Improved dual promotor-driven reverse genetics system for influenza viruses.
作者： Ahmed Mostafa , Pumaree Kanrai , John Ziebuhr , Stephan Pleschka
DOI: 10.1016/J.JVIROMET.2013.07.021
关键词: Virology 、 Biology 、 Vector (molecular biology) 、 Plasmid 、 Viral protein 、 Genetics 、 Cloning vector 、 DNA polymerase I 、 Reverse genetics 、 Gene 、 Complementary DNA
摘要: Reverse genetic systems for influenza A virus (IAV) allow the generation of genetically manipulated infectious from a set transfected plasmid DNAs encoding eight genomic viral RNA segments (vRNA). For this purpose, cDNAs representing these vRNA are cloned into specific vectors that vRNA-like transcripts using polymerase I (Pol I). In addition, plasmids support transcription mRNA by II II), leading to expression protein(s) encoded respective transcripts. an effort develop system further, we constructed bi-directional vector pMPccdB. It is based on pHW2000 (Hoffmann et al., 2000b) but contains additionally (i) ccdB gene whose lethal most Escherichia coli strains and therefore used as negative selection marker (ii) more efficient AarI cloning sites flank either side. Furthermore, modified one-step restriction/ligation protocol insert desired cDNA pMPccdB DNA. Both use improved were shown facilitate engineered IAV illustrated in study rescue 2009 pandemic isolate A/Giessen/6/2009 (Gi-H1N1).
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