JP4579378B2 - Novel polypeptide and its DNA - Google Patents

Description

【００７８】

【００７９】
また、本明細書中で繁用される置換基、保護基および試薬を下記の記号で表記する。

【００８０】
本願明細書の配列表の配列番号は、以下の配列を示す。
［配列番号：１］
実施例１で用いられたアンチセンス鎖プライマーの塩基配列を示す。
［配列番号：２］
実施例１で用いられたセンス鎖プライマーの塩基配列を示す。
［配列番号：４］
配列番号：６で表されるアミノ酸配列を有するヒトＭＬＰ前駆体をコードするＤＮＡの塩基配列を示す。
［配列番号：５］
配列番号：６で表されるアミノ酸配列を有するヒトＭＬＰ前駆体に含まれるシグナル配列のアミノ酸配列を示す。
［配列番号：６］
ヒトＭＬＰ前駆体のアミノ酸配列を示す。
［配列番号：７］
実施例２で用いられたアンチセンス鎖プライマーの塩基配列を示す。
［配列番号：８］
実施例２で用いられたセンス鎖プライマーの塩基配列を示す。
［配列番号：１０］
配列番号：１２で表されるアミノ酸配列を有するマウスＭＬＰ前駆体をコードするＤＮＡの塩基配列を示す。
［配列番号：１１］
配列番号：１２で表されるアミノ酸配列を有するマウスＭＬＰ前駆体に含まれるシグナル配列のアミノ酸配列を示す。
［配列番号：１２］
マウスＭＬＰ前駆体のアミノ酸配列を示す。
［配列番号：１３］
実施例３で用いられたＧ３ＰＤＨ特異的オリゴＤＮＡの塩基配列を示す。
［配列番号：１４］
実施例３で用いられたＧ３ＰＤＨ特異的オリゴＤＮＡの塩基配列を示す。
［配列番号：１５］
実施例３で用いられたアグリカン特異的オリゴＤＮＡの塩基配列を示す。
［配列番号：１６］
実施例３で用いられたアグリカン特異的オリゴＤＮＡの塩基配列を示す。
［配列番号：１７］
実施例３で用いられたII型コラーゲン特異的オリゴＤＮＡの塩基配列を示す。
［配列番号：１８］
実施例３で用いられたII型コラーゲン特異的オリゴＤＮＡの塩基配列を示す。
［配列番号：１９］
実施例３で用いられたＸ型コラーゲン特異的オリゴＤＮＡの塩基配列を示す。
［配列番号：２０］
実施例３で用いられたＸ型コラーゲン特異的オリゴＤＮＡの塩基配列を示す。
［配列番号：２１］
実施例３で用いられたマウスＭＬＰ特異的オリゴＤＮＡの塩基配列を示す。
［配列番号：２２］
実施例３で用いられたマウスＭＬＰ特異的オリゴＤＮＡの塩基配列を示す。
［配列番号：２３］
配列番号：２４で表されるアミノ酸配列を有するヒトＭＬＰをコードするＤＮＡの塩基配列を示す。
［配列番号：２４］
ヒトＭＬＰのアミノ酸配列を示す。
［配列番号：２５］
配列番号：２６で表されるアミノ酸配列を有するマウスＭＬＰをコードするＤＮＡの塩基配列を示す。
［配列番号：２６］
マウスＭＬＰのアミノ酸配列を示す。
［配列番号：２７］
実施例４および実施例６で用いられたプライマーの塩基配列を示す。
［配列番号：２８］
実施例４で用いられたプライマーの塩基配列を示す。
［配列番号：２９］
実施例１で得られたｃＤＮＡ断片の塩基配列を示す。
［配列番号：３０］
実施例２で得られたｃＤＮＡ断片の塩基配列を示す。
［配列番号：３１]
実施例６で用いられた合成ペプチドのアミノ酸配列を示す。
［配列番号：３２］
実施例６でPCRプライマーとして用いられたオリゴDNAの塩基配列を示す。
［配列番号：３３］
実施例６でPCRプライマーとして用いられたオリゴDNAの塩基配列を示す。
［配列番号：３４］
実施例６でPCRプライマーとして用いられたオリゴDNAの塩基配列を示す。
［配列番号：３５］
実施例６でPCRプライマーとして用いられたオリゴDNAの塩基配列を示す。
［配列番号：３６］
実施例６でPCRプライマーとして用いられたオリゴDNAの塩基配列を示す。
［配列番号：３７］
実施例６でPCRプライマーとして用いられたオリゴDNAの塩基配列を示す。
［配列番号：３８］
実施例６でPCRプライマーとして用いられたオリゴDNAの塩基配列を示す。
［配列番号：３９］
実施例９で得られたＤＮＡにコードされるラットＭＬＰ前駆体の部分アミノ酸配列を示す。
［配列番号：４０］
実施例９で得られたラットＭＬＰ前駆体の一部をコードするＤＮＡの塩基配列を示す。
［配列番号：４１］
実施例９で得られたラットＭＬＰ前駆体の一部をコードするＤＮＡを含むＤＮＡの塩基配列を示す。
［配列番号：４２］
実施例９でPCRプライマーとして用いられたオリゴDNAの塩基配列を示す。
［配列番号：４３］
実施例９でPCRプライマーとして用いられたオリゴDNAの塩基配列を示す。
［配列番号：４４］
実施例９でPCRプライマーとして用いられたオリゴDNAの塩基配列を示す。
［配列番号：４５］
実施例９でPCRプライマーとして用いられたオリゴDNAの塩基配列を示す。
［配列番号：４６］
配列番号：４７で表されるアミノ酸配列を有するラットＭＬＰ前駆体をコードするＤＮＡの塩基配列を示す。
［配列番号：４７］
ラットＭＬＰ前駆体のアミノ酸配列を示す。
［配列番号：４８］
配列番号：４９で表されるアミノ酸配列を有するラットＭＬＰをコードするＤＮＡの塩基配列を示す。
［配列番号：４９］
ラットＭＬＰのアミノ酸配列を示す。
［配列番号：５０］
配列番号：４７で表されるアミノ酸配列を有するラットＭＬＰ前駆体に含まれるシグナル配列のアミノ酸配列を示す。
【００８１】
後述の実施例１で得られた形質転換体エシェリヒア コリ（Escherichia coli）XL10-Gold／pDRL128vHは、平成１１年６月１１日から通商産業省工業技術院生命工学工業技術研究所（ＮＩＢＨ）に寄託番号ＦＥＲＭ ＢＰ−６７５０として、平成１１年６月２５日から財団法人・発酵研究所（ＩＦＯ）に寄託番号ＩＦＯ
１６２９２として寄託されている。
後述の実施例２で得られた形質転換体エシェリヒア コリ（Escherichia coli）XL10-Gold／pDRL128vMは、平成１１年６月９日から通商産業省工業技術院生命工学工業技術研究所（ＮＩＢＨ）に寄託番号ＦＥＲＭ ＢＰ−６７４７として、平成１１年６月２５日から財団法人・発酵研究所（ＩＦＯ）に寄託番号ＩＦＯ １６２９３として寄託されている。
寄託されている。
後述の実施例９で得られた形質転換体エシェリヒア コリ（Escherichia coli）XL10-Gold/pDRL128vRは、平成１２年５月１９日から通商産業省工業技術院生命工学工業技術研究所（ＮＩＢＨ）に寄託番号ＦＥＲＭ ＢＰ−７１６７として、平成１２年５月２６日から財団法人・発酵研究所（ＩＦＯ）に寄託番号ＩＦＯ
１６４３９として寄託されている。
【実施例】
以下に、実施例を挙げて本発明を具体的に説明するが、本発明はそれらに限定されるものではない。なお、大腸菌を用いての遺伝子操作は、モレキュラー・クローニング（Molecular cloning）に記載されている方法に従った。
【００８２】
実施例1 ヒトMLP 前駆体タンパク質をコードする cDNA のクローニング
ヒト胎児脳由来 poly(A)⁺ RNA を用いて以下の要領で 5' RACE (Rapid Amplification of cDNA End)、 3' RACE を行うことによりヒトMLP 前駆体タンパク質をコードする cDNA のクローニングを行った。ヒト胎児脳由来 poly(A)⁺ RNA ( Clontech 社 ) 1 μgより、制限酵素部位に続く poly(T) を持つ anchored primerと Super script II MMLV 逆転写酵素 ( Gibco BRL 社 )を用いて1st. strand cDNA を合成後、RNA ligase ( 宝酒造 ) を用いて該1st. strand cDNA の3'末端にanchored primerを付加した。次に、配列番号：1で表されるオリゴDNAをアンチセンス鎖プライマーとして5' RACE を、同じく配列番号：2で表されるオリゴDNAをセンス鎖プライマーとして3' RACE を行い、それぞれ各プライマーを起点とする 5'上流側の配列、3' 下流側の配列を得た。ここで得られた各２本鎖DNAの塩基配列を決定したところ、オーバーラップする共通配列が存在していたことから、両配列は同一遺伝子に由来することが判った。そこで、この5' RACE、3' RACEで得られた各cDNA 断片をこの共通配列部分で接合し、最終的にpoly(A)⁺ 鎖を含む配列番号：29で表される全長 923塩基対（bp）のcDNA 断片を得た。このcDNA 断片には配列番号：5で表される 18 アミノ酸残基の典型的なシグナル配列を含む、 配列番号：6で表される128 個のアミノ酸からなる新規なヒトMLP 前駆体タンパク質がコ−ドされていた。このヒトMLP 前駆体タンパク質はヒトMIA前駆体タンパク質と相同性が最も高く、4 カ所存在するシステイン残基の位置が一致していた（図１）が、その両者間の相同性はアミノ酸レベルで 23.4%にすぎなかった。
本実施例で得られたヒトＭＬＰ前駆体蛋白質をコードするＤＮＡを保持するプラスミドpDRL128vHを大腸菌(Escherichia coli) XL10-Goldに導入して、形質転換体：大腸菌(Escherichia coli) XL10-Gold／pD RL128vHを得た。
【００８３】
実施例２ マウスMLP前駆体タンパク質をコードするcDNAのクローニング
マウスMLP 前駆体タンパク質をコードする cDNA のクローニングは、マウス17.5 日胎児由来 poly(A)⁺ RNA を用いて、ヒトMLP 前駆体タンパク質をコードする cDNA のクローニングの場合と同様にして 5' RACE ( Rapid Amplification of cDNA End )、 3' RACE を行うことにより行った。マウス17.5 日胎児からグアニジンチオシアネ−ト法によって total RNA を分画後、この total RNA をオリゴ (dT) スパンカラム (ファルマシア ) にかけて poly(A)⁺ RNA を調製した。この マウス17.5 日胎児由来 poly(A)⁺ RNA 1 μgより、制限酵素部位に続く poly(T) を持つ anchored primerとSuperscript II MMLV 逆転写酵素 ( Gibco BRL社 )を用いて 1st. strand cDNA を合成後、RNA ligase ( 宝酒造 ) を用いて合成した 1st. strand cDNA の 3' 末端にanchored primer を付加した。5' RACE、3' RACE に用いたプライマーの配列は、実施例１で得られたヒト MLP 前駆体タンパク質をコードするcDNAの塩基配列をクエリー（query） にして public EST (Expressed Sequence Tag)データベースから見いだした、マウス MLP 前駆体タンパク質をコードするcDNAの 3'側の領域を含むと考えられる唯一のESTであるAA222797 の配列を基に作製した。配列番号：7で表されるオリゴDNAをアンチセンス鎖プライマーとして5' RACE を、配列番号：8で表されるオリゴDNAをセンス鎖プライマーとして3' RACE を行い、それぞれ各プライマーを起点とする 5'上流側の配列、3'下流側の配列を得た。ここで得られた各２本鎖DNAの塩基配列を決定したところ、オーバーラップする共通配列が存在していたことから、両配列は同一遺伝子に由来することが判った。そこで、この5'RACE、3' RACEで得られた各cDNA 断片をこの共通配列部分で接合し、最終的にpoly(A)⁺ 鎖を含む全長947 bpの cDNA 断片を得た（配列番号：30）。 この cDNA 断片にはヒト MLP 前駆体タンパク質と同様に、配列番号：11で示される 18 アミノ酸残基の典型的なシグナル配列を含む、配列番号：12で表される128個のアミノ酸からなる新規マウスMLP 前駆体タンパク質がコ−ドされていた。このマウスMLP 前駆体タンパク質は、ヒトおよびマウスのMIA前駆体タンパク質、またヒト MLP前駆体タンパク質で4 カ所存在するシステイン残基の位置がすべて一致していた（図１）。また、マウスMLP 前駆体タンパク質とヒトMLP 前駆体タンパク質とのアミノ酸レベルでの相同性は84.3% に達したが、マウスMIA前駆体タンパク質とマウスMLP前駆体タンパク質との相同性は 22.6% にとどまった。
本実施例で得られたマウスＭＬＰ前駆体蛋白質をコードするＤＮＡを保持するプラスミドpDRL128vMを大腸菌(Escherichia coli)XL10-Goldに導入して、形質転換体：大腸菌(Escherichia coli) XL10-Gold／pDRL128vMを得た。
【００８４】
実施例３ in vitro軟骨分化モデルにおけるMLPの発現
マウス胚性腫瘍由来細胞株ATDC5は、前駆軟骨細胞の性質を極めてよく保持し、またインスリン存在下の培養で高率に軟骨分化が誘導され、その後骨形成においてみられる軟骨分化の全ての段階をシミュレーションすることができることから、in vitro軟骨分化モデルとして用いられている。(Cell Diff. Dev., 30:109-116, 1990, J. Cell Biol., 133:457-468, 1996, J. Bone Min. Res., 1 0:S234, 1995)。そこで、以下の要領でRT-PCR法を行うことで各分化段階での各種遺伝子の発現変化を調べた。まず、分化モデル培養系の各ステージのATDC5細胞から、同細胞をフェノールとチオシアン酸グアニジンを含む均一な液体ISOGEN（ニッポンジーン）に溶解し、クロロホルムを加えて遠心分離することによりRNAを含む水相画分を採取し、さらにイソプロパノールを加えて攪拌後再び遠心し沈殿させることにより精製total RNAを取得した。次にTAKARA RNA PCR Kit (AMV)Ver.2.1（宝酒造）中のAMV Reverse Transcriptase XLとRandom９mersを用いて各被検total RNAを逆転写してcDNAを得、次にそれらを鋳型DNAとして用い、またハウスキーピング遺伝子としてG3PDH（グリセルアルデヒド-３-リン酸脱水素酵素）特異的オリゴDNA（配列番号：13，配列番号：14）、或いは軟骨分化マーカー遺伝子特異的オリゴDNA（アグリカン（配列番号：15，配列番号：16）、II型コラーゲン（配列番号：17，配列番号：18）、 X型コラーゲン（配列番号：19，配列番号：20））をプライマーDNAとして用い、TaKaRa Ex Taq^TM（宝酒造）の反応系でPCR反応を行った。得られた反応産物をアガロースゲル電気泳動で分離し、その生成量を比較した。その結果、各遺伝子についてそれぞれ軟骨細胞の分化段階に対応した表１のような発現パターンを検出することができた。そこで同cDNAサンプル群に対してマウスＭＬＰ前駆体タンパク質をコードするcDNAに特異的なオリゴDNA（配列番号：21、配列番号：22）をプライマーDNAとして用いて、それ以外は全く同一反応条件でRT-PCRを行ったところ、ステージ２からステージ４にかけてマウスＭＬＰ前駆体mRNA量の顕著な亢進が認められた。このことからＭＬＰ前駆体の遺伝子は軟骨分化の初期段階に発現が増加する遺伝子であることがわかった。
【表１】

（表中の＋の数は、各分化ステージでの各々の遺伝子の発現量の差異を表し、その数が多いほど発現量が多いことを示す。）
【００８５】
実施例４ COS7細胞におけるマウスMLP-FLAG融合タンパク質の発現とその検出
ＭＬＰが分泌タンパク質であることを確かめるため、マウスMLPについて、以下の要領でCOS7細胞におけるマウスMLP-FLAG融合タンパク質の発現とその検出を行った。まず実施例２で得たマウスＭＬＰ前駆体ポリペプチドをコードするcDNAの塩基配列 に基づき、２種のプライマーDNAを化学合成した。一つは 5'-CGAATTCCCACCATGGCAAGGATATTGATTCTTTTGCTTG-3'（配列番号：27 ）であり、これは制限酵素EcoRI認識部位を含むアンカー配列を5'末端側に持つ +1〜+28（翻訳開始部位を +1 とする）までのセンス配列を含むオリゴDNAである。もう一つは 5'-GTACAGTCGACTTCACAGAAGAAGTCAATATCCGTGGTTG-3'（配列番号：28）であり、これは制限酵素SalI認識部位を含むアンカー配列の3'側に+355〜+378 までのアンチセンス配列がつながった配列を有するオリゴDNAである。実施例２で得たプラスミドpDRL128vMを鋳型としてこれら２種のプライマーDNAおよびTaKaRa LA Taq^TM（宝酒造）を用い、サーマルサイクラーGeneAmp^TM PCR system 9700（パーキンエルマー社）にて、最初 98℃で 30秒間置いた後、98℃で 10 秒、55℃で 20秒、72℃で 2分を 1反応サイクルとして 25 サイクル増幅反応を繰返し、最後に 72℃で5分間伸長反応を行った。得られたDNA断片を精製後、制限酵素EcoRIとSalIで末端消化の後再精製し、動物細胞用発現ベクターpCAN618FLAGのEcoRI、SalI部位へ挿入、連結した。pCAN618FLAGは、プラスミドベクターpCAN618に由来し、選択マーカーとしてのネオマイシン耐性遺伝子を持つと共に、目的タンパク質をコードするDNA断片をそのクローニング部位であるEcoRI 、SalI部位に挿入することでサイトメガロウイルスの極初期遺伝子エンハンサーとその下流のβ−アクチンプロモーター制御下で該タンパク質を発現させることができるのみならず、SalI部位直後に存在する8アミノ酸のFLAGエピトープ配列(Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys)をコードする塩基配列と終止コドンに読み取り枠を合わせることで該目的タンパク質をFLAG融合タンパク質として発現させることも可能である。上述のPCRクローニングDNA断片のpCAN618FLAGへの挿入もマウスＭＬＰ前駆体全長とFLAGエピトープの融合タンパク質（間にValを１残基挿む）を発現する目的で行い、その結果、発現ベクタープラスミドpMMLP-Fを得た。
次にCOS7 細胞 1.2×10⁵ 細胞を 6 穴プレートを用いて、10%牛胎児血清（ FBS ）を含むダルベッコ変法最小培地（ DMEM ）で 24 時間培養し、この細胞に上記の発現プラスミドpMMLP-F（１ウェルあたり0.4 μg） をリポフェクトアミン（ Gibco BRL ）を用いて導入した。導入 24時間後上記の新しい培地に交換し、さらに 5 時間後に FBS 不含有 Opti-MEM（ Gibco BRL ）に換えて36 時間培養後、その培養上清と細胞抽出液を得た。細胞抽出液は細胞を生理食塩を含むリン酸緩衝液（ PBS ）で 2 回洗浄後、トリスSDSサンプル緩衝液で溶解抽出し、一方、培養上清は限外濾過（分子量 3000 カット）で適宜濃縮後、等容のトリスSDSサンプル緩衝液と混合した。これらのサンプルを熱処理後 15%-25% SDS-ポリアクリルアミドゲルで電気泳動し、さらにそのゲルからPVDF 膜上（ Amersham pharmacia biotech社 ）に転写した。次に該PVDF 膜をブロックエース（雪印乳業）で 1 時間ブロッキングし、0.05% Tween 20 を含む PBS（ PBS-T ）中で抗 FLAGモノクローナル抗体（ 10μg／ml；Kodak社）と 2 時間反応させた。PBS-T で 3 回洗浄後、PBS-T 中で、西洋ワサビ過酸化酵素標識抗マウス IgGヤギ抗体（ Amersham pharmacia biotech社；5000 倍希釈）と 1 時間反応した。PBS-T で 5 回洗浄後、ECLplus 発色キット（ Amersham pharmacia biotech社 ）および ECL film （ Amersham pharmacia biotech社）を用いて化学発光を検出した。その結果、細胞抽出液と共に培養上清中にも約１４kDaの遺伝子産物が検出されたことから、COS7細胞においてマウスMLP-FLAG融合タンパク質が発現し、分泌されていることが明らかとなった。
そこで、次に該マウスMLP-FLAG融合タンパク質の N末端アミノ酸配列の決定を行った。まず、上述の方法に準じて発現させたマウスMLP- FLAGを含むCOS7細胞培養上清から、Anti-FLAG^TM M2-Agarose Affinity Gel (Sigma社)を用いたアフィニティクロマトグラフィーで酸性溶出画分（Glycine-HClバッファー (pH3.5)で溶出） を取得した。該画分を濃縮した後、本実施例既述同様のSDS-ポリアクリルアミドゲル電気泳動、続いてCBB 染色を行ったところ、目的タンパク質であるマウスMLP-FLAGに相当する単一のバンドのみが観察された。そこで同画分の濃縮試料を同様に電気泳動し、そのゲルから該タンパク質をPVDF膜上に転写した後、パルスリキッドアミノ酸シーケンサー Procise CLC491 (ＰＥバイオシステムズ社製)にかけ、Ｎ末端アミノ酸配列を決定した。その結果、N 末端から順に 1.ヒスチジン、2. グリシン、3.バリン、4. フェニルアラニンの各アミノ酸残基が検出され、配列番号：２６で表されるマウスMLPのN末端配列に一致した。以上の結果から、マウスMLP-FLAGタンパク質はマウスMLP-FLAG前駆体タンパク質のN末端の１８アミノ酸残基のシグナル配列が切断され、１９残基目のヒスチジン残基から始まるマウスMLP-FLAG成熟タンパク質としてCOS7細胞より培地中に分泌されることがわかった。
【００８６】
実施例５ マウスMLP−FLAG融合タンパク質発現CHO−K1細胞株の樹立
以下の要領でマウスMLP−FLAG融合タンパク質発現CHO−K1細胞株を樹立した。
CHO−K1細胞3.3ｘ10⁴細胞を、10cm径プラスチックシャーレ上、10%牛胎児血清（FBS）を含むF−12培地（Gibco BRL社）で24時間培養し、これらの細胞に、実施例４で得られた発現プラスミドpMMLP−F（1シャーレあたり1.5μg）をリン酸カルシウム法（ CellPhect Transfection Kit （Amersham Pharmacia Biotech社））を用いて導入した。導入12時間後、細胞をFBSを含まないF−12培地で2回洗浄後、15%グリセロールを含む等張HEPES溶液( pH 7.5) 3mlを用いて3分間グリセロールショックを与えた。 その後再びFBSを含まないF−12培地で2回洗浄し、FBSを含むF−12培地でさらに12時間培養後に500mg / L Geneticin(Gibco BRL社) と10% FBSを含むF−12選択培地（以下、選択培地）に交換した。10日後、シャーレ中で形成されたGeneticin耐性のコロニーをそれぞれ24穴プレートに移し、選択培地で3日間培養した。次に選択培地中で増殖した細胞を6穴プレートに移し、さらに選択培地で4日間培養した後、0.02% CHAPS、0.1mM p−ABSF（和光純薬工業（株））を含むOpti−MEM( Gibco BRL社 )培地1mlに交換し、さらに48時間培養後、その培養上清を回収した。得られた培養上清は、Centricon YM -3限外濾過膜（Amicon社）を用いて濃縮後、等容のトリスSDSサンプル緩衝液と混合した。このサンプルを９５℃、５分間熱処理をおこなった後、18％ SDS−ポリアクリルアミドゲルで電気泳動し、さらにそのゲルから泳動タンパク質をナイロン膜上に転写した。次に該ナイロン膜をブロックエース（雪印乳業（株））で1時間ブロッキングし、0.05% Tween20を含むPBS（PBS−T）中で抗FLAG抗体（ 1 / 2000希釈、SIGMA ）と1時間反応させた。PBS−Tで5回洗浄後、PBS−T中でHRP標識抗マウスIgGヒツジ抗体（ 1 / 2000希釈、Amersham Pharmacia Biotech社）と1時間反応した。 PBS−Tで5回洗浄後、ECL発色キット（Amersham Pharmacia Biotech社）および、Hyperfilm ECL（ Amersham Pharmacia Biotech社 ）を用いて化学発光を検出した。その結果、CHO−K1/mMLP.FLAG#2-1株由来の細胞培養上清中におよそ16kDaの目的遺伝子産物が最も多く検出されたことから、該細胞株をマウスMLP−FLAG融合タンパク質発現CHO−K1細胞株として選択した。
【００８７】
実施例６ MLP抗血清の作製と該抗血清を用いた組換えMLP蛋白質の検出
抗MLP抗血清は以下の要領で作製した。まず、マウスMLP前駆体蛋白質の105残基目のバリンから124残基目のアスパラギン酸までのアミノ酸配列に対応するペプチド鎖のC末端にさらにシステイン１個が付加された配列番号：３１で表される合成ペプチド（Val-Lys-Glu-Gln-Arg-Val-Tyr-Gln-Glu-Ala-Thr-Lys-Glu-Ile-Pro-Thr-Thr-Asp-Ile-Asp-Cys）を公知の方法により化学合成した。このペプチドにキャリアとしてKLH (keyhole limpet hemocyanin)を結合したものを抗原とし、ウサギ(SPF Japanese White Rabbit)に免疫した。計７回の免疫を行った後、全採血を行い、公知の方法により血清画分を取得し、保存剤としてアジ化ナトリウム（最終濃度0.1%）を添加後、該標品を抗MLP抗血清とした。
次に該抗血清の各種組換えタンパク質に対する反応性をウエスタンブロット解析によって調べた。まず、実施例４に記載したマウスMLP-FLAG融合タンパク質に加え、マウスMLPタンパク質（FLAGタグなし） 、ヒトMLP-FLAG融合タンパク質、ヒトMLPタンパク質（FLAGタグなし）、マウスMIA-FLAG融合タンパク質、マウスMIAタンパク質（FLAGタグなし）の各発現ベクタープラスミドを構築した。構築は実施例４同様、pCAN618FLAGのクローニング部位であるEcoRI 、SalI部位に予めPCRクローニングした目的DNA断片を挿入することで行った。各PCR反応に用いたプライマーDNAセットの各塩基配列は、マウスMLPタンパク質（FLAGタグなし）については配列番号：２７と配列番号：３２、ヒトMLP-FLAG融合タンパク質については配列番号：３３と配列番号：３４、ヒトMLPタンパク質（FLAGタグなし）については配列番号：３３と配列番号：３５、マウスMIA-FLAG融合タンパク質については配列番号：３６と配列番号：３７、マウスMIAタンパク質（FLAGタグなし）については配列番号：３６と配列番号：３８で表わされる塩基配列である。また鋳型DNAとしては、マウスMLPタンパク質（FLAGタグなし） は実施例4と同様にpDRL128vMを、ヒトMLP-FLAG融合タンパク質とヒトMLPタンパク質（FLAGタグなし）は実施例1で得られたpDRL128vHを、またマウスMIA-FLAG融合タンパク質とマウスMIAタンパク質（FLAGタグなし）はマウスメラノーマ細胞株Ｂ１６より調製したcDNAを用いた。このようにして得られた各発現ベクタープラスミドのCOS-7細胞への導入、また取得された各培養上清についての抗FLAG抗体を用いたウエスタンブロット解析は実施例４の方法に準拠して行った。また、抗MLP抗血清を用いたウエスタンブロット解析は、１次抗体として該抗血清（1000倍希釈）を、２次抗体として西洋ワサビ過酸化酵素標識抗ウサギ IgG抗体（Amersham pharmacia biotech社；5000 倍希釈）を用いる以外は抗FLAG抗体を用いたウエスタンブロット解析時の方法に準拠して行った。
