Production of capsid proteins of rat hepatitis E virus in Escherichia coli and characterization of self-assembled virus-like particles

•We produced rat HEV ORF2 proteins in E. coli that formed virus-like particles (VLPs).•Truncated ORF2 proteins (amino acids 357–614) self-assembled most efficiently.•The yield of VLPs was 15–20 mg per 100 ml culture medium and the purity was >90%.•The VLPs entered PLC/PRF/5 cells and inhibited the antibody-mediated neutralization.•The VLPs provided high immunogenicity in mice. Rat hepatitis E virus (HEV) has been isolated from wild rats worldwide and the potential of zoonotic transmission has been documented. Escherichia coli (E. coli) is utilized as an effective system for producing HEV-like particles. However, the production of rat HEV ORF2 proteins in E. coli forming virus-like particles (VLPs) has not yet been reported. In this study, nine rat HEV ORF2 proteins of the ratELOMB-131L strain with truncated N- and C-termini (amino acids 339–594, 349–594, 351–594, 354–594, 357–594, 357–599, 357–604, 357–609, and 357–614 of ORF2 protein) were expressed in E. coli and the 357–614 protein self-assembled most efficiently. A bioanalyzer showed that the purified 357–614 protein has a molecular weight of 33.5 kDa and a purity of 93.2%. Electron microscopy revealed that the purified 33.5 kDa protein formed VLPs with a diameter of 21–52 (average 32) nm, and immunoelectron microscopy using an anti-rat HEV ORF2 monoclonal antibody (TA7014) indicated that the observed VLPs were derived from rat HEV ORF2. The VLPs attached to and entered the PLC/PRF/5 cells and blocked the neutralization of rat HEV by TA7014, suggesting that the VLPs possess the antigenic structure of infectious rat HEV particles. In addition, rat HEV VLPs showed high immunogenicity in mice. The present results would be useful for future studies on the development of VLP-based vaccines for HEV prevention in a rat model and for the prevention of rat HEV infection in humans.