| Chinese soft-shelled turtle, Pelodiscus sinensis is one of important reptile species in aquaculture industry of China. The understanding of its immune system is not only important and necessary for developmental and comparative immunity in vertebrates, but also for aquacultural purpose. The present study aims to clone the cDNAs encoding the heavy chains and joining chain of IgM, IgD and IgY in Chinese soft-shelled turtle, and to analyze their expression at mRNA and protein levels. Furthermore, the ontogeny of IgM-, IgD- and IgY- producing cells in lymphoid tissues of the turtle was also analyzed. The cDNA sequence of J chain has been cloned from the Chinese soft-shelled turtle by reverse transcription (RT)-PCR and rapid amplification of cDNA ends (RACE). The cDNA sequence is 2,347 nt in length and contains an open reading frame of 480 nt encoding 160 aa including the signal sequence. The deduced amino acid sequence has a high degree of homology with that of an already reported turtle J chain (80.7%), and of chicken (71.3%), and a low degree of homology with those of amphibians (49.4% and 57.6%); but it is relatively low when compared with the sequence reported from skate (35.8%). It is thus implied that the J chain of P. sinensis is much more similar to avian, as also revealed in the phylogenetic tree. The expression of J chain was detected at 5 days past fertilization (dpf) by RT-PCR, and its expression increased following further developmental stages. The expression of J chain was detected in almost all organs examined at protein levels by Western blotting, except in fat tissue. The J chain-producing cells were detected in lymphoid-related tissues such as in spleen, kidney and intestine by using immunohistochemistry. By using real-time quantitative RT-PCR analysis, a significant up-regulation of J chain transcripts was observed in spleen, kidney and blood of the turtle injected with in-activated Aeromonas hydrophila T4, indicating the important immune role of J chain at early development and in response to bacterial infection.Only three Ig isotypes, IgM, IgD, and IgA, were previously known in reptiles. Here, we describe IgM, IgD and a novel immunoglobulin heavy-chain isotypeÏ…(IgY) in Chinese soft-shelled turtle. The IgM and IgY genes of the turtle are characteristically similar to those described in other species, whereas IgD gene differs from IgD reported in other vertebrates. The turtle IgD contains 6 immunoglobulin heavy chain domains without evidence of intragenic duplications of exons as described in teleost IgD genes. The expression of IgM and IgD was detected early during embryonic development after fertilization, and their expression increased following further developmental stages. However, immunoglobulin Y could not be detected in spleen and kidney until 90 days after hatching out. Western blotting analysis demonstrated that the protein of IgM, IgD and IgY were expressed in almost all organs examined in adult turtles. The IgM-, IgD- and IgY- producing cells in lymphoid-related tissues such as in spleen, kidney and intestine, were investigated by using histochemistry. The transcriptional kinetics of the three Ig molecules in spleen, kidney and blood and antibody response kinetics in serum after bacterial pathogen stimulation were also analyzed by using real-time PCR and by using indirected enzyme-linked immunosorbent assay (ELISA), respectively. A significant up-regulation of J chain was observed at both mRNA and protein levels. Using flow cytometry, a higher proportion of IgM-pruducing cells was observed in spleen, peripheral blood and kidney when compared to proportions of IgD-and IgY- positive cells. The IgM-producing cells was firstly observed in spleen of P. sinensis at 18 dph, and then in kidney and intestine at 28 and 64 dph, respectively. However, IgD and IgY positive cells were detected much later. |
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