JP6152535B2 - 細胞培養組成物及びインフルエンザウイルスの生産方法 - Google Patents
細胞培養組成物及びインフルエンザウイルスの生産方法 Download PDFInfo
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Description
IRF7を標的とするsiRNAとして、配列番号1に示される塩基配列のセンス鎖及び配列番号2に示される塩基配列のアンチセンス鎖からなるsiRNA-1と、配列番号3に示される塩基配列のセンス鎖及び配列番号4に示される塩基配列のアンチセンス鎖からなるsiRNA-2と、配列番号9に示される塩基配列のセンス鎖及び配列番号10に示される塩基配列のアンチセンス鎖からなるsiRNA-3と、を合成した。
48.0℃ 30分
95.0℃ 10分の初期加温に引き続き、
95.0℃ 15秒
60.0℃ 1分(本ステップ終了毎に蛍光を測定)
を40サイクル反復
その結果、下記表1に示されるように、IRF7又はMAVSのノックダウンにより、インフルエンザウイルスの増殖効率は2倍以上増加したことが判明した。
イヌIRF7に対するshRNAを発現するレンチウイルスベクターをMDCK細胞に導入し、安定的にIRF7をノックダウンしたMDCK細胞を作製した。shRNAは、配列番号58に示される塩基配列のセンス鎖及び配列番号59に示される塩基配列のアンチセンス鎖からなるものであった(shRNA-1)。また、配列番号60に示される塩基配列のセンス鎖及び配列番号61に示される塩基配列のアンチセンス鎖からなるshRNA(shRNA-LacZ)を発現するレンチウイルスベクターをMDCK細胞に導入してコントロールMDCK細胞を作製した。次にMDCK細胞にインフルエンザウイルス(A/Narita/1/2009,A(H1N1)pdm09)をm.o.i=0.001で感染させ、このインフルエンザウイルスを感染させたMDCK細胞を34℃、炭酸ガス濃度5%、酸素濃度3ppmで72時間培養した。
イヌIRF7に対するshRNAを発現しているMDCK細胞とコントロールのshRNAを発現しているMDCK細胞を2×105cells/well となるように6ウェルプレートにまき、細胞密度が上限に達するまで培養した。実施例2と同様に、shRNAは、配列番号58に示される塩基配列のセンス鎖及び配列番号59に示される塩基配列のアンチセンス鎖からなるものであった。これらの細胞に表2に示されているA(H1N1)ウイルス、A(H1N1)pdm09ウイルス、A(H3N2)ウイルス、B型ウイルスを、表2に示されている量(100〜1000 pfuまたは100〜1000 TCID50)で感染させ、表2に示されている時間(48時間〜96時間)後にHA価を測定した。
配列番号2,4,6,8,10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54:siRNAアンチセンス配列
配列番号55〜56:プライマー
配列番号57:プローブ
配列番号58,60:shRNAセンス配列
配列番号59,61:shRNAアンチセンス配列
Claims (7)
- IRF7又はMAVSの発現及び/又は機能を抑制させることによりインフルエンザウイルスの増殖効率を高めたMDCK細胞を含む細胞培養組成物。
- 前記IRF7の発現の抑制は、配列番号1に示される塩基配列のオリゴヌクレオチドであるセンス鎖、及び、配列番号1の塩基配列に相補的な塩基配列である配列番号2に示される塩基配列のオリゴヌクレオチドであるアンチセンス鎖、からなる二本鎖RNAであるsiRNAを導入することによりなされることを特徴とする請求項1に記載の細胞培養組成物。
5’- GGCGC CUGGG CAAAU GCAAdT dT-3’ (配列番号1)
5’- UUGCA UUUGC CCAGG CGCCdT dT-3’ (配列番号2) - 前記IRF7の発現の抑制は、配列番号3に示される塩基配列のオリゴヌクレオチドであるセンス鎖、及び、配列番号3の塩基配列に相補的な塩基配列である配列番号4に示される塩基配列のオリゴヌクレオチドであるアンチセンス鎖、からなる二本鎖RNAであるsiRNAを導入することによりなされることを特徴とする請求項1に記載の細胞培養組成物。
5’- CAGAG AAGCU GCUGC AGCAdT dT-3’ (配列番号3)
5’- UGCUG CAGCA GCUUC UCUGdT dT-3’ (配列番号4) - 前記MAVSの発現の抑制は、配列番号5に示される塩基配列のオリゴヌクレオチドであるセンス鎖、及び、配列番号5の塩基配列に相補的な塩基配列である配列番号6に示される塩基配列のオリゴヌクレオチドであるアンチセンス鎖、からなる二本鎖RNAであるsiRNAを導入することによりなされることを特徴とする請求項1に記載の細胞培養組成物。
5’- GUUAG UUCCU GCUGA GGUUdT dT-3’ (配列番号5)
5’- AACCU CAGCA GGAAC UAACdT dT-3’ (配列番号6) - 前記MAVSの発現の抑制は、配列番号7に示される塩基配列のオリゴヌクレオチドであるセンス鎖、及び、配列番号7の塩基配列に相補的な塩基配列である配列番号8に示される塩基配列のオリゴヌクレオチドであるアンチセンス鎖、からなる二本鎖RNAであるsiRNAを導入することによりなされることを特徴とする請求項1に記載の細胞培養組成物。
5’- CCUCC AAGGU GCCUA CCCAdT dT-3’ (配列番号7)
5’- UGGGU AGGCA CCUUG GAGGdT dT-3’ (配列番号8) - 前記IRF7の発現の抑制は、配列番号58に示される塩基配列のオリゴヌクレオチドであるセンス鎖、及び、配列番号58の塩基配列に相補的な塩基配列である配列番号59に示される塩基配列のオリゴヌクレオチドであるアンチセンス鎖、からなる二本鎖RNAであるshRNAを導入することによりなされることを特徴とする請求項1に記載の細胞培養組成物。
5’- GGCGC CTGGG CAAAT GCAA-3’ (配列番号58)
5’- TTGCA TTTGC CCAGG CGCC-3’ (配列番号59) - インフルエンザウイルスの生産方法であって、
請求項1乃至6の何れか1項に記載の細胞培養組成物中のMDCK細胞にインフルエンザウイルスを感染させる工程と、
前記細胞培養組成物をインキュベートする工程と、
前記細胞培養組成物からインフルエンザウイルスを単離する工程と、
を含むインフルエンザウイルスの生産方法。
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