A mutant strain ΔVcrV was constructed by using homologous recombination method for investigating the function of the VcrV gene in Vibrio alginolyticus type Ⅲ secretion system. The genetic stability of ΔVcrV was detected by PCR, and the biological characteristics between the mutant and the wild type strains were compared. ΔVcrV muntat had no significant changes in growth rate and autoagglutination compared with the wild type strain, but the ability to form biofilms was reduced, and the LD₅₀ was increased by 16.5 times. The swimming and swarming motility of the mutant strain ΔVcrV were significantly enhanced, while cell adhesion was significantly reduced than the wild strain (P < 0.01). The tolerance of ΔVcrV mutant to H₂O₂ and NaCl was decreased. Compared with that of the wild type strain, the sensitivity of ΔVcrV mutant to cefuroxime, medimycin and clindamycin was increased, but to amikacin and polymxin B was decreased. The reactive oxygen species (ROS) content of ΔVcrV mutant was significantly decreased (P < 0.01), and the indexes of proline, peptidoglycan, β-lactamase, catalase, superoxide dismutase and glutathione peroxidase of ΔVcrV mutant were significantly increased than that of the wild type strain (P < 0.01). The biological characteristics of ΔVcrV mutant indicated that VcrV gene was involved in pathogenicity and various biological functions of V. alginolyticus type Ⅲ secretion system.
Animals ; Bacterial Proteins/metabolism* ; Fish Diseases ; Hydrogen Peroxide/metabolism* ; Type III Secretion Systems ; Vibrio Infections ; Vibrio alginolyticus/genetics*

In order to develop a recombinant attenuated Salmonella typhimurium as oral live vaccine vector, we constructed recombinant plasmid pYA-sopENt100 by replacing the trc promoter with the sopE promoter and secretion signal sequence sopENt100 of Salmonella typhimurium on the basis of plasmid pYA3493. Then, the complementary plasmid pYA-sopENt100 was transformed into ΔcrpΔasdSL1344 by electroporation to generate attenuated Salmonella typhimurium type III secretion system ΔcrpΔasdSL1344 (pYA-sopENt100). We further characterized ΔcrpΔasdSL1344 (pYA-sopENt100). We also constructed a recombinant strain ΔcrpΔasdSL1344 (pYA-sopENt100-egfp) that harbored the reporter gene-enhanced green fluorescent protein (egfp) gene. Vero cells were infected with ΔcrpΔasdSL1344 (pYA-sopENt100-egfp) and the ability of delivery foreign antigens was tested via Western blotting analysis. The results of PCR, enzyme digestion and sequencing showed that the ΔcrpΔasdSL1344 (pYA-sopENt100) type III secretion system was constructed successfully. The serotype of ΔcrpΔasdSL1344 (pYA-sopENt100) was identical to ΔcrpΔasdSL1344 and SL1344. Compared with wild strain SL1344, the biochemical characteristics of ΔcrpΔasdSL1344 (pYA-sopENt100) had obvious change, but it was basically the same with ΔcrpΔasdSL1344. The growth speed was much slower than that of the wild strain SL1344. The chicken virulence test (LD₅₀) showed that the virulence of ΔcrpΔasdSL1344 (pYA-sopENt100) was 7×10⁴ times lower than SL1344. In addition, we observed the 37 kDa SopENt100-egfp protein in the cultured supernatant of ΔcrpΔasdSL1344 (pYA-sopENt100-egfp) strain by Western blotting analysis. However, both the 37 kDa SopENt100-egfp protein and 27 kDa EGFP protein were detected in ΔcrpΔasdSL1344 (pYA-sopENt100-egfp)-infected Vero cells. These results demonstrated that the recombinant Salmonella typhimurium type III secretion system ΔcrpΔasdSL1344 (pYA-sopENt100) was successfully constructed, and it should be used as a live vaccine vector for expressing foreign genes.
Animals ; Bacterial Proteins ; genetics ; Cercopithecus aethiops ; Mice ; Plasmids ; Promoter Regions, Genetic ; Salmonella typhimurium ; genetics ; Type III Secretion Systems ; genetics ; Vaccines, Attenuated ; genetics ; Vero Cells ; Virulence