Recovery of infectious SV5 from cloned DNA and expression of a foreign gene
Recovery of infectious SV5 from cloned DNA and expression of a foreign gene
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DOI：
10.1006/viro.1997.8801
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发表时间：
1997-10-27
期刊：
影响因子：
3.7
通讯作者：
Lamb, RA
中科院分区：
文献类型：
--
作者：
He, B;Paterson, RG;Lamb, RA
从cDNA构建了副粘病毒SV5基因组的完整cDNA克隆（15,246个核苷酸），使得反基因组RNA可以通过T7 RNA聚合酶转录，并通过使用丁型肝炎病毒核酶切割产生正确的3'末端。将编码反基因组序列的质粒与在 T7 聚合酶启动子控制下表达病毒复制蛋白 NP、P 和 L 的辅助质粒一起转染到先前感染了表达 T7 RNA 聚合酶的重组痘苗病毒的细胞中。通过使用基因组 RNA 的 RT-PCR 和核苷酸测序回收带有标签限制性位点的 SV5，证明了从 DNA 中拯救 RNA 基因组的效果。从 DNA 中拯救 SV5 不需要表达病毒 V 蛋白作为辅助质粒，这表明 V 蛋白对于初始复制不是必需的。 SV5的感染性cDNA也被操纵以在HN和L基因之间引入的SV5转录起始和终止信号的控制下表达绿色荧光蛋白(GFP)。表达的 GFP 量根据新引入的转录信号的性质而变化。 (C) 1997 年学术出版社。
A complete cDNA clone of the genome (15,246 nucleotides) of the paramyxovirus SV5 was constructed from cDNAs such that an anti-genome RNA could be transcribed by T7 RNA polymerase and the correct 3' end generated by cleavage using hepatitis delta virus ribozyme. The plasmid encoding the antigenome sequence was transfected into cells previously infected with recombinant vaccinia virus that expressed T7 RNA polymerase, together with helper plasmids that expressed the viral replication proteins, NP, P, and L, under the control of the T7 polymerase promoter. Rescue of the RNA genome from DNA was demonstrated by recovering SV5 with the tag restriction sites introduced into the DNA clone, using RT-PCR of the genome RNA and nucleotide sequencing. Rescue of SV5 from DNA did not require expression of the viral V protein as a helper plasmid, suggesting that V protein is not essential for initial replication. The infectious cDNA of SV5 was also manipulated to express green fluorescent protein (GFP) under the control of SV5 transcriptional start and stop signals introduced between the HN and L genes. The amount of GFP that was expressed varied depending on the nature of the newly introduced transcription signals. (C) 1997 Academic Press.