Skip to main content

Advertisement

SpringerLink
Log in

Expression and assembly of ApxIIA toxin of Actinobacillus pleuropneumoniae fused with the enterotoxigenic E. coli heat-labile toxin B subunit in transgenic tobacco

  • Original Paper
  • Published:
Plant Cell, Tissue and Organ Culture (PCTOC) Aims and scope Submit manuscript

Abstract

Actinobacillus pleuropneumoniae is a major etiological agent of swine pleuropneumonia, a highly contagious respiratory infection that causes severe economic losses in the swine production industry. ApxIIA, one of the virulence factors in A. pleuropneumoniae, is considered a candidate for the development of a vaccine against this bacterial infection. In this study, a fusion gene consisting of enterotoxigenic Escherichia coli heat-labile enterotoxin B subunit (LTB) and an ApxIIA fragment (amino acids 619–801) (ApxIIA619–801) was fused to the endoplasmic reticulum (ER) retention signal (SEKDEL) and introduced into a plant expression vector under the control of a ubiquitin promoter. The plant expression vector was transformed into tobacco Nicotiana tabacum L. cv. MD609 using an Agrobacterium-mediated transformation procedure. The integration and transcription of the LTB fusion gene were confirmed by genomic DNA PCR amplification and Northern blot analysis, respectively. The synthesis and assembly of LTB fusion protein were evidenced by Western blot analysis and the GM1-ganglioside enzyme-linked immunosorbent assay (GM1-ELISA). A quantitative ELISA used to measure the amount of LTB fusion protein produced in the transgenic plant revealed high levels of the fusion protein, up to 30 μg g−1, in lyophilized leaf tissue. These results demonstrate the feasibility of using a transgenic plant to express the LTB-ApxIIA619–801 fusion protein as a first step towards producing plant-based edible vaccines to elicit immune responses against A. pleuropneumoniae via an oral mucosal delivery system.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Abul-Milh M, Paradis SE, Dubreuil JD, Jacques M (1999) Binding of Actinobacillus pleuropneumoniae lipopolysaccharides to glycosphingolipids evaluated by thin-layer chromatography. Infect Immun 67:4983–4987

    PubMed  CAS  Google Scholar 

  • Arakawa T, Yu J, Chong DK, Hough J, Engen PC, Langridge WH (1998) A plant-based cholera toxin B subunit-insulin fusion protein protects against the development of autoimmune diabetes. Nat Biotechnol 16:934–938

    Article  PubMed  CAS  Google Scholar 

  • Bagdasarian MM, Nagai M, Frey J, Bagdasarian M (1999) Immunogenicity of Actinobacillus ApxIA toxin epitopes fused to the E. coli heat-labile enterotoxin B subunit. Vaccine 17:441–447

    Article  PubMed  CAS  Google Scholar 

  • Blackall PJ, Klaasen HL, van den Bosch H, Kuhnert P, Frey J (2002) Proposal of a new serovar of Actinobacillus pleuropneumoniae: serovar 15. Vet Microbiol 84:47–52

    Article  PubMed  CAS  Google Scholar 

  • Chiang CH, Huang WF, Huang LP, Lin SF, Yang WJ (2009) Immunogenicity and protective efficacy of ApxIA and ApxIIA DNA vaccine against Actinobacillus pleuropneumoniae lethal challenge in murine model. Vaccine 27:4565–4570

    Article  PubMed  CAS  Google Scholar 

  • Dean C, Jones J, Favreau M, Dunsmuir P, Bedbrook J (1988) Influence of flanking sequences on variability in expression levels of an introduced gene in transgenic tobacco plants. Nucleic Acids Res 16:9267–9283

    Article  PubMed  CAS  Google Scholar 

  • Desrosiers RC, Burghoff RL, Bakker A, Kamine J (1984) Construction of replication-competent Herpesvirus saimiri deletion mutants. J Virol 49:343–348

