Transcription Regulation Analysis Of Type Ⅰ IFN Gene By IRF-7in Grass Carp(Ctenopharyngodon Idella)

Interferon (IFNs) are a family of multi-functional glycoprotein that play diverse role in anti-virus, anti-tumor, immune regulation and so on. Interferon regulatory factors (IRFs) owe their name to their discovery as transcription factors for type I interferons (IFNS).IRF-1, IRF-5and IRF-7are transcription mediators which would play an important role in inducing the expression of IFN and IFN-stimulated genes (ISGs). Many studies have identified regulation mechanism of IRFs in mammals, but there are little in fish. Grass carp (Ctenopharyngodon idella) is an important commercial freshwater fish in China. This paper describes the type I interferon regulation mechanism by CiIRF-7.We have cloned and identified the complete genomic sequence and the promoter sequence of grass carp type I interferon. The type I interferon gene promoter sequence of grass carp contains a typical TATA box, four IRF-E binding sites, two NF-κB sites as well as a ATF-2/c-Jun binding sites. According to the structural features of CiIFN promoter, we have constructed nine different length CiIFN promoter sequences CiIFNP1-P9which containing different elements, respectively. Based on the cDNA sequences of CiIRF-7, we detected the expression levels of CiIRF-7in grass carp immunologic tissue liver, spleen and kidney.Real-time PCR analysis revealed that CiIRF-7displayed a low constitutive expression in all the tissues tested. After stimulation by Poly I:C, the expression of CiIRF-7was significantly up-regulated. We expreesed and purified the recombinant protein CiIRF7DBD.The recombinant polypeptides of CiIRF7DBD were analyzed in gel mobility shift assays(EMSA), along with the PCR amplification products of CiIFN promoter sequence CiIFNP2,6and7. The results revealed that CiIRF-7could bind to different regions of CiIFN promoter sequence in vitro. Subsequently, CiIFNPs (CiIFNP2/6/7) were separately transiently co-transfected with CiIRF-7into the mouse myeloma cell lines SP2/0and the grass carp kidney cell lines (CIK), and the impact of CiIRF-7on CiIFN promoter activity was measured by luciferase assays in the transfected cells. These results demonstrated that CiIRF-7acted as a positive regulator on the transcription of CiIFN.We also dected the expression levels of CiIRF-1in grass carp liver, spleen and kidney. CiIRF-1displayed a low constitutive expression in all the tissues tested, and stimulated by Poly I:C. At the same time, we expreesed and purified the recombinant protein CiIRF5DBD. The EMSA experiment show that CiIRF-5can bind to different CiIFN promoter sequences.