Browse > Article

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

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)
Publication Information
Korean Journal of Veterinary Research / v.43, no.2, 2003 , pp. 247-253 More about this Journal
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.
Keywords
Actinobacillus pleuropneumoniae; apxA; DNA seqrence; phylogenetic analysis; expression;
Citations & Related Records
연도 인용수 순위
  • Reference
1 Schaller, A., Kuhn, R., Kuhnert, P., Nicolet, J., Anderson, T. J., Maclnnes, J. I., Segers, R. P. and Frey, J. Characterization of apxIVA, a new RTX determinant of Actinobacillus pluropneumoniae. Microbiology. 1999, 145, 2105-2116
2 Seah, J. N., Frey, J. and Kwang, J. The N-terminal domain of RTX toxin ApxI of Actinobacillus pleura pneumoniae elicits protective immunity in mice. Infect. Immun. 2002, 70, 6464-6467
3 Anderson, C., Potter, A. A. and Gerlach, G. F. Isolation and molecular characterizationof spontaneously occurring cytolysin-negative mutants of Actinobacillus pleuropneumoniae serotype 7. Infect. Immun. 1991, 59, 4110-4116
4 Frey, J. Virulence in Actinobacillus pleuropneumoniae and RTX toxins. Trends Microbiol. 1995, 3, 257-261
5 Kume, K., Nagano, I. and Nakai, T. Bacteriological, seological and pathological examination of Haemophilus pleuropneumoniae infection in 200 slaughtered pigs. Jpn. J. Vet. Sci. 1986, 48, 965-970
6 Min, K. S. and Chae, C. H. Serotype and apx genotype profiles of Actinobacillus pleuropneunoniae field isolates in Konsa. Vet. Rec. 1999, 145, 251-254
7 Prideaux, C. T., Lenghaus, C., Krywult, J. and Hodgson, A. L. Vaccination and protection of pigs against pleuropneumonia with a vaccine strain of Actinobacillus pleuropneumoniaeproduced by site-specific mutagenesos of the Apx II operon. Infect. Immun. 1999, 67, 1962-1966
8 Reimer, D., Frey, J., Jansen, R., Veit, H. P. and Inzana, T. J. Molecular investigation of the role of ApxI and ApxII in the virulence of Actinobacillus pleuropneumoniaeserotype 5. Micro. Pathog. 1995, 18, 197-209
9 Blackall, P. J., Klaasen, H. L., van den Bosch, H., Kuhnert, P. and Frey, J. Proposal of a new serovar of Actinobacillus pleuropneumoniae: serovar 15. Vet. Microbiol. 2002, 84, 47-52
10 Mittal, K. R., Higgins, R. and Lariviere, S. Identificatioon and Serotyping of Haemophilus pleuropneumoniae by coagglutination test. J. Clin. Miciobiol. 1983, 18, 1351-1354
11 Komal, J. P. and Mittal, K. R. Grouping of Actinobacillus pleuropneumoniaestrains of serotypes 1 through 12 on the basis of their virulence in mice. Vet. Microbiol. 1990, 25, 229-240
12 Towbin, H., Staehelin, T. and Gorden, J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedures and some applications. Proc. Nat'l. Acad Sci. 1979, 76, 4350-4354
13 마점술, 전윤성. 양돈단지의 번식율저하에 대한 병인학적 연구. 3. 미생물학적 시험. 서울대학교 수의대논문집. 1979, 4, 120
14 Bosse, J. T., Janson, H., Sheehan, B. J., Beddek, A. J., Rycroft, A. N., Simon Kroll, J. and Langford, P. R. Actinobacillus pluropneumoniae: pathobiology and pathogenesis of infection. Micro. Infect. 2002, 4, 225-235
15 Fales, W.H., Morehouse, L. G., Mittal, K. R, Bean Knudsen, C., Nelson, S. L., Kintner, L. D., Turk, J. R., Turk, M. A., Brown, T. P. and Shaw, D. P. Antimicrobial susceptibility and serotypes cf Actinobacillus (Haemophilus) Pleuropneumoniae recovered from Missouri swine. J. Vet. Diagn. Invest. 1989, 1, 16-19
16 Van Overbeke, I., Chiers, K., Ducatelle, R. and Haesebrouck, F. Effect of endobronchial challenge with Actinobacillus pleuropneumoniae serotype 9 of pigs vaccinated with a vaccine containing Apx toxins and transferring-binding proteins. J. Vet. Med. B. 2001, 48, 15-20
17 Muller, E., Korte, G. and Petzoldt, K. Isolation and serotyping Haemophilus pleuropneumoniae in Northwestern Germany. Proc. Int. Pig Vet. Soc. Congr. 1986, a, 261
18 Fuller, T. E., Martin, S., Teel, J. F., Alaniz, G. R., Kennedy, M. J. and Lowery, D. E. Identification of Actinobacillus pleropneumoniaevirulence. genss using signature-tagged mutagetnesis in a swine infection model. Micro. Pathog. 2000, 29, 39-51
19 Kwn, H. J., Rirk, K. Y., Kim, S. J. and Yoo, R S. Application of necleotide sequence of RNA polymerase $\beta$-subunit gene (rpoB) to molecular differentiation of serovars of Salmonella enterica subsp. enterica, Vet. Mcrobiol. 2001, 82, 121-129
20 Tascon, R. I., Vazquez-Boland, J. A., Gutierrez Martin, C. B., Rodriguez-Barbosa, I. and Rodriguez Ferri, E. F. The RTX haemolysin ApxI and ApxII are major virulence factors of the swine pathogen Actino bacillus pleuropneumoniae: evidence from mutational analysis. Mol. Microbiol. 1994, 14, 207-216
21 Vaillancourt, J. P., Higgins, R., Martineau, G. P., Mittal, K. R, and Lariviere, S. Changes in the susceptibility of Actinobacillus pleuropneumoniae to antimicrobial in Quebec (1981-1986). J. Am. Vet. Med. Assoc. 1988, 193, 470-473
22 Nielsen, R. Haenwphilus pleuropneumoniae serotypescross protection experiments. Nord. Vet. Med. 1984, 36, 221-234
23 Nielson, R. Seroepidemiology of Actinobacillus pleuropneumoniae. Can J. Vet. Res. 1988. 29, 580-582
24 Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227, 680-685
25 Chiers, K., Donne, E., Van Overbeke, L, Ducatelle, R, and Haesebrouck, F. Actinobacillus pleuropnewnoiae infections in closed swine herds: infection patterns and seiological profiles. Vet. Microbiol. 2002, 85, 343-352