Hostname: page-component-586b7cd67f-g8jcs Total loading time: 0 Render date: 2024-11-28T01:07:23.635Z Has data issue: false hasContentIssue false
  • English
  • Français

Cloning, expression and immunogenic characterization of the apxIIA gene of Actinobacillus pleuropneumoniae

Published online by Cambridge University Press:  15 June 2007

Yan Ke-Xia ,
Liu Jian-Jie ,
Wu Bin ,
Tang Xi-Biao ,
Cai Li-Jun ,
Yang Ming-Liu ,
Chen Huan-Chun  and
Zhou Rui
Yan Ke-Xia
Affiliation:
Division of Animal Infectious Disease, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China
Liu Jian-Jie
Affiliation:
Haikou Agriculture, Industry & Trade Luoniushan Co. Ltd.Haikou 570125, China
Wu Bin
Affiliation:
Division of Animal Infectious Disease, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China
Tang Xi-Biao
Affiliation:
Division of Animal Infectious Disease, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China
Cai Li-Jun
Affiliation:
Division of Animal Infectious Disease, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China
Yang Ming-Liu
Affiliation:
Division of Animal Infectious Disease, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China
Chen Huan-Chun
Affiliation:
Division of Animal Infectious Disease, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China
Zhou Rui*
Affiliation:
Division of Animal Infectious Disease, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China
*
*Corresponding author. E-mail: [email protected]
Get access Rights & Permissions [Opens in a new window]

Abstract

The structural gene encoding ApxII toxin (apxIIA) was amplified from the genomic DNA of Actinobacillus pleuropneumoniae (APP) strain HB08 (serotype 2) and cloned into the prokaryotic expression vector pET-28a. SDS-PAGE and Western blot analysis showed that the apxIIA gene was expressed in Escherichia coli BL21 (DE3) and the expressed products could react with ApxII antibodies. The recombinant ApxIIA was purified from the inclusion bodies. Kunming mice were intraperitoneally vaccinated twice, with an interval of 2 weeks, using unfolded/refolded recombinant proteins, the native ApxII toxin extracted from the cultural supernatant of a strain of APP serotype 7 (APP-7) or phosphate-buffered saline (PBS). Serum antibody was examined by ApxIIA-specific enzyme-linked immunosorbent assay (ELISA) 2 weeks after every vaccination. Two weeks after the second vaccination, mice were challenged intraperitoneally with a lethal dose of APP-7 (1.08 × 108 cfu per mouse). The protection rate reached 91.7% in the native ApxII group, 83.3% in the refolded recombinant protein group and 58.3% in the unfolded recombinant protein group, while all mice in the PBS group died within 36 h after challenge. Our data revealed that the refolded recombinant ApxIIA had excellent immunogenicity and could elicit protection against a lethal challenge of APP.

Keywords

Actinobacillus pleuropneumoniaeApxIIcloningprokaryotic expressionimmunogenicity
Type
Research Article
Information
Chinese Journal of Agricultural Biotechnology , Volume 4 , Issue 1 , April 2007 , pp. 63 - 67
Copyright
Copyright © China Agricultural University and Cambridge University Press 2007

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

Footnotes

First published in Journal of Agricultural Biotechnology 2006, 14(4): 493–497

References

Bei, WC, Yan, L, He, QG, Xiao, SB and Chen, HC (2005) Eukaryotic expression and DNA immunization of apxIIA gene of porcine Actinobacillus pleuropneuromoniae. Journal of Agricultural Biotechnology 13(5): 616–619 (in Chinese with English abstract).Google Scholar
Blackall, PJ, Klaasen, HL, van den Bosch, H, Kuhnert, P and Frey, J (2002) Proposal of a new serovar of Actinobacillus pleuropneumoniae: serovar 15. Veterinary Microbiology 84(1–2): 47–52.CrossRefGoogle ScholarPubMed
Bosse, JT, Janson, H, Sheehan, BJ, et al. (2002) Actinobacillus pleuropneumoniae: pathobiology and pathogenesis of infection. Microbes and Infection 4(2): 225–235.CrossRefGoogle ScholarPubMed
Fedorka-Cray, PJ, Huether, MJ, Stine, DL and Anderson, GA (1990) Efficacy of a cell extract from Actinobacillus (Haemophilus) pleuropneumoniae serotype 1 against disease in swine. Infection and Immunity 58(2): 358–365.CrossRefGoogle ScholarPubMed
Frey, J (1995) Virulence in Actinobacillus pleuropneumoniae and RTX toxins. Trends in Microbiology 3(7): 257–261.CrossRefGoogle ScholarPubMed
Frey, J, Bosse, JT, Chang, YF, et al. (1993) Actinobacillus pleuropneumoniae RTX-toxins: uniform designation of haemolysins, cytolysins, pleurotoxin and their genes. Journal of General Microbiology 139(8): 1723–1728.CrossRefGoogle ScholarPubMed
Haga, Y, Ogino, S, Ohashi, S, Ajito, T, Hashimoto, K and Sawada, T (1997) Protective efficacy of an affinity-purified hemolysin vaccine against experimental swine pleuropneumonia. Journal of Veterinary Medical Science 59(2): 115–120.CrossRefGoogle ScholarPubMed
Kamp, EM, Popma, JK, Anakotta, J and Smits, MA (1991) Identification of hemolytic and cytotoxic proteins of Actinobacillus pleuropneumoniae by use of monoclonal antibodies. Infection and Immunity 59(9): 3079–3085.CrossRefGoogle ScholarPubMed
Nielsen, R, van den Bosch, JF, Plambeck, T, Sorensen, V and Nielsen, JP (2000) Evaluation of an indirect enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to the Apx toxins of Actinobacillus pleuropneumoniae. Veterinary Microbiology 71(1–2): 81–87.CrossRefGoogle Scholar
Ŝatrán, P, Nedbalcová, K and Kučerová, Z (2003) Comparison of protection efficacy of toxoid and whole-cell vaccines against porcine pleuropneumonia caused by endotracheal infection with Actinobacillus pleuropneumoniae. Acta Veterinaria 72: 213–219.CrossRefGoogle Scholar
Shin, SJ, Bae, JL, Cho, YW, et al. (2005) Induction of antigen-specific immune responses by oral vaccination with Saccharomyces cerevisiae expressing Actinobacillus pleuropneumoniae ApxIIA. FEMS Immunology and Medical Microbiology 43(2): 155–264.CrossRefGoogle ScholarPubMed
Tascon, RI, Vazquez-Boland, JA, Gutierrez-Martin, CB, Rodriguez-Barbosa, I and Rodriguez-Ferri, EF (1994) The RTX haemolysins ApxI and ApxII are major virulence factors of the swine pathogen Actinobacillus pleuropneumoniae: evidence from mutational analysis. Molecular Microbiology 14(2): 207–216.CrossRefGoogle ScholarPubMed