JP2012511309A - Ec−sodのカルボキシル末端のアポプチンタンパク質導入ドメインの融合蛋白質 - Google Patents
Ec−sodのカルボキシル末端のアポプチンタンパク質導入ドメインの融合蛋白質 Download PDFInfo
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Abstract
【課題を解決する手段】本発明はEC−SODのカルボキシル末端のアポプチンタンパク質導入ドメインの融合タンパク質を提供し、当該融合タンパク質は、SEQ ID NO.1に述べるEC−SODのカルボキシル末端のタンパク質導入ドメイン、もしくはその変異体のアミノ酸配列をSEQ ID NO.2に述べるアポプチン又はその変異体のアミノ酸配列と融合し、前記融合タンパク質は、腫瘍細胞のアポトーシスを誘導する極めて強い能力を有し、腫瘍を治療する薬物を作製することに用いられる。
【選択図】なし
Description
(a)SEQ ID NO.3に記載のアミノ酸配列を有するタンパク質;
(b)(a)のアミノ酸配列と少なくとも60%の相同性があり、またアポトーシスを誘導するタンパク質。
(1)EC−SODのカルボキシル末端のアポプチンタンパク質導入ドメインの融合タンパク質の発現ベクターの構築;
(2)EC−SODのカルボキシル末端のアポプチンタンパク質導入ドメインの融合タンパク質の形質転換体の作製;
(3)EC−SODのカルボキシル末端のアポプチンタンパク質導入ドメインの融合タンパク質の発現;
(4)EC−SODのカルボキシル末端のアポプチンタンパク質導入ドメインの融合タンパク質の精製。
本発明は、EC−SODのカルボキシル末端のアポプチンタンパク質導入ドメインの融合タンパク質をコードするDNA及びこれらのDNAを持つベクターや形質転換体を含む。
a.クローンされたEC−SODのカルボキシル末端のタンパク質導入ドメイン遺伝子を有する発現プラスミドC−pET28aの構築;
b.アポプチン遺伝子をクローン化しているクローン化プラスミドApop−pMD18Tの構築;
c.EC−SODのカルボキシル末端のアポプチンタンパク質導入ドメインの融合タンパク質遺伝子をクローン化している発現プラスミドApopC−pET28aの構築;
d.EC−SODのカルボキシル末端のアポプチンタンパク質導入ドメインの融合タンパク質の発現および精製;
本発明に記載する融合タンパク質は、各種の細胞の過度の増殖により引き起こされる疾患を治療する活性成分として用いることができ、例えば腫瘍:ユーイング肉腫、骨肉腫、軟骨肉腫等の骨癌;聴神経腫、神経芽細胞腫、神経膠腫及び他の脳腫瘍、脊髄腫瘍、乳癌、結腸直腸癌、進行大腸癌などの脳腫瘍と中枢神経系腫瘍;副腎皮質癌、膵癌、脳下垂体癌、甲状腺癌、副甲状腺癌、乳癌、多発性内分泌腫瘍などの内分泌腫瘍類;胃癌、食道癌、小腸癌、肝臓癌、肝臓外胆管癌、消化管カルチノイド、胆嚢癌などの消化管癌類;睾丸癌、陰茎癌、前立腺癌等の泌尿生殖器癌類;子宮頸癌、卵巣癌、膣癌、子宮/子宮内膜癌、陰部癌、妊娠性絨毛腫瘍、卵管癌、子宮肉腫等の婦人科癌類;口腔癌、口唇癌、唾液腺癌、喉頭癌、下咽頭癌、咽頭癌、鼻腔癌、副鼻腔癌、上咽頭癌等の頭頚部癌;小児白血病、急性リンパ芽球性白血病、急性骨髄性白血病、慢性リンパ性白血病、慢性骨髄性白血病、有毛状細胞性白血病、急性前骨髄球性白血病、プラズマ細胞性白血病等の白血病類;骨髄異形成症候群、骨髄増殖性疾患、再生不良性貧血、ファンコニ貧血、特発性マクログロブリン血症等の骨髄の癌類および血液の障害;肺小細胞癌、非小細胞肺癌等の肺癌類;ホッジゴールデン疾患、非ホジキンリンパ腫、皮膚T細胞リンパ腫、末梢T細胞リンパ球腫瘍、AIDS関連リンパ腫等のリンパ腫類;網膜芽細胞腫、ブドウ膜悪性黒色腫等の眼癌類;黒色腫、非黒色腫皮膚癌、メルケル細胞癌の皮膚癌類;小児軟組織肉腫、成人軟組織肉腫、カポジ肉腫等の軟組織肉腫類;腎臓ウィリアムズ癌、膀胱癌、尿道癌等の尿路系の癌、または転移癌等を含むが、これらに限定されるものではない。
