| Aeromonas hydrophila is widely distributed in the world.The diseases caused by the pathogenic strains of this organism have also prevailed in seawater or freshwater culture and brought about a great loss to aquaculture production, which arose great concern about how to control these diseases effectively among scientific researchers.Bacterial ghosts are empty and intact bacterial envelopes of Gram-negative bacteria,which are produced by controlled expression of phage phiX174lysis gene E and devoid of cytoplasmic content. As the surface structure and properties of their original state remained, it is a more ideal vaccine system than conventional vaccines. Current bacterial ghosts production depends on the transformation and induction of plasmids with lysis gene E and its regulatory elements, which brings in the potential risk of antibiotic resistance gene spread and safety problem from the remanent inactivated cells during the ghost preparation.To solve this problem, the lysis gene E and its regulatory elements (cI857/pR/E/rrnb T1T2) were integrated into the aroA gene of A.hydrophila in the present study and the constructed mutants could be used as a vaccine candidate for bacterial ghost production with less virulence and higher safety.1. Identification of Aeromonas hydrophila Strain as Vaccine CandidateA bacterial strain J-1, which was isolated from the moribund crucian carp with the symptom of hemorrhagic septicemia, was identified as aeromonas hydrophila by morpholgy, selective medium,biochemical tests, sequence comparison of16S rDNA and gyrB. Artificial infection experiment showed that the isolate was virulent to allogynogenetic sliver crucian carp with an LD50of1.2X106CFU. 2. A Novel Dual Vector Coexpressing PhiX174Lysis E Gene and Staphylococcal Nuclease A Gene on the basis of Lambda Promoter pR and pL, RespectivelyBacterial Ghost is a novel vaccine platform and its safe and efficient production depends largely upon a suitable and functional vector. In this study, a series of temperature-inducible plasmids, carrying phix174lysis gene E and/or staphylococcal nuclease A (SNA) gene, were constructed and evaluated in Escherichia coli. The results showed that the direct product of SNA (pBV220-SNA) could degrade the plasmid and genomic DNA of E.coli while the fusion product of gene E and partial Cro gene (pKF396M-2) lost the ability to lyse the host strain. The insertion of enhancer T7g10elements and Shine-Dalgarno box (ESD) between them (pKF396M-3) could resume the function of gene E. Using plasmid pKF396M-4with gene E and SNA respectively under the immediate control of promoter pR and pL, the remnant plasmids and genomic DNA of E.coli were eliminated and the rates of inactivation increased by2orders of magnitude over that obtained with the exclusive use of E-mediated lysis plasmid. By substituting these two genes with customized multiple cloning sites (MCS) sequences, the plasmid could be modified to a dual expression vector (pKF396M-5).3. Construction and Characterization of Aeromonas hydrophila aroA-Non-marked Deletion MutantThe aroA gene and its flanked region of aeromonas hydrophila J-l was cloned, and the sequences was determined. The nucleotide sequence of3704bp showed a high degree of identity of97%with the aeromonas hydrophila ATCC7966. On the basis of this the upstream and downstream region of aroA was amplified respectively with redesigned and synthesized primers, and the PCR products were cut with restriction endonuclease and ligated to the corresponding sites of suicide vector pDS132with the MCS modification, therefore constructing a recombinant suicide vector with the1103bp deletion aroA gene. This suicide vector was transformed to aeromonas hydrophila J-l by conjunction and the non-marked aroA deletion mutant which has experienced two homologous recombination was selected by PCR identification.The aroA deletion mutant could normally grow on LB, Rimler-Shoots selective medium and differential medium AHM, but grew slowly in the LB broth than J-l. Among the15kinds of biochemical and physiological tests, only the result of the item VP test was different with J-l (negative). The ECP activity test was positive and the LD50to allogynogenetic sliver crucian carp was2.8×108CFU. 4. Aeromonas hydrophila Ghosts Production by Expression of Chromosomally-Based Lysis Gene EUsing recombinant DNA technology, the lysis gene E and its regulatory elements (cI857-pRM/pR/-E-rrnbT1T2) were intergrated into the aroA gene of A.hydrophila J-l and the plasmid-independent bacterial ghosts were produced sussessfully by induction of temperature upshift from28℃to42℃. The lysis kinetics showed that the OD600value of culture media increased rapidly one hour after induction and then began to decrease gradually two hours later and finally came to a steady state at the time of six hour when the number of the viable cells declined by5orders of magnitude, from6.4X107CFU/ml to1.7×102CFU/ml. Scanning electron microscopy observation proved that most lysed bacteria shrinked and the transmembrane tunnel located either in the middle of the cells or at the polar sites.5. Evaluaion of Induced Immune by Aeromonas hydrophila Ghosts in silver carps, Carassius auratus gibelioAt temperature of27.2℃～29.8℃in water, the immunisity induced by aeromonas hydrophila ghosts (AHG), formalin-killed A.hydrophila (FKC) and PBS were compared in silver carps (Carassius auratus gibelio) by intraperitoneal immunization. The results showed that the serum agglutination titers induced by AHG group (1:277.3) was highest compared to FKC (1:170.7) and PBS control (1:9.6). AHG also induced higher bactericidal activity than either FKC or control, while in lysozyme activity, AHG had similar effect to FKC (0.089),only showing significantly higher level than control (0.032). In challenge test, the AHG-immunized fishes had best protection against A.hydrophila (90%) compared to65%and15%survival in FKC and PBS control, respectively.These findings indicated that AHG could induce stronger immune response and could confer enhanced protection against infection. |
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