A Study on the Virus-Like Particle Formation of Hepatitis E Virus ORF2 and Rotavirus NSP4 Protein in the Eukaryotic and Prokaryotic Expression Systems

Abstract

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.