「組成物」とは、本発明に用いられる組成物を言い、もしくは本発明の融合タンパク質を含む組成物を言う。通常、本発明の組成物が上述の用途に用いられる場合、前記融合タンパク質は、一種以上の薬理学的に許容される担体もしくは賦形剤と混合してもよく、異なる投与経路に用いる投与形態、例えば錠剤、カプセル剤、粉末、顆粒剤、シロップ、溶液、内服液、アルコール剤、チンキ、エアゾール、エアゾール粉剤、注射剤、注射用無菌粉末、坐薬などに製造するできる。
1.EC−SODカルボキシル末端タンパク質導入ドメイン遺伝子のPCR増幅
(1)Genbankで登録されているヒトEC−SODの遺伝子配列に従い、EC−SODカルボキシル末端タンパク質導入ドメイン遺伝子のサブクローンのフォワードとリバースプライマーを設計した:
P1:5’−TAATAAGCTTCCGCTGGAGGCGGTGGAAGCGGGCCCGGGCTC−3’
P2:5’−ATCTCGAGGGCGGCCTTGCACTCGCTCT−3’
フォワードプライマーP1の中に、一つのHindIII制限酵素サイトと8個のアミノ酸残基を有するリンケージペプチド(AlaSerAlaGlyGlyGlyGlySer)を挿入した;一方で、リバースプライマーP2の中に、一つのXhoI制限酵素サイトを導入した。
(2)PCR増幅の反応条件は:94℃において5分間変性させ、次に94℃において30秒間変性させ、さらに60℃において30秒間再生させ、その後72℃において1分間伸長させた。これらの工程を30回で繰り返し、最後72℃を5分間持続した。
(3)PCR産物を回収した後、HindIIIおよびXhoIの二種類の制限酵素で切断し、後で利用するために保存した。
PCR増幅および制限酵素切断により得られた挿入断片を、同じ酵素で加水分解により処理されたpET28aプラスミドの回収されたDNAと結合させた。結合した断片によりE.coli DH5α菌株を形質転換させ、培養した。組み換え体プラスミドDNAを回収し、配列を確認した。
1.アポプチン遺伝子のPCR増幅
(1)Genbankで登録されている鶏貧血ウイルスVP3タンパク質の遺伝子配列に従い、サブクローンアポプチン遺伝子のフォワードとリバースプライマーを設計した:P3:5’−GACATATGATGAACGCTCTCCAAGAAGA−3’
P4:5’−TGGGATCCTTACAGTCTTATACGCCTTTTTG−3’
フォワードプライマーP3の中に一つのNdeI制限酵素サイトを挿入し、リバースプライマーP2の中に一つのBamHI制限酵素サイトと終止コドンを挿入した。
(2)PCR増幅の反応条件は:94℃において5分間変性させ、次に94℃において30秒間変性させ、さらに55℃において30秒間再生させ、その後72℃において45秒間伸長させた。この工程を30回繰り返し、最後72℃の5分間持続した。
(3)PCR産物を回収し、後で利用するために保存した。
PCR増幅から回収した挿入断片を、pMD18Tベクター(タカラバイオ株式会社から購入した)DNAと結合させ、E.coliDH5α菌株を形質転換させ、培養した。液体培地で培養したものを青白検定により陽性クローンを選別した。また液体培地で培養した後、組換え体プラスミドDNAを回収し、配列を確認した。
1.アポプチン遺伝子の挿入断片の獲得
獲得し、シークエンシングにより確認されたApop−pMD18TプラスミドDNAをNdeIおよびBamHIの二種類の制限酵素で切断した。切断された断片は、1%アガロースゲルの電気泳動により分離し、ゲル抽出キットを用いて目的断片DNAを回収した。
