JP2004242644A - Guanosine triphosphate binding protein coupled receptor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To develop a new technique for efficiently extracting the GPCR sequence from the human genome sequence and identify novel GPCR sequences comprehensively. <P>SOLUTION: A system for automatically finding RPCR sequences has been developed and 1215 kinds of new GPCR sequences are successfully identified from the whole human genome by using this system. Among them, a cDNA is isolated in one clone. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

【０１２４】
ここで、各プログラムの閾値の下の括弧内には、その閾値を用いたときの感度（左）と選択性（右）を表す。
【０１２５】
最も信頼の置けるデータ（レベルＡ，最善選択性データセット）は、ＢＬＡＳＴＰ、ＰＦＡＭ、およびＰＲＯＳＩＴＥの最善選択性閾値から得られた配列の和集合によって得られた。これに加え、関係の遠いＧＰＣＲ配列を発見するために、ＴＭＨ予測閾値の３つのレベル（表１）による結果とＢＬＡＳＴＰ、ＰＦＡＭおよびＰＲＯＳＩＴＥの最善感度閾値による結果との和集合を得た。次に、最も感度の高いデータセットを最善感度データセットとして準備した（レベルＤ）。本発明者らが評価した方法によれば、最善選択性データセットにおいて発見された如何なる配列も膜貫通ヘリックス７本を有する蛋白質であり、そのほとんどがグアノシン三リン酸結合タンパク質共役型である可能性が極めて高い。
【０１２６】
［実施例３］ 遺伝子数の精密化
第一の段階において生成した配列から、表１に示す閾値を用いてＧＰＣＲ候補物質をスクリーニングした。しかしこれらの配列は、なおも以下の重複例を含んでいたため、最終的に候補数をより絞り込む必要があった。
【０１２７】
ケース１：同じ遺伝子の位置での完全なマッチまたは重なり。それらは２つの配列生成方法、すなわち（１）６−フレーム翻訳および（２）ＧｅｎｅＤｅｃｏｄｅｒによる予測、の結果である。本発明者らはそれらを同じ遺伝子であると見なした。
【０１２８】
ケース２：異なる染色体間または同じ染色体上の異なる位置の間での多数のコピー。生物学的観点から、本発明者らはそれらを異なる遺伝子と見なした。重複遺伝子の最大数は染色体２と１１とのあいだに多く認められた。
【０１２９】
ケース３：何らかの長い既知の配列に部分的にヒットする２つ以上の配列。これらが生成された理由は、遺伝子発見プログラムによるミススプライシングであると考えられる。本発明者らはそれらを本来は同じ遺伝子として融合するべきとした。
【０１３０】
本発明者らは、上記の各ケースを検討することによってまず候補遺伝子の精度を上げた。具体的なアルゴリズムとして本発見者らは、染色体番号をＣ、フレーム番号をＦおよびゲノム配列上の位置をＲとして配列をＳ（Ｃ、Ｆ、Ｒ）と記述し、２つの配列ｉ，ｊがＣ_ｉ＝Ｃ_ｊ、Ｆ_ｉ＝Ｆ_ｊ、ｎ_ｉ＝ｎ_ｊ、ｅ_ｉ−ｔ_ｊ＜０（ｉ＜ｊ）で、５０残基以上９９％以上の類似度でアラインされた場合に、２つの配列を同じ遺伝子であると見なした。（ここでｎがコンティグ番号であって、ｔ，ｅがコンティグ配列でのＮおよびＣ末端での相対的位置である場合、位置Ｒ＝Ｒ（ｎ，ｔ，ｅ）である）。
【０１３１】
上記のような篩い分けを行った後、さらに、生物学的知見も加えて最終的に本発明者らは８８３および２２９３配列を含む最善選択性および最善感度データセットをそれぞれ得、さらにその他のレベルのデータセットも得た。各データセットに対して染色体ごとのＧＰＣＲ候補体の数を表２にまとめる。
【０１３２】
【表２】

【０１３３】
全てのレベルのデータセットにおいて染色体１１はＧＰＣＲ候補体の最大数を有し、染色体１、６、１９も多数のＧＰＣＲ候補体を示すことがわかる。一方、染色体２１およびＹでは、ＧＰＣＲ候補は極めて少ない。しかもこの傾向は、これまで毎月データを更新する過程でも変わらなかった。
最善選択性データセットに関するさらなる分析を表３に要約する。
【０１３４】
【表３】

【０１３５】
本発明者らは、３０％の配列類似性によって配列を分類した。これは一般的に、進化的に関連したファミリーの閾値であると考えられる。最大のファミリーは５３７メンバーを含む嗅覚受容体であった。２０メンバー以上の主要なファミリーは、アドレナリン、ドーパミンおよびセロトニン受容体（３８）、ファミリー２Ｂ受容体（２０）、ファミリー３Ｃ受容体（３０）ケモカインおよび化学遊走受容体（３１）、ならびにオーファン受容体（７６）であった。
【０１３６】
［実施例４］ 新規配列の抽出
配列をＵＮＩＧＥＮＥ（Ｓｃｈｕｌｅｒ， Ｇ． Ｄ． Ｊ．Ｍｏｌ Ｍｅｄ． ７５， ６９４−６９８ （１９９７）．）およびｎｒ−ａａ（ｆｔｐ：／／ｎｃｂｉ．ｎｌｍ．ｎｉｈ．ｇｏｖ／ｂｌａｓｔ／ｄｂ／ＲＥＡＤＭＥ）データベースに対して検索した。調べた配列中の少なくとも１００残基以上が既知配列に対して連続してアラインされ、その領域のアミノ酸同一性が９６％以上であれば、本発明者らはこの配列を既知の配列であると判断し、この基準で、本発明者らは、新規ＧＰＣＲ候補を得た。これらのデータセットは、定常的な再計算により、将来にわたり、維持、更新されるものである。
【０１３７】
本発明者らは、抽出した新規配列を下記Ａ群、Ｂ群、Ｃ群に分類した（表４から表６）。配列Ａ群、Ｂ群、Ｃ群は、三重分析で得られた配列セットのうち、それぞれ最善選択性データセット（レベルＡ）、レベルＢデータセット、レベルＣデータセットを基にして個数の精密化を行った後、ＵＮＩＧＥＮＥおよびｎｒ−ａａデータベースに対する検索に従って新規配列を求めたものである。
【０１３８】
【表４】

【０１３９】
【表５】

【０１４０】
【表６】

【０１４１】
以下、本発明者が同定したクローンのうち、クローン１３８６０について解析を進めた。
［実施例５］ クローン１３８６０のｃＤＮＡの単離
５．１． ５’−ＲＡＣＥ、３’−ＲＡＣＥ法によるクローン１３８６０ ｃＤＮＡの単離
クローン１３８６０のｃＤＮＡは同様に５’−ＲＡＣＥ、３’−ＲＡＣＥ法を用いて単離した。３’−端側のｃＤＮＡは３’−ＲＡＣＥ法により、Ｈ．Ｈｅｌａ細胞由来のＭａｒａｔｈｏｎ−Ｒｅａｄｙ ｃＤＮＡ（ＣＬＯＮＴＥＣＨ社製）を鋳型とし、ＡＰ１（配列番号：１１）と３’ Ｒ１３８６０（配列番号：１）をプライマーとして第一ＰＣＲを行い、さらに第二ＰＣＲにおいてはＡＰ２（配列番号：１２）と３’ Ｒ１３８６０−ｎｅｓｔ（配列番号：２）をプライマーとして用いた。続いて、５’−端側のｃＤＮＡは５’−ＲＡＣＥ法により、Ｈ．Ｈｅｌａ細胞由来のＭａｒａｔｈｏｎ−Ｒｅａｄｙ ｃＤＮＡを鋳型とし、ＡＰ１と５’ Ｒ１３８６０（配列番号：３）をプライマーとして第一ＰＣＲを行い、さらに第二ＰＣＲにおいてはＡＰ２と５’ Ｒ１３８６０−ｎｅｓｔ（配列番号：４）をプライマーとして用いた。また、それぞれのＰＣＲにおいてはＡｄｖａｎｔａｇｅ ２ Ｐｏｌｙｍｅｒａｓｅ Ｍｉｘ（ＣＬＯＮＴＥＣＨ社製）をＤＮＡポリメラーゼとして用いた。これらのＰＣＲにより得られたＤＮＡ断片はｐＧＥＭ−Ｔ Ｅａｓｙベクター（Ｐｒｏｍｅｇａ社製）に挿入した後、塩基配列の決定を行った。なお、Ａｄｖａｎｔａｇｅ ２ Ｐｏｌｙｍｅｒａｓｅ Ｍｉｘ（ＣＬＯＮＴＥＣＨ社製）を用いたＰＣＲ反応はＭａｒａｔｈｏｎ Ｒｅａｄｙ ｃＤＮＡに添付の方法に従い行った。
【０１４２】
続いて、上記にて単離、同定した塩基配列について５’−側のｃＤＮＡの塩基配列をさらに詳細に解析するために、ヒト脳より調製したｃＤＮＡ（Ｃｌｏｎｔｅｃｈ社製）を鋳型ＤＮＡとして用い、ＰＣＲにより増幅を試みた。すなわち、５０μＬ のＰＣＲ反応液中に５μＬの１０ ｘ ＥｘＴａｑ ｂｕｆｆｅｒ、５μＬのｄＮＴＰｓ（各２．５ ｍＭ）、１０ ｐｍｏｌｅのセンスプライマー１３８６０−１（配列番号：５）ならびに１０ ｐｍｏｌｅのアンチセンスプライマー１３８６０−４（配列番号：６）、２．５μＬのヒト脳 ｃＤＮＡ（Ｃｌｏｎｔｅｃｈ社製）、１μＬのＴａＫａＲａ ＥｘＴａｑ（宝酒造社製）、ならびに１μＬのＴａｑＳｔａｒｔ Ａｎｔｉｂｏｄｙ（宝酒造社製）をそれぞれ含むように調製した後、初めに９６℃で４分インキュベートし、続いて９６℃で２０秒、７２℃で１分の反応からなるサイクルを５回、９６℃で２０秒、７０℃で１分の反応からなるサイクルを５回、さらに９６℃で２０秒、６８℃で１分の反応からなるサイクルを３０回行い、最後に７２℃で２分間インキュベートした。ＰＣＲにより増幅された約８００ ｂｐの産物はｐＧＥＭ−Ｔ Ｅａｓｙベクター（Ｐｒｏｍｅｇａ社製）に挿入し、塩基配列を詳細に決定した（配列番号：１５）。その結果、上記の方法により増幅された領域は単一の読み枠で途中に停止コドンが入ることなくアミノ酸配列（配列番号：１６）を類推することができたことよりクローン１３８６０の開始コドンはさらに上流に存在することが推定された。そこで、再度５’−ＲＡＣＥ法により５’−側のｃＤＮＡの単離、同定を試みた。５’−ＲＡＣＥの際の第一ＰＣＲに用いるＰＣＲプライマー１３８６０−８（配列番号：７）ならびに第二ＰＣＲに用いるＰＣＲプライマー１３８６０−６（配列番号：８）をそれぞれ設計した。ヒト脳由来のＭａｒａｔｈｏｎ−Ｒｅａｄｙ ｃＤＮＡ（Ｃｌｏｎｔｅｃｈ社製）を鋳型として用い、上記と同様に５’−ＲＡＣＥを行った。なお、第一ＰＣＲでは１３８６０−８（配列番号：７）とＡＰ１プライマーを、第二ＰＣＲでは１３８６０−６（配列番号：８）とＡＰ２プライマーをそれぞれ用いた。ＰＣＲにより増幅された約１ ｋｂｐの産物はｐＧＥＭ−Ｔ Ｅａｓｙベクターに挿入し、塩基配列を決定した（配列番号：１７）。その結果、翻訳開始コドンから単一の読み枠でアミノ酸配列が類推され（配列番号：１８）、そのアミノ末端には細胞外分泌シグナル配列と考えられる配列も認められたことより、正しい翻訳開始点が決定できたと考えられた。
【０１４３】
５．２． クローン１３８６０全長ｃＤＮＡの単離
上記のＲＡＣＥ法により決定したクローン１３８６０の塩基配列を基に類推された１３８６０のｃＤＮＡ配列に相当するｍＲＮＡが存在するかどうかを確認するために、１３８６０のコーディング領域と想定される部分のＰＣＲによる増幅、ならびに塩基配列の確認を行った。ＰＣＲに用いるプライマーはセンス方向のプライマーとして翻訳開始コドン近傍の配列にハイブリダイズし、かつ制限酵素ＥｃｏＲＩ認識配列ならびにコザック配列（ＣＣＡＣＣ）を持つように設計した１３８６０−ＡＴＧ（配列番号：９）を、またアンチセンス方向のプライマーとしては１３８６０のカルボキシル末端と想定される配列の下流の配列にハイブリダイズし、且つ制限酵素ＳａｌＩ認識配列を持つように設計した１３８６０−ＴＧＡ２（配列番号：１０）をそれぞれ用い、ＰＣＲの条件は以下のようにして行った。すなわち、５０μＬの反応液中に５μＬの１０ ｘ ＰＣＲ ｂｕｆｆｅｒ（東洋紡社製）、５μＬのｄＮＴＰｓ（各２ ｍＭ）、２μＬの２５ ｍＭ硫酸マグネシウム、各１０ ｐｍｏｌｅずつの１３８６０−ＡＴＧならびに１３８６０−ＴＧＡ２プライマー、５μＬのＨｅｌａ細胞より調製したｃＤＮＡ（Ｃｌｏｎｔｅｃｈ社製）、及び１μＬのＫＯＤ−Ｐｌｕｓ ＤＮＡポリメラーゼ（東洋紡社製）を含むように調製し、初めに９４℃で２分インキュベートした後、９４℃で１５秒、７２℃で２分３０秒からなるサイクルを５回、続いて９４℃で１５秒、７０℃で２分３０秒からなるサイクルを５回、さらに９４℃で１５秒、６８℃で２分３０秒からなるサイクルを２５回行い、最後に７２℃で２分間インキュベートした。ＰＣＲ反応液はＰＣＲ Ｐｕｒｉｆｉｃａｔｉｏｎ Ｋｉｔ（ＱＩＡＧＥＮ社製）を用い脱塩、ＰＣＲプライマーを除去した後、４０μＬのＴＥ溶液で溶出し、再度ＰＣＲによる二次増幅を行った。すなわち、５０μＬの反応液中に５μＬの１０ ｘ ＰＣＲ ｂｕｆｆｅｒ（東洋紡社製）、５μＬのｄＮＴＰｓ（各２ ｍＭ）、２μＬの２５ ｍＭ硫酸マグネシウム、各１０ ｐｍｏｌｅずつの１３８６０−ＡＴＧならびに１３８６０−ＴＧＡ２プライマー、１０μＬの第一ＰＣＲ精製産物、及び１μＬのＫＯＤ−Ｐｌｕｓ ＤＮＡポリメラーゼ（東洋紡社製）を含むように調製し、初めに９４℃で２分インキュベートした後、９４℃で１５秒、６０℃で３０秒、さらに７２℃で２分３０秒からなるサイクルを１０回行い、最後に７２℃で２分間インキュベートした。その結果、約３．４ ｋｂｐの単一のバンドが増幅された。得られたＰＣＲ産物の塩基配列を決定した結果、完全長のアミノ酸配列をコードすることが確認された。最終的に得られたクローン１３８６０の塩基配列を配列番号：１９に、それより類推されるアミノ酸配列を配列番号：２０に示した。
【０１４４】
＜プライマー配列＞

