Construction, Expression And Immunogenicity Of Recombinant Prokaryotic System Expressing Dominant T And B Cell Epitopes Tandem Truncated Cap Gene Of PCV2

Porcine circovirus type 2(PCV2) is currently considered to be one of the most economically important viral pathogens in pig populations globally, diseases associated with PCV2 infection are collectively referred to as porcine circovirus associated disease(PCVAD). Among these conditions, PMWS is the most serious disease. PCV2 mainly threat the body’s immune system, cause severe immunosuppressive, thus prone to secondary or mixed infection of other pathogenic microorganisms, seriously endanger herd health. Since PCV2 vaccines were introduced into the world market in 2006, vaccination became a major tool for the control of PCV2 infection. Four commercial vaccines were developed and currently used worldwide, two of them were based on PCV2 Cap protein as immunogens, experimental and field studies have clearly demonstrated the efficacy of PCV2 vaccines in reducing viremia, eliminating PCVAD, and increasing growth performance. However, PCVAD outbreaks in vaccinated herds do occur, which showed vaccination by existed vaccines did not completely prevent infection or spread of PCV2. In addition, such vaccines have possibilities such as low viral titers, recovery of virulence by incomplete inactivation of viruses, high cost, and poor protective efficacy. Thus, development of novel, safe, and effective vaccines is necessary for control of PCV2 infection.The capsid protein(Cap protein), encoded by ORF2 of the viral gene, is the major structural protein of virus and has type-specific epitopes, and it also has long been a target of the recombinant PCV2 vaccine, however, research into other factors that affect the immunogenicity of Cap protein is rarely. Therefore this study was devoted to express PCV2 truncated Cap and T, B cell antigen epitopes fusion proteins in prokaryotic cells and analyze its immunogenicity, the T, B cell antigen epitopes derived from Rep and Cap protein were selected from previous reports, of which the c DNAs were directly synthesized into the p UC-57 vector and sequenced. Then truncated Cap and synthetic epitope genes sequences were inserted into the vector p ET-32 a sequencely to obtain three recombinant plasmids: p ET-B-cap, p ET-T-cap and p ET-T-B-cap. PCR, restriction analysis and DNA sequencing proved that the three recombinant expression plasmids were constructed successfully, next were transformed into E. coli Bl21(DE3) for expression under induction of IPTG. The expressed products were analyzed by SDS-PAGE and further confirmed by Western blot. Finally, The relative molecular masses of expressed constructs were 49, 46 and 54 k Da, respectively and mainly expression in a form of inclusion body, which showed high reactogenicity. Immunization of SPF BALB / c mice with r B-Cap, r T-Cap, and r T-B-Cap proteins respectively, i ELISA antibody detection demonstrated all could specifically evoked high levels of anti-Cap antibodies, among them, group r T-B-Cap achieved the highest antibody level, which sharply contrasted with r Cap and the whole virus inactivated vaccine groups, indicated that Cap protein tandem dominant T, B cell epitopes could markedly enhance its immunogenicity. Good antigenicity of dominant epitopes tandem Cap protein may highlight the potential significant effect of Cap protein based vaccines as the targets for prevention of PCV2 infection, and it may also provides a method for improving the antigenic of epitope-based vaccines.