JP4676484B2 - 臨床応用のための融合タンパク質の塩基配列決定及び遺伝子発現 - Google Patents
臨床応用のための融合タンパク質の塩基配列決定及び遺伝子発現 Download PDFInfo
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- JP4676484B2 JP4676484B2 JP2007502169A JP2007502169A JP4676484B2 JP 4676484 B2 JP4676484 B2 JP 4676484B2 JP 2007502169 A JP2007502169 A JP 2007502169A JP 2007502169 A JP2007502169 A JP 2007502169A JP 4676484 B2 JP4676484 B2 JP 4676484B2
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
本発明で使用される融合タンパク質は、FP4と称され、配列番号2のアミノ酸配列を有する機能タンパク質若しくは配列番号2から由来のタンパク質、又は配列番号2のアミノ酸配列のアミノ酸を一つ以上増減することによって得られるすべての置換物である。
1)配列番号1:DNA配列
2)配列番号2:アミノ酸配列
3)配列番号1で表されるDNA配列と95%を超える相同性を有し、且つ、同じ機能タンパク質配列をコードするDNA配列。
配列1のDNA配列は、1665塩基対からなる。読み枠は、5末端で始まる1番目の塩基対から1665番目の塩基対までである。
FP4の一対のプライマーでの増幅もまた、本発明の保護の拡張で保護される。
本発明で導入されるFP4の発現方法は、融合タンパク質FP4を発現する陽性クローンを得るためにFP4遺伝子をSP2/0細胞にトランスフォーメーションすることである。
発現ベクターは、通常の方法を使用して構築され、FP4遺伝子がpSecTagベクターにクローニングされる。
必要に応じて、上記の薬に1つ又は複数のキャリアを加えることができる。これらのキャリアは、薬学的な仕様の希釈剤、賦形剤、助剤、結合剤、湿潤剤、粉末化医薬製剤、吸収促進剤、界面活性剤、吸収キャリア、及び潤滑剤等が挙げられる。必要であれば、また、香料添加剤、及び甘味剤も添加可能である。
上記の全ての薬は、概して、用量が0.01〜1ug/kg/日、及び治療期間が10〜20日間である。
図2に示すように、融合タンパク質FP4の発現方法は、以下の工程を含む。ヒト末梢血からのBリンパ球を精製する工程;及び、ゲノムDNAを特異的に直接分離して以下の通りに通常の処理を行う工程。
P1(Fcε):5’-GTGGCCCAGCCGGCCTTCACCCCGCCCACCGTGAAG-3’;
P2(Fcε):5’-GTGGATCCTTTACCGGGATTTACAGACAC-3’;
P3(Fcγ):5’-GGGGATCCGAGCCCAAATCTTGTGAC-3’;
P4(Fcγ):5’-GTGCGGCCGCTCATTTACCCGGAGACAGGGAGAG-3’。
ヌクレオチド配列が正しいことを確認した後、Fcε遺伝子をSfiI−BamHI(NEW ENGLAND BIOLABS)で切断した。Fcγ遺伝子はBamHI−NotIで切断した。その間にpSecTag発現ベクター(インビトロジェン カリフォルニア州)をSfiI−NotIで切断し、Fcε及びFcγをpSecTagFP4にライゲーションした。挿入配列は図1と同じであることを確認した。pSecTagFP4ベクターを、マウス骨髄腫SP2/0細胞にエレクトロポレーションによりトランスフェクションした。ゼオシン(R)で選別後、陽性クローンを酵素免疫測定法(ELISA)によりスクリーニングした(マウス抗ヒトIgEでプレートをコーティングし、クローンの上清を添加し、そしてヤギ抗ヒトIgE−APコンジュゲートを添加する)。FP4タンパク質を高度に発現する陽性クローンを得た。
1×106個のCHO3D10細胞を、精製したFP45μgと4℃で1時間インキュベートし、FITCラベル抗ヒトIgE(カルタグ カリフォルニア)5μlを加え、フローサイトメトリーで分析した。図4に結果を示した。FP4タンパク質はFcεRIレセプターに結合し、CHO3D10のFITC陽性を示す。図4において、SSC−Hは、細胞質の粒状度、FSC−Hは細胞の大きさ、FL2−HはPE、及びFL1−HはFITCである。
1×106個のHMC‐1細胞を、精製したFP4タンパク質5μgと4℃で1時間インキュベートし、FITCラベル抗ヒトIgG(カルタグ カリフォルニア州)5μlを加え、フローサイトメトリーで分析した。結果はFP4タンパク質がFcγRIIレセプターに結合した。HMC‐1細胞はFITC陽性を示し(図5)、SSC−Hは、細胞質の粒状度、FSC−Hは細胞の大きさ、FL2−HはPE、及びFL1−HはFITCである。
ヒト末梢血からのヒト好塩基球を分離精製した。精製した好塩基球(1×106)を、特異的ヒト抗NP−IgE(セロテック)1〜10μgと37℃で2時間インキュベートし、NP−BSAを2μg加えた。ヒスタミン放出をELISAで測定した。結果は、FP4タンパク質が用量依存的にヒト好塩基球でのヒスタミン放出を有意に阻害した(図6)。Hu IgEは、ヒトIgE、NS IgEは非特異的ヒトIgE、NP−BSAはアレルゲン、+は付加、−は付加なしを表す。
