Immunogenicity Study Of The Expressed Trivalent Outer Membrane Protein Of Three Pathogens Isolated From Diseased Eels

Eels,delicious and nutritious,are important commercial fishes in China,with its annual export earnings reaching to 0.7-1 billion dollars.However,Eels culture is valuable to be infected by the bacterial pathogens.Research has shown that Aeromonas hydrophila,Edwardsiella tarda and Vibrio vulnificus are three common pathogens in eels farming.Therefore,vaccines aimed at one bacterial pathogen are difficult to apply in aquaculture since its narrow scope of protection and low protective rate.In recent years,immunogenicity study of different outer membrane proteins had become the focus in fish vaccines.These results indicated that outer membrane proteins of pathogens from one genus of bacteria provided cross protection because of their high homology on gene sequences.However,outer membrane proteins from different genus of bacteria shared low homology.On the basic of our study,three gene fragments selected respectively from A.hydrophila,E.tarda and V.vulnificus,which encoded outer regions of the three omps and showed rich epitopes,were amplified after three pairs of PCR primers were designed,and these fragments were combined by?two-steps?fusion PCR.Then we connected the fusion fragment with expression vector.The triple outer membrane protein,highly expressed by the recombinant expression vector and purified by affinity chromatography,was injected to eels to explore its immunogenicity.Results of this study will lay the foundation for further research of triple vaccines of outer membrane protein.The main research methods and results are as follows:1.Three full length genes of OmpU,OmpA and Omp porin?were selected respectively from V.vulnificus,E.tarda and A.hydrophila.Three gene fragments encoded the outer regions of three omps and showed rich epitopes were amplified after three pairs of PCR primers were designed,and 3 fragments were combined by?two-steps?fusion PCR.According to the character of the expression vector?PGEX-2T-his?,two restriction sites of BamH?and Eco R?were added to 5‘points of two primers which are used to amplify two fragments of OmpU and Omp porin?respectively.The fusion fragment was connected with PGEX-2T-his plasmid and the recombinant expression vector expressed fusion omp was constructed.After the vector transformed into E.coli BL21,the fusion omp with molecular weight of 85 kD was highly expressed by using 0.25 mmol/L IPTG to induce the transformant?E.coli BL21?about 5 hours at16?in a suitable bacteria concentration(OD_{600 nm}=0.8).The expressed potein was eluted and purified by nickel column,and then renaturated by dialysis with gradient of urea.The results of this study prepared vaccine antigen for further study of the immunogenicity of the fusion triple out membrane protein.2.One hundred and nifty-five Anguilla japonica were distributed equally into three groups and then intraperitoneally injected?i.p?with phosphate-buffered saline?PBS group?,formalin killed-whole-cell of V.vulnificus,E.tarda and A.hydrophila?FKC group?,or the triple expressed outer membrane proten?OMP group?.On 14,21,28,42 and 56 days post-vaccination,the serum collected were used to assess the titers of specific antibody,complement activity and lysozyme activity;the lysozyme activities in suspension of kidney,skin mucus were also assessed.The result showed that the complement activity in OMP group was significantly?P<0.05?increased compared to FKC group on 14 days.The lysozyme activities in serum of FKC and OMP group were significantly?P<0.05?enhanced compared to PBS group on 42 days.The lysozyme activity of mucus in FKC group was significantly?P<0.05?increased compared to OMP group on 14 days.The lysozyme activities of kidney in FKC group and OMP group were significantly?P<0.05?enhanced compared to PBS group on 21 days.The antibody titers in serum anti-B11/B79/B88 in FKC group were significantly?P<0.05 or P<0.01?enhanced compared to PBS and OMP group.The antibody titer in serum anti-B79 in OMP group were also enhanced?P<0.05?compared to PBS group on 28 days.3.One hundred and sixty-five Anguilla rostrata were distributed equally into three groups and intraperitoneally injected?i.p?with phosphate-buffered saline?PBS group?,formalin killed-whole-cell of V.vulnificus,E.tarda and A.hydrophila?FKC group?,or the triple OMP?OMP group?.On 14,21,28 and 42 days post-vaccination,the serum collected were used to assess the titers of specific antibody,complement activity and lysozyme activity and the whole blood cells collected were used to evaluate the stimulation index;the lysozyme activities in suspension of liver and kidney,skin mucus were also assessed.On 28 d post-vaccination,eels from three groups were challenged by V.vulnificus,E.tarda and A.hydrophila.The result indicated that the stimulation indexes in OMP group and FKC group were significantly?P<0.05?enhanced compared to PBS group.The complement activity of OMP group was increased compared to PBS and FKC group on 14,21 and 42 days.The lysozyme activities in serum,liver and mucus of FKC group were significantly?P<0.05?enhanced compared to PBS group.The lysozyme activity in mucus of OMP group was significantly?P<0.05 or P<0.01?enhanced compared to PBS group.The antibody titer in serum of OMP and FKC group were significantly?P<0.05 or P<0.01?enhanced compared to PBS group.The relative protection rates of triple omp group to A.hydrophila,E.tarda and V.vulnificus was 0%,70%and 60%,respectively.As a result,the triple outer membrane protein vaccine can stimulate eel‘s non-specific and specific immune system,improve its immune function significantly and strengthen its resistance to E.tarda and V.vulnificus.