JP5593220B2 - C型肝炎ウイルス由来のキメラ遺伝子を含む核酸 - Google Patents
C型肝炎ウイルス由来のキメラ遺伝子を含む核酸 Download PDFInfo
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Description
上記Coreタンパク質のN末端のアミノ酸残基から数えて第328番目のプロリン残基は、プロリン残基以外のアミノ酸残基に置換されている、核酸。
(a) 上記(10)又は(11)に記載の細胞、又は
(b) 上記(9)に記載のキメラC型肝炎ウイルス粒子及びC型肝炎ウイルス感受性細胞、
を培養し、得られる培養物中の前記核酸由来のレプリコンRNA又はウイルス粒子を検出することを含む、抗C型肝炎ウイルス物質をスクリーニングする方法。
上記(9)に記載のキメラC型肝炎ウイルス粒子を回収するステップと、
を含む、キメラC型肝炎ウイルス粒子の作製方法。
上記不活性化又は弱毒化キメラC型肝炎ウイルス粒子をC型肝炎ウイルスワクチンの形態に製剤化するステップと、
を含む、C型肝炎ウイルスワクチンの製造方法。
(1)JFH-1株以外のC型肝炎ウイルス株由来のCoreタンパク質、E1タンパク質、E2タンパク質、及びp7タンパク質、上記JFH-1株若しくは上記JFH-1株以外のC型肝炎ウイルス株由来のNS2タンパク質又は上記JFH-1株由来のNS2タンパク質と上記JFH-1株以外のC型肝炎ウイルス株由来のNS2タンパク質とのキメラNS2タンパク質、並びに、上記JFH-1株由来のNS3タンパク質、NS4Aタンパク質、NS4Bタンパク質、NS5Aタンパク質、及びNS5Bタンパク質、をコードするそれぞれの領域を、5’側から3’側に向かってこの順序で配置してなるC型肝炎ウイルス由来のキメラ遺伝子を含む核酸であって、
(2)上記Coreタンパク質のN末端のアミノ酸残基であるメチオニン残基を1番目としたとき第328番目(或いは、E1タンパク質のN末端のアミノ酸残基を1番目としたとき137番目)のアミノ酸残基が、プロリン残基からプロリン残基以外のアミノ酸残基に置換された複合タンパク質(前駆体タンパク質)をコードする塩基配列を含む、核酸である。
C型肝炎ウイルス(HCV)のゲノムは、約9600ヌクレオチドからなる+鎖の一本鎖RNAである。このゲノムRNAは、5'非翻訳領域(5'NTR又は5'UTRとも表記する)、構造領域と非構造領域とから構成される翻訳領域、及び3'非翻訳領域(3'NTR又は3'UTRとも表記する)からなる。その構造領域にはHCVの構造タンパク質がコードされており、非構造領域には複数の非構造タンパク質がコードされている。
プロモーターの制御下にクローン化したHCV cDNAからRNAを合成し、このRNAを細胞に導入することでキメラHCV粒子を作製することができる。
HCVゲノムの効率的な複製には、ゲノムの塩基配列に変異が起こることが必要であることが示されており(Lohmann, V. ら, J. Virol., 75:1437-1449, 2001)、複製を向上させる変異は適応変異(adaptive mutation)と呼ばれている。上記(2)により作製したHCVゲノムRNAが導入された細胞を継代培養することで、HCV粒子を持続的に生産する細胞株を得ることができる。このように培養を続けることで、HCVゲノムに適応変異が起こり、HCV粒子生産が著しく向上することがある。
HCV粒子はワクチンとしての用途、抗HCV抗体作製のための抗原としての用途に好適である。
(a)キメラHCV粒子を生成する細胞、或いは、
(b)キメラHCV粒子及びC型肝炎ウイルス感受性細胞
を培養し、得られる培養物中の、本発明のキメラHCV粒子が含有する前記核酸に由来するレプリコンRNA又はウイルス粒子を検出することを含む、抗HCV物質をスクリーニングするための方法である。
HCVゲノムRNAのcDNAとして、5’UTRが遺伝子型2aのJFH-1株(GenBankアクセッション番号AB047639、Kato, T.ら, Gastroenterology, 125:1808-1817, 2003)、Coreタンパク質からNS2タンパク質のN末端33アミノ酸残基までが遺伝子型1bのTH株(Wakita, T.ら, J. Biol. Chem., 269:14205-14210, 1994、Moradpour, D.ら, Biochem. Biophys. Res. Commun., 246:920-924, 1998、及び国際公開WO2006/022422)、NS2のN末端34アミノ酸残基から3’UTRまでが遺伝子型2a株のJFH-1株であるTH/JFH-1キメラのcDNAを作製した。
pTH/JFH1をXbaIで切断し、フェノール/クロロホルム抽出、エタノール沈殿を行った。次いで、このXbaI切断断片をMung Bean Nuclease処理し、XbaI認識配列由来の3’末端の余分な塩基配列を除去した。さらにproteinase K処理、フェノール/クロロホルム抽出、エタノール沈殿を行ってDNA断片を精製した。この切断したプラスミドを鋳型として、MEGAscript T7 kit(Ambion社)を用いて37℃、3時間反応させ、HCV RNAの合成を行った。反応後の合成RNAはDNaseI処理後、酸性フェノールによって抽出し、エタノール沈殿を行って精製した。
