Molecular Cloning Of IRF3 And IRF7 Gene Of Black Carp And Preliminary Studying On Their Functions

Vetrebrates take advantage of the immune system to survive from the adverse environment such as the invasion of pathogenic microbes, in which the immunity can be divided into innate immunity and adaptive immunity. The innate immunity refers to the non-specific immune ability, which is endowed with individuals when they are born and functions in a wide range. It plays a significantly important role in defensive mechanism of immune system. Innate immunity contains numerous pattern recognition receptors(PRRs) which could recognize different pathogen-associated molecular patterns(PAMPs)and rapidly activate immune responses of organisms by different signal pathways.Interferons are anti-virus cytokines that possess important functions in innate immunity and adaptive immunity of vertebrates. Interferon Regulatory Factors(IRFs) are transcription factors which regulate IFN expression and participate in the inflammatory response in organisms. IRF3 and IRF7 are significant members of IRF family, which are the key transcription factors regulating the typeⅠinterferon expression. The PRRs in host cells recognize the invading pathogenic microorganisms and activate IKKε(inhibitor of NF-kB kinase ε) and TBK-1(TANK-binding kinase 1) through downstream signaling. The activated IKKε and TBK-1 phosphorylate IRF3 and IRF7, which transfer into nuclear and induce the production of IFN, thus inducing the host antiviral response in innate immunity.Black carp(Mylopharyngodon piceus)is one of the most important commercial fishes as well as one of the “four famous domestic fishes” in China. In this study, black carp IRF3(BC-IRF3) and black carp IRF7(BC-IRF7) were cloned and characterized for the first time. The contents of this paper are shown as below:1. The full length cDNA of BC-IRF3 is 1579 bp and consist of a coding region of 1344 bp, a 5’ untranslated region(UTR) of 138 bp and a 3’UTR of 83 bp. The predicted BCIRF3 contains 448 amino acids.2. The full length cDNA of BCIRF7 is 1901 bp and consist of a coding region of 1275 bp, a 5’ untranslated region(UTR) of 78 bp and a 3’UTR of 538 bp. The predicted BCIRF7 contains 425 amino acids.3. The protein sequence of BC-IRF3 was compared with those of other vertebrates. The comparison result shows that IRF3 is a highly conserved gene in cyprinid fish. BC-IRF3 shares most similarities with Cyprinid fish IRF3 and least similarities with mammals IRF3.4. The protein sequence of BC-IRF7 was compared with those of other vertebrates. The comparison result shows that IRF7 is a highly conserved gene in cyprinid fish. BC-IRF7 shares most similarities with Cyprinid fish IRF7 and least similarities with mammals IRF3.5. The recombinant plasmids expressing BC-IRF3 or BC-IRF7 were constructed respectively, which includes pcDNA5- FRT/TOHA-BC-IRF3, pc DNA5-FRT/TO-BC-IRF3-HA, pcDNA5-FRT/TOFlag-BC-IRF7 and pcDNA5-FRI/TO-BC-IRF7-Flag. HEK293 T cells were transfected with the above plasmid through calcium orthoph osphates method separately and BC-IRF3/7 protein expressions wer e identified by western blot assay. The molecular weight of BCIR F3 is about 60 KDa and that of BCIRF7 is around 60 KDa.6. The result of Dual-luciferase reporter assay shows that BC-IRF3 up-regulate the transcriptional activity of zebra fish IFN1 promoter. The up-regulation folds are from 35 to 130.7. The result of Dual-luciferase reporter assay shows that BC-IRF7 up-regulate the transcriptional activity of zebra fish IFN3 promoter. The up-regulation folds are from 12 to 27.Above all, this study cloned and characterized BC-IRF3 and BCIRF7, which have built a firm foundation for future systematic functional study of IRF3 and IRF7 of black carp and other fishes.