Conclusion
The results indicate BEVS as an appropriate and efficient system for generating CPV-VLPs, and the used method based on two-stage ultracentrifugation was appropriate for purifying these nanoparticles. Produced nanoparticles can be used as the biologic nano-carriers in future studies.
Methods
Sf9 insect cells were transfected with constructed recombinant bacmid shuttle vector encoding an enhanced green fluorescent protein (EGFP) and CPV-VP2 by the cationic lipids of Cellfectin II. Subsequently, two recombinant baculoviruses expressing EGFP and VP2 were produced and expression of VP2 was increased under the optimal condition. In consequence, the CPV-VLP nanoparticles composed of recombinant VP2 subunits were extracted. The purity of VLPs was then evaluated by SDS-PAGE, and the structural integrity and quality of the final product were evaluated by TEM and HA methods. Eventually, the size distribution of the produced biological nanoparticles and their uniformity were determined by the DLS method.
Objective
Canine parvovirus (CPV) is a small virus without an envelope that consists of three viral proteins including VP1, VP2, and VP3. Exclusively, the VP2 can form a typically CPV-sized virus-like particle (CPV-VLP) that can be used as a biological nanocarrier for diagnostic and therapeutic purposes since these VLPs can target cancer cells specially through the transferrin surface receptors (TFRs). Consequently, we aimed to produce these nanocarriers to be used for specific targeting of cancer cells.
Results
The expression of EGFP protein was confirmed by fluorescent microscopy, and the expression of VP2 protein was evaluated by SDS-PAGE and western blotting. Infected Sf9 insect cells also showed cytopathic effects (CPEs), and the maximum expression of VP2 occurred at MOI of 10 (pfu/cell) at the harvest time of 72 h post-infection (hpi). After performing various stages of purification, buffer exchange, and concentration, the quality and structural integrity of the VLP product were confirmed. The results of the DLS technique showed the presence of uniform particles (PdI below 0.5) with an approximate size of 25 nm.