Study On Differential Expression Of Immune-Related Genes From The Skin Of Zebrafish Induced By Staphylococcus

Fish skin is an important part of mucosal immune system. More and moreevidence showed that fish skin mucous could perform intaking, presenting antigenand secreting antibody, and play an extremely important role in resisting pathogeninvasion. However, the expression of immune-related genes of fish skin are notexactly clear. The objective of the study is to explore immune-related genes from theskin of zebrafish during the early period of a primary immune response. Furthermore,the skin of zebrafish induced by lethal dose of Staphylococcus chromogenes anddetected with zebrafish cDNA microarray. The study results could lay the foundationfor expounding the fish skin immune mechanism and developing soak immunevaccines.Firstly, pathogenic Staphylococcus chromogenes was isolated and indentified,which was served as experimental strain for following study. Bacteria were isolatedfrom the intesine samples of Grass carp. The samples were streaked on LB agar platesand incubated at28°C for24h. Preponderant colonies were restreaked on freshmedia to obtain pure cultures, which were then identified by the Gram staining test,and then biochemical characteristics and molecular biological analysis. The resultsshowed that a strain named SC1was isolated. We found it was pathogenic to theexperimental Grass carp and was non-sporing and non-capsule, Gram-positive coccus.The products of16S rRNA and kat genes were1520bp and1232bp, respectively.Combining with the results of sequences analysis, the result showed that SC1wasStaphylococcus chromogenes.Secondly, to establish the infection model of zebrafish, the fish were exploredthe optimum condition, including path of administration, dosage and infection timebased on the mortality rate as well as histopathological changes of zebrafish. Theresults showed that, when adopting immersion infection, setting bacterialconcentration was108cfu/mL and immersing time was10min, all experimentalzebrafish were relatively consistent-swimming slowly and floating head, and themortality was relatively stable. Meanwhile, the pathological changes were clear, wefound particularly evident of kidney necrosis, deepened staining, accompanied byinflammatory cell infiltration; liver cells were loose ordered and deepened staining,also accompanied by inflammatory cell infiltration; while kidney and liver of thecontrol group were not obviously wound. Therefore, the results showed that theinfection conditions above were optimum. Finally, the skin tissue of zebrafish induced by Staphylococcus chromogeneswere explored with Affymetrix zebrafish cDNA microarray, and the differentialexpressing of genes from the early period of a primary immune response were carriedout. Total RNA from skin tissue was extracted, then labeled with biotin. It washybridized with zebrafish cDNA gene chips. The database from the15617genes inzebrafish cDNA array were analyzed by NCBI and MAS. Out of15617genes inzebrafish cDNA chips,272significantly differentiated expressed genes were detected,of which210genes were up-regulated and62were down-regulated. Among272differentiated expressed genes,134of them were known functional genes with theanalysis of Gene Ontology method. The immune-related genes were identified, suchas lgals1l3, mhc1uea、mhc1ufa, gpx1b, tfa, f5, sepw2b, cfb, tnfsf10l4and vtg3wereparticipated in pathogen recognition, antigen processing and presentation,complement activation, immune response. In addition, the analysis of pathwaysshowed that p53signaling pathway, ErbB signaling pathway, Jak-STAT signalingpathway and other6pathways involved in anti-S.chromogenes infection immuneresponse of fish skin.In summary,10immune-related genes were screened and identified in the skin ofzebrafish in present study, moreover, the immune response and several significantsignalling pathways were also clarified.