Molecular Cloning, Expressional Characterization And Functional Analysis Of Ctenopharyngodon Idella PKR(CiPKR)

Innate immunity constitutes the first line of quickly identify invasion of pathogens and anti-microbial host defense, it plays an important role in controlling the spread of pathogens and eliminate pathogens in vivo. Type Ⅰ interferons (IFNs) are primary cytokines produced after viral infections, which induce an antiviral state in neighboring cells by upregulating transcription of IFN-stimulated genes (ISGs), including dsRNA activated protein kinase(PKR),2’,5’-oligoadenylate synthetase, RNase L, ADAR and the Mx protein. Wherein, PKR is a key molecule in the signal transduction process of IFN antiviral function. Stojdl has proved PKR is the one of the effect protein in anti-viral signaling mechanisms of Type Ⅰ IFNs is mediated.In this paper, we have cloned PKR gene from grass carp (CiPKR, JX511974) by homology cloning and RACE. The cDNA of CiPKR has2436bp in length with170bp5’UTR,199bp3’UTR and a largest open reading frame (ORF) of2067bp. The ORF encoded688aa. The sequence analysis of the predicted protein revealed that CiPKR contained three tandem dsRNA-binding motifs (dsRBMs) at N-terminus regulatory region; C-terminus is a conserved Ser/Thr kinase domain. A phylogenetic tree found that CiPKR was highly homology with Carassius auratus and Danio rerio. CiPKR was displayed a low constitutive expression in tissues and cells but it was significantly up-regulated in response to Poly Ⅰ:C challenge.In order to understand more function of CiPKR, we cloned cDNA coding for dsRBMs of CiPKR. Then inserted into the expression vector pET-32a and obtained various purified peptides (PdsRBMs) by using affinity chromatography. The dimerization ability of PdsRBMs was analyszed by a12%native Polyacrylamide gel. The results showed that all of PdsRBMs could form dimer. Howere, the degree of polymerization was difference. Meanwhile, after pcDNA3.1/PKR recombinant plasmids and PGL-3-promoter were transiently cotransfected into CIK cells, we found that CiPKR could obvious restrain the level of luciferase in the cells. On the other hand, after pcDNA3.1/PKR recombinant plasmids were transiently transfected into CIK cells, we found that cell viability was significantly reduced when transfected CiPKR by using MTT assay. These results showed that CiPKR overexpression was able to inhibit protein translation and then induce apoptosis.