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In this research, we identified the translocation signal of <jats:italic>Edwardsiella piscicida<\/jats:italic> T3SS effector EseG and constructed an antibiotic resistance\u2010free balanced\u2010lethal system as attenuated vaccine carrier to present antigens by T3SS. <jats:italic>Edwardsiella piscicida<\/jats:italic> LSE40 <jats:italic>asd<\/jats:italic> gene deletion mutant was constructed and complemented with pYA3342 harbouring the <jats:italic>asd<\/jats:italic> (aspartate \u03b2\u2010semialdehyde dehydrogenase) gene from <jats:italic>Salmonella<\/jats:italic>. Fusion proteins composed of EseG N\u2010terminal 1\u2013108 amino acids and the TEM1\u2010\u03b2\u2010lactamase reporter were inserted in plasmid pYA3342. The fusion protein could secrete into the cell culture, translocate into HeLa cells, and localize in the membrane fraction. Then, the double gene deletion mutant LSE40\u0394<jats:italic>asd<\/jats:italic>\u0394<jats:italic>purA<\/jats:italic> was constructed as an attenuated vaccine carrier, and <jats:italic>Aeromonas hydrophila<\/jats:italic> GapA (glyceraldehyde\u20103\u2010phosphate dehydrogenase) was fused with the translocation signal, instead of the TEM1\u2010\u03b2\u2010lactamase reporter. The bivalent vaccine could protect blue gourami (<jats:italic>Trichogaster trichopterus<\/jats:italic>) against <jats:italic>E<\/jats:italic>. <jats:italic>piscicida<\/jats:italic> and <jats:italic>A<\/jats:italic>. <jats:italic>hydrophila<\/jats:italic>, with the relative per cent survival of 80.77% and 63.83%, respectively. These results indicated that EseG N\u2010terminal 1\u2013108 amino acid peptide was the translocation signal of <jats:italic>E. piscicida<\/jats:italic> T3SS, which could be used to construct bivalent vaccines based on an attenuated <jats:italic>E. piscicida<\/jats:italic> carrier.<\/jats:p>","DOI":"10.1111\/jfd.13338","type":"journal-article","created":{"date-parts":[[2021,3,8]],"date-time":"2021-03-08T08:10:42Z","timestamp":1615191042000},"page":"513-520","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Identification and application of T3SS translocation signal in <i>Edwardsiella piscicida<\/i> attenuated carrier as a bivalent vaccine"],"prefix":"10.1111","volume":"44","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0059-944X","authenticated-orcid":false,"given":"Jie","family":"Li","sequence":"first","affiliation":[{"name":"Laboratory for Marine Fisheries and Aquaculture Qingdao National Laboratory for Marine 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