JP4599354B2 - 大腸菌熱不安定性エンテロトキシンサブユニットに融合した志賀毒素サブユニットまたは志賀毒素様毒素サブユニットを含むハイブリッド毒素およびそのワクチン - Google Patents
大腸菌熱不安定性エンテロトキシンサブユニットに融合した志賀毒素サブユニットまたは志賀毒素様毒素サブユニットを含むハイブリッド毒素およびそのワクチン Download PDFInfo
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- JP4599354B2 JP4599354B2 JP2006520828A JP2006520828A JP4599354B2 JP 4599354 B2 JP4599354 B2 JP 4599354B2 JP 2006520828 A JP2006520828 A JP 2006520828A JP 2006520828 A JP2006520828 A JP 2006520828A JP 4599354 B2 JP4599354 B2 JP 4599354B2
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Description
細菌株およびプラスミド
大腸菌(E.coli)宿主株BL21(DE3)star、HMS174(DE3)およびBL21コドン+RIL(DE3)をNovagen(Madison,Wisconsin,USA)から購入した。大腸菌(E.coli)株TOP1OF’ならびにプラスミドpCR2.1−TOPO TAおよびpCR−bluntII−TOPOをInvitrogen(Groningen,the Netherlands)から購入した。プラスミドpMMB66HEはFurste,J.P.ら,Gene 48:119−131(1986)に記載されている。
大腸菌(E.coli)染色体DNA上でのPCRをSupertaq plus DNAポリメラーゼで行った。該PCR混合物は、20U/ml Supertaq plus(HT Biotechnology Ltd,Cambridge,UK)、8mM dNTP(Promega,Wisconsin,USA)、10pmolのプライマーおよびDNA鋳型としての大腸菌(E.coli)の15ngの染色体DNAを含有するSupertaqバッファー(HT Biotechnology Ltd,Cambridge,UK)を含有していた。DNAの増幅に使用する全てのプライマーのオリゴヌクレオチド配列を表1に示す。PCR産物をアガロースゲル上で分離し、Qiagen PCR精製キット(Qiagen Inc.,California,USA)を使用してゲル精製した。Sambrookら(Maniatis/Sambrook(Sambrook,J.Molecular cloning:a laboratory manual,1989.ISBN 0−87969−309−6))に記載されているとおりに、重複伸長PCRを行った。TOPOクローニングキット(Invitrogen.,Groningen,the Netherlands)を使用して、PCR産物をpCR−bluntII−topo内にクローニングした。クローニング反応は、製造業者の指示に従い行った。
高忠実度ポリメラーゼを使用し、鋳型としてのEDNL50染色体DNAと共にプライマー#1832および#1833(表1を参照されたい)を使用するPCRにより、Stx2eA1を増幅した。離乳後水腫疾患と診断されたブタからEDNL50を単離した。これに関しては、志賀毒素様毒素を産生する任意の他の株も同様に使用可能であったであろう。高忠実度ポリメラーゼを使用し、鋳型としてのプラスミドpMMB66−LTと共にプライマー#1834および#1835(表1を参照されたい)を使用して、ジスルフィド架橋を含むLTA2LTBを増幅した。これに関しては、この場合にも、LTを産生する任意の他の株も同様に使用可能であったであろう。プライマー#1832および#1835を使用して得た重複伸長PCR産物においては、1マイクロリットルの各PCR液を使用した。得られたPCR産物およびpMMB66HEをPstIおよびBamHIで消化し、ついで連結して、pMMB Stx2eA1LTA2Bを得た。該プラスミドをヌクレオチド配列分析により調べたところ、人工産物は見出されなかった。図2は、pMMB Stx2eA1LTA2Bの構築スキームを示す。
組換えタンパク質の発現
tacプロモーターに基づく発現ベクターを含有する大腸菌(E.coli)発現株を、適当な抗生物質および10mM MgSO4を含有する5mlのTB中、37℃、200rpmで一晩増殖させた。翌朝、該一晩培養物を、適当な抗生物質を含有する5mlのTBで1:100希釈した。NovaspecII分光光度計(Pharmacia,Woerden,the Netherlands)による測定でOD600が0.5に達するまで、これらの培養物を同一条件下で成長させた。この時点で、該培養物を、最終濃度1mMまでのIPTGの添加により誘導し、ついで更に37℃で3時間インキュベートした。適当な対照の及び最終インキュベーションの開始時および終了時に、分析のために、100μlのサンプルを採取した。該サンプルをSDS PAGEおよびそれに続くクーマシーブリリアントブルー染色により分析した。残りの培養物を5,000rpmで遠心分離し、ペレットを更なる使用まで−20℃で保存した。
