| Summary: | 碩士 === 國立高雄大學 === 生物科技研究所 === 94 === Actinobacillus pleuropneumoniae can cause haemorrhagic, fibrinous and necrotic pleuropneumonia in pigs that causes critical economic losses in pig farm. Virulence factors of A. pleuropneumoniae include the capsular polysaccharides, lipopolysaccharides, outer membrane proteins, adhesion factors and exotoxins. Virulence is strongly correlated with the Apx toxins. ApxI and ApxⅡ are exotoxins of A. pleuropneumoniae. ApxI is strongly hemolytic and strongly cytotoxic. ApxⅡ is weakly hemolytic and weakly cytotoxic. Pretoxins ApxIA and ApxⅡA are structural proteins of ApxI and ApxⅡ, respectively. ApxIA and ApxⅡA are non-toxic before they are acetylated by ApxIC and ApxⅡC, respectively. Outer membrane proteins are exposed on the surface of pathogen, which are more accessible for immune system to induce immune responses than other proteins and usually be used as candidates for vaccine development. A. pleuropneumoniae needs to obtain iron from iron-containing proteins of the host to express full virulence. These iron-containing proteins include transferrin, haem, and haemoglobin.
In this study, ApxIA, ApxⅡA, OmlA and HgbA were chosen as the target antigens for DNA vaccine development against A. pleuropneumoniae. These genes were cloned to vectors that can express antigens in mammalian system and inserted genes were confirmed by DNA sequencing analysis. The swine kidney cell line LLC-PK1 was transfected with these DNA constructs and detected the expressed proteins by Western blot analysis to demonstrate these proteins can be expressed in mammalian cells.
To evaluate the potency of these DNA vaccines, each DNA vaccine and a mixture vaccine containing above four DNA constructs (4 in 1 vaccine) were administrated into mice by intramuscular injection. A commercial inactivated A. pleuropneumoniae vaccine also used as positive control, empty vector and PBS as negative control. The titers of antisera increased significantly except empty vector and PBS group after second immunization. These mice were challenged with 5x108 colony-forming unit (cfu) of serotype 1 of A. pleuropneumoniae after 24 days of first immunization. The survival rate of each group after 5 days of challenge was as following: 67% for 4 in 1 vaccine; 50% for ApxIA vaccine; 25% for ApxIIA, OmlA and commercial inactivated vaccine; 0% for HgbA, empty vector and PBS group. The results showed that the 4 in 1 DNA vaccine could obtain the best protective efficacy and better than the commercial inactivated vaccine. It indicates that these DNA vaccines used in this study could be a new strategy against infection by A. pleuropneumoniae.
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