Abstract:
The main aim of this work was to develop a time-saving and cost-effective purification
method of infectious plant viral nanoparticles. Virions of Tomato bushy stunt virus
(TBSV), which is a member of Tombusvirus genus, were purified by one-step Biogel HT Hydroxyapatite (HA) column chromatography. Extracts from Nicotiana
benthamiana plants infected with TBSV were directly loaded onto the HA column
and eluted by 10 mM sodium phosphate buffer (pH 6.8). A specificity of virions
has been confirmed by immunoblotting and electron microscopy. Homogeneity of
virions was tested by SDS-PAGE, where only 41 kDa polypeptide bands referring
to the capsid protein of TBSV were detected by Coomassie staining. The biological
infectious activity of a purified material was demonstrated by observing TBSVspecific symptoms observed in N. benthamiana plants at 7‒10 days of postinoculation (dpi). Moreover, purified virions were used for immunization of the
BALb/c mouse to raise primary antibodies against the TBSV virus. Our results
show that in low concentrations of sodium phosphate buffer total proteins extracted
from infected plants adsorb to HA sorbent, while viral particles do not adsorb to the
HA matrix and flow throw column due to Ca2+ ions implicated in TBSV virions’
structure. This highly effective and simple virus purification protocol can also be
used for the isolation of other plant virions.
