Immunogenicity Study Of The Expressed Single And Multiple Outer Membrane Protein Of 3 Pathogens Isolated From Diseased Eels

Aeromonas hydrophila,Edwardsiella anguillarum and Vibrio vulnificus are 3 common pathogens in eel culture.In this study,on the base of good antigen of 3 outer membrane protein genes(Omp,OmpⅡ from A.hydrophila,OmpA from E.anguillarum and OmpU from V.vulnificus),three subunit products of single rOmpⅡ,bivalent rOmpⅡ-A and trivalent rOmpⅡ-UA were expressed and purified after 3 expression vectors were constructed respectively.This study included 3 purposes:whether the immunization of bivalent and trivalent expression products affected their resistance to the infection of A.hydrophila;:whether the immunization of single and bivalent expression products produced cross-protective effects against 3 pathogens;whether the expressed trivalent outer membrane proteins could simultaneously resist the infection of 3 pathogens respectively.The research methods and results were as follows:(1)Based on the previous acquired immunogenicity of these three Omps(OmpII,OmpA and OmpU).Three vectors expressing OmpⅡ,bivalent of rOmpⅡ-A trivalent of rOmp-II-A-U respectively were constructed.These expressed products were purified by nickel column,and then renatured by gradient urea.The molecular weight of three expressed products was 58kDa,56 kDa and 62 kDa respectively.(2)Four hundred European eels(Anguilla anguilla)were divided equally into 5 groups.PBS(PBS group),Freund’s incomplete adjuvant Equal mixed with PBS(PBS+F,adjuvant group),rOmpⅡ(rOmpⅡ+F,single Omp group),rOmp-II-A(rOmpⅡ-A+F,bivlaent Omp group)or rOmpⅡ-U-A(rOmpⅡ-U-A+F,trivalent Omp group)were intraperitoneally injected to European eels,respectively.At 0.5 h,12 h,24 h,48 h and 96 h after the immunization,Liver,spleen,kidney and intestine were collected and used to analyze the expression of immune related genes.On 14,21,28 and 42 day post the immunization(dpi),the whole blood cells were collected to evaluate the stimulation index;the serum and skin mucus were collected to assess the titers of specific antibody,lysozyme activity and the superoxide dismutase(SOD),and SOD activity in the liver and kidney were also detected.The results showed that the mhc2,lysC,sod and igm genes in three Omp groups were significantly up-regulated(p<0.05),but lysC and igm genes were not significantly up-regulated in the intestines and kidneys of these Omp groups.On 28 dpi,the stimulation index of whole blood cells in the single Omp group was significantly(p<0.05)increased compared with the other four groups.On the 14,21,28 and 42 dpi,antibody titers in the serum and mucus of three Omp groups were significantly(p<0.05)increased compared with the PBS and adjuvant group.Lysozyme activity in the skin mucus increased gradually after immunization,while the lysozyme activity in serum peaked at 28 dpi.The SOD activity in liver,kidney,skin mucus and serum of three Omp groups was significantly(p<0.05)increased,and the increasing effect in the serum was more obviously.On 28 dpi,eels from five groups were challenged by A.hydrophila,E.anguillarum or V.vulnificus.The result indicated that the relative percent survival(RPS)of the single Omp group against A.hydrophila,E.anguillarum and V.vulnificus were 83.33%,55.56%and 33.33%.The RPS of the bivalent Omp group against A.hydrophila,E.anguillarum and V.vulnificus were 83.33%,55.56%and44.44%,and the protective effect of the bivalent group against A.hydrophila and E.anguillarum was the same as the single Omp group which showed significantly(p<0.05)higher than the PBS group.The RPS of the trivalent Omp group against A.hydrophila,E.anguillarum and V.vulnificus were 66.67%,22.22%and 44.44%.In conclusion,we found that the single expressed Omp of A.hydrophila and the bivalent expressed Omp of A.hydrophila and E.anguillarum were good antigens,and the good immune protective effect of the two Omps was due to the effective stimulation of the immune-related genes in eel post the immunization,and thus enhanced the antimicrobial capacity of blood cells,serum titers and SOD activity,but the protection effect was not obvious related with the lysozyme activity and the antibody titers in mucus.At the same time,this study also found that the single Omp of A.hydrophila had significant cross-protection effect to E.anguillarum.In addition,as far as A.hydrophila is concerned,the bivalent expressed Omp had no effect on its immune protection,while the triple expressed Omp reduced.its immune protection to a certain extent.