JP2008546399A - サケ科魚類のアルファウイルスのcDNA構築物 - Google Patents
サケ科魚類のアルファウイルスのcDNA構築物 Download PDFInfo
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- JP2008546399A JP2008546399A JP2008517535A JP2008517535A JP2008546399A JP 2008546399 A JP2008546399 A JP 2008546399A JP 2008517535 A JP2008517535 A JP 2008517535A JP 2008517535 A JP2008517535 A JP 2008517535A JP 2008546399 A JP2008546399 A JP 2008546399A
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Abstract
Description
VILLOING et al., J.Virol.74,173−183,2000;WESTON et al., Virology,256,188−195,1999 STRAUSS et STRAUSS,Microbiol.Rev., 58,491−562,1994
−転写プロモーター、
そして該プロモーターの下流に、かつ該プロモーターの転写制御下に、
−少なくとも5個のヌクレオチド、好ましくは10〜100個のヌクレオチドからなるスペーサー配列と、
−サケ科魚類のアルファウイルスのゲノムRNAのcDNA、
を含んでいる。
5’ X1CTGANGARX2B2X’2YGAAAX3B3X’3TH 3’(I)、
という一般式(I)によって定義することができ、
該式において、A、T、GおよびCは通常の意味を有し、Hは、C、TまたはAを表し、YはAまたはGを表し、RはCまたはTを表し、NはA、T、GまたはCを表し、X1は、前記アルファウイルスのゲノムの5’末端の配列に相補的な配列の、少なくとも3個のヌクレオチド、好ましくは6個〜10個のヌクレオチドからなるオリゴヌクレオチドを表し、X2は、任意の配列の、少なくとも3個のヌクレオチド、好ましくは3個〜5個のヌクレオチドからなるオリゴヌクレオチドを表し、B2は、任意の配列の、4個または5個のヌクレオチドからなるオリゴヌクレオチドを表し、X’2はX2に相補的なオリゴヌクレオチドを表し、X3は、任意の配列の、少なくとも2個のヌクレオチド、好ましくは6個〜10個のヌクレオチドからなるオリゴヌクレオチドを表し、B3は、任意の配列の、4個または5個のヌクレオチドからなるオリゴヌクレオチドを表し、そしてX’3は、X3に相補的なオリゴヌクレオチドを表している。
−複製する能力と、自身に導入される非相同性配列を発現させる能力、そして、新しいウイルス粒子を産生するためにパッケージングされる能力をもったベクターであり、一般的には、サブゲノムプロモーターのコピーの制御下に置かれた有益な非相同性配列をアルファウイルスの完全なゲノムに導入することで該アルファウイルスの完全なゲノムから得られるベクターと、
−複製する能力と、自身に導入される非相同性配列を発現する能力は持っているが、新しいウイルス粒子を産生する能力は持たないベクターであり、一般的には、構造タンパク質をコードするアルファウイルスのゲノム領域を、有益な非相同性配列で置換することによって得られるものであり、たとえば、これらのタンパク質を発現する補助のベクターを宿主細胞に導入することにより、または、宿主細胞として、これらのタンパク質を発現する発現カセットによって安定的に形質転換された細胞株を利用することにより、構造タンパク質が宿主細胞内にトランスで存在するときにだけ、ウイルスのパッケージングが行われるベクターである。
ウイルスおよび細胞
以下の実施例で用いたウイルスは、前述したSMVのS49P株に由来するものである(CASTRIC et al., Bulletin of the European Association of Fish Pathologists,17,27−30,1997)。
以下の実施例で用いたプライマーの配列は以下の表1に示している。
SMVのcDNAの完全な構築物であるpBS−SMVはcDNA断片(1〜3として番号を付けた)から得られたものであり、該断片は、SMVの完全なゲノムをカバーし、先行して公開された配列(VILLOING et al.,2000;先掲したWESTON et al.,2002,GENBANKのアクセス番号:NC_003433.1/GI:19352423)から得られたものである。各断片を、SMVのゲノムRNAを鋳型として用いて、逆転写とそれに続くPCR(RT−PCR)によって増幅した。RNAは、PEGで濃縮された、感染したSMV細胞の上清から、精製キットQIAamp Viral RNA(Qiagen)を用いて抽出した。逆転写およびPCR増幅に用いたプライマー(P1〜P6)は表1に示されている。
ハンマーヘッド型リボザイムの配列(HH配列)をSMVのゲノムcDNAの5’末端の最初のヌクレオチドに、以下の要領で融合させた。SMVのcDNAの最初の2Kbを含んでいるp−nsPのHindIII断片を削除し、プラスミドpUC19の中でサブクローニングし、pUC−SDV HindIII構築物を得た。この構築物から、SMVのゲノムの5’末端を含んだBamHI/NaeI断片を削除し、合成DNA断片で置換した。該合成DNAは、SMVゲノムの5’末端に融合しているハンマーヘッド型リボザイムの配列を含んだ、79個および80個からなるヌクレオチドの、部分的に相補的な二つのオリゴヌクレオチドをハイブリダイズし、T4DNAポリメラーゼのクレノウ断片で平滑化することによって得られたものである。これらのオリゴヌクレオチドの配列(5’RIBOおよび3’RIBO)は表1に示されている。
