-●-indicates the case where hGPR8L (1-23) was administered, and -solid square- indicates the case where hGPR8L (1-30) was administered.
FIG. 18 shows the effect of hGPR8L (1-23) continuously administered subcutaneously on food consumption in the light period of rats. In the figure, □ indicates the vehicle group, and black squares indicate the hGPR8L (1-23) group.
FIG. 19 shows the effect of hGPR8L (1-23) continuously administered subcutaneously on food consumption in the dark period of rats. In the figure, □ indicates the vehicle group, and black squares indicate the hGPR8L (1-23) group.
FIG. 20 shows the effect of daily subcutaneous administration of hGPR8L (1-23) on daily food consumption in rats. In the figure, □ indicates the vehicle group, and black squares indicate the hGPR8L (1-23) group.
FIG. 21 shows the effect of hGPR8L (1-23) continuously administered subcutaneously on weight gain in rats. In the figure,-○-indicates the vehicle group, and-□-indicates the hGPR8L (1-23) group.
FIG. 22 shows the effect of hGPR8L (1-23) continuously administered subcutaneously on weight gain in rats. In the figure,-○-indicates the vehicle group, and-□-indicates the hGPR8L (1-23) group.
FIG. 23 shows the effects of hGPR8L (1-23) continuously administered subcutaneously on blood glucose level (a), total blood cholesterol level (b) and blood triglyceride concentration (c) in rats. ○ indicates the numerical value of each individual, and-indicates the average value.
FIG. 24 shows the effect of hGPR8L (1-23) administered intraperitoneally on food intake in rats. In the figure, white indicates the vehicle group, gray indicates the 0.2 mg administration group, and black indicates the 2 mg administration group. In addition, * indicates significant at 5% risk, and ** indicates significant at 1% risk.
FIG. 25 shows the effect of hGPR8L (1-23) administered intraperitoneally on body weight gain in rats. In the figure, white indicates the vehicle group, gray indicates the 0.2 mg administration group, and black indicates the 2 mg administration group.
FIG. 26 [Phe²,¹²⁵I-Tyr¹⁰FIG. 4 shows the binding inhibitory activities of various concentrations of hGPR8L (1-23) and hGPR8L (1-30) on the binding of human GPR8 ligand (1-20) to the cell membrane fraction prepared from human GPR7-expressing CHO cells. Show. In the figure,-○-indicates the case where hGPR8L (1-23) was administered, and-□-indicates the case where hGPR8L (1-30) was administered.
FIG. 27 [Phe²,¹²⁵I-Tyr¹⁰FIG. 7 shows the binding inhibitory activities of various concentrations of hGPR8L (1-23) and hGPR8L (1-30) on the binding of human GPR8 ligand (1-20) to the cell membrane fraction prepared from human GPR8-expressing CHO cells Show. In the figure,-○-indicates the case where hGPR8L (1-23) was administered, and-□-indicates the case where hGPR8L (1-30) was administered.

Claims (23)

A weight gain inhibitor comprising a polypeptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 16, or an amide thereof, an ester thereof, or a salt thereof. A weight reducing agent comprising a polypeptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 16, or an amide or ester thereof, or a salt thereof. A fat mass increase inhibitor comprising a polypeptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 16, or an amide or ester or salt thereof. An antifeedant comprising a polypeptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 16, or an amide thereof, an ester thereof, or a salt thereof. A weight loss inhibitor, a weight loss agent, comprising using a polypeptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 16, or an amide or ester or a salt thereof; A screening method for a fat mass increase inhibitor or an eating inhibitor. Further, a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 126, SEQ ID NO: 138 or SEQ ID NO: 144, a partial peptide thereof, or a salt thereof is used. Item 6. The screening method according to Item 5, A weight gain inhibitor or a weight loss agent comprising a polypeptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 16, or an amide or ester or salt thereof. , A kit for screening for an increase in fat mass or an antifeedant. Furthermore, it contains a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 126, SEQ ID NO: 138 or SEQ ID NO: 144, a partial peptide thereof, or a salt thereof. The screening kit according to claim 7. A weight gain inhibitor, a weight loss agent, a fat mass increase inhibitor or an eating inhibitor obtained by using the screening method according to claim 5 or the screening kit according to claim 7. Inhibition of weight gain comprising a polypeptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 16, or a compound having the activity of an amide or an ester or a salt thereof, or a salt thereof Agents, weight loss agents, fat mass increase inhibitors or food intake inhibitors. It contains an agonist of a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 126, SEQ ID NO: 138 or SEQ ID NO: 144, a partial peptide thereof, or a salt thereof. A weight gain inhibitor, a weight loss agent, a fat mass increase inhibitor, or a food intake inhibitor. Body weight comprising a polypeptide containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 16, or a polynucleotide containing a base sequence encoding an amide or an ester or a salt thereof An increase inhibitor, a weight loss agent, a fat increase inhibitor or an eating inhibitor. A polypeptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 16, or a polynucleotide having a base sequence encoding an amide or an ester or a salt thereof is used. A screening method for a weight gain inhibitor, a weight loss agent, a fat mass increase inhibitor, or a food intake inhibitor. It is characterized by containing a polypeptide containing a polypeptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 16, or a polynucleotide containing a base sequence encoding an amide or an ester or a salt thereof. A screening kit for a weight gain inhibitor, a weight loss agent, a fat mass increase inhibitor, or a food intake inhibitor. A weight gain inhibitor, a weight loss agent, a fat mass increase inhibitor or an eating inhibitor obtained by using the screening method according to claim 13 or the screening kit according to claim 14. A polypeptide comprising the amino acid sequence represented by SEQ ID NO: 149. 17. The polypeptide of claim 16, which is labeled. The screening method according to claim 5, wherein the polypeptide according to claim 16 is used. (I) a polypeptide according to claim 17, and (ii) an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 126, SEQ ID NO: 138 or SEQ ID NO: 144. The screening method according to claim 6, wherein the contained protein, its partial peptide, or a salt thereof is used. (I) a protein containing the polypeptide according to claim 17, and (ii) a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 4 or SEQ ID NO: 144, or a partial peptide thereof, or a salt thereof. 20. The screening method according to claim 19, wherein 21. The screening method according to claim 20, wherein the polypeptide according to claim 17 and a protein containing the amino acid sequence represented by SEQ ID NO: 144, a partial peptide thereof, or a salt thereof are used. For a mammal, (1) a polypeptide containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 16, or an amide or ester thereof, or a salt thereof, (2) the above polypeptide Or a compound having the activity of an amide or an ester or a salt thereof, or a salt thereof, or (3) identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 126, SEQ ID NO: 138 or SEQ ID NO: 144 A method for suppressing body weight gain, a method for suppressing body weight loss, a method for suppressing fat mass increase, or a method for suppressing food intake, comprising administering an effective amount of an agonist of a protein or a partial peptide thereof or a salt thereof, which contains a substantially identical amino acid sequence. (1) For producing a weight gain inhibitor, a weight loss agent, a fat mass increase inhibitor or an antifeedant, (1) contains an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 16 A polypeptide or an amide or an ester thereof or a salt thereof, (2) a compound having the activity of the polypeptide or an amide or an ester thereof or a salt thereof, or (3) SEQ ID NO: 4, SEQ ID NO: 126; Use of an agonist of a protein or a partial peptide thereof or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 138 or SEQ ID NO: 144.

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