Studies On The Immunogenicity Of The Protein Expressed By Reconstrural Two Genes Separately From The Out Membrane Protein Of Vibrio Vulnificus And Edwardsiella Tarda

Eels culture is a large industry in China,but diseased infected by bacterial pathogens is serious.Since Vibrio Vulnificus and Edwardsiella tarda are main pathogens in farming eels,it will be promising to develop vaccines against these pathogens.Conventional vaccine such as inactivated vaccine shows high toxicity,and one pathogen often has different subspecies or serotypes and generally had no cross-protection.Genetic engineering subunit vaccines protect fish from only one pathogen infecting,and it is difficult to be used in aquaculture due to limited protection to pathogen infection.This study was conducted to protect eels from infecting by two above-mentioned bacterial pathogens,by building a splicing of two outer membrane protein(OMP)genes in vitro and expressing a bivalent OMP efficiently after constructing a recombinant expression vector.The main contents and results of this study showed as following:(1)In this study,template DNA was extracted from pathogenic Vibrio Vulnificus and Edwardsiella tarda isolated from diseased eels and confirmed by challenge experiment.According to gene sequences coding omp in GenBank database,two pairs of primers were designed and the whole gene sequences of ompA of Edwardsiella tarda and ompU of Vibrio vulnificus were amplified by PCR.Protein structure,hydrophilicity and immunogenicity of two sequences were analyzed by some relevant software.Two gene fragments,showing good hydrophilicity and immunogenicity and encoding the out part of the membrane region of ompU and ompA respectively,were obtained and combined by fusion PCR.(2)Optimum expression conditions of the recombinant expression vector were studied on different IPTG concentration,temperature,culture time and bacterial concentration.The results indicated different concentration of IPTG,temperature,time and bacterial concentration had no distinct influence on the expression of the bivalent protein.Expression of the bivalent OMP was induced by 0.25 mM IPTG over night at(pGEX-2T-His-Vibrio-Edwa)constructed,and the expressed protein with the molecular weight about 77.6 kD was purified by HisPur Ni-NTA Resin and Kits.(3)One hundred eighty Japanese eels(Anguilla japanica)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 E.tarda(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,E.tarda or Aeromonas hydrophila.The results showed that,Relative Percent Survival(RPS)after challenged by V.vulnificus and E.tarda on 28 days post immunization in OMP group vs.PBS group were significantly(P < 0.05)enhanced from 5 d and 12 d respectively,and RPS of the infection of two pathogens was 50% in the eels of bivalent Omp group.Proliferation of whole blood cells in OMP group was enhanced compared to the PBS group and FKC group on 21 days and Proliferation of whole blood cells in FKC group was significantly(P < 0.05)enhanced compared with the OMP group.Compared with the PBS group,the serum titers of anti-V.vulnificus and anti-E.tarda antibody in eels of FKC and OMP group were significant increased(P < 0.05 or P < 0.01)on 21 and 28 days.On 42 d post immunization,the serum titers of anti-V.vulnificus and anti-E.tarda antibody in eels of OMP group were significant increased(P < 0.01)compared with the PBS and 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.These results suggest that Japanese eel immunized with the bivalent OMP would positively affect specific as well as non-specific immune parameters and protect eels against infection by the two pathogens in freshwater farming.