NuPAGE電気泳動系(Novex,San Diego,USA)からの4〜12% Bis−Trisゲルを使用して、SDS−PAGEを行った。変性タンパク質プロフィールを得るために、分離前に、サンプルを、β−メルカプトエタノールの存在下、サンプルバッファー(サンプル:バッファー=2:1)と共に5分間煮沸した。非変性タンパク質の分離のためには、β−メルカプトエタノールを含有しないサンプルバッファーをサンプルに加えた。これらのサンプルは、加熱することなくゲル上にローディングした。ゲルをクーマシーブリリアントブルーで染色し、または標準的な半乾燥ウエスタンブロット法によりImmobulon−P−メンブレン(Millipore,Bedford,USA)上にブロットした。ウサギ抗LTポリクローナルα0508/09HRPおよびウサギ抗LTポリクローナルα0506/07をホルマリン不活化LTに対して産生させた。抗LT−Aモノクローナル抗体をBiotrend(Koln,Germany)から購入した。陽性対照として使用したLT(K8425)は製造バッチからのものであった。該LTは、培養上清から精製されたガラクトース−シリカであった。溶出に使用したガラクトースは透析により除去した。最終産物は156mg/lのLTを含有していた。
誘導された5mlの培養物をデューティーサイクル50%およびマイクロティップ(microtip)で超音波処理(Branson sonifier,Geneva,Switzerland)して完全に細胞溶解した。ライセートを6,000rpmで5分間遠心分離して不溶性タンパク質を除去した。清澄化上清を1ml ガラクトース−シリカカラムに適用した。カラム物質はOrganon(Oss,the Netherlands)により供給されたものである。このカラムを10容量のTEANバッファー(50mM Tris,1mM EDTA,3mM Na−アジド,200mM NaCl,pH 7.5)で予め平衡化した。該上清の結合後、該カラムを5容量のTEANバッファーで洗浄した。精製タンパク質を0.5M ガラクトースで溶出し、更なる使用まで4℃で保存した。
Stx2eA1LTA2B融合タンパク質の発現
3つの大腸菌(E.coli)発現株を該融合タンパク質の発現に関して試験した。構築物pMMB Stx2eA1LTA2BをBl21star(DE3)、HMS174(DE3)およびJA221に導入し、記載されているとおりに誘導した。発現株Bl21star(DE3)が最高発現レベルを示した(データ非表示)。
前記のSDS−PAAGE−ゲルを標準的な半乾燥ウエスタンブロット法によりImmobulon−P−メンブレン(Millipore,Bedford,USA)上にブロットした。該ブロットを現像するためのウサギ抗LTポリクローナルα0506/07は、ホルマリン不活化LTに対して産生させた。陽性対照として使用したLTは、アフィニティークロマトグラフィー(ガラクトース−シリカ)を用いて培養上清から精製したものであった。図3、レーン2から理解されうるとおり、両方のLTサブユニットがポリクローナル抗血清と反応した(LTA(26kDa)およびLTB(14.1kDa))。この後者のバンドは、予想どおり、pMMB Stx2eA1LTA2Bの発現産物を含有するレーン1においても認められる。Stx2eA1LTA2におけるLTA2断片の存在は、予想サイズ(35.1kDa)のレーン1における明らかに可視性のStx2eA1LTA2バンドを得るのに十分なものであった。
pMMB Stx2eA1LTA2Bを、記載されているとおりに誘導し、Stx2eA1LTA2B融合タンパク質をガラクトース精製により細菌ペレットから精製した。結果を図4に示す。この図は該ガラクトース−シリカカラムの種々の画分中のStx2eA1LTA2B発現産物の量および純度を示している:レーン1:前染色マーカー;レーン2:誘導前の全培養pMMB Stx2eA1LTA2B;レーン3:未結合画分;レーン4:洗浄容量1;レーン5:洗浄容量5;レーン6:精製されたStx2eA1LTA2B溶出物1;レーン7:精製されたStx2eA1LTA2B溶出物2;レーン8:精製されたStx2eA1LTA2B溶出物3;レーン9:精製されたStx2eA1LTA2B溶出物4;レーン10:精製されたStx2eA1LTA2B溶出物5;レーン11:精製されたStx2eA1LTA2B溶出物6;レーン12:精製されたStx2eA1LTA2B溶出物7;レーン13:精製されたStx2eA1LTA2B溶出物8。
Claims (8)
- 大腸菌(Escherichia coli)熱不安定性エンテロトキシンのA2サブユニットに融合した志賀毒素または志賀毒素様毒素のA1サブユニットを含んでなるハイブリッド細菌毒素サブユニット。
- 該A1サブユニットがStx2eのA1サブユニットである、請求項1記載のハイブリッド細菌毒素サブユニット。
- 大腸菌(Escherichia coli)熱不安定性エンテロトキシンの5個のBサブユニットと請求項1または2記載のハイブリッド細菌毒素サブユニットとを含んでなるハイブリッド二成分細菌毒素。
- 請求項1または2記載のハイブリッド細菌毒素サブユニットをコードするヌクレオチド配列を含んでなる核酸分子。
- 請求項4記載の核酸分子を含んでなるDNA断片。
- 請求項4記載の核酸分子または請求項5記載のDNA断片を、機能的に連結されたプロモーターの制御下で含んでなる組換えDNA分子。
- 請求項4記載の核酸分子、請求項5記載のDNA断片または請求項6記載の組換えDNA分子を含んでなる生組換え担体(live recombinant carrier)。
- 請求項4記載の核酸分子、請求項5記載のDNA断片、請求項6記載の組換えDNA分子または請求項7記載の生組換え担体を含んでなる宿主細胞。
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US20030170248A1 (en) * | 1997-12-23 | 2003-09-11 | Jeffrey R. Stinson | Humanized monoclonal antibodies that protect against shiga toxin induced disease |
