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KMID : 0359320030430020247
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Korean Journal of Veterinary Research
2003 Volume.43 No. 2 p.247 ~ p.253
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Cloning, Sequencing and Expression of apxⅠA, ⅡA, ⅢA of Actinobacillus pleuropneumoniae Isolated in Korea
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Abstract
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Actinobacillus pleuropneumoniae causes a highly contagious pleuropneumoniae in swine. The bacterium produces several virulence factors such as exotoxin, LPS, capsular polysaccharide, etc. Among them, the exotoxin, called Apx, has been focused as the major virulence factor, and the toxin consists of 4 gene cluster. apx CABD. apxA is the structural gene of toxin and has four different types, I, II, III and IV. As the first step of development of a new subunit vaccine, the three different types of apxA gene were amplified from A. pleuropneumoniae isolated from Korea by PCR with primer designed based on the N- and C- terminal of the toxin. The sizes of apxIA, IIA and IIIA wre 3,073, 2,971 and 3,159 bps, respectively. The comparison of whole DNA sequences of apxIA, IIA, and IIIA genes with those of the reference strain demonstrated 98%, 99% and 98% homology, respectively. In addition, the phylogenetic analysis was performed based on the amino acid sequences compared with 12 different RTX toxin family using the neighbor-joining method. ApxA proteins of Korean isolates were identical with reference strains in this study.
All ApxA proteins were expressed in E. coli with pQE expression vector and identified using Western block with polyclonal antiblodies against culture supernatants of A. pleuropneumoniae serotype 2 or 5. The sizes of each expressed ApxA protein were about 120, 110, 125 kDa (M.W.), respectively. The results obtained in this study could be used for the future study to develop a new vaccine to porcine pleuropneumoniae.
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