Functional study of intrinsically disordered region of porcine circovirus type 2 capsid protein for fusing foreign epitope.

Abstract

Sixty capsid protein (Cap) subunits of porcine circovirus type 2 (PCV2) can be assembled into a virus-like particle (VLP), serving as versatile nanoplatforms for foreign peptides (such as B-cell epitopes) display. In our previous work, an intrinsically disordered region (IDR) within the Cap was identified and exposed on the surface of the VLP. IDRs are structurally flexible and lack a defined structure, facilitating the integration of foreign peptides while preserving proper protein folding. In this study, we systematically engineered this IDR to fuse varying copy numbers (1-6) of a B-cell epitope (B5-E1) derived from porcine parvovirus 1 structural protein. The mutant Cap subunits were expressed in Escherichia coli and purified via affinity chromatography. Remarkably, these mutant subunits retained the ability to self-assemble into VLPs in vitro, as confirmed by transmission electron microscopy (TEM) and gel filtration chromatography. Notably, these chimeric VLPs (cVLPs) maintained the ability to enter PK15 cells, comparable to their wild-type counterpart. Critically, these cVLPs elicited a robust humoral immune response in a mouse model, and antibodies against both PCV2 (the antibody titers reached 1:204800) and epitope B5-E1 (the antibody titers reached 1:3200) were detected in ELISA. Importantly, cVLPs exhibited optimal configuration-dependent immunogenicity, with cVLPs-I2 (2 epitope copies) inducing the highest epitope B5-E1-specific antibody levels. These results demonstrate the promising potential of PCV2 VLPs as versatile epitope-display platforms, while establishing the IDR as a suitable fusion site that maintains both structural integrity and biological functionality.