図２に抗FLAG抗体を用いたウエスタンブロット解析、図３に抗MLP抗血清を用いたウエスタンブロット解析の結果を示す。抗MLP抗血清は、抗原ペプチドの由来タンパク質であるマウスMLPとともにヒトMLPに対しても交差性を示し、その反応性はほとんど変わらなかった。また、マウスMLP-FLAG融合タンパク質、ヒトMLP-FLAG融合タンパク質も該抗血清に反応したことから、FLAGタグの存在にも影響されなかった。一方、マウスMIA-FLAG融合タンパク質とマウスMIAタンパク質（FLAGタグなし）については全くシグナルが検出されなかったことから、該抗血清がMLP分子種に特異的であることが判った。
さらに、該抗血清を用いて実施例５で取得されたマウスMLP-FLAG発現CHO細胞株に対し公知の方法に準じて免疫染色を行った結果を図４に示す。免疫前ウサギ血清を用いた対照実験では各細胞は染色されていないが、該抗血清使用時にはすべての細胞が染色されたことから、該抗血清は未変性のMLPタンパク質とも反応することが明らかとなった。
【００８８】
実施例７ 軟骨組織中でのMLPタンパク質の発現
実施例６で得られた抗MLP抗血清を用いて軟骨組織中でのMLPタンパク質の検出を試みた。被検試料としては、マウス(BALB/c)大腿骨骨頭軟骨を液体窒素で凍結破砕し、TRIS SDS βME SAMPLE BUFFER（第一化学薬品）で抽出後、遠心して残査を除去したものを用いた。1レーンあたりマウス1匹分由来相当量の試料をSDS-PAGE(15-25%)で電気泳動の後、実施例６と同様の方法で抗MLP抗血清によるウエスタンブロット解析を行った。その結果、マウスMLP組換えタンパク質とほぼ同じ泳動位置にシグナルが検出されたことから、 MLPタンパク質が該軟骨組織中で発現していることが明らかとなった。
また、軟骨組織以外の各種ヒト組織でのヒトMLP mRNAの発現を調べるために、実施例１で得られたヒトMLP前駆体タンパク質をコードするDNA断片と［α−³²Ｐ］ｄＣＴＰ（Amersham pharmacia biotech社：6000 Ci/mmol）を用いて、Multiprime DNA labelling system (Amersham pharmacia biotech社：RPN. 1601Y) の方法でプローブを作製し、該プローブ（比活性1.3×10¹⁰ cpm/μg）を用いて、Human MTE^TM Array（CLONTECH社：＃7775-1）に対してハイブリダイゼーションを行った。ハイブリダイズの条件は該アレイ膜添付のマニュアルに従い、最終的な洗浄は0.1×SSC、0.1％ SDS 55℃で行い、検出はＢＡＳ−２０００（フジフィルム）を用いて行った。その結果、該アレイ上でシグナルが得られたのは、コントロールのヒト染色体DNA (100ng、500ng)とともにわずかに黒質、胎児脳のスポットであったが、いずれのシグナルも検出限界付近であり、その発現量は転写段階で極めて低いと判断された。よって、MLPタンパク質は軟骨組織で特異的に発現していることが強く示唆された。
【００８９】
実施例８ in vitro軟骨分化モデルにおける各種遺伝子発現変動に対するMLP組換えタンパク質の添加効果
実施例３記載のATDC5細胞を用いたin vitro軟骨分化モデルにおいて、各種遺伝子発現変動に対するMLP組換えタンパク質の添加効果を調べた。 MLP組換えタンパク質は、実施例４と同様に、マウスMLP-FLAG融合タンパク質を発現させたCOS-7細胞培養上清より抗FLAG抗体によるアフィニティカラムクロマトグラフィーを公知の方法で行い、精製濃縮された標品を用いた。該タンパク質をモデル系設定初日よりATDC5細胞に2日おきに添加し、10日間培養後細胞を回収して、実施例3同様のRT-PCR法により各遺伝子発現を調べた。その結果、該タンパク質添加細胞群では、 アグリカン、II型コラーゲン、 X型コラーゲン等、分化に伴って発現量が増加する各マーカー遺伝子の発現抑制が認められた。一方、軟骨への分化に抑制的に働いているPTH/PTHr受容体発現にはその変動に有意な影響は見られなかった。このことから、MLPタンパク質はATDC5を用いた本モデル系において軟骨分化に抑制的に作用することが判った。
【００９０】
実施例９ ラットMLP前駆体タンパク質（rMLP）をコードする遺伝子の解析
まず、rMLPの一部をコードするDNA断片を以下のようなPCR法により取得した。
すなわち、配列番号：２１で示されるオリゴDNAをセンス鎖プライマーとして、配列番号：２２で示されるオリゴＤＮＡをアンチセンス鎖プライマーとして各々4 pmol含み、さらに10 x Advantage^TM 2 PCR Buffer （クロンテック社）2μｌ、50 x dNTP mix（クロンテック社）0.4 μl、50 x Advantage 2 Polymerase Mix（クロンテック社）0.4μｌ、鋳型DNAとしてSD(IGS)ラット下垂体由来のcDNA溶液 ２μｌを含む混合液20μｌを調製し、サーマルサイクラー（GeneAmp^TM PCR system model 9700（パーキンエルマー社））を用いて９５℃、1分、続いて９５℃、１０秒→ ５４℃、１０秒→７２℃、１分を３５サイクル繰り返し、さらに７２℃、３分で伸長反応させるプログラムでＰＣＲ反応を行った。反応終了液を２．０％アガロースゲルを用いて電気泳動後、そのゲルをSYBR^TM Green I nucleic acid gel stain（モレキュラープローブ社）で染色したところ、分子量マーカー換算で300bp付近の位置にPCR反応で増幅されたDNAに対応するバンドを確認した。キアクィックゲルエキストラクションキット（キアゲン社）を用いて該ＤＮＡ断片を回収し、塩基配列を決定する為にpCR^TM2.1-Topo（インビトロジェン社）を用いてＴＡクローニングし、該プラスミドを大腸菌（Escherichia coli）Epicurian Coli XL10-Gold^TM株（ストラタジーン社）のコンピテントセルに導入した。アンピシリン含有LB寒天培地上で出現するアンピシリン耐性形質転換株のコロニーの中から、外来ＤＮＡ断片が挿入されていたプラスミドを保持していたクローンを選択し、同クローンから該プラスミドＤＮＡ、pDRL128vRを調製した。 挿入ＤＮＡの塩基配列を決定するため、 pDRL128vRを鋳型ＤＮＡ、市販プライマーＤＮＡ（Bca BEST Primer RV-P（宝酒造(株））をシーケンスプライマーとし、ABI PRISM^TM BigDye Terminator Cycle Sequencing FS Ready Reaction Kit（ パーキンエルマー社 ）を用いたシーケンス反応を添付資料の条件にしたがって、サーマルサイクラー（GeneAmp^TM ＰＣＲ system model 9700（パーキンエルマー社））で行った後、該反応試料をＤＮＡシーケンサーABI PRISM^TM 377（パーキンエルマー社）で分析した。
その結果、 pDRL128vRには、配列番号：３９で示される87個のアミノ酸からなる新規ラットMLP前駆体タンパク質の一部をコードする配列番号：４０で示される261塩基対のDNA断片を含む、配列番号：４１で示される307塩基対のＤＮＡ断片が含まれていた。
次に、該タンパク質をコードする遺伝子について、上記塩基配列よりさらに5’上流側及び3’下流側の構造を調べるために、genome walkingを行った。被検材料としてRat GenomeWalker^TM Kit（クロンテック社）を用い、方法はＰＣＲ反応用の酵素としてTaKaRa Ex Taq^TM（宝酒造）を用いた点を除いては同キットの添付資料に従った。まず、 gene specific primerとして、配列番号：４０で示される塩基配列の一部にあたる２種のオリゴDNA （rMLPGW F1(配列番号：４２)、 rMLPGWF2(配列番号：４３))と該配列の一部に相補的な配列の２種のオリゴDNA （rMLPGWR1(配列番号：４４)、 rMLPGWR2(配列番号：４５))をそれぞれ化学合成した。これらは、3’下流側DNA取得の為の1st PCR反応ではrMLPGWF1、続くnested PCR反応ではrMLPGWF2を用い、また5’上流側DNA取得の為の1st PCR反応ではrMLPGWR1、続くnested PCR反応ではrMLPGWR2を用いた。これらの反応によって得られた各増幅DNA断片について、上記プライマー配列部位を起点としてヒト及びマウスMLP 前駆体タンパク質をコードするcDNAの塩基配列との相同性を比較しながら各々の塩基配列を解析した。その結果、判明したゲノムの一次構造から、ラットMLP前駆体タンパク質は、配列番号：４６で示される384塩基のDNAでコードされる配列番号：４７で示される128個のアミノ酸残基からなるタンパク質であることが明らかとなった。ラットMLP 前駆体タンパク質とヒトMLP前駆体タンパク質及びマウスMLP前駆体タンパク質とのアミノ酸レベルでの相同性は それぞれ84.3%、96.0% に達したが、ラットMIA前駆体タンパク質とラットMLP前駆体タンパク質との相同性は26.5% にとどまった。ラットMLPは、配列番号４８：で示される330塩基のDNAでコードされる配列番号：４９で示される110個のアミノ酸残基からなるタンパク質であり、ラットMLP前駆体タンパク質から配列番号：５０で表される、マウスMLPと同様１８アミノ酸残基からなるシグナルペプチドがプロセスされて生成されるものと考えられる。
本実施例で得られたラットMLP前駆体タンパク質の一部をコードするDN Aを保持するプラスミドpDRL128vRを大腸菌（Escherichia coli）XL10-Goldに導入して、形質転換体：大腸菌（Escherichia coli）XL10-Gold / pDRL128vRを得た。
【００９１】
【発明の効果】
本発明のポリペプチドおよびそれをコードするＤＮＡは、例えば、骨・関節疾患、病的血管新生の診断、治療、予防などに使用することができる。また、本発明のポリペプチドは、本発明のポリペプチドの活性を促進もしくは阻害する化合物またはその塩のスクリーニングのための試薬として有用である。さらに、本発明のポリペプチドに対する抗体は、本発明のポリペプチドを特異的に認識することができるので、被検液中の本発明のポリペプチドの検出、定量、中和等に使用することができる。
さらに本発明のプロモーターを使用することにより、タンパク質（任意の有用遺伝子産物等）を非ヒト温血動物の軟骨で優位に（好ましくは特異的に）しかも大量に発現させることが可能になるので、遺伝子治療の分野に貢献することができる。
【００９２】
【配列表】

【００９３】
【図面の簡単な説明】
【図１】ヒトＭＬＰ前駆体（hMLP）、マウスＭＬＰ前駆体（mMLP）、ヒトＭＩＡ前駆体（hMIA）、マウスＭＩＡ前駆体（mMIA）、ラットＭＩＡ前駆体（rMIA）およびウシＭＩＡ前駆体（bMIA）のアミノ酸配列を示す。
【図２】実施例６で行われたウエスタンブロット解析の結果を示す。一次抗体として抗ＦＬＡＧ抗体を用いた。
図中、レーン１はマウスＭＬＰ（ＦＬＡＧタグなし）、レーン２はマウスＭＬＰ−ＦＬＡＧ、レーン３はマウスＭＩＡ（ＦＬＡＧタグなし）、レーン４はマウスＭＩＡ−ＦＬＡＧ、レーン５はヒトＭＬＰ（ＦＬＡＧタグなし）、レーン６はマウスＭＬＰ−ＦＬＡＧをそれぞれ導入したＣＯＳ−７細胞の培養上清を電気泳動したレーンを示す。
【図３】実施例６で行われたウエスタンブロット解析の結果を示す。一次抗体として抗ＭＬＰ抗血清を用いた。
図中、レーン１はマウスＭＬＰ（ＦＬＡＧタグなし）、レーン２はマウスＭＬＰ−ＦＬＡＧ、レーン３はマウスＭＩＡ（ＦＬＡＧタグなし）、レーン４はマウスＭＩＡ−ＦＬＡＧ、レーン５はヒトＭＬＰ（ＦＬＡＧタグなし）、レーン６はマウスＭＬＰ−ＦＬＡＧをそれぞれ導入したＣＯＳ−７細胞の培養上清を電気泳動したレーンを示す。
【図４】実施例６で行われた免疫染色の結果を示す。左のパネルは免疫前ウサギ血清を用いた対照実験の結果を示し、右のパネルは抗ＭＬＰ抗血清を用いた実験の結果を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel secretory cell function regulatory protein and its DNA.
[0002]
[Prior art]
Regardless of its prokaryotic or eukaryotic nature, cells secrete a wide variety of proteins through their unique mechanisms. In particular, multicellular organisms (living bodies) exchange various information between cells to maintain their differentiation, proliferation, and homeostasis, and many of the various humoral factors that play a central role are secreted proteins. Alternatively, it is a mature form, and is classified into hormones, neurotransmitters, cytokines, growth factors, etc. based on its structural and functional characteristics. Recent advances in recombinant DNA technology and cell culture technology have steadily elucidated the genes and protein structures encoding these secreted proteins. On the other hand, the discovery of these factors dramatically advances the analysis of the receptors expressed on the cell surface, and further elucidates the mechanism of information transmission within each cell, and characterizes its physiological functions. become. In many diseases in humans, or in the pathology of various disease model animals, there are many cases where such abnormal expression of some humoral factors that should remain homeostatic can be the cause, and as a result, it can lead to deterioration, For example, there are phenomena that can be applied in the field of diagnosis of various diseases, such as so-called tumor markers that are specifically upregulated in cancer, and their expression control mechanisms are also important targets for drug discovery research. Yes.
[0003]
The melanoma inhibitory protein MIA (melanoma inhibitory activity) published by Blesh et al. In 1994 is also a secreted protein included in such a category. Initially, as its name suggests, antimelanoma activity against human melanoma cells as an indicator It was isolated from the cell culture supernatant and its gene was also obtained (Cancer Research 54, 5695-5701, 1994). Later, in 1996, Sandell et al. Re-identified the homologous gene of this protein as CD-RAP (cartilage-derived retinoic acid-sensitive protein) produced by bovine chondrocytes, and physiologically forms and maintains joints. (The Journal of Biological Chemistry 271, 3311-3316, 1996). Although the MIA / CD-RAP gene has a high homology of 85% or more at the amino acid level among human, mouse, rat, and bovine species, a known homologous protein has been found so far. In addition, genetic analysis in cattle and rats has suggested that there is no other similar gene to this gene (The Journal of Biological Chemistry) 271, 3311- 3316, 1996).
[0004]
On the other hand, structural analysis of the total DNA of a living organism, that is, the genome, has already been completed in bacteria, and humans are expected to be completed in a few years. Is said to be 100,000. Although the genes encoding many secreted proteins or secreted peptides have been isolated so far, the number is not very comprehensive when viewed from the whole genome. In order to understand the life phenomenon at the individual level, it is necessary to be able to explain the exchange of information between all the cells occurring in the cell, but the humoral function that is not yet known besides these known genes Molecules are likely to play an important physiological role, and the discovery of such substances has been strongly desired.
[0005]
[Problems to be solved by the invention]
The present invention relates to a novel cell function-regulated secreted protein (hereinafter sometimes referred to as MLP protein or MLP), a partial peptide thereof or a salt thereof, a DNA encoding the protein, a recombinant vector, a transformant, a method for producing the protein, It is an object of the present invention to provide a drug containing the protein or DNA, an antibody against the protein, a screening method for a receptor agonist / antagonist and a screening kit, a receptor agonist / antagonist obtained by the screening, a drug thereof, and the like.
[0006]
[Means for Solving the Problems]
Isolation of new secreted proteins that regulate cell function provides new insights into mechanisms such as cell differentiation, proliferation, and canceration, and further advances the elucidation of life phenomena such as individual development and homeostasis. It is possible to develop new pharmaceuticals that exhibit inhibitory activity or promoting activity against the protein and are useful for the prevention, diagnosis and treatment of various diseases.
As a result of intensive studies, the present inventors succeeded in cloning cDNAs having novel base sequences from human fetal brain and mouse fetal cDNA libraries. The present inventors have also found that the protein encoded by the obtained cDNA is a precursor protein of MIA / CD-RAP-like protein MLP having useful cell function-regulating activity, and the signal sequence is cleaved from the MLP precursor. The resulting MLP was found to be a secreted protein. As a result of further studies based on these findings, the present invention has been completed.
[0007]
That is, the present invention
(1) a polypeptide comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 24, an amide thereof, an ester thereof or a salt thereof,
(2) The polypeptide according to (1) above, which contains the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 6, its amide, its ester or its salt,
(3) The polypeptide, its amide, or its ester according to the above (1), wherein the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 24 is the amino acid sequence represented by SEQ ID NO: 26 Or its salt,
(4) The polypeptide according to (2) above, wherein the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 6 is the amino acid sequence represented by SEQ ID NO: 12, its amide, or an ester thereof Or its salt,
(5) The polypeptide according to (1), wherein the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 24 is the amino acid sequence represented by SEQ ID NO: 49, an amide thereof, or an ester thereof Or its salt,
(6) The polypeptide according to (2), wherein the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 6 is the amino acid sequence represented by SEQ ID NO: 47, an amide thereof, or an ester thereof Or its salt,
(7) DNA containing DNA having a base sequence encoding the polypeptide according to (1),
(8) the DNA according to (6), wherein the base sequence encoding the polypeptide according to (1) is the base sequence represented by SEQ ID NO: 23;
(9) The DNA according to (6), wherein the base sequence encoding the polypeptide according to (1) is the base sequence represented by SEQ ID NO: 4,
(10) the DNA according to (6), wherein the base sequence encoding the polypeptide according to (1) is the base sequence represented by SEQ ID NO: 25;
(11) The DNA according to (6), wherein the base sequence encoding the polypeptide according to (1) is the base sequence represented by SEQ ID NO: 10.
