Abstract
BackgroundReporter genes are often used as a selectable marker for generation of recombinant viruses in order to investigate the mechanism of pathogenesis and to obtain candidate vaccine viruses. Routine selection of the recombinant parapoxvirus is time-consuming and labor intensive. Therefore, developing a novel method for selection is critical.ResultsIn this study, we developed a rapid method to generate recombinant Orf viruses (ORFV) based on the enhanced green fluorescent protein (EGFP) reporter gene as a selectable marker. The coding sequence of EGFP gene was amplified from pEGFP-N1 vector and subcloned into the pZIPPY-neo/gus plasmid under the control of the early-late vaccinia virus (VACV) VV7.5 promoter and flanked by two multiple cloning sites (MCS) to generate a novel transfer vector pSPV-EGFP. Using the pSPV-EGFP, two recombination cassettes pSPV-113LF-EGFP-113RF and pSPV-116LF-EGFP-116RF were constructed by cloning the flanking regions of the ORFV113 and ORFV116 and inserted into two MCS flanking the EGFP gene. Using this novel system, two single gene deletion mutants OV-IA82Δ113 and OV-IA82Δ116 were successfully generated.ConclusionsThis approach shortens the time needed to generate recombinant ORFVs (rORFVs). Thus, the pSPV-EGFP vector provides a direct, fast, and convenient way to manipulate the recombinant viruses, indicating that it is highly suited for its designed purpose.
Highlights
Reporter genes are often used as a selectable marker for generation of recombinant viruses in order to investigate the mechanism of pathogenesis and to obtain candidate vaccine viruses
In previous studies [23,24,25], the pZIPPY-neo/gus vector containing the neo/gusA cassette was initially used to construct recombinant cassettes based on the flanking regions of ORFV002, ORFV012, ORFV024, ORFV113, ORFV116, ORFV120 and ORFV121
We have shown that generating rORFVs is easy and efficient using the enhanced green fluorescent protein (EGFP) reporter gene and is faster than conventional neo/gusA methods
Summary
Reporter genes are often used as a selectable marker for generation of recombinant viruses in order to investigate the mechanism of pathogenesis and to obtain candidate vaccine viruses. To investigate the role of these genes during virus infection, one approach to studying uncharacterized genes is to create mutations and/or deletions of specific ORFV genes to disrupt their function. This can be achieved by using transfer vectors designed to insert into site-specific locations of the viral genome via homologous recombination [6]. The experimental procedures to generate and isolate rORFVs are adapted from standard protocols used in generation of the vaccinia virus This protocol is labor intensive and time consuming [7,8]
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