Functional Proteomics Research On Immune Response In Large Yellow Croaker

Large yellow croaker(Pseudosciana crocea)is an economically important marine fish species in China.In recent years,with the rapid development of large yellow croaker culture industry,virus and bacteria-caused infectious diseases become more and more severe,while the knowledge about the genetic and immunological basis of large yellow croaker is limited. The lack of knowledge hinders the establishment of effective measures in diseases control and genetic improvement.In this study,proteomics approaches were utilized to identify the differentially expressed proteins in the spleen tissue of large yellow croaker in response to poly I:C induction at four different time points(0,12,24 and 48h after induction).All together,38 protein spots differentially expressed were identified and further analyzed by PMF or MALDI-TOF-TOF, in which 28 were up-regulated and 10 showed down-regulated.And most changes took place around 12 and 24h post induction.To understand the inter-relationship of these differentially expressed proteins and investigate their roles in disease-resistance mechanism,protein interaction network was build up by Cytoscape software.In this network,PPRAγ,Annexinâ…£and ROCK1 were identified to participate in both immune response and apoptosis control. Annexinâ…£,for its diverse biological functions and huge amount of binding partners, especially drew our attention and was chosen for further investigation.The over-expressed Annexinâ…£was found to cause the increase of the activity of caspase-3 and apoptosis in spleen tissue,also,it up regulated the expression of pro-inflammatory inhibitor IL-10,and down regulated expression of chemokines CXC and CC.These results indicated that up-regulation of Annexinâ…£at 12h after induction with PolyI:C could cause the spleen cell apoptosis and regulate the inflammatory response.On the other hand,some anti-apoptosis proteins such as PRDXâ… and AIP were up-regulated at 48h post-induction.These differential expressed proteins ensured a dynamic balance between cell proliferation and apoptosis so as to maintain the stable inner environment and control the immune response in large yellow croaker.In the differential proteomics analysis of large yellow croaker spleen tissue in response to trivalent bacterium vaccine induction,24 differential expressed proteins spots were identified and further analyzed by PMF or MALDI-TOF-TOF.Three altered proteins,PRDXâ… ,PRDXâ…¡,PRDXâ…£,were found belong to PRDXs superfamily.This indicated PRDXs may play an important role in immune response to the trivalent bacterium vaccine-induction in large yellow croaker.A differential expressed protein(Spot 7)homologous to a mammalian Peroxiredoxinâ…£attracted our attention,for it is reported to mediate the activation of NF-kB in the cytosol by modulating IkBαphosphorylation in mankind.The recombinant large yellow croaker PRDXâ…£exhibited activities of oxidation resistance.Intracellular localization of PRDXâ…£in the spleen cells by immune colloidal gold electron microscopic analysis showed that most of the gold particles exist in the peroxisome,cytoplasm and rough endoplasmic reticulum,and more gold particles in rough endoplasmic reticulum were found in the spleen of trivalent bacterium vaccine-induced fish than that of normal fish.Furthermore, the suppression of PRDXâ…£by siRNA caused the up-regulated expression of TNF-α2,CC, and NF-kB(p65)and down-regulated expression of IL-10.But the over-expression of PRDXâ…£caused the down-regulated expression of TNF-α2,CC,and NF-kB(p65)and up-regulated expression of IL-10.To further corroborate the anti-inflammation activity of PRDXâ…£, twelve hours after the siRNA or protein injection,fish were infected by mixed pathogenic bacterium The result shows that after the PRDXâ…£expression was suppressed,the fish mortality was significantly higher than that of control group,while the enhanced PRDXâ…£could protect large yellow croaker from the bacterium induced death.All these results indicate that PRDXâ…£can inhibit the inflammation and protect large yellow croaker from bacterial infection by inhibiting the expression of NF-kB.Our research reveals a new way of anti-bacterium infection in fish.Two other genes,NCCRP-1 andβ₂-microglobulin were also cloned,their tissue distribution and expression patterns upon stimulation with polyI:C and inactivated trivalent bacterial vaccine were analyzed.These results will help us to further stand immune response and disease-resistance mechanism in large yellow croaker.