(12) the DNA according to (6), wherein the base sequence encoding the polypeptide according to (1) is the base sequence represented by SEQ ID NO: 48;
(13) the DNA according to (6), wherein the base sequence encoding the polypeptide according to (1) is the base sequence represented by SEQ ID NO: 46;
(14) a recombinant vector containing the DNA according to (6),
(15) A transformant transformed with the recombinant vector according to (14),
(16) A method for producing the polypeptide according to (1), its amide, its ester or its salt, wherein the transformant according to (15) is cultured to produce the polypeptide,
(17) An antibody against the polypeptide according to (1), an amide thereof, an ester thereof or a salt thereof,
(18) The compound or salt thereof that promotes or inhibits the activity of the polypeptide or salt thereof according to (1), wherein the polypeptide, amide, ester or salt thereof according to (1) is used Screening method,
(19) Screening of compound or salt thereof that promotes or inhibits activity of polypeptide, amide, ester or salt thereof according to (1), comprising the polypeptide or salt thereof according to (1). For kit,
(20) Promoting the activity of the polypeptide according to (1), its amide, its ester or its salt obtained by using the screening method according to (18) or the screening kit according to (19) An inhibiting compound or a salt thereof,
(21) Promoting or inhibiting the activity of the polypeptide according to (1), its amide, its ester or its salt obtained by using the screening method according to (18) or the screening kit according to (19) A pharmaceutical comprising the compound or a salt thereof,
(22) A medicament comprising the polypeptide according to (1), an amide thereof, an ester thereof or a salt thereof,
(23) A prophylactic / therapeutic agent for bone / joint diseases or pathological angiogenesis, comprising the polypeptide according to (1), an amide thereof, an ester thereof, or a salt thereof,
(24) The present invention relates to a diagnostic agent comprising the antibody according to (17).
[0008]
Furthermore, the present invention provides:
(25) The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 24 is about 50% or more (preferably about 60% or more, more preferably about 50% or more) with the amino acid sequence represented by SEQ ID NO: 24 70% or more, more preferably about 80% or more, particularly preferably about 90% or more, most preferably about 95% or more) the amino acid sequence having the homology, the polypeptide according to the above (1), its amide, or Its ester or its salt,
(26) One or more amino acid sequences substantially identical to the amino acid sequence represented by SEQ ID NO: 24 are (1) one or more in the amino acid sequence represented by SEQ ID NO: 24 (preferably 1 to 30) Amino acid sequence from which amino acids have been deleted, (2) 1 or 2 or more (preferably about 1 to 40, more preferably about 1 to 30) in the amino acid sequence represented by SEQ ID NO: 24 (3) an amino acid sequence in which one or more (preferably about 1 to 30) amino acids in the amino acid sequence represented by SEQ ID NO: 24 are substituted with other amino acids Or (4) the polypeptide of the above (1), its amide, its ester, its salt, or the like, which is an amino acid sequence combining them.
Furthermore, the DNA of the present invention, and polypeptides, amides or esters thereof or salts thereof may be used for basic research such as molecular weight markers, tissue markers, chromosome mapping, identification of genetic diseases, primers, probe designs, etc. is there.
[0009]
DETAILED DESCRIPTION OF THE INVENTION
A polypeptide containing the amino acid sequence represented by SEQ ID NO: 24 of the present invention (hereinafter sometimes referred to as human polypeptide), a polypeptide containing the amino acid sequence represented by SEQ ID NO: 26 (hereinafter referred to as human polypeptide) A mouse polypeptide), a polypeptide containing the amino acid sequence represented by SEQ ID NO: 49 (hereinafter sometimes referred to as a rat polypeptide) and a human polypeptide. Polypeptides containing amino acid sequences (hereinafter, human-type polypeptides and polypeptides containing substantially the same amino acid sequences as human-type polypeptides may be collectively referred to as polypeptides of the present invention) Or cells of warm-blooded animals (eg, guinea pigs, rats, mice, chickens, rabbits, pigs, sheep, cows, monkeys, etc.) , Hepatocytes, spleen cells, nerve cells, glial cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermis cells, epithelial cells, endothelial cells, fibroblasts, fiber cells, muscle cells, adipocytes, immune cells (Eg, macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synoviocytes, chondrocytes, bone cells, osteoblasts , Osteoclasts, mammary cells, or stromal cells, or precursor cells of these cells, stem cells, cancer cells, etc.) or any tissue in which these cells are present, for example, the brain, each part of the brain (eg, olfactory bulb, tonsil) Nucleus, basal sphere, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla, cerebellum), spinal cord, pituitary gland, stomach, pancreas, kidney, liver, gonad, thyroid, gallbladder, bone marrow, adrenal gland, skin, muscle, lung, Digestive tract (eg, large Intestine, small intestine), blood vessel, heart, thymus, spleen, salivary gland, peripheral blood, prostate, testis, ovary, placenta, uterus, bone, cartilage, joint, skeletal muscle, etc. It may be a polypeptide or a synthetic polypeptide.
In addition, when the polypeptide of the present invention has a signal peptide, the polypeptide can be efficiently secreted outside the cell.
[0010]
The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 24 is about 50% or more, preferably about 60% or more, more preferably about 70%, with the amino acid sequence represented by SEQ ID NO: 24. More preferably, amino acid sequences having homology of about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more are mentioned, and specifically, it is represented by SEQ ID NO: 26 Examples include the amino acid sequence or the amino acid sequence represented by SEQ ID NO: 49.
A polypeptide having the amino acid sequence represented by SEQ ID NO: 24 is a human MLP or human MLP protein, a polypeptide having an amino acid sequence represented by SEQ ID NO: 26 is a mouse MLP or mouse MLP protein, and SEQ ID NO: 49. In some cases, the polypeptide having the amino acid sequence represented by the formula: is referred to as rat MLP or rat MLP protein, and these may be collectively referred to as MLP.
Specifically, the polypeptide containing the amino acid sequence represented by SEQ ID NO: 24 or the amino acid sequence substantially identical thereto, for example, the amino acid sequence represented by SEQ ID NO: 6 or amino acids substantially identical thereto Examples thereof include a polypeptide containing a sequence.
The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 6 is about 50% or more, preferably about 60% or more, more preferably about 70%, with the amino acid sequence represented by SEQ ID NO: 6. More preferably, amino acid sequences having homology of about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more are mentioned. Specifically, it is represented by SEQ ID NO: 12. Examples include the amino acid sequence or the amino acid sequence represented by SEQ ID NO: 47.
A polypeptide having the amino acid sequence represented by SEQ ID NO: 6 is a human MLP precursor or human MLP precursor protein, and a polypeptide having the amino acid sequence represented by SEQ ID NO: 12 is a mouse MLP precursor or mouse MLP precursor. The body protein and the polypeptide having the amino acid sequence represented by SEQ ID NO: 47 may be referred to as rat MLP precursor or rat MLP precursor, and these may be collectively referred to as MLP precursor.
[0011]
Examples of the polypeptide having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 24 of the present invention include, for example, an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 24. And a polypeptide having substantially the same properties as the polypeptide having the amino acid sequence represented by SEQ ID NO: 24 is preferred.
In addition, as a polypeptide having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 6, for example, an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 6 is used. Preferred is a polypeptide having substantially the same properties as the polypeptide having the amino acid sequence represented by SEQ ID NO: 6.
The substantially homogeneous property includes, for example, being secreted and acting as a humoral factor. “Substantially homogeneous” means that these properties are qualitatively homogeneous. Therefore, it is preferable that properties such as secretory action and solubility are equivalent (eg, about 0.1 to 100 times, preferably about 0.5 to 10 times, more preferably 0.5 to 2 times). Quantitative factors such as the degree of nature and the molecular weight of the polypeptide may be different.
[0012]
More specifically, for example, (1) SEQ ID NO: 24 or SEQ ID NO: as a polypeptide containing an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 24 or SEQ ID NO: 6 1 or 2 (preferably about 1 to 30, more preferably about 1 to 10, more preferably several (1 to 5)) amino acids in the amino acid sequence represented by 6 have been deleted. 1 or 2 or more (preferably about 1 to 40, more preferably about 1 to 30, more preferably 1) in the amino acid sequence, (2) SEQ ID NO: 24 or SEQ ID NO: 6 An amino acid sequence to which about 10 to 10 (especially, preferably 1 to 5) amino acids are added, (3) 1 or 2 or more in the amino acid sequence represented by SEQ ID NO: 24 or SEQ ID NO: 6 ( Good Or about 1 to 30, more preferably about 1 to 10, more preferably several (1 to 5) amino acids, (4) SEQ ID NO: 24 or SEQ ID NO: 6 1 or 2 or more (preferably about 1 to 30, more preferably about 1 to 10, more preferably several (1 to 5)) amino acids in the amino acid sequence represented by So-called muteins such as polypeptides containing a substituted amino acid sequence or (5) an amino acid sequence combining them are also included.
When the amino acid sequence is inserted, deleted or substituted as described above, the position of the insertion, deletion or substitution is not particularly limited, but the sequence of SEQ ID NO: 24, SEQ ID NO: 26 and SEQ ID NO: 49 Other than the amino acid sequence common to the amino acid sequence represented by each SEQ ID NO., Other than the amino acid sequence common to the amino acid sequence represented by each SEQ ID NO .: SEQ ID NO: 6, SEQ ID NO: 12 and SEQ ID NO: 47 And the like.
[0013]
The polypeptide in the present specification has an N-terminus (amino terminus) at the left end and a C-terminus (carboxyl terminus) at the right end in accordance with the convention of peptide designation. The polypeptide of the present invention including the polypeptide containing the amino acid sequence represented by SEQ ID NO: 24 has a C-terminus that is usually a carboxyl group (—COOH) or a carboxylate (—COO). ^- ) But the C-terminus is an amide (-CONH ₂ ) Or ester (—COOR).
Here, R in the ester is, for example, C such as methyl, ethyl, n-propyl, isopropyl or n-butyl. _1-6 Alkyl groups such as C, such as cyclopentyl, cyclohexyl, etc. _3-8 A cycloalkyl group such as phenyl, α-naphthyl and the like _6-12 Aryl groups such as phenyl-C such as benzyl, phenethyl and the like _1-2 Alkyl groups or α-naphthyl-C such as α-naphthylmethyl _1-2 C such as alkyl group _7-14 In addition to the aralkyl group, a pivaloyloxymethyl group that is widely used as an oral ester is used.
When the polypeptide of the present invention has a carboxyl group (or carboxylate) in addition to the C-terminus, those in which the carboxyl group is amidated or esterified are also included in the polypeptide of the present invention. As the ester in this case, for example, the above C-terminal ester or the like is used.
Furthermore, in the polypeptide of the present invention, the amino group of the N-terminal amino acid residue (eg, methionine residue) is protected with a protecting group (for example, C-form such as formyl group, acetyl group, etc.). _1-6 C such as alkanoyl _1-6 A group protected by an acyl group or the like, an N-terminal glutamine residue produced by cleavage in vivo, pyroglutamine oxidized, a substituent on the side chain of an amino acid in the molecule (eg, —OH, —SH) , Amino group, imidazole group, indole group, guanidino group, etc.) are suitable protecting groups (for example, C such as formyl group, acetyl group, etc.) _1-6 C such as alkanoyl group _1-6 Examples thereof include those protected with an acyl group or the like, or complex polypeptides such as so-called sugar polypeptides to which sugar chains are bonded.
[0014]
As the polypeptide of the present invention or a salt thereof, a salt with a physiologically acceptable acid (eg, inorganic acid, organic acid) or base (eg, alkali metal salt) is used, and particularly physiologically acceptable. The acid addition salts are preferred. Such salts include, for example, salts with inorganic acids (eg hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.
The polypeptide of the present invention or a salt thereof can be produced from the aforementioned human or warm-blooded animal cells or tissues by a known method for purifying a polypeptide (protein), or a DNA encoding a polypeptide described later can be obtained. It can also be produced by culturing the transformant contained therein. Moreover, it can also manufacture according to the below-mentioned peptide synthesis method.
When producing from human or mammalian tissue or cells, the human or mammalian tissue or cells are homogenized and then extracted with acid or the like, and the extract is subjected to chromatography such as reverse phase chromatography or ion exchange chromatography. Can be purified and isolated.
[0015]
For the synthesis of the polypeptide of the present invention or a salt thereof, or an amide thereof, a commercially available polypeptide (protein) synthesis resin can be used. Examples of such resins include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethyl. Examples include phenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ′, 4′-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2 ′, 4′-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin, and the like. it can. Using such a resin, an amino acid in which the α-amino group and the side chain functional group are appropriately protected is condensed on the resin according to various known condensation methods according to the sequence of the target polypeptide. At the end of the reaction, the polypeptide is cut out from the resin, and at the same time, various protecting groups are removed, and an intramolecular disulfide bond forming reaction is carried out in a highly diluted solution to obtain the desired polypeptide or their amides.
Regarding the condensation of the above-mentioned protected amino acids, various activating reagents that can be used for polypeptide synthesis can be used, and carbodiimides are particularly preferable. As the carbodiimide, DCC, N, N′-diisopropylcarbodiimide, N-ethyl-N ′-(3-dimethylaminoprolyl) carbodiimide and the like are used. For activation by these, a protected amino acid is added directly to the resin together with a racemization inhibitor (eg, HOBt, HOOBt), or the protected amino acid is activated in advance as a symmetric anhydride, HOBt ester or HOOB t ester. Can then be added to the resin.
[0016]
The solvent used for the activation of the protected amino acid and the condensation with the resin can be appropriately selected from solvents that are known to be usable for the polypeptide (protein) condensation reaction. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol, dimethyl sulfoxide, etc. Sulfoxides, ethers such as pyridine, dioxane and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, or an appropriate mixture thereof may be used. The reaction temperature is appropriately selected from a range known to be usable for a polypeptide (protein) bond-forming reaction, and is usually appropriately selected from a range of about -20 to 50 ° C.
The activated amino acid derivative is usually used in an excess of 1.5 to 4 times. As a result of a test using the ninhydrin reaction, if the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. When sufficient condensation is not obtained even if the reaction is repeated, acetylation of the unreacted amino acid with acetic anhydride or acetylimidazole can prevent the subsequent reaction from being affected.
[0017]
Examples of the protective group for the amino group of the raw material include Z, Boc, t-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, and trifluoroacetyl. , Phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc, and the like.
[0018]
The carboxyl group is, for example, alkyl esterified (eg, linear, branched or cyclic alkyl ester such as methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc. ), Aralkyl esterification (for example, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarbonylhydrazide, t-butoxy It can be protected by carbonyl hydrazation, trityl hydrazation or the like.
[0019]
The hydroxyl group of serine can be protected, for example, by esterification or etherification. Examples of groups suitable for this esterification include lower (C _1-6 ) Aroyl groups such as alkanoyl group and benzoyl group, groups derived from carbonic acid such as benzyloxycarbonyl group and ethoxycarbonyl group are used. Examples of groups suitable for etherification include benzyl group, tetrahydropyranyl group, t-butyl group and the like.
Examples of protecting groups for the phenolic hydroxyl group of tyrosine include Bzl, Cl ₂ -Bzl, 2-nitrobenzyl, Br-Z, t-butyl and the like are used.
As the imidazole protecting group for histidine, for example, Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like are used.
[0020]
Examples of the activated carboxyl group of the raw material include a corresponding acid anhydride, azide, active ester [alcohol (eg, pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, Cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, ester with HOBt)] and the like. As the activated amino group of the raw material, for example, a corresponding phosphoric acid amide is used.
Examples of the method for removing (eliminating) the protecting group include catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, anhydrous hydrogen fluoride, methanesulfonic acid, trifluoro. Acid treatment with romethanesulfonic acid, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., reduction with sodium in liquid ammonia, and the like are also used. The elimination reaction by the acid treatment is generally performed at a temperature of about −20 to 40 ° C. In the acid treatment, for example, anisole, phenol, thioanisole, metacresol, paracresol, dimethyl sulfide, 1,4- Addition of a cation scavenger such as butanedithiol or 1,2-ethanedithiol is effective. The 2,4-dinitrophenyl group used as the imidazole protecting group of histidine was removed by thiophenol treatment, and the formyl group used as the indole protecting group of tryptophan was the above 1,2-ethanedithiol and 1,4-butane. In addition to deprotection by acid treatment in the presence of dithiol or the like, it can also be removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia or the like.
[0021]
The protection of the functional group that should not be involved in the reaction of the raw materials, the protection group, the removal of the protective group, the activation of the functional group involved in the reaction, and the like can be appropriately selected from known groups or known means.
As another method for obtaining an amide form of a polypeptide, for example, first, the α-carboxyl group of the carboxy terminal amino acid is amidated and protected, and then the peptide (polypeptide) chain is extended to the desired chain length on the amino group side. Then, a polypeptide in which only the N-terminal α-amino protecting group of the peptide chain was removed and a polypeptide in which only the C-terminal carboxyl protecting group was removed were prepared. The condensation is performed in such a mixed solvent. The details of the condensation reaction are the same as described above.
After purifying the protected polypeptide obtained by condensation, all the protecting groups are removed by the above-described method to obtain the desired crude polypeptide. This crude polypeptide can be purified using various known purification means, and the main fraction can be freeze-dried to obtain an amide of the desired polypeptide.
In order to obtain a polypeptide ester, for example, the α-carboxyl group of the carboxy terminal amino acid is condensed with a desired alcohol to form an amino acid ester, and then the desired polypeptide ester is obtained in the same manner as the amide of the polypeptide. You can get a body.
[0022]
The polypeptide of the present invention or a salt thereof can be produced according to a peptide synthesis method known per se. As a peptide synthesis method, for example, either a solid phase synthesis method or a liquid phase synthesis method may be used. That is, the partial peptide or amino acid that can constitute the partial peptide of the present invention is condensed with the remaining portion, and when the product has a protective group, the target peptide can be produced by removing the protective group.
Examples of known condensation methods and protecting group removal include the methods described in (1) to (5) below.
(1) M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966)
(2) Schroeder and Luebke, The Peptide, Academic Press, New York (1965)
(3) Nobuo Izumiya et al., Basics and Experiments of Peptide Synthesis, Maruzen Co., Ltd. (1975)
(4) Haruaki Yajima and Shunpei Sugawara, Biochemistry Experiment Course 1, Protein Chemistry IV, 205, (1977)
▲ 5 ▼ Supervision by Yajima Haruaki, Development of follow-up medicines, Volume 14, Peptide synthesis, Hirokawa Shoten
Further, after the reaction, the polypeptide polypeptide of the present invention can be purified and isolated by combining ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization and the like. When the polypeptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method or a method equivalent thereto, and conversely, when obtained as a salt, the known method or a method equivalent thereto. It can be converted to the free form or other salts by methods.
[0023]
The DNA encoding the polypeptide of the present invention may be any DNA as long as it contains the base sequence encoding the polypeptide of the present invention described above. Further, any of genomic DNA, cDNA derived from the cells and tissues described above, and synthetic DNA may be used.
The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Moreover, it can also be directly amplified by reverse transcriptase polymerase chain reaction (hereinafter abbreviated as RT-PCR method) using a total RNA or mRNA fraction prepared from the cells / tissues described above.
[0024]
Examples of the DNA encoding the polypeptide of the present invention include a DNA containing the base sequence represented by SEQ ID NO: 23, or hybridizes with the base sequence represented by SEQ ID NO: 23 under highly stringent conditions. A DNA encoding a polypeptide having substantially the same quality as the polypeptide of the present invention (eg, immunogenicity, etc.) and substantially the same as the polypeptide of the present invention. Any DNA may be used as long as it encodes a polypeptide having the following properties.
As the DNA containing the base sequence represented by SEQ ID NO: 23, DNA containing the base sequence represented by SEQ ID NO: 4 is used.
Examples of the DNA that can hybridize with the base sequence represented by SEQ ID NO: 23 under highly stringent conditions include, for example, about 60% or more, preferably about 70% or more, with the base sequence represented by SEQ ID NO: 23, More preferably, DNA containing a base sequence having a homology of about 80% or more is used.
Further, as a DNA that can hybridize with the base sequence represented by SEQ ID NO: 23 under highly stringent conditions, specifically, a DNA containing the base sequence represented by SEQ ID NO: 25, or SEQ ID NO: 25, which encodes a polypeptide having a nucleotide sequence that hybridizes under highly stringent conditions with the nucleotide sequence represented by 25, and having substantially the same properties (eg, immunogenicity) as the polypeptide of the present invention. Examples thereof include a DNA encoding a polypeptide having DNA or the like and having substantially the same properties as the polypeptide of the present invention.
Examples of DNA that can hybridize with the base sequence represented by SEQ ID NO: 25 under highly stringent conditions include, for example, about 60% or more, preferably about 70% or more, with the base sequence represented by SEQ ID NO: 25, More preferably, DNA containing a base sequence having a homology of about 80% or more is used, and specifically, DNA containing the base sequence represented by SEQ ID NO: 10 is used.
Further, as a DNA that can hybridize with the base sequence represented by SEQ ID NO: 23 under highly stringent conditions, specifically, a DNA containing the base sequence represented by SEQ ID NO: 48, or SEQ ID NO: 48, which has a base sequence that hybridizes with the base sequence represented by 48 under highly stringent conditions, and encodes a polypeptide having substantially the same properties (eg, immunogenicity) as the polypeptide of the present invention. Examples thereof include a DNA encoding a polypeptide having DNA or the like and having substantially the same properties as the polypeptide of the present invention.
The DNA that can hybridize with the base sequence represented by SEQ ID NO: 48 under highly stringent conditions is, for example, about 60% or more, preferably about 70% or more, with the base sequence represented by SEQ ID NO: 48, More preferably, DNA containing a base sequence having a homology of about 80% or more is used, specifically, DNA containing a base sequence represented by SEQ ID NO: 41 or SEQ ID NO: 46 is used. It is done.
[0025]
Hybridization can be performed according to a method known per se or a method analogous thereto, for example, the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). . Moreover, when using a commercially available library, it can carry out according to the method as described in an attached instruction manual. More preferably, it can be carried out according to highly stringent conditions.
The highly stringent conditions are, for example, conditions in which the sodium concentration is about 19 to 40 mM, preferably about 19 to 20 mM, and the temperature is about 50 to 70 ° C., preferably about 60 to 65 ° C.
