Cloning,Identification And Function Analysis Of Grass Carp(Ctenopharyngodon Idella) Ripk1

RIPK1(receptor-interacting serine/threonine-protein kinase 1)is a kind of serine/threonine kinase.It plays an important role in cell death and inflammation.In mammals,RIPK1 participates in TLR(toll-like receptor)and RLR(RIG-like receptor)pathway to activate NF-κB pathway and induce type I IFN expression.Recent study found that cleavage of RIPK1 by Caspase8 could regulate the related apoptosis pathway and necrosis pathway.Meanwhile,Caspase8 could down-regulated the IFN I expression via cleaving RIPK1.But,compared with mammals,the studies of RIPK1 are limited in lower vertebrates such as fish.Therefore,this study mainly explored the function of RIPK1 in innate immunity.We specifically studied its regulation on the key cytokines IFN1 to understand the molecular machanism of grass carp innate immunity.This study provides the basis for disease resistance and breeding of fish.The open reading frame(ORF)of grass carp ripk1 was cloned by homologous cloning.The ORF is 2019 BP long and encodes 672 amino acids.The phylogenetic tree showed that RIPK1 was relatively conservative in many species,but the similarity between cyprinid fishes RIPK1 and other species RIPK1 was low.Multiple sequence alignment further found that the N-terminal and C-terminal of RIPK1 in grass carp were more conservative,while the intermediate domain was less conservative.The expression of ripk1 in grass carp was detected by q PCR experiment.It was found that Ciripk1 was significantly up-regulated by poly(I:C)in both tissues and Ctenopharyngodon Idella kidney(CIK)cells.Ciripk1 was highly expressed in most tissues at 6 h,while Ciripk1 was highly expressed in cells at 12 h.Overexpression experiment in CIK cells showed that CiRIPK1 could up regulate the expression of ifn1,while Nec-1,a RIPK1 specific inhibitor,inhibited the expression of ifn1.Further studies showed that the overexpression of CiRIPK1 without kinase domain did not upregulate of ifn1,and CiRIPK1 without kinase domain inhibited the up regulation of ifn1 under poly(I:C)stimulation.In order to study whether the regulation of CiRIPK1 on interferon is related to Ci Caspase8,the expression of Cicaspase8 was detected.It was found that Cicaspase8 could respond to poly(I: C)stimulation in all tested tissues.Meanwhile,overexpression of Ci Caspase8 in cells verified its negative regulation on ifn1.Treatment with z-VADfmk,a Caspase8 inhibitor,significantly up-regulated ifn1 in CIK cells,while the upregulation was inhibited by Nec-1.PI-hoechst staining and CCK-8 cell activity test found that the two inhibitors did not cause significant apoptosis or programmed necrosis.The cleavage site of CiRIPK1 was predicted,and a variety of mutant plasmids were constructed by point mutation experiment.Western blot analysis showed that aspartic acid at position 369 of CiRIPK1 might be the key cleavage site of Caspase8.Subcellular localization showed that CiRIPK1 protein existed in the cytoplasm of cells.Co-IP experiments showed that CiRIPK1 could interact with Ci TBK1,suggesting that CiRIPK1 may have the similar regulation funciton to mammals,and it regulated the expression of ifn1 through TBK1.It is worth mentioning that CiRIPK1 overexpression can also up regulate the expression of tnf-α,Z-VAD-fmk can up regulate the expression of tnf-α in CIK cells,and Nec-1 can inhibit this up regulation.