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.

在先前发现的基于人类巨细胞病毒菌株镇基因组中的Hindiii O片段的基础上，对于病毒复制而言，我们构建了一个载体PKM，该向量PKM通过同源重组来指导外国基因，精确地替换了O片段。使用载体，我们构建了菌株衍生的重组病毒，其中插入了与SV40启动子融合的嵌合LacZ基因，并插入了Poly A信号，以代替O片段。获得了两种类型的重组，它们沿相反的方向携带嵌合基因。在整个感染周期中，在感染这些重组剂感染的HEL细胞中表达βGAL，其在感染的早期阶段的合成速率与65 kDa病毒糖蛋白的合成相当，这是一种病毒蛋白，这是一种大量表达的病毒蛋白。引入的嵌合LACZ基因是稳定的，到目前为止尚未观察到LACZ-恢复。

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)。

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 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）。

Masataka Takekoshi: "EB・Cytomegalovirus" Clinical Microbiology. 17. 45-50 (1990)

竹越正孝：“EB·巨细胞病毒”临床微生物学 17. 45-50 (1990)。