As the DNA encoding the polypeptide of the present invention having the amino acid sequence represented by SEQ ID NO: 24, the DNA having the base sequence represented by SEQ ID NO: 23 is the amino acid sequence represented by SEQ ID NO: 6. As the DNA encoding the polypeptide of the present invention having the DNA, the DNA having the base sequence represented by SEQ ID NO: 4 encodes the polypeptide of the present invention having the amino acid sequence represented by SEQ ID NO: 26 As the DNA, the DNA having the base sequence represented by SEQ ID NO: 25 and the like encoding the polypeptide of the present invention having the amino acid sequence represented by SEQ ID NO: 12 are represented by SEQ ID NO: 10. As a DNA encoding the polypeptide of the present invention having the amino acid sequence represented by SEQ ID NO: 49, the DNA having the nucleotide sequence represented by The DNA encoding the polypeptide of the present invention having the amino acid sequence represented by SEQ ID NO: 47, such as the DNA having the base sequence represented by SEQ ID NO: 48, has the base sequence represented by SEQ ID NO: 46 The DNA etc. which it has are used.
[0026]
As a means for cloning DNA that completely encodes the polypeptide of the present invention, DNA amplified by a PCR method using a synthetic DNA primer having a partial base sequence of the polypeptide of the present invention or incorporated into an appropriate vector is used. Can be selected by hybridization with a DNA fragment encoding a part or the entire region of the polypeptide of the present invention or labeled with a synthetic DNA. The method of hybridization can be performed according to the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989), for example. Moreover, when using a commercially available library, it can carry out according to the method as described in an attached instruction manual.
DNA base sequence conversion can be performed by PCR or a known kit such as Mutan. ^TM -G (Takara Shuzo Co., Ltd.), Mutan ^TM -K (Takara Shuzo Co., Ltd.) and the like can be carried out according to a method known per se such as the Gapped duplex method and the Kunkel method or a method analogous thereto.
The DNA encoding the cloned polypeptide can be used as it is depending on the purpose, or digested with a restriction enzyme or added with a linker, if desired. The DNA may have ATG as a translation initiation codon on the 5 ′ end side, and may have TAA, TGA, or TAG as a translation termination codon on the 3 ′ end side. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter.
Examples of the expression vector for the polypeptide of the present invention include (a) excising a target DNA fragment from the DNA encoding the polypeptide of the present invention, and (b) placing the DNA fragment downstream of the promoter in an appropriate expression vector. It can manufacture by connecting.
[0027]
Examples of vectors include E. coli-derived plasmids (eg, pBR322, pBR325, pUC12, pUC13), Bacillus subtilis-derived plasmids (eg, pUB110, pTP5, pC194), yeast-derived plasmids (eg, pSH19, pSH15), λ phage, and the like. In addition to animal viruses such as bacteriophage, retrovirus, vaccinia virus, and baculovirus, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo and the like are used.
The promoter used in the present invention may be any promoter as long as it is suitable for the host used for gene expression. For example, when animal cells are used as a host, SRα promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter, β-actin and the like can be mentioned.
Of these, the CMV (cytomegalovirus) promoter, SRα promoter and the like are preferably used. When the host is Escherichia, trp promoter, lac promoter, recA promoter, λPL promoter, lpp promoter, T7 promoter, etc., and when the host is Bacillus, SPO1 promoter, SPO2 promoter, penP promoter, etc. When the host is yeast, the PHO5 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable. When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable.
[0028]
In addition to the above, an expression vector containing an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40ori) and the like is used as desired. it can. Examples of selection markers include dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [methotrexate (MTX) resistance], ampicillin resistance gene (hereinafter Amp). ^r A neomycin resistance gene (hereinafter Neo) ^r Geneticin resistance) and the like. In particular, when dhfr gene-deficient Chinese hamster cells are used and the dhfr gene is used as a selection marker, the recombinant cells can be selected by a medium not containing thymidine.
If necessary, a signal sequence suitable for the host is added to the N-terminal side of the polypeptide of the present invention. When the host is Escherichia, the PhoA signal sequence, OmpA, signal sequence, etc., and when the host is Bacillus, the α-amylase signal sequence, subtilisin signal sequence, etc. In some cases, MFα • signal sequence, SUC2 • signal sequence, etc. When the host is an animal cell, insulin signal sequence, α-interferon signal sequence, antibody molecule / signal sequence, etc. can be used.
A transformant can be produced using a vector containing the DNA encoding the polypeptide of the present invention thus constructed.
[0029]
As the host, for example, Escherichia, Bacillus, yeast, insect cells, insects, animal cells and the like are used.
Specific examples of the genus Escherichia include, for example, Escherichia coli K12 • DH1 [Procedures of the National Academy of Sciences of the USA (Proc. Natl. Acad. USA, 60, 160 (1968)], JM103 [Nucleic Acids Research, 9, 309 (1981)], JA221 [Journal of Molecular Biology (Journal of Molecular Biology). Molecular Biology)], 120, 517 (1978)], HB101 [Journal of Molecular Biology, 41, 459 (1969)], C600 [Genetics, 39, 440 (1954)] Etc. are used.
Examples of Bacillus bacteria include, for example, Bacillus subtilis MI114 [Gen, 24, 255 (1983)], 207-21 [Journal of Biochemistry, 95, 87 (1984). )] Etc. are used.
Examples of yeast include Saccharomyces cerevisiae AH22, AH22R. ^- NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036, Pichia pastoris KM71, and the like.
[0030]
As insect cells, for example, when the virus is AcNPV, larvae-derived cell lines (Spodoptera frugiperda cells; Sf cells), MG1 cells derived from the midgut of Trichoplusia ni, High Five derived from eggs of Trichoplusia ni ^TM Cells, cells derived from Mamestra brassicae or cells derived from Estigmena acrea are used. When the virus is BmNPV, sputum-derived cell lines (Bombyx mori N cells; BmN cells) and the like are used. Examples of the Sf cells include Sf9 cells (ATCC CRL 1711), Sf21 cells (Vaughn, JL et al., In Vivo, 13, 213-217, (1977)) and the like.
Examples of insects include silkworm larvae [Maeda et al., Nature, 315, 592 (1985)].
Examples of animal cells include monkey cell COS-7, Vero, Chinese hamster cell CHO (hereinafter abbreviated as CHO cell), dhfr gene-deficient Chinese hamster cell CHO (hereinafter referred to as CHO (dhfr). ^- Abbreviated cells), mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells, and the like.
[0031]
For transforming Escherichia, for example, Proc. Natl. Acad. Sci. USA, 69, 2110 (Proc. Natl. Acad. Sci. USA) 1972), Gene, Vol. 17, 107 (1982), and the like.
Transformation of Bacillus can be performed, for example, according to the method described in Molecular & General Genetics, 168, 111 (1979).
To transform yeast, for example, Methods in Enzymology, 194, 182-187 (1991), Processings of the National Academy of Sciences of The method can be carried out according to the method described in Proc. Natl. Acad. Sci. USA, 75, 1929 (1978).
Insect cells or insects can be transformed, for example, according to the method described in Bio / Technology, 6, 47-55 (1988).
In order to transform animal cells, for example, Cell Engineering Annex 8 New Cell Engineering Experiment Protocol. 263-267 (1995) (published by Shujunsha), Virology, Vol. 52, 456 (1973).
In this way, a transformant transformed with an expression vector containing DNA encoding the polypeptide can be obtained.
When cultivating a transformant whose host is an Escherichia bacterium or Bacillus genus, a liquid medium is suitable as a medium used for the culture, including a carbon source necessary for the growth of the transformant, Nitrogen sources, inorganic substances, etc. are contained. Examples of the carbon source include glucose, dextrin, soluble starch, and sucrose. Examples of the nitrogen source include ammonium salts, nitrates, corn steep liquor, peptone, casein, yeast extract, meat extract, soybean cake, potato Examples of inorganic or organic substances and inorganic substances such as extracts include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride. In addition, yeast extract, vitamins, growth promoting factors and the like may be added. The pH of the medium is preferably about 5-8.
[0032]
As a medium for culturing Escherichia, for example, M9 medium containing glucose and casamino acid [Miller, Journal of Experiments in Molecular Genetics] 431-433, Cold Spring Harbor Laboratory, New York 1972]. In order to make the promoter work efficiently as necessary, a drug such as 3β-indolylacrylic acid can be added.
When the host is an Escherichia bacterium, the culture is usually carried out at about 15 to 43 ° C. for about 3 to 24 hours, and if necessary, aeration or agitation can be added.
When the host is Bacillus, the culture is usually performed at about 30 to 40 ° C. for about 6 to 24 hours, and if necessary, aeration or agitation can be added.
When cultivating a transformant whose host is yeast, examples of the medium include a Burkholder minimal medium [Bostian, KL et al., Proceedings of the National Academy of Sciences of Science. USA, Proc. Natl. Acad. Sci. USA, 77, 4505 (1980)] and SD medium containing 0.5% casamino acid [Bitter, GA et al., Proceedings of the National -Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 81, 5330 (1984)]. The pH of the medium is preferably adjusted to about 5-8. The culture is usually performed at about 20 to 35 ° C. for about 24 to 72 hours, and aeration and agitation are added as necessary.
When culturing a transformant whose host is an insect cell or an insect, a medium such as 10% bovine serum immobilized in Grace's Insect Medium (Grace, TCC, Nature, 195,788 (1962)) is added. What added the thing suitably is used. The pH of the medium is preferably adjusted to about 6.2 to 6.4. The culture is usually performed at about 27 ° C. for about 3 to 5 days, and aeration and agitation are added as necessary.
When cultivating a transformant whose host is an animal cell, examples of the medium include a MEM medium containing about 5 to 20% fetal bovine serum [Science, Vol. 122, 501 (1952)], DMEM medium. [Virology, 8, 396 (1959)], RPMI 1640 medium [The Journal of the American Medical Association 199, 519 (1967)], 199 medium [Proceeding of the Society for the Biological Medicine, Volume 73, 1 (1950)] and the like are used. The pH is preferably about 6-8. The culture is usually performed at about 30 to 40 ° C. for about 15 to 60 hours, and aeration and agitation are added as necessary.
As described above, the polypeptide of the present invention can be produced outside or inside the transformant.
[0033]
Separation and purification of the polypeptide of the present invention from the culture can be performed, for example, by the following method.
When the polypeptide of the present invention is extracted from cultured cells or cells, the cells or cells are collected by a known method after culturing, suspended in an appropriate buffer, and subjected to ultrasound, lysozyme and / or freeze-thaw. For example, a method of obtaining a crude polypeptide extract by centrifugation or filtration after destroying cells or cells by, for example, is suitably used. Protein denaturants such as urea and guanidine hydrochloride in the buffer, Triton X-100 ^TM Etc. may be contained. When the polypeptide is secreted into the culture solution, after completion of the culture, the cells or cells and the supernatant are separated by a method known per se, and the supernatant is collected.
Purification of the polypeptide contained in the culture supernatant or the extract thus obtained can be performed by appropriately combining per se known separation and purification methods.
These known separation and purification methods include mainly molecular weights such as methods utilizing solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis. Method using difference in charge, method using difference in charge such as ion exchange chromatography, method using specific affinity such as affinity chromatography, and difference in hydrophobicity such as reverse phase high performance liquid chromatography A method using a difference in isoelectric point, such as a method, isoelectric focusing method, or the like is used.
[0034]
When the polypeptide thus obtained is obtained in a free form, it can be converted into a salt by a method known per se or a method analogous thereto, and conversely when it is obtained as a salt, a method known per se or a method analogous thereto. Depending on the method, it can be converted to the free form or other salts.
The polypeptide produced by the recombinant may be arbitrarily modified or partially removed by allowing an appropriate protein modifying enzyme or proteolytic enzyme to act before or after purification. it can. Examples of these enzymes include trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like.
The presence of the polypeptide of the present invention or a salt thereof thus produced can be measured by enzyme immunoassay using a specific antibody, Western blot analysis or the like.
[0035]
The antibody against the polypeptide of the present invention or a salt thereof may be either a polyclonal antibody or a monoclonal antibody as long as it can recognize the polypeptide of the present invention or a salt thereof.
The antibody against the polypeptide of the present invention or a salt thereof can be produced according to a method for producing an antibody or antiserum known per se, using the polypeptide of the present invention as an antigen.
[Production of monoclonal antibodies]
(A) Preparation of monoclonal antibody-producing cells
The polypeptide of the present invention or a salt thereof is administered to a warm-blooded animal by itself, together with a carrier or a diluent, at a site where antibody production is possible by administration. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. The administration is usually performed once every 2 to 6 weeks, about 2 to 10 times in total. Examples of the warm-blooded animal used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats and chickens, and mice and rats are preferably used.
When producing monoclonal antibody-producing cells, select an individual with a recognized antibody titer from a warm-blooded animal immunized with an antigen, such as a mouse, and collect the spleen or lymph nodes 2 to 5 days after the final immunization. Monoclonal antibody-producing hybridomas can be prepared by fusing the antibody-producing cells to be fused with allogeneic or xenogeneic myeloma cells. The antibody titer in the antiserum can be measured, for example, by reacting the labeled polypeptide described below with the antiserum and then measuring the activity of the labeling agent bound to the antibody. The fusion operation can be performed according to a known method, for example, the method of Kohler and Milstein [Nature, 256, 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus. Preferably, PEG is used.
[0036]
Examples of myeloma cells include warm-blooded animal myeloma cells such as NS-1, P3U1, SP2 / 0, AP-1, and P3U1 is preferably used. The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG 1000 to PEG 6000) is added at a concentration of about 10 to 80%. The cell fusion can be efficiently carried out by incubating at about 20 to 40 ° C., preferably about 30 to 37 ° C. for about 1 to 10 minutes.
Various methods can be used to screen for monoclonal antibody-producing hybridomas. For example, the hybridoma culture supernatant is added to a solid phase (eg, microplate) on which a polypeptide antigen is adsorbed directly or with a carrier, and then radioactive material is added. An anti-immunoglobulin antibody labeled with an enzyme or an enzyme (when the cell used for cell fusion is a mouse, an anti-mouse immunoglobulin antibody is used) or protein A to detect a monoclonal antibody bound to a solid phase, Examples include a method in which a hybridoma culture supernatant is added to a solid phase on which an immunoglobulin antibody or protein A is adsorbed, a polypeptide labeled with a radioactive substance or an enzyme is added, and a monoclonal antibody bound to the solid phase is detected.
The selection of the monoclonal antibody can be performed according to a method known per se or a method analogous thereto. Usually, it can be performed in a medium for animal cells supplemented with HAT (hypoxanthine, aminopterin, thymidine). As a selection and breeding medium, any medium may be used as long as the hybridoma can grow. For example, RPMI 1640 medium containing about 1-20%, preferably about 10-20% fetal calf serum, GIT medium (Wako Pure Chemical Industries, Ltd.) or hybridoma culture containing about 1-10% fetal calf serum Serum-free medium (SFM-101, Nissui Pharmaceutical Co., Ltd.) etc. can be used. The culture temperature is usually about 20 to 40 ° C, preferably about 37 ° C. The culture time is usually 5 days to 3 weeks, preferably 1 to 2 weeks. Culturing can usually be performed under 5% carbon dioxide gas. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the antibody titer in the above antiserum.
[0037]
(B) Purification of monoclonal antibody
Separation and purification of the monoclonal antibody can be performed by a method known per se, for example, by an immunoglobulin separation and purification method [eg, salting-out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, DEAE). Absorption / desorption method, ultracentrifugation method, gel filtration method, specific purification method to obtain antibody by collecting only antibody with antigen-binding solid phase or active adsorbent such as protein A or protein G and dissociating the binding] Can do.
[0038]
[Preparation of polyclonal antibody]
The polyclonal antibody of the present invention can be produced according to a method known per se or a method analogous thereto. For example, an immune antigen (polypeptide antigen) itself or a complex thereof and a carrier protein is prepared, and a warm-blooded animal is immunized in the same manner as the above-described monoclonal antibody production method. An antibody-containing substance against the salt can be collected and the antibody can be separated and purified.
Regarding the complex of immunizing antigen and carrier protein used to immunize warm-blooded animals, the type of carrier protein and the mixing ratio of carrier and hapten are such that antibodies are more effective against hapten immunized by cross-linking to carrier. As long as it is possible, any substance may be cross-linked at any ratio. For example, bovine serum albumin, bovine thyroglobulin, hemocyanin and the like are about 0.1 to 20, preferably about 1 with respect to hapten 1 by weight ratio. A method of coupling at a rate of ˜5 is used.
In addition, various condensing agents can be used for coupling of the hapten and the carrier, and active ester reagents containing glutaraldehyde, carbodiimide, maleimide active ester, thiol group, and dithiobilidyl group are used.
The condensation product is administered to a warm-blooded animal by itself, together with a carrier and a diluent, at a site where antibody production is possible. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. The administration is usually performed once every about 2 to 6 weeks and about 3 to 10 times in total.
Polyclonal antibodies can be collected from blood, ascites, etc., preferably blood of warm-blooded animals immunized by the above method.
The polyclonal antibody titer in the antiserum can be measured in the same manner as the above-described measurement of the antibody titer in the antiserum. Separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as the above-described monoclonal antibody separation and purification.
[0039]
An antisense DNA having a base sequence complementary to or substantially complementary to the DNA encoding the polypeptide of the present invention (hereinafter sometimes referred to as the DNA of the present invention) is complementary to the DNA of the present invention. Any antisense DNA may be used as long as it has a specific or substantially complementary base sequence and has an action capable of suppressing the expression of the DNA.
The base sequence substantially complementary to the DNA of the present invention is, for example, about 70 of the total base sequence or partial base sequence of the base sequence complementary to the DNA of the present invention (that is, the complementary strand of the DNA of the present invention). % Or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more of a base sequence having homology. In particular, of the total base sequence of the complementary strand of the DNA of the present invention, about 70% or more of the complementary strand of the base sequence encoding the N-terminal site of the polypeptide of the present invention (for example, the base sequence near the start codon, etc.) Antisense DNA having a homology of preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more is suitable. These antisense DNAs can be produced using a known DNA synthesizer or the like.
[0040]
When the polypeptide of the present invention has a signal peptide, it is efficiently secreted extracellularly and exhibits an important biological activity as a humoral factor for signal transmission, self-defense, and the like.
Hereinafter, the polypeptide of the present invention or a salt thereof (hereinafter sometimes abbreviated as the polypeptide of the present invention), the DNA encoding the polypeptide of the present invention (hereinafter sometimes abbreviated as the DNA of the present invention). The use of an antibody against the polypeptide of the present invention or a salt thereof (hereinafter sometimes abbreviated as the antibody of the present invention) and antisense DNA will be described.
[0041]
(1) Since the polypeptide of the present invention is expressed specifically in cartilage tissue, it can be used as a tissue marker. That is, it is useful as a marker for detecting tissue differentiation, disease state, cancer metastasis, and the like. It can also be used for fractionation of corresponding receptors, ligands, binding polypeptides and the like. Furthermore, it can be used to examine biological activity in a panel known per se for high-throughput screening. Chromosome mapping can also be used to study genetic diseases.
(2) Agents for treating / preventing various diseases related to the polypeptide of the present invention
The polypeptide of the present invention exists as a humoral factor in vivo (particularly cartilage tissue), and has a function of suppressing cartilage differentiation. Therefore, the polypeptide of the present invention or the DNA of the present invention has an abnormality, When it is deficient or when the expression level is abnormally decreased or increased, various diseases develop.
When the DNA or the like of the invention is deficient or the expression level is abnormally decreased, for example, osteoarthritis, rheumatoid arthritis, bone / joint diseases such as marble disease, pathological angiogenesis, etc. Disease develops.
Therefore, the polypeptide of the present invention and the DNA of the present invention can be used as pharmaceuticals for treating and preventing various diseases such as osteoarthritis, rheumatoid arthritis, bone / joint diseases such as marble disease, pathological angiogenesis and the like. Can be used as
For example, when there is a patient whose information transmission in cells is not fully or normally performed due to a decrease or deficiency of the polypeptide of the present invention in vivo, (i) the DNA of the present invention is transferred to the patient By administering and expressing the polypeptide of the present invention in vivo, (b) inserting the DNA of the present invention into a cell, expressing the polypeptide of the present invention, and then transplanting the cell to a patient Or (c) The role of the polypeptide of the present invention in the patient can be fully or normally exerted by administering the polypeptide of the present invention to the patient.
When using the DNA of the present invention as the above-mentioned therapeutic / prophylactic agent, after inserting the DNA alone or into an appropriate vector such as a retrovirus vector, adenovirus vector, adenovirus associated virus vector, etc., according to conventional means, It can be administered to humans or warm-blooded animals. The DNA of the present invention can be formulated as it is or with a physiologically recognized carrier such as an auxiliary agent for promoting ingestion and administered by a gene gun or a catheter such as a hydrogel catheter.
When the polypeptide of the present invention is used as the above-mentioned therapeutic / prophylactic agent, a polypeptide purified to at least 90%, preferably 95% or more, more preferably 98% or more, and further preferably 99% or more is used. Is preferred.
[0042]
The polypeptide of the present invention is aseptically obtained, for example, orally as a tablet, capsule, elixir, microcapsule, etc., with sugar coating as necessary, or with water or other pharmaceutically acceptable liquid. It can be used parenterally in the form of an injection solution such as an aqueous solution or suspension. For example, the polypeptide of the present invention is admixed in a unit dosage form required for the practice of a generally accepted formulation with physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, and the like. Can be manufactured. The amount of active ingredient in these preparations is such that an appropriate dose within the indicated range can be obtained.
Examples of additives that can be mixed into tablets, capsules and the like include binders such as gelatin, corn starch, tragacanth and gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, and alginic acid. Leavening agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, red mono oil or cherry. When the dispensing unit form is a capsule, a liquid carrier such as fats and oils can be further contained in the above-mentioned type of material. Sterile compositions for injection can be formulated according to conventional pharmaceutical practice, such as dissolving or suspending active substances in vehicles such as water for injection, naturally produced vegetable oils such as sesame oil, coconut oil and the like.
Examples of aqueous solutions for injection include isotonic solutions (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) containing physiological saline, glucose and other adjuvants, and suitable solubilizing agents. For example, alcohol (for example, ethanol), polyalcohol (for example, propylene glycol, polyethylene glycol, etc.), nonionic surfactant (for example, polysorbate 80) ^TM , HCO-50, etc.). Examples of the oily liquid include sesame oil and soybean oil, and may be used in combination with benzyl benzoate, benzyl alcohol or the like as a solubilizing agent. Buffers (eg, phosphate buffer, sodium acetate buffer, etc.), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.), You may mix | blend with preservatives (for example, benzyl alcohol, phenol, etc.), antioxidant, etc. The prepared injection solution is usually filled in a suitable ampoule.
