Differential Expression And Identification Of Skin Immune-related Proteins And Their Genes Of Cynoglossus Semilaevis Against Vibrio Vulnificus Infection

Half-smooth tongue sole(Cynoglossus semilaevis)is an important marine culture species in China and frequently suffered from skin ulcers and ascites caused by pathogenic Vibrio infection.In this study,half-smooth tongue sole infected with Vibrio vulnificus was used as an experimental animal.Isobaric tags for relative and absolute quantification(i TRAQ)technique was used to screen and identify the immune-related proteins of half-smooth tongue sole skin,and real-time quantitative PCR(qRT-PCR)was used to verify the expression of the target protein genes.The results showed that 23 immune-related proteins were obtained from i TRAQ sequencing data,including complement proteins(C3,C5,C8β,C9,etc.),prothrombin,heat shock proteins,alpha-2-macroglobulin-like,mucins and parvalbumin beta-like.Heat shock cognate 71 k Da protein-like and parvalbumin beta-like mRNA were up-regulated,while complement C3-like,complement C5 and complement component C9 mRNA were downregulated.Further analysis of mRNA expression temporally of differential protein genes were performed by qRT-PCR method.The results showed that the complement C3,C9,C5 and complement component C8 beta chain mRNA were up-regulated at 6 h,but significantly downregulated at 12 h,and then reached highest expression at 24 h.The heat shock cognate 71 k Da protein-like mRNA was up-regulated after 6 h of infection and reached the highest expression at 36 h.Prothrombin,heparin cofactor protein 2,alpha-2-macroglobulin-like and parvalbumin beta-like mRNA reached the highest expression at 24 h.The data of the differentially expressed proteins at 12 h and 36 h by i TRAQ were basically consistent with the results of qRT-PCR gene analysis(r=0.746).RT-PCR method was used to amplify the CsMUC18,CsMUC5 AC and CsMLP genes using the cDNA of the half-smooth tongue sole skin as a template,and their protein structures,antigenic surface and phylogenetic trees were analyzed.The results showed that the CsMUC18,CsMUC5 AC and CsMLP gene fragments were 1934 bp,2771 bp and 2989 bp respectively,which were predicted to encode 635 aa,917 aa and 996 aa.CsMUC5 AC and CsMLP was predicted to contain five and six major antigenic surfaces respectively and contained C8,VWD and others structural domains.CsMUC18 contained 7 major antigenic surfaces,immunoglobulin superfamily domains,etc.Phylogenetic tree analysis showed that CsMUC5 AC and CsMLP were closely related to secreted mucin,while CsMUC18 were closely related to membrane-bound mucin.Further analysis of the tissue distribution and time-series expression of CsMLP gene showed that CsMLP was abundantly expressed in gills and skin tissues.CsMLP was up-regulated significantly at 12 h after V.vulnificus infection,and significantly down-regulated after 36 h of infection(p<0.01).These results above provide a scientific reference for the skin immune response mechanism of Vibrio infection on half-smooth tongue sole and proteomics.