SMVの感染性cDNAクローンを以下の要領で構築した。
組換えSMVの感染力を確認するため、50匹の若い健康なニジマス(Oncorhynchus mykiss)を、感染したBF−2細胞から得た5×104UFP/mlの野生型SMVまたは組換えSMVを含む10℃の水で満たした水槽に2時間にわたって浸漬することで感染させた。次に、水槽を30リットルの新鮮な水で満たした。対照として用いた魚は、ウイルス懸濁液の代わりに培養培地を用いて、同じ条件で処理した。
GFPを発現する感染性の組換えウイルスを産生するため、感染性のcDNAであるpHH−SDV−T7tを二つの異なる様式で修飾することで、GFPを発現する追加の発現カセットを導入した。
pHH−nsP−GFP構築物を、別個の二つのPCR増幅産物を生成するための鋳型として用いた。産物「GFP PCR」は5’GFPプライマーおよび3’GFPプライマーを用いることで得られ(表1)、産物「SMVサブゲノムPCR」は5’nsP4プライマー(7706〜7750)および3’Junプライマーを用いて得られた(表1)。SMVのサブゲノムプロモーターの正確な位置と最小のサイズはまだ判定されていないため、nsP4の配列の終端と結合領域とを含む約100個のヌクレオチドからなる断片を用いた。次に、SMVのサブゲノムプロモーターを、第一の増幅に起因する二つの産物を混合し、5’GFPプライマーと3’Junプライマーを用いることで、GFPをコードする配列の3’末端においてPCRによってライゲートした。
この構築物において、GFPの発現カセットを構造遺伝子の下流に導入した。
−5’ProGFPおよび3’ProGFPをプライマーとして用い(表1)、pHH−nsP−GFP構築物を鋳型として用いることで、GFPと融合したSMVのサブゲノムプロモーター(産物PCR1)と、
−3’UTRおよびT7tをプライマーとして用い(表1)、pHHSDV−T7t構築物を鋳型として用いることで、ポリ(A)テールとT7ターミネーターと融合したSMVの3’非翻訳領域(産物PCR2)である。
Claims (13)
- サケ科魚類のアルファウイルスのゲノムに由来する組換えDNAであり、
−転写プロモーター、
そして該プロモーターの下流に、かつ該プロモーターの転写制御下に、
−少なくとも5個のヌクレオチドからなるスペーサー配列と、
−サケ科魚類のアルファウイルスのゲノムRNAのcDNA、
を含んでいる、組換えDNA。 - スペーサー配列が、
5’ X1CTGANGARX2B2X’2YGAAAX3B3X’3TH 3’ (I)、
という一般式(I)によって定義され、
該式において、A、T、GおよびCは通常の意味を有し、Hは、C、TまたはAを表し、YはAまたはGを表し、RはCまたはTを表し、NはA、T、GまたはCを表し、X1は、前記アルファウイルスのゲノムの5’末端の配列に相補的な配列の、少なくとも3個のヌクレオチド、好ましくは6個〜10個のヌクレオチドからなるオリゴヌクレオチドを表し、X2は、任意の配列の、少なくとも3個のヌクレオチド、好ましくは3個〜5個のヌクレオチドからなるオリゴヌクレオチドを表し、B2は、任意の配列の、4個または5個のヌクレオチドからなるオリゴヌクレオチドを表し、X’2はX2に相補的なオリゴヌクレオチドを表し、X3は、任意の配列の、少なくとも2個のヌクレオチド、好ましくは6個〜10個のヌクレオチドからなるオリゴヌクレオチドを表し、B3は、任意の配列の、4個または5個のヌクレオチドからなるオリゴヌクレオチドを表し、そしてX’3は、X3に相補的なオリゴヌクレオチドを表していることを特徴とする、請求項1に記載の組換えDNA。 - サケ科魚類のアルファウイルスのcDNAインサートが一つまたは複数の発現カセットを含んでおり、該発現カセットのそれぞれが、前記サブゲノムプロモーターのコピーと、前記サブゲノムプロモーターの下流かつ該プロモーターの転写制御下に、発現させようとする非相同性配列またはこの配列の導入を可能にするクローニング部位を含んでいることを特徴とする、請求項1または請求項2に記載の組換えDNA。
- サケ科魚類のアルファウイルスのRNAレプリコンの調製方法であり、宿主細胞に、請求項2または請求項3に記載の組換えDNAを導入することと、前記宿主細胞を培養することを含むことを特徴とする方法。
- 請求項4に記載の方法によって得ることのできるサケ科魚類のアルファウイルスのRNAレプリコン。
- サケ科魚類の組換えアルファウイルスの調製方法であり、請求項2または請求項3に記載の組換えDNAまたは請求項5に記載のRNAレプリコンを、自身のパッケージングに不可欠な前記アルファウイルスの構造タンパク質の全てが発現する宿主細胞に導入することと、前記宿主細胞を培養することを含む方法。
- 前記構造タンパク質の発現を可能にする全遺伝情報が前記組換えDNAまたは前記RNAレプリコンに保持されていることを特徴とする、請求項6に記載の方法。
- 前記構造タンパク質の発現を可能にする遺伝情報の全部または一部が宿主細胞によってトランスで提供されることを特徴とする、請求項6に記載の方法。
- 請求項6〜請求項8のいずれか一つに記載の方法によって得ることのできる、サケ科魚類の組換えアルファウイルス。
- 請求項1〜請求項3のいずれか一つに記載の組換えDNA、請求項5に記載のサケ科魚類のアルファウイルスのRNAレプリコン、または、請求項9に記載のサケ科魚類の組換えアルファウイルスの、ワクチンを得るための利用方法。
- 請求項1〜請求項3のいずれか一つに記載の組換えDNAを含むワクチン。
- 請求項5に記載のサケ科魚類のアルファウイルスのRNAレプリコンを含むワクチン。
- 請求項9に記載のサケ科魚類の組換えアルファウイルスを含むワクチン。
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