【０１４５】
［実施例６］ クローン１３８６０の発現解析
クローン１３８６０のヒト各臓器ならびに腫瘍組織におけるｍＲＮＡの発現をＲＴ−ＰＣＲ法により解析した。鋳型としてはＭｕｌｔｉｐｌｅ Ｔｉｓｓｕｅ ｃＤＮＡ （ＭＴＣ） Ｐａｎｅｌｓ（Ｈｕｍａｎ Ｉ、Ｈｕｍａｎ ＩＩ、Ｈｕｍａｎ Ｔｕｍｏｒ、ＣＬＯＮＴＥＣＨ社製）をそれぞれ用い、以下のＰＣＲ条件で目的のクローンを増幅した。
【０１４６】
ＰＣＲプライマーとして、１３８６０−１「ＣＴＣＡＣＴＧＧＴＣＴＴＣＴＧＧＧＡＴ（配列番号：１３）」と１３８６０−２「ＴＣＴＴＧＴＴＣＣＧＣＡＣＣＡＣＧＡＣ（配列番号：１４）」を用い、９４℃で１分反応させた後、「９４℃で３０秒、５６℃で３０秒、７２℃で３０秒」からなるサイクルを４０サイクル行った。
その結果、クローン１３８６０は脳、小腸、大腸において発現が高いことが明らかになった。
【０１４７】
【発明の効果】
本発明により、新規なＧＰＣＲ、該ポリペプチドをコードするポリヌクレオチド、該ポリヌクレオチドを含むベクター、該ベクターを含む宿主細胞、該ポリペプチドの製造方法が提供された。さらに、該ポリペプチドに結合するあるいはその活性を修飾する化合物の同定方法が提供された。本発明のポリペプチドやポリヌクレオチド、または本発明のポリペプチドに結合若しくはその活性を修飾する化合物は、本発明のポリペプチドが関連する疾患の新しい予防薬や治療薬の開発への利用が期待される。 さらに本発明によって、本発明のポリペプチドをコードする遺伝子の変異や発現を検出することを含む疾患の検査方法が提供された。ＧＰＣＲは医薬品開発や医療の分野において最も重要かつ注目されている分子の一つであり、本発明において新規ＧＰＣＲが網羅的に提供されたことにより、これら分野の飛躍的な発展が期待される。ＧＰＣＲの研究者にとっても、本発明は貴重な情報源となろう。
【０１４８】
【配列表】

