Molecular Cloning of Recombinant ORF2 and Infectious Clone of Porcine Circovirus Type II
Date Issued
2006
Date
2006
Author(s)
Chiu, Jui-Yu
DOI
zh-TW
Abstract
Post-weaning multisystemic wasting syndrome (PMWS) becomes a major problem in many pig-producing countries throughout the world. Porcine circovirus type 2 (PCV2) has been identified as an essential component of PMWS. Approximately 83.5% of the pigs in Taiwan were seropositive to PCV2. The open reading frame 2 (ORF2) encoding the major capsid protein with epitopes to induce neutralizing antibodies is the potential target gene for vaccine development in against PCV2 infection. However the difficulty to effectively express ORF2 seems to limit the development of PCV2 vaccine. In this study, we expressed full-length, N-terminal, middle and C-terminal ORF2 covering high antigenic epitopes by E. coli. Only ORF2M and ORF2C could be overexpressed and purified. The yield of purified ORF2M or ORF2C was about 20 mg/L. Mouse antisera and monoclonal antibodies produced by ORF2M or ORF2C recognized PCV2 under Western blotting and immunofluorescence assay. The neutralization titer of ORF2M-induced serum was about 32X, suggesting that ORF2M is a potential candidate of PCV2 subunit vaccine. We also tried but failed to express ORF2 in protozoa expression system. It is hard to isolate PCV2 from field sample and to propagate it in cell culture. We constructed the infectious clone of PCV2 to generate a biologically pure and homogeneous infectious virus stock to study viral behaviors. In PCV2-infected PK-15 cells ORF2’ was detected at 12 hours post inoculation (h.p.i.) by Western blotting, but ORF1 was detected at 24 h.p.i.. The expression of ORF2 seemed to be earlier than that of ORF1. On the other hand, overexpression of ORF1, ORF2 or ORF3 had no effect on viral propagation. However, overexpression of ORF2 advanced, but ORF1 delayed the appearanceof cytopathic effect (CPE). The ORF1-delayed CPE increased the life span of PCV2-infected cells and then viral propagation. The results provide useful information for efficiently producing PCV2 virus stock in PK-15 cell model.
Subjects
第二型豬環狀病毒
外鞘蛋白
複製相關蛋白
次單位疫苗
感染性質體
重組蛋白
porcine circovirus type II
capsid protein
Rep protein
subunit vaccine
infectious clone
recombinant protein
SDGs
Type
thesis
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