Molecular cloning and characterization of secretory and membrane-bound IgM of turbot

Abstract

In recent years, increasing diseases especially bacterial diseases have brought a host of losses with the expansive cultivation of turbot (Scophthalmus maximus). In order to do more research about the immune system of turbot for better understanding the mechanism of resisting diseases, the immunoglobulin genes related to secretory and membrane-bound IgM (s-IgM and m-IgM) of turbot were cloned using homology sequences cloning and SMART RACE PCR method. The heavy chain of s-IgM cDNA is 1900 bp in length including a leader region, a variable region, four constant regions (CH1, CH2, CH3 and CH4) and a C-terminal while the cDNA of m-IgM is 1795 bp with the same leader region, variable region, three constant regions (CH1, CH2 and CH3) and two transmembrane regions (TM1 and TM2). The sequence of IgM gene was also obtained and the structure consisted of V–CH1–CH2–CH3–CH4-TM1-TM2 is similar to other fishes. The highest level of s-IgM expression was observed in spleen, followed by kidney, gills, eyes, skin of the healthy turbot whereas the same profile of m-IgM expression is found with low level. And s-IgM takes up dominant proportion of total IgM expression. Also the relative expressions of s-IgM and m-IgM were analyzed in turbot vaccinated with the live attenuated vaccine Vibrio anguillarum. Not only the transcriptions of both s-IgM and m-IgM in liver, spleen and kidney of turbot injected with V. anguillarum MVAV6203 were up-regulated but also the expressions of s-IgM and m-IgM in spleen, kidney, gut, skin and gills of bath-vaccinated turbot were increased. Comparing the ratio changes of relative expression of m-IgM and s-IgM in vaccinated turbot, we found that the proportion of m-IgM were increasing in both administration routes, which probably indicated that the increasing expression of m-IgM strengthen the phagocytic ability of B cells.