TH/JFH-1 RNAを導入した細胞の継代時に、HCV抗原ELISAテストキット(オーソ社)を用いて培養上清中に含まれるHCV Coreタンパク質を定量し、HCV粒子産生の確認を行った。その結果、培養上清中のHCV Core量は導入23日目までは経時的に減少していったが、導入から26日を経過した時点でHCV Core量が上昇し始め、34日経過時点で一定の高い産生量を示した(図2)。このことから、TH/JFH-1 RNAは、Huh7細胞に導入当初は高いウイルス産生能を有していないが、後にウイルスゲノム内にウイルス産生に必要な適応変異が導入され、高いウイルス産生能を有するようになると考えられた。
TH/JFH-1ウイルスが高い産生量を有するために必要な適応変異を調べるために、RNA導入から34日目の感染細胞内のtotal RNAを抽出し、この中に含まれるHCVゲノムの配列解析を行った。
実施例4で示した、TH/JFH-1ウイルスが高い産生量を有するために必要な適応変異を持つプラスミドの構築を行った。プラスミドの作製方法を図3に示す。
実施例5で作製したプラスミドをそれぞれXbaIで切断し、フェノール/クロロホルム抽出、エタノール沈殿を行った。次いで、このXbaI切断断片をMung Bean Nuclease処理し、XbaI認識配列由来の3’末端の余分な塩基配列を除去した。さらにproteinase K処理、フェノールクロロホルム抽出、エタノール沈殿を行ってDNA断片を精製した。この切断したプラスミドを鋳型にMEGAscript T7 kit(Ambion社)を用いて各HCV RNAの合成を行った。
TH/JFH-1(PA) RNAを導入した細胞から産生されるウイルスの感染性を野生型TH/JFH-1と比較した。各RNAを導入後4、24、48、72及び96時間までの細胞内及び培養上清のHCV Coreタンパク質の変動を調べ、その培養上清の感染性を調べた。
Claims (15)
- JFH-1株以外のC型肝炎ウイルス株由来のCoreタンパク質、E1タンパク質、E2タンパク質、及びp7タンパク質、前記JFH-1株若しくは前記JFH-1株以外のC型肝炎ウイルス株由来のNS2タンパク質又は前記JFH-1株由来のNS2タンパク質と前記JFH-1株以外のC型肝炎ウイルス株由来のNS2タンパク質とのキメラNS2タンパク質、並びに、前記JFH-1株由来のNS3タンパク質、NS4Aタンパク質、NS4Bタンパク質、NS5Aタンパク質、及びNS5Bタンパク質、をコードするそれぞれの領域を、5’側から3’側に向かってこの順序で配置してなるC型肝炎ウイルス由来のキメラ遺伝子を含む核酸であって、
前記Coreタンパク質のN末端のアミノ酸残基から数えて第328番目のプロリン残基は、アラニン又はトレオニンに置換されている、核酸。 - 前記Coreタンパク質をコードする領域の5’側に前記JFH-1株の5’非翻訳領域を含み、前記NS5Bタンパク質をコードする領域の3’側に前記JFH-1株の3’非翻訳領域を含む、請求項1記載の核酸。
- 前記JFH-1株以外のC型肝炎ウイルス株は、遺伝子型1a、1b又は2aに属する株である、請求項1又は2記載の核酸。
- 前記JFH-1株以外のC型肝炎ウイルス株は、TH株、Con1株、J1株、及びそれらの派生株からなる群から選択される、請求項1〜3のいずれか一項記載の核酸。
- 請求項1〜4のいずれか一項記載の核酸であって、前記核酸は、配列表の配列番号1で示される塩基配列若しくは該塩基配列と90%以上の同一性を有する塩基配列を含むDNA、又は配列表の配列番号3で示される塩基配列若しくは該塩基配列と90%以上の同一性を有する塩基配列を含むRNAであり、かつ該DNA又はRNAにおいて配列番号1又は3で示される塩基配列の第1322番目〜第1324番目の塩基がアラニンをコードしている、核酸。
- 請求項1〜4のいずれか一項記載の核酸であって、前記核酸は、配列表の配列番号2で示される塩基配列若しくは該塩基配列と90%以上の同一性を有する塩基配列を含むDNA、又は配列表の配列番号4で示される塩基配列若しくは該塩基配列と90%以上の同一性を有する塩基配列を含むRNAであり、かつ該DNA又はRNAにおいて配列番号2又は4で示される塩基配列の第1322番目〜第1324番目の塩基がトレオニンをコードしている、核酸。
- 配列表の配列番号1で示される塩基配列を含むDNA、配列表の配列番号3で示される塩基配列を含むRNA、又は、配列表の配列番号6で示されるアミノ酸配列をコードする塩基配列を含むDNA若しくはRNAである、請求項1〜4のいずれか一項記載の核酸。
- 配列表の配列番号2で示される塩基配列を含むDNA、配列表の配列番号4で示される塩基配列を含むRNA、又は、配列表の配列番号7で示されるアミノ酸配列をコードする塩基配列を含むDNA若しくはRNAである、請求項1〜4のいずれか一項記載の核酸。
- 請求項1〜8のいずれか一項記載の核酸を含む、ベクター。
- 請求項1〜8のいずれか一項記載の核酸をウイルスゲノムとして含有するキメラC型肝炎ウイルス粒子。
- 請求項10に記載のキメラC型肝炎ウイルス粒子を生産する細胞。
- 前記細胞は、Huh-7又はその派生株である、請求項11記載の細胞。
- 被検物質の存在下で、
(a) 請求項11又は12記載の細胞、又は
(b) 請求項10記載のキメラC型肝炎ウイルス粒子及びC型肝炎ウイルス感受性細胞、
を培養し、得られる培養物中の前記核酸由来のレプリコンRNA又はウイルス粒子を検出することを含む、抗C型肝炎ウイルス物質をスクリーニングする方法。 - 請求項10記載のキメラC型肝炎ウイルス粒子を含む、C型肝炎ウイルスワクチン。
- 請求項10記載のキメラC型肝炎ウイルス粒子を抗原として非ヒト動物に投与することを特徴とする、抗C型肝炎ウイルス抗体の製造方法。
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