    PubMed  CAS  Google Scholar 

  • Dom P, Hommez J, Castryck F, Devriese LA, Haesebrouck F (1994) Serotyping and quantitative determination of in vitro antibiotic susceptibility of Actinobacillus pleuropneumoniae strains isolated in Belgium (July 1991–August 1992). Vet Q 16:10–13

    PubMed  CAS  Google Scholar 

  • Dubreuil JD, Jacques M, Mittal KR, Gottschalk M (2000) Actinobacillus pleuropneumoniae surface polysaccharides: their role in diagnosis and immunogenicity. Anim Health Res Rev 1:73–93

    Article  PubMed  CAS  Google Scholar 

  • Frey J (1995) Virulence in Actinobacillus pleuropneumoniae and RTX toxins. Trends Microbiol 3:257–261

    Article  PubMed  CAS  Google Scholar 

  • Fuller TE, Thacker BJ, Duran CO, Mulks MH (2000) A genetically-defined riboflavin auxotroph of Actinobacillus pleuropneumoniae as a live attenuated vaccine. Vaccine 18:2867–2877

    Article  PubMed  CAS  Google Scholar 

  • Haesebrouck F, Chiers K, Van Overbeke I, Ducatelle R (1997) Actinobacillus pleuropneumoniae infections in pigs: the role of virulence factors in pathogenesis and protection. Vet Microbiol 58:239–249

    Article  PubMed  CAS  Google Scholar 

  • Haesebrouck F, Pasmans F, Chiers K, Maes D, Ducatelle R, Decostere A (2004) Efficacy of vaccines against bacterial diseases in swine: what can we expect? Vet Microbiol 100:255–268

    Article  PubMed  CAS  Google Scholar 

  • Jacques M, Belanger M, Roy G, Foiry B (1991) Adherence of Actinobacillus pleuropneumoniae to porcine tracheal epithelial cells and frozen lung sections. Vet Microbiol 27:133–143

    Article  PubMed  CAS  Google Scholar 

  • Kang TJ, Yang MS (2004) Rapid and reliable extraction of genomic DNA from various wild-type and transgenic plants. BMC Biotechnol 4:20

    Article  PubMed  Google Scholar 

  • Kang TJ, Loc NH, Jang MO, Jang YS, Kim YS, Seo JE, Yang MS (2003) Expression of the B subunit of E. coli heat-labile enterotoxin in the chloroplasts of plants and its characterization. Transgenic Res 12:683–691

    Article  PubMed  CAS  Google Scholar 

  • Kang TJ, Han SC, Jang MO, Kang KH, Jang YS, Yang MS (2004) Enhanced expression of B-subunit of Escherichia coli heat-labile enterotoxin in tobacco by optimization of coding sequence. Appl Biochem Biotechnol 117:175–187

    Article  PubMed  CAS  Google Scholar 

  • Kang TJ, Kim YS, Jang YS, Yang MS (2005) Expression of the synthetic neutralizing epitope gene of porcine epidemic diarrhea virus in tobacco plants without nicotine. Vaccine 23:2294–2297

    Article  PubMed  CAS  Google Scholar 

  • Kang TJ, Han SC, Yang MS, Jang YS (2006) Expression of synthetic neutralizing epitope of porcine epidemic diarrhea virus fused with synthetic B subunit of Escherichia coli heat-labile enterotoxin in tobacco plants. Protein Express Purif 46:16–22

    Article  CAS  Google Scholar 

  • Kilian M, Mestecky J, Russell MW (1988) Defense mechanisms involving Fc-dependent functions of immunoglobulin A and their subversion by bacterial immunoglobulin A proteases. Microbiol Rev 52:296–303

    PubMed  CAS  Google Scholar 

  • Kim JM, Jung DI, Eom YJ, Park SM, Yoo HS, Jang YS, Yang MS, Kim DH (2010) Surface-displayed expression of a neutralizing epitope of ApxIIA exotoxin in Saccharomyces cerevisiae and oral administration of it for protective immune responses against challenge by Actinobacillus pleuropneumoniae. Biosci Biotechnol Biochem 74:1361–1367

