[Expression,Purification,Structure Determination and Immunogenicity Assay of Hepatitis E Virus Capsid Protein p495 Derived from Baculovirus-based Insect Cell].

Our objective was to establish a robust method for the expression and purification of hepatitis E virus(HEV)p495protein using a baculovirus-based insect cell expression system; to determine the properties and cryo-EM structure of the resulting virus-like particles(VLPs);and to compare their immunogenicity with p239 particles in the commercial hepatitis E vaccine (Hecolin). The sequence spanning HEV ORF2 amino acids 112-606 in the genotype I HEV isolate was cloned into baculovirus to express recombinant p495 protein. ELISA, analytical ultracentrifugation, size-exclusion chromatography and negative-staining transmission electron microscopy(TEM)were carried out to characterize the physicochemical properties of p495.Recombinant p495 VLPs were obtained successfully from the insect cell expression system with purity of>95%and yield of 15mg/L.The recombinant HEV p495 protein was homogeneous in solutions. The 3Dstructure of p495 VLPs was determined by cryo-EM;it was icosahedral with T=1arrangement,and showed good congruency with the crystal structure in the literature(PDB ID:2ZZQ).In mouse vaccination experiments,p495 conferred comparable immunogenicity with that of p239 antigen in Hecolin. Thus, a robust and scalable approach to obtain homogeneous, immunogenic HEV p495 VLPs has been established. This study may assist investigations of HEV receptors, epitope mapping, vaccine improvement and so on.