| Summary: | 碩士 === 國立中興大學 === 分子生物學研究所 === 100 === Porcine circovirus type 2 (PCV2) was the mainly infectious agent of postweaning multisystemic wasting syndrome (PMWS), one of the most important swine disease with a negative impact concerning on economic losses worldwide. PMWS was clinically characterized by wasting and growth retardation, although other signs such as fever, pallor of the skin, respiratory distress and diarrhoea could be observed as well. The PCV2 capsid proteins (Cap) were the major structural viral proteins encoded by ORF2 could induce host-specific neutralizing antibody. Recombinant baculovirus expression systems have been exploited for the production of vaccines. However, the higher costs in eukaryotic expression systems will be the obstacles when the products are undertaked to the commercially application in the future. Thus, the aim of this study was to construct high efficient recombinant baculoviruses, improving the target protein expression level and immunogenicity in a cost-effective manner. The baculovirus surface display system was used as the platform. After infection, the proteins were expressed and anchored on the plasma membrane of Sf-9 cells. In this study, recombinant baculovirus, BacSC-Cap(d41) with deletion of 41 amino acids in N-terminal side of the Cap protein, hvae been confirmed to express more stably in insect cells than BacSC-Cap(d41)-mcherry with Cap(d41) fused with mCherry by Western blot analysis. Furthermore, four different recombinant baculovirus shuttle vectors, pBacSC-Cap(d41), pBacDD-2Cap(d41), pBacDD-3Cap(d41) and pBacDD-4Cap(d41) for construction of recombinant baculoviruses, namely BacSC-Cap(d41), BacDD-2Cap(d41), BacDD-3Cap(d41) and BacDD-4Cap(d41) respectively, hve been constructed. The results indicated that BacDD-4Cap (d41) was able to express the highest level of Cap(d41) paoteins by Western blot analysis. For the highest efficiency of protein expression, we have tested different conditions, such as infection days, mutiplicity of infection (MOI) and cell numbers. Our results reveal that three days post infection under MOI 5 or 10 showed the highest protein expression level. In vivo experiments, the cell numbers of 10^7, 10^6 and 10^5 infected with recombinant baculoviruses were used as the antigens to immunize the mice, and the results showed all the BacSC-Cap(d41), BacDD-4Cap(d41) and commercial vaccine could elicit the immune response by ELISA analysis. The Cap(d41) protein produced from recombinant baculovirus BacDD-4Cap(d41) could elicit anti-PCV2 neutralizing antibodies, as confirmed by virus neutralization test. Importantly, it also induces IFN-γ further confirming cellular immunity mediated at PCV2-Cap(d41) protein. Our results suggest that the PCV2-Cap(d41) protein can elicite both cellular and humoral immune responses. Taken together, the high efficiency of recombinant baculovirus expression vectors and recombinant baculoviruses have been constructed to develope the PCV2 Cap subunit vaccine.
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