| Eel is an important economic farming fish,but large-scale intensive farming increased injections of different pathogens.Diseased infected by Bacterial pathogens is common in farmed eels in China,and have caused significant economic losses.Many fish pathogens had been reported and one species of pathogen often showed different subspecies or serotypes.Generally,subunit vaccines protect fish from one pathogen infecting,but it is difficult to be used in aquaculture due to limited protection to the infection of different pathogens.In this study,template DNA was extracted from pathogenic Vibrio vulnificus and Aeromonas hydrophila isolated from diseased eels in recent 10 years.Designing primers according to gene sequences coding the Outer Membrane Protein(OMP)in GenBank database,the whole gene sequences of ompU and porin II were amplified.After sequence analysis,two gene fragments coding the outer part of the OMP of two pathogens(B82 and B11)were combined by fusion PCR in vitro.The expressed vector(pGEX-2T-His-Vibrio-Aero)contained two above-mentioned gene fragments was constructed and highly expressed.After the expressed protein was purified,it was used to immunize American eel to study the immunogenicity of the expressed product by detecting the activity of the lysozyme,Stimulation Index(SI)of whole blood cells,titers of serum antibody and protect effect of challenge to the expressed protein in eels.The main research contents and results showed as following:1.According to whole and OMP gene sequences of Vibrio vulnificus and Aeromonas hydrophila in the Genebank database,we designed primers according to up and down ssequences near the whole OMP gene.After sequence analysis,genes were used for protein structure,hydrophilicity and immunogenicity analysis by some relevant softwares.Two gene fragments,encoding outer membrane region of the OmpU and porin II respectively,which showed good hydrophilicity and immunogenicity were combined by the “two-steps” fusion PCR.According to the cut sites of BamH?and EcoR?of the expression vector(pGEX-2T-His),two restriction enzymes were added in the combined sequence contained two fragments of ompU and porin II and the recombinant expression vector(pGEX-2T-His-Vibrio-Aero)contained two fragments of outer membrane protein was constructed.2.One hundred eighty American eels were distributed into 3 equal groups and intraperitoneal(i.p)injection with phosphate-buffered saline(PBS group),formalinkilled-whole-cell(FKC)of V.Vulnificus and A.hydrophila(FKC group)or the bivalent OMP(OMP group).The whole blood cells collected on 14,21,28 and 42 days post-vaccination were used to evaluate the stimulation index(SI)and the sera collected on 14,21,28 and 42 days were used to assize the titers of specific antibody as well as lysozyme activity.Lysozyme activities in skin mucus,suspension of liver and kidney were also recorded on 14,21,28 and 42 days.On 28 d post-vaccination,eels from all three groups were challenged by i.p injection of live V.vulnificus or Aeromonas hydrophila.The results showed that,Relative Percent Survival(RPS)after challenged by V.vulnificus and A.hydrophila on 28 days post immunization in OMP group vs.PBS group were significantly(P < 0.05)enhanced from 1 d,and RPS of the infection of V.vulnificus and A.hydrophila were all 50% in the eels of bivalent Omp group.Proliferation of whole blood cells in OMP group was enhanced compared to the PBS group on 28 days.Compared with the PBS group,the serum titers of OMP group was significantly increased during the whole experiment time,and on day 42,the serum titers of OMP group was significantly higher than the FKC group.Activity of the lysozyme in serum,skin mucus,liver and kidney were significantly changed(P < 0.05 or P < 0.01)between the three groups.The results showd that the bivalent expressed OMP could significantly improve the specificity and non-specific immune function of American eels,and strengthen the resistance of eels to the infection of two bacterial pathogens. |
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