Stable Insertion into the Human Cytomegalovirus Genome of a Foreign Gene.

外源基因稳定插入人类巨细胞病毒基因组。

• 批准号: 01480182
• 负责人: WATANABE Yasushi
• 金额: $ 4.29万
• 依托单位: Tokai University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1989
• 资助国家: 日本
• 起止时间: 1989 至 1990
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-01480182/
• 关键词: Human cytomegalovirus; Expression vector; Recombinant virus; Vaccine

On the bases of previous finding that the HindIII O fragment of 7.8-kb in the genome of human cytomegalovirus strain Towne is nonessential for viral replication, we constructed a vector pKM that directs introduction of foreign genes by homologous recombination precisely replacing the O fragment. Using the vector, we constructed strain Towne-derived recombinant viruses in which a chimeric lacZ gene fused to the SV40 promoter and poly A signals was inserted in place of the O fragment. Two types of recombinants were obtained which carry the chimeric gene in opposite directions. Beta Gal was expressed in HEL cells infected with these recombinants throughout the infection cycle, and the rate of its synthesis in early stages of infection was comparable to that of synthesis of a 65 kDa viral glycoprotein, one of viral proteins expressed abundantly. The chimeric lacZ gene introduced was stable and no lacZ- revertant has been observed so far.

在前人发现人巨细胞病毒Towne株基因组中7.8-kb的HindIII O片段对病毒复制不是必需的基础上，我们构建了一种通过同源重组精确取代O片段的载体pKM来指导外源基因的导入。利用该载体构建了融合了SV40启动子的LacZ嵌合基因和多聚A信号的Towne株重组病毒。获得了两种不同方向携带嵌合基因的重组子。β-半乳糖在感染HEL细胞的整个感染周期中都有表达，在感染早期其合成速度与65 kDa病毒糖蛋白的合成速度相当，后者是病毒中大量表达的蛋白之一。导入的嵌合LacZ基因是稳定的，到目前为止还没有观察到LacZ回复突变。

项目成果

Masataka Takekoshi: "Site-specific stable insertion into the human cytomegalovirus genome of a foreign gene under the control of the SV40 promoter" Gene. (1991)

Masataka Takekoshi：“在 SV40 启动子控制下，外源基因在人类巨细胞病毒基因组中进行位点特异性稳定插入”基因。

竹腰 正隆: "EB・サイトメガロウイルス" 臨床と微生物. 17. 45-50 (1990)

Masataka Takekoshi：“EB/巨细胞病毒”临床微生物学 17. 45-50 (1990)。

Masataka Takekoshi: "Siteーspecific stable insertion into the human cytomegalovirus genome of a foreign gene under control of the Sv_<40> premoter" gene. (1991)

Masataka Takekoshi：“在 Sv_<40> 前驱基因的控制下，外源基因在人类巨细胞病毒基因组中进行位点特异性稳定插入”（1991）。

竹腰正隆: "EB・サイトメガロウイルス" 臨床と微生物. 17. 45-50 (1990)

Masataka Takekoshi：“EB/巨细胞病毒”临床和微生物学 17. 45-50 (1990)。

Masataka Takekoshi: "Siteーspecific stable insertion into the human cytomegalovirus genome of a foreign gene under control of the SV40 promoter" gene. (1991)

Masataka Takekoshi：“在 SV40 启动子的控制下，外源基因在人类巨细胞病毒基因组中进行位点特异性稳定插入”（1991）。