Expression Of CaPKR-like Za Domains And Its Affinity To Nucleic Acids

PKR is an dsRNA-dependent protein kinase that is induced by interferon treatment.It was found to be present in almost all mammalian cells.A fish PKR-like gene,named CaPKR-like,was the first identified fish gene most similar to mammalian PKRs.It has 2192 base pairs in length with a largest open reading frame (ORF) encoding a protein (CaPKR-like )of 513 amino acids residues. CaPKR-like possesses a conserved C-terminal catalytic domain. Within its N-terminus, there is no dsRNA-binding domains conserved in mammalian PKRs instead of two putative Z-DNA binding domains (Za).In order to realize the function of its N-terminus, three kind of Za cDNAs were cloned from SMART-cDNA library induced by UV-inactivated GCHV(grass carp hemorrhage virus).Three kind of fusion peptides (P_Zα1, P_Zα2, P_Zα1Zα2 were expressed to heel and purified by affinity chromatography. Gel retardation assay showed: neither P_Zα1 nor P_Zα2 could bind to Poly I:C and DNA, but P_Zα1Zα2 could bind to Poly I:C and DNA strongly. In vitro, P_Zα2 and P_Zα1Zα2were much easier to dimerize than P_Zα1.Multiclonal antibodies was prepaired from rabbit immunized by purified P_Zα1Zα2 Western blotting showed that CaPKR-like had middle strong expression in almost all tissues and CAB cells treated by Poly I:C. But there was low level expression in CAB cells without treated by Poly I:C.