Cloning, expression and functional analysis of PKR from grass carp (Ctenopharyngodon idellus)
- Authors
- Hu, Y.S., Li, W., Li, D.M., Liu, Y., Fan, L.H., Rao, Z.C., Lin, G., and Hu, C.Y.
- ID
- ZDB-PUB-131105-8
- Date
- 2013
- Source
- Fish & shellfish immunology 35(6): 1874-81 (Journal)
- Registered Authors
- Keywords
- CiPKR, dsRNA, Antiviral activity, Interferon, Grass carp
- MeSH Terms
-
- Amino Acid Sequence
- Animals
- Base Sequence
- Carps/genetics*
- Carps/immunology*
- Cloning, Molecular
- Cytokine-Induced Killer Cells/cytology
- Cytokine-Induced Killer Cells/virology
- DNA, Complementary/genetics
- DNA, Complementary/metabolism
- Fish Proteins/chemistry
- Fish Proteins/genetics*
- Fish Proteins/metabolism
- Gene Expression Regulation
- Molecular Sequence Data
- Organ Specificity
- Phylogeny
- RNA, Messenger/genetics
- RNA, Messenger/metabolism
- Real-Time Polymerase Chain Reaction/veterinary
- Reoviridae
- eIF-2 Kinase/chemistry
- eIF-2 Kinase/genetics*
- eIF-2 Kinase/metabolism
- PubMed
- 24084043 Full text @ Fish Shellfish Immunol.
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.