De Zoysa Mahanama, Jung Sungju, Lee Jehee
Department of Biotechnology, College of Ocean Science, Cheju National University, Ara-Dong, Jeju 690-756, Republic of Korea.
Fish Shellfish Immunol. 2009 Apr;26(4):625-31. doi: 10.1016/j.fsi.2008.10.004. Epub 2008 Oct 18.
Tumor necrosis factor alpha (TNF-alpha) is considered as a multifunctional immune modulator that plays an important role in the innate and adaptive immune systems in vertebrates. Here, we described the characterization and expression analysis of the first TNF-alpha homologue in mollusk abalone, named as AbTNF-alpha. It has 930-bp full length with a 717-bp open reading frame (ORF), encoding 239 amino acids. The AbTNF-alpha amino acid sequence shows the characteristic TNF family signature, N-terminal transmembrane domain consisting of a hydrophobic amino acid cluster and cell attachment sequence at (155)RGD(157). Phylogenic analysis results showed that AbTNF-alpha is more related to the invertebrate Ciona savignyi TNF superfamily ligand member (CsTL). Quantitative real-time PCR expression results showed that AbTNF-alpha was constitutively expressed in both immune and non-immune tissues in a tissue specific manner. The highest constitutive expression was in the gill tissue with a 1.5-fold compared to hemocytes expression. The AbTNF-alpha mRNA expression in gill tissue was monitored in vivo stimulated by a mixture of pathogenic bacteria (Vibrio alginolyticus, Vibrio parahemolyticus, and Lysteria monocytogenes), viral haemorrhagic septicaemia virus (VHSV) and lipopolysaccharide (LPS). The AbTNF-alpha expression was significantly (p<0.05) induced by bacteria, VHSV and LPS compared to the control animals. Moreover, the highest level expressions of each induction were at 24 h (5.2-fold), 48 h (2.8-fold), and 48 h (3.3-fold) by bacteria mixture, VHSV and LPS, respectively. These results indicate that AbTNF-alpha could respond to pathogenic infection or stimulation and may play an important role in the abalone immune system.
肿瘤坏死因子α（TNF-α）被认为是一种多功能免疫调节剂，在脊椎动物的先天性和适应性免疫系统中发挥着重要作用。在此，我们描述了软体动物鲍鱼中首个TNF-α同源物（命名为AbTNF-α）的特性及表达分析。它全长930 bp，开放阅读框（ORF）为717 bp，编码239个氨基酸。AbTNF-α氨基酸序列显示出TNF家族的特征性信号，N端跨膜结构域由一个疏水氨基酸簇组成，且在（155）RGD（157）处有细胞附着序列。系统发育分析结果表明，AbTNF-α与无脊椎动物萨氏海鞘TNF超家族配体成员（CsTL）的关系更为密切。定量实时PCR表达结果显示，AbTNF-α在免疫和非免疫组织中均以组织特异性方式组成性表达。组成性表达最高的是鳃组织，与血细胞表达相比有1.5倍的差异。在体内，用病原菌混合物（溶藻弧菌、副溶血性弧菌和单核细胞增生李斯特菌）、病毒性出血性败血症病毒（VHSV）和脂多糖（LPS）刺激后，监测鳃组织中AbTNF-α mRNA的表达。与对照动物相比，细菌、VHSV和LPS显著（p<0.05）诱导了AbTNF-α的表达。此外，细菌混合物、VHSV和LPS诱导的最高表达水平分别在24 h（5.2倍）、48 h（2.8倍）和48 h（3.3倍）。这些结果表明，AbTNF-α能够对病原体感染或刺激作出反应，可能在鲍鱼免疫系统中发挥重要作用。