A vector into which the DNA of the present invention has been inserted is formulated in the same manner as described above, and is usually used parenterally.
[0043]
The preparations thus obtained are safe and have low toxicity, so that, for example, humans or warm-blooded animals (eg rats, mice, guinea pigs, rabbits, birds, sheep, pigs, cows, horses, cats, dogs, monkeys) Etc.).
The dosage of the polypeptide of the present invention varies depending on the target disease, administration subject, administration route, etc. For example, when the polypeptide of the present invention is administered orally for the purpose of treating bone / joint diseases, it is generally an adult. In (60 kg), the polypeptide of the present invention is administered at about 1 mg to 1000 mg, preferably about 10 to 500 mg, more preferably about 10 to 200 mg per day. When administered parenterally, the single dose of the polypeptide of the present invention varies depending on the administration subject, target disease, etc., but for example, the polypeptide of the present invention is administered as an injection for the purpose of treating bone / joint diseases. In the case of administration to an adult (with a body weight of 60 kg), the polypeptide is administered by injecting about 1 to 1000 mg, preferably about 1 to 200 mg, more preferably about 10 to 100 mg of the polypeptide into the affected area per day. It is convenient to do. In the case of other animals, an amount converted per 60 kg can be administered.
[0044]
(2) Screening of drug candidate compounds for disease
Since the polypeptide of the present invention exists as a humoral factor in vivo (particularly cartilage tissue etc.) and has a function of suppressing cartilage differentiation, a compound or a salt thereof that promotes the function of the polypeptide of the present invention is, for example, It can be used as a medicine for treatment / prevention of osteoarthritis, rheumatoid arthritis, bone / joint diseases such as marble disease, and pathological angiogenesis.
On the other hand, a compound that inhibits the function of the polypeptide of the present invention or a salt thereof may cause a disease caused by excessive production of the polypeptide of the present invention, such as osteoarthritis, rheumatoid arthritis, osteogenesis imperfecta, osteoporosis, fracture. In addition, it can be used as a medicine such as a therapeutic / preventive agent for bone and joint diseases such as femoral head necrosis and chondrogenesis dysplasia, and pathological angiogenesis.
Therefore, the polypeptide of the present invention is useful as a reagent for screening a compound or a salt thereof that promotes or inhibits the function of the polypeptide of the present invention.
That is, the present invention
(1) A compound or salt thereof that promotes the function of the polypeptide of the present invention or a salt thereof, characterized by using the polypeptide of the present invention or a salt thereof (hereinafter sometimes abbreviated as an accelerator), or the present Provided is a screening method for a compound that inhibits the function of the polypeptide of the invention or a salt thereof (hereinafter sometimes abbreviated as an inhibitor).
The screening kit of the present invention contains the polypeptide of the present invention or a salt thereof.
[0045]
The compound or salt thereof obtained using the screening method or screening kit of the present invention is, for example, a peptide, protein, non-peptide compound, synthetic compound, fermentation product, cell extract, plant extract, animal tissue extract. Or a compound selected from plasma and the like, which promotes or inhibits the function of the polypeptide of the present invention.
As the salt of the compound, the same salts as those of the polypeptide of the present invention described above can be used.
[0046]
When the compound obtained by using the screening method or screening kit of the present invention is used as the above-mentioned therapeutic / prophylactic agent, it can be carried out according to conventional means. For example, in the same manner as the pharmaceutical containing the polypeptide of the present invention described above, a tablet, a capsule, an elixir, a microcapsule, a sterile solution, a suspension, and the like can be obtained.
Since the preparation thus obtained is safe and has low toxicity, it can be used, for example, in humans or warm-blooded animals (eg, mice, rats, rabbits, sheep, pigs, cows, horses, birds, cats, dogs, monkeys, etc.). It can be administered orally or parenterally.
The dose of the compound or a salt thereof varies depending on its action, target disease, administration subject, administration route, etc. For example, a compound that promotes the function of the polypeptide of the present invention for the purpose of treating bone / joint disease When administered orally, generally for an adult (with a body weight of 60 kg), the compound is administered at about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day. . When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, etc., but for example, a compound that promotes the function of the polypeptide of the present invention for the purpose of treating bone / joint diseases is used. When administered to an ordinary adult (as 60 kg) in the form of an injection, the compound is about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day. Is conveniently administered by intravenous injection. In the case of other animals, an amount converted per 60 kg can be administered.
On the other hand, when a compound that inhibits the function of the polypeptide of the present invention is orally administered, generally in an adult (with a body weight of 60 kg), the compound is about 0.1 to 100 mg, preferably about 1.0, per day. -50 mg, more preferably about 1.0-20 mg. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, etc., but the compound that inhibits the function of the polypeptide of the present invention is usually administered in the form of an injection (as 60 kg). ), It is convenient to administer about 0.1 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg of the compound per day by intravenous injection. is there. In the case of other animals, an amount converted per 60 kg can be administered.
[0047]
(3) Quantification of the polypeptide of the present invention or a salt thereof
Since the antibody against the polypeptide of the present invention (hereinafter sometimes abbreviated as the antibody of the present invention) can specifically recognize the polypeptide of the present invention, the polypeptide of the present invention in the test solution It can be used for quantitative determination, particularly quantitative determination by sandwich immunoassay.
That is, the present invention
(I) Competitively reacting the antibody of the present invention with a test solution and the labeled polypeptide of the present invention, and measuring the ratio of the labeled polypeptide of the present invention bound to the antibody A method for quantifying the polypeptide of the present invention in a test solution, and
(Ii) The test solution and the antibody of the present invention insolubilized on the carrier and the labeled another antibody of the present invention are reacted simultaneously or successively, and then the activity of the labeling agent on the insolubilized carrier is measured. The method for quantifying the polypeptide of the present invention in a test solution is provided.
[0048]
In addition to quantification of the polypeptide of the present invention using a monoclonal antibody against the polypeptide of the present invention (hereinafter sometimes referred to as the monoclonal antibody of the present invention), detection by tissue staining or the like can also be performed. For these purposes, the antibody molecule itself may be used, or the F (ab ′) of the antibody molecule. ₂ , Fab ′, or Fab fractions may be used.
The method for quantifying the polypeptide of the present invention using the antibody of the present invention is not particularly limited, and the antibody, antigen or antibody corresponding to the amount of antigen in the solution to be measured (for example, the amount of polypeptide of the present invention) -Any measurement method can be used as long as it is a measurement method in which the amount of antigen complex is detected by chemical or physical means and calculated from a standard curve prepared using a standard solution containing a known amount of antigen. Also good. For example, nephrometry, competition method, immunometric method and sandwich method are preferably used, but the sandwich method described later is particularly preferable in view of sensitivity and specificity.
Examples of the labeling agent used in the measurement method using the labeling substance include radioisotopes, enzymes, fluorescent substances, luminescent substances, and the like. Examples of radioisotopes include [ ¹²⁵ I], [ ¹³¹ I], [ ^Three H], [ ¹⁴ C] and the like are used. As the enzyme, a stable enzyme having a large specific activity is preferable. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used. Furthermore, a biotin-avidin system can also be used for binding of an antibody or antigen and a labeling agent.
[0049]
For insolubilization of the antigen or antibody, physical adsorption may be used, or a method using a chemical bond usually used to insolubilize or immobilize a polypeptide or an enzyme may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, or glass.
In the sandwich method, the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further labeled with another monoclonal antibody of the present invention (secondary reaction), and then on the insolubilized carrier. By measuring the activity of the labeling agent, the amount of the polypeptide of the present invention in the test solution can be quantified. The primary reaction and the secondary reaction may be performed in the reverse order, or may be performed simultaneously or at different times. The labeling agent and the insolubilization method can be the same as those described above.
In the immunoassay by the sandwich method, the antibody used for the solid phase antibody or the labeling antibody is not necessarily one type, and a mixture of two or more types of antibodies is used for the purpose of improving measurement sensitivity. May be.
In the method for measuring the polypeptide of the present invention by the sandwich method of the present invention, the monoclonal antibody of the present invention used for the primary reaction and the secondary reaction is preferably an antibody having a different site to which the polypeptide of the present invention binds. It is done. That is, the antibody used in the primary reaction and the secondary reaction is preferably an antibody used in the primary reaction when the antibody used in the secondary reaction recognizes the C-terminal part of the polypeptide of the present invention. For example, an antibody that recognizes the N end other than the C end is used.
[0050]
The monoclonal antibody of the present invention can be used in measurement systems other than the sandwich method, for example, competitive methods, immunometric methods, nephrometry, and the like.
In the competition method, the antigen in the test solution and the labeled antigen are reacted competitively with the antibody, and then the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated. (B / F separation), the amount of labeling of either B or F is measured, and the amount of antigen in the test solution is quantified. In this reaction method, a soluble antibody is used as an antibody, B / F separation is performed using polyethylene glycol, a liquid phase method using a second antibody against the antibody, and a solid-phased antibody is used as the first antibody, or As the first antibody, a soluble method is used, and a solid phase method using a solid phase antibody as the second antibody is used.
In the immunometric method, the antigen in the test solution and the immobilized antigen are subjected to a competitive reaction with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated, or the antigen in the test solution is separated. Are reacted with an excess amount of labeled antibody, and then a solid phased antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Next, the labeled amount of any phase is measured to quantify the amount of antigen in the test solution.
In nephrometry, the amount of insoluble precipitate produced as a result of antigen-antibody reaction in a gel or solution is measured. Laser nephrometry using laser scattering is preferably used even when the amount of antigen in the test solution is small and only a small amount of precipitate is obtained.
[0051]
In applying these individual immunological measurement methods to the quantification method of the present invention, special conditions, operations and the like are not required to be set. What is necessary is just to construct | assemble the measurement system of the polypeptide of this invention, adding the usual technical consideration of those skilled in the art to the usual conditions and operation methods in each method. For the details of these general technical means, it is possible to refer to reviews, books and the like.
For example, Hiroshi Irie “Radioimmunoassay” (Kodansha, published in 1974), Hiroshi Irie “Continue Radioimmunoassay” (published in Kodansha, 1979), “Enzyme Immunoassay” edited by Eiji Ishikawa et al. 53)), edited by Eiji Ishikawa et al. "Enzyme Immunoassay" (2nd edition) (Medical Shoin, published in 1982), edited by Eiji Ishikawa et al. "Enzyme Immunoassay" (3rd edition) (Medical Shoin, Showa) 62), “Methods in ENZYMOLOGY” Vol. 70 (Immunochemical Techniques (Part A)), Id. Vol. 73 (Immunochemical Techniques (Part B)), Id. Vol. 74 (Immunochemical Techniques (Part C)), Id. 84 (Immunochemical Techniques (Part D: Selected Immunoassays)), ibid.Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)), ibid. )) (From Academic Press ) And the like can be referred to.
As described above, the polypeptide of the present invention can be quantified with high sensitivity by using the antibody of the present invention.
Further, by quantifying the concentration of the polypeptide of the present invention using the antibody of the present invention, (1) when an increase in the concentration of the polypeptide of the present invention is detected, for example, bone / joint diseases (for example, , Osteoarthritis, rheumatoid arthritis, osteogenesis imperfecta, osteoporosis, fractures, femoral head necrosis, chondrogenic dysplasia, etc.), pathological angiogenesis (eg tumor angiogenesis, etc.) or future It can be diagnosed that there is a high possibility of being affected. (2) When a decrease in the concentration of the polypeptide of the present invention is detected, for example, bone / joint diseases (for example, osteoarthritis, rheumatoid arthritis, marble disease, etc.), pathological angiogenesis (for example, , Tumor angiogenesis, etc.) or can be diagnosed as having a high possibility of suffering in the future.
The antibody of the present invention can be used for detecting the polypeptide of the present invention present in a subject such as a body fluid or tissue. In addition, production of an antibody column used for purifying the polypeptide of the present invention, detection of the polypeptide of the present invention in each fraction during purification, analysis of the behavior of the polypeptide of the present invention in test cells, etc. Can be used for.
[0052]
(4) Gene diagnostic agent
The DNA of the present invention can be used, for example, as a probe in humans or warm-blooded animals (eg, rats, mice, guinea pigs, rabbits, birds, sheep, pigs, cows, horses, cats, dogs, monkeys, etc.). Since abnormality (gene abnormality) of DNA or mRNA encoding the polypeptide of the invention can be detected, for example, damage, mutation or expression decrease of the DNA or mRNA, increase or overexpression of the DNA or mRNA, etc. It is useful as a gene diagnostic agent.
The above gene diagnosis using the DNA of the present invention includes, for example, known Northern hybridization and PCR-SSCP method (Genomics, Vol. 5, pp. 874-879 (1989), Processings of. The National Academy of Sciences of USA (Proceedings of the National Academy of Sciences of the United States of America), Vol. 86, 2766-2770 (1989)), DNA microarray, etc. Can do.
For example, when a decrease in expression is detected by Northern hybridization or DNA microarray, or when a DNA mutation is detected by PCR-SSCP method or DNA microarray, for example, bone / joint disease (for example, osteoarthritis, Rheumatoid arthritis, marble disease, etc.) and pathological angiogenesis (for example, tumor angiogenesis, etc.) are highly likely to be diagnosed.
[0053]
(5) Medicine containing antisense DNA
An antisense DNA that can complementarily bind to the DNA of the present invention and suppress the expression of the DNA can suppress the function of the polypeptide of the present invention or the DNA of the present invention in vivo. Diseases caused by excessive expression of the polypeptide of the present invention (for example, osteoarthritis, rheumatoid arthritis, osteogenesis imperfecta, osteoporosis, fracture, femoral head necrosis, chondrosplasia, etc., It can be used as a therapeutic / preventive agent for pathological angiogenesis such as tumor angiogenesis.
The antisense DNA can be used as the above therapeutic / prophylactic agent in the same manner as the above-described therapeutic / preventive agents for various diseases containing the DNA of the present invention.
For example, the antisense DNA can be administered alone or after insertion into a suitable vector such as a retrovirus vector, adenovirus vector, adenovirus associated virus vector, etc., and then administered according to conventional means. The antisense DNA can be formulated as it is or with a physiologically recognized carrier such as an adjuvant for promoting intake, and can be administered by a gene gun or a catheter such as a hydrogel catheter.
Furthermore, the antisense DNA can also be used as a diagnostic oligonucleotide probe for examining the presence and expression status of the DNA of the present invention in tissues and cells.
[0054]
(6) Medicament containing the antibody of the present invention
The antibody of the present invention having the action of neutralizing the activity of the polypeptide of the present invention can be used, for example, for diseases caused by excessive expression of the polypeptide of the present invention (for example, osteoarthritis, rheumatoid arthritis, osteogenesis imperfecta). , Osteoporosis, fractures, bone and joint diseases such as femoral head necrosis, and chondrogenesis dysplasia, and pathological angiogenesis such as tumor angiogenesis).
The therapeutic / prophylactic agent for the above-mentioned diseases containing the antibody of the present invention can be used as a solution or as a pharmaceutical composition of an appropriate dosage form as a human or mammal (eg, rat, rabbit, sheep, pig, cow, cat, It can be administered orally or parenterally to dogs, monkeys, etc.). The dose varies depending on the administration subject, target disease, symptom, administration route and the like, but for example, the antibody of the present invention is usually about 0.01 to 20 mg / kg body weight for the purpose of treating bone / joint diseases. Preferably, about 0.1 to 10 mg / kg body weight, more preferably about 0.1 to 5 mg / kg body weight is administered by intravenous injection about 1 to 5 times a day, preferably about 1 to 3 times a day. Is convenient. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If symptoms are particularly severe, the dose may be increased according to the symptoms.
The antibody of the present invention can be administered per se or as an appropriate pharmaceutical composition. The pharmaceutical composition used for the administration comprises the above or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient. Such compositions are provided as dosage forms suitable for oral or parenteral administration.
That is, for example, compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (soft capsules). ), Syrups, emulsions, suspensions, and the like. Such a composition is produced by a method known per se, and contains a carrier, diluent or excipient usually used in the pharmaceutical field. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.
[0055]
As a composition for parenteral administration, for example, injections, suppositories and the like are used. Injections are dosage forms such as intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, infusions, and the like. Is included. Such an injection is prepared according to a method known per se, for example, by dissolving, suspending or emulsifying the above-described antibody or a salt thereof in a sterile aqueous or oily liquid usually used for injections. As an aqueous solution for injection, for example, an isotonic solution containing physiological saline, glucose and other adjuvants, and the like are used, and suitable solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants [eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)] and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent. The prepared injection solution is usually filled in a suitable ampoule. A suppository used for rectal administration is prepared by mixing the above-described antibody or a salt thereof with a normal suppository base.
The above oral or parenteral pharmaceutical compositions are conveniently prepared in dosage unit form to suit the dosage of the active ingredient. Examples of the dosage form of such a dosage unit include tablets, pills, capsules, injections (ampoules), suppositories and the like, and usually about 5 to 500 mg per dosage unit dosage form, especially 5 to 100 mg for injections. In other dosage forms, about 10 to 250 mg of the above antibody is preferably contained.
Each of the above-described compositions may contain other active ingredients as long as they do not cause an unfavorable interaction by blending with the antibody.
[0056]
(7) DNA transfer animals
The present invention has a DNA encoding an exogenous polypeptide of the present invention (hereinafter abbreviated as the exogenous DNA of the present invention) or a mutant DNA thereof (sometimes abbreviated as the exogenous mutant DNA of the present invention). A non-human mammal is provided.
That is, the present invention
(1) a non-human mammal having the foreign DNA of the present invention or a mutant DNA thereof,
(2) the animal according to (1), wherein the non-human mammal is a rodent;
(3) The animal according to (2), wherein the rodent is a mouse or a rat, and
(4) Provided is a recombinant vector containing the exogenous DNA of the present invention or a mutant DNA thereof and capable of being expressed in mammals.
A non-human mammal having an exogenous DNA of the present invention or a mutant DNA thereof (hereinafter abbreviated as a DNA-transferred animal of the present invention) can be used for embryos including unfertilized eggs, fertilized eggs, sperm and their progenitor cells, etc. Preferably, at the stage of embryonic development in non-human mammal development (more preferably at the single cell or fertilized egg cell stage and generally prior to the 8-cell stage), the calcium phosphate method, electric pulse method, lipofection method, aggregation method, It can be produced by transferring the target DNA by the microinjection method, particle gun method, DEAE-dextran method or the like. Further, by the DNA transfer method, the target exogenous DNA of the present invention can be transferred to somatic cells, living organs, tissue cells, etc., and can be used for cell culture, tissue culture, etc. The DNA-transferred animal of the present invention can also be produced by fusing the above-mentioned embryo cells with a cell fusion method known per se.
[0057]
Examples of non-human mammals include cows, pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, mice, rats and the like. Among them, rodents, especially mice (for example, C57BL / 6 strain, DBA2 strain, etc. as pure strains), which have relatively short ontogeny and biological cycle from the viewpoint of creating a disease animal model system, and which are easy to reproduce, are crossed. As a system, B6C3F ₁ System, BDF ₁ System, B6D2F ₁ Strain, BALB / c strain, ICR strain, etc.) or rats (eg, Wistar, SD, etc.) are preferred.
Examples of the “mammal” in the recombinant vector that can be expressed in mammals include humans and the like in addition to the non-human mammals described above.
The exogenous DNA of the present invention is not the DNA of the present invention inherently possessed by a non-human mammal but the DNA of the present invention once isolated and extracted from the mammal.
Examples of the mutant DNA of the present invention include those in which a mutation (for example, mutation or the like) has occurred in the base sequence of the original DNA of the present invention, specifically, addition of a base, deletion, substitution to another base, etc. The resulting DNA or the like is used, and abnormal DNA is also included.
The abnormal DNA means a DNA that expresses an abnormal polypeptide of the present invention. For example, a DNA that expresses a polypeptide that suppresses the function of the normal polypeptide of the present invention is used.
The exogenous DNA of the present invention may be derived from mammals of the same or different species as the target animal. In transferring the DNA of the present invention to a target animal, it is generally advantageous to use the DNA as a DNA construct bound downstream of a promoter that can be expressed in animal cells. For example, when transferring the human DNA of the present invention, the expression of DNA derived from various mammals (for example, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.) having the DNA of the present invention having high homology thereto. The DNA of the present invention is highly expressed by microinjecting a DNA construct (eg, vector, etc.) in which the human DNA of the present invention is bound downstream of various promoters into a fertilized egg of a target mammal, such as a mouse fertilized egg. DNA transfer mammals can be created.
[0058]
Examples of the expression vector for the polypeptide of the present invention include plasmids derived from Escherichia coli, plasmids derived from Bacillus subtilis, plasmids derived from yeast, bacteriophages such as λ phage, retroviruses such as Moloney leukemia virus, animals such as vaccinia viruses and baculoviruses. Virus or the like is used. Of these, plasmids derived from Escherichia coli, plasmids derived from Bacillus subtilis, plasmids derived from yeast, and the like are preferably used.
Examples of promoters that regulate the above DNA expression include: (1) promoters of DNA derived from viruses (eg, simian virus, cytomegalovirus, Moloney leukemia virus, JC virus, breast cancer virus, poliovirus, etc.); ▼ Promoters from various mammals (human, rabbit, dog, cat, guinea pig, hamster, rat, mouse, etc.), such as albumin, insulin II, uroplakin II, elastase, erythropoietin, endothelin, muscle creatine kinase, glial fibrillary acidity Protein, glutathione S-transferase, platelet-derived growth factor β, keratin K1, K10 and K14, collagen type I and type II, cyclic AMP-dependent protein kinase βI subunit, dystrophy Tartrate-resistant alkaline phosphatase, atrial natriuretic factor, endothelial receptor tyrosine kinase (commonly abbreviated as Tie2), sodium potassium adenosine 3 kinase (Na, K-ATPase), neurofilament light chain, metallothionein I and IIA, metalloproteinase 1 tissue inhibitor, MHC class I antigen (H-2L), H-ras, renin, dopamine β-hydroxylase, thyroid peroxidase (TPO), polypeptide chain elongation factor 1α (EF-1α), β Actin, α and β myosin heavy chain, myosin light chain 1 and 2, myelin basic protein, thyroglobulin, Thy-1, immunoglobulin, heavy chain variable region (VNP), serum amyloid P component, myoglobin, troponin C, smooth muscle α Emissions, preproenkephalin A, promoters vasopressin, etc. and the like are used. Among these, a cytomegalovirus promoter capable of being highly expressed throughout the body, a human polypeptide chain elongation factor 1α (EF-1α) promoter, human and chicken β-actin promoters, and the like are preferable.
