TY - JOUR
T1 - Generation of recombinant Orf virus using an enhanced green fluorescent protein reporter gene as a selectable marker
AU - Ning, Zhangyong
AU - Peng, Yongzheng
AU - Hao, Wenbo
AU - Duan, Chaohui
AU - Rock, Daniel L.
AU - Luo, Shuhong
N1 - Funding Information:
We thank Diego G Diel (USDA, ARS, Southeast Poultry Research Laboratory) and Gustavo Delhon (Department of Veterinary and Biomedical Sciences) for the technique support. This work was supported by the grant (No. 31070138 and No. 31170147 to Shuhong Luo) from the National Natural Science Foundation of China (NSFC).
PY - 2011/12/22
Y1 - 2011/12/22
N2 - Background: 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. Routine selection of the recombinant parapoxvirus is time-consuming and labor intensive. Therefore, developing a novel method for selection is critical.Results: In 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.Conclusions: This 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.
AB - Background: 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. Routine selection of the recombinant parapoxvirus is time-consuming and labor intensive. Therefore, developing a novel method for selection is critical.Results: In 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.Conclusions: This 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.
UR - http://www.scopus.com/inward/record.url?scp=84055219457&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84055219457&partnerID=8YFLogxK
U2 - 10.1186/1746-6148-7-80
DO - 10.1186/1746-6148-7-80
M3 - Article
C2 - 22192523
AN - SCOPUS:84055219457
SN - 1746-6148
VL - 7
JO - BMC Veterinary Research
JF - BMC Veterinary Research
M1 - 80
ER -