【図面の簡単な説明】
【図１】図１は、既知のＧＰＣＲ配列を、ＧＰＣＲ配列１，０５４個および非ＧＰＣＲ配列６４，１５４個を含む評価用データベースに対して検索した際、Ｅ値に対してプロットしたＧＰＣＲ配列同士対の数およびＧＰＣＲ配列と非ＧＰＣＲ配列の対の数を示す図である。
【図２】図２は、クローン１３８６０の各臓器における発現を解析した結果を示す電気泳動写真である。グラフは、左から、心臓、脳、胎盤、肺、肝臓、骨格筋、腎、膵臓、脾臓、胸腺、前立腺、精巣、卵巣、小腸、大腸、白血球、肺癌、大腸癌、肺癌、前立腺癌、大腸癌、卵巣癌、膵癌である。
【図３】図３は、図２の結果をグラフで示した。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel polypeptide belonging to the guanosine triphosphate binding protein coupled receptor (hereinafter referred to as “GPCR”) family, a polynucleotide encoding the polypeptide, and production and use of these molecules.
[0002]
[Prior art]
To date, more than 90% of drugs that have been created by pharmaceutical companies around the world are targeted for interaction in the extracellular space, and among them, GPCR (Baldwin, JM Curr, which has seven transmembrane helices). Opin.Cell Biol.6, 180-190 (1994)., Strader, CD, et al. FASEB.J.9, 745-754 (1995)., Bocaert, J., Pin, J.P. EMBO, J. 18, 1723-1729 (1999). For this reason, GPCR is one of the most important targets for discovering genes for drug design. GPCRs are involved in signal transduction induced by specific ligands such as adrenaline and acetylcholine, and the characteristics of their binding mechanism have been energetically investigated by experiments (Watson, S & Arkinsrallall, S. The G). -Protein Linked receptor Facts Book (Academic Press, London)).
[0003]
However, even with vast data sources such as cDNA, EST, and microarray analysis, the new sequences discovered so far are only a limited family (Lee, DK et al. Brain Res. Mol. Brain Res.31, 13-22 (2001)., Mishimashima, K., et al. Genomics.69, 314-321 (2000)., Matsumoto, M. et al. Gene.28, 183-189 (2000) , Marches, A., et al. Trends Pharmacol Sci. 20, 447 (1999)., Lee, DK, FEBS, Lett. 446, 103-107 (1999), Yonger, R. M et. al. ome Research. 11, 519-530 (2001)., Horn, F., et al. Nucleic Acids Res. 29, 346-349 (2001).). GPCRdb (Lee, DK et al. Brain Res. Mol. Brain Res. 31, 13-22 (2001)) and the collection by PSI-BLAST (Josefson, L. G. Gene. 239, 333-340 ( Even a large-scale classification of known GPCR sequences such as 1999).) Has not yet led to a broad interpretation at the genome level.
[0004]
Therefore, more than 90% of the whole human genome sequence has already been determined (International Human Sequencing Consortium. Initial sequencing of analysis of the human genome. Nature, 409, 860-921. al. Science 291, 1304- 1351 (2001).) It is important to elucidate the entire GPCR family.
[0005]
[Problems to be solved by the invention]
The present invention has been made in view of such a situation, and an object thereof is to develop an automatic method for efficiently extracting a GPCR sequence from a human genome sequence, thereby comprehensively identifying a new GPCR. It is in.
[0006]
Another object of the present invention is to provide the use of the novel GPCR thus identified. As one aspect of a preferable use of the novel GPCR, an application for screening a drug candidate compound such as a ligand is provided. Furthermore, as another aspect of the preferred use of the novel GPCR, the present invention provides a disease testing method using mutation or expression abnormality of the novel GPCR as an index.
Furthermore, the present invention provides uses for the treatment of diseases of novel GPCRs or molecules that modulate their activity.
[0007]
[Means for Solving the Problems]
In order to solve the above-mentioned problems, the present inventors first performed sequence search (Altschul, SF et al. Nucleic Acids Res. 25, 3389-3402 (1997).), Motif and domain attribution (Bateman, A Nucleic Acids Res. 28, 263-266 (2000), Bairch, A. Nucleic Acids Res. 20, 2013-2018 (1992)), transmembrane helix prediction (Hirokawa, T., et. al. Bioinformatics. 14, 378-379 (1998).), an automated system for the discovery of GPCR sequences in the entire human genome was developed while carefully evaluating the analytical method. This automatic system consists of the following three stages.
[0008]
The first step is gene prediction, ie translation from genomic sequence to amino acid sequence. Many of the known GPCR genes do not contain introns, so 6-frame expansion of nucleotide sequences can be supported to some extent. On the other hand, sequences with multiple exons predict the entire gene structure using a gene discovery program. There is a need to.
[0009]
The second stage consists of a triple analysis of the amino acid sequence. Namely, (1) sequence search against a known GPCR database, (2) motif and domain assignment, and (3) transmembrane helix (TMH) prediction. The former two techniques are used to find closely related GPCR homologues, while TMH prediction is used to deal with distantly related GPCR homologues. Next, candidate sequences are screened by taking the union of the results of each of the three analyses. We used union at the screening stage to maximize the number of candidate sequences.
[0010]
The third stage is to further refine the quality of the gene candidate by eliminating overlapping sequences or by fusing fragment sequences separated by misprediction.
[0011]
According to this automatic system, GPCR sequences can be found efficiently and comprehensively. A great advantage of this automatic system is that it can be detected even in the case of GPCR sequences consisting of multi-exons and distantly related homolog sequences that were difficult to find by conventional methods.
[0012]
The present inventors succeeded in identifying 1215 new GPCR sequences guaranteed with high reliability from the entire human genome by using this automatically developed automated system, and cDNA was isolated from one clone among them. Released. The discovery of new GPCR sequences enables screening for ligands, antagonists or agonists that are expected to be useful as pharmaceuticals. Moreover, GPCR is considered to have an important function in the living body, and abnormal expression or function can cause various diseases. Therefore, it is possible to test for such diseases by using inappropriate GPCR activity or expression as an index. Identified GPCRs, polynucleotides encoding them, and ligands, antagonists or agonists for the identified GPCRs would be suitable therapeutics for these diseases.
[0013]
The present invention relates to a novel GPCR and its gene, and their production and use. More specifically, the present invention provides the following (1) to (32).
(1) The polynucleotide according to any one of (a) to (d) below, which encodes a guanosine triphosphate binding protein-coupled receptor.
(A) a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 20
(B) a polynucleotide comprising the coding region of the base sequence set forth in SEQ ID NO: 19
(C) a polynucleotide encoding a polypeptide comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence set forth in SEQ ID NO: 20
(D) a polynucleotide that hybridizes under stringent conditions to the DNA comprising the nucleotide sequence set forth in SEQ ID NO: 19
(2) A polynucleotide encoding a fragment of the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 20.
(3) A vector comprising the polynucleotide according to (1) or (2).
(4) A host cell carrying the polynucleotide according to (1) or (2) or the vector according to (3).
(5) A polypeptide encoded by the polynucleotide according to (1) or (2).
(6) The method for producing the polypeptide according to (5), comprising a step of culturing the host cell according to (4) and recovering the produced polypeptide from the host cell or a culture supernatant thereof.
(7) An antibody that binds to the polypeptide according to (5).
(8) A method for identifying a ligand for the polypeptide according to (5),
(A) contacting the candidate compound with a cell expressing the polypeptide according to (5) or the polypeptide according to (5) or a cell membrane thereof, and
(B) A method comprising detecting whether the candidate compound binds to the polypeptide according to (5) or the cell expressing the polypeptide according to (5) or a cell membrane thereof.
(9) A method for identifying an agonist for the polypeptide according to (5),
(A) contacting a cell expressing the polypeptide according to (5) with a candidate compound; and
(B) A method comprising detecting whether a candidate compound generates a signal that serves as an indicator of activation of the polypeptide according to (5).
(10) A method for identifying an antagonist to the polypeptide according to (5),
(A) contacting a cell expressing the polypeptide according to (5) in the presence of a candidate compound with an agonist for the polypeptide according to (5);
(B) A method comprising detecting whether or not a signal serving as an indicator of activation of the polypeptide according to (5) is decreased as compared with a case where detection is performed in the absence of a candidate compound.
(11) A ligand identified by the method according to (8).
(12) An agonist identified by the method according to (9).
(13) An antagonist identified by the method according to (10).
(14) A kit for use in the method according to (8) to (10), comprising at least one molecule according to (a) or (b) below.
(A) the polypeptide according to (5)
(B) The host cell or cell membrane thereof according to (4)
(15) A pharmaceutical composition for treating a patient who needs to increase the activity or expression of the polypeptide according to (5), wherein the molecule according to (a) to (c) below is therapeutically effective A pharmaceutical composition comprising a suitable amount.
(A) an agonist for the polypeptide according to (5)
(B) the polynucleotide according to (1) or (2)
(C) The vector according to (3)
(16) A pharmaceutical composition for treating a patient who needs to suppress the activity or expression of the polypeptide according to (5), wherein the molecule according to (a) or (b) below is therapeutically effective A pharmaceutical composition comprising a suitable amount.
(A) an antagonist to the polypeptide according to (5)
(B) a polynucleotide that suppresses the expression of a gene encoding the polypeptide according to (5) that is endogenous in vivo.
(17) A method for examining an abnormality in expression of a gene encoding the polypeptide according to (5) or a disease associated with an abnormality in activity of the polypeptide according to (5), wherein the gene or Detecting a mutation in the expression control region.
(18) The inspection method according to (17), including the following steps (a) to (d):
(A) A step of preparing a DNA sample from a subject
(B) a step of isolating a DNA encoding the polypeptide according to (5) or an expression control region thereof.
(C) determining the base sequence of the isolated DNA
(D) a step of comparing the base sequence of the DNA determined in step (c) with a control
(19) The inspection method according to (17), including the following steps (a) to (d):
(A) A step of preparing a DNA sample from a subject
(B) A step of cleaving the prepared DNA sample with a restriction enzyme
(C) A step of separating DNA fragments according to their sizes
(D) a step of comparing the size of the detected DNA fragment with a control
(20) The inspection method according to (17), including the following steps (a) to (e):
(A) A step of preparing a DNA sample from a subject
(B) Amplifying the DNA encoding the polypeptide according to (5) or its expression control region
(C) cleaving the amplified DNA with a restriction enzyme
(D) A step of separating DNA fragments according to their sizes
(E) comparing the size of the detected DNA fragment with a control
(21) The inspection method according to (17), including the following steps (a) to (e):
(A) A step of preparing a DNA sample from a subject
(B) Amplifying the DNA encoding the polypeptide according to (5) or its expression control region
(C) Dissociating the amplified DNA into single-stranded DNA
(D) Separating the dissociated single-stranded DNA on a non-denaturing gel
(E) A step of comparing the mobility of the separated single-stranded DNA on the gel with the control
(22) The inspection method according to (17), including the following steps (a) to (d).
(A) A step of preparing a DNA sample from a subject
(B) Amplifying the DNA encoding the polypeptide according to (5) or its expression control region
(C) A step of separating the amplified DNA on a gel in which the concentration of the DNA denaturant is gradually increased
(D) A step of comparing the mobility of the separated DNA on the gel with the control.
(23) A method for examining a disease associated with abnormal expression of a gene encoding the polypeptide according to (5), comprising detecting the expression level of the gene in a subject.
(24) The inspection method according to (23), including the following steps (a) to (c).
(A) A step of preparing an RNA sample from a subject
(B) a step of measuring the amount of RNA encoding the polypeptide according to (5) contained in the RNA sample
(C) comparing the measured amount of RNA with a control
(25) The inspection method according to (23), including the following steps (a) to (d).
(A) A step of providing a substrate on which a cDNA sample prepared from a subject and a nucleotide probe that hybridizes with a DNA encoding the polypeptide described in (5) are immobilized.
(B) contacting the cDNA sample with the substrate
(C) The expression level of the gene encoding the polypeptide according to (5) contained in the cDNA sample is measured by detecting the strength of hybridization between the cDNA sample and the nucleotide probe immobilized on the substrate. Process
(D) a step of comparing the measured expression level of the gene encoding the polypeptide according to (5) with a control
(26) The inspection method according to (23), including the following steps (a) to (c).
(A) A step of preparing a protein sample from a subject
(B) a step of measuring the amount of the polypeptide according to (5) contained in the protein sample
(C) comparing the measured amount of polypeptide to a control
(27) An oligonucleotide having a chain length of at least 15 nucleotides, which hybridizes to the DNA encoding the polypeptide of (5) or an expression control region thereof.
(28) A diagnostic agent for a disease associated with abnormal expression of a gene encoding the polypeptide according to (5) or abnormal activity of the polypeptide according to (5), comprising the oligonucleotide according to (27) .
(29) A test agent for a disease associated with abnormal expression of a gene encoding the polypeptide according to (5) or abnormal activity of the polypeptide according to (5), comprising the antibody according to (7).
(30) A mammal into which the polynucleotide according to (1) or (2) has been introduced.
(31) A mammal in which the expression of the endogenous polynucleotide according to (1) is artificially suppressed.
(32) The mammal according to (30) or (31), which is a mouse.
[0014]
The definitions of the terms defined in the present specification are shown below, but these are described for the purpose of facilitating understanding of terms used in the present specification, and are intended to limit the present invention. It should be understood that it should not be used.
[0015]
As used herein, “guanosine triphosphate binding protein coupled receptor (GPCR)” refers to a cell membrane receptor that transmits a signal into a cell through activation of a GTP binding protein.
[0016]
As used herein, “polynucleotide” means a ribonucleotide or deoxynucleotide, which is a polymer composed of a plurality of bases or base pairs. The polynucleotide includes single-stranded and double-stranded DNA. A polynucleotide is intended to include both those that are not modified from the naturally occurring state and those that are modified. Examples of modified bases include tritylated bases and special bases such as inosine.
[0017]
As used herein, “polypeptide” means a polymer composed of a plurality of amino acids. Thus, oligopeptides and proteins are also included in the polypeptide concept. Polypeptide is meant to include both unmodified and modified from the naturally occurring state. Modifications include acetylation, acylation, ADP-ribosylation, amidation, flavin covalent bond, heme moiety covalent bond, nucleotide or nucleotide derivative covalent bond, lipid or lipid derivative covalent bond, phosphatidylinositol covalent bond , Crosslinking, cyclization, disulfide bond formation, demethylation, covalent bridge formation, cystine formation, pyroglutamate formation, formylation, γ-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, Examples include methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer RNA-mediated addition of amino acids to proteins, ubiquitination, and the like.
[0018]
As used herein, “isolated” refers to a substance (eg, a polynucleotide or polypeptide) that has been removed from its original environment (eg, the natural environment if it occurs naturally). It has been changed by “Isolated” is meant to include compounds present in a sample substantially enriched in the compound of interest and / or compounds present in a sample in which the compound of interest is partially or substantially purified. As used herein, the term “substantially purified” refers to a compound that is separated from its natural environment and does not contain at least 60%, preferably 75%, and most preferably 90% of other components that are naturally associated. (Eg, polynucleotide or polypeptide).
[0019]
As used herein, “mutation” refers to an amino acid change in an amino acid sequence or a base change in a base sequence (ie, single or multiple amino acid or nucleotide substitutions, deletions, additions or insertions). Accordingly, “mutant” as used herein refers to an amino acid sequence in which one or more amino acids are changed or a base sequence in which one or more bases are changed. Changes in the base sequence of this variant may or may not alter the amino acid sequence of the polypeptide encoded by the reference polynucleotide. The mutant may be a naturally occurring mutant such as an allelic mutant or a mutant that is not known to exist naturally. A variant may have conservative changes in which a substituted amino acid has similar structural or chemical properties. More rarely, the variant may have non-conservative substitutions. Guidance on determining which and how many amino acid residues to substitute, insert, or delete without inhibiting biological or immunological activity are computer programs well known in the art, such as Can be discovered using DNA Star software.
[0020]
A “deletion” is any amino acid or nucleotide sequence in which one or more amino acid or nucleotide residues are not present compared to the amino acid sequence or nucleotide sequence of a naturally occurring GPCR or GPCR-related polypeptide, respectively. Is a change.
[0021]
An “insertion” or “addition” is a change in an amino acid or nucleotide sequence to which one or more amino acid residues or nucleotide residues are added, respectively, compared to the amino acid sequence or nucleotide sequence of a naturally occurring GPCR or GPCR-related polypeptide. It is.
[0022]
A “substitution” is a change in an amino acid or nucleotide sequence in which one or more amino acids or nucleotides are each replaced by a different amino acid or nucleotide compared to the amino acid sequence or nucleotide sequence of a naturally occurring GPCR or GPCR-related polypeptide. is there.
[0023]
As used herein, “hybridization” refers to the process by which a nucleic acid strand binds to a complementary strand through base pairing.
[0024]
“Treatment” as used herein generally means obtaining a pharmacological and / or physiological effect. The effect may be preventive in that the disease or symptom is completely or partially prevented, or may be therapeutic in that the symptom of the disease is completely or partially treated. The term “treatment” as used herein includes all treatment of diseases in mammals, particularly humans. In addition, the term includes prevention of the onset of a subject who is predisposed to the disease but has not yet been diagnosed, suppressing the progression of the disease, or reducing the disease.
[0025]
As used herein, “ligand” means a molecule that binds to a polypeptide of the invention. Ligand includes natural and synthetic ligands. “Agonist” means a molecule that binds to and activates a polypeptide of the invention. On the other hand, “antagonist” means a molecule that inhibits activation of the polypeptide of the present invention.
[0026]
DETAILED DESCRIPTION OF THE INVENTION
<Polypeptide>
The present invention provides novel polypeptides belonging to the GPCR family. The nucleotide sequence of a human-derived polynucleotide identified by the present inventors and included in the present invention is shown in SEQ ID NO: 19, and the amino acid sequence of the polypeptide encoded by the polynucleotide is shown in SEQ ID NO: 20.
[0027]
GPCRs have the activity of signal transduction into cells through the activation of G proteins by the action of their ligands, including genetic diseases, cranial nervous system, circulatory system, digestive system, immune system, exercise It is associated with a large number of diseases, including the genital and urogenital systems. Therefore, the polypeptide of the present invention can be used for screening ligands, agonists or antagonists that regulate its function, and is an important target for the development of pharmaceuticals for the above diseases.
[0028]
The present invention also provides polypeptides that are functionally equivalent to the polypeptides identified by the inventors. Here, “functionally equivalent” means that the polypeptide of interest has biological properties equivalent to those of the polypeptide identified by the present inventors. The biological properties of GPCRs include the activity of signal transduction into cells through the binding activity with ligands and the activation of trimeric GTP-binding proteins. The trimeric GTP-binding protein is activated by the type of intracellular transmission system that is activated. ²⁺ Is classified into three categories: Gq type that increases cAMP, Gs type that increases cAMP, and Gi type that suppresses cAMP (Trends Pharmacol. Sci. (99) 20: 118). Therefore, whether or not the polypeptide of interest has the same biological characteristics as the polypeptide identified by the present inventors can be determined, for example, by changes in intracellular cAMP concentration or calcium concentration due to its activation. It is possible to evaluate by detecting.
[0029]
One embodiment of a method for preparing a polypeptide functionally equivalent to the polypeptide identified by the present inventors includes a method for introducing a mutation into an amino acid sequence in a protein. Such methods include, for example, site-directed mutagenesis (Current Protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. Jon Wily & Sons Section 8.1-8.5)). In addition, amino acid mutations in polypeptides may occur in nature. The present invention includes substitution or deletion of one or more amino acids in the amino acid sequence of the polypeptide identified by the present inventors (SEQ ID NO: 20), whether artificial or naturally occurring. Proteins that are mutated due to loss, insertion, and / or addition, and that are functionally equivalent to the polypeptides identified by the present inventors.
[0030]
The amino acid to be substituted is preferably an amino acid having properties similar to the amino acid before substitution from the viewpoint of maintaining the function of the protein. For example, Ala, Val, Leu, Ile, Pro, Met, Phe, and Trp are all classified as nonpolar amino acids, and thus are considered to have similar properties. Further, examples of non-chargeability include Gly, Ser, Thr, Cys, Tyr, Asn, and Gln. Examples of acidic amino acids include Asp and Glu, and examples of basic amino acids include Lys, Arg, and His.
[0031]
The number of amino acid mutations and mutation sites in these polypeptides are not limited as long as their functions are maintained. The number of mutations is typically considered to be within 10% of all amino acids, preferably within 5% of all amino acids, and more preferably within 1% of all amino acids.
[0032]
Another embodiment of the method for preparing a polypeptide functionally equivalent to the polypeptide identified by the present inventors includes a method using a hybridization technique or a gene amplification technique. That is, a person skilled in the art can use a hybridization technique (Current Protocols in Molecular Biology edit. Ausubel et al. (1987) Publisher. Jon Wily & Sons Section 6.3-6.4) by the present inventors. Based on the DNA sequence (SEQ ID NO: 19) encoding the identified polypeptide or a part thereof, a DNA sample having high homology with this DNA is isolated from the DNA sample derived from the same species or a heterogeneous organism, Obtaining a polypeptide that is functionally equivalent to the polypeptide identified by the inventors is usually possible. Thus, a polypeptide encoded by a DNA that hybridizes with a DNA encoding a polypeptide identified by the present inventors, which is functionally equivalent to the polypeptide identified by the present inventors Are also included in the polypeptides of the present invention.
[0033]
Examples of organisms for isolating such polypeptides include, but are not limited to, rats, mice, rabbits, chickens, pigs, cows, and the like in addition to humans.
[0034]
As stringent hybridization conditions for isolating a DNA encoding a polypeptide functionally equivalent to the polypeptide identified by the present inventors, “1 × SSC, 0.1% SDS, 37 ° C.” is usually used. The more severe condition is “0.5 × SSC, 0.1% SDS, 42 ° C.”, and the more severe condition is “0.2 × SSC, 0.1% SDS, 65 ° C.” It is a condition of the degree. Thus, isolation of DNA having high homology with the probe sequence can be expected as the hybridization conditions become more severe. However, combinations of the above SSC, SDS, and temperature conditions are exemplary, and those skilled in the art will recognize the above or other factors that determine the stringency of hybridization (eg, probe concentration, probe length, hybridization reaction). It is possible to achieve the same stringency as above by appropriately combining the time and the like.
[0035]
A polypeptide encoded by DNA isolated using such a hybridization technique usually has high homology in amino acid sequence with the polypeptide identified by the present inventors. High homology means at least 40% or more, preferably 60% or more, more preferably 80% or more, more preferably 90% or more, more preferably at least 95% or more, and more preferably at least 97% or more (for example, 98% or more). ~ 99%) sequence homology. The identity of amino acid sequences can be determined, for example, by the algorithm BLAST (Proc. Natl. Acad. Sei. USA 87: 2264-2268, 1990, Proc. Natl. Acad. Sei. USA 90: 5873-5877, 1993) by Karlin and Altschul. Can be determined by. Based on this algorithm, a program called BLASTX has been developed (Altschul et al. J. Mol. Biol. 215: 403-410, 1990). When the amino acid sequence is analyzed by BLASTX, the parameters are, for example, score = 50 and wordlength = 3. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific techniques for these analysis methods are known (http://www.ncbi.nlm.nih.gov.).
[0036]
In addition, the present inventors identified using polyamplification technology (PCR) (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publ. John Wiley & Sons Section 6.1-6.4). A primer is designed based on a part of the DNA sequence encoding the peptide (SEQ ID NO: 19), and a DNA fragment highly homologous to the DNA sequence encoding the polypeptide identified by the present inventors is isolated, It is also possible to obtain a polypeptide functionally equivalent to the polypeptide identified by the present inventors based on DNA.
[0037]
The polypeptides of the present invention may be in the form of a “mature” protein or may be part of a larger protein, such as a fusion protein. The polypeptide of the present invention may contain secretory or leader sequences, pro sequences, sequences useful for purification such as multiple histidine residues, or additional sequences that ensure stability during recombinant production. Good.
[0038]
<Polypeptide fragments>
The present invention also provides fragments of the polypeptides of the present invention. Such a fragment is a polypeptide having an amino acid sequence which is entirely the same as a part of the amino acid sequence of the polypeptide of the present invention but not the same. The polypeptide fragment of the present invention is a polypeptide fragment consisting of a sequence of usually 8 amino acid residues or more, preferably 12 amino acid residues or more (for example, 15 amino acid residues or more). Suitable fragments include, for example, deletion of a series of residues including the amino terminus or a series of residues including the carboxyl terminus, or a series of residues including the amino terminus and a series of residues including the carboxyl terminus. A truncation polypeptide having the amino acid sequence deleted of Α helix and α helix formation region, β sheet and β sheet formation region, turn and turn formation region, coil and coil formation region, hydrophilic region, hydrophobic region, α amphiphilic region, β amphiphilic region, Also suitable are fragments characterized by structural or functional properties, such as fragments comprising a variable region, a surface forming region, a substrate binding region, and a high antigen index region. Other suitable fragments are biologically active fragments. Biologically active fragments are those that mediate the activity of a polypeptide of the invention, including fragments with similar activity, fragments with increased activity, or fragments with reduced undesirable activity. For example, the fragment | piece which has the activity which a ligand couple | bonds and performs signal transmission in a cell is mentioned. Further included are fragments that are antigenic or immunogenic in animals, particularly humans. These polypeptide fragments preferably retain the biological activity of the polypeptide of the present invention, including antigenic activity. Variants of the identified sequences and fragments also form part of the present invention. Preferred variants are those that differ from the subject by conservative amino acid substitutions, i.e., one residue is replaced with another residue of similar nature. Typical such substitutions are between Ala, Val, Leu and Ile, between Ser and Thr, between acidic residues Asp and Glu, between Asn and Gln, between basic residues Lys and Arg, or aromatic. Occurs between the family residues Phe and Tyr.
[0039]
In addition, a fragment that binds to a ligand and does not cause intracellular signal transduction is useful because it can be a competitive inhibitor of the polypeptide of the present invention, and such a fragment is also included in the present invention.
[0040]
<Production of polypeptide>
