Design And Construction Of Bacterial Vector Vaccine In Escherichia Coli And Vibrio Anguillarum Based On In Vivo Lysis System

Recombinant bacterial vector vaccine is an attractive vaccination strategy to induce the host immune response to a carried protective antigen. The superiority of live bacterial vectors includes mimicry of a natural infection, intrinsic adjuvant properties, and the potential for administration by mucosal routes and carrying multi-foreign antigens. This work focused on the design of efficient and safe multivalent bacterial vector vaccine.Firstly, Escherichia coli BL21(DE3) was used as a vaccine vector, and PviuB, with its efficient, strict and self-inducible by sensing iron-limiting signal in vivo characteristics, was selected from23iron-regulated promoter candidates. PviuB was fused with lysis gene E to establish an in vivo inducible lysis circuit. The resultant in vivo lysis circuit was introduced into a strain also carrying a PT7-controlled antigen synthesis circuit, forming a novel E. coli vector vaccine based on in vivo inducible lysis system. The recombinant E. coli produced a large amount of antigen in vitro, and could lyse in response to the iron-limiting signal in vivo, implementing antigen release and biological containment. The gapA gene, encoding the protective antigen GAPDH from fish pathogen Aeromonas hydrophila LSA34, was introduced into the E. coli-based antigen delivery system, and the resultant recombinant vector vaccine was evaluated in turbot (Scophtalmus maximus). Over80%of the vaccinated fish survived challenge with A. hydrophila LSA34, suggesting that E. coli vector vaccine based on in vivo inducible lysis system had great potential in bacterial vector vaccine application.The strictness of expression system is a great concern to keep the stability of bacterial vector vaccine based on in vivo lysis system. So a tightly regulated expression vector PYPfhuAl was constructed. In order to improve the strictness of PfhuA regulation in the purpose of lysis gene expression, Fur box region in promoter sequence and fur expression were refined through five different approaches. Eventually, through substituting E. coli consensus Fur box for original one of PfhuA, the induction ratio of modified PfhuA (named PfhuAl) was improved from3to101. In practice, under the control of Pn1UAl, strong toxic gene E could be successfully expressed, and other two toxic proteins, Gef and MazF, were also functionally svnthesized without E. coli death before induction.Moreover, in V. anguillarum, an iron-limiting regulated promoter PvjuA was screened with its high efficiency and strictness by the way used in E. coli. Four lysis strategies were designed using wild type MVM425and attenuated live vaccine stain MVM6203as the hosts, PviuA as promoter, stable plasmid pUTat as vector, and lysis genes from different phages. It was tested that the recombinant strains425/pAVOE,425/pAVP22,6203/pAVOE, and6203/pAVP22could lyse after induction. The lysis genes of the strains were from the optimized lysis gene E (named OE) and the lysis apparatus13-19-15of Salmonella phage P22. The lysis efficiency was about90%. As observed by the scanning electron microscope, the lysed bacteria were in debris state, indicating that the optimized E gene and13-19-15could lyse V. anguillarum under the control of PViuA.At last, some studies were performed on the background of Flavobacterium columnure ATCC23463, a pathogen of freshwater fish, including its iron-uptake system. In this work, part of the iron uptake machinery of F. columnare was identified and analyzed. During in vitro growth in iron limited conditions, synthesis of an outer membrane protein of～86kDa was up-regulated in F. columnare. Chrome azurol S (CAS) agar plate assay showed that the synthesis and secretion of F. columnare siderophores were constitutive and independent of iron levels. A putative ferric uptake regulator (Fur) protein was also identified in the F. columnare genome, which was similar to Fur proteins involved in iron uptake regulation of other bacteria. Furthermore. Salmonella typhimurium fur mutants were partially complemented by the F. columnare fur gene. We concluded that a siderophore-mediated iron uptake system existed in E. columnare, and fur from E columnare could partially complemented S. typhimurium fur mutant.