構築、増幅により作製されたC−pET28aプラスミドのDNAを、同様にNdeIおよびBamHIの二種類の制限酵素で切断した。得られた切断断片は、1%アガロースゲルの電気泳動により分離し、ゲル抽出キットを用いて目的DNA断片を回収した。
回収したアポプチンのDNA断片をC−pET28aプラスミドDNAと結合させ、E.coli BL21(DE3)菌株を形質転換させて、37℃で培養した。プラスミドDNAを回収し、シークエンシングにより確認した。
1.ApopC−pET28aプラスミドを持つE.coli BL21(DE3)組み換え体合成バクテリアをカナマイシンが入ったLBアガープレートで培養した。クローンを一つ選び、37℃で50ug/mlのカナマイシンを含有するLB培地で、一晩培養した。次に、この培養液を、1%の接種量を使って新しい50ug/mlのカナマイシンを含むLB培地に移し、OD600= 0.3〜0.6に到達するまで37℃で培養した。導入のために0.5〜3mMのIPTG加え、3〜5時間、37℃で引き続き培養した。バクテリアを回収して、超音波処理し、遠心分離にて封入体を収集した。
1.HeLa細胞を96穴の細胞培養プレートで細胞密度が60%程度になるまで37℃で培養した。発現した後に分離および精製して得た組み換え体アポプチン−EC−SOD−PTDサンプルを繰り返し透析し、イミダゾールを除去した。滅菌濾過を行った後、異なる量のDMEM細胞培地を加えてタンパク質を希釈した。最も高いタンパク質濃度から濃度を半減していき、タンパク質濃度がゼロになるまで段階的に希釈した。そして、タンパク質を含まない溶液をネガティブコントロールとし、各タンパク質濃度を3〜4個のウェルに取って、並列コントロールとした。同時に、類似する方法を採用して、同様の方法で分離、精製した原核細胞の発現システムから得られた組み換え体アポプチンタンパク質をコントロールとして同様の実験操作を行った。2セットのサンプルを24時間培養し、培地を除去し、100μlの5ug/mlのMTTを含有するDMEMを加え、37℃において4時間培養し、培地を除去し、100μlのDMSOを加えで紫の結晶を溶解し、37℃において10分間培養し、マイクロプレートリーダーを使って、490nmでの吸光度を測定した。組み換え体アポプチン−EC−SOD−PTD濃度が100μg/mlの場合、HeLa細胞の生存率は47%(IC50=105μg/ml)であった。ただし、同一の条件下における対照群の組み換え体アポプチン(100μg/ml)とネガティブコントロールとしての緩衝液は、HeLa細胞の生長に及ぼす影響が著しくなかった。
In vitroで培養した正常なヒト肝臓セルラインL02に対しての組み換え体アポプチン−EC−SOD−PTDのIC50をMTTで測定し、投与量と細胞毒性の関係を観察した。
1.正常なヒト肝臓セルラインL02をRPMI1640培地(10%ウシ胎児血清を含む)で培養した。膵酵素で分解した後、培養細胞は、1×105/mlの細胞濃度の単個細胞浮遊液を1640培養液で作成した。96穴の細胞培養プレートに100μl接種した。37℃、5%CO2のインキュベーターで4時間培養した。
組み換え体アポプチン−EC−SOD−PTDの腫瘍抑制をC57BL/6マウスLewis肺癌モデルを使って証明した。ブランクコントロール、ポジティブコントロール、組み換え体アポプチン−EC−SOD−PTD試験グループ、組み換え体ポリアルギニン−アポプチン試験グループ(Poly Arg−Apoptin)の四つのグループを設け、各グループに対して8匹の動物をテストした。腫瘍を接種した後に、ブランクコントロールに生理食塩水を腹腔注入した。一方で、ポジティブコントロールには、シクロホスファミド溶液(20mg/kgBW/日)を注射した。この二つの試験グループに、それぞれに精製、透析、滅菌濾過された組み換え体アポプチン−EC−SOD−PTD(10mg/kgBW/日)と組み換え体Poly Arg−アポプチン(40mg/kgBW/日)を注射した。