The polypeptide of the present invention can be produced by any appropriate method. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Is included. Means for the production of such polypeptides are well understood in the art. A recombinant polypeptide can be prepared, for example, by introducing a vector inserted with the polynucleotide of the present invention into a suitable host cell and purifying the polypeptide expressed in the transformant. On the other hand, naturally-derived polypeptides can be prepared, for example, using an affinity column to which an antibody against the polypeptide of the present invention described later is bound (Current Protocols in Molecular Biology edit. Ausubel et al. (1987) Publishing). John Wiley & Sons Section 16.1-16.19). The antibody used for affinity purification may be a polyclonal antibody or a monoclonal antibody. Further, in vitro translation (see, for example, “On the fidelity of mRNA translation in the nuclease-treated rabbit recycle lysate system. Dasso, M. C., Jackson-3, R. JN. 9: 1989, 1989, N.J. It is also possible to prepare the polypeptide of the present invention by such means. A fragment of the polypeptide of the present invention can be produced, for example, by cleaving the polypeptide of the present invention with an appropriate peptidase.
[0041]
<Polynucleotide>
The present invention also provides a polynucleotide encoding the polypeptide of the present invention. The polynucleotide of the present invention includes a polynucleotide encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 20, a polynucleotide comprising the coding region of the base sequence set forth in SEQ ID NO: 19, and due to the degeneracy of the genetic code A polynucleotide encoding a polypeptide consisting of a nucleotide sequence different from the nucleotide sequence set forth in SEQ ID NO: 19 but consisting of the amino acid sequence set forth in SEQ ID NO: 20 is included. The polynucleotide of the present invention further encodes a polypeptide functionally equivalent to the polypeptide encoded by these polynucleotides, and is at least 40% or more, preferably 60% or more in the sequence and the total length of the polynucleotide. More preferably 80% or more, more preferably 90% or more, more preferably 95% or more, and still more preferably 97% or more (for example, 98 to 99%). The identity of the base sequence is determined, for example, by the algorithm BLAST by Karlin and Altschul (Proc. Natl. Acad. Sei. USA 87: 2264-2268, 1990, Proc. Natl. Acad. Sei. USA 90: 5873-5877, 1993). Can be determined by. Based on this algorithm, a program called BLASTN has been developed (Altschul et al. J. Mol. Biol. 215: 403-410, 1990). When analyzing a base sequence by BLASTN, parameters are set, for example, score = 100 and wordlength = 12. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific techniques for these analysis methods are known (http://www.ncbi.nlm.nih.gov.). The polynucleotide of the present invention includes a polynucleotide having a base sequence complementary to the base sequence of the above polynucleotide.
[0042]
The polynucleotides of the invention can be obtained by standard cloning and screening, for example, from a cDNA library derived from mRNA in cells. In addition, the polynucleotide of the present invention can be obtained from a natural source such as a genomic DNA library, and can also be synthesized using known techniques that are commercially available.
[0043]
A polynucleotide comprising a nucleotide sequence having significant homology with the polynucleotide sequence (SEQ ID NO: 19) identified by the present inventors can be obtained by, for example, a hybridization technique (Current Protocols in Molecular Biology edit. Ausubel et al. (1987) Publ. John Wiley & Sons Section 6.3-6.4) and Gene Amplification Technology (PCR) (Current protocols in Molecular Biology edit. Ausubel et al. (1987) PublS. -6.4). That is, using a hybridization technique, a DNA sample derived from the same species or a heterogeneous organism based on the polynucleotide sequence (SEQ ID NO: 19) identified by the present inventors or a part thereof is homologous to this. High DNA can be isolated. Furthermore, by using gene amplification technology, a primer is designed based on a part of the polynucleotide sequence (SEQ ID NO: 19) identified by the present inventors, and a polynucleotide having high homology with the polynucleotide sequence Can be isolated. Therefore, the present invention includes a polynucleotide that hybridizes with the polynucleotide having the base sequence set forth in SEQ ID NO: 19 under stringent conditions. Stringent hybridization conditions are usually about “1 × SSC, 0.1% SDS, 37 ° C.”, and more stringent conditions are about “0.5 × SSC, 0.1% SDS, 42 ° C.” More severe conditions are about “0.2 × SSC, 0.1% SDS, 65 ° C.”. Thus, isolation of DNA having high homology with the probe sequence can be expected as the hybridization conditions become more severe. However, combinations of the above SSC, SDS, and temperature conditions are exemplary, and those skilled in the art will recognize the above or other factors that determine the stringency of hybridization (eg, probe concentration, probe length, hybridization reaction). It is possible to achieve the same stringency as above by appropriately combining the time and the like.
[0044]
A polynucleotide comprising a nucleotide sequence having significant homology with the polynucleotide sequence identified by the present inventors is a method for introducing a mutation into the nucleotide sequence set forth in SEQ ID NO: 19 (for example, site-directed mutagenesis). (Current Protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons Section 8.1-8.5)). Such polynucleotides may also be caused by natural mutations. In the present invention, a polynucleotide encoding a polypeptide in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence of SEQ ID NO: 20 due to such a mutation of the base sequence Is included.
[0045]
When the polynucleotide of the present invention is used for recombinant production of the polypeptide of the present invention, the polynucleotide includes a mature polypeptide coding sequence or a fragment thereof alone, other coding sequences (eg, leader or secretory sequences, A coding sequence for a mature polypeptide or fragment thereof in the same reading frame as that encoding a pre-, pro- or pre-pro-protein sequence, or other fusion peptide moiety). For example, a marker sequence that facilitates purification of the fusion polypeptide can be encoded. In a preferred embodiment of this aspect of the invention, the marker sequence is provided by the pcDNA3.1 / Myc-His vector (Invitrogen) and Gentz et al., Proc. Natl. Acad. Sci. USA (1989) 86: 821-824, a hexa-histidine peptide, or Myc tag. The polynucleotide may also include 5 ′ and 3 ′ non-coding sequences, such as transcribed but not translated sequences, splicing and polyadenylation signals, ribosome binding sites, and mRNA stabilizing sequences.
[0046]
<Probe / Primer / Antisense / Ribozyme>
The present invention provides a nucleotide having a chain length of at least 15 nucleotides, which is complementary to the polynucleotide identified by the present inventors (polynucleotide consisting of the base sequence described in SEQ ID NO: 19 or its complementary strand). . Here, “complementary strand” refers to the other strand with respect to one strand of a double-stranded nucleic acid consisting of A: T (U in the case of RNA) and G: C base pairs. In addition, “complementary” is not limited to the case where the sequence is completely complementary in at least 15 consecutive nucleotide regions, and is at least 70%, preferably at least 80%, more preferably 90%, and even more preferably 95%. What is necessary is just to have the homology on the above base sequences. The algorithm described in the present specification may be used as an algorithm for determining homology. Such a nucleotide can be used as a probe for detecting and isolating the polynucleotide of the present invention and as a primer for amplifying the nucleotide of the present invention. When used as a primer, it usually has a chain length of 15 to 100 nucleotides, preferably 15 to 35 nucleotides. When used as a probe, nucleotides having a chain length of at least 15 nucleotides, preferably at least 30 nucleotides, including at least a part or all of the sequence of the DNA of the present invention are used. Such nucleotides preferably hybridize specifically to DNA encoding the polypeptide of the present invention. “Specifically hybridize” means to hybridize with the nucleotide (SEQ ID NO: 19) identified by the present inventors under normal hybridization conditions, preferably under stringent conditions, It means that it does not hybridize with DNA encoding the peptide.
[0047]
These nucleotides can be used for examining and diagnosing abnormal activity of the polypeptide of the present invention and abnormal expression of the gene encoding the polypeptide.
[0048]
In addition, these nucleotides include polynucleotides that suppress the expression of a gene encoding the polypeptide of the present invention. Such polynucleotides include antisense DNA (DNA encoding antisense RNA complementary to the transcription product of the gene encoding the polypeptide of the present invention) and ribozyme (transcription of gene encoding the polypeptide of the present invention). DNA encoding an RNA having ribozyme activity that specifically cleaves the product.
[0049]
There are several factors as described below for the action of antisense DNA to suppress the expression of a target gene. Inhibition of transcription by triplex formation, suppression of transcription by hybridization with a site where an open loop structure is locally created by RNA polymerase, transcription inhibition by hybridization with RNA that is undergoing synthesis, intron and exon Of splicing by hybrid formation at the junction with the protein, suppression of splicing by hybridization with the spliceosome formation site, suppression of transition from the nucleus to the cytoplasm by hybridization with mRNA, hybridization with capping sites and poly (A) addition sites Suppression by formation, suppression of translation initiation by hybridization with a translation initiation factor binding site, translation suppression by hybridization with a ribosome binding site in the vicinity of the initiation codon, hive with mRNA translation region and polysome binding site Head forming outgrowth inhibitory peptide chain by, and the like gene silencing by the formation of a hybrid with an interaction site between a nucleic acid and protein. They inhibit transcription, splicing, or translation processes and suppress target gene expression (Hirashima and Inoue "Neurochemistry Experiments 2 Nucleic acid IV gene replication and expression", Japan Biochemical Society, Tokyo Kagaku Dojin , Pp. 319-347, 1993).
[0050]
The antisense DNA used in the present invention may suppress the expression of the target gene by any of the actions described above. In one embodiment, designing an antisense sequence complementary to the untranslated region near the 5 ′ end of the mRNA of a gene would be effective for inhibiting translation of the gene. However, sequences complementary to the coding region or the 3 ′ untranslated region can also be used. Thus, the DNA containing the antisense sequence of the non-translated region as well as the translated region of the gene is also included in the antisense DNA used in the present invention. The antisense DNA to be used is linked downstream of a suitable promoter, and preferably a sequence containing a transcription termination signal is linked on the 3 ′ side. The sequence of the antisense DNA is preferably a sequence complementary to the target gene or a part thereof, but may not be completely complementary as long as the gene expression can be effectively inhibited. The transcribed RNA preferably has a complementarity of 90% or more, most preferably 95% or more, with respect to the transcription product of the target gene. In order to effectively inhibit the expression of a target gene using an antisense sequence, the antisense DNA has a chain length of at least 15 bp, preferably 100 bp, more preferably 500 bp or more in order to cause an antisense effect. The chain length is usually within 3000 bp, preferably within 2000 bp.
[0051]
Such antisense DNA may be applied to gene therapy for diseases caused by abnormality (functional abnormality or expression abnormality) of the polypeptide of the present invention. The antisense DNA is, for example, based on the sequence information of DNA encoding the polypeptide of the present invention (for example, SEQ ID NO: 19) (Stein, 1988 Physicochemicals of phosphodeoxynucleotides Nucleoids. Nucleoids. , 3209-21 (1988)) and the like.
[0052]
Suppression of endogenous gene expression can also be carried out using DNA encoding a ribozyme. Ribozyme refers to an RNA molecule having catalytic activity. Some ribozymes have various activities. Among them, research on ribozymes as enzymes that cleave RNA has made it possible to design ribozymes for site-specific cleavage of RNA. Some ribozymes have a group I intron type and a size of 400 nucleotides or more like M1RNA contained in RNaseP, but some have an active domain of about 40 nucleotides called hammerhead type or hairpin type ( Makoto Koizumi and Eiko Otsuka, (1990) Protein Nucleic Acid Enzymes, 35: 2191).
[0053]
For example, the self-cleaving domain of hammerhead ribozyme cleaves on the 3 ′ side of C15 of G13U14C15, but it is important for activity that U14 forms a base pair with A at position 9, and the base at position 15 is In addition to C, it has been shown to be cleaved by A or U (M. Koizumi et al., (1988) FEBS Lett. 228: 225). If the ribozyme substrate binding site is designed to be complementary to the RNA sequence near the target site, it is possible to create a restriction enzyme-like RNA-cleaving ribozyme that recognizes the UC, UU or UA sequence in the target RNA. (M. Koizumi et al., (1988) FEBS Lett. 239: 285, Makoto Koizumi and Eiko Otsuka, (1990) Protein Nucleic Acid Enzyme, 35: 2191, M. Koizumi et al., (1989) Nucleic Acids Res. 17: 7059. ). In the polynucleotide (SEQ ID NO: 19) identified by the present inventors, there are a plurality of sites that can be targeted.
[0054]
Hairpin ribozymes are also useful for the purposes of the present invention. Hairpin ribozymes are found, for example, in the minus strand of satellite RNA of tobacco ring spot virus (JM Buzyan Nature 323: 349, 1986). It has been shown that this ribozyme can also be designed to cause target-specific RNA cleavage (Y. Kikuchi and N. Sasaki (1992) Nucleic Acids Res. 19: 6751, Hiroshi Kikuchi, (1992) Chemistry and Biology 30 : 112).
[0055]
When the polynucleotide that suppresses the expression of the gene encoding the polypeptide of the present invention is used for gene therapy, for example, a viral vector such as a retroviral vector, an adenoviral vector, an adeno-associated viral vector, or a non-viral such as a liposome It is conceivable to administer to a patient by an ex vivo method or an in vivo method using a vector or the like.
[0056]
<Production of vector, host cell, polypeptide>
The present invention also provides a vector containing the polynucleotide of the present invention, a polynucleotide of the present invention or a host cell carrying the vector, and a method for producing the polypeptide of the present invention using the host cell.
[0057]
The vector of the present invention is not particularly limited as long as it can stably hold the inserted DNA. For example, if E. coli is used as a host, a pBluescript vector (manufactured by Stratagene) is preferred as a cloning vector. An expression vector is particularly useful when a vector is used for the purpose of producing the polypeptide of the present invention. The expression vector is not particularly limited as long as it is a vector that expresses a polypeptide in vitro, in E. coli, in cultured cells, or in an individual organism. For example, in the case of in vitro expression, pBEST vector (manufactured by Promega), E. coli PET vector (manufactured by Invitrogen), pME18S-FL3 vector (GenBank Accession No. AB009864) for cultured cells, pME18S vector (Mol Cell Biol. 8: 466-472 (1988)) for organisms, etc. Is preferred. The insertion of the DNA of the present invention into a vector can be carried out by a conventional method, for example, by a ligase reaction using a restriction enzyme site (Current protocol in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley &). Sons.Section 11.4-11.11).
[0058]
The host cell into which the vector of the present invention is introduced is not particularly limited, and various host cells can be used depending on the purpose. Examples of cells for expressing the polypeptide include bacterial cells (eg, Streptococcus, Staphylococcus, E. coli, Streptomyces, Bacillus subtilis), fungal cells (eg, yeast, Aspergillus), insect cells (eg, Drosophila S2). , Spodoptera SF9), animal cells (eg, CHO, COS, HeLa, C127, 3T3, BHK, HEK293, Bowes melanoma cells) and plant cells. Vector introduction into a host cell can be performed by, for example, calcium phosphate precipitation method, electric pulse perforation method (Current protocol in Molecular Biology edit. Ausubel et al. (1987) Publisher. John Wiley & Sons. 9-9. 9.1). It can be carried out by a known method such as a lipofectamine method (GIBCO-BRL) or a microinjection method.
[0059]
In order to secrete the polypeptide expressed in the host cell into the lumen of the endoplasmic reticulum, into the periplasmic space, or into the extracellular environment, an appropriate secretion signal can be incorporated into the polypeptide of interest. These signals may be endogenous to the polypeptide of interest or may be heterologous signals.
[0060]
The polypeptide of the present invention is recovered when the polypeptide of the present invention is secreted into the medium. When the polypeptide of the present invention is produced intracellularly, the cell is first lysed, and then the polypeptide is recovered.
[0061]
To recover and purify the polypeptides of the invention from recombinant cell culture, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, Known methods including hydroxylapatite chromatography and lectin chromatography can be used.
[0062]
<Inspection method>
The present invention provides a method for examining a disease associated with abnormal expression of a gene encoding the polypeptide of the present invention or abnormal activity of the polypeptide of the present invention. GPCRs are considered to have important functions in vivo, and abnormal expression or function can cause various diseases. Therefore, it is possible to examine such diseases by using inappropriate activity or expression of the polypeptide of the present invention as an index.
[0063]
In the present invention, “disease test” is performed not only for a test for establishing a treatment strategy for a subject exhibiting a symptom of a disease, but also for determining whether or not the subject is susceptible to the disease. Also included are preventive tests.
[0064]
One embodiment of the test method of the present invention is a method comprising detecting a mutation in a gene encoding the polypeptide of the present invention or an expression control region thereof in a subject.
[0065]
One method is a method in which a test is performed by directly determining the base sequence of the gene encoding the polypeptide of the present invention or the expression control region thereof in a subject. In this method, first, a DNA sample is prepared from a subject. The DNA sample can be prepared based on chromosomal DNA or RNA extracted from a subject's cells, such as blood, urine, saliva, tissue biopsy or autopsy material. In order to prepare a DNA sample of this method from chromosomal DNA, for example, chromosomal DNA may be cleaved with an appropriate restriction enzyme and cloned into a vector to prepare a genomic library. In order to prepare the DNA sample of this method from RNA, for example, a cDNA library may be prepared from RNA using reverse transcriptase. In this method, next, DNA containing the gene encoding the polypeptide of the present invention or its expression control region is isolated. The DNA can be isolated by screening a genomic library or cDNA library using a probe that hybridizes to a gene encoding the polypeptide of the present invention or a DNA containing its expression control region. . It can also be isolated by genomic DNA library, cDNA library, or PCR using RNA as a template, using a primer that hybridizes to the gene encoding the polypeptide of the present invention or DNA containing its expression control region. it can. In this method, the base sequence of the isolated DNA is then determined. The base sequence of the selected DNA can be determined by a method known to those skilled in the art. In this method, the determined DNA base sequence is then compared with a control. The “control” in the present method refers to a base sequence of DNA containing a normal (wild type) gene encoding the polypeptide of the present invention or an expression control region thereof. As a result of such comparison, if the base sequence of the subject's DNA is different from that of the control, the subject is determined to be affected or at risk of developing the disease.
[0066]
The test method of the present invention can use various methods other than the method for directly determining the base sequence of DNA derived from a subject as described above.
[0067]
In one method, a DNA sample is first prepared from a subject. Next, the prepared DNA sample is cleaved with a restriction enzyme. Next, the DNA fragments are separated according to their sizes. The size of the detected DNA fragment is then compared to a control. In another embodiment, a DNA sample is first prepared from a subject. Subsequently, the gene encoding the polypeptide of the present invention or DNA containing its expression control region is amplified. Furthermore, the amplified DNA is cleaved with a restriction enzyme. Next, the DNA fragments are separated according to their sizes. The size of the detected DNA fragment is then compared to a control.
[0068]
Examples of such a method include a method using a restriction fragment length polymorphism / RFLP, a PCR-RFLP method, and the like. Specifically, if there is a mutation at the restriction enzyme recognition site, or if there is a base insertion or deletion in the DNA fragment produced by the restriction enzyme treatment, the size of the fragment produced after the restriction enzyme treatment is compared with the control. Change. By amplifying a portion containing this mutation by the PCR method and treating with each restriction enzyme, these mutations can be detected as a difference in band mobility after electrophoresis. Alternatively, the presence or absence of a mutation can be detected by treating chromosomal DNA with these restriction enzymes, performing electrophoresis, and then performing Southern blotting using the probe DNA of the present invention. The restriction enzyme used can be appropriately selected according to each mutation. In this method, in addition to genomic DNA, RNA prepared from a subject can be converted to cDNA using reverse transcriptase, and this can be cleaved with a restriction enzyme as it is, followed by Southern blotting. It is also possible to amplify a gene encoding the polypeptide of the present invention or a DNA containing its expression control region by PCR using this cDNA as a template, cleave it with a restriction enzyme, and then examine the difference in mobility. .
[0069]
In another method, a DNA sample is first prepared from a subject. Subsequently, the gene encoding the polypeptide of the present invention or DNA containing its expression control region is amplified. Furthermore, the amplified DNA is dissociated into single-stranded DNA. The dissociated single-stranded DNA is then separated on a non-denaturing gel. The mobility of the separated single-stranded DNA on the gel is compared with the control.
[0070]
As such a method, for example, the PCR-SSCP (single-strand conformation polymorphism, single-strand conformation morphisms). (Cloning and polymerase chain reformation-single-molecule formation polymorphism 11) 1992 Jan 1; 12 (1): 139-146, Detection of p53 gene mutations in human brains by single-strand conformation polymorphism of polymorphism. erase chain reaction products. Oncogene. 1991 Aug 1; 6 (8): 1313-1318., Multiple fluorescence-based PCR-SSCP analysis with post labeling. ). This method is suitable for screening a large number of DNA samples because it has advantages such as relatively simple operation and a small amount of test sample. The principle is as follows. When a double-stranded DNA fragment is dissociated into single strands, each strand forms a unique higher-order structure depending on its base sequence. When the dissociated DNA strand is electrophoresed in a polyacrylamide gel not containing a denaturing agent, single-stranded DNA having the same complementary strand length moves to a different position according to the difference in each higher-order structure. The higher-order structure of this single-stranded DNA also changes by substitution of a single base, and shows different mobility in polyacrylamide gel electrophoresis. Therefore, by detecting this change in mobility, it is possible to detect the presence of mutations due to point mutations, deletions or insertions in the DNA fragment.
[0071]
Specifically, first, a DNA encoding a gene encoding the polypeptide of the present invention or an expression control region thereof is amplified by a PCR method or the like. As a range to be amplified, a length of about 200 to 400 bp is usually preferable. Those skilled in the art can perform PCR by appropriately selecting reaction conditions and the like. During PCR ³² By using a primer labeled with an isotope such as P, a fluorescent dye, or biotin, the amplified DNA product can be labeled. Or in the PCR reaction solution ³² It is also possible to label the amplified DNA product by performing PCR by adding a substrate base labeled with an isotope such as P, a fluorescent dye, or biotin. Furthermore, using a Klenow enzyme after the PCR reaction, ³² Labeling can also be performed by adding a substrate base labeled with an isotope such as P, a fluorescent dye, or biotin to the amplified DNA fragment. The labeled DNA fragment thus obtained is denatured by applying heat or the like, and subjected to electrophoresis using a polyacrylamide gel not containing a denaturing agent such as urea. In this case, the conditions for separating DNA fragments can be improved by adding an appropriate amount (about 5 to 10%) of glycerol to the polyacrylamide gel. Electrophoretic conditions vary depending on the nature of each DNA fragment, but are usually carried out at room temperature (20 to 25 ° C.), and when a favorable separation cannot be obtained, a temperature of 4 to 30 ° C. gives optimum mobility. Review. After electrophoresis, the mobility of the DNA fragment is analyzed by autoradiography using an X-ray film, a scanner for detecting fluorescence, or the like. If a band with a difference in mobility is detected, this band can be directly excised from the gel, amplified again by PCR, and directly sequenced to confirm the presence of the mutation. Even when the labeled DNA is not used, the band can be detected by staining the gel after electrophoresis with ethidium bromide or silver staining.
[0072]
In yet another method, a DNA sample is first prepared from a subject. Subsequently, the gene encoding the polypeptide of the present invention or DNA containing its expression control region is amplified. Furthermore, the amplified DNA is separated on a gel where the concentration of the DNA denaturant is gradually increased. The mobility of the separated DNA on the gel is then compared to the control.
[0073]
Examples of such a method include denaturant gradient gel electrophoresis (DGGE method). The DGGE method is a method in which a mixture of DNA fragments is run in a polyacrylamide gel having a concentration gradient of a denaturing agent, and the DNA fragments are separated according to differences in instability. When a mismatched unstable DNA fragment migrates to a certain denaturant concentration in the gel, the DNA sequence around the mismatch is partially dissociated into single strands due to its instability. The mobility of this partially dissociated DNA fragment becomes very slow and can be separated from the mobility of complete double-stranded DNA without a dissociated portion. Specifically, a gene encoding the polypeptide of the present invention or a DNA containing its expression control region is amplified by a PCR method or the like using the primer of the present invention, and the concentration of a denaturing agent such as urea is transferred. Electrophorese in a polyacrylamide gel that gradually increases according to and compare to the control. In the case of a DNA fragment with mutation, the DNA fragment becomes single-stranded at a lower denaturant concentration position, and the movement speed becomes extremely slow. Therefore, the presence or absence of mutation is detected by detecting the difference in mobility. be able to.
[0074]
In addition to the above method, an allele specific oligonucleotide (ASO) hybridization method can be used for the purpose of detecting only a mutation at a specific position. When an oligonucleotide containing a base sequence that is considered to have a mutation is prepared and hybridized with the sample DNA, the efficiency of hybridization is reduced when the mutation exists. It can be detected by Southern blotting, a method using the property of quenching by intercalating a special fluorescent reagent into the hybrid gap, or the like. Moreover, the detection by the ribonuclease A mismatch cutting | disconnection method is also possible. Specifically, DNA containing a gene encoding the polypeptide of the present invention is amplified by the PCR method or the like, and hybridized with labeled RNA prepared from control cDNA or the like incorporated into a plasmid vector or the like. Since the hybrid has a single-stranded structure in the portion where the mutation exists, the presence of the mutation can be detected by cleaving this portion with ribonuclease A and detecting this by autoradiography or the like.
[0075]
Another embodiment of the test method of the present invention is a method comprising detecting the expression level of a gene encoding the polypeptide of the present invention. Herein, “gene expression” includes transcription and translation. Thus, “expression product” includes mRNA and protein.
[0076]
In the test method at the transcription level of the gene encoding the polypeptide of the present invention, an RNA sample is first prepared from a subject. Next, the amount of RNA encoding the polypeptide of the present invention contained in the RNA sample is measured. The measured polypeptide of the invention is then compared to the control for the amount of RNA encoded.
[0077]
Such methods include Northern blotting using a probe that hybridizes to a polynucleotide encoding the polypeptide of the present invention, or RT- using a primer that hybridizes to a polynucleotide encoding the polypeptide of the present invention. A PCR method etc. can be illustrated.
[0078]
