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

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) (ApxIIA 619-801 ) 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 G M1 -ganglioside enzyme-linked immunosorbent assay (G M1 -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 lg g -1 , in lyophilized leaf tissue. These results demonstrate the feasibility of using a transgenic plant to express the LTB-ApxIIA 619-801 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.Keywords Enterotoxigenic Escherichia coli heat-labile enterotoxin B subunit Á Actinobacillus pleuropneumoniae Á Plant-based edible vaccine Á ApxIIA

Expression and functional validation of heat-labile enterotoxin B (LTB) and cholera toxin B (CTB) subunits in transgenic rice (Oryza sativa)

“…CTB has been expressed in a number of plant species, including potato (Arakawa et al 1997 ), tomato (Jani et al 2002 ), tobacco (Jani et al 2004 ), carrot (Kim et al 2009 ), rice (Oszvald et al 2008 ; Kajiura et al 2013 ), and maize (Karaman et al 2012 ). Similarly, LTB was expressed in transgenic tobacco (Kang et al 2003 ; Kim et al 2011 ), rice callus (Kim et al 2010 ) and carrot (Rosales-Mendoza et al 2007 , 2008 ), including the hairy roots of Solanaceous species (Guzman et al 2011 ), and transgenic lettuce (Kim et al 2007 ). However, the majority of plant expression systems pose challenges for vaccine production.…”

Oral immunization with a lettuce-derived Escherichia coli heat-labile toxin B subunit induces neutralizing antibodies in mice

“…In recent years, plants have been used to express antigenic proteins against myriad diseases as they serve as low-cost ''biofactories'' and vehicles for these functional protective antigens (Hoffmeisterová et al 2008;Kim et al 2006Kim et al , 2011Landell et al 2008;Lau and Korban 2009). These alternative subunit vaccines have several advantages, such as low-cost of production and friendly administration .…”

Expression of a KDEL-tagged dengue virus protein in cell suspension cultures of Nicotiana tabacum and Morinda citrifolia

“…In particular, recombinant proteins produced in the whole plant or in plant cell cultures can be targeted to different subcellular compartments. The addition of the endoplasmic reticulum (ER) retention/retrieval sequence KDEL (Lys-Asp-Glu-Leu) to the C-terminal end of different recombinant proteins has been studied largely (Kim et al 2006(Kim et al , 2010aLoc et al 2010). It has been shown that KDEL increases recombinant protein accumulation levels in different plant expression systems, such as scFv antibodies in tobacco plants (Fiedler et al 1997) and rice callus tissues (Torres et al 1999), the 14D9 catalytic antibody in hairy roots of tobacco (Martínez et al 2005) and the spider silk protein in tobacco and potato plants (Scheller et al 2001).…”