Construction And Efficacy Evaluation Of Porcine Circovirus 2d Virus-like Particles And The Cap-based Chimeric VLPs

Porcine circovirus type 2(PCV2)is the pathogen of post-weaning piglet multisystem failure syndrome(PMWS),which leads to severe immunosuppression and subsequent coinfection with other pathogens.This mixed infection causes huge economic losses to the pig industry.Currently,vaccination is the most effective way to prevent PCV2 infection.There are 8 subtypes of PCV2(PCV2a?h).With the increased immune pressure,the main epidemic genotypes of PCV2 in China have shifted from PCV2b to PCV2d in recent years.However,the vaccine strains currently used in commercial vaccines are mainly PCV2a and PCV2b.The cross protection against PCV2d has not been well studied yet.As the main structural protein of PCV2,Cap protein is the target for genetic engineering vaccine development.We have studied the immunogenicity,epitopes and the potential to allow foreign peptides insertion of PCV2d VLPs.All provides new strategies for the research and development of PCV2d VLPs subunit vaccines.This study are as follows:1.The soluble expression of PCV2d Cap protein in E.coliBased on PCV2d subtype strain HLJ1501(GenBank:KY940534.1),the coding sequence without N-terminal 16 amino acids of ORF2 protein was synthesized after sequence optimization according to prokaryotic codon preference.The recombinant expression plasmid pET28a-2dCap-16 was transformed into Rosetta expression strain,and the expression conditions(IPTG,temperature,induction time)were optimized.SDS-PAGE and Western blot results confirmed that these recombinant proteins were successfully secreted and had the correct immunogenicity against PCV2,which could provide experimental materials for the diagnosis of PCV2 and the preparation of VLPs vaccine.2.Generation of PCV2d Cap protein VLPs vaccine and preliminary evaluation of the immunogenicity.The imidazole solution of purified Cap protein was replaced by Cap VLP assembly buffer using ultrafiltration dialysis system,a large number of virus-like particles with regular shape and diameter of about 17 nm could be seen under transmission electron microscope.Mice were immunized with assembled Cap VLPs and monomeric Cap protein respectively,commercial vaccine and PBS control group were also included.The results of serum ELISA detection showed that the same dose of Cap VLPs could induce higher PCV2 specific antibodies than monomeric Cap,and the low dose VLPs vaccine could induce the same antibody level as the commercial vaccine.These results indicate that the Cap VLPs prepared in this study has better immunogenicity than the monomer Cap protein,and it can be used as a subunit vaccine candidate for PCV2d prevention and control.3.The effect of C-terminal fusion with foreign protein on PCV2d Cap VLPs assembly.In order to explore the ability of Cap VLPs as a protein delivery vector,using the constructed pET28a-2dCap-d16 as a backbone,two copies of neutralizing epitopes(ep,TAVSPTTLR)derived from classical swine fever virus envelope protein E2 were inserted into the C-terminal of Cap gene,The recombinant plasmid pET28a-2dCap-d16-2ep was transformed into Rosetta strain for protein expression.SDS-PAGE and Western blot showed that Cap-2ep VLPs could react not only with PCV2 polyclonal antibody but also with E2 monoclonal antibody.Transmission electron microscopy revealed that the Cap-2ep fusion protein could form virus-like particles of uniform size,which was similar to the shape and size of wild-type VLPs.When the mice were immunized with Cap-2ep VLPs,ELISA analysis showed that Cap-2ep VLPs induced the same level of antibody as wild-type VLPs.Both VLPs could induce a high level of specific immune response in mice.Therefore,the C-terminal insertion of foreign protein has no effect on the ability of Cap self-assembly into VLPs,and the immunogenicity of Cap was completely retained.These results provide new ideas for the development of bivalent vaccine based on PCV2d VLPs4.Production and characterization of monoclonal antibodies against PCV2 Cap protein.In order to expand the understanding of the antigenic epitopes of PCV2d Cap protein,BALB/c mice was immunized with the PCV2d Cap protein.Combining with traditional hybridoma technique and ELISA screening,six hybridoma cell lines 4D4,4H8,5E3,5H7,6F3 and 3E3 were obtained.All the 6 McAbs could react specifically with PCV2b strains by IFA and could be used to identify PCV2 strains.Using different truncated protein of Cap protein,the reactivity analysis showed that all the McAbs recognized the antigenic epitope 207YDQDYN212,which belonged to a newly discovered PCV2 epitope in this study.Combined with the amino acid sequence alignment analysis of Cap protein,the D210E mutation in the epitope has no effect on the recognition of PCV2b by monoclonal antibody,which further indicates that this kind of monoclonal antibody is suitable for the detection and diagnosis of PCV2.