[0059]
The vector preferably has a sequence (generally referred to as a terminator) that terminates transcription of a target messenger RNA in a DNA-transferred mammal. For example, the sequence of each DNA derived from viruses and various mammals Preferably, a simian virus SV40 terminator or the like is used.
In addition, the splicing signal of each DNA, enhancer region, part of intron of eukaryotic DNA, etc., 5 ′ upstream of the promoter region, between the promoter region and the translation region or between the translation regions for the purpose of further expressing the target foreign DNA It is also possible to connect it 3 'downstream of the target.
The translation region can be prepared as a DNA construct that can be expressed in a metastasized animal by an ordinary DNA engineering technique in which it is linked downstream of the promoter and optionally upstream of the transcription termination site.
The transfer of the foreign DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the subject mammal. The presence of the foreign DNA of the present invention in the embryo cells of the produced animal after DNA transfer means that all the progeny of the produced animal retain the foreign DNA of the present invention in all the germ cells and somatic cells. means. The offspring of this type of animal that has inherited the foreign DNA of the present invention has the foreign DNA of the present invention in all germ cells and somatic cells.
[0060]
The non-human mammal to which the exogenous normal DNA of the present invention has been transferred can be subcultured in a normal breeding environment as a DNA-bearing animal after confirming that the exogenous DNA is stably retained by mating. .
The transfer of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be excessively present in all germ cells and somatic cells of the target mammal. Excessive exogenous DNA of the present invention is present in the embryo cells of the produced animal after the DNA transfer. This means that all the offspring of the produced animal have the exogenous DNA of the present invention in all the germ cells and somatic cells. Means. The offspring of this type of animal that has inherited the foreign DNA of the present invention has an excess of the foreign DNA of the present invention in all germ cells and somatic cells.
By obtaining a homozygous animal having the introduced DNA in both homologous chromosomes and mating the male and female animals, all the offspring can be bred and passaged so as to have the DNA in excess.
In the non-human mammal having the normal DNA of the present invention, the normal DNA of the present invention is highly expressed, and finally the function of the endogenous normal DNA is promoted to finally promote hyperfunction of the polypeptide of the present invention. Can be used as a model animal of the disease state. For example, using the normal DNA-transferred animal of the present invention, elucidation of the hyperfunction of the polypeptide of the present invention and the pathologic mechanism of the disease associated with the polypeptide of the present invention and examination of the treatment method for these diseases It is possible.
In addition, since the mammal to which the exogenous normal DNA of the present invention has been transferred has an increased symptom of the released polypeptide of the present invention, it is also used in screening tests for therapeutic agents for diseases related to the polypeptide of the present invention. Is possible.
[0061]
On the other hand, the non-human mammal having the exogenous abnormal DNA of the present invention can be subcultured in a normal breeding environment as the DNA-bearing animal after confirming that the exogenous DNA is stably retained by mating. Furthermore, the target foreign DNA can be incorporated into the aforementioned plasmid and used as a raw material. A DNA construct with a promoter can be prepared by a normal DNA engineering technique. The abnormal DNA transfer of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal. The presence of the abnormal DNA of the present invention in the embryo cell of the produced animal after DNA transfer means that all the offspring of the produced animal have the abnormal DNA of the present invention in all the germ cells and somatic cells. The offspring of this type of animal that has inherited the foreign DNA of the present invention has the abnormal DNA of the present invention in all of its germ cells and somatic cells. By obtaining a homozygous animal having the introduced DNA in both homologous chromosomes and mating the male and female animals, all the offspring can be bred to have the DNA.
In the non-human mammal having the abnormal DNA of the present invention, the abnormal DNA of the present invention is highly expressed, and finally the function of the polypeptide of the present invention is inactivated by inhibiting the function of the endogenous normal DNA. It can become type refractory and can be used as a model animal for the disease state. For example, by using the abnormal DNA-transferred animal of the present invention, it is possible to elucidate the pathological mechanism of the functional inactive refractory of the polypeptide of the present invention and to examine a method for treating this disease.
Further, as a specific applicability, the abnormally high-expressing animal of the present invention is capable of inhibiting the function of the normal polypeptide (dominant negative) by the abnormal polypeptide of the present invention in the functional inactive refractory of the polypeptide of the present invention. Model).
Further, since the mammal to which the foreign abnormal DNA of the present invention has been transferred has an increased symptom of the released polypeptide of the present invention, it is also used in a therapeutic drug screening test for the functional inactive refractory of the polypeptide of the present invention. Is available.
[0062]
Further, as other applicability of the above-mentioned two kinds of DNA transfer animals of the present invention, for example,
(1) Use as a cell source for tissue culture,
(2) Specific expression or activation by the polypeptide of the present invention by directly analyzing DNA or RNA in the tissue of the DNA-transferred animal of the present invention or by analyzing the polypeptide tissue expressed by the DNA Analysis of the relationship to the polypeptide
(3) Cell of tissue having DNA is cultured by a standard tissue culture technique, and using these, research on the function of cells from tissue generally difficult to cultivate,
(4) Screening for drugs that enhance cell function by using the cells described in (3) above, and
(5) Isolation and purification of the mutant polypeptide of the present invention and production of an antibody thereof can be considered.
Furthermore, using the DNA-transferred animal of the present invention, clinical symptoms of diseases related to the polypeptide of the present invention, including functional inactive refractories of the polypeptide of the present invention, can be examined. More detailed pathological findings in each organ of a disease model related to the polypeptide can be obtained, which can contribute to the development of a new treatment method and further to the study and treatment of secondary diseases caused by the disease.
Further, each organ is taken out from the DNA-transferred animal of the present invention, and after minced, free DNA-transferred cells are obtained, cultured or systematized by using a polypeptide (protein) degrading enzyme such as trypsin. Is possible. Furthermore, the polypeptide of the present invention and its action can be investigated by examining the relationship between the specification, apoptosis, differentiation or proliferation of the polypeptide-producing cells of the present invention, or their signal transduction mechanisms, and their abnormalities. It becomes an effective research material for elucidation.
Further, in order to develop a therapeutic agent for a disease related to the polypeptide of the present invention, including the functionally inactive refractory of the polypeptide of the present invention, using the DNA-transferred animal of the present invention, In addition, it is possible to provide an effective and rapid screening method for the therapeutic agent for the disease using a quantitative method and the like. Moreover, it is possible to examine and develop a DNA therapy for a disease associated with the polypeptide of the present invention using the DNA-transferred animal of the present invention or the exogenous DNA expression vector of the present invention.
[0063]
(8) Knockout animals
The present invention provides a non-human mammal embryonic stem cell in which the DNA of the present invention is inactivated and the DNA expression-deficient non-human mammal of the present invention.
That is, the present invention
(1) a non-human mammalian embryonic stem cell in which the DNA of the present invention is inactivated,
(2) The embryonic stem cell according to (1), wherein the DNA is inactivated by introducing a reporter gene (eg, β-galactosidase gene derived from E. coli),
(3) The embryonic stem cell according to (1), which is neomycin resistant,
(4) The embryonic stem cell according to item (1), wherein the non-human mammal is a rodent.
(5) The embryonic stem cell according to (4), wherein the rodent is a mouse,
(6) the DNA expression-deficient non-human mammal in which the DNA of the present invention is inactivated,
(7) The DNA can be inactivated by introducing a reporter gene (eg, β-galactosidase gene derived from E. coli), and the reporter gene can be expressed under the control of a promoter for the DNA of the present invention (6) The non-human mammal according to Item,
(8) The non-human mammal according to (6), wherein the non-human mammal is a rodent,
(9) The non-human mammal according to item (8), wherein the rodent is a mouse, and
(10) A screening method for a compound or a salt thereof that promotes or inhibits promoter activity against the DNA of the present invention, which comprises administering a test compound to the animal according to item (7) and detecting the expression of a reporter gene I will provide a.
[0064]
The non-human mammal embryonic stem cell in which the DNA of the present invention is inactivated refers to suppressing the DNA expression ability by artificially adding mutation to the DNA of the present invention possessed by the non-human mammal, or By substantially losing the activity of the polypeptide of the present invention encoded by the DNA, the DNA has substantially no ability to express the polypeptide of the present invention (hereinafter referred to as the knockout DNA of the present invention). And non-human mammal embryonic stem cells (hereinafter abbreviated as ES cells).
As the non-human mammal, the same ones as described above are used.
As a method for artificially adding a mutation to the DNA of the present invention, for example, a part or all of the DNA sequence can be deleted or another DNA can be inserted or replaced by a genetic engineering technique. With these mutations, for example, the knockout DNA of the present invention may be prepared by shifting the reading frame of codons or destroying the function of the promoter or exon.
Specific examples of non-human mammalian embryonic stem cells in which the DNA of the present invention is inactivated (hereinafter abbreviated as the DNA inactivated ES cell of the present invention or the knockout ES cell of the present invention) A DNA of the present invention possessed by a non-human mammal is isolated and a neomycin resistance gene, a drug resistance gene typified by a hygromycin resistance gene, or lacZ (β-galactosidase gene), cat (chloramphenicol acetyl) Inserting a reporter gene typified by a transferase gene) or the like, or inserting a DNA sequence (for example, a polyA addition signal) that terminates the transcription of the gene into an intron between exons, By preventing the synthesis of complete messenger RNA As a result, a DNA strand having a DNA sequence constructed so as to destroy the gene (hereinafter abbreviated as a targeting vector) is introduced into the chromosome of the animal by, for example, homologous recombination, and the obtained ES cells are By Southern hybridization analysis using the DNA sequence of the invention or the vicinity thereof as a probe, or by a PCR method using the DNA sequence of the targeting vector and the DNA sequence of the neighboring region other than the DNA of the invention used for the preparation of the targeting vector as primers. It can be obtained by analyzing and selecting the knockout ES cell of the present invention.
[0065]
Moreover, as the original ES cell for inactivating the DNA of the present invention by homologous recombination method, for example, those already established as described above may be used, or according to the methods of known Evans and Kaufma. Newly established may be used. For example, in the case of mouse ES cells, currently 129 ES cells are generally used, but since the immunological background is unclear, an alternative purely immunologically genetic background For example, for the purpose of obtaining ES cells with clear clarification, for example, B57 improved by reducing the number of eggs collected in C57BL / 6 mice and C57BL / 6 by crossing with DBA / 2 ₁ Mouse (F of C57BL / 6 and DBA / 2 ₁ The one established using) can be used well. BDF ₁ In addition to the advantage that the number of eggs collected is high and the eggs are strong, the mouse has C57BL / 6 mice as a background. Therefore, when ES cells obtained using these mice are used to produce pathological model mice, C57BL / 6 mice can be advantageously used in that they can be backcrossed to replace their genetic background with C57BL / 6 mice.
In addition, when establishing ES cells, blastocysts on the 3.5th day after fertilization are generally used, but in addition to this, many blastocysts can be efficiently obtained by collecting 8-cell embryos and culturing them to blastocysts. Early embryos can be obtained.
Although both male and female ES cells may be used, male ES cells are usually more convenient for producing germline chimeras. In addition, it is desirable to discriminate between males and females as soon as possible in order to reduce troublesome culture work.
As a method for determining sex of male and female ES cells, for example, a method of amplifying and detecting a gene in the sex-determining region on the Y chromosome by PCR can be mentioned as an example. If this method is used, it is conventionally about 10 times for karyotype analysis. ⁶ In contrast to the number of cells required, the number of ES cells of about 1 colony (about 50) is sufficient, so primary selection of ES cells in the early stage of culture can be performed by male / female discrimination. Yes, the early labor of the culture can be greatly reduced by enabling the selection of male cells at an early stage.
[0066]
The secondary selection can be performed, for example, by confirming the number of chromosomes by the G-banding method. The number of chromosomes of the obtained ES cell is preferably 100% of the normal number. However, if it is difficult due to physical operations at the time of establishment, the gene of the ES cell is knocked out, and then normal cells (for example, chromosomes in mice) It is desirable to clone again into cells where the number is 2n = 40.
The embryonic stem cell line obtained in this way is usually very proliferative, but it tends to lose its ontogenic ability, so it needs to be subcultured carefully. For example, in a carbon dioxide incubator (preferably about 5% carbon dioxide, about 95% air, or about 5% oxygen) in the presence of LIF (1-10000 U / ml) on a suitable feeder cell such as STO fibroblast. Culturing at about 37 ° C. in about 5% carbon dioxide gas, about 90% air), and at the time of passage, for example, trypsin / EDTA solution (usually about 0.001-0.5% trypsin / about 0.1-5 mM EDTA (preferably about 0.1% trypsin / about 1 mM EDTA) is made into a single cell and treated on a newly prepared feeder cell. Such passage is usually carried out every 1-3 days. At this time, it is desirable to observe the cells, and when morphologically abnormal cells are found, the cultured cells should be discarded.
ES cells can be differentiated into various types of cells such as parietal muscle, visceral muscle, and myocardium by monolayer culture to high density or suspension culture until a cell conglomerate is formed under appropriate conditions. [MJ Evans and MH Kaufman, Nature 292, 154, 1981; GR Martin Proceedings of National Academy of Sciences USA (Proc. Natl. Acad. Sci. USA) 78, 7634, 1981; TC Doetschman et al., Journal of Embrology and Experimental Morphology, 87, 27, 1985], ES cells of the present invention. The DNA expression-deficient cells of the present invention obtained by differentiation are useful in cell biology of the polypeptide of the present invention in vitro.
The DNA expression deficient non-human mammal of the present invention can be distinguished from normal animals by measuring the mRNA level of the animal using a known method and comparing the expression level indirectly.
As the non-human mammal, those similar to the above can be used.
[0067]
The DNA expression-deficient non-human mammal of the present invention is a DNA in which, for example, a targeting vector prepared as described above is introduced into mouse embryonic stem cells or mouse egg cells, and the DNA of the targeting vector of the present invention is inactivated by the introduction. The DNA of the present invention can be knocked out by homologous recombination in which the sequence replaces the DNA of the present invention on the chromosome of mouse embryonic stem cells or mouse egg cells by gene homologous recombination.
A cell in which the DNA of the present invention is knocked out is obtained by analyzing the DNA sequence of the Southern hybridization analysis or targeting vector using the DNA sequence of or near the DNA of the present invention as a probe and the mouse used for the targeting vector of the present invention. It can be determined by analysis by a PCR method using a DNA sequence in a neighboring region other than DNA as a primer. When a non-human mammalian embryonic stem cell is used, a cell line in which the DNA of the present invention is inactivated by gene homologous recombination is cloned, and the cell is placed at a suitable time, for example, an 8-cell non-human mammal. It is injected into animal embryos or blastocysts, and the produced chimeric embryos are transplanted into the uterus of the non-human mammal that is pseudopregnant. The produced animal is a chimeric animal composed of both cells having the normal DNA locus of the present invention and cells having the artificially mutated DNA locus of the present invention.
When some of the germ cells of the chimeric animal have the mutated DNA locus of the present invention, all the tissues are artificially mutated from the population obtained by mating such a chimeric individual with a normal individual. It can be obtained by selecting an individual composed of cells having the added DNA locus of the present invention, for example, by determining the coat color. The individuals thus obtained are usually individuals with deficient hetero-expression of the polypeptide of the present invention, and individuals having deficient hetero-expression of the polypeptide of the present invention are mated with each other, and the polypeptide of the present invention is derived from their offspring. Can be obtained.
When using an egg cell, for example, a transgenic non-human mammal having a targeting vector introduced into the chromosome can be obtained by injecting a DNA solution into the nucleus of the egg cell by a microinjection method. Compared to mammals, it can be obtained by selecting those having a mutation at the DNA locus of the present invention by gene homologous recombination.
[0068]
Thus, an individual in which the DNA of the present invention is knocked out can be reared in a normal breeding environment after confirming that the individual animal obtained by mating has also been knocked out.
In addition, germline acquisition and retention may be followed in accordance with conventional methods. That is, a homozygous animal having the inactivated DNA in both homologous chromosomes can be obtained by mating male and female animals possessed by the inactivated DNA. The obtained homozygous animal can be efficiently obtained by rearing the mother animal in a state where there are 1 normal individual and multiple homozygous animals. By crossing male and female heterozygous animals, homozygous and heterozygous animals having the inactivated DNA are bred and passaged.
The non-human mammal embryonic stem cell in which the DNA of the present invention is inactivated is very useful for producing the non-human mammal deficient in DNA expression of the present invention.
In addition, since the non-human mammal deficient in DNA expression of the present invention lacks various biological activities that can be induced by the polypeptide of the present invention, diseases caused by inactivation of the biological activity of the polypeptide of the present invention Therefore, it is useful for investigating the causes of these diseases and for examining therapeutic methods.
[0069]
(8a) Screening method for compounds having therapeutic / preventive effects on diseases caused by deficiency or damage of the DNA of the present invention
The non-human mammal deficient in the expression of DNA of the present invention is a disease caused by deficiency or damage of the DNA of the present invention (bone / joint diseases (for example, osteoarthritis, rheumatoid arthritis marble disease, etc.), pathological blood vessels. It can be used for screening for compounds having therapeutic / prophylactic effects on neoplasia (for example, neovascularization of tumors, etc.).
That is, the present invention is attributable to the deficiency or damage of the DNA of the present invention, which comprises administering a test compound to the non-human mammal deficient in DNA expression of the present invention and observing and measuring changes in the animal. Provided is a screening method for a compound having a therapeutic / preventive effect on a disease or a salt thereof.
Examples of the non-human mammal deficient in DNA expression of the present invention used in the screening method include those described above.
Examples of the test compound include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma and the like, and these compounds are novel compounds. It may be a known compound.
Specifically, the DNA expression-deficient non-human mammal of the present invention is treated with a test compound, compared with a non-treated control animal, and tested using changes in each organ, tissue, disease symptom, etc. of the animal as an index. The therapeutic / prophylactic effect of the compound can be tested.
As a method for treating a test animal with a test compound, for example, oral administration, intravenous injection, and the like are used. The method is appropriately selected according to the symptom of the test animal, the nature of the test compound, and the like.
Can. The dosage of the test compound can be appropriately selected according to the administration method, the properties of the test compound, and the like.
For example, when screening for a compound having a therapeutic / prophylactic effect on tumor angiogenesis, cancer cell transplantation is performed on the non-human mammal deficient in DNA expression of the present invention, and a test compound is administered before or after transplantation of the cancer cells, The tumor marker value and tumor mass size of the animal are measured over time.
[0070]
The compound obtained using the screening method of the present invention is a compound selected from the test compounds described above, and is caused by a defect or damage of the polypeptide of the present invention (bone / joint disease (for example, degenerative joint). , Rheumatoid arthritis marble disease, etc.), pathological angiogenesis (eg tumor angiogenesis, etc.), etc., and therefore, pharmaceuticals such as safe and low-toxic therapeutic / preventive agents for the disease Can be used as Furthermore, compounds derived from the compounds obtained by the above screening can be used as well.
The compound obtained by the screening method may form a salt. Examples of the salt of the compound include physiologically acceptable acids (eg, inorganic acids, organic acids), bases (eg, alkali metals) and the like. In particular, physiologically acceptable acid addition salts are preferred. Such salts include, for example, salts with inorganic acids (eg hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.
A medicament containing the compound obtained by the screening method or a salt thereof can be produced in the same manner as the aforementioned medicament containing the polypeptide of the present invention.
Since the preparation thus obtained is safe and has low toxicity, it can be used, for example, in humans or mammals (eg, rats, mice, guinea pigs, rabbits, sheep, pigs, cows, horses, cats, dogs, monkeys, etc.). Can be administered.
The dose of the compound or a salt thereof varies depending on the target disease, administration subject, administration route, etc. For example, when the compound is administered orally for the purpose of treating bone / joint diseases, generally an adult (with a body weight of 60 kg) is used. ), The compound is administered at about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day. When administered parenterally, the single dose of the compound varies depending on the administration subject, the target disease, etc. For example, for the purpose of treating bone / joint disease, the compound is usually administered in the form of an injection (60 kg). The compound is administered at about 0.01 to 30 mg per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg by intravenous injection per day. It is. In the case of other animals, an amount converted per 60 kg can be administered.
[0071]
(8b) Screening method for compounds that promote or inhibit the activity of the promoter for the DNA of the present invention
The present invention provides a compound that promotes or inhibits the activity of a promoter for the DNA of the present invention, which comprises administering a test compound to a non-human mammal deficient in DNA expression of the present invention and detecting the expression of a reporter gene, or a compound thereof A salt screening method is provided.
In the above screening method, the DNA expression deficient non-human mammal of the present invention is inactivated by introducing a reporter gene among the DNA deficient non-human mammals of the present invention described above, A gene capable of expressing the reporter gene under the control of a promoter for the DNA of the present invention is used.
Examples of the test compound are the same as described above.
As the reporter gene, the same ones as described above are used, and a β-galactosidase gene (lacZ), a soluble alkaline phosphatase gene, a luciferase gene or the like is preferable.
In non-human mammals with deficient DNA expression of the present invention in which the DNA of the present invention is replaced with a reporter gene, the reporter gene exists under the control of the promoter for the DNA of the present invention, so the expression of the substance encoded by the reporter gene is traced. By doing so, the activity of the promoter can be detected.
[0072]
For example, when a part of the DNA region encoding the polypeptide of the present invention is substituted with the β-galactosidase gene (lacZ) derived from E. coli, the tissue of the present invention is originally expressed in the tissue expressing the polypeptide of the present invention. Β-galactosidase is expressed instead of the peptide. Therefore, for example, by staining with a reagent that is a substrate of β-galactosidase such as 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal), The expression state of the polypeptide of the present invention in an animal body can be observed. Specifically, the polypeptide-deficient mouse of the present invention or a tissue section thereof is fixed with glutaraldehyde or the like, washed with phosphate buffered saline (PBS), and then stained with X-gal at room temperature or 37 ° C. After reacting in the vicinity for about 30 minutes to 1 hour, the tissue specimen is washed with a 1 mM EDTA / PBS solution to stop the β-galactosidase reaction and observe the coloration. Moreover, you may detect mRNA which codes lacZ according to a conventional method.
[0073]
The compound obtained by using the screening method or a salt thereof is a compound selected from the above-described test compounds, and is a compound that promotes or inhibits the promoter activity for the DNA of the present invention.
The compound obtained by the screening method may form a salt, and as a salt of the compound, a salt with a physiologically acceptable acid (eg, inorganic acid), base (eg, organic acid), etc. And physiologically acceptable acid addition salts are particularly preferred. Such salts include, for example, salts with inorganic acids (eg hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.
