Department of Bioscience, College of Life Science and Food Engineering, Nanchang University, Nanchang 330031, China; Medical College, Jinggangshan University, Ji'an 343009, China.
Fish Shellfish Immunol. 2013 Dec;35(6):1874-81. doi: 10.1016/j.fsi.2013.09.024. Epub 2013 Sep 29.
The interferon-induced, dsRNA-activated protein kinase (PKR) is considered as an important component of innate immune system and as a representative effector protein of interferon system. In the present study, PKR gene (CiPKR, JX511974) from grass carp (Ctenopharyngodon idellus) was isolated and identified using homology-based PCR. CiPKR shares high sequence identity with the counterparts of goldfish (Crucian carp) and zebrafish (Danio rerio). The full-length cDNA of CiPKR was found to be 2436 bp, with an ORF of 2067 bp that encodes a polypeptide of 688 amino acids. The deduced polypeptide CiPKR contains three tandem dsRNA-binding motifs (dsRBMs) at the N-terminus and a conserved Ser/Thr kinase domain at the C-terminus. CiPKR was expressed ubiquitously at a low-level under normal conditions, but it could be up-regulated after intraperitoneal (ip) injection with grass carp haemorrhagic virus (GCHV). CiPKR was dramatically up-regulated at 6 h post-injection and then recovered rapidly to normal levels within 24 h; however, it was obviously up-regulated once again at 48 h or 72 h post-injection. It seemed that CiPKR could respond to GCHV infection in an IFN-independent as well as an IFN-dependent pathway. To further investigate its mechanism of biological actions, we constructed a series of recombinant plasmids including pcDNA3.1/PKR-wt, pcDNA3.1/PKR-K430R, pcDNA3.1/PKR-C (deletion of dsRBD sequence) and pcDNA3.1/PKR-C-K430R, and then each recombinant plasmid was transfected into CIK cells. In comparison with those of controls, a 79% and a 64% decrease of luciferase activities were detected in the tested cells transfected with CiPKR and CiPKR-C, respectively; however, luciferase activities were increased in those cells transfected with PKR-K430R and PKR-C-K430R, with a 160% and 115% up-regulation, respectively. Similarly, MTT colorimetric assay indicated that cell viabilities of CIK cells transfected with pcDNA3.1/PKR-wt, pcDNA3.1/PKR-K430R, pcDNA3.1/PKR-C and pcDNA3.1/PKR-C-K430R were 49%, 90%, 54% and 100%, respectively. Our observations suggested that the expression of CiPKR could be up-regulated following viral infection, and then resulted in the inhibition of protein synthesis and the induction of potential apoptosis.
干扰素诱导的双链 RNA 激活蛋白激酶（PKR）被认为是先天免疫系统的重要组成部分，也是干扰素系统的代表性效应蛋白。本研究采用基于同源性 PCR 的方法，从草鱼（Ctenopharyngodon idellus）中分离并鉴定了 PKR 基因（CiPKR，JX511974）。CiPKR 与金鱼（Carassius auratus）和斑马鱼（Danio rerio）的对应物具有很高的序列同一性。CiPKR 的全长 cDNA 为 2436 bp，ORF 为 2067 bp，编码 688 个氨基酸的多肽。推定的 CiPKR 多肽在 N 端包含三个串联的双链 RNA 结合基序（dsRBMs），在 C 端包含一个保守的丝氨酸/苏氨酸激酶结构域。正常情况下，CiPKR 低水平广泛表达，但经腹腔（ip）注射草鱼出血病毒（GCHV）后可被上调。注射后 6 小时 CiPKR 显著上调，24 小时内迅速恢复正常水平；然而，在 48 小时或 72 小时再次明显上调。CiPKR 似乎可以通过 IFN 非依赖性和 IFN 依赖性途径对 GCHV 感染做出反应。为了进一步研究其生物学作用机制，我们构建了一系列重组质粒，包括 pcDNA3.1/PKR-wt、pcDNA3.1/PKR-K430R、pcDNA3.1/PKR-C（dsRBD 序列缺失）和 pcDNA3.1/PKR-C-K430R，然后将每个重组质粒转染至 CIK 细胞。与对照相比，转染 CiPKR 和 CiPKR-C 的细胞的荧光素酶活性分别降低了 79%和 64%；然而，转染 PKR-K430R 和 PKR-C-K430R 的细胞中的荧光素酶活性增加，分别上调了 160%和 115%。同样，MTT 比色法测定表明，转染 pcDNA3.1/PKR-wt、pcDNA3.1/PKR-K430R、pcDNA3.1/PKR-C 和 pcDNA3.1/PKR-C-K430R 的 CIK 细胞的细胞活力分别为 49%、90%、54%和 100%。我们的观察表明，病毒感染后 CiPKR 的表达可被上调，从而导致蛋白质合成抑制和潜在凋亡诱导。
