Cloning, Sequencing and Expression of apxIA, IIA, IIIA of Actinobacillus pleuropneumoniae Isolated in Korea

국내 분리 흉막폐렴균의 apxIA, IIA, IIIA 유전자 Cloning, 염기서열 분석 및 단백질 발현

  • Shin, Sung-jae (Department of Infectious Diseases, College of Veterinary Medicine and School of Agricultural Biotechnology, Seoul National University) ;
  • Cho, Young-wook (Department of Infectious Diseases, College of Veterinary Medicine and School of Agricultural Biotechnology, Seoul National University) ;
  • Yoo, Han-sang (Department of Infectious Diseases, College of Veterinary Medicine and School of Agricultural Biotechnology, Seoul National University)
  • 신성재 (서울대학교 수의과대학 및 농생명공학부) ;
  • 조영욱 (서울대학교 수의과대학 및 농생명공학부) ;
  • 유한상 (서울대학교 수의과대학 및 농생명공학부)
  • Accepted : 2003.05.03
  • Published : 2003.06.30

Abstract

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 were 3,073, 2,971 and 3,159bps, 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 blot with polyclonal antibodies 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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