The compound or the salt thereof that promotes the promoter activity for the DNA of the present invention can promote the expression of the polypeptide of the present invention and promote the function of the polypeptide. It is useful as a pharmaceutical agent such as a safe and low toxic therapeutic / preventive agent for diseases such as osteoarthritis, rheumatoid arthritis marble disease, etc.) and pathological angiogenesis (eg, tumor angiogenesis).
Furthermore, compounds derived from the compounds obtained by the above screening can be used as well.
[0074]
The medicament containing the compound or salt thereof obtained by the screening method can be produced in the same manner as the medicament containing the polypeptide of the present invention or salt thereof.
Since the preparation thus obtained is safe and has low toxicity, it can be used, for example, in humans or mammals (eg, rats, mice, guinea pigs, rabbits, sheep, pigs, cows, horses, cats, dogs, monkeys, etc.). Can be administered.
The dose of the compound or a salt thereof varies depending on the target disease, administration subject, administration route, etc. For example, for the purpose of treating bone / joint diseases, a compound that promotes promoter activity against the DNA of the present invention is orally administered. In general, in an adult (with a body weight of 60 kg), the compound is administered at about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, etc., but for example, a compound that promotes the promoter activity for the DNA of the present invention for the purpose of treating bone / joint disease is used. When administered to an ordinary adult (as 60 kg) in the form of an injection, the compound is about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day. Is conveniently administered by intravenous injection. In the case of other animals, an amount converted per 60 kg can be administered.
On the other hand, for example, when a compound that inhibits promoter activity against DNA of the present invention is orally administered, generally in an adult (with a body weight of 60 kg), the compound is about 0.1 to 100 mg, preferably about 1 per day. The dose is from 0 to 50 mg, more preferably from about 1.0 to 20 mg. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, etc., but the compound that inhibits the promoter activity against the DNA of the present invention is usually administered in the form of an injection (as an adult 60 kg). ), It is convenient to administer about 0.1 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg of the compound per day by intravenous injection. is there. In the case of other animals, an amount converted per 60 kg can be administered.
[0075]
Thus, the non-human mammal deficient in DNA expression of the present invention is extremely useful in screening for compounds or salts thereof that promote or inhibit the activity of the promoter for the DNA of the present invention. It can greatly contribute to the investigation of the causes of various diseases caused or the development of preventive / therapeutic drugs.
Furthermore, since the gene encoding the polypeptide of the present invention is expressed in a very large amount in mouse and human, particularly in cartilage tissue, the promoter sequence of the gene is the target protein (any useful gene product etc.). It is convenient as a promoter for large-scale expression in cartilage tissue of non-human warm-blooded animals. Examples of non-human warm-blooded animals include those similar to those exemplified as the above-mentioned warm-blooded animals.
That is, the present invention relates to the downstream (3 ′ end) of the promoter region of a gene encoding a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 12 or SEQ ID NO: 47. The target protein (any useful gene product, etc.) is linked to the non-human animal, and the target protein (any useful gene product, etc.) is expressed predominantly in the cartilage tissue of non-human warm-blooded animals. provide.
Examples of the target protein (any useful gene product) include, for example, cytokines (eg, interleukins, interferons, chemokines, hematopoietic factors), growth factors (eg, EGF (epidermal growth factor)) or substantially the same activity as it. (For example, EGF, haregulin (HER2 ligand), etc.), insulin or a substance having substantially the same activity (for example, insulin, IGF (insulin-like growth factor) -1, IGF-2, etc.), FGF (fibroblast growth factor) or one having substantially the same activity (for example, aFGF, bFGF, KGF (Keratindcyte Growth Factor), HGF (Hepatocyte Growth Factor), FGF-10, etc.), other cell growth factors ( For example, CSF (colony stimulating factor), EPO (erythropoietin), IL-2 (interleukin-2), NGF (nerve growth factor), PDGF (platelet-derived growth factor), TGFβ (transforming grow th factorβ))), hormones (eg, luteinizing hormone releasing hormone (LH-RH), growth hormone, growth hormone releasing hormone (GH-RH), prolactin, melanocyte stimulating hormone, thyroid hormone releasing hormone, thyroid stimulating hormone, Luteinizing hormone, luteinizing hormone, follicle stimulating hormone, gastrin, motilin, somatostatin, secretin, glucagon, PACAP, VIP, etc., digestive enzymes (eg, amylase, pepsinogen, lipase, etc.), antibodies against pathogens (eg, pathogenic Salmonella, etc.) Antibodies against pathogenic bacteria, antibodies against pathogenic viruses such as influenza, antibodies against parasites such as echinocox), antibacterial polypeptides (eg, cecropin, histatin, indolicidin, protegrin, defensin, lysozyme) ) Useful gene product, etc., and the like.
Of the above target proteins,
(1) By expressing cytokines specifically in cartilage, for example, enhancement and regulation of immune activity of non-human warm-blooded animals can be achieved.
(2) By expressing the growth factor in a cartilage-specific manner, for example, protection of the cartilage tissue of a non-human warm-blooded animal can be achieved.
[0076]
The downstream of the promoter region of the gene encoding the polypeptide characterized by containing the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 12 or SEQ ID NO: 47 (3 ′ end side) Expressing the target protein (any useful gene product, etc.) specifically in the cartilage of a non-human warm-blooded animal by ligating DNA or RNA encoding the target protein (any useful gene product, etc.) and introducing it into a non-human animal The method of making it describe more specifically.
First, a promoter of a gene encoding a polypeptide containing the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 12 or SEQ ID NO: 47 is colony hybridization, plaque hybridization or PCR. And the like (for example, the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989)). Identification of a region having promoter activity can be obtained by a method known per se such as a reporter assay (for example, the method described in Analytical Biochemistry, 188, 245 (1990)). it can.
Next, in order to link the target protein (any useful gene product, etc.) downstream (3 ′ end side) of the promoter obtained by the above method, it is known per se for constructing a plasmid using T4 DNA ligase. The object can be achieved by a method (for example, the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989)).
In order to introduce a DNA encoding a target protein (any useful gene product, etc.) downstream of the promoter (3 ′ end side) into a non-human warm-blooded animal, a method using electroporation, a gene A method using a gun, a method using a retroviral vector (for example, the method described in Blood Cells, Vol. 17, 407 (1991)), a method using an adenoviral vector (for example, Pathology) , 30, 335 (1998), etc.).
[0077]
In the present specification and drawings, bases, amino acids, and the like are represented by abbreviations based on abbreviations by IUPAC-IUB Commission on Biochemical Nomenclature or conventional abbreviations in the field, examples of which are described below. In addition, when there is an optical isomer with respect to an amino acid, the L form is shown unless otherwise specified.

[0078]

[0079]
In addition, substituents, protecting groups and reagents frequently used in the present specification are represented by the following symbols.

[0080]
The sequence numbers in the sequence listing in the present specification indicate the following sequences.
[SEQ ID NO: 1]
The base sequence of the antisense strand primer used in Example 1 is shown.
[SEQ ID NO: 2]
The base sequence of the sense strand primer used in Example 1 is shown.
[SEQ ID NO: 4]
This shows the base sequence of DNA encoding the human MLP precursor having the amino acid sequence represented by SEQ ID NO: 6.
[SEQ ID NO: 5]
This shows the amino acid sequence of the signal sequence contained in the human MLP precursor having the amino acid sequence represented by SEQ ID NO: 6.
[SEQ ID NO: 6]
The amino acid sequence of a human MLP precursor is shown.
[SEQ ID NO: 7]
The base sequence of the antisense strand primer used in Example 2 is shown.
[SEQ ID NO: 8]
The base sequence of the sense strand primer used in Example 2 is shown.
[SEQ ID NO: 10]
This shows the base sequence of DNA encoding the mouse MLP precursor having the amino acid sequence represented by SEQ ID NO: 12.
[SEQ ID NO: 11]
This shows the amino acid sequence of the signal sequence contained in the mouse MLP precursor having the amino acid sequence represented by SEQ ID NO: 12.
[SEQ ID NO: 12]
The amino acid sequence of a mouse MLP precursor is shown.
[SEQ ID NO: 13]
The base sequence of G3PDH specific oligo DNA used in Example 3 is shown.
[SEQ ID NO: 14]
The base sequence of G3PDH specific oligo DNA used in Example 3 is shown.
[SEQ ID NO: 15]
The base sequence of the aggrecan-specific oligo DNA used in Example 3 is shown.
[SEQ ID NO: 16]
The base sequence of the aggrecan-specific oligo DNA used in Example 3 is shown.
[SEQ ID NO: 17]
The base sequence of the type II collagen-specific oligo DNA used in Example 3 is shown.
[SEQ ID NO: 18]
The base sequence of the type II collagen-specific oligo DNA used in Example 3 is shown.
[SEQ ID NO: 19]
The base sequence of the type X collagen specific oligo DNA used in Example 3 is shown.
[SEQ ID NO: 20]
The base sequence of the type X collagen specific oligo DNA used in Example 3 is shown.
[SEQ ID NO: 21]
The base sequence of mouse | mouth MLP specific oligo DNA used in Example 3 is shown.
[SEQ ID NO: 22]
The base sequence of mouse | mouth MLP specific oligo DNA used in Example 3 is shown.
[SEQ ID NO: 23]
This shows the base sequence of DNA encoding human MLP having the amino acid sequence represented by SEQ ID NO: 24.
[SEQ ID NO: 24]
The amino acid sequence of human MLP is shown.
[SEQ ID NO: 25]
This shows the base sequence of DNA encoding mouse MLP having the amino acid sequence represented by SEQ ID NO: 26.
[SEQ ID NO: 26]
The amino acid sequence of mouse MLP is shown.
[SEQ ID NO: 27]
The base sequence of the primer used in Example 4 and Example 6 is shown.
[SEQ ID NO: 28]
The base sequence of the primer used in Example 4 is shown.
[SEQ ID NO: 29]
The base sequence of the cDNA fragment obtained in Example 1 is shown.
[SEQ ID NO: 30]
The base sequence of the cDNA fragment obtained in Example 2 is shown.
[SEQ ID NO: 31]
The amino acid sequence of the synthetic peptide used in Example 6 is shown.
[SEQ ID NO: 32]
The base sequence of the oligo DNA used as a PCR primer in Example 6 is shown.
[SEQ ID NO: 33]
The base sequence of the oligo DNA used as a PCR primer in Example 6 is shown.
[SEQ ID NO: 34]
The base sequence of the oligo DNA used as a PCR primer in Example 6 is shown.
[SEQ ID NO: 35]
The base sequence of the oligo DNA used as a PCR primer in Example 6 is shown.
[SEQ ID NO: 36]
The base sequence of the oligo DNA used as a PCR primer in Example 6 is shown.
[SEQ ID NO: 37]
The base sequence of the oligo DNA used as a PCR primer in Example 6 is shown.
[SEQ ID NO: 38]
The base sequence of the oligo DNA used as a PCR primer in Example 6 is shown.
[SEQ ID NO: 39]
The partial amino acid sequence of the rat MLP precursor encoded by the DNA obtained in Example 9 is shown.
[SEQ ID NO: 40]
The base sequence of DNA which codes a part of rat MLP precursor obtained in Example 9 is shown.
[SEQ ID NO: 41]
The base sequence of DNA containing DNA which codes a part of rat MLP precursor obtained in Example 9 is shown.
[SEQ ID NO: 42]
The base sequence of the oligo DNA used as a PCR primer in Example 9 is shown.
[SEQ ID NO: 43]
The base sequence of the oligo DNA used as a PCR primer in Example 9 is shown.
[SEQ ID NO: 44]
The base sequence of the oligo DNA used as a PCR primer in Example 9 is shown.
[SEQ ID NO: 45]
The base sequence of the oligo DNA used as a PCR primer in Example 9 is shown.
[SEQ ID NO: 46]
This shows the base sequence of DNA encoding rat MLP precursor having the amino acid sequence represented by SEQ ID NO: 47.
[SEQ ID NO: 47]
The amino acid sequence of a rat MLP precursor is shown.
[SEQ ID NO: 48]
This shows the base sequence of DNA encoding rat MLP having the amino acid sequence represented by SEQ ID NO: 49.
[SEQ ID NO: 49]
The amino acid sequence of rat MLP is shown.
[SEQ ID NO: 50]
This shows the amino acid sequence of the signal sequence contained in the rat MLP precursor having the amino acid sequence represented by SEQ ID NO: 47.
[0081]
The transformant Escherichia coli XL10-Gold / pDRL128vH obtained in Example 1 described later has been deposited with the National Institute of Biotechnology, NIBH from June 11, 1999. No. FERM BP-6750, Deposit No. IFO to the Foundation Fermentation Research Institute (IFO) from June 25, 1999
Deposited as 16292.
The transformant Escherichia coli XL10-Gold / pDRL128vM obtained in Example 2, which will be described later, has been deposited with the National Institute of Biotechnology and Industrial Technology (NIBH), Ministry of International Trade and Industry from June 9, 1999. Deposit number IFO 16293 has been deposited with the Foundation Fermentation Research Institute (IFO) from June 25, 1999 under the number FERM BP-6747.
It has been deposited.
The transformant Escherichia coli XL10-Gold / pDRL128vR obtained in Example 9, which will be described later, has been deposited with the National Institute of Biotechnology, NIBH from May 19, 2000. No. FERM BP-7167, Deposit No. IFO to the Foundation Fermentation Research Institute (IFO) from May 26, 2000
Deposited as 16439.
【Example】
Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto. In addition, gene manipulation using E. coli was performed according to the method described in Molecular cloning.
[0082]
Example 1 Cloning of cDNA encoding human MLP precursor protein
Human fetal brain poly (A) ⁺ Using 5 'RACE (Rapid Amplification of cDNA End) and 3' RACE in the following manner, RNA was used to clone cDNA encoding human MLP precursor protein. Human fetal brain poly (A) ⁺ From 1 μg of RNA (Clontech), 1st.strand cDNA was synthesized using anchored primer with poly (T) following the restriction enzyme site and Superscript II MMLV reverse transcriptase (Gibco BRL), then RNA ligase (Takara Shuzo) ) Was used to add an anchored primer to the 3 ′ end of the 1st strand cDNA. Next, 5 ′ RACE was performed using the oligo DNA represented by SEQ ID NO: 1 as an antisense strand primer, and 3 ′ RACE was performed using the oligo DNA represented by SEQ ID NO: 2 as a sense strand primer. The 5 ′ upstream sequence and 3 ′ downstream sequence as starting points were obtained. When the base sequence of each double-stranded DNA obtained here was determined, it was found that both sequences were derived from the same gene because there was an overlapping common sequence. Therefore, the cDNA fragments obtained by 5 'RACE and 3' RACE were joined at this common sequence, and finally poly (A) ⁺ A cDNA fragment having a full length of 923 base pairs (bp) represented by SEQ ID NO: 29 including the chain was obtained. This cDNA fragment contains a novel human MLP precursor protein consisting of 128 amino acids represented by SEQ ID NO: 6, including a typical signal sequence of 18 amino acid residues represented by SEQ ID NO: 5. Had been. This human MLP precursor protein had the highest homology with the human MIA precursor protein, and the positions of the four cysteine residues were the same (Fig. 1), but the homology between them was 23.4 at the amino acid level. It was only%.
The plasmid pDRL128vH carrying the DNA encoding the human MLP precursor protein obtained in this example was introduced into Escherichia coli XL10-Gold, and the transformant: Escherichia coli XL10-Gold / pD RL128vH Got.
[0083]
Example 2 Cloning of cDNA encoding mouse MLP precursor protein
Cloning of the cDNA encoding the mouse MLP precursor protein was performed using mouse 17.5 day fetal poly (A) ⁺ RNA was used to perform 5 'RACE (Rapid Amplification of cDNA End) and 3' RACE in the same manner as the cloning of cDNA encoding human MLP precursor protein. After total RNA was fractionated from mouse 17.5 day fetus by guanidine thiocyanate method, this total RNA was applied to oligo (dT) span column (Pharmacia) and poly (A) ⁺ RNA was prepared. This mouse 17.5 days fetal poly (A) ⁺ 1st strand cDNA was synthesized from 1 μg of RNA using anchored primer with poly (T) following the restriction enzyme site and Superscript II MMLV reverse transcriptase (Gibco BRL), and then synthesized using RNA ligase (Takara Shuzo). An anchored primer was added to the 3 ′ end of the first strand cDNA. The primer sequences used for 5 ′ RACE and 3 ′ RACE were obtained from the public EST (Expressed Sequence Tag) database using the base sequence of the cDNA encoding the human MLP precursor protein obtained in Example 1 as a query. Based on the sequence of AA222797, the only EST that was found to contain the 3 'region of the cDNA encoding the mouse MLP precursor protein. 5 'RACE is performed using the oligo DNA represented by SEQ ID NO: 7 as an antisense strand primer and 3' RACE is performed using the oligo DNA represented by SEQ ID NO: 8 as a sense strand primer. An 'upstream sequence, 3' downstream sequence was obtained. When the base sequence of each double-stranded DNA obtained here was determined, it was found that both sequences were derived from the same gene because there was an overlapping common sequence. Therefore, the cDNA fragments obtained by 5'RACE and 3'RACE were joined at this common sequence, and finally poly (A) ⁺ A cDNA fragment having a full length of 947 bp including the strand was obtained (SEQ ID NO: 30). This cDNA fragment is a novel mouse consisting of 128 amino acids represented by SEQ ID NO: 12, including a typical signal sequence of 18 amino acid residues represented by SEQ ID NO: 11, as well as the human MLP precursor protein. The MLP precursor protein was coded. In this mouse MLP precursor protein, the positions of the cysteine residues present at four positions in the human and mouse MIA precursor proteins and the human MLP precursor protein all coincided (FIG. 1). In addition, the homology at the amino acid level between mouse MLP precursor protein and human MLP precursor protein reached 84.3%, but the homology between mouse MIA precursor protein and mouse MLP precursor protein was only 22.6%. .
The plasmid pDRL128vM carrying the DNA encoding the mouse MLP precursor protein obtained in this Example was introduced into Escherichia coli XL10-Gold, and a transformant: Escherichia coli XL10-Gold / pDRL128vM was obtained. Obtained.
[0084]
Example 3 Expression of MLP in an in vitro cartilage differentiation model
The mouse embryonic tumor-derived cell line ATDC5 retains the properties of progenitor chondrocytes very well, and induces cartilage differentiation at a high rate in culture in the presence of insulin, and then undergoes all stages of cartilage differentiation observed in bone formation. Since it can be simulated, it is used as an in vitro cartilage differentiation model. (Cell Diff. Dev., 30: 109-116, 1990, J. Cell Biol., 133: 457-468, 1996, J. Bone Min. Res., 10: S234, 1995). Therefore, RT-PCR was performed as follows to examine changes in the expression of various genes at each differentiation stage. First, from the ATDC5 cells at each stage of the differentiation model culture system, the cells are dissolved in a homogeneous liquid ISOGEN (Nippon Gene) containing phenol and guanidine thiocyanate, and chloroform is added to the mixture, followed by centrifugation, and an aqueous phase fraction containing RNA. Minutes were collected, further isopropanol was added, and the mixture was stirred and then centrifuged again to precipitate, thereby obtaining purified total RNA. Next, each test total RNA was reverse-transcribed using AMV Reverse Transcriptase XL and Random9mers in TAKARA RNA PCR Kit (AMV) Ver.2.1 (Takara Shuzo) to obtain cDNA, then used as template DNA, and house G3PDH (glyceraldehyde-3-phosphate dehydrogenase) specific oligo DNA (SEQ ID NO: 13, SEQ ID NO: 14) or cartilage differentiation marker gene specific oligo DNA (aggrecan (SEQ ID NO: 15, SEQ ID NO: 16), type II collagen (SEQ ID NO: 17, SEQ ID NO: 18), type X collagen (SEQ ID NO: 19, SEQ ID NO: 20)) are used as primer DNA, TaKaRa Ex Taq ^TM PCR reaction was carried out in a (Takara Shuzo) reaction system. The obtained reaction products were separated by agarose gel electrophoresis, and the amounts produced were compared. As a result, it was possible to detect the expression pattern shown in Table 1 corresponding to the differentiation stage of chondrocytes for each gene. Therefore, for the same group of cDNA samples, oligo DNA (SEQ ID NO: 21, SEQ ID NO: 22) specific for cDNA encoding mouse MLP precursor protein was used as a primer DNA, and other conditions were the same under the same reaction conditions. As a result of -PCR, a marked increase in the amount of mouse MLP precursor mRNA was observed from stage 2 to stage 4. From this, it was found that the gene of MLP precursor is a gene whose expression increases in the early stage of cartilage differentiation.
[Table 1]

(The number of + in the table represents the difference in the expression level of each gene at each differentiation stage, and the greater the number, the greater the expression level.)
[0085]
Example 4 Expression and detection of mouse MLP-FLAG fusion protein in COS7 cells
In order to confirm that MLP is a secreted protein, mouse MLP-FLAG fusion protein was expressed in COS7 cells and detected for mouse MLP as follows. First, based on the base sequence of the cDNA encoding the mouse MLP precursor polypeptide obtained in Example 2, two types of primer DNA were chemically synthesized. One is 5'-CGAATTCCCACCATGGCAAGGATATTGATTCTTTTGCTTG-3 '(SEQ ID NO: 27), which has an anchor sequence containing a restriction enzyme EcoRI recognition site on the 5' end side, +1 to +28 (translation start site is +1 and This is an oligo DNA containing the sense sequence up to The other is 5'-GTACAGTCGACTTCACAGAAGAAGTCAATATCCGTGGTTG-3 '(SEQ ID NO: 28), which is a sequence in which an antisense sequence from +355 to +378 is connected to the 3' side of the anchor sequence containing the restriction enzyme SalI recognition site. Is an oligo DNA having Using the plasmid pDRL128vM obtained in Example 2 as a template, these two kinds of primer DNA and TaKaRa LA Taq ^TM (Takara Shuzo), thermal cycler GeneAmp ^TM In PCR system 9700 (PerkinElmer), first place at 98 ° C for 30 seconds, then repeat 25 cycles of amplification reaction with 98 ° C for 10 seconds, 55 ° C for 20 seconds, 72 ° C for 2 minutes as one reaction cycle, Finally, an extension reaction was performed at 72 ° C. for 5 minutes. The resulting DNA fragment was purified, end-digested with restriction enzymes EcoRI and SalI, repurified, inserted into the EcoRI and SalI sites of the animal cell expression vector pCAN618FLAG, and ligated. pCAN618FLAG is derived from the plasmid vector pCAN618, has a neomycin resistance gene as a selection marker, and inserts a DNA fragment encoding the target protein into its cloning site, EcoRI and SalI sites, so that the very early gene of cytomegalovirus Not only can the protein be expressed under the control of an enhancer and its downstream β-actin promoter, but also an 8 amino acid FLAG epitope sequence (Asp-Tyr-Lys-Asp-Asp-Asp-Asp) immediately after the SalI site. It is also possible to express the target protein as a FLAG fusion protein by matching the reading frame to the base sequence encoding -Lys) and the stop codon. The above PCR cloning DNA fragment was also inserted into pCAN618FLAG in order to express a fusion protein of the entire mouse MLP precursor and the FLAG epitope (with one Val inserted between them). As a result, the expression vector plasmid pMMLP-F Got.
