| Nile tilapia(Oreochromis niloticus)is one of the major aquatic species in southern China.However,in recent years,various bacterial and viral diseases occur frequently,especially the newly discovered virus--Tilapia Lake Virus(Ti LV)has brought huge impacts and challenges to the Tilapia breeding industry.Therefore,it is urgent to sthudy the immune system of tilapia.In this study,we carried out a basic study on the innate immune molecule Cluster of Diffentiation 94(On CD94)of Nile tilapia.CD94,a type of C-type lectin(CTL),belongs to the CTL Ⅴ family,which are known as the natural killer cell receptor(NKR)family.We tested the expression pattern of CD94 in various tissues and organs of Nile tilapia and its immune-related function.Our results are as follows:1.The Open Reading Frame(ORF)of OnCD94 was cloned by PCR,which is 681 bp and encodes 227 amino acids.On CD94 has no signal oeptide,and there is a carbohydrate recognition domain at positions 99-223 of the extracellular segment.Multiple sequence alignment showed that the amino acid sequence similarity of On CD94 to other species’ is17.39-91.59 %.Phylogenetic tree analysis showed that fish CD94 clustered into one category.2.The result of Fluorescence quantitative PCR(q RT-PCR)showed that the transcript of CD94 can be detected in all tested tissues of Nile tilapia.Among them,the expression level was higher in the spleen,blood,and head and kidney,and the lowest expression was observed in the brain.In addition,its expression level in non-specific toxic cells(NCC)is remarkable.With Streptococcus agalactiae infection,the expression of On CD94 in the spleen,blood and head and kidney were increased.Subcellular localization analysis of On CD94 expression on the cell membrane.The result of the dual luciferase reporter system analysis showed that On CD94 could significantly up-regulate the activity of the NF-κB pathway.3.The rolaryotic expression vector of OnCD94 was construted,and the vector was imported into the expression system for protein expression.Then On CD94 fusion protein(r On CD94)with his tag was purified.The results of Western blot showed that r On CD94 can significantly bind to Streptococcus agalactiae,Aeromonas hydrophila,Streptococcus inus,Vibrio harveyi and Staphylococcus aureus.ELISA was used to detect the binding ability of r On CD94 to LPS and PGN,which are the main polysaccharides on the cell wall of bacteria,and the results showed that r On CD94 could significantly bind LPS and PGN.Meanwhile,r On CD94 had agglutinating effect on Streptococcus agalactiae,Aeromonas hydrophila,Streptococcus inus,Vibrio harveyi and Staphylococcus aureus.In addition,r On CD94 could significantly inhibit the growth of Streptococcus agalactiae and Aeromonas hydrophila and promote the phagocytosis of macrophages in vitro.Finally,after incubating NCC with r On CD94,the expression of NCC effector including granzyme,perforin-1,CAS,FADD,TNF-α,Fas L and lymphphotoxin-α were up-regulated.These results indicated that OnCD94 participates in the anti-bacterial immune response and the regulation of NCC activity of Nile tilapia.Our findings lay a theoretical foundation for the study of fish anti-bacterial immune response and non-specific cytotoxic cells regulatory mechanism. |
