Ontogeny Of B Cells And Cloning Of TCR Genes For Mandarin Fish Siniperca Chuatsi

Mandarin fish Siniperca chuatsi, as an important species in aquaculture industry of China, was threatened seriously by many kinds of diseases in recent years. It is thus important and necessary to understand its immune system. The present study aims to claim the ontogeny of IgM-producing cells in lymphoid tissues and expression changes of IgM, IgD and IgZ in lymphoid tissues of mandarin fish stimulated with Flavobacterium columnare G4. In addition, cDNA sequences and structure characters of TCRα,β,γconstant region were analyzed in mandarin fish. In situ hybridization indicated that IgM-producing cells firstly occurred in mandarin fish head kidney at 20 dph. Then IgM positive cells were discovered in spleen and thymus at 26 and 39 dph. In gill and intestine positive signals were detected later by 3 mph.The expression of IgD and IgZ was also detected in adult mandarin fish lymphoid tissues by in situ hybridization. IgD was detected in head kidney and spleen with small amount. No IgD positive signals were detected in gill and intestine. IgZ staining signals were detected weakly in head kidney and spleen. In gill IgZ positive cells scattered like cells producing IgM.The expression changes over a period of 8 weeks of three Igs were also determined in blood, head kidney, spleen and gill of mandarin fish injected with formalin-killed Flavobacterium columnare G4 by quantitive real-time PCR.Significant up-regulation was observed for IgM gene especially in boosted group in almost all the target tissues although with different degrees. For IgD and IgZ, statistically significant increases were detected at 1 or 2 weeks after injection. The constant regions of TCRα,β,γgene were cloned in mandarin fish for the first time. The result indicated that mandarin fish express typical TCRα,β,γC chains which included immunoglobulin (Ig), connecting peptide (CPS), transmembrane (TM) and cytoplasmic (CYT) domains. Some amino acids motifs playing important roles were conserved such as Cys in TM, which was important for TCRα/βheterodimers, conserved antigen receptor transmembrane motif (CART) and characteristic motif of Ig and MHC so on.The organ distribution of the three genes was analyzed by RT-PCR. The three TCR chains could be detected in all the sampled tissues, but prevalent expression was detected in the lymphoid tissues head kidney, spleen, thymus and gill. The expression changes of TCRα,γwere detected in blood, head kidney, spleen, thymus, intestine and gill of mandarin fish injected with formalin-killed Flavobacterium columnare G4 by quantitive real-time PCR. Obvious up-regulation was detected for both genes in group I, especially in thymus, gill and intestine theγgene increased greatly after the first injection.