Study On The Cloning, Expression And Immunogenicity Of The Main Virulence-Related Genes From Vibrio Alginolyticus

Vibrio alginolyticus, a halophilic Gram-negative bacterium, inhabiting inmarine and estuarine environments, is one of the major Vibrio pathogenscommun to human and marine animals. Pseudosciaena crocea is widelydistributed the east-southern coast of China. It is known as one of four popularsea fishes in China owing to its good dicicious and nutrition. V. alginolyticusis one main pathogen to Pseudosciaena crocea, which causes it woundinfections, gastroenteritis and septicemia, and leads to large economic damage.Thus, it is essential to explore an effective protective pathway against theinfection of this microorganism. In this study, six main virulence related geneswere cloned from the DNA of V. alginolyticus strain ZJ04107, and theircharacterization was analyzed, then expressed in E.coli. The purified proteinwas evaluated if it could serve as an effective vaccine candidate against V.alginolyticus.The bacterial strain ZJ04107 isolated from the liver of the diseasedPseudosciaena Crocea as well as the typical stain 17700 as control,artificially infected the health Pseudosciaena Crocea, the result indicated thatthe isolated bacterium ZJ04107 was the pathogen of PseudosciaenaCrocea. Based on the physiological and biochemical characteristic analysisand 16S rRNA gene sequence comparison, the strain ZJ04107 was identifiedas Vibrio alginolyticus. The LD₅₀ of strain ZJ04107 is 2.0×10⁷ CFU, thetypical stain 17700 is 8.0×10⁷ CFU. The sensitive tests of antibacterial agentsshowed that the pathogen is sensitive to Ceftriaxon, Sulfamethoxazole,Norfloxacinum, Ofloxacin, Tetracycline and Spectinomycine.Six main virulence related genes(fur, flaA, valC, aspA, ompK and ompW)were cloned from V. alginolyticus strain ZJ04107. The above gene fragmentswhich encode 16.8 kDa, 39.8 kDa, 89 kDa, 56 kDa, 31.3 kDa and 23.3kDa mature protein respectively, were subcloned into pET-30a(+) to constructexpression plasmids (pET-Fur, pET-FlaA, pET-ValC, pET-AspA, pET-OmpKand pET-OmpW), and got over-expressed in E.coli BL21(DE3). Therecombinant proteins were purified by affinity chromatography on Ni-NTAresin. Western blot analysis revealed that the recombinant proteins (Fur, FlaA,ValC, AspA, OmpK and OmpW) had immunoreactivity. Based on the methodof bioinformation and biosoftware, six proteins were constructed molecularmodel on SWISS-MODEL, which will aid to further study on the functions ofthe proteins.Based on the techniques of splicing by overlapping extension (SOEing)and three-times PCR, aspA and ompW was fused together into pET-30a(+), byidentification of enzyme digestion, PCR and sequcencing, two recombinantprokaryotic expression vectors pET-AspA-OmpW and pET-OmpW-AspA weresuccessfully constructed. SDS-PAGE indicated the fusion genes aspA-ompWand ompW-aspA got over-express in E. coli BL21 (DE3), the recombinantproteins were purified by affinity chromatography on Ni-NTA resin. Westernblot analysis revealed that the fusion proteins had immunoreactivity, whichcould provide a base for further study on the immunogenecity of the fusionproteins.Psedosiaena Crocea was immunized with different expressedrecombinant proteins (FlaA, AspA, OmpW, OmpK and OmpW-AspA), themixing FAWK (averagely mixed with FlaA, AspA, OmpW and OmpK) and thewhole-cells of formalin killed Vibrio alginolyticus(FKC). The results of theantibody titers of sera tested by ELISA and the relative percentage survival(RPS) revealed that specific antibody titers against different vaccines weredetected in all sera of the vaccinated large yellow croakers, the antibodylevels were statistically significant in the vaccinated groups in relation to thenegative control (P＜0.05), and reached to the highest on the 5th week aftervaccinated; the highest RPS is FAWK(85.2%), next is OmpW-AspA and FKC (81.5%), then is FlaA and OmpW (77.8%), the lowest is AspA (63.0%).This study provided a good base for explore new style subunit vaccinesagainst the infection of Vibrio alginolyticus.Analysis was carried on part characterization and function of the fur. Itcontained 994 bp, including an open reading frame (ORF) of 450 bp, whichencodes 150 amino acid residues with 16.8 kDa protein. The ribosome-bindingsequence, the putative-10 and-35 sequences and two potential transcriptionalterminators were identified in the fur. Highly conserved sequence from G46 toD104 existed in the middle of the translated Fur amino acid sequence,including an iron-binding motif (H₃-D-H-L-V-C-L-D-C-G) with richHistidines. No 'Fur-box' homology to the E. coli was identified. The fur of V.alginolyticus functionly complemented the fur mutant E. coli H1681, whichprovided a base for further study on the structure and functions of fur and Fur.