Study On The Immunogenicity Of Vibrio Harveyi Outer Membrane Protein (OmpK And GAPDH) And Screening Of Cross Protective Immunogens Of Outer Membrane Protein From Several Main Marine Pathogenetic Vibrios

Marine vibrios are Gram-negative bacteria ubiquitous in marine environment, someof which are main pathogenic organisms responsible to the bacterial ulcer of marineanimals. In view of the bad aftermath like the drug residue in the fish flesh and/or thedevelopment of drug-resistance in pathogens due to the misuse of antibiotics, the curepolicy to vibriosis has to change from the application of chemotherapy to theimmunotherapy. In recent years, people attached more and more importance to the outermembrane proteins (OMPs) of Gram-negative bacteria for their potential to serve asprotective antigens. This research separated three common pathogens from the infectedlarge yellow croaker: Vibrio harveyi, Vibrio parahaemolyticus, Vibrio alginolyticus. WithV. harveyi as main object, two of its OMP genes were cloned and their immunogenicity assubunit vaccines was detected. By the immunoproteomics involving 2-DE andMALDI-TOF-MS, the components of OMP that can induce cross-immunoreaction amongthree vibrios were identified, the probability of developing poly-value vaccines wasexplored.The ORF sequence of ompK from V. harveyi comprised 804 nucleotides encodingOmpK of 267 amino acids with a calculated molecular mass of 29 kDa while the ORFsequence of gapdh from K harveyi comprised 996 nucleotides encodingglyceraldehyde-3-phosphate dehydrogenase (GAPDH) of 331 amino acids with acalculated molecular mass of 35 kDa. Blastn indicates that nucleotide homology of ompKis 83-99% among the Vibrios, and gapdh is more than 91%. The nucleotide sequences ofboth ompK and gapdh were deposited in the GenBank database under the accessionnumbers of DQ279075 and DQ184650, respectively.The analysis with LipoP software showed that OmpK included a signal peptide of 20amino acids in the N-terminal domain, but it was not found in the GAPDH. It is stillunclear how GAPDH travels to the cell surface, ompK gene, of which the signal peptidesequence was omitted, was fused with gapdh by SOE-PCR into ompK-gapdh, and clonedinto pET-30a(+). The expression systems were constructed, named pET-30a-ompK,pET-30a-gapdh and pET-30a-ompK-gapdh, respectively. The recombinant proteins wereexpressed in large scale in E.coliBL21 and purified. The anti-r-OmpK serum and theanti-r-GAPDH serum could specifically recognize the r-OmpK and the r-GAPDHrespectively. Moreover, both antisera could effectively recognize the native OMPsextracted from the fresh-cultured V. harveyi, indicating that r-OmpK and r-GAPDH can induce the same immunoreaction as the native OMPs. The anti-r-OmpK andanti-r-GAPDH can agglutinate the fresh-cultured V. harveyi, indicating that both OmpKand GAPDH all appeared on the surface of the cells.The simulation of the three-dimensional structure of OmpK showed the presence of12 anti-parallelβ-sheets, which organize theβ-cylinder, 6 surface-exposed loops and 5short periplasmic turns, indicating that OmpK structurally belonged to the porin. Themembrane-spanning anti-parallelβ-strands connected by short periplasmic turns andsurface-exposed loops made up a pore inside the cylinder. Different from OmpK, thethree-dimensional structure of GAPDH was mainly composed of two domains that wereconnected by a chain. Each domain contained 9 anti-parallelβ-strands and 6α-helices,apparently not belonging to porin family. Two bands occurred when the anti-r-OmpKserum reacted with the OMPs, meaning that OmpK was the heat modifiable protein likeOmpA. When membrane porins were heated at 100℃for long periods of time, thesecondary structure of the porins was relaxed and a second band appeared, probably due toconformation modifications of the protein like the lost of theβ-barrel structure. Whileonly one band occurred for anti-r-GAPDH, meaning that GAPDH was not heat modifiableprotein.The r-OmpK-GAPDH, r-OmpK, r-GAPDH and their mixture were served as antigensto immunize large yellow croaker respectively. Although the ELISA for the titers of thesera showed a significant difference (p＜0.05) between the trial groups and the controlgroup, the antibody value was not high, suggesting that the humoral immunity acted lesson the protection against viriosis in fish. Percentage of phagocytosis in cells immunizedwith r-OmpK, r-GAPDH, r-OmpK-GAPDH or the mixture of r-OmpK and r-GAPDHshowed variation in comparison with the controls and was statistically significant (P＜0.05). Similar results were obtained for the phagocytic index. Cells treated withr-OmpK-GAPDH had significantly higher values than those in other three treated groups,but these three treated groups were not significantly different from each other. Theseresults demonstrated that the cellular immunity played more important role in theprotection against viriosis in fish. Fish in every group were challenged with the dose of500 LD₅₀ strains (5×10⁶ cfu per fish). The fish immunized with r-OmpK-GAPDHremained high survival percentage (69.1%) compared not only with the control group, butalso with r-OmpK (37.7%) and r-GAPDH (40.0%), even with the mixture of OmpK andGAPDH (41.9%), suggesting a synergistic effect was created between OmpK andGAPDH when they were produced as a fusion protein. By common physiological and biochemical methods and the phylogenetic analysisbased on the partial sequences of HSP60 genes, eight strains of pathogens separated frominfected large yellow croakers were identified distinctly to the different strains whichbelonged to V. harveyi, V. parahaemolyticus and V. alginolyticus. The antiserum againstthe whole V. harveyi was prepared and used to screen OMPs from V. parahaemolyticusand V. alginolyticus that could cross-react with it. By the immunoproteomics methodinvolving 2-DE and MALDI-TOF-MS, the components of OMP that can inducecross-immunoreaction among three vibrios were identified as the maltoporin of V.parahaemolyticus and a putative porin of V. alginolyticus. Gene engineering vaccinesbased on these protein antigens are expected to protect simultaneously from the infectionof sereral different vibrios, and will give an important suggestion to the development ofpoly-value gene engineering vaccines.