    Google Scholar 

  • Kozak M (1981) Possible role of flanking nucleotides in recognition of the AUG initiator codon by eukaryotic ribosomes. Nucleic Acids Res 9:5233–5252

    Article  PubMed  CAS  Google Scholar 

  • Kunkel EJ, Butcher EC (2003) Plasma-cell homing. Nat Rev Immunol 3:822–829

    Article  PubMed  CAS  Google Scholar 

  • Lee KY, Kim DH, Kang TJ, Kim J, Chung JH, Yoo HS, Arntzen CJ, Yang MS, Jang YS (2006) Induction of protective immune responses against the challenge of Actinobacillus pleuropneumoniae by the oral administration of transgenic tobacco plant expressing ApxIIA toxin from the bacteria. FEMS Immunol Med Microbiol 48:381–389

    Article  PubMed  CAS  Google Scholar 

  • Liljeqvist S, Stahl S, Andreoni C, Binz H, Uhlen M, Murby M (1997) Fusions to the cholera toxin B subunit: influence on pentamerization and GM1 binding. J Immununol Methods 210:125–135

    Article  CAS  Google Scholar 

  • Loc NH, Song NV, Tien NQD, Minh TH, Nga PTQ, Kim TG, Yang MS (2010) Expression of the Escherichia coli heat-labile enterotoxin B subunit in transgenic watercress (Nasturtium officinale L.). Plant Cell Tiss Organ Cult. doi: 10.1007/s11240-010-9835-0

  • Ma JK, Drake PM, Christou P (2003) The production of recombinant pharmaceutical proteins in plants. Nat Rev Genet 4:794–805

    Article  PubMed  CAS  Google Scholar 

  • Marillonnet S, Thoeringer C, Kandzia R, Klimyuk V, Gleba Y (2005) Systemic Agrobacterium tumefaciens-mediated transfection of viral replicons for efficient transient expression in plants. Nat Biotechnol 23:718–723

    Article  PubMed  CAS  Google Scholar 

  • Millar DG, Hirst TR, Snider DP (2001) Escherichia coli heat-labile enterotoxin B subunit is a more potent mucosal adjuvant than its closely related homologue, the B subunit of cholera toxin. Infect Immun 69:3476–3482

    Article  PubMed  CAS  Google Scholar 

  • Mitragotri S (2005) Immunization without needles. Nat Rev Immunol 5:905–916

    Article  PubMed  CAS  Google Scholar 

  • Mora JR, Bono MR, Manjunath N, Weninger W, Cavanagh LL, Rosemblatt M, Von Andrian UH (2003) Selective imprinting of gut-homing T cells by Peyer’s patch dendritic cells. Nature 424:88–93

    Article  PubMed  CAS  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Neutra MR, Kozlowski PA (2006) Mucosal vaccines: the promise and the challenge. Nat Rev Immunol 6:148–158

    Article  PubMed  CAS  Google Scholar 

  • Park SM, Choi EJ, Kwon TH, Jang YS, Yoo HS, Choi W, Park BK, Kim DH (2005) Expression of the Apx toxins of Actinobacillus pleuropneumoniae in Saccharomyces cerevisiae and its induction of immune response in mice. Biotechnol Bioprocess Eng 10:362–366

    Article  CAS  Google Scholar 

  • Peach C, Velten J (1991) Transgene expression variability position effect of CAT and GUS reporter genes driven by linked divergent T-DNA promoters. Plant Mol Biol 17:49–60

    Article  PubMed  CAS  Google Scholar 

  • Pilette C, Ouadrhiri Y, Godding V, Vaerman JP, Sibille Y (2001) Lung mucosal immunity: immunoglobulin-A revisited. Eur Respir J 18:571–588

