Multi-epitope Vaccines Against Porcine Circoviruse Type2

Porcine Circovirus type2（PCV2）, as the main pathogen causing porcine circovirus associativediseases（PCVAD）such as of PMWS, has taken major economic losses to the pig industry worldwide.Nowadays, Vaccine is the most effective method against the infection and spread of PCV2, so PCV2vaccine has been the focus of scientists around the world. Additionally, because multi-epitope vaccineshave many advantages compared with traditional vaccines, the PCV2multi-epitope vaccine was thepurpose of this research.The effect of the multi-epitope vaccines are determined by the vector and epitopes in some way.BCG heat shock protein65(the heat shock protein65) was used to the vector and the molecularadjuvant. Hsp65can advance antigenic peptides entering into the APC (antigen presenting cells) in theimmune response, and antigenic peptides will be processed, bind to MHC-I moleculesand present toAPC’s surface, in order to stimulate and activate specific CTLs mediated immune response. In this studysix epitopes were chose, including five epitopes from the Cap protein and one epitope from the Repprotein. These six epitopes were directly used to build five series, which were cloned into pGEM-Teasy vector together with BCG Hsp65gene by three restriction sites （Nco I and Spe I and Sal I）.And then the recombination of fragments were transferred into the expression vector pET30a by tworestriction sites （Nco I and Sal I） and induced expression in E. coli BL21（DE3）. Finally, these fivepurified fusion proteins were inject into mice, and relative data were collected such as the detection ofspecific antibodies, the lymphocyte proliferation and the typing of antibodies and levels of severalcytokines.Results showed that five kinds of fusion protein expression were able to reach more than80%and protein expression by western-blot analysis found that five kinds of fusion protein has goodreactionogenicity. The data indicated that five kinds of proteins could induce mouse high levels ofspecific antibodies and neutralizing antibody against PCV2, of which HSP e-2and HSP-e-3were betterthan others; these five kinds of proteins can stimulate lymphocyte proliferation in some degree, ofwhich effect of HSP-e-3, HSP-e-4and HSP-e-5were better than others. In summary, HSP-e-3has thebest effect as a single recombinant protein, in spite that the effect of HSP-e-3induced cellular immunityless than HSP-e-4and HSP-e-5. We can take the mixed immune with HSP-e-2or HSP-e-3and HSP-e-4or HSP-e-5for advance in practice. The total result suggested that the fusion proteins were valueable toidentify the effect of pig experiments in advance, and this research provided basic data for latermulti-epitope vaccine research against PCV2.