The Immunomodulation Of A Novel Tumor Necrosis Factor(CgTNF-1) In Oyster Crasssostrea Sisas

Tumor necrosis factor (TNF) is an important cytokine which can regulate manyphysiological processes such as development, repair, hemopoiesis and inflammationin vertebrate. However, the research of TNF in invertebrate is just at the beginning. Inthis article, a new molecule with a typical TNF domain has been screened from oysterCrassostrea gigas and relative researches on function has been conducted after geneclone and prokaryotic expression by using the method of molecular biology and cellbiology.CgTNF-1can encode a polypeptide of337amino acid with a ORF of1014bp.Thepredicted molecular weight of CgTNF-1is38.01kDa, and the isoelectric point is8.25.CgTNF-1shares a characteristic transmembrane structure in the N terminal and atypical TNF domain in the C terminal connecting with an unknown domain. CgTNF-1shares higher similarity with TNF in Ciona savignyi, and they first clustered inphylogenetic analysis, but the similarity between CgTNF-1and other TNFs invertebrate and Drosophila melanogaster is very low. Morever, CgTNF-1mRNA canbe examined in all tested tissues such as gill, gonad, hemolymph, hepatopancrease etal by Real-time PCR, and there are higher expressions in gill, heart andhepatopancrease. The analysis by immunofluorescence indicated that the soluble formof CgTNF-1locates around the nucleus of hemocyte.The expression of CgTNF-1mRNA has an obvious increase at12h and the proteinof CgTNF-1has also significant increase during6to12h after LPS stimulation. Bothof the apoptosis and phagocytosis rate have an obvious increase after in vitro injectionwith LPS and CgTNF-1(10ng mL-1) during6to12h, as well as the concentration of NO, the activity of phenoloxidase and lysozyme and the anti-bacterial activity during6to12h. After incubation with human A549cells and mouse L929cells for12to24h, CgTNF-1could obviously inhibit the proliferation of these cells. CgTNF-1proteincould directly bond PGN (P/N＞2.1).The results indicated that tumor necrosis factor in oyster can not only involve inimmune response after LPS stimulation but also participate in regulating cellularimmunity through activating cell apoptosis and phagocytosis as well as humoralimmunity by regulating NO system and immune related enzyme, which has a criticalfunction on the immune response in molluscs. These results lay the foundation ofexploring the structure and function of CgTNF-1.