點帶石斑魚介白素-6之基因選殖、蛋白表現及功能分析

Abstract

Epinephelus coioides (orange-spotted grouper) is an economic marine fish of aquaculture in Asia. But the intensive rearing increases the susceptibility of infection and disease, which can cause heavy loss. Therefore, in order to control disease, it is essential to understand the immune system background of the grouper. The cytokines, interleukin 6 (IL-6), produced by T cell and macrophages plays an important role in immune response. It regulates the proliferation and differentiation of T cells and elicits immunoglobulin produced by B cells. In this study, we try to figure the biofunction of grouper IL-6. First, we obtain the il-6 of grouper. The deduced grouper IL-6 protein was a 223 amino acid protein and the amino acid sequence is about 30% identical to mammalian IL-6 and 43% to 63% identical to other available teleost IL-6. The putative conformation was composed by a signal peptide and four conserved alpha helices, which are the same as their counterparts of mammalian and teleost. In the mRNA expression pattern, peripheral blood leukocytes had a high expression of il-6 at 8 hours after LPS stimulation in the in vitro condition. In the in vivo condition, il-6 was continuously expressed in gill, head kidney, brain and muscle without LPS stimulation; however, il-6 was expressed in all immune organ in the fish injected with LPS. In the in vitro condition, il-6 expression was high at 8 hours after LPS stimulation in peripheral blood lymphocytes. In the immunohistochemistry, the greatest numbers of cells were recognized by grouper IL-6 antiserum in the head kidney at 8 hours after LPS stimulation. The prokaryotically produced recombinant grouper IL-6 (rIL-6) was a 28 KDa protein. At the end of this study, an E. coli expressed rIL-6 was injected into the test fish, and the immune related gene expression at different times and in various organs was evaluated and to determine the biofunction of grouper IL-6. The data showed the pre-inflammatory cytokines, tnf-α1, tnf-α2 and il-1β, as well as the Th1 specific transcription factor, t-bet, were down-regulated. The Th2 specific transcription factors, c-maf and gata, as well as the Ig were up-regulated in immune organ after rIL-6 injection. We also observed that the acute-phase protein gene, crp, was up-regulated in live. These results indicate that grouper IL-6 plays an important role in the immune system and is a regulator for naїve T helper cell differentiation towards Th2 cells, because it elicited antibody production.