Further, in the examination at the transcription level of the gene encoding the polypeptide of the present invention, a DNA array (new genetic engineering handbook, Masaaki Muramatsu / Masaka Yamamoto, Yodosha, p280-284) can also be used. Specifically, first, a substrate on which a cDNA sample prepared from a subject and a polynucleotide probe that hybridizes with a polynucleotide encoding the polypeptide of the present invention is immobilized is provided. There may be a plurality of polynucleotide probes immobilized on the substrate in order to detect a plurality of polynucleotides encoding the polypeptides of the present invention. Preparation of a cDNA sample from a subject can be performed by methods well known to those skilled in the art. In a preferred embodiment of preparing a cDNA sample, first, total RNA is extracted from a subject's cells. Examples of the cells include blood, urine, saliva, tissue biopsy or autopsy material cells. Extraction of total RNA can be performed, for example, as follows. Any existing method and kit can be used as long as the method can prepare high-purity total RNA. For example, after pretreatment using Ambion “RNA later”, total RNA is extracted using Nippon Gene “Isogen”. Specific methods may follow those attached protocols. Next, using the extracted total RNA as a template, cDNA is synthesized using reverse transcriptase to prepare a cDNA sample. Synthesis of cDNA from total RNA can be performed by methods well known to those skilled in the art. The prepared cDNA sample is labeled for detection as necessary. The labeling substance is not particularly limited as long as it can be detected, and examples thereof include fluorescent substances and radioactive elements. The labeling can be carried out by a method generally performed by those skilled in the art (L Luo et al., Gene expression profiles of laser-captured adjutant neuronal subtypes. Nat Med. 1999, 117-122).
[0079]
In the present invention, the “substrate” means a plate-like material on which a polynucleotide can be immobilized. The substrate of the present invention is not particularly limited as long as the polynucleotide can be immobilized, but a substrate generally used in DNA array technology can be preferably used.
[0080]
The advantage of DNA array technology is that the amount of hybridization solution is very small and a highly complex target containing cDNA derived from total cellular RNA can be hybridized to immobilized nucleotide probes. In general, a DNA array is composed of thousands of nucleotides printed on a substrate at high density. Usually, these DNAs are printed on the surface of a non-porous substrate. The surface layer of the substrate is generally glass, but a porous film such as a nitrocellulose membrane can be used. There are two types of nucleotide immobilization (array), one is a polynucleotide-based array developed by Affymetrix, and the other is a cDNA array developed mainly at Stanford University. In an array of polynucleotides, the polynucleotides are usually synthesized in situ. For example, in situ synthesis methods for polynucleotides using photolithographic technology (Affymetrix) and ink-jet (Rosetta Informatics) technology for immobilizing chemical substances are already known. Can be used. The polynucleotide probe immobilized on the substrate is not particularly limited as long as it specifically hybridizes with the gene encoding the polypeptide of the present invention. The polynucleotide probe of the present invention includes a polynucleotide or cDNA. Here, “specifically hybridizes” means substantially hybridizing with a polynucleotide encoding the polypeptide of the present invention and not substantially hybridizing with other polynucleotides. As long as specific hybridization is possible, the polynucleotide probe need not be completely complementary to the base sequence of the polynucleotide to be detected. The length of the polynucleotide probe bound to the substrate is usually 100 to 4000 base, preferably 200 to 4000 base, and more preferably 500 to 4000 base when cDNA is immobilized. When immobilizing a synthetic polynucleotide, it is usually 15 to 500 base, preferably 30 to 200 base, and more preferably 50 to 200 base. The process of immobilizing the polynucleotide to the substrate is also commonly referred to as “printing”. Specifically, for example, printing can be performed as follows, but the present invention is not limited to this. Several polynucleotide probes are printed in one area of 4.5 mm x 4.5 mm. In doing so, it is possible to print each array using a single pin. Thus, with a 48-pin tool, it is possible to print 48 repeated arrays on one standard microscope slide.
[0081]
In this method, the cDNA sample is then brought into contact with the substrate. By this step, the cDNA sample is hybridized to the nucleotide probe on the substrate that can specifically hybridize with the DNA encoding the polypeptide of the present invention. The hybridization reaction solution and reaction conditions may vary depending on various factors such as the length of the nucleotide probe immobilized on the substrate, but can generally be performed by methods well known to those skilled in the art.
[0082]
In this method, the expression level of the gene encoding the polypeptide of the present invention contained in the cDNA sample is then measured by detecting the strength of hybridization between the cDNA sample and the nucleotide probe immobilized on the substrate. To do. Furthermore, the measured expression level of the gene encoding the polypeptide of the present invention is compared with the control.
[0083]
In the present invention, when cDNA derived from the gene encoding the polypeptide of the present invention is present in the cDNA sample, the cDNA is hybridized with the nucleotide probe immobilized on the substrate. Therefore, the expression level of the gene encoding the polypeptide of the present invention can be measured by detecting the strength of hybridization between the polynucleotide probe and the cDNA. The detection of the hybridization intensity between the polynucleotide probe and the cDNA can be appropriately performed by those skilled in the art depending on the type of the substance labeled with the cDNA sample. For example, when cDNA is labeled with a fluorescent substance, it can be detected by reading a fluorescent signal with a scanner.
[0084]
In the method of the present invention, a cDNA sample derived from a subject and a control (healthy person) is labeled with a different fluorescent substance, whereby expression of a gene encoding the polypeptide of the present invention in each measurement is performed. The quantity can be measured simultaneously. For example, one of the above cDNA samples can be labeled with Cy5, which is a fluorescent substance, and the other with Cy3. The relative intensity of each fluorescent signal indicates the relative amount according to the expression level of the gene encoding the polypeptide of the present invention in the subject and the control (Duggan et al., Nat. Genet. 21: 10-14). , 1999).
[0085]
On the other hand, in the examination at the translation level of the gene encoding the polypeptide of the present invention, first, a polypeptide sample is prepared from the subject. Next, the amount of the polypeptide of the present invention contained in the polypeptide sample is measured. The measured amount of the polypeptide of the invention is then compared to a control.
[0086]
Such methods include SDS polyacrylamide electrophoresis and Western blotting, dot blotting, immunoprecipitation, enzyme linked immunoassay (ELISA) using antibodies that bind to the polypeptides of the present invention, and An immunofluorescence method can be exemplified.
[0087]
In the above method, when the expression level of the gene encoding the polypeptide of the present invention is significantly changed compared to the control, the subject suffers from a disease associated with abnormal expression of the gene. Or is at risk of developing the disease.
[0088]
<Testing agent>
The present invention also provides a diagnostic agent for a disease associated with abnormal expression of a gene encoding the polypeptide of the present invention or abnormal activity of the polypeptide of the present invention.
[0089]
One aspect thereof is a test agent comprising an oligonucleotide having a chain length of at least 15 nucleotides, which hybridizes to a polynucleotide encoding the above-described polypeptide of the present invention or a DNA containing an expression control region thereof. The oligonucleotide comprises a gene encoding the polypeptide of the present invention or an expression control region thereof as a probe for detecting the gene encoding the polypeptide of the present invention or an expression control region thereof in the test method of the present invention. It can be used as a primer for amplification. The oligonucleotide of the present invention can be prepared, for example, by a commercially available oligonucleotide synthesizer. The probe can also be prepared as a double-stranded DNA fragment obtained by restriction enzyme treatment or the like. When the oligonucleotide of the present invention is used as a probe, it is preferably used after appropriately labeling. As a labeling method, T4 polynucleotide kinase is used to remove the 5 ′ end of the oligonucleotide. ³² A method of labeling by phosphorylation with P, and a DNA polymerase such as Klenow enzyme, and using random hexamer oligonucleotides as primers ³² Examples thereof include a method (such as a random prime method) of incorporating a substrate base labeled with an isotope such as P, a fluorescent dye, or biotin.
[0090]
Another embodiment of the test drug of the present invention is a test drug containing an antibody that binds to the polypeptide of the present invention described later. The antibody is used for detecting the polypeptide of the present invention in the above-described test method of the present invention. The form of the antibody is not limited as long as it can detect the polypeptide of the present invention. Antibodies for testing include polyclonal antibodies and monoclonal antibodies. The antibody may be labeled as necessary.
[0091]
In the above test agents, in addition to the active ingredients such as oligonucleotides and antibodies, for example, sterilized water, physiological saline, vegetable oil, surfactants, lipids, solubilizers, buffers, protein stabilizers (such as BSA and gelatin) ), Preservatives and the like may be mixed as necessary.
[0092]
<Antibody>
The present invention provides antibodies that bind to the polypeptides of the present invention. “Antibodies” as used herein include polyclonal and monoclonal antibodies, chimeric antibodies, single chain antibodies, humanized antibodies, as well as Fab fragments containing the products of Fab or other immunoglobulin expression libraries.
[0093]
The polypeptides of the invention or fragments or analogs thereof, or cells expressing them can also be used as immunogens to produce antibodies that bind to the polypeptides of the invention. The antibody is preferably immunospecific for a polypeptide of the invention. “Immunospecific” means that the antibody has a substantially higher affinity for a polypeptide of the invention than its affinity for another polypeptide.
[0094]
Antibodies that bind to the polypeptides of the present invention can be prepared by methods known to those skilled in the art. If it is a polyclonal antibody, it can obtain as follows, for example. Serum is obtained by immunizing a small animal such as a rabbit with the polypeptide of the present invention or a fusion protein thereof with GST. This is prepared by, for example, purification by ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, affinity column coupled with the polypeptide of the present invention, and the like. In the case of a monoclonal antibody, for example, the polypeptide of the present invention is immunized to a small animal such as a mouse, the spleen is removed from the mouse, and this is ground to separate cells, such as mouse myeloma cells and polyethylene glycol. A clone that produces an antibody that binds to the polypeptide of the present invention is selected from the fused cells (hybridoma) formed by fusion with a reagent. Subsequently, the obtained hybridoma is transplanted into the abdominal cavity of the mouse, and ascites is collected from the mouse, and the obtained monoclonal antibody is obtained by, for example, ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, It can be prepared by purification using an affinity column coupled with a polypeptide.
[0095]
The antibody of the present invention can be used for isolation, identification, and purification of the polypeptide of the present invention and cells expressing the polypeptide. The antibody that binds to the polypeptide of the present invention can also be used to measure the expression level of the polypeptide of the present invention in the examination of diseases associated with abnormal expression of the polypeptide of the present invention.
[0096]
<Identification of ligand, agonist and antagonist>
The polypeptides of the present invention can be used in identifying their ligands, agonists or antagonists. These molecules to be identified may be naturally derived or artificially synthesized structural or functional mimetics. The polypeptides of the present invention are responsible for many biological functions, including many pathologies. Accordingly, it is desirable to discover compounds that activate the polypeptides of the present invention and compounds that can inhibit activation of the polypeptides of the present invention.
[0097]
In identifying a ligand for the polypeptide of the present invention, first, the polypeptide of the present invention is brought into contact with the candidate compound, and then whether or not the candidate compound binds to the polypeptide of the present invention is detected.
[0098]
The test sample is not particularly limited, and for example, known compounds and peptides (for example, those registered in a chemical file) or phage display methods (J. Mol. Biol) whose ligand activity of various GPCRs is unknown. (1991) 222, 301-310) can be used. In addition, culture supernatants of microorganisms, natural components derived from plants and marine organisms, and the like are also subject to screening. Other examples include, but are not limited to, biological tissue extracts such as brain, cell extracts, and expression products of gene libraries.
[0099]
In this method, binding to a candidate compound can be detected using the purified polypeptide of the present invention. For the detection of binding, for example, many known methods such as a method of purifying a compound that binds to the polypeptide of the present invention by contacting a test sample with the affinity column of the polypeptide of the present invention or a West Western blotting method are used. be able to. When using these methods, the candidate compound is appropriately labeled, and binding to the polypeptide of the present invention can be detected using this label. Further, by preparing a cell membrane that expresses the polypeptide of the present invention, immobilizing it on the chip, and dissociating the trimeric GTP-binding protein upon ligand binding, a change in surface plasmon resonance (surface plasmon resonance) It is also possible to use a detection method (Nature Biotechnology (99) 17: 1105). Furthermore, the binding activity between the candidate compound and the polypeptide of the present invention can be detected using a signal serving as an index of activation of the polypeptide of the present invention as an index. Examples of such signals include intracellular Ca. ²⁺ Examples include, but are not limited to, changes in levels, changes in intracellular cAMP levels, changes in intracellular pH, changes in intracellular adenylate cyclase levels.
[0100]
As one example of this method, a cell membrane in which the polypeptide of the present invention is expressed is treated with 20 mM HEPES (pH 7.4), 100 mM NaCl, 10 mM MgCl. ₂ , In 50 μM GDP solution, ³⁵ It is possible to use a technique of mixing with STP-labeled GTPγS 400 pM, incubating in the presence and absence of the test sample, filtering, and comparing the radioactivity of the bound GTPγS.
[0101]
GPCRs also share a system that transmits signals into cells through the activation of trimeric GTP-binding proteins. The trimeric GTP-binding protein is activated by the type of intracellular transmission system that is activated. ²⁺ Are classified into three types: Gq type that increases cAMP, Gs type that increases cAMP, and Gi type that suppresses cAMP. Gq protein α subunit is chimerized with other G protein α subunits by applying this, or positive signal in ligand screening using promiscuous Gα protein, Gα15, Gα16 Is Ca ²⁺ It can result in an increase. Increased Ca ²⁺ The level can be detected using a reporter gene system having a TRE (TPA reactive element) or MRE (multiple reactive element) upstream, a staining indicator such as Fura-2, Fluo-3, and a change in the fluorescent protein aequorin as an index. Similarly, Gs protein α subunit and other G protein α subunit are chimerized, a positive signal is caused to increase cAMP, which is an intracellular transmission pathway of Gs, and CRE (cAMP-responsible element) is upstream. It is also possible to use changes in the reporter gene system as an index (Trends Pharmacol. Sci. (99) 20: 118).
[0102]
There are no particular limitations on the host cell that expresses the polypeptide of the present invention in this screening system, and various host cells are used depending on the purpose. For example, mammalian cells such as COS cells, CHO cells, HEK293 cells, Examples include yeast, Drosophila-derived cells, or E. coli cells. Examples of vectors for expressing the polypeptide of the present invention in vertebrate cells include a promoter located upstream of the gene encoding the polypeptide of the present invention, an RNA splice site, a polyadenylation site, a transcription termination sequence, a replication origin, and the like. What has can be used suitably. For example, pSV2dhfr having the early promoter of SV40 (Mol. Cell. Biol. (1981) 1,854-864), pEF-BOS (Nucleic Acids Res. (1990) 18, 5322), pCDM8 (Nature (1987) 329). , 840-842), pCEP4 (Invitrogen), etc. are useful vectors for expressing GPCRs. The DNA encoding the polypeptide of the present invention can be inserted into a vector by a ligase reaction using a restriction enzyme site by a conventional method (Current protocol in Molecular Biology edit. Ausubel et al. (1987) Publisher. John Wiley). & Sons.Section 11.4-11.11). In addition, vector introduction into a host cell can be performed by, for example, calcium phosphate precipitation method, electric pulse perforation method (Current protocol in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. 9-9. ), Lipofectamine method (GIBCO-BRL), FuGENE6 reagent (Boehringer Mannheim), and microinjection.
[0103]
In identifying an agonist for the polypeptide of the present invention, a cell expressing the polypeptide of the present invention is brought into contact with the candidate compound, and the candidate compound generates a signal that serves as an indicator of activation of the polypeptide of the present invention. Whether or not is detected. That is, in the above-described method for identifying a ligand using a cell that expresses the polypeptide of the present invention, a compound that generates a signal serving as an index of activation of the polypeptide of the present invention by the action of the candidate compound is identified. Such compounds are candidates for agonists for the polypeptides of the present invention.
[0104]
In the identification of an antagonist for the polypeptide of the present invention, a cell expressing the polypeptide of the present invention is contacted with an agonist for the polypeptide of the present invention in the presence of the candidate compound, and detection is performed in the absence of the candidate compound. In comparison with the case (control), it is detected whether or not the signal serving as an indicator of the activation of the polypeptide of the present invention is decreased. That is, in the above-described method for identifying a ligand using a cell that expresses the polypeptide of the present invention, an agonist is allowed to act on the cell in addition to the candidate compound, and the activity of the polypeptide of the present invention in response to agonist stimulation A compound that suppresses the generation of a signal that is an indicator of oxidization is identified. Such compounds are candidates for antagonists to the polypeptides of the present invention. Examples of potential antagonists of the polypeptides of the invention include antibodies, in some cases polypeptides closely related to the ligand (eg, a fragment of a ligand), or a response that binds to a polypeptide of the invention. A small molecule that does not induce (and thus interferes with the activity of the receptor).
[0105]
The present invention also provides a kit for use in the identification method. This kit includes the polypeptide of the present invention or a cell expressing the polypeptide of the present invention or a cell membrane thereof. The kit may contain compounds that are candidates for GPCR ligands, agonists, or antagonists.
[0106]
<Pharmaceutical composition for treatment of disease>
The present invention provides a pharmaceutical composition for treating a patient in need of increasing or suppressing the activity or expression of the polypeptide of the present invention.
[0107]
As an active ingredient of a pharmaceutical composition for increasing the activity or expression of the polypeptide of the present invention, an agonist for the polypeptide of the present invention, the polynucleotide of the present invention, or a vector into which the polynucleotide of the present invention has been inserted is used. be able to. On the other hand, the active ingredient of the pharmaceutical composition for suppressing the activity or expression of the polypeptide of the present invention includes an antagonist to the polypeptide of the present invention, a gene encoding the endogenous polypeptide of the present invention in vivo. Polynucleotides that suppress the expression of can be used. Antagonists include soluble forms of the polypeptide of the invention capable of binding the ligand in competition with the endogenous polypeptide of the invention. A typical example of such a competitor is a fragment of the polypeptide of the present invention. Examples of the polynucleotide that suppresses the expression of the gene encoding the polypeptide of the present invention include the above-described antisense DNA and ribozyme.
[0108]
When the therapeutic compound is used as a pharmaceutical, it can be administered as a pharmaceutical composition formulated by a known pharmaceutical method, in addition to directly administering the compound itself to a patient. For example, pharmaceutical compositions or tablets or pills obtained by mixing with pharmacologically acceptable carriers (excipients, binders, disintegrants, flavoring agents, flavoring agents, emulsifiers, diluents, solubilizers, etc.) , Powders, granules, capsules, troches, syrups, solutions, emulsions, suspensions, injections (solutions, suspensions, etc.), suppositories, inhalants, transdermal absorption agents, eye drops, eye ointments Etc., and is formulated in a form suitable for oral or parenteral use.
[0109]
Administration to a patient can be generally performed by methods known to those skilled in the art, such as intraarterial injection, intravenous injection, and subcutaneous injection. The dose varies depending on the weight and age of the patient, the administration method, etc., but those skilled in the art can appropriately select an appropriate dose. In addition, if the compound can be encoded by DNA, it may be possible to incorporate the DNA into a gene therapy vector and perform gene therapy.
[0110]
Examples of the gene therapy vector include a viral vector such as a retroviral vector, an adenoviral vector, and an adeno-associated viral vector, and a non-viral vector such as a liposome. Utilizing this vector, the target DNA can be administered to a patient by ex vivo method or in vivo method.
[0111]
<Transgenic animals, knockout animals>
The present invention provides a mammal into which the polynucleotide of the present invention has been introduced or a mammal in which the expression of the endogenous polynucleotide is artificially suppressed. These mammals can be produced by methods known to those skilled in the art. These mammals are preferably rodents, most preferably mice. These mammals can be used as, for example, model animals for diseases associated with the polynucleotide of the present invention.
[0112]
【Example】
The following examples further illustrate details for identifying the polypeptides of the present invention.
[0113]
[Example 1] Extraction of amino acid sequence from human genome data
In the first step for GPCR gene discovery, ie, the sequence extraction step, the inventors selected all candidates for the 6-frame translation sequence present between the start and stop codons from the human genome sequence. (6F expansion sequence). When many initiation codons (ATG) were found on the same sequence, the longest sequence was selected. On the other hand, in order to detect a sequence having a plurality of exons, a gene discovery program (GeneDecoder) (Asai, K., et al. Pacific Symposium on Biocomputing 98, pp.228-239 (PSB98, 1998).) Is used. The protein coding region was discovered (GD sequence). Since the GPCR protein has 7 transmembrane helices of about 20 residues in length, it was required that both sequences required 150 or more residues (> 20 * 7).
[0114]
375, 412 sequence by 6-frame translation. And 95,900 sequences were predicted by GeneDecoder. The former sequence corresponds to the case where no intron is included, and the latter is mainly composed of a plurality of exons.
[0115]
GeneDecoder is a gene discovery program using a hidden Markov model (HMM), and also uses information on sequence similarity and exon length distribution. The program was evaluated using Genset 98 (http // bioinformatics weizmann.ac.il/databases/gensets/Human/) containing 462 sequences of exons and 2,843 exons. It was found to show 6% sensitivity and 40.4% selectivity, while 64.2% sensitivity and 21.3% selectivity to detect the correct exon boundary.
[0116]
[Example 2] Triple analysis
At the triple analysis stage, we used BLASTP (Altschul, SF et al. Nucleic Acids Res. 25, 3389-3402 (1997).) For domain searches and sequence searches. PFAM database (Bateman, A., et al. Nucleic Acids Res. 28, 263-266 (2000).) And PROSITE (Bairoch, A. Nucleic Acids Res. 20, 2013-2018 (1992).), And For TMH prediction, TMWindows, which is our original algorithm, is used, and Mitaku's method (Hirokawa, T., et al. Bioinformatics. 14, 78-379 (1998).) Was taken into account. Specifically, it is as follows.
(1) Using BLASTP, the amino acid sequence (6F development sequence, GD sequence) obtained in the sequence extraction step is searched against the SWISSPROT database, and E value <10 ^-10 Or 10 ^-50 Lists sequences that match known GPCR sequences.
(2) Using the HMMER program, the domain specific to the GPCR of the PFAM database note is assigned to the 6F expansion sequence, GD sequence, E value <1.0 or <10 ^-10 The sequences that could be assigned in were listed. At the same time, the motif pattern peculiar to GPCR in the PROSITE (Bairoch, A. Nucleic Acids Res. 20, 2013-2018 (1992).) Database has a P value <2 × 10 ^-3 Or <10 ^-5 The sequences that could be assigned in were listed.
(3) The number of transmembrane helices of 6F expansion sequence and GD sequence was predicted using the method of TMWindows and Mitaku. For example, the relationship between the result predicted by TMWindows and 7 predicted by Mitaku's method in the range of 6 to 8 is described as {TMWindows (7) or Mitaku (6-8)}. {TMWindows (7) or Mitaku (6-8)}, {TMWindows (7) or Mitaku (7)}, and {TMWindows (7) and Mitaku (7)} and sequences that match each condition prepared are listed.
[0117]
Describe the details of the programs and databases used in the above analysis. PFAM is a protein domain database described by a Hidden Markov Model (HMM), and HMMER (Bateman, A., et al. Nucleic Acids Res. 28, 263-266 (2000).) Assigns them to sequences. And score its significance by E value. On the other hand, PROSITE is a motif pattern described by a regular expression. In order to score the significance of attribution, the present inventors used (P value) multiplied by the appearance probability of each residue as an index. For example, if the regular expression pattern is A- [T, S] -G, the P value is P _A * {Pt + Ps} * P _G It is.
[0118]
TMWindows is a program unique to the present inventor relating to TMH prediction. (Angelman, DM, et al. Annual Review of Biophysics and Biophysical Chemistry. 15, 321-353.) (1986). The entire sequence is scanned with 9 different window widths (19-27 residues). This index was determined as the most suitable index for membrane protein analysis through comparison of all indices included in the AAindex database (Tomii, K. & Kanehisa, M. Protein Eng. 9, 27-36 (1996)). For each window width, a continuous region with an average hydrophobicity index> 2.5 is predicted as a transmembrane helix. The number predicted for each different window set means the range of the number of helices. On the other hand, Mitaku's method predicts the number of helices using physicochemical parameters.
[0119]
The threshold values used in these analyzes are obtained by the inventor evaluating each method. The reference data set used for the evaluation is a sequence obtained by excluding a fragment sequence from SWISSPROT version 39 (Bairoch, A. & Apweiler, R. Nucleic Acids Res. 28, 45-48 (2000).). This includes 1054 known GPCR sequences and 64,154 non-GPCR sequences. The specific evaluation procedure for the analysis method is shown below.
(1) Using BLASTP, 1054 known GPCR sequences were searched against the evaluation data set, and the sensitivity and selectivity for accurate and inaccurate pair identification were calculated for each E value.
(2) Using HMMER, the PFAM domain specific to GPCR was assigned to the sequence of the evaluation data set, and the sensitivity and selectivity of the E value were calculated with respect to the number of correct and inaccurate assignments. On the other hand, for the PROSITE pattern, the sensitivity and selectivity of the P value were calculated with respect to the number of exact and inaccurate assignments.
(3) TMH prediction tools are generally not very accurate in predicting the true number of helices. However, if the predicted number of helices is as wide as 6-8, 5-9, 4-10, etc., the sensitivity for detecting true seven transmembrane helix types is clear. Can be increased. For both TMWindows and Mitaku methods, we consider four ranges, 7, 6-8, 5-9, 4-10, and true 7 for all combinations of each other (16 ways). The sensitivity and selectivity to detect the transmembrane helix of the book were calculated.
[0120]
Throughout the evaluation, we focused on two thresholds: the best sensitivity threshold and the best selectivity threshold. The former threshold is intended to minimize false positives and obtain a sensitivity of almost 100%, while the latter is intended to minimize false negatives and obtain a selectivity of almost 100%.
[0121]
For example, the evaluation of the BLASTP threshold is shown in FIG. The line indicated by the left arrow indicates the number of pairs of GPCRs, and the line indicated by the right arrow indicates a pair of GPCR and non-GPCR sequence. E value is 10 ^-50 In regions that are less than, almost all pairs were between GPCR sequences except for some unrelated pairs around the border. This corresponds to the best selectivity threshold. Interestingly, these false positives were caused by a match with the LDL receptor domain or the EGF factor domain characteristic of receptors with only one transmembrane helix. E value is 10 ^-10 If it was less than 115, there were 115 false positives, but almost all GPCRs were included. This boundary area corresponds to the best sensitivity threshold.
[0122]
Similarly, as summarized in Table 1, we evaluated the thresholds of each tool and created a four-level data set based on them.
[0123]
[Table 1]

