Molecular cloning and expression analysis of immunoglobulin M heavy chain gene of blunt snout bream (Megalobrama amblycephala)

摘要

Immunoglobulins (Igs), which bind antigens with high specificity, are essential molecules in adaptive immune system of jawed vertebrates. In this study, cDNA encoding the secreted form of the immunoglobulin heavy chain of IgM (sIgM) was cloned from the mesonephros of blunt snout bream (Megalabrama amblycephala) using RT-PCR and rapid amplification of cDNA ends (RACE). The full-length cDNA of sIgM heavy chain gene has 1961 nucleotides encoding a putative protein of 569 amino acids, constant region shares high amino acid identity with that of Ctenopharyngodon idella (80%), Carassius auratus langsdorfii (65%) and Danio rerio (59%). Multiple protein sequence alignment revealed that blunt snout bream sIgM was clustered with the homologues of cyprinid fish and constructed one clade. Using quantitative real-time PCR (qRT-PCR) analysis, the level of sIgM mRNA was determined, with a V-shape change pattern: decreased initially from unfertilized egg stage to 4 cells stage and increased from 16 cells stage to prelarva. This sharp drop indicates that sIgM mRNA is maternally transferred, and was continuously degraded until 16 cells stage. The drastic rising in sIgM level from blastula stage to prelarva might be attributed to embryonic stem cell differentiation procedure. Compared with juvenile fish, the expression of sIgM was significantly higher in pronephros, liver, spleen, gill and muscle of adult fish. After the injection of Aeromonas hydrophila, the expression pattern of sIgM was found first down-regulated at 4 h, then up-regulated and reached the peak at 7 d and 21 d in mesonephros, spleen, liver and gill, respectively