腫瘍への抑制率=(ブランクコントロールの腫瘍の平均重量−試験グループの腫瘍の平均重量)/ブランクコントロールの腫瘍の平均重量X100%
Claims (19)
- EC−SODのカルボキシル末端のアポプチンタンパク質導入ドメインの融合タンパク質であって、SEQ ID NO.1のアミノ酸配列もしくはその変異体の中にEC−SODカルボキシル末端のタンパク質導入ドメインと、アミノ酸配列SEQ ID NO.2またはその変異体におけるアポプチンとを有することを特徴とするEC−SODのカルボキシル末端のアポプチンタンパク質導入ドメインの融合タンパク質。
- 請求項1に記載のEC−SODのカルボキシル末端のアポプチンタンパク質導入ドメインの融合タンパク質であって、前記融合タンパク質は、式:R2−R1、R1−R2、R1−L−R2−L−R1、R1−L−R2、R2−L−R1もしくはR2−L−R1−L−R1を含み、R1は、SEQ ID NO.1もしくはその変異体のアミノ酸配列の中のEC−SODカルボキシル末端のタンパク質入ドメインであり、Lは、リンケージペプチドであり、R2は、SEQ ID NO.2に述べるアミノ酸もしくはその変異体の少なくとも一つを有するアポプチンであることを特徴とするEC−SODのカルボキシル末端のアポプチンタンパク質導入ドメインの融合タンパク質。
- 請求項1に記載のEC−SODのカルボキシル末端のアポプチンタンパク質導入ドメインの融合タンパク質であって、前記EC−SODカルボキシル末端のタンパク質導入ドメインのSEQ ID NO.1に述べるアミノ酸配列もしくはその変異体は、前記融合タンパク質のカルボキシル末端に位置することを特徴とするEC−SODのカルボキシル末端のアポプチンタンパク質導入ドメインの融合タンパク質。
- 請求項1に記載のアEC−SODのカルボキシル末端のアポプチンタンパク質導入ドメインの融合タンパク質であって、前記EC−SODのカルボキシル末端のアポプチンタンパク質導入ドメインの融合タンパク質は、次の(a)もしくは(b)のタンパク質であることを特徴とする:
(a)アミノ酸配列が例えばSEQ ID NO.3に述べるようなタンパク質;
(b)(a)のアミノ酸配列と少なくとも60%の相同性を有し、またアポトーシス作用を誘導する活性があるタンパク質。 - 請求項1〜4のいずれか1つに記載のEC−SODのカルボキシル末端のアポプチンタンパク質導入ドメインの融合タンパク質をコードするポリヌクレオチド分子。
- 請求項5に記載のポリヌクレオチド分子であって、SEQ ID NO.4に述べる核酸配列を有するポリヌクレオチド分子。
- 請求項5に記載のポリヌクレオチド分子を含む組み換え体発現ベクター。
- 請求項7に記載の組み換え体発現ベクターであって、当該ポリヌクレオチド分子の核酸配列がSEQ ID NO.4に記載のものであることを特徴とする発現ベクター。
- 請求項7に記載の組み換え体発現ベクターを有する形質転換体。
- 請求項9に記載の形質転換体であって、前記形質転換体が大腸菌であることを特徴とする形質転換体。
- 請求項1〜4のいずれか1つに記載のアEC−SODのカルボキシル末端のアポプチンタンパク質導入ドメインの融合タンパク質の製造方法であって、下記の工程を含む:
(1)請求項1に記載の前記EC−SODのカルボキシル末端のアポプチンタンパク質導入ドメインの融合タンパク質の発現ベクターの構築;
