RESCUE OF MEASLES VIRUSES FROM CLONED DNA
RESCUE OF MEASLES VIRUSES FROM CLONED DNA
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DOI：
10.1002/j.1460-2075.1995.tb00266.x
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发表时间：
1995-12-01
期刊：
影响因子：
11.4
通讯作者：
BILLETER, MA
中科院分区：
文献类型：
--
作者：
RADECKE, F;SPIELHOFER, P;BILLETER, MA
已经建立了一个系统，允许从克隆的 DNA 中拯救复制的麻疹病毒 (MV)。一方面，构建了质粒，由噬菌体 T7 RNA 聚合酶转录具有正确末端的 MV 反基因组 RNA。另一方面，源自人胚胎肾293细胞系的辅助细胞被产生，其组成型表达T7 RNA聚合酶以及MV核衣壳蛋白和磷蛋白。在T7启动子的指导下，用MV反基因组质粒和编码MV聚合酶的质粒同时转染辅助细胞，导致合胞体的形成，从中很容易回收MV，子代病毒中存在包含三个核苷酸变化的遗传标签，作为反向遗传学的首次应用，删除了融合基因5'非编码区的504个核苷酸片段，产生了其复制行为Vero 细胞与实验室埃德蒙斯顿 B 株的细胞没有区别，由于不涉及辅助病毒，该系统原则上应该适用于拯救大病毒目单链病毒目的任何成员，即具有不分段负链 RNA 基因组的病毒。
A system has been established allowing the rescue of replicating measles viruses (MVs) from cloned DNA, On one hand, plasmids were constructed from which MV antigenomic RNAs with the correct termini are transcribed by phage T7 RNA polymerase. On the other hand, helper cells derived from the human embryonic kidney 293 cell line were generated constitutively expressing T7 RNA polymerase together with MV nucleocapsid protein and phosphoprotein. Simultaneous transfection of the helper cells with the MV antigenomic plasmid and with a plasmid encoding the MV polymerase under direction of a T7 promoter led to formation of syncytia from which MVs were easily recovered, A genetic tag comprising three nucleotide changes was present in the progeny virus, As a first application of reverse genetics, a segment of 504 nucleotides from the 5' non-coding region of the fusion gene was deleted,leading to an MV variant whose replication behaviour in Vero cells was indistinguishable from that of the laboratory Edmonston B strain, Since no helper virus is involved, this system, in principle, should be applicable to the rescue of any member of the large virus order Mononegavirales, i.e. viruses with a non-segmented negative-strand RNA genome.