Preparation And Immunogenicity Evaluation Of Porcine Circovirus Virus Type 3 Virus-like Particles

Porcine circovirus type 3(PCV3),a new circovirus first discovered in the United States in 2016,is now widespread epidemic worldwide.PCV3 is susceptible to pigs of all ages and can cause respiratory disease,digestive disease,reproductive disorders and various inflammatory reactions,posing a huge threat to the global pig industry and related industries.PCV3 is currently difficult to isolate from cell lines,which has severely limited research into the pathogenic characteristics,pathogenesis and vaccine development of the virus.However,virus like particles(VLP)can mimic the invasion process of native virus particles and reveal the characteristics of the virus,which has important research significance and potential application.In this study,we expressed the capsid(Cap)protein of PCV3,which has good immunogenicity and can self-assemble into VLP,laying the preliminary basis for the research of PCV3.To evaluate the PCV3 VLP,it is needed to prepare an anti-PCV3 monoclonal antibody.In this study,the truncatedCap gene(dCap),which deleted the nuclear localization signal,was amplified by PCR from clinical samples.The dCap gene was then cloned into the prokaryotic expression vector pET32 a.The recombinant plasmids were transformed into BL21(DE3)competent cells,and the expression conditions were optimized and the solubility was analyzed using SDS-PAGE.In addition,the dCap protein was purified using nickel affinity chromatography and employed as an antigen to immunize 6-week-old BALB/c mice.Finally,the monoclonal antibody against PCV3 dCap was prepared using ELISA,subcloning,ascites preparation and purification.The results showed that PCV3 dCap protein was successfully expressed in E.coli,which was mainly expressed in the form of inclusion bodies.Three monoclonal antibody,3C2-1A,4F9-6D and 9B5-3H,were screened.These results indicate that the PCV3 monoclonal antibody was successfully prepared in this study,laying the basis for the subsequent identification of VLP.To prepare PCV3 VLP,the full-length Cap gene of PCV3 was codon optimized according to Sf9 insect cells,and then cloned into baculovirus transfer vector pYBDM-IM.Positive recombinant plasmids were transformed into SW106 Am competent cells to obtain recombinant Bacmid-IM-PCV3 Cap.Positive colonies were screened and transfected into Sf9 cells to obtain recombinant baculovirus BV-IM-PCV3 Cap.The virus titer was detected using endpoint dilution method,and the expression of Cap protein was detected using Western blot,the VLP was purified by density gradient centrifugation and examined using transmission electron microscopy.The PCV3 VLP was prepared as vaccine to immune the 6-week-old BALB/c mice,and the specific antibody level in the serum was measured by indirect ELISA.The result showed that recombinant baculoviruses expressing PCV3 Cap protein were successfully constructed.Western blot analysis revealed that the expressed product was approximately 27 k Da.PCV3 VLP can stimulated high level of specific antibodies in mice,and the antibody titer could reach 12,800.In summary,PCV3 virus-like particles were successfully prepared using a baculovirus expression system,and it showed good immunogenicity in mice,which laid the foundation for further develgopment of PCV3 VLP vaccine.