(2)請求項1に記載のEC−SODのカルボキシル末端のアポプチンタンパク質導入ドメインの融合タンパク質の形質転換体の作製;
(3)請求項1に記載のEC−SODのカルボキシル末端のアポプチンタンパク質導入ドメインの融合タンパク質の発現;
(4)請求項1に記載のEC−SODのカルボキシル末端のアポプチンタンパク質導入ドメインの融合タンパク質の精製。 - 請求項11に記載の製造方法であって、前記アEC−SODのカルボキシル末端のアポプチンタンパク質導入ドメインの融合タンパク質の形質転換体が、原核遺伝子発現システムもしくは真核遺伝子発現システムであることを特徴とする方法。
- 請求項1〜4のいずれか1つに記載のEC−SODのカルボキシル末端のアポプチンタンパク質導入ドメインの融合タンパク質を有し、薬理学的に許容される担体を含むことを特徴とする組成物。
- 細胞の過度の増殖により引き起こされる疾患の治療薬として請求項1〜4のいずれか1つに記載のEC−SODのカルボキシル末端のアポプチンタンパク質導入ドメインの融合タンパク質を使用する使用方法。
- 請求項14に記載の使用方法であって、前記過度の細胞の増殖により引き起こされる疾患が腫瘍であることを特徴とする使用方法。
- 請求項15に記載の使用方法であって、前記腫瘍が子宮頸癌、肝臓癌もしくは肺癌からなる群のいずれか一つであることを特徴とする使用方法。
- 過度の細胞の増殖により引き起こされる疾患や障害を予防、治療もしくは改善する方法であって、前記方法は、請求項1〜4のいずれか1つに記載されたEC−SODのカルボキシル末端のアポプチンタンパク質導入ドメインの融合タンパク質を哺乳動物へ効果的な量で投与することを特徴とする方法。
- 請求項17に記載の方法であって、前記過度の細胞の増殖により引き起こされた疾患が腫瘍であることを特徴とする方法。
- 請求項18に記載の方法であって、前記腫瘍は、子宮頸癌、肝臓がん、もしくは肺癌であることを特徴とする方法。
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- 2009-04-07 WO PCT/CN2009/000379 patent/WO2010066090A1/zh active Application Filing
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KR20190033914A (ko) * | 2017-09-22 | 2019-04-01 | 가톨릭대학교 산학협력단 | N 말단 또는 c 말단의 알부민 접합을 이용한 세포외 분비 슈퍼옥사이드 디스뮤테이즈(ec-sod) 단백질의 안정화 방법 |
KR102223576B1 (ko) * | 2017-09-22 | 2021-03-05 | 가톨릭대학교 산학협력단 | N 말단 또는 c 말단의 알부민 접합을 이용한 세포외 분비 슈퍼옥사이드 디스뮤테이즈(ec-sod) 단백질의 안정화 방법 |
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CN101747437A (zh) | 2010-06-23 |
EP2380914A4 (en) | 2013-02-27 |
WO2010066090A1 (zh) | 2010-06-17 |
CN101747437B (zh) | 2012-09-26 |
EP2380914A1 (en) | 2011-10-26 |
US20110230421A1 (en) | 2011-09-22 |
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