    Article  PubMed  CAS  Google Scholar 

  • Prideaux CT, Lenghaus C, Krywult J, Hodgson AL (1999) Vaccination and protection of pigs against pleuropneumonia with a vaccine strain of Actinobacillus pleuropneumoniae produced by site-specific mutagenesis of the ApxII operon. Infect Immun 67:1962–1966

    PubMed  CAS  Google Scholar 

  • Ramjeet M, Deslandes V, Goure J, Jacques M (2008) Actinobacillus pleuropneumoniae vaccines: from bacterins to new insights into vaccination strategies. Anim Health Res Rev 9:25–45

    Article  PubMed  Google Scholar 

  • Reimer D, Frey J, Jansen R, Veit HP, Inzana TJ (1995) Molecular investigation of the role of ApxI and ApxII in the virulence of Actinobacillus pleuropneumoniae serotype 5. Microb Pathog 18:197–209

    Article  PubMed  CAS  Google Scholar 

  • Rybicki EP (2009) Plant-produced vaccines: promise and reality. Drug Discov Today 14:16–24

    Article  PubMed  CAS  Google Scholar 

  • Shin SJ, Bae JL, Cho YW, Lee DY, Kim DH, Yang MS, Jang YS, Yoo HS (2005) Induction of antigen-specific immune responses by oral vaccination with Saccharomyces cerevisiae expressing Actinobacillus pleuropneumoniae ApxIIA. FEMS Immunol Med Microbiol 43:155–164

    Article  PubMed  CAS  Google Scholar 

  • Snider DP (1995) The mucosal adjuvant activities of ADP-ribosylating bacterial enterotoxins. Crit Rev Immunol 15:317–348

    PubMed  CAS  Google Scholar 

  • Tascón RI, Vazquez-Boland JA, Gutierrez-Martin CB, Rodriguez-Barbosa I, Rodriguez-Ferri EF (1994) The RTX haemolysins ApxI and ApxII are major virulence factors of the swine pathogen Actinobacillus pleuropneumoniae: evidence from mutational analysis. Mol Microbiol 14:207–216

    Article  PubMed  Google Scholar 

  • Tremblay R, Wang D, Jevnikar AM, Ma S (2010) Tobacco, a highly efficient green bioreactor for production of therapeutic proteins. Biotechnol Adv 28:214–221

    Article  PubMed  CAS  Google Scholar 

  • Van Haute E, Joos H, Maes M, Warren G, Van Montagu M, Schell J (1983) Intergeneric transfer and exchange recombination of restriction fragments cloned in pBR322: a novel strategy for the reversed genetics of the Ti plasmids of Agrobacterium tumefaciens. EMBO J 2:411–417

    PubMed  Google Scholar 

  • Woof JM, Kerr MA (2006) The function of immunoglobulin A in immunity. J Pathol 208:270–282

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This study was supported by a grant from Biogreen 21 Program (20080401034048), RDA, Republic of Korea.

Author information

Authors and Affiliations

  1. Department of Molecular Biology, Chonbuk National University, Jeonju, 561–756, Republic of Korea

    Mi-Young Kim, Tae-Geum Kim & Moon-Sik Yang

  2. College of Veterinary Medicine, KRF Zoonotic Disease Priority Research Institute and BK21 for Veterinary Science, Seoul National University, Seoul, 151–742, Korea

    Han-Sang Yoo

  3. Jeonju Center, Korea Basic Science Institute, Jeonju, 561–756, Republic of Korea

    Moon-Sik Yang

Authors
  1. Mi-Young Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tae-Geum Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Han-Sang Yoo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Moon-Sik Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Moon-Sik Yang.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kim, MY., Kim, TG., Yoo, HS. et al. Expression and assembly of ApxIIA toxin of Actinobacillus pleuropneumoniae fused with the enterotoxigenic E. coli heat-labile toxin B subunit in transgenic tobacco. Plant Cell Tiss Organ Cult 105, 375–382 (2011). https://doi.org/10.1007/s11240-010-9877-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11240-010-9877-3

Keywords

Search

Navigation