ウイルスライブラリーを用いた新しいHSVワクチンおよび遺伝子治療ベクターの開発

• Project/Area Number: 14704027
• Research Category: Grant-in-Aid for Young Scientists (A)
• Allocation Type: Single-year Grants
• Research Field: Virology
• Research Institution: Nagoya University
• Principal Investigator: 川口 寧 名古屋大学, 大学院・医学系研究科, 助教授 (60292984)
• Project Period (FY): 2002 – 2004
• Project Status: Completed (Fiscal Year 2004)
• Budget Amount: ¥25,220,000 (Direct Cost: ¥19,400,000、Indirect Cost: ¥5,820,000); Fiscal Year 2004: ¥8,060,000 (Direct Cost: ¥6,200,000、Indirect Cost: ¥1,860,000); Fiscal Year 2003: ¥8,060,000 (Direct Cost: ¥6,200,000、Indirect Cost: ¥1,860,000); Fiscal Year 2002: ¥9,100,000 (Direct Cost: ¥7,000,000、Indirect Cost: ¥2,100,000)
• Keywords: HSV / BAC system / RecA法 / RecE&T法 / FRT / FLP系 / 組み換えウイルス作製法 / FLP / FRT系 / 大腸菌 / bac system / ワクチン / 遺伝子治療ベクター

Research Abstract

本研究の目的は、申請者が独自に開発中である単純ヘルペスウイルス(herpes simplex virus : HSV)全ゲノムを保持する大腸菌を用いた新しいHSV組み換え法を利用して、新しいHSVワクチンおよび遺伝子治療用ベクター開発に向けての基礎的な知見を蓄積することにある。
独自に開発したHSV全ゲノムを有する大腸菌(YEbac102)内でウイルスゲノムに変異を導入する系をいくつか開発してきた。(i)RecA法、(ii)RecE&T法とFRT/FLP系を組み合わせた方法、(iii)RecE&T法+FRT/FLP系とRecA法を組み合わせる方法の3つの系を開発した。これらの系を用いることによって、HSVゲノムに、欠損変異、点変異、挿入変異といった如何なる変異も簡便かつ迅速に導入できるようになった。具体的には、遺伝子発現制御因子であるウイルス因子ICPO、ウイルス特異プロテインキナーゼUL13,Us3、機能不明なウイルス因子UL51の変異体を作製することに成功している。また、これらの変異体を利用して、(i)UL51遺伝子産物がウイルスの成熟過程に寄与していること、(ii)UL13のプロテインキナーゼ活性が従来報告されているUL13の機能である遺伝子発現制御には関係ないことを明らかにし、UL13はプロテインキナーゼ以外の機能を有する多機能因子であることを明らかにした。本研究で確立されたHSVの組み換え系は、HSVの基礎研究、ワクチン開発、ベクター開発に有用であると考えられる。

Research Products

• [Journal Article] Construction of recombinant herpes simplex virus type 1 expressing green fluorescence protein without loss of any viral genes. (2004)
  • Author(s): M.Tanaka et al.
  • Journal Title: Microbes and Infection 6 Pages: 485-493
• [Journal Article] Epstein-Barr Virus Nuclear Antigen Leader Protein Induces Expression of Thymus and Activation-Regulated Chemokine in B Cells. (2004)
  • Author(s): M.Kanamori et al.
  • Journal Title: J.Virol. 78 Pages: 3984-3993
• [Journal Article] Cell lines that support replication of a novel herpes simplex 1 UL31 deletion mutant can properly target UL34 protein to the nuclear rim in the absence of UL31. (2004)
  • Author(s): L.Liang et al.
  • Journal Title: Virology 329 Pages: 68-76
• [Journal Article] Formation of aggresome-like structures in herpes simplex virus type 2-infected cells and a potential role in virus assembly. (2004)
  • Author(s): N.Nozawa et al.
  • Journal Title: Exp.Cell Res. 299 Pages: 486-497
• [Journal Article] The HSV-2 membrane protein UL56 associates with the kinesin motor protein KIF1A.
  • Author(s): T.Koshizuka et al.
  • Journal Title: J.Gen.Virol. 印刷中
• [Journal Article] Herpes Simplex Virus Type 1 UL51 Protein Is Involved in Maturaion and Egress of Virus Particles.
  • Author(s): N.Nozawa et al.
  • Journal Title: J.Virol. 印刷中
• [Publications] Y.Kawaguchi et al.: "Protein kinases conserved in herpesviruses potentially share a function mimicking the cellular protein kinase cdc2."Reviews in Medical Virology. 13. 331-340 (2003)
• [Publications] K.Kato et al.: "Identification of protein kinases responsible for phosphorylation of Epstein-Barr virus nuclear antigen leader protein at serine 35 that regulates its coactivator function."J.Gen.Virol.. 84. 3381-3392 (2003)
• [Publications] M.Kanamori et al.: "Epstein-Barr Virus Nuclear Antigen Leader Protein Induces Expression of Thymus and Activation-Regulated Chemokine in B Cells."J.Virol.. (印刷中).
• [Publications] M.Tanaka et al.: "Construction of recombinant herpes simplex virus type 1 expressing green fluorescence protein without loss of any viral genes."Microbes and Infection. (印刷中).
• [Publications] M.Tanaka et al.: "Construction of an Excisable Bacterial Artificial Chromosome Containing a Full-Length Infectious Clone of Herpes Simplex Virus Type 1 : Viruses Reconstituted from the Clone Exhibits Wild-Type Properties In Vitro and In Vivo"Journal of Virology. 77. 1382-1391 (2003)
• [Publications] M.Tanaka et al.: "The Conserved region CR2 of Epstein-Barr Virus Nuclear Antigen Leader Protein Is a Multifunctional Domain that mediates Self-Association As Well As Nuclear Localization and Nuclear Matrix Association"Journal of Virology. 76. 1025-1032 (2002)
• [Publications] Y.Kawaguchi et al.: "Conserved Protein Kinases Encoded by Herpesviruses and a Cellular Protein Kinase Cdc2 Target the Same Phosphorylation Site In Eukaryotic Elongation Factor 1δ"Journal of Virology. 77. 2359-2368 (2003)
• [Publications] M.Igarashi et al.: "Physical interaction of Epstein-Ban Virus (EBV) nuclear antigen leader protein (EBNA-LP) with human estrogen-related receptor I (hERR1) : hERR1 interacts with a conserved domain of EBNA-LP that is critical for EBV-induced B-cell immortalization"Journal of General Virology. 84. 319-327 (2003)
• [Publications] G.Matsuda et al.: "Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) forms complexes with a cellular anti-apoptosis protein bcl-2 or its EBV counterpart BHRF1 through HS1-associated protein X-1"Microbiology and Immunology. 49. 91-99 (2003)
• [Publications] K.Kato et al.: "Characterization of the regulatory function of the Marek's disease virus serotype 1 ICP22 homologue"Veterinary Microbiology. 85. 305-313 (2002)