Cloning, Identification Of Cytokine Receptor Family B Subunits CRFB1 And CRFB5 From Grass Carp (Ctenopharyngodon Idella)

Grass carp is one of the most economically important species in China’s freshwater aquaculture. In breeding process, the high mortality of viral infection has been tormented aquaculture workers. IFN is a cytokine which has multifunction and various biological activities. The recombinant IFN producted by genetic engineering technique has been widely applied to control viral diseases of human and economic animals. Among them, type I IFN plays a vital role in cell antiviral activity. Similar to the mammalian counterparts, fish type I interferon(IFN) performs its potential biological activities by combining with its corresponding receptor on cell membrane. Fish type I IFN receptor, a kind of enzyme-linked receptor, consists of two subunits. Insight into the grass carp I IFN receptor will help us better understand fish IFN signaling pathway and provide theoretical basis for the healthy breeding of fish.Based on zebrfish type I interferon receptor subunits CRFB1 and CRFB5, we cloned and identified two putative grass carp(Ctenopharyngodon idella) type I interferon receptor subunits(termed Ci CRFB1 and Ci CRFB5) by homology cloning techniques. The full length c DNA sequences of Ci CRFB1 and Ci CRFB5 are 2945 bp and 1517 bp respectively. Through the sequences analysis,we found that Ci CRFB1 and Ci CRFB5 exhibits 74 %, 70 % identity to the known Dr CRFB1(EF014952), Dr CRFB5(EF014955) respectively. Phylogenetic analysis results also showed that Ci CRFB1 and Ci CRFB5 had higher homology to Danio rerio CRFB1 and CRFB5 respectively compared to the counterparts of the other species. Structurally, both Ci CRFB1 and Ci CRFB5 are composed of a relatively conserved extracellular domain of about 200 amino acids containing two fibronectin type III motifs connected by a linker. Near the extracellular domain, there is a single transmembrane domain behind it. The intracellular domain of Ci CRFB1(279 aa) is far longer than that of Ci CRFB5(129 aa). Ci CRFB1 and Ci CRFB5 were up-regulated after two triggering stimulus, Grass Carp Hemorrhagic Virus(GCHV) and Polyinosinic-polycytidylic acid(Poly I:C), indicating that they are associated with the intracellular antiviral activity.In order to further understanding the roles of Ci CRFB1 and Ci CRFB5 in antiviral activity, the extracellular domains of Ci CRFB1(Ci CRFB1-EC) and Ci CRFB5(Ci CRFB5-EC), as well as grass carp type I IFN(Ci IFN) were expressed in Escherichia coli BL21, and purified through affinity chromatography using the Ni-NTA His-Bind Resin. Cross-linking reactions were employed to analyze the affinity of the ligand(Ci IFN) with the two putative receptor subunits(Ci CRFB1-EC and Ci CRFB5-EC). The result suggested the formation of(Ci CRFB5)2 homodimer was more easily than that of(Ci CRFB1)2 under the induction of Ci IFN in vitro. However, Ci IFN was inclined to bind to(Ci CRFB1)2 homodimer. Interestingly, although Ci IFN seemed unable to facilitate the formation of(Ci CRFB1+Ci CRFB5) heterodimer in the absence of DSS cross linker, however it can bind to the heterodimer in the present of DSS. This indicated that the homodimer and the heterodimer were the potential receptor for Ci IFN.