ヒトFcεRIα鎖トランスジェニックマウス(カリフォルニア大学サンディエゴ校)に、ヒト抗NP−IgE(セロテック)を皮内注射し、4時間後にNP−BSA 100μg+Evans blue(シグマ)染料1%を静脈注射した。アレルギー反応が起これば、血管から染料が漏れるため皮膚が局所的に青く変色する。同量の融合タンパク質(FP4)を加えた場合、アレルギー反応は完全に阻止され、皮膚が局所的に青く変色しなかった(図6)。この実験結果により、FP4タンパク質がin vivoでアレルギー反応が阻止することが更にわかった。
Claims (10)
- 配列番号2で表されるアミノ酸配列を有するタンパク質及び、
前記配列番号2で表されるアミノ酸配列に由来し、且つ、前記配列番号2で表されるアミノ酸配列を有するタンパク質と同じ抗アレルギー活性を有し、且つ、前記配列番号2で表されるアミノ酸配列に対する一つ以上のアミノ酸残基の置換、欠失、又は付加によって生成されるタンパク質
のいずれかであることを特徴とする融合タンパク質。 - 配列番号2で表されるアミノ酸配列を有する請求項1に記載の融合タンパク質。
- 融合タンパク質コード遺伝子であって、
1)配列番号1で表されるDNA配列、
2)配列番号2で表されるアミノ酸配列を有するタンパク質をコードするDNA配列、及び
3)配列番号1で表されるDNA配列と90%を超える同一性を有し、且つ、前記配列番号1で表されるDNA配列にコードされるタンパク質と同じ抗アレルギー活性を有するタンパク質をコードするDNAのDNA配列
のいずれか1つのDNA配列を有することを特徴とする融合タンパク質コード遺伝子。 - 前記融合タンパク質コード遺伝子が、配列番号1で表されるDNA配列からなる遺伝子であることを特徴とする請求項3に記載の融合タンパク質コード遺伝子。
- 請求項3に記載の融合タンパク質コード遺伝子を含有する、発現ベクター及び細胞株。
- 請求項3に記載の融合タンパク質コード遺伝子を増幅するための一対のプライマー。
- 請求項1に記載の融合タンパク質を発現させる方法であって、
融合タンパク質コード遺伝子を含む発現ベクターをSP2/0細胞にトランスフォーメーションして陽性クローンを得る工程と、
該陽性クローンを培養して融合タンパク質FP4を発現させる工程と
を含み、
該融合タンパク質コード遺伝子は
1)配列番号1で表されるDNA配列、
2)配列番号2で表されるアミノ酸配列を有するタンパク質をコードするDNA配列、及び
3)配列番号1で表されるDNA配列と90%を超える同一性を有し、且つ、前記配列番号1で表されるDNA配列にコードされたタンパク質と同じ抗アレルギー活性を有するタンパク質をコードするDNAのDNA配列
のいずれか1つのDNA配列を有することを特徴とする融合タンパク質を発現させる方法。 - 前記融合タンパク質コード遺伝子を含む発現ベクターが、発現ベクターpSecTagへの前記融合タンパク質コード遺伝子のライゲーションによって得られる請求項7に記載の方法。
- 請求項1に記載の融合タンパク質を有効成分とした抗アレルギー薬。
- 抗アレルギー治療用薬剤の調製への請求項1に記載の融合タンパク質の使用。
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PCT/CN2004/000449 WO2005085291A1 (fr) | 2004-03-10 | 2004-05-08 | Proteine fusionnee, gene codant pour elle, sa methode d'expression et ses utilisations |
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JP5854432B2 (ja) * | 2012-09-28 | 2016-02-09 | 森永乳業株式会社 | アレルギー反応検出法 |
US8961992B1 (en) * | 2014-04-02 | 2015-02-24 | Tunitas Therapeutics, Inc. | Epsigam fusion protein |
US10293059B2 (en) * | 2015-04-09 | 2019-05-21 | Cornell University | Gene therapy to prevent reactions to allergens |
CN104922670A (zh) * | 2015-05-06 | 2015-09-23 | 武汉奥斯梅得生物医药有限公司 | 融合免疫蛋白在制备治疗多发性硬化症药物中的应用 |
CN107383200B (zh) * | 2016-05-17 | 2021-04-09 | 深圳先进技术研究院 | 鼠源抗人IgE单克隆抗体的制备方法及其应用 |
CN111454366B (zh) * | 2019-01-21 | 2023-06-16 | 中国科学院深圳先进技术研究院 | 一种融合蛋白及其应用 |
CN111574608B (zh) * | 2020-04-21 | 2021-11-19 | 沈阳农业大学 | 牛细粒棘球蚴病的特异性检测抗原及其应用 |
CN112023062A (zh) * | 2020-09-18 | 2020-12-04 | 北京基因安科技有限公司 | 一个用可溶性IgE受体抑制过敏反应的方法 |
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AU2004316866A1 (en) | 2005-09-15 |
US7544783B2 (en) | 2009-06-09 |
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