Antigenic Analysis of ORF1, OR2 and ORF3 of Porcine Circovirus Type 2 Expressed Viral Peptides

• Main Authors: Duan-Liang Shyu, 徐敦良
• Other Authors: Shiow-Suey Lai
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/76665853457948582976

碩士 === 國立臺灣大學 === 獸醫學研究所 === 92 === Porcine circovirus type 2 (PCV2)cause PMWS in weaning piglets, and was found highly infectious in pug farms. Polymerase chain reactions (PCR) was used to amplify ORF1, ORF2 and ORf3 of PCV2 from infected lymph node emulsion by using specific primers. E. coli expression vector pRSET A and DNA vaccine vactor pcDNA3 were used as the plasmid to clone the 3 ORFs of PCV2, respectively. Recombinant proteins of ORF1, ORF2 and ORF3 of PCV2 were expressed in E. coli were analyzed by SDS-PAGE and western blotting. The DNA vector vaccine were used to immulized rabbits by intramuscular injection. MagicShuttle protein(MGT), a kind of DNA binding protein, were also used to increase the efficiency of DNA transfection. PCV2 antibody and viral neutralizing antibody of immunized rabbits serum were tested by indirect immunofluorescent assay (IFA). The result of E. coli expression showed that the soluble form of ORF1 and ORF2 recombinant proteins, and the inclusion form of ORF3 recombinant protein could be recognized by PCV2 polyclonal antibody but not by PCV2 ORF2 monoclonal antibody. The size of ORF1 and ORF2 recombinant proteins were correct, but that of ORF3 was bigger than the expected size. Sera from DNA vector vaccine immunized rabbits showed that ORF2 and ORF3 immunized sera had PCV2 antibody titers, but not found in ORF1 immunized sera. Viral neutralizing antibody activities were only detected in sera collected in ORF2 and PCV2 infected lymoh node emulsion immunized sera：the neutralizing activities was only found in using low amount of PCV2 (10 TCID50).