[0124]
Here, the sensitivity (left) and selectivity (right) when the threshold is used are shown in parentheses below the threshold of each program.
[0125]
The most reliable data (level A, best selectivity data set) was obtained by union of sequences obtained from BLASTP, PFAM, and PROSITE best selectivity thresholds. In addition, in order to find distantly related GPCR sequences, a union of the results from the three levels of TMH prediction thresholds (Table 1) and the results from the best sensitivity thresholds of BLASTP, PFAM and PROSITE was obtained. Next, the most sensitive data set was prepared as the best sensitivity data set (level D). According to the method evaluated by the inventors, any sequence found in the best selectivity data set is a protein having seven transmembrane helices, most of which may be guanosine triphosphate binding protein coupled. Is extremely high.
[0126]
[Example 3] Refinement of the number of genes
GPCR candidate substances were screened from the sequences generated in the first stage using the threshold values shown in Table 1. However, since these sequences still included the following duplicate examples, it was necessary to finally narrow down the number of candidates.
[0127]
Case 1 : Perfect match or overlap at the same gene position. They are the result of two sequence generation methods: (1) 6-frame translation and (2) prediction by GeneDecoder. We considered them to be the same gene.
[0128]
Case 2 : Multiple copies between different chromosomes or between different positions on the same chromosome. From a biological point of view, we considered them as different genes. The maximum number of duplicate genes was found between chromosomes 2 and 11.
[0129]
Case 3 : Two or more sequences that partially hit some long known sequence. The reason why these were generated is thought to be mis-splicing by the gene discovery program. We decided that they should be fused as the same gene.
[0130]
The present inventors first raised the accuracy of candidate genes by examining each of the above cases. As a specific algorithm, the present inventors describe the sequence as S (C, F, R), where C is the chromosome number, F is the frame number, and R is the position on the genome sequence. C _i = C _j , F _i = F _j , N _i = N _j , E _i -T _j Two sequences were considered to be the same gene when <0 (i <j) and aligned with a similarity of 50 residues or more and 99% or more. (Where n is the contig number and t, e are the relative positions at the N and C terminus in the contig sequence, position R = R (n, t, e)).
[0131]
After sieving as described above, further adding biological knowledge, we finally obtained the best selectivity and best sensitivity datasets containing 883 and 2293 sequences, respectively, and at other levels. We also got the data set. Table 2 summarizes the number of GPCR candidates per chromosome for each data set.
[0132]
[Table 2]

