In the framework of the International Atomic Energy Agency (IAEA) Technical Cooperation Project TUN5032 (http://www.aquavac-ir.tn/), recombinant VLPs were expressed in prokaryotic systems under the control of different radiation-inducible promoters (AquaVac-ir process-2).
Briefly, the total RNA was used for all-in-one step amplification of the transcript (1040 bp) encoding the Nodavirus P-capsid protein with high fidelity Reverse Transcriptase. Recombinant expression vectors were designed (15 distinct constructions), synthetized and used to transform electrocompetent Escherichia coli strains. Each recombinant clone was cultured in a selective medium and recombinant P-capsid expression was induced under optimized UV irradiation conditions. The apparent molecular weight (MW) of the P-capsid protein, estimated at 42 kDa, was observed in vitro through SDS-PAGE and subsequently detected via immunoblotting. The assay utilized a rabbit anti-Nodavirus polyclonal antibody, followed by an HRP-conjugated anti-rabbit secondary antibody for visualization. Upstream process and downstream processes were optimized for successful carrying out production and purification steps at lab scale.
The primary results demonstrated that P-capsid production driven by a UV-inducible promoter (Construction c-1) reached a yield comparable to that obtained using synthetic IPTG induction (Construction c-2). The recombinant virus-like particles (VLP) were successfully isolated throughout ultracentrifugation, as confirmed by Transmission Electronic Microscopy (TEM). These findings represent a significant advancement in the development of a recombinant vaccine, using radiation-inducible promoters, against Nodavirus-induced encephalopathy, contributing to improved health management in aquaculture.