Construction of fowlpox virus vector carrying viral 2A-peptide interlinked immunogenic genes of Avian Avulavirus-1 and IL-18 cytokine
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Date
2020-02
Authors
Rajasekaran, Ranjani
John Kirubaharan, J
TANUVAS
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Abstract
In the present study, a Fowlpox virus (FWPV) plasmid vector was developed to carry three heterologous
genes, which included two immunogenic genes of Avian Avulavirus-1 (AAv1) namely Fusion (F) and
Haemagglutinin-neuraminidase (HN), and IL-18 cytokine. The FWPV plasmid vector, pJFA was constructed
in two intermediate plasmid constructs, pJF7F9 and pJFHNIL18G. The construction of pJF7F9 involved
cloning of F7 and F9 genes of FWPV with modifications in the F7-F9 intergenic region to carry AscI-SalI
restriction enzyme (RE) sites. The construction of pJFHNIL18G involved sequential cloning of F, HN and IL-
18 genes under a synthetic early late promoter (P E/L). These three heterologous genes were interlinked with
two viral 2A-peptides P2A and T2A. Immediately downstream of these three heterologous genes, a marker
gene AcGFP was cloned under a late promoter (P11). This entire construct PE/L-F-P2A-HN-T2A-IL18-P11-
AcGFP in pJFHNIL18G was released and was cloned into AscI-SalI RE site of pJF7F9 to obtain pJFA plasmid.
ThepJFA plasmid upon transfection in chicken embryo fibroblast (CEF) cells previously infected with wild
FWPVshowed AcGFP fluorescence from 48 hours post transfection. The expression of immunogenic genes
F and HN was observed at 72 hours post transfection by western blotting at 66 kDa and 74 kDa respectively.
Similarly, the expression of IL-18 cytokine was confirmed by ELISA. Thus, the expression of F, HN and
IL-18 in the constructed viral 2A-peptide based FWPV vector confirms the usage of this strategy for future
poultry vector vaccine constructions.
Description
TNV_20thMVC_PP_Feb-2020_YSA03
Keywords
Veterinary Science