Expression And Immunogenicity Of Capsid Protein Of Porcine Circovirus Type Ⅱ Chimeric Neutralizing Epitopes Of Foot-and-mouth Disease Virus

Porcine Circovirus2(PCV2) is one of the primary etiologies of postweaning multisystemicwasting syndrome (PMWS) and has been associated with porcine dermatitis and nephropathy syndrome(PDNS) as well as porcine reproductive disorders. Circoviruses are small nonenveloped icosahedralviruses, named after their circular single-stranded DNA (ssDNA) genome (1767bases). PCV2has onlyone kind of capsid protein (Cap),60copies of which could self-assemble to virus-like particles withicosahedral symmetry. And it has been proved that virus-like particles of PCV2assembled in insect cellsand E.coli have similar biological structure. Our study aims to express Caps with PCV2Caps chimericneutralizing epitopes of foot-and-mouth disease virus (FMDV), and detect the immunogenicity ofchimeric Caps, and further lay foundation of showing exogenous epitopes and studyingepitope-showing vaccines based on PCV2Caps.Caps of PCV2have a nuclear localization sequence (NLS) with41amino acids at N-terminal,which is the non-essential area of Caps assembly. The Monomer of capsid protein PCV2is composed of8β-strands. Loops connecting strands BC/DE/FG/HI are four to nine residues, while loops connectingstrands CD/EF/GH are21to36residues. The long loops contribute to the capsid stabilization.According to above research background, we built five reconstructive PCV2Caps: one chimericCap with neutralizing epitopes of FMDV (132~160aa of O/Mya/98FMDV VP1G-H loop and200~213aa at C-terminal) replacing PCV2NLS; three chimeric Caps with FMDV VP1G-H loopreplacing loops connecting strands CD/EF/GH of PCV2and with common T-cell epitope replacingPCV2NLS; one chimeric Cap with G-H loops from different topologies of FMDV (O/Mya/98, PanAsiaand Cathy) replacing loops connecting strands CD/EF/GH of PCV2at the same time. All the fivechimeric Caps were expressed in E.coli and named ORF2-VP1(N), ORF2-VP1(CD), ORF2-VP1(EF),ORF2-VP1(GH) and ORF2-VP1(All), respectively. The integral PCV2Cap was also expressed inE.coli.The expressed proteins were purified through C-terminal hexahistidine tag using affinitychromatography method and renatured using GSH-GSSG oxidation-reduction system. Oil adjuvantvaccines were prepared using refolding proteins with206as adjuvant and polyinosinic polycytidylicacid (Poly I: C) to immune mice, and evaluate both humoral and cellular immune responses againstFMDV and PCV2, and evaluate the immunogenicity of different chimeric Caps.The detection of specific antibody responses against FMDV of different chimeric Caps showed thatORF2-VP1(N) failed to produce neutralizing antibody against FMDV, indicating N-terminal was notsuitable to insert FMDV B-cell epitope; ORF2-VP1(CD)+Poly（I:C），ORF2-VP1(EF)+Poly（I:C）,ORF2-VP1(GH)+Poly（I:C），ORF2-VP1(All)+Poly（I:C）only produced low-level neutralizingantibody against FMDV, indicating loops CD/EF/GH of PCV2ORF2chimera with FMDV G-H loop were conducive to FMDV-epitope showing and neutralizing antibody production; Only ORF2-VP1(All)without adding Poly（I:C）produced high-level neutralizing antibody against FMDV (P<0.05), indicatingaddition of Poly（I:C）restrained the production of neutralizing antibody against FMDV which still needsfurther validation. And the detection of cytokines including IFN-γ、TNF-α、IL-2、IL-4、IL-6、IL-10、IL12testified Th1-type cellular immune dominated the cellular immune response.