| Summary: | 碩士 === 國立臺灣大學 === 獸醫學研究所 === 100 === The capsid (Cap) protein encoded by the PCV2 ORF2 gene may be an potential candidate for vaccine development. The objective of the study was to evaluate the immunogenicity of porcine circovirus type 2 (PCV2) DNA vaccines in mice model. Previously studies showed native PCV2 ORF2 encoded capsid protein(Cap)demonstrated low transformation yield and almost undetectable PCV2-Cap expression levels in either prokaryotic or eukaryotic system. It has been noted that deletion of the nuclear localization signal (NLS) region of the Cap protein led to a higher level expression of the dCap protein. Therefore, these constructs were performed in the present study. We have constructed the pcDNA3.1/V5-His-TOPO, a eukaryotic expression vector containing the human cytomegalovirus promoter,and a pET 21,a Escherichia coli expression vector containing the T7 RNA polymerase promoter, respectively. Further,the recombinant expression plasmids were constructed. Protein expression and its immunogenicity was confirmed by Western blot analysis and mice model. Four DNA constructs included (1) PCV2a native cap gene(pcDNA-2a); (2)PCV2b native cap gene (pcDNA-2b);(3)PCV2a modified cap gene (pcDNA-Δ2a);(4)PCV2b modified cap gene (pcDNA-Δ2b).One hundred μg of constructed DNA were injected into mice intramuscularly for four times at a 2-week interval. Serum samples were collected at various designated time points for the measurement of PCV2-specific antibodies and splenocytes were isolated at time of sacrifice for lymphocyte blastogenesis assay. The results showed that Coomassie blue staining of cell pellets of pET-Δ2a and pET-Δ2b were noted in E.coli system after 0.1 and 0.5 mM IPTG induction. The size of each expression protein were 24 kDa and 22 kDa respectively, and only pET-Δ2a showed weak expression in the Western blot analysis. However, weak results were both noted in the Coomassie blue staining and Western blot analysis in all recombinant subunit protein expressed in transfected PK-15 cells. In addition, weak intracytoplasmic positive signals were noted in PK-15 cells transfected with various recombinant constructs by IIFA. The immunogenicity assay of four constructs in mice model reveals no significant cell-mediated response in MTS assay and flow cytometry, and no significant humoral response in blocking ELISA assay and IIFA.
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