Direct molecular cloning of a modified poxvirus genome
    2.
    发明授权
    Direct molecular cloning of a modified poxvirus genome 失效
    改良痘病毒基因组+ B的直接分子克隆

    公开(公告)号:US5445953A

    公开(公告)日:1995-08-29

    申请号:US750080

    申请日:1991-08-26

    摘要: A method is disclosed for producing a modified eukaryotic cytoplasmic DNA virus by direct molecular cloning of a modified DNA molecule comprising a modified cytoplasmic DNA virus genome. The inventive method comprises the steps of (I) modifying under extracellular conditions a DNA molecule comprising a first cytoplasmic DNA virus genome to produce a modified DNA molecule comprising the modified cytoplasmic DNA virus genome; (II) introducing the modified DNA molecule into a first most cell which packages the modified DNA molecule into infectious virions; and (III) recovering from the host cell virions comprised of the modified vital genome. The host cell is infected with a helper virus which is expressed to package the modified viral genome into infectious virions. Examples of packaging a modified poxvirus genome by a helper poxvirus of the same or different genus are described. Also disclosed are novel poxvirus vectors for direct molecular cloning of open reading frames into a restriction enzyme cleavage site that is unique in the vector. In one model poxvirus vector, the open reading frame is transcribed by a promoter located in the vector DNA upstream of a multiple cloning site comprised of several unique cleavage sites.

    摘要翻译: 公开了通过直接分子克隆包含修饰的细胞质DNA病毒基因组的修饰的DNA分子来产生修饰的真核细胞质DNA病毒的方法。 本发明的方法包括以下步骤:(I)在细胞外条件下修饰包含第一细胞质DNA病毒基因组的DNA分子以产生包含修饰的细胞质DNA病毒基因组的修饰的DNA分子; (II)将修饰的DNA分子引入到将修饰的DNA分子包装成感染性病毒粒子的第一大多数细胞中; 和(III)从由修饰的重要基因组组成的宿主细胞病毒粒子中回收。 宿主细胞感染辅助病毒,其被表达为将经修饰的病毒基因组包装成感染性病毒粒子。 描述了通过相同或不同属的辅助痘病毒包装修饰的痘病毒基因组的实例。 还公开了用于将开放阅读框直接分子克隆到在载体中独特的限制酶切割位点的新型痘病毒载体。 在一个模型痘病毒载体中,开放阅读框架由位于由多个独特的切割位点组成的多克隆位点上游的载体DNA中的启动子转录。