Olecular Cloning And Expression Analysis Of Immunoglobulin D Heavy Chainin Scophthalmus Maximus

In this paper, full length cDNA sequence of mIgD was cloned from the spleen of Scophthalmus maximus, Sequence analysis showed that it contained the classic domain of the immunoglobulin family. RT-PCR was used to analyze of gene expression in different tissues, quantitative real-time PCR was used to analyze gene expression after intraperitoneal injection with formalin inactivated Vibrio anguillarum in the pronephros, suggested the fish mIgD may play an important role immune signal transduction and humoral immunity. On the basis of the sequence analysis, mIgD δ domain recombinant expression vector was constructed, segment of mIgD was expressed in E.coli, and the polyclonal antibody against mIgD was prepared. IFA experiment showed that the polyclonal antibody could bind specifically to peripheral leukocytes of Scophthalmus maximus, indicating that the Membrane-bounded Ig exists on the cell membrane of leukocytes in peripheral blood.1The full length cloning of Immunoglobulin D heavy chain gene in Scophthal-mus maximusAfter obtaining the conserved sequence by degenerate primers, a3357bp full-length cDNA sequence of membrane-bounded IgD (mIgD) heavy chain gene in the turbot Scophthalmus maximus was obtained by rapid amplication of cDNA ends (RACE), containing a2997bp open reading frame, which encodes999animo acids. The gene structure of turbot mIgD was VDJ-μ1-δ1-δ2-δ3-δ4-δ5-δ6-δ7-TM. BLAST result based on the mIgD heavy chain amino sequence of δ1～δ7domain showed that turbot mIgD shared high similarities with Hippoglossus hippoglossus (78%), Siniperca chuatsi (71%) and Paralichthys olivaceus (68%), and the result of multiple sequence alignment showed that the deduced amino acid sequence of mIgDs possessed two highly conserved tryptophans and cysteines sites in each δ constant domain. Phylogenetic tree based on immunoglobulin heavy chain amino acid sequences of different teleosts was constructed using the neighbor-joining method, which exhibited that all the teleost IgDs gathered in one branch, and the turbot mIgD was clustered with the mIgDs of H. hippoglossus and P. olivaceus.2The expression of Immunoglobulin D heavy chain gene in Scophtha/mus maximusThe tissue distribution of IgD in trout was analyzed by RT-PCR, the result showed that mIgD of turbot was expressed mainly in peripheral blood, speen, pronephros and mesonephros.The expression response of mIgD in pronephros was investigated after intraperitoneal injection with formalin inactivated Vibrio anguillarum by quantitative real-time PCR, and the result showed that the expression of mIgD was significantly down-regulated after injection and decreased to the lowest level at8h with approximate1/28relative expression amount of that in control fishes, and then it exhibited an up-regulation, till48h post infection, there was no significant difference in relative expression of mIgD between injection and control groups.3The expression of cDNA fragment of Scophthalmus maximus and Production of monoclonal antibodies anti-mIgDThe cDNA fragment encoding δ region of mIgD were amplified and inserted into pET32a Vector, then transformed into E. coli BL21(DE3). The recombinant proteins were successfully expressed in positive clones with molecular masses of99.5kDa (δ region of mIgD) in SDS-PAGE. Then mouse polyclonal antibody against mIgD was prepared, ELISA experiment displayed binding capability to recombinant proteins. Furthermore, indirect immunofluorescence results showed that the polyclonal antibody could bind specifically to peripheral leukocytes of Scophthalmus maximus, indicating that the Membrane-bounded Ig exists on the cell membrane of leukocytes in peripheral blood.