Cloning,Identification And Immune Function Of Trachinotus Ovatus TNFSF6

Tumor necrosis factor ligand superfamily member 6(TNFSF6)is a member of the TNF ligand,which is essential for maintaining the body's immune homeostasis.It can cause programmed cell death by binding to its receptor TNFRSF6.However,the current reports on TNFSF6 in fish are relatively scarce.In order to study the antibacterial and immune-related functions of Trachinotus ovatus TNFSF6(Tro TNFSF6),this study cloned and identified the T.ovatus Tro TNFSF6 and conducted a preliminary research on its antibacterial activity.The results are as follows:(1)The results of sequence analysis in this study indicated that Tro TNFSF6 open reading frame sequence is 687 bp in length,encoding 228 amino acids,and the predicted protein size is 25.5 k Da.The structural characteristics of the protein showed that Tro TNFSF6 is a type ? transmembrane protein,which contains a transmembrane region,a domain unique to TNF family,two cysteine residues capable of forming disulfide bonds,a potential N-glycosylation site and a tyrosine phosphorylation site.The results of sequence homology alignment showed that TNFSF6 is less conserved,the homology of Tro TNFSF6 with TNFSF6 of other teleost fish is 25.64% ? 58.79%.Among them,it has the highest homology with the Collichthys lucidus(58.79%)and the lowest homology with the Gadus morhua(25.64%).Phylogenetic tree revealed that Tro TNFSF6 is clustered with other species belonging to the teleost such as C.lucidus,Maylandia zebra and Oreochromis niloticus,there is a closest relationship between them,and it has a furthest relationship with Homo sapiens and Mus musculus.(2)Tissue-specific analysis showed that Tro TNFSF6 was expressed in the 11 tissues tested,with the highest expression in the liver,followed by the intestine and gills,and the least in muscle.In addition,the expression of Tro TNFSF6 in liver,spleen,and head kidney was significantly up-regulated after the infection of Edwardsella tarda.The expression of Tro TNFSF6 in liver and head kidney reached its peak at 24 hours after infection,they were up-regulated by 84 times and 33.8 times,respectively.The peak of Tro TNFSF6 expression in the spleen appeared at 12 hours after infection,and the expression was up-regulated by5.5 times.(3)TNFSF6 expressed on the surface of the cell membrance could combine with the receptor TNFRSF6 to trigger a series of biological functions.Subcellular localization showed that Tro TNFSF6 is localized on the cell membrane of T.ovatus snout cells(GPS).In this study,the Tro TNFSF6 overexpression plasmid was constructed for exploring thefunction of Tro TNFSF6 in fish at the individual level.It was found that when Tro TNFSF6 was overexpressed in fish,the activity of alkaline phosphatase and acid phosphatase in the serum of T.ovatus was significantly increased.In order to study the in vitro activity of Tro TNFSF6,a prokaryotic recombinant expression plasmid of Tro TNFSF6 was constructed in this study and its protein r Tro TNFSF6 was obtained.The results showed that r Tro TNFSF6 can significantly induce the proliferation of lymphocyte in a dose-dependent manner.In order to test the role of r Tro TNFSF6 in the proliferation of cancer cells,r Tro TNFSF6 was added into mouse liver cancer cells Hepa1-6 for incubation,and flow cytometry was used to detect the apoptosis level of the cells after 24 h of culture.It was found that the apoptosis rate of Hepa1-6 cells with r Tro TNFSF6 significantly increased by 14.05% compared with the control group.When the expression of TNFSF6 in Hepa1-6 cells was interfered with,the apoptosis rate was significantly reduced by 13.44%compared with the control group.The results show that Tro TNFSF6 can promote the apoptosis of Hepa1-6.In summary,Tro TNFSF6 plays an important role in the antibacterial immune process of T.ovatus and can promote the apoptosis of Hepa1-6.The results of this experiment will facilitate the future in-depth study on TNFSF6 and the excavation of the immune mechanism of T.ovatus.