| Edwardsiella is one of the most common pathogenic bacteria in aquatic animals,and it shows strong pathogenicity to freshwater and seawater fishes.It is easy to cause a large number of deaths and huge economic losses after intensively cultured fish are infected with this bacterial pathogen.Therefore,the prevention and treatment of diseases caused by this kind of bacteria has become a research hotspot of aquatic disease prevention and controling.In our previous study,outer membrane protein A(OmpA)from Edwardsiella anguillarum had been proved to be a good antigen.In this study,we intends to explore the reason why OmpA has good immunogenicity from the identification of B cell linear epitope of OmpA,and to find the mechanism of its immunoprotective effect on Eruopean eel(Anguilla anguilla)post the immunization from the transcriptome analysis.The main research methods and results are as follows:1.Gene cloning and expressing of the outer membrane protein A(OmpA)according to the DNA temperate of the strain of E.anguillarum.New Zealand white rabbit and European eels were immunized by the OmpA after it was purified,and the serum of rabbit was collected when the anti-OmpA serum ELISA titer was 1:40000,then IgG in the rabbit serum was separated by proteinA column affinity chromatography.The theoretical epitopes of the OmpA were analyzed by the ABCpred Prediction Server and 19 B-cell linear epitopes were ascertained,then hese epitopes were synthesized and coated on the ELISA plate.Three epitopes were identified by the indirect ELISA using the purified rabbit IgG,and 2 epitopes were identified by indirect ELISA when the eel serum was used,then a novel common epitope,VETKRFTLKSDVLFNF(215-230),was found when the OmpA reacted with rabbit and eel antibodies respectively.The B-cell epitope was identified for the first time using rabbit and fish serum simultaneously,and results of this study will provide the reason of good immunogenicity of OmpA from the peptide level of B cell linear epitope.2.The eels were divided into three groups:blank control group(control group),1.0 ×107cfu/mL of E.anguillarum challenging group(0.2mL/tail,challenge group)and 1.0×107cfu/mL E.anguillarum challenging group(0.2mL/tail,immune group)post the immunization of OmpA.The eels in the immune group were immunized twice with Freund’s complete and incomplete adjuvants and equal volume OmpA(1.Omg/mL)respectively,and were challenged by E.anguillarum 14 days after the boost immunization;the eels in the challenge group were challenged by the same method,and the eels in the control group were keep unchallenged.Eels from three groups were collected for anatomical and histopathological observation after 48 h of challenge.At the same time,total RNA of livers from three groups of eels and E.anguillarum cultured in vitro was extracted.The different expression genes(DEGs)between the transcriptome of E.anguillarum cultured in vivo(in the liver)and cultured in vitro were analyzed by high-throughput transcriptome sequencing,and the gene expression of the transcriptome in the liver of three groups were compared and verified by fluorescence quantitative PCR(qPCR).Finally,interaction betveen the genes of E.anguillarum and the genes of the eel liver was analyzed by dual RNA-seq to explore the co expression of genes between the pathogen and the host in the process of infection and anti-infection.The results showed that,compared with the control group and the immune group,the livers of eel in the challenge group had serious congestion and/or bleeding,and there were a lot of thrombosis in the liver vessels;compared with E.anguillarum cultured in vitro,the gene expression of E.anguillarum in the liver of challenge group eels was generally down regulated;there were few up and down regulated genes between the transcriptome of eel liver in the control group and the immune group;compared with the control and the immune group,a lot of genes in the liver of the challenge group was significant up and down regulated,and some of the genes were confirmed by fluorescence quantitative PCR.Therefore,the results of this study elucidated the reasons for the immunoprotective effect of OmpA on European eels at the transcriptome level.3.Go and KEGG analysis showed that the down regulated genes of E.anguillarum in the liver were mainly involved in cellular amino acid metabolic process,organic acid metabolic process,mRNA metabolic process and metabolic process in the GO function,and these genes are mainly enriched in the biosynthesis of secondary metabolites,the resistance of cationic antimicrobial peptide(cAMP),the biosynthesis of antibiotics,the citrate cycle and the biosynthesis of amino acids ACEDS)and other KEGG pathway.Compared with the control and the immune group,a large number of up-regulated genes were mainly involved in small molecule binding,nucleotide binding,catalytic activity and oxidation-reduction process in the GO function,and these genes were mainly enriched in the RIG-Ⅰ-like receptor signaling pathways,NF kappa B signaling pathway,TNF signaling pathway and Toll-like receptor signaling pathway and other KEGG pathway;A large number of down regulated genes in the challeged group were mainly involved in metabolic process,coenzyme binding and iron ion binding in the GO funtion,and these genes were mainly enriched in the Drug metabolism-cytochrome P450,Vitamin B6 metabolism and Fat digestion and absorption pathway of KEGG.Referring to the database of interaction between pathogen and host from HPIDB 3.0,the interaction analysis of the successfully annotated proteins of pathogen and host DEGs showed that 1145 pathogen genes and 1713 host genes participated in the interaction between pathogen and host,of which 54 pathogenic genes and 111 host genes were significantly up or down regulated.These pathogen and host significantly up or down regulated genes expressed proteome will provide reference for further molecular mechanism study of pathogenic infection and host anti-infection. |
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