PREPARATION OF POLYCLONAL ANTIBODY AND VIRUS2INDUCED TISSUE EXPRESSION OF PARALICHTHYS OLIVACEU HRI

The heme2regulated initiation factor 2α (eIF2α) kinase (HRI) is one of Ser/ Thr kinases that phosphorylate the α-sub- unit of eIF to inhibit protein synthesis in eukaryotic cells under various stress conditions, including heme deficiency and heat shock. HRI is firstly discovered in rabbit reticulocytes as an inhibitor of globin synthesis under heme deficiency, but so far little is known about the importance of HRI in stress conditions other than heme deficiency and the roles of HRI in nonerythroid tissues. For example, there was an argument on the tissue specificity of HRI expression due to the fact that no HRI protein expression was reported in nonerythroid tissues and a finding that in HRI knockout mice, only red blood cells(RBCs) and their precursors were directly affected by the lack of HRI. Paralichthys olivaceu HRI is the first identified fish homologue, and a previous study showed for the first time thatPoHRI could be induced by virus infection in vitro. In order to further investigate whetherPoHRI protein is also synthesized in nonerythroid tissues and can be induced by virus infection in vivo, here a anti-PoHRI polyclonal antibody was prepared and subsequently used to detect the expression ofPoHRI in different tissues of flounder. Firstly,the cDNA fragment encoding 12200 amino acids ofPoHRI was cloned and integrated into a prokaryotic expression vector pET32a. Next, a recombinant protein similar to the expected size was induced and then purified by Ni+2-NTA chromatography. The anti-PoHRI polyclonal antibody was then prepared by immunization of mice. The specific recognition of anti-PoHRI polyclonal antibody toPoHRI was further verified by a test in which anti-PoHRI antiserum, after pre-adsorbed with recombinantPoHRI peptide, cannot recognize the corresponding polypeptide. In healthy flounder tissues, the constitutive expression ofPoHRI was able to be detected at both mRNA and proteins levels, and SMRV infection was able to induce the upregulation ofPoHRI in the corresponding tissues, especially in head kidney and spleen. These results support the notion thatPoHRI is ubiquitously expressed in flounder tissues, which indicates that it may play an important role in cellular antiviral immune response. It is well known that PKR is the primary eIF2αkinase responsive to virus infection; however, previous reports also showed that the phosphorylation of eIF2αin cultured mammalian cells in response to heat shock was apparently due to the activation of eIF2α kinases including HRI and PKR, indicating that HRI and PKR were likely redundant and could functionally replace one another under some conditions. In fact, a recent study revealed that GCN2, another eIF2α kinase, participate in inhibition of sindbis virus (SV) replication by phosphorylating eIF2αto block early viral translation of genomic SV RNA. However, GCN2 was originally characterized as being required for amino-acid control of GCN4 mRNA. Therefore, the upregulation of PoHRI by virus infection indicates thatPoHRI may exert inhibition of protein synthesis in virus-infected flounder, a function similar to mammalian PKR. In addition,PoHRI might exhibit a new uncharacterized, heme-independent function other than translational shutoff under virus infection, since mammalian NF-κB can be activated by HRI through phosphorylation of inhibitor IκB. Therefore, further studies will be needed to understand the importance ofPoHRI in flounder under virus infection.