Development Of Display Systems And Balanced-lethal Host-vector System In Vibrio Angillarum

Vibrio anguillarum is an important bacterial fish pathogen, responsible for both marine and freshwater fish epizootics throughout the world. In our previous work, several attenuated V. anguillarum strains derived from wild-type V. anguillarum strain MVM425 were constructed with recombinant DNA technology and proved to be excellent live vaccine candidates against Vibrio pathogens.For developing potential multivalent recombinant vaccines based on attenuated V. anguillarum live vaccine, efficient surface display systems are needed to display protective antigens onto the surface of live carrier. In this work, a series of novel cell surface display systems were examined by employing V. anguillarum outer membrane protein as anchoring motifs. These display systems consist of two parts. The first is the signal sequence and first eleven N-terminal amino acids of V. anguillarum outer membrane lipoprotein Wza, or the signal sequence and first nine N-terminal amino acids of the mature major Escherichia coli lipoprotein Lpp, and the second part is the transmembrane domains of V. anguillarum outer membrane proteins Omporfl, OmpU or Omp26La. Green fluorescent protein (GFP) was inserted to the systems and the results of GFP surface localization confirmed that four of the six surface display systems could successfully display GFP on Escherichia coli surface. The major capsid protein (MCP) of large yellow croaker iridovirus (LYCIV) was fused with Wza-Omporfl system and was displayed on the surface of an attenuated V. anguillarum MVAV6203, and a putative antigen protein EseB from pathogenic Edwardsiella tarda was successfully expressed on the surface of MVAV6203 to get multivalent vaccine candidates. The immune protection evaluation in zebra fish (Danio rerio) demonstrated that the V. anguillarum EseB-display strain AV/pW-26La-B could trigger a full protection against V. anguillarum infection and an early protection against E. tarda infection in the immunized fish. These results suggest that surface display of heterologous protective antigens in attenuated V. anguillarum could be used as a tool to develop potential V. anguillarum vector vaccine.At the same time, the secretion of MCP based on the E. coliα-haemolysin transport system was investigated in both E. coli and attenuated V. anguillarum MVAV6203. With the aid of E. coli a-haemolysin transport system, about 400μg/L MCP was secreted into culture supernatant in E. coli, while intracellularly expressed MCP in MVAV6203 was not secreted. In order to optimize the V. anguillarum vaccine strain and develop an antibiotic-sensitive recombinant attenuated Vibrio vaccine, we try to develop a balanced-lethal host-vector system by delete the V. anguillarum relA gene, aspartate-semialdehyde dehydrogenase gene asd and insert TTaraC PBAD c2. We found that V. anguillarum contained two asd genes, asdA and asdB. Each of two genes functioned to complement different asd mutants from other bacterial species, and double deletions of asdA and asdB in V. anguillarum were not lethal. In the 3D structure analysis of AsdA and AsdB it was shown that AsdB isoform is quite different from the AsdA isoform and in fact more closely resembles the Gram-positive spASD structure. The result was confirmed in their alignment analysis. We also found that in V. anguillarum the glyceraldehyde-3-phosphate dehydrogenase can be secreted to the supernatant.