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Acta Virologica Vol.58, No.3, p.274-277, 2014 |
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Title: Transient expression of the influenza A virus PB1-F2 protein using a plum pox virus-based vector in Nicotiana benthamiana | ||
Author: M. KAMENCAYOVÁ, I. KOŠÍK, J. HUNKOVÁ, Z. W. ŠUBR | ||
Abstract: PB1-F2 protein of influenza A virus (IAV) was cloned in a plum pox virus (PPV) genome-based vector and attempts to express it in biolistically transfected Nicotiana benthamiana plants were performed. The vector-insert construct replicated in infected plants properly and was stable during repeated passage by mechanical inoculation, as demonstrated by disease symptoms and immunoblot detection of PPV capsid protein, while PB1-F2-specific band was more faint. We showed that it was due its low solubility. Modification of sample preparation (denaturation/solubilization preceding the centrifugation of cell debris) led to substantial signal enhancement. Maximal level of PB1-F2 expression in plants was observed 12 days post inoculation (dpi). Only 1% SDS properly solubilized the protein, other detergents were much less efficient. Solubilization with 8M urea released approximately 50% of PB1-F2 from the plant tissues, thus the treatment with this removable chaotropic agent may be a good starting point for the purification of the protein for eventual functional studies in the future. |
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Keywords: viral vector; potyvirus; biolistics; heterologous expression | ||
Year: 2014, Volume: 58, Issue: 3 | Page From: 274, Page To: 277 | |
doi:10.4149/av_2014_03_274 |
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