| Fish,cold blooded vertebrates,have some very unique immune factors and immune system.For example,there are PKR and its duplication PKZ co-exist in fish genomes.PKR(double-stranded RNA-dependent protein kinase)is a dsRNA receptor in cell,and PKZ(protein kinase containg Z-DNA binding domain)is a newly discovered Z-DNA/Z-RNA recognition receptors.PKR and PKZ belong to ISGs(interferon stimulated genes)family,Both of them work synergistically in the antiviral defense by inhibiting intracellular proteins translation.IRF3(interferon regulatory factor 3)and IRF7(interferon regulatory factor 7)belong to IRFs(interferon regulatory factors)family,The transcriptional factor IRF3 and IRF7 acts as a key regulator of type I IFN(Interferon)and ISGs.IRF3 and IRF7 play a critical role in innate immune response defense against DNA and RNA virus infection.In order to understand the molecular mechanisms of Ctenopharyngodon idella IRF3 and IRF7 on the transcriptional regulation of CiPKR and CiPKZ,we examine the possible virus-induced change of CiPKR and Ci PKZ mRNA,RT-PCR data display the relative mRNA level of CiPKR and CiPKZ in CIK cells stimulated with poly I:C for 0 h,3 h,6 h,12 h,24 h,48 h and 72 h.The inducers resulted in the up-regulation of CiPKR and CiPKZ in the similar trend.In detail,CiPKR was up-regulated at 3 h post-poly I:C stimulation,peaked at 6 h(5.0-fold increase than the control),subsequently returned to the control level at 48 h post-induction.CiPKZ expression was up-regulated gradually from 3 h to 24 h,and peaked at 24 h post-treatment(which was 29.3-fold increase than the control),and then dropped and returned to the control level gradually at 48 h.Full length of CiPKZ promoter(KJ704844)and CiPKR(KJ704845)were obtained and stored in our lab.In this paper,the TRANSFAC database make a predictive analysis,discovering there were two ISRE elements in CiPKR promoter,one ISRE element in CiPKZ promoter.To study the affinity of CiIRF3 and CiIRF7 with promoters of CiPKR,CiPKZ and two mutational promoters(CiPKR-nISRE and CiPKZ-nISRE).we obtain the type CiPKR and CiPKZ,and the truncated mutants,CiPKR-nISRE-pro and CiPKR-nISRE-pro which lacked ISRE element according to the promoter sequences of CiPKR(KJ704845)and CiPKZ(KJ704844).On the basis of the cloned CiIRF3 and CiIRF7 previously,CiIRF3 and CiIRF7 with His-tag was over-expressed in BL21 Escherichia coli,and the expressed protein was purified by affinity chromatography with Ni-NTA His-Bind Resin.In vitro,Gel mobility shift assays demonstrated that CiIRF3 and CiIRF7 could combine CiPKR and CiPKZ promoters with high affinity,However,CiIRF3 and CiIRF7 bound to the mutants CiPKR-nISRE and CiPKZ-nISRE faintly.Whereafter,the recombinant plasmids of pGL3-Ci PKR-luc and pGL3-CiPKZ-luc were transiently co-transfected with pcDNA3.1-CiIRF3,pcDNA3.1-CiIRF7 respectively into C.idella kidney(CIK)cells.Cell transfection assays indicated that CiIRF3 and CiIRF7 up-regulated the transcriptional level of CiPKR and CiPKZ.We guess CiIRF3 and CiIRF7 may regulate PKR and PKZ by combining to the ISRE element.In order to comprehend the effect of ISRE element on the transcriptional regulation of CiPKR and CiPKZ further,we also constructed the mutant(pGL3-CiPKR-nISRE-luc,pGL3-CiPKZ-nISRE-luc)reporter gene vectors according to the CiPKR-nISRE-pro and CiPKR-n ISRE-pro which lacked ISRE element.Subsequently,the recombinant plasmids of pGL3-CiPKR-luc,pGL3-CiPKZ-luc,pGL3-Ci PKRnISRE-luc and pGL3-CiPKZ-nISRE-luc were transiently co-transfected with pcDNA3.1-CiIRF3,pcDNA3.1-CiIRF7 respectively into C.idella kidney(CIK)cells.The results revealed that the mutant CiPKR-nISRE and CiPKR-nISRE promoters which lacked ISRE element,reduced these genes transcription activity obviously.It explains the consensus sequence of ISRE element was an important regulatory element for the transcriptional initiation of Ci PKR and CiPKZ,And fish IRF3 and IRF7 was a kind of transcriptional regulator of ISG transcription and performed in an IFN-independent manner. |
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