Objective The capsid protein (Cap) of eel circovirus (EeCV) was prepared using a prokaryotic expression system for the development of an EeCV subunit vaccine and related biological products.
Method The genome sequence of EeCV-Cap (GenBank accession number: NC_023421.1) was used as the reference for codon optimization and plasmid synthesis. The Cap gene was amplified with designed specific primers and cloned into pET-32a vector to construct the pET32a-EeCV-Cap recombinant expression plasmid. EeCV-Cap recombinant protein was obtained by transforming the recombinant plasmid into the host bacterium BL21 (DE3) followed by an IPTG induction. Western blot was performed to confirm expression of the recombinant Cap.
Result The recombinant pET32a-EeCV-Cap was mainly expressed as a soluble protein in E. coli. A purified target protein was obtained using nickel affinity chromatography with the highest expression achieved with the final IPTG concentration of 0.5mM for the induction at 20°C in 16h. A single band with the calculated molecular weight of 31kDa was detected by Western blot. Under a transmission electron microscope, the negative phosphotungstic acid-stained protein suspension showed numerous regular virus-like particles (VLPs) that were 20nm in diameter.
Conclusion The solubility expression of EeCV-Cap in E. coli was realized in this study to visualize the VLPs. Using the modified nickel affinity chromatography, a single target protein was obtained that could spontaneously assemble into VLPs in vitro for the development of an EeCV subunit vaccine and related products.
DownLoad: