Expression of recombinant classical swine fever virus glycoprotein E2 and subunit pasteurella multocida toxin Tox1

• Main Authors: Yung-Yi Chen, 陳詠宜
• Other Authors: 簡茂盛
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/00016499707050007644

碩士 === 國立中興大學 === 獸醫病理學研究所 === 90 === Abstract Classical swine fever (CSF) is a highly contagious and fatal viral disease of swine. Glycoprotein E2 is one of the three glycoproteins on the envelope of classical swine fever virus (CSFV) and is also the most immunogenic antigen of CSFV. Because the marker vaccine can be used for discriminating vaccinated from infected pigs, the recombinant glycoprotein E2 has been constructed in the baculovirus expression vector system (BEVS) and tested for its immunogenicity. In order to make the subunit vaccine more proficient, we also tried to fuse the E2 glycoprotein gene to another pathogenic antigen gene for developing a bivalent subunit vaccine based on the finding that the folding and immunogenicity of the fusion protein could be still recognized correctly. Tox1 is the N-terminal subunit of Pasteurella multocida toxin (PMT), and is the major virulence factor associated with progressive atrophic rhinitis (PAR). The N-terminal of recombinant subunit PMT showed highly protection in pigs for vaccine efficacy from prior trials. In this study, transfer vectors that were inserted the genes of E2, Tox1, and E2Tox1 respectively were first applied to obtain recombinant baculoviruses with linearlized baculovirus DNA by homologous recombination in Spodoptera frugiperda (Sf9) cells. After repeat screening with blue plaque assays and confirming the inserted genes by DNA sequencing, the recombinant baculoviruses were purified and successfully expressed recombinant proteins in Sf9 cells. The calculated molecular weights of expressed proteins were 39 kDa (E2), 60 kDa (Tox1), and 95 kDa (E2Tox1) and all could be recognized by the Western blotting method. In addition, these recombinant proteins were utilized for immunization in mice and swine and both could induce neutralizing antibodies against CSFV and PMT as well. The large scales of animal trails will still be needed to apply for further evaluation the efficacy of these recombinant subunit vaccines in the near future.