Simplified recombinational approach for influenza A virus reverse genetics
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- 作者：Shuai Wang ; Qinfang Liu ; Juan Pu ; Yishan Li ; Liya Keleta ; Yu-Wen Hu ; Jinhua Liu ; Earl G. Brown
- 关键词：Influenza virus ; Reverse genetics ; Recombination
- 刊名：Journal of Virological Methods
- 出版年：2008
- 出版时间：July 2008
- 年：2008
- 卷：151
- 期：1
- 页码：74-78
- 全文大小：299 K
文摘
Influenza A virus (FLUAV) reverse genetics requires the cloning of all eight viral genome segments into genomic expression plasmids using restriction enzyme cleavage and ligation. Herein is described the construction of a pair of plasmid vectors and their use in RecA Escherichia coli for direct recombination with influenza cDNA for reverse genetics. This approach is simpler; avoiding restriction digestion and ligation while maintaining the required orientation of genome segments. For this recombinational approach two plasmid constructs were generated, pHH21A and pHH21G, that both possess a 25 nucleotide recombination cassette comprised of the consensus 5′ and 3′ ends of the negative strand divided by a StuI cleavage site, but that differ at position 4 from the 3′ end due to the presence of an A or G nucleotide (plus sense) to correspond to differences among genome segments. Using the described procedure it was possible to clone viral cDNA genomes of several avian and human FLUAVs into genomic expression plasmids in a single recombination step. This novel approach to generating sets of genomic plasmid constructs for reverse genetics reduces the time and complexity of procedures thus avoiding complications that would delay rescue of viral genomes for vaccine production or biological characterization and analysis.