Next, COS7 cells 1.2 × 10 ^Five Cells are cultured for 24 hours in Dulbecco's modified minimal medium (DMEM) containing 10% fetal bovine serum (FBS) using a 6-well plate, and the above expression plasmid pMMLP-F (0.4 μg per well) is added to the cells. Was introduced using Lipofectamine (Gibco BRL). After 24 hours from the introduction, the medium was replaced with the above-mentioned new medium, and further 5 hours later, it was replaced with FBS-free Opti-MEM (Gibco BRL) and cultured for 36 hours to obtain the culture supernatant and cell extract. The cell extract was washed twice with phosphate buffer (PBS) containing physiological saline and then dissolved and extracted with Tris SDS sample buffer. On the other hand, the culture supernatant was concentrated appropriately by ultrafiltration (molecular weight 3000 cut). Later mixed with an equal volume of Tris SDS sample buffer. These samples were electrophoresed on a 15% -25% SDS-polyacrylamide gel after heat treatment, and further transferred from the gel onto a PVDF membrane (Amersham pharmacia biotech). Next, the PVDF membrane was blocked with Block Ace (Snow Brand Milk Products) for 1 hour, and reacted with anti-FLAG monoclonal antibody (10 μg / ml; Kodak) for 2 hours in PBS containing 0.05% Tween 20 (PBS-T). . After washing 3 times with PBS-T, the mixture was reacted with horseradish peroxidase-labeled anti-mouse IgG goat antibody (Amersham pharmacia biotech; diluted 5000 times) in PBS-T for 1 hour. After washing 5 times with PBS-T, chemiluminescence was detected using an ECLplus coloring kit (Amersham pharmacia biotech) and ECL film (Amersham pharmacia biotech). As a result, a gene product of about 14 kDa was detected in the culture supernatant together with the cell extract. This revealed that the mouse MLP-FLAG fusion protein was expressed and secreted in COS7 cells.
Therefore, next, the N-terminal amino acid sequence of the mouse MLP-FLAG fusion protein was determined. First, from the COS7 cell culture supernatant containing mouse MLP-FLAG expressed according to the above method, Anti-FLAG ^TM An acidic elution fraction (eluted with Glycine-HCl buffer (pH 3.5)) was obtained by affinity chromatography using M2-Agarose Affinity Gel (Sigma). After concentrating the fraction, SDS-polyacrylamide gel electrophoresis similar to that described in this example, followed by CBB staining, only a single band corresponding to the target protein mouse MLP-FLAG was observed. It was done. Thus, a concentrated sample of the same fraction was electrophoresed in the same manner, and the protein was transferred from the gel onto a PVDF membrane, and then subjected to a pulse liquid amino acid sequencer Procise CLC491 (PE Biosystems) to determine the N-terminal amino acid sequence. . As a result, amino acid residues of 1. histidine, 2. glycine, 3. valine, 4. phenylalanine were detected in order from the N terminus, and matched with the N terminal sequence of mouse MLP represented by SEQ ID NO: 26. From the above results, mouse MLP-FLAG protein is a mature mouse MLP-FLAG protein starting from the 19th histidine residue after cleaving the N-terminal 18 amino acid residue signal sequence of mouse MLP-FLAG precursor protein. It was found that it was secreted into the medium from COS7 cells.
[0086]
Example 5 Establishment of mouse MLP-FLAG fusion protein-expressing CHO-K1 cell line
A mouse MLP-FLAG fusion protein-expressing CHO-K1 cell line was established as follows.
CHO-K1 cells 3.3x10 ^Four The cells were cultured for 24 hours in an F-12 medium (Gibco BRL) containing 10% fetal bovine serum (FBS) on a 10 cm plastic dish, and the expression plasmid pMMLP- obtained in Example 4 was added to these cells. F (1.5 μg per petri dish) was introduced using the calcium phosphate method (CellPhect Transfection Kit (Amersham Pharmacia Biotech)). Twelve hours after introduction, the cells were washed twice with F-12 medium containing no FBS, and then subjected to glycerol shock for 3 minutes using 3 ml of isotonic HEPES solution (pH 7.5) containing 15% glycerol. Then, it was again washed twice with F-12 medium containing no FBS, and further cultured for 12 hours in F-12 medium containing FBS, followed by F-12 selective medium containing 500 mg / L Geneticin (Gibco BRL) and 10% FBS ( Hereinafter, the medium was replaced with a selective medium. Ten days later, Geneticin-resistant colonies formed in the petri dish were transferred to 24-well plates and cultured in a selective medium for 3 days. Next, after the cells grown in the selective medium were transferred to a 6-well plate and further cultured in the selective medium for 4 days, Opti-MEM containing 0.02% CHAPS and 0.1 mM p-ABSF (Wako Pure Chemical Industries, Ltd.) Gibco BRL) The medium was replaced with 1 ml, and the culture supernatant was recovered after further incubation for 48 hours. The obtained culture supernatant was concentrated using Centricon YM-3 ultrafiltration membrane (Amicon) and then mixed with an equal volume of Tris SDS sample buffer. This sample was heat-treated at 95 ° C. for 5 minutes, then electrophoresed on an 18% SDS-polyacrylamide gel, and the electrophoretic protein was transferred from the gel onto a nylon membrane. Next, the nylon membrane is blocked with Block Ace (Snow Brand Milk Products Co., Ltd.) for 1 hour and reacted with anti-FLAG antibody (1/2000 dilution, SIGMA) for 1 hour in PBS containing 0.05% Tween20 (PBS-T). It was. After washing 5 times with PBS-T, the mixture was reacted with HRP-labeled anti-mouse IgG sheep antibody (1/2000 dilution, Amersham Pharmacia Biotech) for 1 hour in PBS-T. After washing 5 times with PBS-T, chemiluminescence was detected using an ECL coloring kit (Amersham Pharmacia Biotech) and Hyperfilm ECL (Amersham Pharmacia Biotech). As a result, the largest amount of the target gene product of approximately 16 kDa was detected in the cell culture supernatant derived from the CHO-K1 / mMLP.FLAG # 2-1 strain. -Selected as K1 cell line.
[0087]
Example 6 Preparation of MLP antiserum and detection of recombinant MLP protein using the antiserum
Anti-MLP antiserum was prepared as follows. First, it is represented by SEQ ID NO: 31 in which one cysteine is further added to the C-terminus of the peptide chain corresponding to the amino acid sequence from the valine at the 105th residue to the aspartic acid at the 124th residue of the mouse MLP precursor protein. Known synthetic peptide (Val-Lys-Glu-Gln-Arg-Val-Tyr-Gln-Glu-Ala-Thr-Lys-Glu-Ile-Pro-Thr-Thr-Asp-Ile-Asp-Cys) Was chemically synthesized. Rabbits (SPF Japanese White Rabbit) were immunized with antigens obtained by binding KLH (keyhole limpet hemocyanin) as a carrier to this peptide. After a total of 7 immunizations, whole blood is collected, a serum fraction is obtained by a known method, sodium azide (final concentration 0.1%) is added as a preservative, and the sample is anti-MLP antiserum. It was.
Next, the reactivity of the antiserum to various recombinant proteins was examined by Western blot analysis. First, in addition to the mouse MLP-FLAG fusion protein described in Example 4, mouse MLP protein (no FLAG tag), human MLP-FLAG fusion protein, human MLP protein (no FLAG tag), mouse MIA-FLAG fusion protein, mouse Each expression vector plasmid of MIA protein (without FLAG tag) was constructed. As in Example 4, the construction was performed by inserting a DNA fragment of interest that had been previously PCR-cloned into the EcoRI and SalI sites, which are cloning sites of pCAN618FLAG. Each base sequence of the primer DNA set used for each PCR reaction is SEQ ID NO: 27 and SEQ ID NO: 32 for mouse MLP protein (without FLAG tag), and SEQ ID NO: 33 and SEQ ID NO for human MLP-FLAG fusion protein. : 34, for human MLP protein (without FLAG tag), SEQ ID NO: 33 and SEQ ID NO: 35, for mouse MIA-FLAG fusion protein, SEQ ID NO: 36 and SEQ ID NO: 37, for mouse MIA protein (without FLAG tag) Are base sequences represented by SEQ ID NO: 36 and SEQ ID NO: 38. As template DNA, mouse MLP protein (without FLAG tag) is pDRL128vM as in Example 4, and human MLP-FLAG fusion protein and human MLP protein (without FLAG tag) are pDRL128vH obtained in Example 1, For mouse MIA-FLAG fusion protein and mouse MIA protein (without FLAG tag), cDNA prepared from mouse melanoma cell line B16 was used. The introduction of each expression vector plasmid thus obtained into COS-7 cells, and Western blot analysis using an anti-FLAG antibody for each culture supernatant obtained were performed according to the method of Example 4. It was. In addition, Western blot analysis using anti-MLP antiserum was performed by using the antiserum (diluted 1000 times) as a primary antibody and horseradish peroxidase-labeled anti-rabbit IgG antibody (Amersham pharmacia biotech; 5000 times) as a secondary antibody. The method was performed according to the method for Western blot analysis using an anti-FLAG antibody except that (dilution) was used.
FIG. 2 shows the results of Western blot analysis using an anti-FLAG antibody, and FIG. 3 shows the results of Western blot analysis using an anti-MLP antiserum. The anti-MLP antiserum showed cross-reactivity to human MLP as well as mouse MLP, which is a protein derived from the antigen peptide, and its reactivity was hardly changed. In addition, mouse MLP-FLAG fusion protein and human MLP-FLAG fusion protein also reacted with the antiserum and were not affected by the presence of the FLAG tag. On the other hand, since no signal was detected for mouse MIA-FLAG fusion protein and mouse MIA protein (no FLAG tag), it was found that the antiserum was specific to MLP molecular species.
Furthermore, FIG. 4 shows the result of immunostaining according to a known method for the mouse MLP-FLAG-expressing CHO cell line obtained in Example 5 using the antiserum. In a control experiment using preimmune rabbit serum, each cell was not stained, but all cells were stained when the antiserum was used, indicating that the antiserum also reacts with native MLP protein. became.
[0088]
Example 7 Expression of MLP protein in cartilage tissue
Using the anti-MLP antiserum obtained in Example 6, detection of MLP protein in cartilage tissue was attempted. As the test sample, mouse (BALB / c) femoral head cartilage was frozen and crushed with liquid nitrogen, extracted with TRIS SDS βME SAMPLE BUFFER (Daiichi Kagaku), and centrifuged to remove the residue. . A sample equivalent to one mouse per lane was electrophoresed by SDS-PAGE (15-25%), and then subjected to Western blot analysis using anti-MLP antiserum in the same manner as in Example 6. As a result, since a signal was detected at almost the same migration position as that of the mouse MLP recombinant protein, it was revealed that the MLP protein was expressed in the cartilage tissue.
Further, in order to examine the expression of human MLP mRNA in various human tissues other than cartilage tissue, a DNA fragment encoding the human MLP precursor protein obtained in Example 1 and [α- ³² A probe was prepared by the method of Multiprime DNA labeling system (Amersham pharmacia biotech: RPN. 1601Y) using P] dCTP (Amersham pharmacia biotech: 6000 Ci / mmol), and the probe (specific activity 1.3 × 10 6). ^Ten cpm / μg), Human MTE ^TM Hybridization was performed on Array (CLONTECH: # 7775-1). Hybridization conditions were in accordance with the manual attached to the array membrane. Final washing was performed at 0.1 × SSC and 0.1% SDS at 55 ° C., and detection was performed using BAS-2000 (Fuji Film). As a result, the signals obtained on the array were slightly substantia nigra and fetal brain spots with the control human chromosomal DNA (100 ng, 500 ng), but both signals were near the detection limit, The expression level was judged to be extremely low at the transcription stage. Therefore, it was strongly suggested that MLP protein is specifically expressed in cartilage tissue.
[0089]
Example 8 Effect of MLP recombinant protein addition on various gene expression changes in in vitro cartilage differentiation model
In the in vitro cartilage differentiation model using ATDC5 cells described in Example 3, the effect of addition of MLP recombinant protein on various gene expression changes was examined. In the same manner as in Example 4, the MLP recombinant protein was purified and concentrated by performing affinity column chromatography using an anti-FLAG antibody from a COS-7 cell culture supernatant expressing mouse MLP-FLAG fusion protein by a known method. A standard was used. The protein was added to ATDC5 cells every 2 days from the first day of setting the model system, and the cells were collected after 10 days of culture. The expression of each gene was examined by the RT-PCR method as in Example 3. As a result, in the protein-added cell group, suppression of the expression of each marker gene such as aggrecan, type II collagen, type X collagen and the like whose expression level increases with differentiation was observed. On the other hand, the expression of PTH / PTHr receptor, which suppresses cartilage differentiation, was not significantly affected. From this, it was found that MLP protein has an inhibitory effect on cartilage differentiation in this model system using ATDC5.
[0090]
Example 9 Analysis of gene encoding rat MLP precursor protein (rMLP)
First, a DNA fragment encoding a part of rMLP was obtained by the following PCR method.
That is, 4 pmol each of the oligo DNA represented by SEQ ID NO: 21 as a sense strand primer and the oligo DNA represented by SEQ ID NO: 22 as an antisense strand primer, respectively, and 10 x Advantage ^TM 2 PCR Buffer (Clontech) 2 μl, 50 x dNTP mix (Clontech) 0.4 μl, 50 x Advantage 2 Polymerase Mix (Clontech) 0.4 μl, template DNA containing 2 μl of SD (IGS) rat pituitary cDNA solution Prepare 20 μl of the mixture and use a thermal cycler (GeneAmp ^TM PCR system model 9700 (Perkin Elmer)), 95 ° C., 1 minute, then 95 ° C., 10 seconds → 54 ° C., 10 seconds → 72 ° C., 1 minute repeated 35 cycles, then at 72 ° C. for 3 minutes A PCR reaction was performed using a program for extension reaction. After the reaction completed solution was electrophoresed on a 2.0% agarose gel, the gel was SYBR ^TM When stained with Green I nucleic acid gel stain (Molecular Probes), a band corresponding to DNA amplified by the PCR reaction was confirmed at a position near 300 bp in terms of molecular weight marker. In order to recover the DNA fragment and determine its base sequence using the Chiaquick Gel Extraction Kit (Qiagen) ^TM TA-cloning using 2.1-Topo (Invitrogen), the plasmid was Escherichia coli Epicurian Coli XL10-Gold ^TM It was introduced into a competent cell of the strain (Stratagene). From the ampicillin-resistant transformant colonies appearing on the ampicillin-containing LB agar medium, a clone that had retained the plasmid into which the foreign DNA fragment had been inserted was selected, and the plasmid DNA, pDRL128vR was prepared from the clone. . To determine the nucleotide sequence of the inserted DNA, pDRL128vR was used as a template DNA, and commercial primer DNA (Bca BEST Primer RV-P (Takara Shuzo Co., Ltd.)) was used as a sequence primer. ^TM The sequencer reaction using the BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (Perkin Elmer) was performed according to the conditions in the attached document. ^TM PCR system model 9700 (Perkin Elmer)), and then the reaction sample was DNA sequencer ABI PRISM ^TM 377 (PerkinElmer).
As a result, pDRL128vR contains a DNA fragment of 261 base pairs represented by SEQ ID NO: 40 encoding a part of a novel rat MLP precursor protein consisting of 87 amino acids represented by SEQ ID NO: 39. : A 307 base pair DNA fragment represented by 41 was contained.
Next, the gene encoding the protein was subjected to genome walking in order to examine the structure further 5 ′ upstream and 3 ′ downstream from the base sequence. Rat GenomeWalker as test material ^TM Kit (Clontech) is used, and the method is TaKaRa Ex Taq as an enzyme for PCR reaction. ^TM Except for the use of (Takara Shuzo), we followed the attachment of the kit. First, as a gene specific primer, two kinds of oligo DNAs (rMLPGW F1 (SEQ ID NO: 42), rMLPGWF2 (SEQ ID NO: 43)) corresponding to a part of the base sequence shown in SEQ ID NO: 40 and a part of the sequence Two oligo DNAs (rMLPGWR1 (SEQ ID NO: 44) and rMLPGWR2 (SEQ ID NO: 45)) having complementary sequences were chemically synthesized. These are rMLPGWF1 in the 1st PCR reaction for 3 ′ downstream DNA acquisition, rMLPGWF2 in the subsequent nested PCR reaction, rMLPGWR1 in the 1st PCR reaction for 5 ′ upstream DNA acquisition, and rMLPGWR2 in the subsequent nested PCR reaction. Using. With respect to each amplified DNA fragment obtained by these reactions, each base sequence was analyzed while comparing homology with the base sequences of cDNAs encoding human and mouse MLP precursor proteins starting from the above primer sequence site. As a result, from the revealed primary structure of the genome, the rat MLP precursor protein is a protein consisting of 128 amino acid residues represented by SEQ ID NO: 47 encoded by 384 base DNA represented by SEQ ID NO: 46. It became clear that there was. The homology at the amino acid level between rat MLP precursor protein and human MLP precursor protein and mouse MLP precursor protein reached 84.3% and 96.0%, respectively. The homology was only 26.5%. Rat MLP is a protein consisting of 110 amino acid residues represented by SEQ ID NO: 49 encoded by 330 base DNA represented by SEQ ID NO: 48: and represented by SEQ ID NO: 50 from rat MLP precursor protein. Like the mouse MLP, it is considered that a signal peptide consisting of 18 amino acid residues is processed and produced.
Plasmid pDRL128vR carrying DNA encoding part of the rat MLP precursor protein obtained in this example was introduced into Escherichia coli XL10-Gold, and transformant: Escherichia coli XL10- Gold / pDRL128vR was obtained.
[0091]
【The invention's effect】
The polypeptide of the present invention and the DNA encoding the same can be used, for example, for diagnosis, treatment, or prevention of bone / joint diseases and pathological angiogenesis. In addition, the polypeptide of the present invention is useful as a reagent for screening for compounds or salts thereof that promote or inhibit the activity of the polypeptide of the present invention. Furthermore, since the antibody against the polypeptide of the present invention can specifically recognize the polypeptide of the present invention, it can be used for detection, quantification, neutralization, etc. of the polypeptide of the present invention in a test solution. it can.
Furthermore, by using the promoter of the present invention, proteins (any useful gene product etc.) can be expressed predominantly (preferably specifically) in non-human warm-blooded animal cartilage and in large quantities, Can contribute to the field of gene therapy.
[0092]
[Sequence Listing]

[0093]
[Brief description of the drawings]
FIG. 1 Human MLP precursor (hMLP), mouse MLP precursor (mMLP), human MIA precursor (hMIA), mouse MIA precursor (mMIA), rat MIA precursor (rMIA) and bovine MIA precursor (bMIA) ) Amino acid sequence.
FIG. 2 shows the results of Western blot analysis performed in Example 6. Anti-FLAG antibody was used as the primary antibody.
In the figure, lane 1 is mouse MLP (no FLAG tag), lane 2 is mouse MLP-FLAG, lane 3 is mouse MIA (no FLAG tag), lane 4 is mouse MIA-FLAG, lane 5 is human MLP (no FLAG tag) ), Lane 6 shows a lane in which the culture supernatant of COS-7 cells into which mouse MLP-FLAG has been introduced are electrophoresed.
FIG. 3 shows the results of Western blot analysis performed in Example 6. Anti-MLP antiserum was used as the primary antibody.
In the figure, lane 1 is mouse MLP (no FLAG tag), lane 2 is mouse MLP-FLAG, lane 3 is mouse MIA (no FLAG tag), lane 4 is mouse MIA-FLAG, lane 5 is human MLP (no FLAG tag) ), Lane 6 shows a lane in which the culture supernatant of COS-7 cells into which mouse MLP-FLAG has been introduced are electrophoresed.
FIG. 4 shows the result of immunostaining performed in Example 6. The left panel shows the results of a control experiment using preimmune rabbit serum, and the right panel shows the results of an experiment using anti-MLP antiserum.

Claims (17)

SEQ ID NO: polypeptide comprising the amino acid sequence represented by 24, its amide or its ester, or salts thereof. SEQ ID NO: polypeptide consisting of the amino acid sequence represented by 6,
The amide or ester or salt thereof. SEQ ID NO: 26 polypeptide consisting of the amino acid sequence represented by, its amide or its ester, or salts thereof. SEQ ID NO: 12 polypeptide consisting of the amino acid sequence represented by, its amide or its ester, or salts thereof.   DNA containing DNA consisting of a base sequence encoding the polypeptide according to any one of claims 1 to 4.   6. The DNA according to claim 5, wherein the base sequence is the base sequence represented by SEQ ID NO: 23.   The DNA according to claim 5, wherein the base sequence is a base sequence represented by SEQ ID NO: 4.   The DNA according to claim 5, wherein the base sequence is the base sequence represented by SEQ ID NO: 25.   The DNA according to claim 5, wherein the base sequence is the base sequence represented by SEQ ID NO: 10.   A recombinant vector containing the DNA according to claim 5.   A transformant transformed with the recombinant vector according to claim 10.   The transformant according to claim 11 is cultured to produce the polypeptide according to any one of claims 1 to 4, the polypeptide according to any one of claims 1 to 4, its amide, or its A method for producing an ester or a salt thereof.   An antibody against the polypeptide according to any one of claims 1 to 4, an amide thereof, an ester thereof or a salt thereof.   The polypeptide according to any one of claims 1 to 4, an amide thereof, an ester thereof or a salt thereof is used, and the function of inhibiting the differentiation of cartilage of the polypeptide according to any one of claims 1 to 4 or a salt thereof is provided. A screening method for a compound or a salt thereof that promotes or inhibits.   The polypeptide according to any one of claims 1 to 4, or a salt thereof, comprising the polypeptide according to any one of claims 1 to 4, its amide, or an ester thereof, or a salt thereof, promoting a cartilage differentiation inhibiting function or A kit for screening an inhibitory compound or a salt thereof.   A composition comprising the polypeptide according to any one of claims 1 to 4, an amide thereof, an ester thereof or a salt thereof.   A composition comprising the antibody according to claim 13.

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