Primary Study On Immune Function Of TNFSF10(TRAIL) In Tilapia,Oreochromis Niloticus

TNFSF10(Tumor necrosis factor superfamily member 10),also called TRAIL(TNF related apoptosis inducing ligand),is an important member of the TNF super family.TRAIL expresses in a variety of cells of the immune system such as NK cells,T cells,NK T cells,dendritic cells and macrophages.It plays important role in immune regulation,pathogen defense,tumor surveillance.Bying activating apoptosis signaling pathways,TRAIL can promote apoptosis in tumor cells and infected cells in a short time,which could effectively prevent the spread of pathogens and improve the host immunity.TRAIL gene has been cloned from Danio rerio,Ctenopharyngodon idellus,Siniperca chuats and Takifugu rubripes.But the function of TRAIL protein was barely studied.In this paper,recombinant tilapia TNFSF10 mature peptide was purified and used to prepare of mouse polyclonal antibody and rabbit polyclonal antibody.The expression level of TNFSF10 in tilapia spleen and head kidney were detected after Streptococcus agalactiae infection.Finally,we incubated head kidney leukocyte with different concentration of TNFSF10-1 recombinant proteins and then observed the changes of respiratory burst.The main results are as follows:1.Recombinant tilapia TNFSF10 mature peptide was obtained through prokaryotic expression and was used to prepare of polyclonal antibody.Amplified thefragment encoding the tilapia TNFSF10 mature peptide and inserted into expression vector PET32 a.The recombinant plasmid was transformed into Rosetta-gami.Soluble protein was obtained after IPTG stimulation and followed by purification of recombinant tilapia TNFSF10.We prepared the polyclonal antibody of tilapia TNFSF10 after immunization in mice and rabbit.2.The mRNA and protein expression of three TNFSF10 was detected in spleen and head kidney after IP injection of S.agalactiae to tilapia.The results showed that mRNA expression of three TNFSF10 was up-regulated in one week after infection in both spleen and head kidney.All those three TNFSF10 were increased within 6 h in head kidney,while the upregulation was delayed in spleen.However,the protein level of three TNFSF10 were up-regulated to different extent in the two days in both spleen and head kidney.3.Results of head kidney leucocyte respiratory burst experiments exhibited that100 ng/ml TNFSF10 recombinant protein can induce high strength breathing outbreak and the result is dose-dependent.