JP2009536818A (ja) * | 2006-04-20 | 2009-10-22 | ザ ヘンリー エム. ジャクソン ファウンデーション フォー ザ アドヴァンスメント オブ ミリタリー メディシン インコーポレイテッド | 志賀毒性1型タンパク質に基づく方法および組成物 |
US9023352B2 (en) | 2007-02-20 | 2015-05-05 | Tufts University | Methods, compositions and kits for treating a subject using a recombinant heteromultimeric neutralizing binding protein |
JP5392528B2 (ja) * | 2008-03-25 | 2014-01-22 | 独立行政法人産業技術総合研究所 | 毒素またはウィルス含有汚染液の固相抽出方法および無毒化方法、並びにそれに用いる固相抽出用捕集剤および除染剤 |
EP2287300B1 (en) * | 2008-05-02 | 2018-06-27 | Idemitsu Kosan Co., Ltd. | Bacterial toxin vaccine |
CN102292098A (zh) * | 2009-01-23 | 2011-12-21 | 杰克孙M.亨利基金会先进军事医学有限公司 | 基于志贺毒素2型蛋白的方法和组合物 |
US20120269842A1 (en) * | 2009-10-09 | 2012-10-25 | South Dakota State University | Enterotoxigenic e. coli fusion protein vaccines |
CN105899227A (zh) * | 2013-11-26 | 2016-08-24 | 出光兴产株式会社 | 大肠杆菌病用疫苗 |
WO2015100409A2 (en) | 2013-12-26 | 2015-07-02 | Tufts University | Methods, compositions and kits for treating a subject using a recombinant neutralizing binding protein |
WO2016164407A2 (en) * | 2015-04-07 | 2016-10-13 | Polyskope Labs | Detection of one or more pathogens |
US10973908B1 (en) | 2020-05-14 | 2021-04-13 | David Gordon Bermudes | Expression of SARS-CoV-2 spike protein receptor binding domain in attenuated salmonella as a vaccine |
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SE9501682D0 (sv) * | 1995-05-05 | 1995-05-05 | Jan Holmgren | Hybrid molecules between heat-labile enterotoxin and cholera toxin B subunits |
EP1057895A1 (de) * | 1999-06-04 | 2000-12-06 | Lohmann Animal Health GmbH & Co. KG | Fusionsprotein das das Fragment B des Shigatoxins enthält, dieses enthaltende (Impf-)Stoffzusammensetzung und Verfahren zu dessen Herstellung |
US20010031485A1 (en) | 2000-03-22 | 2001-10-18 | Sibtech, Inc. | Recombinant proteins containing Shiga-like toxin and vascular endothelial growth factor fragments |
AU2001271268A1 (en) * | 2000-05-19 | 2001-12-03 | The Administrators Of The Tulane Educational Fund | Hybrid lt-a/ct-b holotoxin for use as an adjuvant |
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US7807184B2 (en) | 2010-10-05 |
WO2005011733A1 (en) | 2005-02-10 |
JP2006527990A (ja) | 2006-12-14 |
CA2531441A1 (en) | 2005-02-10 |
DE602004032143D1 (de) | 2011-05-19 |
ES2365035T3 (es) | 2011-09-20 |
CA2531441C (en) | 2013-04-02 |
ATE504312T1 (de) | 2011-04-15 |
US20080107653A1 (en) | 2008-05-08 |
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