Summary: | 碩士 === 國立中興大學 === 獸醫學系 === 93 === Porcine circovirus (PCV), classified in the family Circoviridae, is a small nonenveloped virus with a circular ssDNA genome. This family includes chicken anemia virus (CAV), beak and feather disease virus (BFDV), porcine circovirus (PCV), and pigeon circovirus (PiCV). PCV2 was noted in 1991and the first case was reported in Canada. PCV2 cause a diaease called postweaning multisystemic wasting syndrome (PMWS), clinical signs including dyspneia, enlarged lymph node, and sometimes jaundice and diarrhea cases. In this work, virus DNA was extracted from the cultured cell, and the PCV2 full length genomic DNA was amplified by polymerase chain reaction (PCR). The PCR product was cloned and sequenced ( 1767 bp) . From complete nucleotide sequencc comarison, we found it showed similarity (about 98 %) with No.AY484407 in GenBank. The Rep gene of PCV2 was further cloned into a prokaroyte expression vector pET32c. The recombinant Rep protein was expressed by host BL21 (DE3). The protein with expected size about 54 kDa was obviously produced. The recombinant Rep protein was identified by Western blot assay with antibody against His-tag. Expressed Rep protein was purified by passing through the Ni-NTA superflow column (QIAGEN) , and was analyzed by SDS-PAGE. Finally purified proteins were identified by mass spectrometry, and the result confirmed the purified protein is Rep protein of porcine circovirus type 2.
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