Yellow Catfish IgM Molecules And Its Expression Studies

Yellow catfish Pelteobagrus fulvidraco, as one of important commercial fishes in aquaculture industry of China, was threatened seriously by many kinds of diseases in recent years. It is thus important and necessary to understand developmental and comparative immunity in vertebrates and immune system in Yellow catfish for disease controlling. In this paper, the cDNAs encoding IgM heavy chain and light chains were cloned and analyzed, and expression pattern, maternal transfer and ontogeny of IgM were studied in yellow catfish.cDNAs encoding IgM heavy chain (ycIgM) and light chains (ycIgL) were cloned using RACE-PCR and RT-PCR methods in P. fulvidraco. The full-length ycIgM gene cDNA contains1993nucleotides, including42nucleotides of5'-untranslated region (UTR),229nucleotides of3'-UTR, and1722nucleotides of the open reading frame encoding a putative protein of573amino acids; ycIgL1gene cDNA contains1021nucleotides, including39nucleotides of5'-UTR,259nucleotides of3'-UTR, and723nucleotides of the open reading frame encoding a putative protein of240amino acids; ycIgL2gene cDNA contains953nucleotides, including57nucleotides of5'-UTR,164nucleotides of3'-UTR, and732nucleotides of the open reading frame encoding a putative protein of243amino acids; ycIgL3gene cDNA contains965nucleotides, including58nucleotides of5'-UTR,184nucleotides of3'-UTR, and723nucleotides of the open reading frame encoding a putative protein of240amino acids. Compared with those of other teleost species ycIgM, L1, L2and L3shared the highest identity with that in channel catfish (52.3%,90.%,80.2%, and68.9%), respectively. Phylogenetic tree based on some teleost IgM heavy chain amino acids suggested IgM in P. fulvidraco was clustered closely with that of I. punctatus.The abundance of ycIgM transcripts, was higher in the head kidney, kidney, blood cells and spleen than that in heart, muscle, and brain. Higher abundance of ycIgL1transcripts was found in the head kidney, spleen, gill, intestine and blood cells, whereas very little expression in the muscle, skin, and brain; Higher abundance of ycIgL2transcripts was found in the head kidney, spleen, gill, intestine and blood cells, whereas very little expression in the heart and brain; Higher abundance of ycIgL3transcripts was found in the spleen, gill, liver, intestine, and blood cells, whereas very little expression in the heart and brain.The result of the real-time PCR also revealed that IgM mRNA expression increased significantly in blood cells, spleen, head kidney, liver, and intestine after injection of Aeromonas hydrophila. In situ hybridization results showed that ycIgM-producing cells were located in all the tissues investigated, such as head kidney, spleen and intestine, and increased in density in them post injection of A. hydrophila. This was the reason why the IgM mRNA expression increased significantly in the organs mentioned above after stimulation of the bacterial pathogen.Using prokaryotic expression vector pQE30and induction of IPTG, the fusion protein of ycIgM heavy chain was produced in E. coli M15. Using the recombinant ycIgM protein as antigen, The anti-ycIgM polyclonal antibody was gotten by means of immuning a New Zealand white rabbit. The result of the Enzyme-linked Immunosorbent Assay (ELISA) showed that the highest titer of anti-serum was1:512000. Western blotting analysis showed IgM protein, about72kDa was detected in head kidney, spleen, serum, skin, intestine, gill, liver and kidney. Immuno-histochemical analysis showed that ycIgM protein positive signal appeared in head kidney, spleen, gill, intestine and liver tissue, and increased significantly at15d after infection of A. hydrophila.Immunohistochemical and immuno-fluorescence analysis showed that the rich positive protein signals of ycIgM appear in head kidney, spleen, intestine, gill and liver tissue, and changeded significantly in the signals at15d after infection of A. hydrophila.Using RT-PCR and Real-time PCR technology, ycIgM mRNA was detected in eggs, embryos and larva at14d after hatching, and wasn't detected in larva at3-7d after hatching. ELISA analysis showed that ycIgM protein was detected in eggs, embryos and larva in all phases. ycIgM protein decreased gradually in egg, embryos and early larva, and increased at14d after hatching. These studies showed synthesis of ycIgM mRNA and protein did not appear in embryos and early larva, and suggested that ycIgM proteins in them came from maternal yellow catfish.