Abstract
Hepatitis E virus (HEV) is associated with acute hepatitis disease, which may lead to chronic disease in immunocompromised individuals. The disease is particularly severe among pregnant women (20–30% mortality). The only licensed vaccine against HEV, which is available in China, is the Escherichia coli purified recombinant virus-like particles (VLPs) encompassing the 368–660 amino acids (aa) of the viral ORF2 protein. The viral capsid is formed by the ORF2 protein, which harbors three glycosylation sites. Baculo virus expression system has been employed to generate a glycosylated VLP, which encompasses 112–608aa of the ORF2 protein. Here, we sought to produce a recombinant VLP containing 112–608aa of the ORF2 protein in Pichia pastoris (P. pastoris) expression system. The cDNA sequence encoding 112–608aa of the ORF2 protein was fused with the α-mating factor secretion signal coding sequence (for release of the fusion protein to the culture medium) and cloned into the yeast vector pPICZα. Optimum expression of recombinant protein was obtained at 72 h induction in 1.5% methanol using inoculum density (A600) of 80 and at pH-3.0 of the culture medium. Identity of the purified protein was confirmed by mass spectrometry analysis. Further studies revealed the glycosylation pattern and VLP nature of the purified protein. Immunization of BALB/c mice with these VLPs induced potent immune response as evidenced by the high ORF2 specific IgG titer and augmented splenocyte proliferation in a dose dependent manner. 112–608aa ORF2 VLPs produced in P. pastoris appears to be a suitable candidate for development of diagnostic and prophylactic reagents against the hepatitis E.
Highlights
Hepatitis E virus (HEV) is a single-stranded, positive-sense RNA virus with a size of 27–34 nm, belonging to the family Hepeviridae
Level of 112–608aa ORF2 protein in the culture medium at different time point was measured by enzyme linked immunosorbent assay (ELISA), which showed that clone D1 expressed more 112–608aa ORF2 compared to clone C1 (Figure 1A)
ELISA data showed that the highest yield of 112–608aa ORF2 was obtained by 72 h incubation with 1.5% methanol (Figure 1C). pH analysis of the culture medium demonstrated that pH 3.0 is optimal for the maximum yield of the 112–608aa ORF2 (Figure 1D)
Summary
Hepatitis E virus (HEV) is a single-stranded, positive-sense RNA virus with a size of 27–34 nm, belonging to the family Hepeviridae. It is a major cause of acute viral hepatitis (Smith et al, 2014; Nan and Zhang, 2016). The mortality rate ranges from 0.5 to 3% in young adults and increases up to 30% in pregnant women (Chaudhry et al, 2015). Genotype 3 and genotype 4 are highly diverse and zoonotic with an expanded host range. All the genotypes are antigenically conserved and there is only one serotype, making the development of a univalent hepatitis E vaccine reasonable (Schlauder and Mushahwar, 2001; Emerson et al, 2006; Lu et al, 2006)
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