Novel Expression System for Combined Vaccine Production in Edwardsiella tarda Ghost and Cadaver Cells

Abstract

To develop combined vaccine systems, we have generated Edwardsiella tarda ghosts (ETG) displaying a foreign protein on the outer membrane and also Ed. tarda cadaver (ETC) expressing a heterologous protein in the cytoplasm. Green fluorescent protein (GFP) was used as a model foreign protein. A constitutive promoter (EtPR C28-1) cloned newly from Ed. tarda was used as a promoter for the expression of foreign protein. Comparison of the strength of the new promoter with a commercially available constitutive promoter (P(HCE)) showed higher expression levels of the novel expression system. The N-terminal domain of ice nucleation protein (InaN), an outer membrane protein of Pseudomonas syringae, was used as an anchor motif for surface display of GFP. By transformation of Ed. tarda with the constructed vectors, GFP was successfully expressed on the surface of ETG and in the cytoplasm of ETC. When compared to P(HCE) driven expression, approximately more than 2 times of GFP was expressed on ETG and in ETC by EtPR C28-1 promoter when judged by fluorescent spectrophotometry. Furthermore, significantly higher expression of GFP on the surface of ETG by EtPR C28-1 than by P(HCE) was demonstrated by serum agglutination assay. These results suggest that the newly cloned Ed. tarda constitutive promoter is capable to express foreign proteins not only on the surface of Ed. tarda ghosts but also in the cytoplasm of Ed. tarda cadavers, and can be used as an efficient promoter for the expression of heterologous antigens of the ETG and ETC-based combined vaccines.

Generation of two auxotrophic genes knock-out Edwardsiella tarda and assessment of its potential as a combined vaccine in olive flounder (Paralichthys olivaceus)

“…The asd gene expression was driven by a weak constitutive promoter (G02) that was selected from the previously constructed E. tarda promoter trap library [40]. Briefly, the putative promoter region (G02) was PCR-amplified by using a pair of primers (G02 AatII F and G02 SpeI R), cloned into pGEM-T vector, and sequenced for confirmation.…”

“…The antibiotic resistance gene (Amp R ) in pG02-ASD was removed by digestion of the plasmid with DraI and NarI, then a sequence containing multiple cloning sites (DraI-BglII-NcoI-ScaI-NsiI-NarI) was inserted into the digested plasmid, resulting in pG02-ASD-MCS. The GFP expressing cassette driven by a strong constitutive promoter (EtPR C28-1) of E. tarda [40] was cut by digestion of plasmid pEtPR-GFP with ApaI and NsiI, then, ligated to the pG02-ASD-MCS, and designated as pG02-ASD-EtPR-GFP.…”

“…Vector pG02-ASD-EtPR-GFP contains two cassettes. One has a strong constitutive promoter (EtPR) cloned from Edwardsiella tarda[40] and the green fluorescent protein (GFP) gene as a model foreign protein. The other cassette consists of a weak constitutive promoter (G02) of E. tarda, aspartate semialdehyde dehydrogenase (ASD) gene, and a transcriptional terminator (rrnBT1).…”

Differential resistance to edwardsiellosis in rohu (Labeo rohita) families selected previously for higher growth and/or aeromoniasis-resistance

“…Now, researchers are looking into developing vaccines to tackle its epizootics. Recently, a few effective vaccines have been developed viz., E. tarda ghosts (Kwon et al 2006;Choi et al 2010), rifampicin-resistant strains (Evans et al 2006;Sun et al 2010), recombinant GAPDH (Liu et al 2007), and mutants for esrB gene (Lan et al 2007), with variable success but these are yet to be commercialized. In such conditions, selective breeding seems to provide the most promising solution to the disease problem.…”

Autotransporter-Based Antigen Display in Bacterial Ghosts

“…For surface display of antigens in bacterial ghosts, the ice nucleation protein (6) and various outer membrane proteins, such as outer membrane protein A, have been used as anchors (12,13). Recently, it has been shown that antibody responses to heterologous antigens that are secreted by Salmonella cells or exposed at the surface of Salmonella-derived outer membrane vesicles (OMVs) are higher than antibody responses to antigens that are present intracellularly or in the lumen of the OMVs, respectively (e.g., see references 14 to 16).…”