[0133]
It can be seen that chromosome 11 has the largest number of GPCR candidates in all levels of the data set, and chromosomes 1, 6 and 19 also represent a large number of GPCR candidates. On the other hand, on chromosomes 21 and Y, there are very few GPCR candidates. Moreover, this trend has not changed in the process of updating data every month.
Further analysis on the best selectivity data set is summarized in Table 3.
[0134]
[Table 3]

[0135]
We classified the sequences by 30% sequence similarity. This is generally considered to be an evolutionarily related family threshold. The largest family was olfactory receptors containing 537 members. Major families of more than 20 members include adrenaline, dopamine and serotonin receptors (38), family 2B receptors (20), family 3C receptors (30) chemokines and chemotactic receptors (31), and orphan receptors. (76).
[0136]
[Example 4] Extraction of new sequence
The sequence was assigned to UNIGENE (Schuler, GD J. Mol Med. 75, 694-698 (1997).) And nr-aa ( ftp: // ncbi. nlm. nih. gov / blast / db / README ) Searched against the database. If at least 100 residues or more in the examined sequence are continuously aligned with a known sequence and the amino acid identity of the region is 96% or more, the present inventors consider this sequence to be a known sequence. Judging and on this basis, we obtained new GPCR candidates. These data sets will be maintained and updated in the future by routine recalculation.
[0137]
The present inventors classified the extracted new sequences into the following groups A, B, and C (Tables 4 to 6). The sequence A group, the B group, and the C group are refined based on the best selectivity data set (level A), the level B data set, and the level C data set, respectively, among the sequence sets obtained by the triple analysis. Then, a new sequence was determined according to a search against UNIGENE and nr-aa databases.
[0138]
[Table 4]

