| Summary: | 碩士 === 中國文化大學 === 生物科技研究所 === 102 === Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome in pigs and has impact on swine-producing industry worldwide. The open reading frame 3 (ORF3) of PCV2 which encodes an 11.9 kDa ORF3 protein has been found to be involved in the pathogenesis of PCV2 infection by its apoptotic activity. The ORF3–based antigens thus represent a traditional approach to develop efficacious oral vaccines for immunization against PCV2. In this study, various ORF3-containing expression vectors, namely pGKF/U-ORF3, pGKF/U-L-ORF3, pGKF/U-ORF2-3b, and pGKF/U-L-ORF2-3b, were transformed to tobacco plants in order to produce the respective subunit recombinant vaccines. Following the Agrobacterium tumefaciens-mediated transformation of 200 leaf samples, 20 ORF3, 15 L-ORF3, 14 ORF2-3b, and 14 L-ORF2-3b putative transgenic tobacco plants were regenerated on the selective medium supplied with 200 mg/L kanamycin and showed GUS expression. The expected sequences of respective ORF3-containing trangenes, 359 bp, 369 bp, 1056 bp, and 1439 bp were amplified from genomic DNA of 19 ORF3 (9.5%), 14 L-ORF3 (7.0%), 12 ORF2-3b (6.0%), and 12 L-ORF2-3b (6.0%) GUS-positive transgenic plants, using specific pair of primers in PCR analysis. On the other hand, integration of 1-2 copies of independent ORF3-containing transgenes into tobacco genome was determined by means of Southern hybridization. The mRNA transcripts of particular transgenes were also found in the PCR-positive transgenic plants. Particularly, expression of respective ORF3 fusion proteins was qualitatively examined via immunobloting (Western blot) assay. However, the results have not clearly shown presence of target proteins, due to either low level of expression or interfering background.
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