| Edwardsiella tarda is an enteric gram-negative and invasive intracellular fish pathogen which causes enteric septicemia in fish. This bacterium can be applied to develop a recombinant attenuated E. tarda vaccine for fish aquaculture industry. Since live vaccine strains (attenuated by natural selection or genetic engineering) can be potentially released into the environment by the vaccines, safety issues concerning the medical as well as environmental aspects must be considered, for example, the spread of undesired genes like antibiotic resistance genes. However, deletion of asd in E. tarda EIB202results in a DAP dependent mutant that cannot grow in the environment where no DAP exists. Asd is an essential gene for the synthesis of cell wall in most of the gram-negative bacterials. The wild type asd gene was used to replace the antibiotic gene in the plasmids in order to generate non-antibiotic plasmids. Several pUTxa expression vectors with different replicon were transformed into attenuated E. tarda (WEDâ–³asdB) to develop a balanced-lethal system. To obtain a highly efficient growth rate, the SD sequence and start codon of asdB gene were changed to construct various expression systems. The optimized system was tested in vivo and in vitro via a reporter gene gfp and an in vivo inducible promoter Pdps selected by the lab, and the evaluation of the system received anticipated results. The gapA34gene, encoding the Glyceraldehyde-3-phosphate dehydrogenase from the fish pathogen A. hydrophila LSA34, was introduced into the antibiotic-free expression system to generate recombinant vector vaccine WEDâ–³asdB/pUTta4DGap, which was evaluated in zebrafish (Denio rerio) and turbot (Scophtalmus maximus). Here based on the in vivo inducible promoter a non-antibiotic antigen delivery system was constructed and applied to the development of new multivalent vaccine in E. tarda. |
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