High-level Expression Of Porcine Circovirus Type 2 Cap Protein By Recombinant Baculovirus And Immunological Characteristic

Porcine circovirus type 2?PCV2?is the causative agent of postweaning multisystemic wasting syndrome?PMWS?.High PCV2 viremia and viral load in tissues,granulomatous inflammation,depletion of lymphocytes and dysfunction of the lymphoid system causing immunosuppression were characterized as the hallmarks of severe PCV2 infection.Since its discovery in 1991,PCV2 has caused a serious economic problem for the swine industry around the world.The vaccination is the main way to prevent PCV2 infection.The PCV2commercial vaccines consist of inactivated vaccines,virus-like particles vaccine using prokaryotic expression system and subunit vaccines using baculovirus expression system.However,inactive vaccines and subunit vaccines expressed by prokaryotic expression system may have insufficient antigen concentration,or more endotoxin traces,which results in immunity failure and remarkable side effects.Most large pig farms tend to use imported subunit vaccines of high price.Therefore,the recombinant baculovirus that express high-level Cap protein were constructed by Bac-to-Bac^TMM baculovirus expression system.The main work and results are as follows:1 The pFastBacI vector was modified with an promoter and homologous enhancer sequence cis-linked to the downstream and upstream of polh promoter,respectively.2 The optimized Cap gene deleted by N-terminal nuclear location signal?NLS?sequence that was added to 6×His tag at C-terminal was cloned into multiple cloning site?MCS?.3 The recombinant baculovirus were rescued by transfection recombinant bacmids into Spodoptera frugiperda 9?Sf9?cells and the recombinant protein was expressed efficiently in High Five^TM?H5?cells under optimized expression condition.4 The immunogenicity of the recombinant protein was evaluated by vaccination in mice using intramuscular injection route.A booster injection was carried out at an interval of two weeks and serums were separated at 21,28,35,42days respectively.Simultaneously,commercial inactived vaccine,subunit vaccine,wild baculovirus and PBS were set up as controls at the same dose and way.Results:1 The recombinant baculovirus that efficiently expressed PCV2 Cap protein were constructed via the modified Bac-to-Bac^TM expression cassette.2 The expression of Cap protein reached 150mg/L and above in H5 cells under optimized conditions.3 Comparing immune effect between the Cap protein and commercial vaccines,the former posesess significant advantages.