[0139]
[Table 5]

[0140]
[Table 6]

[0141]
Hereinafter, among the clones identified by the present inventor, analysis was advanced on clone 13860.
[Example 5] Isolation of cDNA of clone 13860
5.1. Isolation of clone 13860 cDNA by 5'-RACE and 3'-RACE methods
Clone 13860 cDNA was similarly isolated using the 5'-RACE and 3'-RACE methods. The 3′-terminal cDNA was obtained by H.R. A first PCR is performed using Marathon-Ready cDNA (manufactured by Clontech) derived from Hela cells as a template, AP1 (SEQ ID NO: 11) and 3 ′ R13860 (SEQ ID NO: 1) as primers, and AP2 in the second PCR. (SEQ ID NO: 12) and 3 ′ R13860-next (SEQ ID NO: 2) were used as primers. Subsequently, the 5′-end cDNA was obtained by H.R. First PCR was performed using Marathon-Ready cDNA derived from Hela cells as a template, AP1 and 5 ′ R13860 (SEQ ID NO: 3) as primers, and AP2 and 5 ′ R13860-next (SEQ ID NO: 4) in the second PCR. ) Was used as a primer. In each PCR, Advantage 2 Polymerase Mix (manufactured by CLONTECH) was used as a DNA polymerase. The DNA fragments obtained by PCR were inserted into pGEM-T Easy vector (manufactured by Promega), and the nucleotide sequence was determined. The PCR reaction using Advantage 2 Polymerase Mix (manufactured by CLONTECH) was performed according to the method attached to Marathon Ready cDNA.
[0142]
Subsequently, in order to analyze the 5′-side cDNA base sequence in more detail for the base sequence isolated and identified above, cDNA prepared from human brain (Clontech) was used as a template DNA, PCR Amplification was attempted. That is, 5 μL of 10 × ExTaq buffer, 5 μL of dNTPs (2.5 mM each), 10 pmole of sense primer 13860-1 (SEQ ID NO: 5) and 10 pmole of antisense primer 13860- in 50 μl of PCR reaction solution. 4 (SEQ ID NO: 6), 2.5 μL of human brain cDNA (Clontech), 1 μL TaKaRa ExTaq (Takara Shuzo), and 1 μL TaqStart Antibody (Takara Shuzo), respectively, First, incubate at 96 ° C for 4 minutes, followed by 5 cycles consisting of 96 ° C for 20 seconds and 72 ° C for 1 minute, 5 cycles of 96 ° C for 20 seconds and 70 ° C for 1 minute. Cycle, 30 cycles of 96 ° C for 20 seconds and 68 ° C for 1 minute And finally incubated at 72 ° C. for 2 minutes. A product of about 800 bp amplified by PCR was inserted into pGEM-T Easy vector (manufactured by Promega), and the nucleotide sequence was determined in detail (SEQ ID NO: 15). As a result, the region amplified by the above method was able to infer the amino acid sequence (SEQ ID NO: 16) without any stop codon in the middle of a single reading frame, and therefore the start codon of clone 13860 was further Presumed to exist upstream. Then, isolation and identification of 5'-side cDNA were tried again by 5'-RACE method. PCR primer 13860-8 (SEQ ID NO: 7) used for the first PCR during 5′-RACE and PCR primer 13860-6 (SEQ ID NO: 8) used for the second PCR were designed. Using a human brain-derived Marathon-Ready cDNA (Clontech) as a template, 5′-RACE was performed in the same manner as described above. In the first PCR, 13860-8 (SEQ ID NO: 7) and AP1 primer were used, and in the second PCR, 13860-6 (SEQ ID NO: 8) and AP2 primer were used. The product of about 1 kbp amplified by PCR was inserted into the pGEM-T Easy vector, and the nucleotide sequence was determined (SEQ ID NO: 17). As a result, the amino acid sequence was inferred from the translation initiation codon in a single reading frame (SEQ ID NO: 18), and a sequence considered to be an extracellular secretory signal sequence was also found at the amino terminus. It was thought that it was able to be decided.
[0143]
5.2. Isolation of full-length clone 13860 cDNA
In order to confirm whether mRNA corresponding to the 13860 cDNA sequence analogized based on the base sequence of the clone 13860 determined by the RACE method is present, amplification of the 13860 coding region and the supposed portion by PCR In addition, the base sequence was confirmed. The primer used for PCR was 13860-ATG (SEQ ID NO: 9) designed to hybridize to a sequence in the vicinity of the translation initiation codon as a primer in the sense direction and to have a restriction enzyme EcoRI recognition sequence and a Kozak sequence (CCACC). In addition, as a primer in the antisense direction, 13860-TGA2 (SEQ ID NO: 10) designed to hybridize to a sequence downstream of the expected nucleotide end of 13860 and to have a restriction enzyme SalI recognition sequence was used. The PCR conditions were as follows. That is, 5 μL of 10 × PCR buffer (manufactured by Toyobo Co., Ltd.), 5 μL of dNTPs (2 mM each), 2 μL of 25 mM magnesium sulfate, 10 pmole of 13860-ATG and 13860-TGA2 primer in 50 μL of reaction solution, Prepared to contain 5 μL of Hela cells (Clontech) and 1 μL of KOD-Plus DNA polymerase (Toyobo), first incubated at 94 ° C. for 2 minutes, then at 94 ° C. for 15 seconds , 5 cycles of 2 minutes 30 seconds at 72 ° C, followed by 5 cycles of 94 ° C 15 seconds, 70 ° C 2 minutes 30 seconds, 94 ° C 15 seconds, 68 ° C 2 minutes 30 A cycle of 25 seconds was performed, and finally incubated at 72 ° C. for 2 minutes. The PCR reaction solution was desalted using PCR Purification Kit (manufactured by QIAGEN), after removing PCR primers, eluted with 40 μL of TE solution, and subjected to secondary amplification by PCR again. That is, 5 μL of 10 × PCR buffer (manufactured by Toyobo Co., Ltd.), 5 μL of dNTPs (2 mM each), 2 μL of 25 mM magnesium sulfate, 10 pmole of 13860-ATG and 13860-TGA2 primer in 50 μL of reaction solution, Prepared to contain 10 μL of the first PCR purified product and 1 μL of KOD-Plus DNA polymerase (Toyobo), first incubated at 94 ° C. for 2 minutes, then 94 ° C. for 15 seconds, 60 ° C. for 30 seconds Further, 10 cycles of 2 minutes 30 seconds were performed at 72 ° C., and finally, incubation was performed at 72 ° C. for 2 minutes. As a result, a single band of about 3.4 kbp was amplified. As a result of determining the base sequence of the obtained PCR product, it was confirmed that it encodes the full-length amino acid sequence. The nucleotide sequence of the finally obtained clone 13860 is shown in SEQ ID NO: 19, and the amino acid sequence deduced therefrom is shown in SEQ ID NO: 20.
[0144]
<Primer sequence>

[0145]
[Example 6] Expression analysis of clone 13860
The expression of mRNA in human organs and tumor tissues of clone 13860 was analyzed by RT-PCR. As a template, Multiple Tissue cDNA (MTC) Panels (Human I, Human II, Human Tumor, manufactured by CLONTECH) were used, and the target clone was amplified under the following PCR conditions.
[0146]
As PCR primers, 13860-1 “CTCACTGGTCTTCTGGGGAT (SEQ ID NO: 13)” and 13860-2 “TCTTGTTCCGCAACCACGAC (SEQ ID NO: 14)” were reacted at 94 ° C. for 1 minute, then “94 ° C. for 30 seconds, 56 ° C. 40 cycles of “30 ° C. for 30 seconds and 72 ° C. for 30 seconds” were performed.
As a result, it was revealed that clone 13860 is highly expressed in the brain, small intestine and large intestine.
[0147]
【The invention's effect】
The present invention provides a novel GPCR, a polynucleotide encoding the polypeptide, a vector containing the polynucleotide, a host cell containing the vector, and a method for producing the polypeptide. Further provided are methods of identifying compounds that bind to or modify the activity of the polypeptide. The polypeptide or polynucleotide of the present invention, or a compound that binds to or modifies the activity of the polypeptide of the present invention is expected to be used for the development of new preventive or therapeutic agents for diseases associated with the polypeptide of the present invention. The Furthermore, the present invention provides a method for examining a disease comprising detecting mutation or expression of a gene encoding the polypeptide of the present invention. GPCR is one of the most important and attracting attention in the fields of drug development and medicine, and the comprehensive development of new GPCRs in the present invention is expected to make dramatic progress in these fields. Even for GPCR researchers, the present invention will be a valuable source of information.
[0148]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a diagram showing GPCR sequences plotted against E values when a known GPCR sequence is searched against an evaluation database including 1,054 GPCR sequences and 64,154 non-GPCR sequences. It is a figure which shows the number of the number of pairs, and the pair of GPCR sequence and a non-GPCR sequence.
FIG. 2 is an electrophoretogram showing the results of analyzing the expression of clone 13860 in each organ. Graph from left to right: heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, large intestine, white blood cell, lung cancer, colon cancer, lung cancer, prostate cancer, large intestine Cancer, ovarian cancer, pancreatic cancer.
FIG. 3 is a graph showing the result of FIG.

Claims (32)

The polynucleotide according to any one of (a) to (d) below, which encodes a guanosine triphosphate binding protein-coupled receptor.
(A) a polynucleotide encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 20 (b) a polynucleotide comprising the coding region of the base sequence set forth in SEQ ID NO: 19 (c) described in SEQ ID NO: 20 A polynucleotide encoding a polypeptide comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence (d), which is stringent to DNA comprising the nucleotide sequence set forth in SEQ ID NO: 19 A polynucleotide that hybridizes under conditions A polynucleotide encoding a fragment of the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 20. A vector comprising the polynucleotide according to claim 1 or 2. A host cell carrying the polynucleotide of claim 1 or 2 or the vector of claim 3. A polypeptide encoded by the polynucleotide of claim 1 or 2. The method for producing a polypeptide according to claim 5, comprising culturing the host cell according to claim 4 and recovering the produced polypeptide from the host cell or a culture supernatant thereof. An antibody that binds to the polypeptide of claim 5. A method for identifying a ligand for the polypeptide of claim 5, comprising:
(A) contacting the candidate compound with a cell expressing the polypeptide according to claim 5 or the polypeptide according to claim 5 or a cell membrane thereof; and (b) the candidate compound according to claim 5. Detecting whether or not it binds to a cell expressing the polypeptide or a cell membrane expressing the polypeptide according to claim 5 or a cell membrane thereof. A method for identifying an agonist for the polypeptide of claim 5, comprising:
(A) a step of bringing a cell expressing the polypeptide of claim 5 into contact with the candidate compound, and (b) a signal that serves as an indicator of activation of the polypeptide of claim 5 by the candidate compound. Detecting whether or not to generate. A method for identifying an antagonist to the polypeptide of claim 5, comprising:
(A) contacting a cell expressing the polypeptide of claim 5 in the presence of the candidate compound with an agonist for the polypeptide of claim 5, and (b) in the absence of the candidate compound. The method of detecting whether the signal used as the parameter | index of activation of the polypeptide of Claim 5 decreases compared with the case where it detects by (6). 9. A ligand identified by the method of claim 8. An agonist identified by the method of claim 9. An antagonist identified by the method of claim 10. A kit for use in the method according to claim 8 to 10, comprising at least one molecule according to (a) or (b) below.
(A) The polypeptide according to claim 5 (b) the host cell according to claim 4 or a cell membrane thereof A pharmaceutical composition for treating a patient in need of increasing the activity or expression of the polypeptide according to claim 5, comprising a therapeutically effective amount of the molecule according to (a) to (c) below Pharmaceutical composition.
(A) an agonist for the polypeptide according to claim 5; (b) a polynucleotide according to claim 1 or 2; and (c) a vector according to claim 3. A pharmaceutical composition for treating a patient in need of suppressing the activity or expression of the polypeptide of claim 5, comprising a therapeutically effective amount of the molecule according to (a) or (b) below Pharmaceutical composition.
(A) an antagonist to the polypeptide of claim 5 (b) a polynucleotide that suppresses the expression of a gene encoding the polypeptide of claim 5 that is endogenous in vivo. A method for examining an abnormality in expression of a gene encoding the polypeptide according to claim 5 or a disease associated with an abnormality in activity of the polypeptide according to claim 5, wherein the gene or expression control thereof in a subject is performed. Detecting a mutation in the region. The inspection method according to claim 17, comprising the following steps (a) to (d):
(A) a step of preparing a DNA sample from a subject (b) a step of isolating the DNA encoding the polypeptide of claim 5 or its expression control region (c) determining the base sequence of the isolated DNA (D) A step of comparing the base sequence of the DNA determined in the step (c) with a control. The inspection method according to claim 17, comprising the following steps (a) to (d):
(A) a step of preparing a DNA sample from a subject (b) a step of cleaving the prepared DNA sample with a restriction enzyme (c) a step of separating a DNA fragment according to its size (d) a detected DNA fragment Comparing the size of the control with the control The inspection method according to claim 17, comprising the following steps (a) to (e):
(A) a step of preparing a DNA sample from a subject (b) a step of amplifying the DNA encoding the polypeptide of claim 5 or its expression control region (c) a step of cleaving the amplified DNA with a restriction enzyme (D) A step of separating the DNA fragments according to their sizes (e) A step of comparing the size of the detected DNA fragments with the control The inspection method according to claim 17, comprising the following steps (a) to (e):
(A) a step of preparing a DNA sample from a subject (b) a step of amplifying the DNA encoding the polypeptide of claim 5 or its expression control region (c) dissociating the amplified DNA into single-stranded DNA (D) separating the dissociated single-stranded DNA on a non-denaturing gel (e) comparing the mobility of the separated single-stranded DNA on the gel with a control The inspection method according to claim 17, comprising the following steps (a) to (d):
(A) a step of preparing a DNA sample from a subject (b) a step of amplifying the DNA encoding the polypeptide of claim 5 or its expression control region (c) the amplified DNA at a concentration of a DNA denaturant (D) the step of comparing the mobility of the separated DNA on the gel with the control A method for examining a disease associated with abnormal expression of a gene encoding the polypeptide according to claim 5, comprising detecting the expression level of the gene in a subject. The inspection method according to claim 23, comprising the following steps (a) to (c):
(A) a step of preparing an RNA sample from a subject (b) a step of measuring an amount of RNA encoding the polypeptide according to claim 5 contained in the RNA sample (c) an amount of the measured RNA Step to compare with control The inspection method according to claim 23, comprising the following steps (a) to (d):
(A) providing a substrate on which a cDNA sample prepared from a subject and a nucleotide probe that hybridizes with the DNA encoding the polypeptide of claim 5 are immobilized (b) the cDNA sample and the substrate; The step of contacting (c) the expression level of the gene encoding the polypeptide of claim 5 contained in the cDNA sample by detecting the strength of hybridization between the cDNA sample and the nucleotide probe immobilized on the substrate (D) A step of comparing the measured expression level of the gene encoding the polypeptide of claim 5 with a control The inspection method according to claim 23, comprising the following steps (a) to (c):
(A) preparing a protein sample from a subject (b) measuring the amount of the polypeptide according to claim 5 contained in the protein sample (c) comparing the measured amount of the polypeptide with a control Process An oligonucleotide that hybridizes to the DNA encoding the polypeptide of claim 5 or an expression control region thereof and has a chain length of at least 15 nucleotides. A test for a disease associated with abnormal expression of a gene encoding the polypeptide according to claim 5 or abnormal activity of the polypeptide according to claim 5, comprising the oligonucleotide according to claim 27. A test agent for a disease associated with abnormal expression of a gene encoding the polypeptide according to claim 5 or abnormal activity of the polypeptide according to claim 5, comprising the antibody according to claim 7. A mammal into which the polynucleotide according to claim 1 or 2 has been introduced. A mammal in which expression of the polynucleotide according to claim 1 is artificially suppressed. The mammal according to claim 30 or 31, which is a mouse.

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