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The expression of two novel orange-spotted grouper (Epinephelus coioides) TNF genes in peripheral blood leukocytes, various organs, and fish larvae

Lam, Freda Wai-San, Wu, Szu-Yin, Lin, Shih-Jie, Lin, Chin-Chou, Chen, Yi-Ming, Wang, Han-Ching, Chen, Tzong-Yueh, Lin, Han-Tso, Lin, John Han-You
Fish & shellfish immunology 2011 v.30 no.2 pp. 618-629
lipopolysaccharides, mammals, heart, farmed fish, brain, leukocytes, gene expression, intestines, pollution, spleen, molecular weight, introns, cysteine, apoptosis, pathogens, messenger RNA, amino acid sequences, tumor necrosis factor-alpha, fish larvae, immune response, kidneys, exons, cell proliferation, proteins, rearing, thymus gland, proteinases, inflammation, grouper, Epinephelus coioides, hatcheries
The tumour necrosis factor (TNF) super-family is a group of important cytokines involved in inflammation, apoptosis, cell proliferation, and the general stimulation of the immune system. The TNF gene has been cloned in some bony fish; however, its counterparts are still unidentified in the majority of fish species. In this study, we cloned gTNF-1 and gTNF-2 from the orange-spotted grouper (Epinephelus coioides), an economically important farmed fish. Both genes include 4 exons and 3 introns and encoded 253 and 241 amino acid proteins with a molecular weight of approximately 27 and 26 kDa, respectively. The identity of the putative amino acid sequences between gTNF-1 and gTNF-2 was only 38%. The positions of cysteine residues, a protease cleavage site, and a transmembrane domain sequence derived from gTNF-1 and gTNF-2 were similar to those in other fish and mammalian TNF-α. The mRNA expression levels of the 2 gTNF molecules were evaluated in unstimulated/stimulated peripheral blood leukocytes, various organs, and fish larvae. Following lipopolysaccharide (LPS) treatment, gTNF-2 was expressed at higher levels, was up-regulated more quickly, and was more sensitive to the immune response than gTNF-1. gTNF-1 was constitutively expressed in the thymus, brain, and spleen, but it was also expressed in the heart, head kidney, and trunk kidney after LPS stimulation. gTNF-2 was constitutively expressed in the thymus, head kidney, trunk kidney, spleen, and intestine; further, gTNF-2 was highly expressed in all organs post-LPS stimulation. Finally, the gTNF expression levels were evaluated at various developmental stages in grouper larvae. A higher variation of gTNF expression levels was observed in fish larvae from a contaminated hatchery. This study revealed the different expression patterns of gTNF-1 and gTNF-2. In addition, gTNF-2 was more sensitive to pathogens than gTNF-1; therefore, it may be an appropriate marker for pathogen invasion and the evaluation of the larval rearing environment.