Identification Of A Nkef Homolog From Nile Tilapia (Oreochromis Niloticus) And Functional Analysis Of Activation Regulatory In NCC

NKEF?Natural Killer Enhancing Factor?-A/B is an antioxidant protein of Peroxiredoxin?Prxs?family which first identified in yeast,and named by enhancing cytotoxic of NK cells.NKEF play essential roles in physiology and pathology process and involved in inflammation regulation,cancer development and development,and redox reactions.NCC?Nonspecific Cytotoxic Cell?is the evolutionary precursor of mammalian NK cell,which has multiple important functions in innate immunity of teleost.But it is far unknown of activation regulatory in tilapia NCC.In this study,a NKEF homolog from Nile tilapia has been cloned and made an investigation about the function of activation regulatory in NCC,and achieved the following results:1.The full nkef cDNA?Accession No.MK584553,named On-NKEF?was cloned from Nile tilapia,which was 1247 bp,composed a 57 bp length 5'untranslated region?5'-UTR?,620 bp 3'-UTR and 597 bp ORF encoded a putative protein consisting 198 amino acid with a calculated molecular mass of 22.085 kDa and a pI of 6.51.The conserved domain analysis revealed that On-NKEF contains a typical 2-Cys family domain,two active sites were at 51_st and 172 _nd.This protein was very conserved in mammals,birds,amphibians and fish,the homology is above 80%.Phylogenetic analysis showed that it had the closest relationship with Zebra mbuna?Maylandia zebra?.2.The mRNA expression of On-NKEF could be tested in all selected tissues or organs,the relative highest expression was found in head kidney lymphocytes and NCCs,and in organs,the highest expression was found in gill and spleen,the lowest was in skin.After the stimulation of Streptococcus agalactiae,it was significantly up-regulated in head kidney,spleen,gill and skin.We also isolated NCCs from head kidney which cultured in medium.On-NKEF was also significantly up-regulated at 24 hours post Streptococcus agalactiae,LPS and poly?I:C?stimulation in NCCs.The recombinant plasmid was transfected in FHM cells,it could be observed that On-NKEF was the punctate expression in the cytoplasm.3.Through transfection,we found that On-NKEF could induce cell proliferation and significantly inhibit the NF-?B pathway.rOn-NKEF could induce cell proliferation in B cells,macrophages and NCCs,moreover it could up-regulate NCC effector molecules,such as perforin-1,granzyme,FasL,TNF?,lymphotoxin?,FADD and CAS,it is inferred that on-NKEF could enhance the cytotoxicity of NCC by up-regulating the expression of effector molecules.The above results indicate that On-NKEF plays an important role in the regulation of tilapia NCC activity,providing new ideas and theoretical support for further exploration of the antibacterial immune mechanism of fish.