Purification of Tomato Bushy Stunt Virus Particles by One-Step Hydroxyapatite Column Chromatography

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 Bio-gel 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 TBSV-specific symptoms observed in N. benthamiana plants at 7‒10 days of post-inoculation (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.