Key result
Truncated ORF2-NSP4 protein forms virus-like particles in Baculovirus systems but fails in E. coli.
The truncated ORF2-NSP4 protein successfully forms virus-like particles in the Baculovirus expression system (Sf9 cells) but not in E. coli, suggesting its potential as a mucosal vaccine candidate.
Supports baculovirus VLP production for further study; leaves open immunogenicity and protection in animal models before clinical consideration.
Background: Hepatitis E virus (HEV) is able to induce fulminant hepatitis E infection in immunosuppressed individuals. Previous studies showed that the Baculovirus expression system (BES) could be used for developing hepatitis E virus-like particles (VLPs). Objectives: In the present study, the formation of VLPs of the recombinant proteins of HEV truncated open reading frame 2 (ORF2; 112 - 607) and Rotavirus non-structural protein 4 (NSP4) (OSU-a) were investigated in BES and Escherichia. coli. Methods: To construct VLPs, the truncated ORF2-NSP4 protein was expressed in BES. The expression of protein was confirmed by western blot. This protein was expressed in E. coli. The truncated ORF2-NSP4 gene was subcloned in pET28a, and then transformed into E. coli DH5α. The confirmed colonies were transformed into E. coli BL21. The solubility of protein were checked by SDS-PAGE and western blot analysis. In the final step, to verify the VLP formation in BES and E. coli, the recombinant proteins were stained with 2% uranyl acetate and checked by transmission electron microscopy (TEM). Results: The 75 KDa truncated ORF2-NSP4 protein was successfully expressed in Sf9 cells, assembled, and formed VLP according to TEM results. In the prokaryotic expression system (E. coli), the ORF2-NSP4 gene was successfully subcloned and expressed in BL21 but VLP was not detected in TEM analysis. Conclusions: The truncated ORF2-NSP4 VLP were efficiently expressed in the SF9 cells, as a potential mucosal vaccine against HEV and Rotavirus. In the prokaryotic expression system (E. coli), the ORF2-NSP4 gene was successfully expressed.
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Makvandi et al. (2017) studied Hepatitis E virus and Rotavirus. Baculovirus expression system (BES) vs. Escherichia coli (prokaryotic expression system) was evaluated on Virus-like particle (VLP) formation. The truncated ORF2-NSP4 protein successfully formed virus-like particles in the Baculovirus expression system (Sf9 cells) but not in the prokaryotic E. coli system.
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