Generation of recombinant occlusion bodies containing the capsid of porcine circovirus type 2 for developing safe vaccine against PCV2

• Main Authors: Chun-kuang Lin, 林俊光
• Other Authors: Jin-Ching Lee
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/90901327472578436325

碩士 === 高雄醫學大學 === 生物科技學系碩士班 === 99 === Porcine circovirus type 2 (PCV2) is concerned with the causative agnet of postweaning multisystemic wasting syndrome (PMWS), which is an important economical disease affecting the swine industry worldwide. Effectively controlling PMWS has been driven toward developing protective vaccine against the virus, and the PCV2 capsid protein encoded by the PCV2 ORF2 gene has been indentified as a candidate for developing vaccine. In this study, the occlusion bodies of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) were constituted by polyhedron protein, and the occlusion bodies were incorporate with PCV2 capsid and enhanced GFP protein to form recombinant occlusion bodies (polh-cap-egfp). The component of recombinant occlusion bodies were confirmed by SDS-PAGE and western blotting with anti-GFP and anti-capsid antibody. The immunogenicity was determined by indirect ELISA assay. These results indicated that the recombinant occlusion bodies were able to elicit immune responses in mice with high titer. In order to eliminate the virion in the occlusion bodies, we develop a controllable CreER/loxP system, which could mediate the expression of the target gene microRNA, for producing safely occlusion bodies. The CreER/loxP system contains two components: One is stably expressing CreER Sf9 cell line, which could be activated by 4-hydroxytamoxifen (4OH-TM), and the other is recombinant baculovirus containing a LacZ expression cassette between two loxP sites, microRNA and EmGFP gene. When the activated CreER recognized loxP sites resulting recombination, upstream ie1 promoer would express the target gene microRNA and EmGFP gene. The infection of vAci-loxZ-Gp35miR viruses suppressed luciferase activity with does-dependent manner in p35-Fluc reporter plasmid transfected and 4OH-TM-treated Sf9-CreER cells, but the expression of EmGFP protein was increased at 48 hrs. In addition, The RNA level of p35 gene and the replication of virus DNA was also reduced by the p35 microRNA of vAci-loxZ-Gp35miRNA viruse. These results suggested that the CreER/loxP system was a well-controllable gene expression system, and we would combine the controllable CreER/loxP system and recombinant occlusion bodies-stimulated candidate vaccine in the future.