牙鲆HRI多克隆抗体的制备及病毒诱导的组织表达分析

PREPARATION OF POLYCLONAL ANTIBODY AND VIRUS-INDUCED TISSUE EXPRESSION OF PARALICHTHYS OLIVACEU HRI

血红素调节的eIF2α激酶HRI是一种丝氨酸/苏氨酸蛋白激酶,在血红素缺乏和热休克等胁迫存在时,通过磷酸化底物eIF2α调控真核细胞的蛋白合成。牙鲆PoHRI是在鱼类鉴定的第一个HRI基因。PCR克隆编码PoHRI蛋白1-200氨基酸的cDNA片段,定向插入pET32a构建表达载体。IPTG诱导,随后利用Ni2+-NTA亲和层析柱纯化,成功获得预期大小的融合蛋白。将纯化的融合蛋白免疫小鼠获得抗PoHRI多克隆抗体,抗体中和实验进一步证实了抗PoHRI多克隆抗体对PoHRI蛋白的特异性识别。在健康的牙鲆组织中能检测到PoHRI mRNA和蛋白的组成型表达。人工感染大鳞鲆弹状病毒SMRV诱导相应组织PoHRI的表达明显上调。结果表明,PoHRI是一种在牙鲆组织中广泛表达的蛋白,可能在抗病毒免疫反应中发挥某种功能。

The heme-regulated initiation factor 2a （elF2a） kinase （HRI） is one of Ser/Thr kinases that phosphorylate the a-subunit of elF2 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 that Po HRI could be induced by virus infection in vitro. In order to further investigate whether PoHRI 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 of PoHRI in different tissues of flounder. Firstly, the cDNA fragment encoding 1-200 amino acids of PoHRI 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 Ni2^＋ -NTA chromatography. The anti-Po HRI polyclonal antibody was then prepared by immunization of mice. The specific recognition of anti-PoHRI polyclonal antibody to PoHRI was further verified by a test in which anti-PoHRI antiserum, after pre-adsorbed with recombinant PoHRI peptide, cannot recognize the corresponding polypeptide. In healthy flounder tissues, the constitutive expression of PoHRI was able to be detected at both mRNA and proteins levels, and SMRV infection was able to induce the upregulation of Po HRI in the corresponding tissues, especially in head kidney and spleen. These results support the notion that Po HRI 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 eIF2a kinase responsive to virus infection; however, previous reports also showed that the phosphorylation of eIF2a in cultured mammalian ceils in response to heat shock was apparently due to the activation of eIF2a 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 eIF2a kinase, participate in inhibition of sindbis virus （SV） replication by phosphorylating eIF2a to 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 Po HRI by virus infection indicates that Po HRI may exert inhibition of protein synthesis in virus-infected flounder, a function similar to mammalian PKR. In addition, Po HRI 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 of Po HRI in flounder under virus infection.